key: cord- -yjaiybwf authors: sachsenröder, jana; twardziok, sven o.; scheuch, matthias; johne, reimar title: the general composition of the faecal virome of pigs depends on age, but not on feeding with a probiotic bacterium date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: yjaiybwf background: the pig faecal virome, which comprises the community of viruses present in pig faeces, is complex and consists of pig viruses, bacteriophages, transiently passaged plant viruses and other minor virus species. only little is known about factors influencing its general composition. here, the effect of the probiotic bacterium enterococcus faecium (e. faecium) ncimb on the pig faecal virome composition was analysed in a pig feeding trial with sows and their piglets, which received either the probiotic bacterium or not. results: from pooled faecal samples derived from the feeding trial, dna and rna virus particles were prepared and subjected to process-controlled next generation sequencing resulting in , sequence reads. in average, % of the reads showed significant sequence identities to known viruses. the percentage of detected mammalian virus sequences was highest ( – %) in the samples of the youngest piglets and lowest ( – %) in the samples of the sows. in contrast, the percentage of bacteriophage sequences increased from – % in the youngest piglets to approximately % in the sows. the dominating mammalian viruses differed remarkably among day-old piglets (kobuvirus), day-old piglets (boca-, dependo- and pig stool-associated small circular dna virus [pigscv]) and the sows (pigscv, circovirus and “circovirus-like” viruses cb-a and rw-a). in addition, the shannon index, which reflects the diversity of sequences present in a sample, was generally higher for the sows as compared to the piglets. no consistent differences in the virome composition could be identified between the viromes of the probiotic bacterium-treated group and the control group. conclusion: the analysis indicates that the pig faecal virome shows a high variability and that its general composition is mainly dependent on the age of the pigs. changes caused by feeding with the probiotic bacterium e. faecium could not be demonstrated using the applied metagenomics method. the viral community present in faeces is composed of a variety of viruses originating from the gut tissue, from intestinal microorganisms or from ingested food. the totality of viruses present in faeces has also been frequently designated as the faecal virome [ , ] . the functions of the faecal virome are supposed to be manifold, which include roles for the viruses as pathogens, regulators of bacterial growth, gene-transfer vehicles and modulators of the immune system [ ] [ ] [ ] [ ] . early insights into the composition of the human faecal virome were provided by random cloning strategies [ , ] . later on, the availability of deep sequencing methods lead to more comprehensive analyses of faecal viromes [ , ] , including the development of processcontrolled techniques enabling comparison of different analyses [ ] . the composition of the faecal virome of pigs has been studied recently [ , , ] . although samples derived from different continents had been analysed in these studies, the general composition was found to be similar. the majority of the detected virus sequences belonged to bacteriophages and pig viruses. only a few sequences belonged to plant viruses as well as other viruses. most of the bacteriophage sequences originated from viruses belonging to the families siphoviridae, microviridae and myoviridae [ ] . the most abundant porcine viruses were kobuvirus, rotavirus, pig stool-associated small circular dna virus (pigscv), astrovirus, sapovirus and enterovirus b. most of them represent widely distributed enteric viruses of pigs [ , , ] . whereas rotaviruses are well-known pathogens of piglets, which may lead to diarrhoea [ , , ] , the clinical importance of the other viruses is a subject of controversy [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . only little is known about the stability and dynamics of the faecal virome under different conditions. for the human faecal virome, reyes et al. [ ] investigated the intra-and interpersonal variation by analysing faeces of monozygotic twins and their mothers at different time-points. by this, it was found that the viromes were unique to the individuals regardless of their degree of genetic relatedness. minot et al. [ ] analysed the inter-individual variation of the human faecal virome and its dynamic response to diet. it was shown that the largest source of variance among the viromes was caused by interpersonal variations and not by the diet. a high interpersonal diversity of gut bacteriophages was also described in two humans which were monitored over a . year period [ ] . in another study, a much lower diversity of the virus community was found in infants as compared to adults [ ] . although this study has been conducted by cloning followed by classical sequencing, mathematical modelling of the derived sequence data indicated that the virome of adults was composed of approximately genotypes as compared to only genotypes in one week-old infants. the observed beneficial effects of probiotic bacteria on enteric virus infections have been recently reviewed by colbere-garapin et al. [ ] . this includes clinical studies showing beneficial effects of probiotic bacteria in children with rotavirus-caused diarrhoea [ ] [ ] [ ] . feeding with probiotic microorganisms such as lactobacillus rhamnosus gg, saccharomyces boulardii or bifidobacterium lactis resulted in milder clinical symptoms, reduced virus shedding and shortened the duration of diarrhoea in children [ ] [ ] [ ] . in pigs, enterococcus (e.) faecium ncimb is a commonly used probiotic bacterium [ , ] . it has been shown recently, that feeding of pigs with this probiotic bacterium affected shedding of enteric viruses dependent on the virus species [ ] . especially, rotavirus was shed later and in lower amounts in the group of piglets that received e. faecium ncimb as compared to the control group. the specific mechanisms responsible for this effect are not known so far. however, changes in the mucosal and systemic immunity due to feeding with e. faecium ncimb have been described [ ] [ ] [ ] [ ] . in addition, direct interactions of this bacterium with enteric virus particles have been observed in in vitro studies [ ] . however, it is not known so far, whether probiotic bacteria can also influence the general composition of the faecal virome, e.g. by changing the composition of the bacterial community, which represents the host population for bacteriophages, or by direct interactions with specific viruses. the primary aim of the presented study was to analyze the effect of the probiotic bacterium e. faecium ncimb on the general composition of the faecal virome in pigs. faecal samples from sows and their piglets experimentally fed with or without the probiotic bacterium were analyzed using a process-controlled deep sequencing method. the populations of the detected virus sequences were compared between the feeding groups as well as the age groups and general insights into the stability and dynamics of the pig faecal virome under different age-related and feeding conditions were generated. the animal experiment (pig feeding trial) was approved by the local state office of occupational health and technical safety ''landesamt für gesundheit und soziales berlin'' (lageso reg. nr. / ). animal experiment and sampling scheme the design of the pig feeding trial has been described in detail by martin et al. [ ] and is schematically shown in figure . briefly, sows and their piglets received either no probiotic bacterium or approximately cfu/g e. faecium ncimb , which was fed with their diet starting at days ante partum. the sows received a commercial diet (una-hakra, hamburg, germany). additional feeding of the piglets started at days of age with a non-medicated non-commercial pre-starter diet [ ] . after weaning at days of age, they were fed with a non-commercial mash starter diet [ ] . the homogenous distribution of the probiotic in feed has been previously demonstrated by a colony hybridization assay [ ] . the faeces of sows of each group were sampled at day ante partum (before e. faecium diet) and at day post partum. faeces of their piglets ( from each group) were collected at day and at day of age (end of the experiment). piglets were euthanized at the end of the experiment by intracardial injection of a lethal dose of tetracaine hydrochloride, mebezonium iodide and embutramide (t , intervet, unterschleißheim, germany). although the whole experiment included a larger number of animals [ , ] , only faeces of piglets were analyzed, for which the faeces of their mother sows had also been analysed. the faeces of each group and time-point were pooled. the samples were stored at uc until analysis. dna was extracted from faecal samples and subsequently analyzed by real-time pcr specific for e. faecium ncimb as previously described [ ] . the standards used for quantification were prepared from negative pig faecal samples spiked with known amounts of cultured e. faecium ncimb cells as described by starke et al. [ ] results are expressed as log of cell numbers per g faeces. three different bacteriophages (m , ms , t ) were grown, titrated and used as process controls for monitoring the efficiency of the virome analysis procedure as described previously [ ] . a total of ml of the bacteriophage mixture containing approximately plaque-forming units of each bacteriophage was added per g faeces. virus particles were purified from the faecal samples by a combination of tangential flow filtration (tff) and caesium chloride (cscl) density gradient ultracentrifugation, and concentrated by centrifugal filtration and tff as described [ , ] . a total of g of pooled faecal samples from the sows were used. due to limited availability of faeces in the youngest age group, . g of the pooled faecal samples from the piglets was used. the samples were spiked with test-phages and resuspended : in smbuffer ( mm nacl, mm mgso , mm tris-hcl ph . ) by magnetic stirring. the sample was centrifuged at , g for min in order to remove the large particulate debris and the supernatant was collected. the procedure was repeated by centrifugation for hours at , g to remove smaller particular structures. afterwards, a first tff was performed using a . mm filter (pall corporation, middleton; ma, usa) to remove bacterial and eukaryotic cells and debris. the remaining filtrate was subjected to a second tff with a kda filter (pall corporation, middleton; ma, usa) in order to concentrate the virus particles. the viral preparations were further concentrated by centrifugation through vivaspin , mwco concentrators (sartorius stedim biotech gmbh, götting, germany) at , g resulting in a final volume of ml. the preparation was divided into two fractions of ml, which were added separately onto preformed stepwise caesium chloride (cscl) density gradients with density layers of . , . , . and . g/ml ( ml each) and ultracentrifuged at , g for hours at uc. the . - . g/ml layers were collected from the gradients using a syringe. to eliminate free dna present in the virus concentrate, an aliquot of ml cscl purified virus solution was treated with units dnase i ( , u/mg, bovine pancreas grad ii; roche diagnostics gmbh, mannheim, germany) for min at uc, followed by heat inactivation for min at uc. thereafter, dna and rna were extracted simultaneously using nuclisens magnetic extraction (biomerieux, nürtingen, germany). the extracted nucleic acids ( ng per reaction) were randomly primed for cdna synthesis using the transplexh complete whole transcriptom amplification kit (wta , sigma-aldrich, st. louis, mo, usa) according to the protocol recommended by the supplier; however, the annealing temperature was decreased to uc ( cycles) and uc ( cycles) in order to enable the simultaneous amplification of dna and rna [ ] . aliquots of ml each were removed from the wta reaction at different cycle numbers, purified and size-selected using mobispin s- columns (mobitec, göttingen, germany). the dna concentration was measured from the preparations using a nanodrop spectrometer (analytic jena, jena, germany) and the preparation derived from a minimum of amplification cycles with a dna concentration above ng/ml was chosen for deep sequencing. a total of mg dna was used for deep sequencing on a / plate of the gs-flx sequencer titanium (gs titanium sv empcr kit (lib-l) v ; gs titanium picotiterplate kit ; gs titanium sequencing kit xlr t; life sciences, roche, branford, usa) according to the manufacturer's protocol. the raw sequence data have been submitted to the sequence read archive (sra) at genbank as bioproject prjna with sra accession numbers srp (srx -srx ). primary sequence analysis was performed in two steps: identification of all virus species included in the samples and analysis of species abundances regarding selected sets of species. raw sequence reads were subjected to primer/adaptor trimming using seqman (dnastar, lasergene, usa) and selection for a minimum length of nt. in parallel, all primary reads were subjected to de novo contig assemble using the newbler assembler [ ] software, with criteria of % minimum overlap identity and a minimum overlap length of nt. in order to create a local database containing all virus sequences with significant homologies to the sequence reads, homology searches for all primary reads were performed with blastx [ ] against the non-redundant nucleotide database of ncbi [ ] . in parallel, homology searches for the contigs were performed with clc main workbench . [ ] against the viral genome nonredundant reference sequence nucleotide database [ ] and additional sequences from recently discovered viruses using the tblastx algorithms [ ] . from both approaches, all blast results with an e-value , = were selected and used for creation of the local sequence database. using this database, abundances of species were calculated. for bray curtis dissimilarity (see below), specific subsets, which consisted of mammalian viruses, bacteriophages or enterococcus phages, were used. in all cases, trimmed reads were mapped against the sequences of the local database to calculate species abundances with the readmapper bowtie . . [ ] . thereafter, numbers of mapped reads were corrected for multiple read assignments. the reads of the bacteriophages used as process control were subtracted from the number of the virus reads in subsequent analyses. shannon index [ ] was calculated to compare the diversity of the species identified by primary reads. the shannon index is maximal for a sample with a balanced species distribution and it has a low value for a sample with an uneven species distribution; e.g. if some single species are a highly abundant. the maximal value depends on the number of species in a sample. bray curtis dissimilarity [ ] was calculated for pairwise comparisons of samples and dendrograms were constructed by hierarchical clustering with the average linkage method. this analysis included counting of detected species and determination of their taxonomy, which was also used to determine the virus hosts (bacteria, vertebrates, plants etc.). a total of pooled faecal samples were derived from sows and their piglets from an experimental feeding trial with the probiotic bacterium e. faecium ncimb . four of the samples were derived from animals receiving the probiotic bacterium (group p) and four samples originated from the control group that did not receive probiotics (group c). a detailed scheme of the feeding trial is presented in figure . the presence of e. faecium ncimb in the faeces of sows and their piglets was analyzed by quantitative real-time pcr. as shown in table , e. faecium ncimb was not detected in the samples of the control group. also, no e. faecium ncimb was detectable in the sample taken from the sows of the probiotic group immediately at the beginning of the experiment ( day ante partum) as well as in the samples from the day-old piglets of this group, which were still suckled at this time-point. considerable amounts of e. faecium ncimb were demonstrated in the samples taken from the sows at day post partum and from the day-old piglets, both belonging to the probiotic group. the pooled faecal samples were analyzed by processcontrolled deep sequencing. in total, , reads were generated, with an average of , reads per sample. the efficiency of the whole method was monitored by a process control consisting of three bacteriophages, which were added in constant amounts to the samples. in all samples the three test-phages could be detected representing . % to . % of all generated reads. the numbers of totally generated reads, test-phage reads and other virus reads is summarized for the individual samples in table . the number of the test-phage reads in relation to the total virus reads ranged from . % to . % and is shown in figure . using a cut-off e-value of , = for the blastx homology search of the sequences, the viral reads could be assigned to known virus families. only of these families dominated the faecal viromes representing more than % in at least one of the samples. as shown in figure and table s , the composition of the faecal viromes according to virus families varied remarkably among the samples. a grouping of the virus families according to the taxonomic kingdom of hosts of the contained viruses revealed that the main detected groups were mammalian viruses (colored red in fig. ) and bacteriophages (colored blue in fig. ). in contrast, viruses from other hosts (insects, plants, amphibians and fungi) ranked together between . % and . % only. a closer inspection of the proportion of the read numbers from mammalian viruses compared to that from bacteriophages revealed marked differences between the samples derived from different age groups. in the youngest piglet group ( days of age), the main fraction consisted of mammalian viruses with % (control group) and % (probiotic group). in the group of dayold piglets, the proportion of mammalian viruses was reduced to % (control group) and % (probiotic group). within the four groups of the sows (one year old) the amount of mammalian viruses ranged from % to %. in contrast to those findings, the proportion of bacteriophages increased with the age of the pigs. in the day-old piglets, % (control group) and % (probiotic group) of the reads relate to bacteriophages. the percentage of bacteriophages increases in the day-old piglets to % (control group) and % (probiotic group), whereas approximately % of the virus reads belong to bacteriophages in the four sow groups. no differences in the general composition of virus families or the respective hosts were evident, when the probiotic group was compared to the control group. in overall, sequences with significant identities to known bacteriophage species were detected. the bacteriophages most abundant in the eight samples are shown in figure and table s . in all cases, the bacteriophage population is dominated by to species, which represent - % of all bacteriophage reads of the respective sample. the most abundant phages as identified by the highest number of reads with sequence identities to known bacteriophage genomes are lactococcus phage , dragonflyassociated microphage , chlamydia phages , chp and chp , bdellovibrio phage phimh k, spiroplasma phage , microvirus ca as well as enterococcous phages efap- and efrm . a comparison between the bacteriophage populations of the specific samples indicated that many of the most abundant bacteriophage species are present in all samples, however, with different relative frequency. apart from that, the composition of the faecal virome with regard to bacteriophage species was relatively variable between the samples and every sample contained its unique collection of bacteriophages. no consistent differences between age groups and feeding groups were obvious when the abundance of bacteriophage species was analyzed. interestingly, the sample taken at day post partum from the enterococcus faecium-fed sows contained relatively high amounts of the enterococcous phages efap- ( . %) and efrm ( . %), which were only sporadically detected in the other groups ( . % to . %). by analysis of all virus reads excluding the bacteriophages, sequences with significant identities to known virus species were detected. among that, . % of the sequences belonged to viruses infecting mammalian animals. this percentage of mam- malian viruses decreased with age, with an average of . % in the day-old piglet group, . % in the day-old piglet group and . % in the sows. the relative percentage of the most abundant mammalian virus genera in the eight samples is shown in figure and dant mammalian viruses. among the ''circovirus-like'' viruses, sequences with highest identities to the viruses cb-a and rw-a were most often detected. no consistent differences were obvious between the group fed with the probiotic bacterium and the control group. however, a relatively high proportion of mamastrovirus sequences ( . %) was detected in the sample derived from the day-old piglets; while this virus was not detected in the other groups (less than reads per sample). the calculation of the shannon index was used to assess the diversity of the sequences detected in the specific samples (table ) . generally, comparison of shannon indexes between piglets and sows indicated that the diversity increased with age. when only the bacteriophage sequences were analysed, the average shannon index of the piglet groups was . and that of the sows . . for the mammalian virus sequences, the average shannon index for the piglets was . and that for the sows . . calculation of bray-curtis distances determined similarities of the faecal viromes detected in the specific samples. figure illustrates clustering of samples on the basis of bray curtis distance calculated by abundances of species-specific subsets. as shown in the dendrogram based on abundances of all virus species (including bacteriophages), a grouping according to age is evident (fig. a) . the two samples taken from the day-old piglets cluster closely together and are separated from the other samples. among these other samples, the two samples taken from the day-old piglets form one separate branch, whereas the four samples of the sows are all contained in the other branch. a branching according to the feeding group is not evident from this dendrogram. the same grouping is evident, when only the mammalian virus sequences are analysed (fig. b) . the analysis of the bacteriophages shows no evident grouping according to age or feeding group (fig. c) . also, no grouping according to age or feeding group was evident, when only the sequences of the enterococcus phages were used for the analysis (fig. d ). comparisons of the composition of intestinal viromes from different samples have been only scarcely described so far. a few studies investigated individual differences of faecal viromes and the influence of diet and age in humans [ , , ] , whereas similar studies on faecal viromes of pigs are almost missing. technical problems with the use of deep sequencing methods for comparative virome analyses may represent one major problem in this context [ , ] . here, we tried to overcome some of these problems by using a process-controlled deep sequencing approach [ ] . by this, the efficiency of the analysis can be estimated for each sample, thereby enabling identification of major differences due to different performances of the method. we could show here, that all types of the bacteriophages used as process control could be detected in the final data sets of all samples. this indicates that the method has a reproducible performance and the generated data can generally be used for comparative analyses. however, the detection rates of the process control bacteriophages varied between the samples from . % to . %. as the detection rate of the bacteriophages is -besides technical factors -also dependent on the amount of viruses initially present in the analyzed sample, improved deep sequencing methods enabling quantitative analyses should be developed in future for comparative virome investigations. in the eight investigated pooled samples, the overall composition of the virus community was similar to that described for other pig faecal viromes [ , , ] . the two major virus groups were bacteriophages and porcine viruses, whereas plant viruses and viruses with other hosts were only rarely detected. however, large differences were detected in the ratio between bacteriophages and mammalian viruses in the distinct samples; in addition, the diversity of detected virus species varied between the analysed viromes. these data indicate that the faecal virome of pigs is not uniform and static, but shows a remarkable variability. for human faecal viromes, a high variability even between the analyzed individuals has been described [ , ] . as only pooled faecal samples have been analyzed in the study presented here, future investigations are necessary in order to assess the inter-individual variability of faecal viromes of pigs. the most obvious factor influencing the composition of the pig faecal virome was the age. the percentage of porcine viruses, which comprised the most abundant group in the youngest piglet samples, decreased dramatically in the samples from the older pigs. in parallel, the percentage of bacteriophages as well as the diversity of detected virus species increased by age. interestingly, porcine kobuvirus and pig scv, which both had been discovered only recently [ , ] , were among the most frequently detected viruses in the faecal viromes of the youngest and oldest age groups, respectively. this underlines the importance of unfocused detection systems in order to get an undistorted picture of the abundance of viruses in a sample. as all samples analyzed here originated from an experimental feeding trial, the detected virus composition may vary in comparison to field-origin samples. however, the age-specific effect was strong and very similar in both analyzed groups, which were held completely separate during the whole period of the experiment. the differences may be explained by an age-related susceptibility to specific virus infections as well as by an increasing immunity to porcine viruses due to completed virus infections with higher age. in addition, the progressive diversification of the bacterial enteric flora, which serves as the host pool for bacteriophages, would also explain the increasing diversification of the virus flora by age. an increasing diversity of the virus species in faeces of humans has already been described [ ] . in contrast to the age-related effect, no clear differences could be detected in the composition of the faecal viromes according to feeding with the probiotic bacterium e. faecium ncimb . a relatively high percentage of enterococcus phages in the sample derived from an e. faecium-feeded group may indicate multiplication of the phage due to the application of its host. this explanation may indicate that a larger amount of the probiotic bacteria may be lysed by the bacteriophages and are therefore not available for the probiotic therapy; however, this interpretation is questionable as the bacteriophages were only found in one of the samples. also, a relative high proportion of astrovirus was found in one of the samples of the control group. interestingly, real-time rt-pcr analyses of samples derived from the same feeding trial confirmed the presence of astrovirus exclusively in the control group [ ] . however, the same study indicated later shedding of rotaviruses with lower amounts in the probiotic group as compared to the control group, which was not detected by our virome analysis. a closer inspection of the data shows that up to astroviruses per gram faeces were present in the samples, whereas only up to rotaviruses per gram were detected [ ] . therefore, a lower sensitivity of the virome analysis method may explain the discrepancies and still deeper sequencings may be necessary in future to detect more subtle changes in the faecal virome composition due to probiotic feeding. the composition of the identified bacteriophage species in the different samples revealed no consistent pattern. however, most of the detected sequence reads showed only moderate identities to the known bacteriophage sequences present in the database. therefore, it has to be considered that the majority of the detected sequences belong to so far unknown bacteriophages and that the identified bacteriophage species represent only their next relatives. a definitive assignment of a host to these sequences is therefore currently not possible. the quality of the database with regard to genomic sequences of bacteriophages is crucial for virome analyses. for example, the high proportion of the detected lactococcus phage may reflect the disproportionately high abundance of those phage sequences in the database as a consequence of intensive research on these bacteriophages, which are problematic agents for the diary cheese product industry [ ] . in contrast, for another highly abundant bacteriophage, the dragonfly-associated microphage, the specific bacterial host is still unknown [ ] . an increase of annotated bacteriophage sequences in the databases is therefore a prerequisite for studies on the interactions between bacteriophages and their host bacteria in the gut in future. alternatively, a deeper sequencing may enable the assembly of complete bacteriophage genome sequences from the metagenomic data set. by this, an assignment to their hosts was possible by identification of inserted host-related sequences as recently described [ ] . in summary the data show a high variability of the pig faecal virome. most obvious are age-related differences in the proportion between pig viruses and bacteriophages as well as an increasing diversification of virus species by age. consistent differences due to probiotic feeding could not be identified by our metagenomic analysis. the results of comparative pig virome analyses may help to understand the complex interactions between viruses, bacteria and the pig within the intestinal tract. future research should focus on the optimization of the method in order to increase its sensitivity and on the improvement of the sequence databases, especially regarding annotated bacteriophage sequences. it will be interesting to apply the optimized techniques to analyse the diversity of faecal viromes in individuals and to identify further factors like geographical origin or disease-related changes influencing its composition. table s relative abundance of virus families in the analyzed faecal viromes. the table shows the number of reads with sequence identities to a certain virus family in relation to all virus reads (in %). families showing an abundance of less than % in a distinct faecal virome are subsumed to other families. families which were not classified by the international committee on taxonomy of viruses (ictv) are subsumed to not assigned. the group p received the probiotic bacterium e. faecium ncimb (p) and the group c (c) received no probiotic. table s relative abundance of bacteriophage species among all bacteriophages detected in the analyzed faecal viromes. the table shows the number of reads with sequence identities to a certain bacteriophage species in relation to all bacteriophage reads (in %). bacteriophage species showing an abundance of less than % in a distinct faecal virome are subsumed (, %). the group p received the probiotic bacterium e. faecium ncimb (p) and the group c (c) received no probiotic. (pdf ) table s relative abundance of mammalian virus genera among all animal viruses detected in the analyzed faecal viromes. the table shows the number of reads with sequence identities to a certain mammalian virus genus in relation to all animal virus reads (in %). mammalian viruses, which are so far not assigned to a certain genus, are indicated in apostrophes. mammalian virus genera showing an abundance of less than % in a distinct faecal virome are subsumed (genera , %). viruses from non-mammalian hosts are subsumed to non mammalian viruses. the group p received the probiotic bacterium e. faecium ncimb (p) and the group c (c) received no probiotic. 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african straw-coloured fruit bats: detection of a chiropteran poxvirus and isolation of a novel adenovirus candidate new species of kobuvirus in porcine hosts characterization of , a virulent phage from lactococcus lactis with similarities to prophages from other firmicutes diverse circular ssdna viruses discovered in dragonflies (odonata: epiprocta) we thank robert pieper and his colleagues at the institute for animal nutrition (free university berlin, germany) for the coordination of the animal experiment and paul wrede (charité, berlin, germany) for helpful discussion. we also thank wilfried vahjen and ingo starke (institute for animal nutrition, free university berlin, germany) for their support in detection of e. faecium ncimb in the samples. key: cord- -ggtxuulg authors: mauk, michael g.; liu, changchun; song, jinzhao; bau, haim h. title: integrated microfluidic nucleic acid isolation, isothermal amplification, and amplicon quantification date: - - journal: microarrays (basel) doi: . /microarrays sha: doc_id: cord_uid: ggtxuulg microfluidic components and systems for rapid (< min), low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (naats) are described. a microfluidic point-of-care (poc) diagnostics test to quantify hiv viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” naat devices. a portable, miniaturized poc naat with performance comparable to conventional pcr (polymerase-chain reaction)-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: ( ) nucleic acids (nas) are extracted from relatively large (~ml) volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase (“membrane”) to capture sample nas in a flow-through, filtration mode; ( ) nas captured on the membrane are isothermally (~ °c) amplified; ( ) amplicon production is monitored by real-time fluorescence detection, such as with a smartphone ccd camera serving as a low-cost detector; and ( ) paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. limits of detection (lod) better than ( ) virons/sample can be achieved. a modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample na template. in addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed. in the last decade there has been considerable interest and research in addressing the needs and opportunities for on-site nucleic acid-based tests (nats) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these tests are also termed nucleic acid amplification tests (naats) when incorporating sequence-specific enzymatic amplification of nucleic acids such as pcr (polymerase chain reaction), or molecular diagnostics when used in a medical context. such in vitro diagnostic tests implemented with low-cost, easy-to-operate, portable instruments and miniaturized sample processing/analysis devices can be used outside of medical laboratories for detecting pathogens in blood, urine, saliva and other sample types. naats provide more timely results compared to traditional culturing methods for diagnosis, and also work with the many pathogens that cannot be cultured. more generally, pervasive, cheap, and rapid naats will foster new paradigms for improved and sustainable medical care (e.g., mobile healthcare using smartphones) and new tools for research and discovery. for example, point-of-care (poc) diagnostics devices have been developed for quantifying hiv viral load with finger-prick blood samples. closely related microfluidics technologies have been demonstrated for food and water safety testing [ , ] , rapid genetic tests [ ] , environmental monitoring [ ] , surveillance for bioterrorism agents [ , ] , cancer screening [ ] [ ] [ ] , assuring the health and hygiene of laboratory animals [ ] , analyzing biopsies [ , ] , testing of livestock and pets for parasites [ ] , examining insects for genotype and infections [ , ] , and first responder-administrated triage tests for stroke biomarkers [ ] . to date, most rapid tests for biomarkers are immunoassays [ ] . the lateral flow strip test, or immunochromatographic assay, such as found in home pregnancy tests, is an elegantly simple implementation of immunoassays employed to detect antigens or antibodies in urine, blood, food, and water. molecular assays for dna and rna targets offer many important advantages over immunoassays, including greater sensitivity (typically by a factor of or more) and higher specificity. the high sensitivity is due to the ~million fold amplification of the target sequence. the amplicons can be conveniently detected with intercalating dyes or reporters that specifically label the amplicons, allowing measurements with relatively simple fluorescence detectors such as ccd cameras or photodiodes, or less commonly with electrochemical sensors. further, unlike immunoassays which rely on monoclonal antibodies and may require weeks or months of development time, naats utilize nucleic acid oligos for hybridization or as primers for enzymatic amplification, which can be designed, synthesized, and validated in a short timeframe-in some cases as short as one week-once the pathogen has been at least partially sequenced [ ] . while immunoassays can be performed on crude, unprocessed samples (e.g., whole blood or raw food) at room temperature, more elaborate sample processing and instrumentation is required for molecular diagnostics. in particular, ( ) virons and cells must be lysed to release nucleic acid (unless cell-free nas are the target); ( ) substances, such as heme in blood specimens, that may inhibit downstream processes such as reverse transcription and amplification, must be removed; ( ) amplification reaction temperatures must be closely regulated (typically within ± °c); ( ) the target na must be concentrated so to reduce amplification reaction volume to to µl in order to economize reagents and facilitate precise temperature control and uniformity; and ( ) amplicon detection schemes should provide a statistically reliable means to discriminate between true and false positives, and true and false negatives. thus, in general, although molecular diagnostics provide greater sensitivity and selectivity than immunoassays, they require more sample preparation, more instrumentation, more labor and expertise, and longer test times. fortunately, naats can be implemented in a microfluidics system for manual, semi-automated, or fully-automated operation that can be readily adapted for point-of-care use. a broad aim of microfluidics poc efforts is to make molecular diagnostics nearly as easy to use as lateral flow strip immunoassays. microfluidics refers to various methods and technologies wherein small quantities ( nl to ml) of liquids are manipulated, processed, and analyzed using miniaturized, e.g., palm-sized, devices. in biotechnology, microfluidic components and devices have been demonstrated for, among other things, serial dilutions; cell fractionation, sorting, and enrichment; cell and virus lysis; isolation, concentration, and purification of nucleic acids; purification of proteins; immunoassays; reverse transcription; labeling of biomolecules with reporters; enzymatic amplification (e.g., pcr); electrophoresis; micro total analytical systems (µtas) combine these functions into an integrated sample-to-report device for applications in medical diagnostics; environmental, food, and water monitoring; detection of bioterrorism over conventional benchtop processing agents, and as tools for research, discovery, and quality assurance. the potential advantages of microfluidics include lower cost, faster results, effective containment of hazardous or infection material, reduced cross-contamination from other samples, better utilization of smaller samples sizes, reduced consumption of reagents and reduced generation of waste, portability, miniaturization, and ease of use for operation by minimally trained people. here, we review microfluidic "lab on a chip" (loc) technology integrating sample preparation and analysis for point-of-care molecular diagnostics and other on-site naats. as such, microfluidic systems can support and complement microarrays. while microarrays offer a high degree of parallel processing for multiplex detection (hundreds or thousands of targets), microfluidics can streamline and automate the serial sample processing steps starting with crude, heterogeneous samples and yielding the requisite concentrated and purified nucleic acids for enzymatic amplification, microarray analysis, and sequencing. a -to -fold concentration of target for amplification is often needed. for example, the viral nucleic acid in ml of blood plasma needs to be concentrated to a volume of about µl for amplification. the "backend" functions of loc molecular diagnostics systems which include amplification, labeling, and detection are well established. however, the "front end" functions of interfacing with the outside world through sample collection devices, as well as sample preparation and processing (lysis and na isolation) of variegated heterogeneous samples are less developed. the "backend" functions are generic and largely independent of sample type and application, but the "front end" functions need to be tailored to specific applications (e.g., viral load measurements in blood vs. bacteria detection in food samples) according to the required sample size determined by the concentration of target and the sample and the desired limits of detection (lods) [ ] . other factors related to sample processing include the need for and difficulty of lysis of tissue, cells and viruses containing the target na, the presence of inhibitors intrinsic to the sample or added to the sample to effect lysis, and the stability of analytes, e.g., labile rnas. sample processing steps may also include deactivation of nucleases, proteases, infectious agents with reagents or heat. some degree of multiplexing is feasible in even simple microfluidic devices, and, as such, loc devices may be suitable as low-cost, convenient alternatives to microarrays when multiplex detection of tens of targets suffices. this can be accomplished, for example, by incorporating multiple amplification reactors on a single chip, each containing primers for a specific target. the sample lysate is split and loaded on the several membrane/chamber channels, which can be imaged together using a ccd camera. multiplexing in a single chamber would require probes specific to a target and with a unique fluorescence emission wavelength, combined with a color-sensitive imager. to support, supplement, or complement microarrays, standardized or customized microfluidic devices can be useful for rapid sample preparation and screening of samples where labile targets may suffer degradation (especially mrnas) or low yield (e.g., micrornas), quality control of reagents, detecting the presence of inhibitor substances and other interfering components, before the samples are subjected to more costly and time-consuming microarray analysis or sequencing. further, knowledge of sequences gained with microarrays can be used to design amplification primers and probes for microfluidics-based naats. molecular diagnostics is comprised of the sequential steps of lysis (when the target nucleic acids are sequestered in cells or viruses); nucleic acid isolation (extraction, concentration, and purification of dna and rna from the lysate); reverse transcription for rna targets, enzymatic amplification of target dna or rna with sequence-specific primers; and real-time or endpoint detection of amplification products. in the laboratory, these unit operations are typically carried out in benchtop instruments. first, the sample is incubated with lysing agents (salts, detergents, enzymes, chaotropic agents), sometimes supplemented with mechanical grinding, sonication, freeze-thaw, and heating, to release and solubilize nucleic acids. next, nucleic acids are isolated from the lysate, commonly using a spin column kit for solid-phase extraction, where nucleic acids are captured on a silica membrane. lysing agents such as chaotropic salts promote preferential binding of nucleic acids to silica glass or cellulose fibers of the porous membrane. the membrane is then washed with ethanol:water salt solutions to remove proteins and other debris. the captured na is then desorbed and eluted from the membrane with water. at this stage, the captured nas are concentrated and of sufficiently purity for enzymatic amplification, typically by pcr in a thermal cycler instrument. in the last five years, many new isothermal amplification methods, e.g., lamp (loop mediated amplification), helicase dependent amplification, nasba (nucleic acid sequence-based amplification), and rpa (recombinase polymerase amplification) have gained prominence for poc diagnostics [ ] . traditionally, amplification products are detected and assessed by gel electrophoresis, but nowadays, real-time monitoring of the amplification process, using fluorescent intercalating dyes (sybr ® green, syto ® green, evagreen ® ) or molecular probes (e.g., taqman ® ) is preferred due convenience and shorter protocols (obviating the need for post-amplification analysis), the feasibility of relative quantification, and lower risk of laboratory contamination by amplicons. alternatives for detection of amplification products include lateral flow strips analysis of amplicons made with primers conjugated with antigens, or other real-time methods based on monitoring various products or by-products of amplification such as changes in ph, turbidity, electrical conductivity, phosphate or atp concentration, some of which can be monitored by luminescence reactions using, e.g., luciferase/luciferin. generally, this naat laboratory protocol requires six or more hours, and is carried out by skilled technicians in dedicated facilities equipped with hoods, centrifuges, temperature baths, and benchtop thermal cycler instruments. the labile reagents (polymerases, reverse transcriptases, proteases for lysis)-and often the sample too-need to be maintained in a cold chain, and the laboratory work area and procedures must minimize cross contamination of samples. the foregoing benchtop procedure can be implemented with a credit-card sized microfluidic cassette "chip", with total sample processing times of under one hour [ ] . a photo of a representative chip is shown in figure . the chip hosts three parallel reaction chambers for the isothermal amplification of nucleic acids. a porous cellulose (for dna) or silica glass fiber (for rna) "membrane" disc (~ . -mm thick and mm in diameter) is placed at the inlet of each chamber. during the sample loading stage, nas are captured on the porous membrane solid phase, as sample lysate perfuses through the membrane. the nas bound on the membrane are then washed with ethanol-based solutions to remove proteins and other contaminants. the chamber is then filled with water and heated to the amplification temperature, typically - °c, depending on the primer annealing temperatures and optimum temperature of the polymerase enzyme. during the heating to amplification temperature, the lyophilized reagents, enzymes, and dye that were pre-stored in the reaction chamber are released and hydrated. by encapsulating the reagents in paraffin that has a melting temperature below the amplification temperature, the stored reagents are shielded from the sample loading and wash solutions that flow through the chamber prior to amplification, and are reconstituted during the heated amplification step after the chamber is filled with water. the target nas captured on the membrane are then amplified. rna targets are simultaneously reverse transcribed into cdna templates by reverse transcriptase, or an rna/dna dependent polymerase is used [ ] . this approach avoids the need for a separate elution step as include in the benchtop spin column protocol. specificity is dictated by appropriate design of the amplification primers. as with benchtop real-time pcr, the increasing amplicon concentration is monitored in real time during the amplification process. the fluorescence emission can be detected with an inexpensive usb microscope, photomultiplier, or smartphone (see section ). the amplification time required for the emitted fluorescence intensity to cross a specified threshold intensity is correlated with the concentration of target na in the sample. the chip performs multi tasks to simplify its design, operation, and flow control and to reduce its cost. to summarize, the chip combines the following unit operations in a single reactor: ( ) a porous silica or cellulose na binding "membrane" is used to load sample lysate in a flow-through mode, allowing na to be isolated from large sample volumes, such that the amplification volume is thus decoupled from the sample size, which is crucial for detecting low abundance targets. this feature is absent in many previously-reported poc naat devices; ( ) in situ amplification of na captured on the membrane, which eliminates the need for a separate elution step and simplifies flow control; ( ) isothermal amplification such as loop mediated isothermal amplification (lamp), which simplifies the instrumentation compared to pcr and is less energy demanding than thermal cycling. lamp appears more tolerant of inhibitors compared to pcr; ( ) pre-storing in the amplification chamber paraffin-encapsulated, lyophilized amplification reagents (polymerases, reverse transcriptases, buffer salts, nucleotides, oligo primers, and dna-binding dyes) further reduces the number of flow control operations, thereby simplifying the chip. further, use of lyophilized reagents relieves both the need to add reagents at the time of use, and the need for any cold storage of reagents. as an added benefit, the method provides a "hot start" to amplification in that reagents are released only once the reactor has reached its operating temperature, reducing non-specific amplification. moreover, it is feasible to store different reagents with different primer sets in separate parallel reactors, enabling the detection of multiple targets as well as providing control and calibration reactors; ( ) our chips enable real-time detection of the amplicon production, which provides more information than merely end-point detection, and allows one to terminate the test based on the target concentration, i.e., when targets are available in high abundance, the test can be concluded in as little as - min; ( ) the fluorescence emission can be detected with a ccd camera which readily enables concurrent monitoring of a large number of reactors and the utilization of smartphone cameras for cost reduction. figure . chip using single-chamber for na isolation, isothermal amplification, and real-time detection. photo of chip with three parallel chambers for multiplex detection (inset). molecular diagnostics process flow schematic (right): sample is mixed with lysis/binding reagent (guanidium hcl), sample is injected into chip, ethanol wash is injected into chip, washing na (nucleic acid) capture membrane (silica glass fiber or cellulose disc embedded in chip), chamber is filled with water and sealed. chamber is heated to amplification temperature ( °c), melting paraffin encapsulation layer and reconstituting lyophilized enzymes, reagents, and dna intercalating fluorescent dye, blue light excitation from led generates fluorescent signal (proportional to amount of dna amplicon) detected through filter (to block excitation light) by ccd camera (smarthphone). as a specific application example, we used the chip described above to assess viral load in plasma samples (see below). several hundred microliters of plasma were mixed with an equal part of lysing buffer ( m guanidinium hcl), briefly incubated (~ min), and pipetted into the chip, through the embedded nucleic-acid isolation membrane. next, µl of ethanol:water ( : ) was pipetted into the chip to wash the membrane. finally, the chip amplification chamber was filled with water. the chip inlet and outlet ports were sealed with tape, and the chip was then inserted into a portable palm-sized instrument with a heated stage and mounting fixture for a cellphone. figure shows the results for plasma samples spiked with three different concentrations of hiv virons plus a negative control, indicating a lod (limit of detection) of copies per ml of plasma sample. alternatives to real-time monitoring of the amplification process for quantifying nucleic acids that are less demanding of imaging and computational capabilities could prove more amenable to low-cost poc diagnostics in resource-limited settings and may open new opportunities for naats for identification of bacterial flora and gene expression profiles. one such method that has been recently developed by our group consists of an amplification-diffusion conduit (adc), which provides a spatial indication of the concentration of target nucleic acids in the sample (figure ) [ ] . this "nuclemeter" chip, shown in figure a , hosts four adcs to illustrate a serial dilution effect of target concentration on device operation. briefly, the adc extends from the embedded silica membrane that captures na, as described above, and which provides the source of template na for amplification and diffusion. the adcs are typically µm wide × µm deep × mm long and are pre-filled with a stabilized gel (hydroxypropyl)methyl cellulose (hpmc) to retard diffusion and with all the ingredients needed for the amplification reaction, sans the target itself. the adc is maintained at the amplification temperature ( °c for the lamp process). as the target diffuses into the conduit, it amplifies. eventually, the amplification process runs its course (i.e., saturates). after a short time from the beginning of the process, the reaction-diffusion conduit can be viewed as consisting of two regions: a region in which the reaction has been completed and a region into which the target nucleic acids have not yet diffused and the amplification reaction has not yet started. when an intercalating dye is used, the first region of the adc, in which the reaction has been completed, emits fluorescence and appears as a column of light, while the second region, in which the amplification reaction has not yet occurred, appears dark (figure b ) which can be imaged with a smartphone camera (figure c) . the two regions are separated by a relatively sharp reaction front. once the initial reaction region has saturated, the reaction front moves with a constant velocity v (figures d- ,d- ). that is, the position of the front is given by the equation t ) . x is the front location when the amplification process near the adc's entrance has saturated and t is time (figure d (figure d- ) , it serves a similar role to that of the threshold time in real time machines. adcs operating with known quantities of target molecules can serve to calibrate the length of the fluorescent column to enable quantification. the emission from multiple columns can be conveniently recorded with a smart phone camera. microfluidic devices can leverage smartphone technology to image the chip and detect the increasing level of fluorescence due to the amplification process [ ] [ ] [ ] [ ] , as well as for computation and control functions, communication of test results (including gps), data logging, and connect to sources of medical advisory information to the tested person. in one implementation [ ] , a blue led (@ ma current) excites the dna-binding fluorophore, and the longer-wavelength fluorescence is detected by the ccd camera after optically filtering out the excitation light. the ccd camera of a smartphone proves to be an adequate, and widely-available, low-cost detector of green fluorescence (using syto green, evagreen™ dye, or similar dna intercalating dyes) for real-time monitoring of amplification on the chip. the digitized image of the amplification chamber, generated by custom app (mobile application) software, can quantitatively assess the increasing intensity of fluorescence during the course of amplification to determine a threshold time which correlates with the starting na template concentration captured on the membrane. further, many parallel chambers can be simultaneously imaged for multiplex detection, including several analytes, positive and negative controls, and calibration standards. figure shows a fixture for mounting the cellphone over the chip docked on a custom made, electricity-free heating stage. d figure . nucleometer chip. (a) the chip having a similar structure to that described in figure . the amplification-diffusion column is pre-filled with all the ingredients needed for the amplification process and a polymer to reduce diffusion rate; (b) as the target diffuses into the column it amplifies. after a short time that depends on initial target concentration, the column consists of two regions. a region in which the reaction has been completed (column of light) and a region in which the reaction has not yet started (dark region); (c) the test results can be monitored, documented, and transmitted with a smartphone. the front position of the fluorescence region is extended as a function of time and can be used to estimate target (na template) concentration; (d- ) relative concentration of amplicon as function of distance along the conduit, as determined by fluorescence; (d- ) relative concentration of amplicon along the conduit, as determined by fluorescence, as a function of time; (d- ) distance along conduit (xf) as a function of time for different starting template concentrations (copies); (d- ) threshold time of fluorescence region extension vs. rna template copy number [ ] . . smartphone mounted on portable instrument that provides the chip with temperature-regulated heating, optical excitation with a blue led, a filter to block the excitation light from the smartphone camera and detection. the heating is provided with an exothermic reaction in which a magnesium alloy powder interacts with water. the temperature is regulated with a phase change material (pcm, e.g., paraffin) that acts as a ballast to maintain the reaction chambers at the pcm melting temperature, since excess heat production is absorbed as latent heat, maintaining the reaction chamber at the pcm melting temperature [ ] . molecular tests for bloodborne pathogens are usually based on detection of na biomarkers in cell-free plasma. whole blood sample processing is problematic due to cells clogging the na binding membrane, heme from lysis of red blood cells inhibiting enzyme action, and genomic dna from white blood cells, for example, containing integrated retroviruses, confounding and complicating test results. furthermore, prevailing standards for viral loads are based on virus counts in plasma rather than in whole blood. in a laboratory setting, the cell-free plasma component of blood is typically prepared as the supernatant of centrifuged (e.g., , rpms for min) whole blood. many poc venues do not have access to centrifuges, and therefore a simpler, instrument-free method for extracting plasma from whole blood samples is highly desired. various microfluidic approaches for poc plasma extraction often involve supporting equipment such as electric motors and actuators, or vacuum pumps. non-instrumented microfluidic devices rely on capillary flow or filtration, but these techniques either have low yield (volume of plasma extracted/volume of blood sample) or are limited to small blood sample sizes (~ µl). in our work [ ] , we have developed a plasma extraction device based on combination of gravitational sedimentation and filtration through a porous membrane to achieve practical performance levels for field use (figure ). our device is a rectangular cartridge ( cm × cm × cm) with a vertically oriented center chamber open at the top, and in which two opposing sidewalls featuring arrays of micropillars ( µm × µm in cross section, and µm tall), machined or stamped, support polysulfone membranes. the membrane has asymmetric pores (larger diameter facing the center chamber) such that blood cells are gently trapped without lysis. the advantage of our approach is in allowing cells to sediment to the bottom of the chamber while plasma effuses through the vertical membranes, trickles trough the pillar arrays into trench channels, from where it can be collected by a pipette. the advantage of our approach is in reducing the amount of cells that come into contact with the separation membrane, thereby increasing separation membrane capacity. for example, . ml of whole blood loaded into this device yielded nearly µl of plasma in min. in a newer version of the device (not shown), we have nearly tripled the yield. an assay for heme in the extract indicated negligible lysis of red blood cells. quantitative pcr of the plasma extracted from whole blood spiked with hiv indicated that the plasma is of sufficient quality for enzymatic amplification and that the virus recovery is over %. figure (shown earlier) provides amplification results of a serial dilution sensitivity data for hiv lamp in the chip, using plasma samples extracted with our plasma separator as described above. the microfluidic devices described above can be fabricated by several prototyping methods [ ] . indeed, this is one of the advantages of microfluidics: custom design and rapid prototyping of components within a timeframe of one to two days is feasible. chips are made as bonded laminate structures from plastic sheet materials, including pmma (polymethylacrylate, "acrylic"), polycarbonate, polystryrene or cyclic olefin copolymer. the fluidic circuits are drawn using computer aided design (cad) software (solidworks™ or autocad™). a middle layer is patterned with the fluidic circuit by a cnc milling machinine, a desktop engraver, or a co laser cutter. alternatively, the chip can be directly formed using a d printer with clear acrylic-like polymer alloys. the smallest feature size (i.e., channel width) is about . mm. smaller depths can be obtained with thin laminates. the component sheets are aligned and bonded using adhesives, solvent bonding, thermal pressure bonding, or ultrasonic welding. both resistive heating or peltier cooling modules can be used for temperature regulation, along with a thermocouple temperature sensors. the power consumption is on the order of watt. a commercial temperature controller or microntroller can be used to regulate the temperature, or even simpler analog electronics control circuits are sufficient. reagents are from commercial lamp kits (eiken chemical, tokyo, japan). freeze-dried lamp reagents (including primers and fluorescent dyes) as lyophilized spheres can be preloaded into the chip during assembly to simplify operation. the feasibility and utility of microfluidic lab-on-a-chip implementations of component subprocesses for naats, including plasma extraction, lysis, na isolation, amplification, and real-time detection and end-point quantification, have been well demonstrated for representative sample types and varied applications. poc devices and operation are significantly simplified by ( ) a multifunctional reaction chamber that includes an embedded membrane for the capture of dna and rna, storage of reagents, and the in situ amplification of na on the membrane which circumvents the need for a separate elution step and decouples sample size from amplification reaction volume; and ( ) isothermal (constant temperature) amplification such as lamp (loop mediated amplification) with real-time detection, using for example, a smartphone ccd camera as detector. when blood samples are used, the hybrid sedimentation-filtration plasma extraction device can replace centrifugation enabling plasma separation from whole blood specimens at the point of care. the plasma separator can be integrated with the nucleic acid amplification chip for seamless operation. poc molecular diagnostics devices were first reported in the mid- s [ ] , single-chamber pcr chips made in glass or silicon. in the last twenty years, these chips have evolved to more viable polymer materials, have integrated sample prep functions, included on-chip storage of reagents and buffers, and accommodate low-cost detection schemes, such as smartphone cameras, and as such now offer convenient, low-cost diagnostics suitable for use outside of traditional clinical laboratories, including resource-limited settings throughout the world. we also describe a new paradigm in target nucleic acid quantification through the use of a chip-based amplification-diffusion channel. this method facilitates end point detection and enables the estimation of target (na template) concentration from the lengths of emitting columns, akin to reading temperature in a mercury thermometer from the length of the mercury column. the various modules described herein can be combined into total microanalytical systems for sample-to-report poc diagnostics devices. in this review, we focused on naat of hiv in blood, suggesting the application of similar designs for other nucleic acid based tests where time, convenience, cost constraints would preclude the use of a microarrays, while still achieving multiplexing capability and quantification of both rna and dna targets with only modest or no instrumentation. we use a solid-phase extraction for na isolation based on the common chaotrope-silica method used in commercial spin columns. related na isolation methods based on chargeswitch ® technology (cst) and solid-phase reversible immobilization (spri) are also potential alternatives. these point-of care molecular diagnostics devices complement microarrays and provide an alternative to expensive laboratory-based instruments. as a comparison, commercial pcr-based and related systems, such as the cobas ampliprep/taqman hiv- (roche), versant hiv- rna (siemens), rna qt (biomerieux), and realtime m hiv (abbott) can process close to samples in a two-hour time span. limits of detection typically range from to copies/ml plasma [ ] . these instruments cost over $ , and their use is restricted to modern clinical laboratory facilities. self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and dna microarray detection monitoring systems and quantitative measurement of biomolecules for the management of trauma microfluidic technology for molecular diagnostics advances in microfluidic pcr for point-of-care infectious disease diagnostics antibody production, design and use for biosensor-based applications point-of-care diagnostics: an advancing sector with nontechnical issues present technology and future trends in point-of-care microfluidic diagnostics point-of-care technologies for molecular diagnostics using a drop of blood a handheld flow genetic analysis system (fgas): towards rapid, sensitive, quantitative and multiplex molecular diagnosis at the point-of-care level smartphones for cell and biomolecular detection microfluidic chip for molecular amplification of influenza a rna in human respiratory specimens miniaturized nucleic acid amplification systems for rapid and point-of-care diagnostics: a review anal chim advances in developing hiv- viral load assays for resource-limited settings elevating sampling rapid development of nucleic acid diagnostics point of care technologies for hiv isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review an isothermal amplification reactor with an integrated isolation membrane for point-of-care detection of infectious diseases simultaneous quantification of multiple food-and waterborne pathogens by use of microfluidic quantitative pcr lab-on-a-chip pathogen sensors for food safety a handheld point-of-care genomic diagnostic system application of microfluidics in waterborne pathogen monitoring: a review point-of-care diagnostics for ricin exposure a lab-on-a-chip for detection of nerve agent sarin in blood capturing cancer: emerging microfluidic technologies for the capture and characterization of circulating tumor cells point-of-care rare cell cancer diagnostics microfluidics for research and applications in oncology nucleic-acid testing, new platforms and nanotechnology for point-of-decision diagnosis of animal pathogens a microfluidic technique for quantification of steroids in core needle biopsies microfluidic biosensor for β-hydroxybutyrate (βhba) determination of subclinical ketosis diagnosis microfluidics for food, agriculture and biosystems industries a low-cost microfluidic chip for rapid genotyping of malaria-transmitting mosquitoes a microfluidic platform to isolate avian erythrocytes infected with plasmodium gallinaceum malaria parasites based on surface morphological changes simultaneous detection of c-reactive protein and other cardiac markers in human plasma using micromosaic immunoassays and self-regulating microfluidic networks microfluidic chips for immunoassays rapid development of real-time rt-pcr assays and positive controls in response to emerging coronavirus a novel thermostable polymerase for rna and dna loop-mediated isothermal amplification (lamp) nuclemeter: a reaction-diffusion based method for quantifying nucleic acids undergoing enzymatic amplification biosensing with cell phones transformation of personal computers and mobile phones into genetic diagnostic systems gene-z: a device for point of care genetic testing using a smartphone smart cup: a minimally-instrumented, smartphone-based point-of-care molecular diagnostics device membrane-based, sedimentation-assisted plasma separator for point-of-care applications microfluidic devices for nucleic acid (na) isolation, isothermal na amplification, and real-time detection pcr in a silicon microstructure past, present and future molecular diagnosis and characterization of human immunodeficiency virus infections the work reported here was supported, in part, by nih grants u de (haim h. bau, michael g. mauk) and k ai (changchun liu), and nih sstr phase i grant r ai - a via ac diagnostics, inc. (fayetteville, ar) (changchun liu). michael g. mauk contributed to the sections relating to background, literature review, and chips for na isolation, amplification and detection, changchun liu contributed to the sections on plasma extraction and nuclemeter design operation, and data analysis, jinzhao song contributed to the sections on dry storage and cellphone detection, and haim h. bau contributed to the introduction and discussion, and technical descriptions, and modeling of nuclemeter. the authors declare no conflict of interest. key: cord- -psnec qp authors: mbareche, hamza; veillette, marc; pilote, jonathan; létourneau, valérie; duchaine, caroline title: bioaerosols play a major role in the nasopharyngeal microbiota content in agricultural environment date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: psnec qp background: bioaerosols are a major concern for public health and sampling for exposure assessment purposes is challenging. the nasopharyngeal region could be a potent carrier of long-term bioaerosol exposure agents. this study aimed to evaluate the correlation between nasopharyngeal bacterial flora of swine workers and the swine barns bioaerosol biodiversity. methods: air samples from eight swine barns as well as nasopharyngeal swabs from pig workers (n = ) and from a non-exposed control group (n = ) were sequenced using s rrna gene high-throughput sequencing. wastewater treatment plants were used as the industrial, low-dust, non-agricultural environment control to validate the microbial link between the bioaerosol content (air) and the nasopharynxes of workers. results: a multivariate analysis showed air samples and nasopharyngeal flora of pig workers cluster together, compared to the non-exposed control group. the significance was confirmed with the permanova statistical test (p-value of . ). unlike the farm environment, nasopharynx samples from wastewater workers did not cluster with air samples from wastewater treatment plants. the difference in the microbial community of nasopharynx of swine workers and a control group suggest that swine workers are carriers of germs found in bioaerosols. conclusion: nasopharynx sampling and microbiota could be used as a proxy of air sampling for exposure assessment studies or for the determination of exposure markers in highly contaminated agricultural environments. the microbial flora of aerosols, referred to as bioaerosols, consists of a combination of viable and non-viable microorganisms (e.g., bacteria, fungi and viruses) and derived compounds of biological origin (e.g., animal and plant debris, endotoxins, exotoxins, and other microbial metabolites) [ ] [ ] [ ] . bioaerosols are ubiquitous in indoor and outdoor environments and are generated from various natural and/or anthropogenic sources. composed of particles ranging in size from a few nanometers to µm in diameter, bioaerosols remain suspended in the air for long periods of time and may travel many kilometers depending on the size of the particle [ , [ ] [ ] [ ] [ ] [ ] . therefore, the dispersal of bioaerosols may impact the air quality of extensive areas that are far from the source and can create public health issues, due to the presence of highly diverse and dynamic microbial communities. beyond affecting the time that particles remain suspended and the distances they travel, particle size plays a role in human diseases, as it dictates which pathway the particle follows in the respiratory tract after inhalation [ ] . for example, particles with a size of µm to µm tend to get deposited in the upper airways, while larger particles may remain in the nasal cavity [ , ] . bioaerosols can be a transmission vector for infectious diseases and are responsible for a variety of health problems, principally through inhalation [ , [ ] [ ] [ ] [ ] . human exposure to bioaerosols is associated with a wide variety of acute and chronic diseases ranging from allergies, asthma, rhinitis, sinusitis and bronchitis, mostly due to occupational exposure [ , [ ] [ ] [ ] [ ] . however, health risks from bioaerosols also exist just from living in close proximity to an intensive source of airborne biological particles [ , ] . additionally, other health problems linked to bioaerosols include fatigue, headache, mucous membrane irritation syndrome, nasal congestion, sore throat, and irritation of the nose and eyes [ , , ] . the industrial environment is the main source of occupational health issues, due to the presence of raw organic materials, the prevalence of operations releasing harmful bioaerosols (e.g., mechanical operations such as wood planning, straw chopping, animal bedding, hay handling, and compost pile turning) and the eventual large amounts of bioaerosols present in confined spaces. for example, biowaste facilities are characterized by notable concentrations of bioaerosols, due to the intense microbial activity involved in waste degradation and the activities performed by workers [ ] [ ] [ ] [ ] . wastewater treatment plants (wwtps) represent another environment where workers are subject to bioaerosol exposure, due to the steps required for the treatment of discharged municipal and industrial effluents [ , ] . intensive animal farming practices in confined buildings that hold a large number of animals (e.g., pigs, poultry, cattle) are also associated with extreme exposure to airborne microbes. the variety of possible sources (e.g., animals, feces, feed, litter) present at farms leads to the emission of complex mixtures of biological particles [ ] [ ] [ ] [ ] [ ] [ ] . moreover, the dynamic nature of the microbial composition makes health risk evaluation complicated for farm workers and nearby residents [ , ] . environmental hygienists continue to insist that insufficient exposure assessments are a primary reason for the absence of bioaerosol exposure limits and strategies to mitigate risk [ , ] . there are several challenges and limitations to measuring bioaerosol exposure. the type of sampler, time and duration of sampling, meteorological conditions [ ] , and geographical positions all affect bioaerosol sampling efficiency, making it difficult to compare studies and limiting collaboration efforts in bioaerosol exposure studies. an added challenge in terms of measuring microbial diversity is that culture-dependent analytical approaches recuperate the cultural/viable portion of bioaerosols exclusively. in contrast, high-throughput sequencing (hts) methods are associated with a more in-depth characterization of the microbial content of a sample [ , , [ ] [ ] [ ] [ ] . to overcome the aforementioned challenges, the scientific community that studies aerosols has identified the need to explore new alternatives and complementary approaches for assessing bioaerosol exposure, such as identifying markers that can help classify environments based on human health risks [ ] [ ] [ ] . the upper respiratory tract, which includes the nasal cavity and the nasopharynx and is a primary pathway for inhaled air, is an important niche for transient environmental bacteria and for colonization. previous studies have already used nasopharynx or nasal cavity samples to look for specific microorganisms, using culture-based approaches [ , ] , or revealed the presence of bacterial resistance genes, using molecular biology approaches [ ] . recently, an hts approach was used on samples from the anterior and posterior cavity of the nose to study the bacterial diversity of individuals [ ] . however, because the microflora in the nasal cavity is dynamic and fluctuant [ ] , a region like the nasopharynx may represent a more long-term reservoir of inhaled bioaerosols. to the best of our knowledge, no study has ever used a s rrna amplicon-based hts approach to investigate the microbial diversity of nasopharyngeal flora, in order to assess occupational exposure. microbiome studies that use upper respiratory tract tissue and focus on specific pathologies (such as asthma, chronic obstructive pulmonary disease and chronic rhinosinusitis) may underestimate the effect of occupational exposure on the microbiomes of the subjects examined in those studies. we hypothesis that the microbiome of patients with respiratory disease is most likely affected by exposure to their work environment in addition to disease, rather than to disease alone. this study aims to contribute to the development of a new alternative method for assessing bioaerosol exposure, by linking the nasopharyngeal flora of pig farm workers to the bioaerosol microbial composition of their occupational environment, and to explore the role of bioaerosols in the nasopharyngeal microbial content. the s rrna amplicon-based hts approach was used to determine bacterial diversity, while qpcr was used to evaluate the presence of human pathogenic agents and bacterial resistance genes in bioaerosols and in nasopharyngeal samples from pig workers. additionally, wwtps were used as the industrial, low-dust, non-agricultural environment control to validate the microbial link between the bioaerosol content (air) and the nasopharynxes of workers. although the experiment focused on pig farmers and the buildings that they work in, the results may have implications for a wider population of agricultural workers. consequently, nasopharynx samples could be used as proxies for air samples in exposure assessment studies and for the determination of exposure markers in agricultural settings. in addition, the study advocates for the need of systematic bioaerosol exposure study when evaluating the nasopharyngeal microbiota. air samples were collected from eight confined pig buildings in eastern canada during fall/winter . inside each farm building, a sampling site was designated based on worker activities and bioaerosol exposure. the buildings visited were mechanically ventilated and contained between - pigs each weighing between - kg. there were no obvious signs of illness affecting the animals. air samples were collected from eight wwtps in the province of quebec, located in eastern canada, the during summer and winter seasons. summer visits occurred between september and july , and winter visits occurred between february and march . four sampling sites were chosen depending on the wastewater treatment process (screening, de-gritting/degreasing, settling tank, and bio-filtration), workers' daily tasks, and the level of confinement of the space. a liquid cyclonic impactor coriolis µ biological air sampler (bertin corp., rockville, md, usa) was used for collecting air samples. the samplers were set at l/min for min ( m of air per sample), placed within - m of the bioaerosol source and away from any turbulent air flow (e.g., away from building exhaust fans). fifteen milliliters of a phosphate buffer saline (pbs) solution ( . m, ph . , lonza, walkersville, md, usa) were used as the collecting solution. nasopharyngeal samples were taken from controls (university students and staff never exposed to animal farms), pig farmers and wwtp workers between the fall of and spring . all controls, pig farmers, and wwtp workers were non-smokers and none were taking antibiotics. the protocol was approved by the ethics committee of the institut universitaire de cardiologie et de pneumologie de québec (cer ). nasopharyngeal samples were collected by a nurse using swabs (puritan ® hydraflock ® collection devices, guilford, ma, usa) with a dry secure transport system. briefly, a swab was inserted into the nose until a resistance was felt and then turned a few times before it was removed. samples were transported to the laboratory at • c. from the ml collecting solution of the coriolis µ biological air sampler (bertin corp.), a . ml aliquot was centrifuged for min at , × g (j. pilote protocol = ml aliquot, min, , × g). the supernatant was discarded and the pellets were kept at − • c until dna extraction. likewise, the tips of the swabs were cut and vortexed thoroughly in ml pbs (lonza) and discarded. suspensions were then centrifuged for min at , × g, the supernatants were discarded and the pellets were stored at − • c until dna extraction. total genomic dna from air and nasopharyngeal samples was extracted with a powerlyser ® powersoil isolation dna kit (mo bio laboratories, carlsbad, ca, usa) following the manufacturer's instructions. dna samples were stored at − • c until subsequent analyses. the amplification of targeted genes, equimolar pooling, and sequencing were performed at the plateforme d'analyses génomiques (ibis, université laval, québec, qc, canada). the s rrna v -v region was amplified using the sequence-specific regions described in comeau et al. using a two-step dual-indexed pcr approach, specifically designed for illumina ® instruments, san diego, ca, usa [ ] . the gene-specific sequence was first fused to the illumina ® truseq sequencing primers and pcr was carried out in a total volume of µl containing × q buffer (neb, ipswish, ma, usa), . µm of each primer, µm of each of the dntps, u of q high-fidelity dna polymerase (neb) and µl of template dna. pcr thermoprotocol began with an initial denaturation at • c for s followed by cycles of denaturation at • c for s, annealing at • c for s, extension at • c for s and a final extension at • c for min. the pcr reaction was purified using the axygen pcr cleanup kit (axygen ® , waltham, ma, usa). the quality of the purified pcr product was checked on a % agarose gel. a fifty to -fold dilution of the purified product was used as a template for a second round of pcr in order to add barcodes (dual-indexed) and for missing sequences required for illumina sequencing. the thermoprotocol for the second pcr was identical to the first one but with cycles. pcr reactions were purified again in the same way as above, checked for quality on a dna bioanalyzer chip (agilent ® , santa clara, ca, usa) and then quantified spectrophotometrically with the nanodrop ® (thermo fisher scientific, waltham, ma, usa). barcoded amplicons were pooled in equimolar concentrations for sequencing on the illumina ® miseq machine. the oligonucleotide sequences that were used for pcr amplification are presented in table . briefly, after de-multiplexing the raw fastq files, the reads generated from the paired end sequencing were combined using the make.contigs script from mothur [ ] . quality filtering was also performed with mothur, using the screen.seqs script to discard homopolymers, reads with ambiguous sequences, and reads with suspiciously short lengths. similar sequences were gathered together in order to reduce the computational burden, and the number of copies of the same sequence was displayed to monitor the abundance of each sequence. this de-replication step was performed with vsearch [ ] . the sequences were then aligned with the bacterial reference silva core alignment using the qiime script align_seqs.py [ ] . operational taxonomic units (otus), with a % similarity cut-off, were clustered using the uparse method implemented in vsearch. uchime was used to identify and remove the chimeric sequences [ ] . qiime was used to assign taxonomy to otus based on the silva database reference training dataset for taxonomic assignment and to generate an otu table. a metadata-mapping file was produced that includes information about air and nasopharyngeal samples. the microbial diversity analyses, including statistical analyses, conducted in this study, were achieved using qiime plugins in version . . as described in qiime scripts (http://qiime.org/scripts/). the names of the scripts used are mentioned in the results section of each analysis. pcr was performed with cfx- and cfx- touch™ real-time pcr detection systems (bio-rad laboratories, mississauga, on, canada) to evaluate the presence of six human pathogens (clostridium difficile, listeria monocytogenes, mycobacterium avium, salmonella spp., staphylococcus aureus, and methicillin-resistant staphylococcus aureus (mrsa)), and antibiotic and metal resistance genes (cephalosporin, colistin, zinc). the pcr mixture contained µl of dna template, - nm for each primer, - nm probe and µl of × iq™ supermix or iq™ sybr ® green supermix (bio-rad laboratories) in a µl reaction mixture. the results were analyzed using bio-rad cfx manager software, version . (bio-rad laboratories). positive control and standard curves ranging from × to one copy of the targeted genes were used for each protocol using genomic dna or synthetic genes as templates (integrated dna technologies, coralville, ia, usa). negative controls were included in the plates as ntc (non-template controls). all primers and probes were purchased from integrated dna technologies. the primers, probes, hybridization temperatures, amplicon sizes and original references for all targeted genes are listed in pilote et al. [ ] . for alpha diversity measures, the normality was verified using the d'agostino and pearson omnibus normality test. the assumption of data normality was not fulfilled. non-parametric mann-whitney u tests (two-tailed) were performed to highlight that there are significant differences in diversity measures between the groups of samples. a p-value ≤ . was considered statistically significant. all of the results were analyzed using the software graphpad prism . (graphpad software, inc., san diego, ca, usa). to determine the statistical significance of the variation in the observed microbial community composition with multivariate analyses (pcoa), a permanova test was performed on the unweighted unifrac matrix. the compare_categories.py qiime script was used to generate the statistical results. because permanova is a non-parametric test, significance is determined through permutations. in this case, permutations were used. a p-value ≤ . was considered to be statistically significant. detailed information about the performance of the test is presented in the multivariate section of the results. the non-parametric mann-whitney u test was used to ascertain whether or not differences in otu abundances were statistically significant between the controls and pig farmers. to test otu differential abundance, the null hypothesis was that the populations that the two groups of samples were collected from have equal means. the range of p-values obtained for the most differentially abundant otus between the control samples and the pig farmer samples are presented in the differential abundance section of the results. this study used both positive and negative controls. the negative controls include unused swabs that underwent the same extraction protocol as the swabs collected from the subjects of this study. a pcr amplification targeting the s rrna genes allowed us to confirm the very low biomass of the negative controls compared to the nasopharyngeal swabs from the pig workers and non-exposed controls. for this reason, negative controls did not pass the next step of illumina hts. additional negative controls consisted of outdoor air samples that were taken outside the pig buildings sampled in this study. these samples showed enough concentration of bacterial biomass with the pcr amplification. thus, outdoor negative controls were sequenced. however, the number of reads and subsequent otu clustering was low compared to the indoor air samples. during the rarefaction step, the negative controls were not included in the analyses due to a low number of sequences. the goal is to have a number of sequences per sample deep enough to cover most of the bacterial diversity. positive controls consisted of a mock community containing equal concentrations of bacteria purchased from atcc ( strain even mix genomic material atcc ® msa- tm ). sequencing of the mock community showed a taxonomic profile resembling the expected microbial community, but with different relative abundances. in total, air samples from pig buildings, nasopharynx samples from farmers and nasopharynx samples from the non-exposed control group resulted in , , sequences (air samples = , ; farmers = , , , controls = , , ). following quality filtering and the discarding of singletons, , , unique sequences clustered into otus. representing the non-agricultural control environment (wwtps), , , sequences came from air samples ( , ) and nasopharynx samples ( , , ) from plant workers. after quality filtering and the removal of singletons, , unique sequences clustered into otus. a rarefaction analysis was performed to validate the sequencing depth and to confirm the effective sampling of the microbial diversity using the alpha_rarefaction.py qiime script. the lowest-depth sample parameter was used for the rarefaction analyses, allowing equal numbers of sequences for all samples. therefore, the samples with a sequencing depth lower than the reference sample were excluded from the analyses. the higher the sequencing depth, the more likely that the full diversity coverage is attained. the sequencing depth was , sequences for all the groups of samples: the air samples from pig buildings, the nasopharyngeal samples of pig farmers and non-exposed controls. the points shown in figure were calculated as follows: ten values from to , analyzed sequences were randomly selected. for each of these values, the corresponding number of otus observed, was noted for all of the samples. then, the average number of otus observed, plus or minus one standard deviation, were calculated for each of the ten values. the samples were divided into three groups: air from pig buildings, pig farmers and non-exposed controls. the slope of the curves shows sufficient sequencing depth and good bacterial coverage in all samples. moreover, pig farmers and air samples showed the highest average number of otus compared to non-exposed controls. four indexes were used to measure alpha diversity using the alpha_diversity.py script: chao richness estimator (the higher the number of otus in a sample, the higher the value of the chao index). for a more detailed explanation about richness estimate calculation, please refer to http://chao.stat.nthu.edu.tw/wordpress/paper/ .pdf. in shannon and simpson diversity measures, richness is combined with abundance to obtain an evenness measure. simpson values are bounded between and , where represents the most diverse case. shannon values are bounded between and , where represent the highest diversity) and phylogenetic diversity (pd) whole tree (quantitative measure of phylogenetic diversity; the higher the value, the higher the diversity; no limit value). the nasopharynx samples from pig farmers consistently showed the highest richness estimates and diversity measure values, whereas non-exposed controls displayed the lowest values (figure a-d) . the difference between the two groups of samples was significant (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). the richness estimates and diversity measures in the air samples were nearly as high as the pig farmer nasopharynx samples, although the measures from the pig farmer samples were statistically higher (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). the difference between the air samples from pig buildings and non-exposed controls (nasopharynx) was significant as well (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). four indexes were used to measure alpha diversity using the alpha_diversity.py script: chao richness estimator (the higher the number of otus in a sample, the higher the value of the chao index). for a more detailed explanation about richness estimate calculation, please refer to http://chao.stat.nthu.edu.tw/wordpress/paper/ .pdf. in shannon and simpson diversity measures, richness is combined with abundance to obtain an evenness measure. simpson values are bounded between and , where represents the most diverse case. shannon values are bounded between and , where represent the highest diversity) and phylogenetic diversity (pd) whole tree (quantitative measure of phylogenetic diversity; the higher the value, the higher the diversity; no limit value). the nasopharynx samples from pig farmers consistently showed the highest richness estimates and diversity measure values, whereas non-exposed controls displayed the lowest values (figure a-d) . the difference between the two groups of samples was significant (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). the richness estimates and diversity measures in the air samples were nearly as high as the pig farmer nasopharynx samples, although the measures from the pig farmer samples were statistically higher (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). the difference between the air samples from pig buildings and non-exposed controls (nasopharynx) was significant as well (chao p = . ; shannon p = . ; simpson p = . ; pd whole tree p = . ). an ecological analysis was conducted to reveal the variation in the community composition between the three sample groups (nasopharynx of pig farmers and non-exposed controls and air from pig farms). the weighted unifrac distance metric was used to calculate the pairwise distances between samples using the beta_diversity.py script. the distance matrix was then transformed into coordinates using the principal_coordinates.py script and inter-samples distances were represented in a two-dimensional ( d) space using ordination. the samples closer to one another were more similar than those ordinated further apart. the principal coordinate analysis (pcoa) was used to visualize bacterial community variation (make_ d_plots.py). figure a shows the two principal coordinate axes capturing a total of . % of the variation observed. a distinct clustering of pig farmers, nonexposed controls, and air samples from pig buildings is also illustrated in that figure. the profiles of pig farmers were more similar to the profiles of air samples than to the profiles of non-exposed controls. the distinct clustering was confirmed by the per-mutational multivariate analyses of variance (permanova p = . ). the same statistical test was used to confirm the non-significant clustering of air and pig farmer (nasopharynx) samples, as the test showed a non-significant difference (permanova p = . ). interestingly, air samples from the pig buildings seemed to display less dispersion amongst its individuals than the farmers and non-exposed groups, indicating a more homogenous bacterial community structure. we used a phylogram that displays sample clustering, using the unweighted pair group method, with arithmetic mean to confirm the sample clustering observed with the pcoa analyses ( figure b ). an ecological analysis was conducted to reveal the variation in the community composition between the three sample groups (nasopharynx of pig farmers and non-exposed controls and air from pig farms). the weighted unifrac distance metric was used to calculate the pairwise distances between samples using the beta_diversity.py script. the distance matrix was then transformed into coordinates using the principal_coordinates.py script and inter-samples distances were represented in a two-dimensional ( d) space using ordination. the samples closer to one another were more similar than those ordinated further apart. the principal coordinate analysis (pcoa) was used to visualize bacterial community variation (make_ d_plots.py). figure a shows the two principal coordinate axes capturing a total of . % of the variation observed. a distinct clustering of pig farmers, non-exposed controls, and air samples from pig buildings is also illustrated in that figure. the profiles of pig farmers were more similar to the profiles of air samples than to the profiles of non-exposed controls. the distinct clustering was confirmed by the per-mutational multivariate analyses of variance (permanova p = . ). the same statistical test was used to confirm the non-significant clustering of air and pig farmer (nasopharynx) samples, as the test showed a non-significant difference (permanova p = . ). interestingly, air samples from the pig buildings seemed to display less dispersion amongst its individuals than the farmers and non-exposed groups, indicating a more homogenous bacterial community structure. we used a phylogram that displays sample clustering, using the unweighted pair group method, with arithmetic mean to confirm the sample clustering observed with the pcoa analyses ( figure b) . the clustering (air from pig buildings and pig farmers together, versus the non-exposed controls) was statistically significant as confirmed by the permanova test (p-value = . ). given the observed difference in the number of bacterial otus, evenness, and evolutionary distance (alpha diversity) and in the bacterial community composition (beta diversity) in samples of the nasopharyngeal flora of farmers and non-exposed individuals and bioaerosols, collected in pig buildings, the next step was to reveal the taxonomic profiles of the three groups. figure shows the taxonomic distribution of the bacterial phyla across the three groups of samples. overall, actinobacteria, proteobacteria, bacteriotedes, and firmicutes dominated the three profiles, representing more than % of the taxonomic abundance. however, major differences distinguished the pig farmer samples from the non-exposed controls. in the latter, actinobacteria and proteobacteria were the most abundant phyla (relative abundances of %, and %, respectively). however, in farmers, firmicutes and bacteriotedes were the most dominant phyla with relative abundances of %, and %, respectively. consistent with the previous analyses, air samples from pig farms had different relative abundances values, but comparable profiles (with the same conclusions) to the nasopharyngeal flora of farmers, with a dominance of firmicutes ( %), followed by bacteriotedes ( %). actinobacteria, and proteobacteria had a relative abundance of less than % in bioaerosol samples. notably, spirochaetes, tenericutes, and verrumicrobia were detected only in farmers and the air from pig buildings. the clustering (air from pig buildings and pig farmers together, versus the non-exposed controls) was statistically significant as confirmed by the permanova test (p-value = . ). given the observed difference in the number of bacterial otus, evenness, and evolutionary distance (alpha diversity) and in the bacterial community composition (beta diversity) in samples of the nasopharyngeal flora of farmers and non-exposed individuals and bioaerosols, collected in pig buildings, the next step was to reveal the taxonomic profiles of the three groups. figure shows the taxonomic distribution of the bacterial phyla across the three groups of samples. overall, actinobacteria, proteobacteria, bacteriotedes, and firmicutes dominated the three profiles, representing more than % of the taxonomic abundance. however, major differences distinguished the pig farmer samples from the non-exposed controls. in the latter, actinobacteria and proteobacteria were the most abundant phyla (relative abundances of %, and %, respectively). however, in farmers, firmicutes and bacteriotedes were the most dominant phyla with relative abundances of %, and %, respectively. consistent with the previous analyses, air samples from pig farms had different relative abundances values, but comparable profiles (with the same conclusions) to the nasopharyngeal flora of farmers, with a dominance of firmicutes ( %), followed by bacteriotedes ( %). actinobacteria, and proteobacteria had a relative abundance of less than % in bioaerosol samples. notably, spirochaetes, tenericutes, and verrumicrobia were detected only in farmers and the air from pig buildings. the relative abundance of taxa was more thoroughly analyzed by examining the most abundant bacterial classes across the three groups of samples ( figure ). similar to the phyla profiles, the class profiles showed notable differences between non-exposed controls and farmers/air from pig buildings. in the former, actinobacteria ( %), saprospirae ( %), bacilli ( %), gammaproteobacteria ( %) and betaproteobacteria ( %) represented more than % of the taxonomic profile. however, the profile from farmers was more evenly distributed. clostridia had the highest relative abundance ( %) followed by saprospirae ( %), bacilli ( %) and actinobacteria ( %). unlike the non-exposed control group, gammaproteobacteria and betaproteobacteria represented less than % of the profile, whereas bacteroidia represented % of the relative abundance. in the non-exposed control group, bacteroidia represented . % of the taxonomic profile. in air samples, clostridia, bacilli and bacteroidia dominated the profile representing more than % of the relative abundance, thus confirming the previous observations about the similarity between the flora from pig farmers and air samples. interestingly, the presence of coriobacteria, erysipelotrichi, spichaetes, mollicutes, sphyngobacteria, epsilonproteobacteria, and verruco- was exclusive to samples from the nasopharynx of pig farmers and sampled bioaerosols. the relative abundance of taxa was more thoroughly analyzed by examining the most abundant bacterial classes across the three groups of samples ( figure ). similar to the phyla profiles, the class profiles showed notable differences between non-exposed controls and farmers/air from pig buildings. in the former, actinobacteria ( %), saprospirae ( %), bacilli ( %), gammaproteobacteria ( %) and betaproteobacteria ( %) represented more than % of the taxonomic profile. however, the profile from farmers was more evenly distributed. clostridia had the highest relative abundance ( %) followed by saprospirae ( %), bacilli ( %) and actinobacteria ( %). unlike the non-exposed control group, gammaproteobacteria and betaproteobacteria represented less than % of the profile, whereas bacteroidia represented % of the relative abundance. in the non-exposed control group, bacteroidia represented . % of the taxonomic profile. in air samples, clostridia, bacilli and bacteroidia dominated the profile representing more than % of the relative abundance, thus confirming the previous observations about the similarity between the flora from pig farmers and air samples. interestingly, the presence of coriobacteria, erysipelotrichi, spichaetes, mollicutes, sphyngobacteria, epsilonproteobacteria, and verruco- was exclusive to samples from the nasopharynx of pig farmers and sampled bioaerosols. a non-parametric mann-whitney u test, was used to analyze count data and determine the species most significantly associated with farming. the test compares otu frequencies in groups of samples and ascertains if there are statistically different otu abundances between the two groups of samples. the mann-whitney u test uses absolute data counts rather than relative abundances. more specifically, the output of the test contains the test statistic, the p-value corrected for multiple comparisons, and a mean count for each otu in the given sample group. this test was used following instructions from the group_significance.py qiime script. the thirty taxa (identified to the species or genera) with the greatest significant differences in counts between samples from pig farmers and non-exposed controls are presented in figure . however, to better visualize and emphasize the most striking cases of differential abundance, the list is not exhaustive. the complete results output of differential abundance is presented in additional file (supplementary material). it represents the results of the mann-whitney u test to determine the statistical differential abundance of taxa in nasopharynx of workers and non-exposed controls. the test was applied to sequences using qiime script (group_significance.py) with the mann-whitney u test option. the taxonomy represent bacteria from the nasopharynx samples. notably, some taxa were identified only to class or family, as those were the highest levels of identification possible using the silva database. p-values were corrected for multiple comparisons using the bonferroni correction. values ranged from . to . for the differentially abundant taxa, from pig farmer samples, and from . to . for the differentially abundant taxa, from non-exposed controls. the most notable imbalance was observed for the class clostridia with a mean count of more than sequences in pig farmer samples and less figure . taxonomic profile showing the relative abundance of each bacterial class across nasopharyngeal flora samples from pig farmers, non-exposed controls and air samples from pig buildings. taxa written in bold type were specific to farmers and air from pig buildings. a non-parametric mann-whitney u test, was used to analyze count data and determine the species most significantly associated with farming. the test compares otu frequencies in groups of samples and ascertains if there are statistically different otu abundances between the two groups of samples. the mann-whitney u test uses absolute data counts rather than relative abundances. more specifically, the output of the test contains the test statistic, the p-value corrected for multiple comparisons, and a mean count for each otu in the given sample group. this test was used following instructions from the group_significance.py qiime script. the thirty taxa (identified to the species or genera) with the greatest significant differences in counts between samples from pig farmers and non-exposed controls are presented in figure . however, to better visualize and emphasize the most striking cases of differential abundance, the list is not exhaustive. the complete results output of differential abundance is presented in additional file (supplementary materials). it represents the results of the mann-whitney u test to determine the statistical differential abundance of taxa in nasopharynx of workers and non-exposed controls. the test was applied to sequences using qiime script (group_significance.py) with the mann-whitney u test option. the taxonomy represent bacteria from the nasopharynx samples. notably, some taxa were identified only to class or family, as those were the highest levels of identification possible using the silva database. p-values were corrected for multiple comparisons using the bonferroni correction. values ranged from . to . for the differentially abundant taxa, from pig farmer samples, and from . to . for the differentially abundant taxa, from non-exposed controls. the most notable imbalance was observed for the class clostridia with a mean count of more than sequences in pig farmer samples and less than sequences in the non-exposed controls. staphylococcus epidermis was present with a mean count of sequences in non-exposed individuals and less than sequences in pig farmers. other important examples related to human health include, the greater differential abundance of haemophilus influenzae in non-exposed controls ( sequences in non-exposed control samples vs. in samples from pig farmers), and the differential abundance of klebsiella in samples from pig farmers ( sequences in pig farmer samples vs. in non-exposed controls). than sequences in the non-exposed controls. staphylococcus epidermis was present with a mean count of sequences in non-exposed individuals and less than sequences in pig farmers. other important examples related to human health include, the greater differential abundance of haemophilus influenzae in non-exposed controls ( sequences in non-exposed control samples vs. in samples from pig farmers), and the differential abundance of klebsiella in samples from pig farmers ( sequences in pig farmer samples vs. in non-exposed controls). figure . taxa identified to highest possible taxonomic level with statistically significant differential abundances across pig farmers and non-exposed controls. from the bottom to the top: the first taxa were the most abundant in samples from farmers and the last were more abundant in nonexposed controls. the taxa written in bold type affect human health. a non-agricultural low-dust control environment (wwtps) was used as a control to validate the link between the microbial composition of nasopharyngeal flora of exposed workers and that of bioaerosols released in the workplace. the nasopharynx samples of the non-exposed controls figure . taxa identified to highest possible taxonomic level with statistically significant differential abundances across pig farmers and non-exposed controls. from the bottom to the top: the first taxa were the most abundant in samples from farmers and the last were more abundant in non-exposed controls. the taxa written in bold type affect human health. a non-agricultural low-dust control environment (wwtps) was used as a control to validate the link between the microbial composition of nasopharyngeal flora of exposed workers and that of bioaerosols released in the workplace. the nasopharynx samples of the non-exposed controls (subjects not previously exposed to any animal farm) were again used for comparison with the nasopharynx samples from wastewater workers and air samples from wwtps. the distances between the groups of samples were compared and visualized using the pcoa approach. similar to the pig farm environment, the pairwise distances were calculated using the weighted unifrac distance metric. figure shows the two principal coordinate axes capturing a total of . % of the variation observed. unlike the farm environment, the nasopharynx samples from wastewater workers did not cluster with air samples from wwtps. in fact, nasopharyngeal flora of wastewater workers and non-exposed controls had similar microbial compositions. the difference between air and nasopharynx samples (controls and wastewater workers) was statistically significant (permanova p = . ). as shown in figure , the difference between non-exposed controls and wastewater workers was not significant (permanova p = . ). (subjects not previously exposed to any animal farm) were again used for comparison with the nasopharynx samples from wastewater workers and air samples from wwtps. the distances between the groups of samples were compared and visualized using the pcoa approach. similar to the pig farm environment, the pairwise distances were calculated using the weighted unifrac distance metric. figure shows the two principal coordinate axes capturing a total of . % of the variation observed. unlike the farm environment, the nasopharynx samples from wastewater workers did not cluster with air samples from wwtps. in fact, nasopharyngeal flora of wastewater workers and non-exposed controls had similar microbial compositions. the difference between air and nasopharynx samples (controls and wastewater workers) was statistically significant (permanova p = . ). as shown in figure , the difference between non-exposed controls and wastewater workers was not significant (permanova p = . ). principal coordinate analysis plot. the plot shows the distances for the microbiota of three groups of samples: nasopharynx samples from wastewater workers and from non-exposed controls and bioaerosols from wastewater treatment plants (wwtps). the pairwise distances were calculated using the weighted unifrac distance metric. the presence of human pathogens was investigated in the nasopharynx of pig farmers. as noted in table , all of the pathogens were more frequently detected in the pig farmer nasopharynx samples, compared to non-exposed controls, with the exception of salmonella spp. striking examples include, mrsa and clostridium difficile, which were present in the nasopharyngeal flora of %, and % of pig farmers, respectively. they were found in %, and % of the non-exposed controls, respectively. principal coordinate analysis plot. the plot shows the distances for the microbiota of three groups of samples: nasopharynx samples from wastewater workers and from non-exposed controls and bioaerosols from wastewater treatment plants (wwtps). the pairwise distances were calculated using the weighted unifrac distance metric. the presence of human pathogens was investigated in the nasopharynx of pig farmers. as noted in table , all of the pathogens were more frequently detected in the pig farmer nasopharynx samples, compared to non-exposed controls, with the exception of salmonella spp. striking examples include, mrsa and clostridium difficile, which were present in the nasopharyngeal flora of %, and % of pig farmers, respectively. they were found in %, and % of the non-exposed controls, respectively. listeria monocytogenes was detected in % of non-exposed controls and in % of pig worker samples. mycobacterium avium was not detected in the nasopharynx samples of either group. likewise, antibiotic and zinc resistance genes were present at a higher frequency among pig farmers compared to non-exposed controls. moreover, cephalosporin, and colistin resistance genes were exclusively detected in the nasopharyngeal flora of pig farmers. table . human pathogens and antibiotic and zinc resistance genes in the nasopharyngeal flora of pig workers compared to non-exposed controls. given the many potential microbial sources, animal farmers inhale a variety of aerosolized bacteria that impact their health [ , , ] . in this study, the bacterial populations in bioaerosols from pig buildings were compared to those of the nasopharyngeal flora of farmers using bioinformatics tools to determine if nasopharynx sampling could be used as a proxy for air sampling in exposure assessment studies. systemic microbial ecology analyses led to unequivocal results with identical conclusions throughout the analyses. the alpha diversity of bacterial species in the air from pig buildings and the nasopharyngeal flora of farmers were not statistically different. the evaluation of species diversity was introduced by whittaker and defined as the number of species and their proportional abundance within one sampling site [ ] . there are different ways to measure alpha diversity and an extensive list of indexes has been presented by magurran and mcgill [ ] . in addition to the usual chao richness estimates and shannon/simpson diversity measures [ ] [ ] [ ] [ ] , pd whole tree was also used to analyze the alpha diversity in samples in this study. pd stands for phylogenetic diversity and is defined as the minimum length of all phylogenetic branches required to span a given set of taxa on the phylogenetic tree [ ] . all four of the alpha diversity measures revealed greater bacterial richness and diversity in the nasopharyngeal samples from pig farmers compared to non-exposed individuals. the observed similarity between bioaerosols from pig buildings and the nasopharyngeal flora from farmers is indicative of occupational exposure and, consequently, a transient presence and/or possible colonization of the upper respiratory tract regions by environmental bacteria. these findings are even more interesting given that the majority of pig farmers recruited for this study do not work in the eight pig buildings selected for air analysis. this suggests that airborne bacteria associated with pig buildings can take over the microbiota in farmers' nasopharynxes. the establishment of this "new" microbial community could represent a microbial signature for the nasopharynx of pig farmers. also, a higher prevalence of viruses in the nasopharynxes of farmers compared to the non-exposed control group could play a role in the increased alpha-diversity [ ] . beta diversity analyses revealed that long-term exposure, such as occupational exposure to bioaerosols in the air of pig buildings, appeared to modify the nasopharyngeal microbiota of farmers. common approaches to evaluating changes in the community composition (beta diversity) rely on the creation of a (dis)similarity matrix to calculate the distance between samples. dis(similarity) matrices may be calculated using different methods depending on the type of dataset, analyses, and the objectives of the study, as some metrics are more suitable than others [ ] [ ] [ ] [ ] . the unifrac distance metric was used as efficacy was proven with s rrna bacterial genes [ ] . in addition, pcoa coupled with permanova offers a robust statistical significance of sample grouping using distance matrices. this non-parametric multivariate analysis of variance separates the distance matrix into sources of variation to describe the robustness and significance of a variable in explaining the variations observed between samples. it is based on the anova experimental design but analyzes the variance and determines the significance by permutations, as it is a non-parametric test [ ] . whereas, anova/manova assumes normal distributions and a euclidean distance, permanova can be used with any distance measure. the two analyses led to the same conclusions for this study. therefore their usefulness when used together as a tool to visualize and evaluate sample clustering was confirmed. the distinct clusters formed between the combination of pig farmers, and the air from pig buildings, and non-exposed individuals, is clearly linked to a strong divergence in the nasopharyngeal microbiota of farmers compared to other non-exposed individuals. mechanical deposition of < µm diameter inhaled particles on nasopharyngeal surfaces by inertial impaction [ ] , represents a continuous source of environmental bacteria to the nasopharynx. this continuous source of bacterial exposure may therefore be responsible for the establishment of a reservoir of bacteria reflecting long-term exposure (e.g., occupational exposure). a thorough understanding of the established bacterial community may then lead to a better evaluation of the risks associated with an environment. for example, domestic animals share some of their microbiota with their human cohabitants, supposedly through frequent and direct contact [ , ] . song et al., ( ) mention that airborne microbiota plays an important role in microbial transfer to the human upper respiratory tract. ten thousand litres of air are inhaled daily and the bioaerosols in the air may have an effect on the human nasal microbial community [ ] . finally, as different farm animals are associated with different microbiota, the normal nasopharyngeal flora of farmers may be differently disturbed. in other words, farmers working with different animals may have a different disturbances of their natural nasopharyngeal flora. therefore, the microbial fingerprint of the nasopharynx may be directly linked to a specific type of farming environment and the potential long-term health effects on farmers. the high abundance of firmicutes and bacteriotedes in pig buildings has been shown [ ] [ ] [ ] . interestingly, the results of this study are consistent with the literature, but with the added information indicating that, these same phyla colonize the nasopharynxes of farmers. more particularly, other studies have previously shown clostridia to be the most abundant class of bacteria in bioaerosols from pig buildings [ ] . this study not only confirmed the abundance of clostridia in air samples, but also that this class was the dominant class found in nasopharynx samples, while it was practically absent in non-exposed individuals. clostridium spp., identified as differentially abundant in the farmer nasopharyngeal samples in the present study, comprise potentially pathogenic species [ ] . specifically, clostridium butyricum, also differentially abundant in samples from farmers, has been identified as an emerging pathogen by public health authorities. some c. butyricum pathogenic strains were associated with the occurrence of necrotizing enterocolitis, a bowel disease [ ] . in addition, prevotella spp., which was strikingly more abundant in nasopharynx samples, is a well-known agent involved in upper respiratory tract infections [ ] [ ] [ ] . moraxella is another genus identified by the differential abundance analyses as being predominant in the nasopharynxes of farmers. like for other taxa identified in this study, strains of moraxella spp. were previously detected as airborne bacteria in pig buildings [ ] . the rate of colonization of moraxella spp. in healthy adult populations is around % [ ] . species of this genus are opportunistic pathogens responsible for upper and lower respiratory tract infections [ ] . taxa identified exclusively in nasopharyngeal samples and air samples from pig buildings could be candidates for new markers to assess exposure to bioaerosols in pig farming environments. although, actinobacteria are most abundant in the non-exposed controls, no pathogen was identified in the most abundant taxa presented in figure (except haemophilus influenza). an example of the most abundant actinobacteria in non-exposed control is micrococcus luteus that was differentially more abundant in controls compared to farmers. another example of firmicutes is staphylococcus epidermis that was more abundant in the controls compared to the farmers. the respiratory health of farmers has been of great interest for testing the hygiene hypothesis that stipulates that exposure to microbes from intensive farming during early life could be beneficial to health in adulthood [ , ] . however, the acceptance of the hygiene hypothesis is not unanimous in the scientific community [ ] . in this study, taxa identified as differentially abundant among farmers could hypothetically play a role in the prevention of allergy and the development of atopic diseases. indeed, some bacteria identified through this investigation (e.g., pedobacter, pelomonas, and megasphaera) have been linked to healthy respiratory conditions [ ] . a recent study, conducted by kraemer and colleagues, also found a distinct clustering between samples from the nasal cavities of pig farmers and air samples from their workplace, when compared to the nasal cavities of non-exposed individuals [ ] . moreover, samples from the nasal cavities of cow farmers clustered separately from pig worker samples and air samples from pig buildings [ ] . although the nasal cavity is a more transient environment for environmental bacteria than the nasopharynx, it confirms the hypothesis of a microbial fingerprint specific to the farming environment. it supports the idea of creating a worldwide database, that lists potential markers specific to certain environments, and to the nasopharynxes of the people working in them. this database could represent an important asset for associating bioaerosol exposure with health problems [ ] . the lack of a correlation between the nasopharynx of wastewater workers and bioaerosols from wwtps could be explained by the nature and duration of exposure. workers wear personal protective devices (e.g., masks) at certain working sites (e.g., biofiltration), which may affect the establishment of a 'new' environmental microflora. supporting this idea, the most abundant bacteria, shared by non-exposed controls and wastewater workers, are naturally occurring skin bacteria like propionibacterium, corynebacterium, staphylococcus, streptococcus, and cutibacterium. these taxa were not present (relative abundance less than %) in air samples from wwtps (data not shown). farmers do not usually wear respiratory protection and, moreover, they often live in the farming environment (e.g., in a house located near farms) and are consequently continuously exposed to the microbes generated from farming activities (occupational and residential exposures). therefore, the exposure that farmers are subjected to is more likely to modify their natural nasopharyngeal flora. the agricultural/non-agricultural hypothesis presented in this work regarding the nasopharyngeal flora of exposed workers should be validated in other agricultural and industrial environments. recent studies of the microbiome of the upper respiratory tract may have underestimated the influence of occupational exposure when considering the effect of a particular disease on the natural flora of upper respiratory tract tissue [ ] [ ] [ ] . the fact that the work environment may affect the natural flora of an exposed person on a long-term scale is a crucial consideration when he/she becomes a patient whose upper respiratory tract microbiota is the target of the disease. the results obtained in this work emphasize the importance of considering the environment of the nasopharyngeal flora of exposed workers, who are or could become patients suffering from chronic respiratory diseases. in the same way that recent advances in methods for identifying microbes has helped implicate the upper respiratory tract microbiome in inflammatory respiratory diseases, evaluating bioaerosol exposure can help us support the roles of resident microbes in both healthy and diseased tissues. specific human pathogens and antibiotic and zinc resistance genes were detected in the nasopharynxes of pig farm workers as well as in bioaerosols of pig buildings [ ] . for bacterial diversity analyses, the qpcr approach supports the use of the nasopharynx as an alternative to air sampling. the presence of zinc and antibiotic resistance genes, in the nasopharynxes of farmers, is explained by the use of zinc and antibiotics in animal farming (for therapeutic or sub-therapeutic use, such as for growth promotion) and implies possible human health risks. extensive use of zinc in pig feed is responsible for the proliferation of zinc-resistant bacterial communities at farms [ ] . cephalosporin is a commonly used antimicrobial drug in human infections and the spread of its resistance constitutes part of the antibiotic resistance crisis [ ] . finally, the mcr- gene was found in the nasopharynxes of half of the pig farmers in this study, although the use of colistin is extremely regulated in north america and limited to multi-drug resistant microbes [ ] . future studies should include detailed health information on the sampled individuals to investigate the nasopharynx microbiota associated with certain occupational health problems. additionally, a longitudinal study of the bacterial diversity, in the nasopharynxes of farmers and in bioaerosols from pig buildings, could unveil a long-term variation in microbial content. finally, information about the diet of exposed human (or animal) and antibiotic use could be added to the analyses as important factors influencing the microbiota. this is the first study to link the nasopharyngeal flora of exposed humans with the source of the exposure in an agricultural setting, using bacterial diversity analyses and the detection of specific pathogens and resistance genes. the results suggest that workers are carriers of bioaerosol-associated bacteria and that nasopharynx sampling could be used as a proxy for air sampling in exposure assessment studies. furthermore, pig farmers are also carriers of specific human pathogens and resistance genes 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open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- - jh lvm authors: loureiro, natália i. v.; viana, henrique v.; rodrigues, carlos r.; cabral, lúcio mendes; silva, thaís d. n.; cardoso, fernanda serpa; santos, dilvani oliveira; castro, helena c. title: solving an ethical issue involved in experimentation with animals in a brazilian teaching laboratory date: - - journal: biochem mol biol educ doi: . /bmb. . sha: doc_id: cord_uid: jh lvm changes are occurring within brazilian institutes of higher education; currently several universities are reviewing their course offerings and teaching approaches to determine if they meet the needs of today's undergraduate students. when changes are made to the curriculum of experimental courses, there should be an understood guarantee that all efforts to avoid ethical and biosafety issues have been diligently considered. ethical considerations lead us to create an alternative experimental session to be conducted that eliminated the use of rats, the conventional in vivo model employed for learning metabolism of glycogen in our university. to avoid possible biosafety issues, we prepared an alternative sample to simulate human urine, which we called guarurine. using our new method, it is possible to verify positive results imitating a diabetic and starving people samples for detection of glucose and ketone. the alternative tool described herein is not only particularly suited to bypass the ethics of using animals for teaching, but also permits the discussion of significant aspects of pathological and physiological situations such as diabetics and starvation in a simple, safe, and interesting way. the use of animals for research and teaching is an issue of great concern worldwide. in contrast to the legislative systems in united states, britain, germany, scandinavia, and many other countries [ ] [ ] [ ] [ ] [ ] [ ] , brazilian scientists and teachers still can pursue research projects and teaching class with relative freedom [ ] . in brazil, animal research ethical committees were created only in the s. the federal law , which provides for the didactic-scientific practice of animal vivisection, was approved in may but still waits for regulation. in addition, some drafts that provide regulation for the use of animals for teaching and research purposes are still being analyzed by the brazilian congress [ , ] . in the brazilian fluminense federal university, the study of biochemistry in the department of molecular and cell biology includes laboratory activities, which mainly allow students to actively explore key concepts in biochemistry in greater depth and acquire skill in scientific reasoning [ - ] . animals (rats) have been widely used as models for teaching the catabolism of glycogen (glycogenolysis) in this discipline. however, in the last years, the student teaching assistants and some students of several different undergraduate courses that attend this class had considered this experimental session as inappropriate. according to them, there was an "unnecessary sacrifice" of rats to demonstrate the use of glycogen as a fuel by an organism. thus, the refusal to attend this specific session by some of these undergraduate students, including those from the faculty of veterinary school, became common. because ethics is not a matter of adhering to absolute rules, but rather of doing what will have best consequences, given the constraints under which we act, the ethics of using a specific experimentation will depend on if the goal can be reached while causing the animal less suffering, using fewer animals, or without using animals at all. anyway, due to ethical and others concerns, we were facing the challenge of changing this teaching session. in resolving this issue, understanding the reasons why students object to animal laboratories was an important step. in their point of view, this experimental class represented a serious religious/ethical dilemma because the life and death of laboratory animals becomes trivialized in the process of exemplifying only one topic of metabolism. therefore, based on their opinion and using the principle of our self-regulation, rules were set up to conduct the selection of a future practical laboratory experiment. these were i) avoid the use of laboratory animals that would be sacrificed; ii) the inclusion of other topics of metabolism such as glycolysis, citric acid cycle, fatty acid and amino acid synthesis and catabolism, and ketogenesis in the experimental discussion; iii) the experiment should have low cost and be performed after the relevant theory material is studied; and finally iv) it also should be easy and fast, due to the limited time of the practical class. consequently, the whole laboratory experience should not last more than h, according to the scheduled time available for the class. given these limitations, a previous laboratory class was considered to be an ideal candidate for replacement of the class in question. "urinalysis" is a nonanimal laboratory experiment basically consisting of assays to determine glucose and ketone levels in the urine from a diabetic and a healthy people. its experimental work does not last more than h, and all necessary reagents were already part of the stockroom, avoiding any further expenses. this class permits the discussion of several topics of metabolism including those previous established; and, most of all, it allows aligning classroom practices with professional practices. hence "urinalysis" met the main criteria that had been determined. however, despite passing these rules, "urinalysis" raises a biosafety issue, e.g. the risk of using body fluids at the undergraduate level. according to the world health organization, the body fluids and substances of all persons should be considered to contain potentially infectious agents [ ] . no distinction may be made between body fluids and substances from individuals with a known disease or infection and those from asymptomatic or undiagnosed individuals [ ] . consequently, infection control practices shall be present in the urinalysis class including the use of gloves and masks, frequent hand washing, proper cleaning, and good disinfectant practice. these procedures greatly raise the expense of the exercise, which could be a problem in less economically developed countries such as brazil [ ] . but an extreme example of the necessity for these protocols is the sars virus, which can survive in urine for at least h [ ] . another virus that can also be transmitted by this body fluid is the cytomegalovirus [ ] . in fact, the risk factors due to using body fluids were the reason for the replacement of the "urinalysis" session in this discipline in the first place. however, due to the several advantages of the "urinalysis" session theme, we decided to keep it, creating a harmless substitute for urine, averting the original animal ethical and biosafety issues. additionally, we also planned a different way of class presentation using a problembased learning-like approach, stimulating the student to be more active in the laboratory class [ , ] . in this article, we will present the protocol and approach used in this practice class, also including the evaluation by student teaching assistants and undergraduate students from nine different courses ("biological science," "pharmacy," "medicine," "veterinary medicine," "nutrition," "nursing," "odontology," "chemistry," and "industrial chemistry"). normal urine is a clear straw-colored liquid, which under normal conditions does not contain sugars, yeast cells, protein, ketones, bacteria, or parasitic organisms. in order to select a substance that imitates perfectly the visual aspect of urine, the famous brazilian guarana (paullinia spp.) was tested. the guarana seeds are used mainly to produce a soft drink that present similar visual appearance of urine [ ] . hence the soft drink in its diet version was utilized to prepare samples to be tested on the urine diagnostic assays. these assays include the detection of glucose by the classic benedict's copper reduction reaction and of acetoacetic acid (the physiological ketone) by the reaction with sodium nitroprusside in a strongly basic medium. in order to prepare the guarurine samples, a -ml cup of diet guarana was diluted twice with distilled and deionized water and three samples of ml were obtained, which we named guarurines (fig. ) . on the first sample, glucose ( % w/v) and acetone ( % v/v) were added, representing the diabetic urine (fig. ) . to the second sample, only acetone ( % v/v) was used to reproduce the urine from a starving person with prolonged diarrhea and vomiting (fig. ) . the last ml was considered the normal urine, therefore no glucose or acetone were added (fig. ) . usually the urine specimens should not remain unrefrigerated for longer than h, but in the case of guarurine samples they resisted several weeks out of the fridge, probably due to the presence of preservative in its formula. this aspect permits the storage of guarurine samples for longer periods than urine. classical benedictЈs copper reaction (glucose)-in the glass tubes, ml of benedict's reagent was added to ml of the guarurine samples. the test tubes were immediately placed into a beaker of boiling water and left for min. when heated with benedict's reagent, guarurine sample containing glucose (sample ) formed an orange-brown precipitate similar to the diabetic urine (fig. ) . sodium nitroprusside reaction in a strongly basic medium (ketone)-two drops of sodium nitroprusside reagent were added in ml of the guarurine samples in a glass tube. the test tube was mixed and tilted to about a °angle, and the samples were slowly overlaid with % ammonium hydroxide. a pink ring appeared at the junction of the two liquids in the guarurine samples containing acetone (samples and ) after min, similar to that observed for starving and diabetic people's urine. therefore, guarurine samples showed to be adequate as a harmless substitute for human urine to be used in the "urinalysis" session, because they act similar to the respective human samples (fig. ) . after the preparation of the protocol and arranging all necessary laboratory material including the guarurine, it was possible to evaluate this new practical class with the group of student teaching assistants from the biochemistry discipline (n ϭ ). the new problem-based learning-like approach used consisted of the presence of an initial situation where three unlabeled "urine" samples from three different people (a diabetic, a starving, and a healthy person) needed to be identified (fig. ) . the main purpose was to correctly identify this "biological" material according to the testing results that would be obtained during the experimental class. this approach would lead to the discussion about the initial expected results and those finally found. by providing specific reagents and the three guarurine samples, the students were invited to use the laboratory reagents in order to find out about the origin of these samples. the goal of this practical class was mainly to provide to these students the opportunity to develop their own reasoning and acquire knowledge from the experi-mental results obtained. the support given during this experimental session at this moment consists of aiding in clarifying the objectives of the experiments, organizing the content, and in the "diagnosing" the clinical cases. consequently, these student teaching assistants were also being prepared to coordinate future "guarurinalysis" session for undergraduate students. in the end, all six students were able to complete the whole session on time and also identify correctly the physiological "source" of the given material. an interview at the end of the experimental session revealed that all six student teaching assistants voted unanimously to the inclusion of "guarurinalysis" in the discipline. aspects such as students safety; the nonobligatory use of gloves and mask but keeping other safety procedures such as frequent hand washing, which would permit the discussion about biosafety issues; and most of all, the opportunity of simulating a "body fluid" clinical analysis in a safer way, were pointed as the main advantages in this experimental session during their interview. this experimental protocol was also tested with students from different undergraduate courses ("biological science," "pharmacy," "medicine," "veterinary medicine," "nutrition," "odontology," "chemistry," and "industrial chemistry") using the help of the students teaching assistants. at the beginning, undergraduate students were divided into groups for the organization of the experiments. however, everyone was stimulated to participate of the discussion during the whole teaching session. at the end, most of students could correctly identify the samples, increasing their knowledge about the biochemistry topics discussed. in case of the students, this practical session was evaluated by the application of a questionnaire consisting of simple and objective questions raised about four important issues; the acceptance level of the practical session; its permanence in the discipline for the next years; its direct correlation with biochemical topics discussed on the lecture part; and the clear applicability of this knowledge on their future professional career ( table i) . the survey results indicated that nearly all students find this experimental session interesting because all of them attributed good ( %) or very good ( %) acceptance level for it. in accordance with this data, % of the students voted for the permanence of this practical session in the biochemistry discipline for the next years (table i) . the undergraduate students noticed the direct correlations of this experimental simulation with the biochemical theoretical topics ( %) and their future professional life ( %) ( table i) . although these are positive results, the opinion about the professional-related topic radically changes if the "odontology" undergraduate course is analyzed individually. surprisingly, "odontology" students did not agree ( %) or partially agree ( %) that the knowl-edge offered by this practical session will be useful on their professional career, seeing no direct correlation between them (not shown). this result emphasizes the necessity of an improvement of the lectures, including professional themes in the biochemistry discipline to the understanding of the undergraduate student [ ] . in case of the "odontology" class, clinical cases involving topics such as the problems with the healing process in teeth extraction procedures on diabetic persons could be a good example on how important is the knowledge about this disease and consequently of this experiment for the dentist. it is important to emphasize the major improvement in the student participation during the learning process using this specific practical approach. in fact, some students asked during the class for testing their own urine ( - %), which was not allowed. this specific students behavior indicate a direct interpretation about the applicability of those assays in quotidian. this is probably due to the fact that they were more required to do critical analysis of experiment's result when a problem-based learning-like approach is used. conclusion various pressures are driving changes on brazilian higher education. they are from government, students, professional bodies, and employers; from changes in teaching styles and in the discipline itself; and from ethical/ animal rights considerations. as these demanded changes occur, an alteration in the knowledge, skills, and attitudes of academic professor will be required, which includes the way teaching is delivered and learning is facilitated [ , ] . herein we described a laboratory class experiment that can be applied in any biochemistry course avoiding ethical and biosafety issues. through the correct application of our established alternative tool and the understanding of the technical and clinical details of this practical session, the students could acquire knowledge not only about biochemistry but also about specific problems related to handling of the samples and reagents and how clinical results are obtained and interpreted. other advantages of this practical class are that it is fast, reasonably safe, and have low cost, requiring fewer resources to simulate clinical cases. this economical advantage is important to a country that struggles to maintain its own public academic institutions such as brazil. historical issues concerning animal experimentation in the united states research with animals: requirement, responsibility, welfare guidelines for animal surgery in research and teaching animal ethical committees the regulations concerning animal experiments in education by the german animal rights law animal experimentation in europe: from its origins to its future aspectos é ticos da pesquisa com animais normas de pesquisa em saú de ethics and animal experiments skills and processes in science education laboratory work in biochemical education: purpose and practice the link between laboratory and learning summit to put education at the heart of brazil's future severe acute respiratory syndrome (sars) multi-country outbreak update ; studies of sars virus survival, situation in china cytomegalovirus (cmv) infection, available at /www the boyer commission on educating undergraduates in the research university for the carnegie foundation for the advancement of teaching ( ) reinventing undergraduate education: a blueprint for america's research universities approaches to cell biology teaching: learning content in context-problem-based learning species of paullinia with economic potential, available at www the integrative nature of biochemistry: challenges of biochemical education in the u learning to become a teacher: the wheelock way successful teacher education programs in the era of reform (an essay review of studies of excellence in teacher education ( vols.): preparation in the undergraduate years; preparation in a five-year program; preparation at the graduate level acknowledgments-we thank the fluminense federal university for the honorable mention for practical and teaching approach development in the coordinators awards. we also thank professor cícero carlos de freitas, mr. hugo rodolfo de oliveira barbosa filho and mrs. dione m. silva for their help and technical assistance. key: cord- -ovuofl q authors: chen, xinguang; hu, hui; xu, xiaohui; gong, jie; yan, yaqiong; li, fang title: probability sampling by connecting space with households using gis/gps technologies date: - - journal: j surv stat methodol doi: . /jssam/smx sha: doc_id: cord_uid: ovuofl q sampling methods for survey studies are challenged by the replacement of landline telephones with mobile phones, the lack of timely census data, and the growing need for studies to address new health challenges. gis/gps-assisted methods provide a promising alternative, but these methods need further improvement. we established a stratified -stage gis/gps-assisted sampling method in which residential areas of a target population are divided into mutually exclusive cells – geographic units (geounits) as the primary sampling frame (psf). geounits with residential households were randomly selected from the psf with a semi-automatic algorithm implemented in r. novel methods were used to sample households and participants. simulations and application studies indicated adequate feasibility, efficiency and validity of the method in sampling rural-to-urban migrants from a large city with complex residential arrangements. with this method, researchers can determine sample size and number of geounits, households and participants to be sampled; optimally allocate geounits; determine area size of sampled geounits and estimate sample weights; and complete sampling for field data collection in a short period. our method adds an integrative approach for gis/gps-assisted random sampling with a de facto population assumption. additional evaluation studies are needed to assess the utility of this method in different settings. modern empirical sciences in public health and medicine have been established largely with survey data collected from random or probability samples. design-based statistical inferences require data collected from a probability sample, and results from a sample of participants can be generalized to a large population only when individual participants are selected with a known probability (kish ; cochran ; chen, yin, and peng ; levy and lemeshow ; groves fowler, couper, lepkowski, and singer ) . despite its importance, a review of the literature indicates that probability sampling is infrequent in studies published in even very prestigious peer-reviewed journals. for example, a review of articles published in journal of acquired immune deficiency syndrome and aids and behavior during june-december of , we found only two out of seventy-two ( %) and six out of ( %) of the population-based survey studies, respectively, used a probability sample for data collection. one primary barrier preventing researchers from using a probabilistic sample design could be the lack of appropriate methods (landry and shen ; shannon, hutson, kolbe, stringer, and haines ; wampler, rediske, and molla ; escamilla, emch, dandalo, miller, martinson et al. ; chen, yu, zhou, zhou, gong et al. ) . one approach is landline telephone number-based random digit dialing (kish ; cochran ) . although this method is very efficient for sampling, incomplete population coverage has been an issue (groves et al. ). approximately - % of the households in the united states do not have a landline telephone and thus are not included in the sampling frame (groves et al. ). people living in these households are more likely to be low in socioeconomic status, drug users, sex workers, and/or undocumented migrants (groves et al. ; singh and clark ) , all of whom are at increased risk for poor health. more threatening than incomplete coverage is the replacement of landline telephones by wireless communication technologies that makes it impossible to implement the random digital dialing method. most methods attempt to achieve randomness by using census data with detailed demographic information at the household level to construct the sampling frame (kish ; cochran ; groves et al. ). however, such data are often not available in a timely manner in all developed countries and unavailable in many resource-limited lower-and middle-income countries. in developed countries, census data are collected only for selected years (usually five or ten years apart), and many resource-limited countries do not collect population census data on a regular basis. even if census data are available, they may fail to count people who are at high risk for poor health, such as temporary and undocumented immigrants (groves et al. ; singh et al. ) . in survey research, sometimes a study population can be operationally defined: for example, non-institutional residents; gender; racial/ethnic minority groups in a country; high school students in a state; or hospitalized patients in a region. in this case, methods are readily available to draw probability samples, such as the multi-stage random sampling methods for national inhousehold surveys supported by census data (cochran ) , telephone surveys supported by random digit dialing with published telephone numbers (groves et al. ), and school surveys using system sampling methods with complete lists of schools and classes (eaton, kann, kinchen, shanklin, flint et al. ) . however, sometimes the sampling frame for a study population may be clear conceptually but hard to define operationally. challenging examples for drawing probability samples include mobile migrants, sex workers, drug users, and persons living with hiv (groves et al. ; singh et al. ) . timing can be another challenge for random sampling when survey studies are needed to address an urgent public health and medical issue (heeringa and o'muircheartaigh a; heeringa and ziniel ) . typical examples include studies of outbreaks and vaccination of infectious diseases, such as hiv/ aids, severe acute respiratory syndrome (sars) (tong ; he, zhuang, zhao, dong, peng et al. ) , ebola (weyer, grobbelaar, and blumberg ; boisen, hartnett, goba, vandi, grant et al. ) , and zika (boeuf, drummer, richards, scoullar, and beeson ; deseda ) . innovative methods have been attempted for timely sampling without using a sampling frame. well-known examples include venue-day-time sampling, where participants are selected from locations within time ranges when participants are often present (mansergh, naorat, jommaroeng, jenkins, jeeyapant et al. ) ; the capture-recapture method derived from agriculture and wild life studies (tilling ) ; and respondent-driving sampling (rds), in which study participants are selected by working with a few seed persons to nominate others within their network connections (heckathorn (heckathorn , . although these methods allow for timely sampling of study participants, their validity in ensuring probability and representative samples is unclear. technological advances in geographic information systems (gis) and global positioning systems (gps) have encouraged numerous researchers to develop speedy probabilistic sampling methods with adequate geographic and population coverage, with minimal data requirements (murray, o'green, and mcdaniel ; landry et al. ; galway, bell, sae, hagopian, burnham et al. ; shannon et al. ; chen et al. ) . a number of gis/gpsassisted probability sampling methods have been developed to deal with specific settings, such as sampling in remote rural areas (wampler et al. ; escamilla et al. ; kondo, bream, barg, and branas ; haenssgen ; pearson, rzotkiewicz, and zwickle ) , mobile populations (landry et al. ; singh and clark ; chen et al. ) , and other special conditions (murray et al. ; galway et al. ) . a review of the published studies reveals that most gis/gps-assisted sampling methods can be characterized as geographically stratified multi-stage sampling. these methods can be summarized in seven steps: ( ) define targeted study population and geographic area, ( ) construct primary sampling frame (psf) and define residential area to determine the primary sampling units (psus), ( ) randomly select psu with a probabilistic procedure (simple random, proportion to or stratified by population density), ( ) select households from each sampled psu through random routes or other methods, and enumerate households to construct the secondary sampling frame (ssf), ( ) randomly select a pre-determined number of participants from ssf, ( ) compute sample weights across all sampling stages, ( ) estimate descriptive statistics for the study population, taking into account the sample design and sampling weights. despite much progress, additional research is needed on gis/gps-assisted sampling methods. first, it is challenging to pre-determine the sample size for several reasons (landry et al. ; singh et al. ; valliant, dever, and kreuter b) . the method consists of two steps: sample geographic areas, then sample participants in selected areas. sample size is easy to determine if a study only needs to draw geographic samples (balch, drapeau, bowler, booth, goes et al. ; chen, zhao, gao, henkelmann, and schramm ; conway ; daly, lei, teixeira, muir, castillo et al. ; huang, zhao, shi, yu, zhao et al. ; valliant et al. b : pearson et al. . however, it is not possible to know exactly how many persons are present in a randomly sampled geographic area before the area is selected (landry et al. ; shannon et al. ; valliant et al. b; chen et al. ) . one solution is to enumerate all households in a randomly sampled area after a geographic area is selected. this method has often proved infeasible (landry et al. ; escamilla et al. ; chen et al. ) because of high and variable population density, complex residential structure, and presence of high-rise and multi-function buildings in selected geographic areas. second, gis/gps-assisted sampling method needs to distinguish residential from non-residential housing. methods for distinguishing between the two have proven very time-consuming to implement (chen et al. ; singh et al. ; escamilla et al. ; kondo et al. ; pearson et al. ) . recent methods haven been developed to recognize visually or digitally residential areas/housing with widely available aerial images (chang et al. ; wampler et al. ; escamilla et al. ; haenssgen ; pearson et al. ) . these methods are fast, inexpensive, and highly feasible. however, correctly recognizing residential houses remains a big problem even with the assistance of people from local communities. for example, pearson et al. ( ) conducted a study to determine residential households using aerial images. with assistance of local experts after computerized sampling, five out of determined residential household structures were verified in the field to be nonresidential. although the error rate is not high, this study was conducted in a semi-nomadic pastoral area, a setting much simpler for random sampling than that of a modern urban area. more research is needed to improve this method for use in sampling complex residential areas (escamilla et al. ; chen et al. ; haenssgen ) . third, stratification has been used in gis/gps-assisted sampling to deal with heterogeneities in population density (kumar ; galway et al. ; valliant et al. b; kondo et al. ). galway and colleagues have used this approach in their studies and generated promising results (galway et al. ). however, for stratification to be effective, detailed demographic data at the population level by individual grid cells across a jurisdiction are needed (galway et al. ; valliant et al. b) , and often such data are not available in resource-limited, low-and middle-income countries. night-time satellite images provide information regarding population density, but this approach does not work for rural areas and resource-limited countries, and places with no electricity (sutton ; schneider, friedl, and potere ) . last but not least, geographic sampling weights are difficult to determine because of the lack of clear boundaries between residential and nonresidential areas and lack of information on the number of persons living in sampled geographic units at a specific date and time (landry et al. ; kumar ; shannon et al. ; valliant et al. b; kondo et al. ). in this study, we report on our attempts to overcome the challenges described above. our goal is to promote the use of gis/gps-assisted sampling method in survey studies with probability samples to better address medical and public health questions. . spatial sampling . . principle and geographic data. geographic data for locations where the study population resides (often by country or jurisdictions within a country) can be obtained from different sources, mostly free of charge (e.g., google maps and openstreetmap) (haklay and weber ) . the area will be divided into mutually exclusive cells (geographic units, or geounits for short) for further sampling. this spatial sampling process is often realized by creating and laying a grid over the target area (figure ) and then randomly drawing geounits. a of a geounit is critical to create the grid network described in the previous and next sections. in traditional spatial sampling, a is simply calculated using the sampling ratio (stehman and overton ; maguire, batty, and goodchild ; valliant et al. b ). for example, if a researcher plans to sample geounits to cover . % ( À ) of a geographic area with the total area size of , , ( ) km , the area for individual geounits is . km (¼  À = ). a more complex method is needed to draw geographic samples for population-based survey studies, because a is determined not by sampling ratio but by the likelihood of covering an appropriate number of households and eligible persons. using a larger a increases the chance of sampling adequate numbers of households and eligible subjects, but also increases the workload for household enumeration. the appropriate size a can be determined through pilot studies, considering population density and the number of subjects to be recruited per geounit. for example, when conducting a survey targeting the rural-to-urban migrants temporarily living in wuhan, china, a ¼ m  m was determined through intensive pilot tests in the field. this number was estimated by counting all households located in a geographic area with different sizes being measured manually using tape rulers and/or laser scales. this value of a has approximately % probability of covering an adequate number of households, ensuring at least subjects per geounit in a city like wuhan (chen et al. ) . after a is determined, a grid network is then created and overlaid on the target geographic area, dividing the area into mutually exclusive cells (with cell size of a). these cells are the primary sampling frame (psf) for further sampling (figure ). different methods are available for grid network creation; it is often simpler to use geographic coordinate systems rather than side length, and the differences between the two approaches are often small for geographic areas on the scale of a city or a state. for studies involving very large geographic areas like a country (e.g., russia, canada, china, or the united states), continents, or the globe, distance defined through appropriate projection systems should be employed. after the psf is created, a pre-determined number of geounits (to be discussed next) are randomly selected from the psf. unlike pearson's method, which uses a set of randomly scattered points as place marker (pearson et al. ) , we randomly sample a set of geounits with size a in the geographic area where the study population resides. given large variations in population density across the geographic area, a stratified strategy is used to sample geounits with more geounits being allocated to areas with higher population density. this step is conducted following an optimum allocation approach to enhance work efficiency (cochran ) . another issue confronted in practice is that a randomly selected geounit has a sizable likelihood of being located in nonresidential areas such as lakes, bridges, highways, and commercial buildings. to overcome this problem and to enhance feasibility while maintaining a probabilistic sampling process, we devised a semi-automatic, computer-assisted, stepwise algorithm with no replacement procedure for implementing the geounit sampling protocol (figure ). more details and the r codes for implementation are provided in appendix of the online supplementary material. before sampling, the number of geounits to be sampled g has to be determined. obviously g depends on the sample size n and the average number of participants to be sampled per geounit m. there is a lack of efficient methods to determine g in reported studies (landry et al. ; kondo et al. ) . to overcome this limitation, we used the same m for all geounits. with m and sample size n determined through conventional statistical power analysis, g can simply be calculated as: for example, assuming a researcher plans to draw a sample of n ¼ , . if twenty subjects are to be sampled from each geounit, based on equation , the number of geounits to be sampled: g¼ , / ¼ . if thirty subjects per geounit are to be sampled, g¼ , / ¼ . . . locating the sampled geounits. after completing spatial sampling steps as described in the previous two sections, detailed geographic data for the selected geounits are available and can be directly uploaded to gps receivers. in this study, we tested our method using the oregon gps from garmin, but any gps receiver can be used if it can upload sampled geographic units with coordinates and areal images and is able to track specified areas manually. . . sequential household access and random participant selection. after a sampled geounit is located in the field with the assistance of a gps receiver, data collectors go to all households within the sampled geounit to prepare for subject sampling and recruitment. this procedure is completed in three steps. step one: approach individual households sequentially following natural order with the first household being selected randomly from the main entrance of a street in urban areas or the beginning of a village in rural areas. we used this random route approach to ensure each household has a known probability of being selected (de rada and martin ; bauer ). step two: list all eligible participants for each selected household. the list for a household constitutes the secondary sampling frame (ssf). step three: select participants randomly from the ssf. if only one person in a household is eligible, this person will be included. if more than one is eligible, only one will be randomly selected using the kish table or other random digit method (kish ) . households not available at the time of sampling are revisited to reduce missingness. an innovation of our method is that data collectors can determine the number of households to be enumerated for a geounit. this is because data collectors have already been told the number of households h and the number of subjects per household s to be sampled. assuming m ¼ and only one subject per household is to be sampled (s ¼ ), approximately twenty households are enumerated (h ¼ ). in addition to minimizing work load, this approach moves the sampling probability of a geounit closer to being proportionate to the population density. this is because the ratio of h to the total households in a same-sized geounit will be smaller in a more populated area and larger in a less populated area. after household enumeration, participant recruitment, and data collection for a given geounit, the following complementary data must be collected. ) actual geounit area size a g for the g th sampled geounit (g ¼ ; ; . . . ; g) where households are accessed and participants are sampled. areas where households are not enumerated must be excluded. the actual area size a g is determined by gps receiver recorded tracking data. ) total number of households t g in the accessed area a g and number of households from which participants are sampled h g . ) households and individuals who refused to participate. to estimate the overall sample weights, the true area size r where the target population resides must be determined to estimate geographic sample weights. although the concept of residential area has no ambiguity, it is often difficult to determine r (shannon et al. ; singh et al. ; valliant et al. b; kondo et al. ) . we have devised two alternative methods for use in different settings. . . . method i: estimate residential area with population and geographic sample data. if a g ¼ actual geounit area size for the g th sampled geounit (g ¼ ; ; . . . ; g) where households are accessed and participants are sampled (described in section . . ), then the total area of g geounits b ¼ p g a g . let r ¼ the total residential area, p ¼ the total population known to reside in the target district, and q ¼ the total population covered by all g sampled geounits (the total population q g for g th geounit is estimated using total households t g and demographic data from the enumerated households h g ). both b and q are calculated based on area size and population data obtained from the randomly selected geounits. if g is adequately large (e.g., twenty or more), the ratio of the two provides an unbiased and reliable estimate of the ratio of r over p. that is, ( ) so we set this approximating method relies on the expectation that households in a sampled geounit are associated only with that geounit. in practice, this can be achieved by carefully determining the appropriate grid size a through pilot studies. . . method ii: estimate residential area with monte carlo method. if data for total population p is not available, the residential area r can be estimated using a monte carlo method (metropolis and ulam ; mathews ) . the size of a target area d can be obtained through many gis packages as described in section . . with the monte carlo method, a target area is uploaded to computer. a total number of n points (i.e., several hundred) are randomly selected within the whole area. if n r points fall on residential areas, and n nr non-residential areas, then n ¼ n r þ n nr . since all points are randomly selected, n r =n provides an unbiased estimate of r=d. thus, r ¼ n r n r þ n nr d ( ) the following equation is used to compute sample weights following the principles for stratified, multistage, and disproportionate probability sampling (kish ; cochran ; groves et al. ; valliant et al. b) . where w g represents the sample weight for g th geounit and equals r=a g , where r is the size of total residential area and a g is the size of g th sampled geounit; w gh represents the sample weight for household h in geounit g and equals t g =h g , where t g ¼ total households in geounit g and h g ¼ number of households sampled in geounit g; and lastly, w ghi ¼ sample weight for individual subject i from household h and equals n gh =n jh , where n gh ¼ ¼ total number of eligible persons in household h within geounit g, and n gh ¼ number of persons sampled in the household h, h ¼ ; ; . . . ; h g . if only one person per household is sampled, n gh ¼ and w ghi ¼ n gh . variance estimation methods are needed to correctly account for the variance inflation due to weighting, (kish ) and the design effect attributable to the clustering of observational units within the sampled area psus also needs to be considered (kish ; heeringa, west, and berglund b; valliant, dever, and kreuter a) . the supplementary appendix online describes a simulation study conducted to validate this method using both the jackknife replication and the bootstrap for variance estimation (valliant et al. a ). we tested the integrative gis/gps-assisted sampling method in wuhan, china, when conducting an nih-funded project (r mh , pi: chen x) to investigate the relationship between social capital and hiv risk behaviors among rural-to-urban migrants. wuhan is the capital of hubei province with a total population of approximately ten million, per capita gdp of $ , , and a large number of rural-to-urban migrants (statistical bureau of wuhan ). the field work for sampling and data collection was completed during - . many migrants do not have a permanent urban residence, and all of them are scattered all over the city. in this case, it is not possible to construct a sampling frame using conventional methods. following the procedure described in this study, a district boundary file of wuhan was obtained using the arcgis. based on pilot studies for field work efficiency, a grid-system with m  m cells was created and imposed on the map to divide the geographic area of wuhan into small and mutually exclusive cells as geounits (see figure ). these mutual exclusive cells consist of the psf for further sampling. a total of sixty geounits with residential housing were randomly drawn from the psf and stratified by population density following the steps described in section . . a sample size of sixty geounits was chosen to achieve a total sample of , with approximately twenty participants per geounit. allocation of the sixty geounits to districts was optimized, considering traveling cost and cost for field data collection. figure shows the geographic distribution of the sampled geounits. households within each of these sampled geounits were then accessed and participants recruited following the steps described in section . . actual geounit area size a g was determined with data collected during sampling (see appendix in the online supplementary material for more details). overall, sixty sampled geounits covered , households, of which approximately - % were occupied by rural migrants. households were selected following their natural order on a street with the beginning of a main entrance street as the start point, and the first household was determined using random numbers. of the migrant-occupied households, , were available and agreed to participate at the time of data collection. a total of , participants were recruited from these households with one participant per gender per household. the total number of households per geounit varied from thirty in least populated areas to , in the most populated areas with a median [quartile , quartile ] of one hundred [ , ] and mean (sd) ¼ ( ). the number of households agreeing to participate per geounit varied from twelve to forty with median [quartile , quartile ] of twenty [ , ] and mean (sd) of twenty-one ( ). table shows the detailed results from the sampling. applying this method to another study conducted in - , we estimated that approximately fifty-eight thousand [ % ci: , , , ] rural-tourban migrants in wuhan were msm with , [ % ci: , , , ] being tested hiv positive (chen et al. ) . official surveillance data from wuhan indicated that a total of , (primarily msm) persons were living with hiv in (wuhan center for disease prevention and control (cdc) ). the observed result is within the estimated % ci, and the relatively small difference provides some evidence supporting the validity of our method. in this study, we reported a geographically stratified -stage (geographic unit, household, and participants) gis/gps-assisted sampling method. this method is developed by integrating various reported gis/gps-assisted sampling methods (chang et al. ; wampler et al. ; escamilla et al. ; haenssgen ; pearson et al. ) , particularly the methods with a stratified cluster sampling approach (cochran ; groves et al. ). innovations include methods to determine residential area and methods for sample weight calculation. our method enhances existing approaches to drawing probability samples for local, national, cross-national, and global survey studies (heeringa et al. a; heeringa et al. ). our method is based on sound theories for population and geographic sampling, and has minimal data requirements. conventional stratified sampling continued strategies can be used in optimizing geounit allocation to deal with large variations in population density and to increase field-work efficiency (cochran ) . the size of geounits can be determined through pilot testing to ensure adequate household/participant coverage, while taking work efficiency into account (chen et al. ) . the random route method (bauer ) can be used to ensure an equal probability household sample. data collected using our method can be analyzed with design-based survey methods (kish ; cochran ; lohr ; groves et al. ; heeringa et al. b; valliant et al. b ). these methods are available in many software packages, including sudaan, sas, stata (survey module), spss, and "survey" package in r. many of the sampling tasks of our method can be implemented on computer with open-source software r and free google imagery data. a more detailed discussion of the application of our methods is provided in appendix of the online supplementary material. in addition to general survey studies, the increased efficiency may make our method an option to draw probability samples for studying sudden outbreaks of a disease, such as sars, ebola, and zika. gis/gps-assisted sampling methods are becoming increasingly available. if a target study population is located in sparsely populated and less developed rural areas, methods with satellite images to identify households for random sampling are a better choice. typical examples include methods reported by haenssgen (haenssgen ) , wampler (wampler et al. ) , and escamilla and colleagues (escamilla et al. ) . however, if a researcher wants to conduct studies in highly developed urban settings with more complicated residential arrangements, our method would be a better choice than many other methods to ensure probability samples (landry et al. ; galway et al. ; kondo et al. ) . to ensure successful application of our method in drawing a probability sample to represent a study population, researchers must pay additional attention to the following three aspects. the first aspect is related to variations in population density. the fundamental mechanism of our method is to link geographic area with varying population density to households using numerous small geounits for further sampling. therefore, one natural approach to deal with varying population density is application of the classic stratified sampling strategy to optimize geounit allocation (cochran ) , as have been commonly used in this and other studies (galway et al. ; chen et al. ) . our method also offers other possibilities to deal with varying population density issues. for example, instead of using a fixed geounit size and sampling grid, with our method researchers can determine the geounit size disproportionate to population density after randomly selecting the pre-determined number of geounits to be selected. although determination of population density could remain be a challenge resource-limited areas, we may be able to deal with it with satellite imagery that is widely available. the second aspect is the determination of area size of a geounit. larger sizes have greater probability of covering an adequate number of households for sampling. however, if a large-sized geounit is randomly selected in a highly populous area, it will prevent researchers from completing the sampling due to high costs of time and money (landry et al. ) . we recommend that researchers conduct adequate pilot studies to determine geounit size, considering variations in population density, time, and resources available for sampling. the third aspect is household selection within a sampled geounit. although each selected geounit is not large in area size with a relatively fewer number of households, household arrangement can still be complex. in this study, we used the random route approach (bauer ) , by randomly selecting one household as starting point and then following natural order to select other households until the pre-determined number of households was reached. however, our method may lead to biased estimates of parameters that are related to physical distance. this can happen even with carefully planned and well-tested instructions (bauer ) . if conditions permit, an ideal approach would be to list all households in a sampled geounit first and then randomly select the pre-determined number of household for further sampling. in this study, we only demonstrate our method in sampling rural migrants in urban china. a full assessment of the value of our approach requires its application to different populations in diverse geographic and residential settings. like any multistage sampling method, it is a challenge to ensure an equal probability sample of households. the random route provides a good option, but attention must be paid to instructions to the data collectors and random selection of the starting household (bauer ) . data on the size of a geographic unit is often not directly available, and can be obtained only through repeated pilot tests. given large variations in household and population density in urban settings, large variations in estimated sample weights are anticipated. such variations may reduce the precision of sample estimates. a multi-year record of hydrographic and bio-optical properties in the gulf of maine: i. spatial and temporal variability biases in random route survey the global threat of zika virus to pregnancy: epidemiology, clinical perspectives, mechanisms, and impact epidemiology and management of the 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systems of a peri-urban interface a procedure for objective respondent selection within household a random spatial sampling method in a rural developing nation spatial sampling design for a demographic and health survey reaching migrants in survey research: the use of the global positioning system to reduce coverage bias in china sampling of populations: methods and applications sampling: design and analysis gis, spatial analysis and modeling adaptation of venue-day-time sampling in southeast asia to access men who have sex with men for hiv assessment in bangkok monte carlo estimate for pi the monte carlo method development of a gis database for ground-water recharge assessment of the palouse basin using remote, spatial techniques to select a random household sample in a dispersed, semi-nomadic pastoral community: utility for a longitudinal health and demographic surveillance system a new map of global urban extent from modis satellite data choosing a survey sample when data on the population are limited: a method using global positioning systems and aerial and satellite photographs creating a frame: a spatial approach to random sampling of immigrant households in inner city johannesberg wuhan statistical yearbook- modeling population density with night-time satellite imagery and gis capture-recapture methods-useful or misleading? emerging infectious disease, , practical tools for designing and weighting survey samples using arcmap, google earth, and global positioning systems to select and locate random households in rural haiti ebola virus disease: history, epidemiology and outbreaks report of hiv/aids epidemic in wuhan, china supplementary materials are available online at academic.oup.com/jssam. key: cord- -pue q wp authors: moreno, paloma s.; wagner, josef; mansfield, caroline s.; stevens, matthew; gilkerson, james r.; kirkwood, carl d. title: characterisation of the canine faecal virome in healthy dogs and dogs with acute diarrhoea using shotgun metagenomics date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: pue q wp the virome has been increasingly investigated in numerous animal species and in different sites of the body, facilitating the identification and discovery of a variety of viruses. in spite of this, the faecal virome of healthy dogs has not been investigated. in this study we describe the faecal virome of healthy dogs and dogs with acute diarrhoea in australia, using a shotgun metagenomic approach. viral sequences from a range of different virus families, including both rna and dna families, and known pathogens implicated in enteric disease were documented. twelve viral families were identified, of which four were bacteriophages. eight eukaryotic viral families were detected: astroviridae, coronaviridae, reoviridae, picornaviridae, caliciviridae, parvoviridae, adenoviridae and papillomaviridae. families astroviridae, picornaviridae and caliciviridae were found only in dogs with acute diarrhoea, with astroviridae being the most common family identified in this group. due to its prevalence, characterisation the complete genome of a canine astrovirus was performed. these studies indicate that metagenomic analyses are useful for the investigation of viral populations in the faeces of dogs. further studies to elucidate the epidemiological and biological relevance of these findings are warranted. interest in the virome, or the entire population of viruses present in a biological sample, has increased recently due to improved availability of high throughput sequencing or next generation sequencing (ngs) technologies, and improved metagenomic analytical methods [ , ] . a a a a a the virome comprises all types of viruses, including those that infect prokaryotic and eukaryotic organisms, dna or rna viruses, and viruses that cause acute or chronic infections. many of these viruses are difficult or impossible to propagate in cell culture, and molecular detection is difficult as no common gene such as the ribosomal s gene that is present in bacterial species exists in viruses. these limitations have hindered the identification and characterisation of uncultured viruses [ , ] . recently, due to the advent of molecular enrichment protocols, high throughput sequencing and new metagenomic analytical methods we are now able to explore, identify and characterise viruses from different biological and environmental samples with a greater capacity [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] in studies of human faeces, the virome has been shown to include viruses that infect eukaryotic organisms and viruses that infect prokaryotes (bacteriophages) [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . bacteriophages have been reported in many studies to be the most frequently detected viral constituent in the gut of humans [ , , , , , , ] . the faecal virome has been characterised for several animal species including pigs, bats, cats, pigeons, horses and ferrets [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in dogs, the presence of enteric viral pathogens such as canine parvovirus, coronavirus, rotavirus and distemper virus (paramyxoviridae) have been identified only through targeted studies [ ] [ ] [ ] [ ] . to date, only one published study has used high throughput sequencing to investigate the faecal viral population in diarrhoeic dogs [ ] . these investigators analysed faeces from dogs with acute diarrhoea and detected two new virus species, canine sapovirus and canine kobuvirus; known canine enteric viruses such as canine coronavirus, canine parvovirus, canine rotavirus as well as plant and insect viruses were also reported [ ] . the aim of this study was to describe the faecal virome of samples collected from healthy dogs, and compare these findings to the faecal virome of dogs with acute diarrhoea in australia, using an illumina miseq shotgun metagenomic sequencing approach. a total of faecal samples ( from healthy and from diarrhoeic dogs) were subjected to viral nucleic acid extraction, followed by nucleic acid enrichment, reverse transcription, random amplification and the creation of two libraries for each sample (dna and cdna), before being sequenced by illumina miseq platform (table ) . after sequencing, a total of , , raw sequences were generated. all raw sequences are available in ncbi, (bioproject id: prjna ). after trimming by quality , , high quality reads (hqrs) were available. all sequences corresponding to dog and cellular organisms ( , and , , respectively) were removed and the resultant reads were de novo assembled (fig ) generating in total , , contigs and singletons (reads). from these contigs/singletons , , ( . %) had no hits in the database (s table) . further analysis of contigs/singletons with no hits confirmed most sequences had no hits, while a limited number matched bacterial, human or animal sequences with a very low coverage. in addition to the contigs/singletons with no hit in the database, some contigs/singletons matching to cellular organisms and some with low complexity were identified, however, were not analysed any further (s fig) . sequences similar to twelve viral families were identified in faecal samples from healthy and diarrhoeic dogs after analyses with two different bioinformatic pipelines and comparison against viral and ncbi databases (fig ) . eight of these viral families infect eukaryotic organisms, and the remaining four infect prokaryotes. despite the known bias of sispa in the resultant sequences after de novo assembly [ ], we report the number of contigs/singletons matching viral families and the subsequent analysis of alignments with the lowest common ancestor according to megan v . . [ ]. faecal samples were collected from eight healthy dogs (table ) . genetic analyses identified , contigs/singletons with no hits and contigs/singletons were classified as viral, matching to five viral families that infect eukaryotes and four that infect prokaryotes. . % ( contigs/singletons) of the total number of viral contigs/singletons were classified as bacteriophages in the healthy canine faecal virome. bacteriophages were detected in the faeces of all dogs in this group and belonged to caudovirales order and microviridae family. viral contigs/singletons from five eukaryotic virus families were identified in faecal samples from of the healthy dogs (table ) . three out of five viral families detected were dna viruses. adenoviridae and papillomaviridae were detected in a single sample containing only (table ) , however, these were all detected in one sample. after analysis, only contigs/singletons matched the reference sequence of alphacoronavirus (feline infectious peritonitis virus, nc_ . ) and covered only . % of the complete genome (minimum match: % and minimum overlap: ), which represented . % of fipv_gp (receptor binding molecule) region. contigs/singletons belonging to the reoviridae family were found in only one sample. genetic analysis revealed they covered between %- . % of vp , vp , vp and vp genes of reference sequences of rotavirus a (nc_ -nc_ ). another eukaryotic viral family found in one healthy dog sample was parvoviridae, genetic analysis of the contigs/singletons showed a coverage of approximately . % of the complete genome of canine parvovirus reference sequence (nc_ ), or . % of the polyprotetin ns -ns . in eight faecal samples from dogs with acute diarrhoea, a total of , contigs/singletons had no hits and , were identified as viral contigs/singletons comprising eukaryotic and prokaryotic viral families (table ) . bacteriophages comprised . % of the total of viral contigs/singletons and they were present in all individuals and were identified as belonging to order caudovirales and microviridae family. eukaryotic families found in this group were coronaviridae, parvoviridae, reoviridae, caliciviridae, astroviridae, and picornaviridae ( table ) . the most common eukaryotic viruses identified were rna viruses ( / viral families). interestingly, all samples in this group contained at least one eukaryotic family each. co-infection was identified in individual dog samples from this group. from the samples from dogs with acute diarrhoea, different eukaryotic virus families were detected in five samples and eukaryotic families were detected in one sample ( table ). the most prevalent family identified was astroviridae, present in dogs followed by reoviridae present in of dogs with acute diarrhoea (table ) . astroviridae contigs/singletons from dogs were compared with reference sequence of canine astrovirus (nc_ . ), the lowest common ancestor according to megan, and they covered between . % and % of compete genome and between . % and . % of the orf . the sample with the most contigs/singletons was later characterised. reoviridae contigs/singletons found in dogs were compared with reference sequence of rotavirus a (nc_ . ) and of them covered between . % and . % of the vp gene and the other sample covered . % of the vp gene. furthermore, contigs/singletons matching to canine parvovirus, were found in dog samples with acute diarrhoea. one of the samples covered approximately . % of the complete genome of canine parvovirus reference sequence (nc_ ), corresponding to . % of vp . the other sample contained contigs/singletons that matched to this same reference sequence and in total they cover % of vp , . % of polyprotein ns and ns (cpvgp ) and . % of vp genes. in this group were also found contigs/singletons matching to the coronaviridae family, covering between . % and . % of the complete genome of reference sequence alphacoronavirus (fipv, nc_ . ). one dog with acute diarrhoea contained contigs/singletons similar to a canine norovirus (jf . ), covering approximately . % of the complete genome. other dog sample had contigs/singletons similar to a canine kobuvirus (jn . ), covering . % of complete genome. to further explore the high abundance of contigs/singletons from astroviridae family in dogs with acute diarrhoea and their absence in healthy dogs, a near complete full genome of a representative canine astrovirus was generated through sanger sequencing (dd , table ). the genome encoded the complete three open reading frames (orfs): orf a, orf b and orf . the total length was nucleotides, excluding the ' poly (a) tail and the nucleotide composition was % a, % g, % t, % c. the g/c composition was %. the genbank accession number for the canine astrovirus sequence is kx . a phylogenetic tree was constructed using the protein alignment from the conserved region of the capsid (orf ) of the astrovirus characterised in this study (dd ) and other canine astrovirus orf , together with mamastrovirus sequences from different mammalian species, including a chicken astrovirus as an outgroup. the phylogenetic analysis grouped our canine astrovirus within the canine astrovirus clade. the closest canine astroviruses to our australian sample were from uk and china with an identity between . %- . % (fig ) . using next generation sequencing and metagenomics analysis, the virome in faecal samples from healthy dogs and dogs with acute diarrhoea is described. only a single previous shotgun metagenomic study investigating the faecal virome of dogs with diarrhoea has been reported. in that study, mammalian viruses were found in samples and two new virus species were described [ ] . our study analysed faecal samples from dogs ( healthy and diarrhoeic), and identified eukaryotic viruses in samples, including all diarrhoeic samples and % of the healthy samples. thus, % of canine faeces contained eukaryotic viruses, suggesting that mammalian viruses are a common component of the enteric microbial population in dogs. our results must be interpreted with caution, due to bias created by sispa. areas of exaggerated depth appear when the sispa method is used, creating artefacts during de novo assembly. this results in regions of repetitive sequences [ ] . in order to overcome this bias, all contigs/singletons were analysed at family level and only for viral eukaryotic families were the results further analysed to evaluate what percentage they covered to some specific viral species. the most common viral contigs/singletons identified in both groups were bacteriophages, similar to previous findings from human and other animal faecal virome studies [ , , [ ] . bacteriophages modify diversity of bacterial populations due to their lytic life cycle and also promote different characteristics in the bacterial population due to their lysogenic life cycle transferring genes such as encoding toxins or resistance to antibiotic [ ] . this life-cycle may lead to bacteriophages conferring advantage to some bacterial species in the environmental niche [ ]. therefore, it is possible that the greater amount of contigs/singletons corresponding to bacteriophages identified in the group of dogs with acute diarrhoea, when compared to healthy dogs, means a higher amount of bacteriophages. if so, bacteriophages could have generated a change in the normal balance of bacterial population resulting in dysbiosis, and ultimately causing diarrhoea. conversely, it could be that an initial change in the bacterial population in these dogs resulted from the acute diarrhoea [ , ] is the cause of the variation in the bacteriophage population. in our sample population, the latter explanation is most likely, because in a shelter environment a higher number of circulating pathogens, changes in diet and a stressful environment could contribute to the dysbiosis associated with acute diarrhoea [ ] . the bacterial microbiome and the analysis of contigs/singletons matching specific bacteriophages were not assessed in this study, therefore further microbiome/virome cross analysis is necessary to elucidate the association between bacteria and bacteriophages in dogs. however, even this analysis would be unlikely to determine the cause or effect relationship between bacteriophages and dysbiosis at a single point in time. the analysis of the lowest common ancestor of eukaryotic viral families, according to megan, identified eight eukaryotic virus species (table ) . however, each of these results require validation by targeted pcr, or whole genome characterisation of each species, as the ngs results after sispa amplification may be biased and not accurate depiction at a species level [ ] . sequences matching those of human viruses (adenovirus and papillomavirus) were found in one sample from a healthy dog. only one contig from each virus covering a very small percentage of the genome in both cases. this finding could suggest contamination during collection or processing. known enteric pathogenic families parvoviridae and coronaviridae were identified in samples from both healthy dogs and dogs with acute diarrhoea. interestingly, almost all positive samples were from puppies (between - months) that had been vaccinated less than one month prior to sampling. the lowest common ancestry analysis in megan of the contigs/singletons matching parvoviridae family, suggested they were canine parvovirus (cpv), but as cpv positive dogs (as tested by faecal antigen tests) were not included in this study it is highly likely these results represent vaccine derived sequences not detected by the cpv antigen detection kit, or represent a virus load below the level of detection. previous studies have demonstrated that modified live vaccine virus can be detected in faecal samples for extended periods of time after vaccination [ ] . further genome characterisation of these canine parvovirus is warranted to confirm this hypothesis. three individual samples contained coronaviridae contigs/singletons, two of which were from puppies [nd and dd ] ( table ) and one from an adult dog [dd ] ( table ). our results are consistent with li et al , who also reported the highest number of coronaviridae reads in one sample collected from a puppy [ ] . canine coronavirus can be shed in faeces in high numbers for up to days [ , ] . these findings validate the affinity of the coronaviridae viral family to infect young individuals [ ] , and present as a common enteric pathogen in a shelter environment [ , , ] . the uncommon viruses, canine kobuvirus and canine norovirus, were identified only in samples from dogs with acute diarrhoea. previous studies have suggested these viruses may have some association with enteric disease in dogs, however, both viral species have been detected in both healthy dogs and dogs with diarrhoea [ , ] our shotgun metagenomic sequence data indicated that the most frequent rna viral family in dog samples with acute diarrhoea was astroviridae, being identified in more than half of the diarrheal samples. [ ] . in dogs, astrovirus has been previously detected mainly in puppies with diarrhoea, but has also been occasionally reported in healthy dogs [ ] [ ] [ ] [ ] [ ] . the only previous report of a possible canine astrovirus in australia was described in canine faeces in the , where astrovirus-like particles were detected using electronic microscopy in healthy dogs [ ] . to date, canine astrovirus has been reported in usa [ ] , china [ ] , italy [ , , ] , uk [ ] , france [ ] , brazil [ ] , korea [ ] and japan [ ] . the first description of the complete genome of two canine astroviruses was reported by a group of researchers from the uk in [ ] . the current study contributes the first description of the complete genome of a canine astrovirus identified in australia. in our study, using sanger sequencing a near complete genome of a canine astrovirus was assembled from one dog with acute diarrhoea. a phylogenetic tree, analysing the capsid region (orf ) of this australian canine astrovirus and other astrovirus sequences present in genbank, determined that it belonged to the canine astrovirus clade, very closely related to the canine astrovirus strains from the uk and china (fig ) . it is interesting to note that all canine astrovirus positive samples, were collected from the same shelter and obtained within a short period of time (sept-nov ). we could infer that this virus was endemic at that time in that shelter, and or maybe could represent an outbreak of diarrhoea in the shelter within that period of time. a more sensitive test (i.e.: quantitative pcr) in a larger number of samples from cases and controls may be useful to better understand the potential role of astroviruses as an aetiological agent in acute diarrhoea of dogs. in this study we analysed the faecal virome in healthy dogs and compared these findings with the faecal virome of dogs with acute diarrhoea. known dna and rna viruses were found, together with different proportions of bacteriophages in each group. in addition, we described and characterised the first complete genome of a canine astrovirus in australia. future longitudinal studies analysing viruses, bacteria and other potential pathogens should be performed to assess the aetiology of diarrhoea in dogs and further elucidate the pathological importance of viruses found in dog intestines. faecal samples from a total of dogs were obtained between september and march . all dogs were aged between . months and years; and comprised females and males of various breeds ( table ) . all faecal samples were collected from a single shelter in melbourne (lost dogs home), australia. all samples were maintained at ˚c for up to hrs, then were transported on dry ice before storing up to five aliquots of mg of faeces each at - ˚c until further analyses. information about age, sex, breed, diet, vaccination and deworming status was recorded for each dog (university of melbourne animal ethics committee approval ids . and . ). animals were determined to be healthy based on physical examination by a veterinarian and absence of any clinical signs of disease. faecal consistency was considered normal as per published criteria (faecal scoring chart, purina), and all dogs had been treated with deworming drugs for prophylaxis (ilium pyraquantal, troy or milbemax, novartis). all samples were lifted from the floor, first thing in the morning before cleaning, during november . faecal samples from dogs with an acute onset of diarrhoea (less than days of duration), were collected by a veterinarian from within the animal's enclosure. all dogs with acute diarrhoea were tested for the presence of canine parvovirus antigen in faeces using the anigen rapid cpv/ccv ag test kit, (bionote). positive samples were excluded from the study. none of the dogs had been treated with antimicrobial drugs within the previous weeks of sample collection. the majority of healthy dogs were receiving commercial dry food and some of the dogs with diarrhoea were being fed a high-fibre prescription veterinary diet (hill's i/d diet). faecal samples were processed as described previously [ , ] . briefly, aliquots of mg of faecal sample were thawed and re-suspended in saline buffer ( . m tris solution (ph . ), . m nacl, . m cacl ) at : ratio of solid mass. one mm zirconia/silica beads were added to the stool solution, filling around μl of an eppendorf tube, and vortexed vigorously for minutes. the samples were then centrifuged at x g for min, collecting the supernatant and repeating this step three more times. to reduce solid faecal matter and bacterial contamination, μl of this solution was filtered through a . μm tube filter (corning costar spin x) by centrifugation at x g for minutes, then the filtrate was transferred to ml tubes. to enrich for viral dna and rna, a dnase/rnase step was incorporated using a modified protocol described previously [ , ] . each sample was treated with a cocktail of dnases (turbo dnase, from ambion, baseline-zero from epicentre, benzonase from novagen and dnase i from roche) and rnase a (qiagen). this mixture was incubated in a water bath at ˚c for hours. to stop the enzymatic activity, edta (amresco) was added in a final concentration of mm to each sample and incubated at ˚c for min. viral dna/rna protected from digestion within viral capsids were extracted using qiaamp viral rna mini kit (qiagen), according to manufacturer's recommendations. a second dnase/rnase step was performed on the extracted viral rna for elimination of genomic dna, using dnase i recombinant, rnase free ( u/μl) (roche) and protector rnase inhibitor ( u/μl) (roche). after digestion of the dna, the viral rna was transcribed with sensiscript reverse transcriptase kit (qiagen; sensiscript rt kit) to generate cdna, according to manufacturer's instructions with minor modifications. briefly, for a more sensitive detection in the subsequent pcr, a mixture of oligo-dt primers (oligo (dt) primer, promega) and random primers (random hexamers, taqman reverse transcription reagents, roche, applied biosystems) were used and a rna denaturation step ( ˚for minutes) was added. viral cdna and genomic dna were randomly amplified using a modified sispa protocol [ , ] . briefly, a second strand synthesis was performed with large (klenow) fragment (new england biolabs) and random hexamers (roche, biosystems, μm) followed by digestion of the second strand product with the restriction enzyme cviqi (csp . ), (new england biolabs). then a csp /nbam adaptor was ligated to the digested dna using t dna ligase (invitrogen) followed by pcr amplification of the adaptor-ligated product with nbam pcr primers. an aliquot of the pcr product was validated on a % agarose tbe gel, where a positive smear with multiple bands confirmed the random sispa amplification of nucleic acid products. the amplified pcr products were cleaned up using wizard sv gel and pcr clean-up system (promega) following manufacturer's recommendations and two libraries with dual indexing for each sample were generated (dna and cdna) with illumina nextera xt dna sample preparation kit, according to manufacturer indications. after visualise it with agilent tape station system (agilent technologies), the libraries were submitted to the australian genome research facility (agrf) for a bases paired-end sequencing on the miseq illumina platform. all raw sequences were deposited under bioproject id: prjna at ncbi database. raw sequences were trimmed by quality score with prinseq software (v . . ) [ ] , filtering for low quality reads from both ends using the dust score [ ] with a threshold of . poly a/t tails in both ends (ten nucleotides of each end) and sispa primers sequences were also removed using this software. the mothur software v. . . [ ] was applied and the sequences were trimmed again, eliminating homopolymers, ambiguous bases and sequences less than bp. after these trimming steps, high quality reads (hqr) were obtained and all bad quality reads were removed from the group file (fig ) . subsequently, these dog free sequences were compared against a bacterial database (cam-era prokaryotic nucleotide database .v , nov ; http://camera.calit .net/) [ ] to eliminate bacterial sequences, using the blastn (blast . . + standalone) algorithm with an % identity cut off. to extract cellular organism sequences from the group file, megan v . . and mothur software were used as described above (fig ) . the host and bacteria free sequence reads, were de novo assembled with metavelvet (velvet . . , kmer ) [ ] using kmer size and contigs and singletons were created. these singletons were clustered with a % similarity using cd-hit-est (version. . . ) [ ] (fig ) . all contigs and singletons clusters were analysed through two pipelines. ( ) contigs and singletons clusters were compared against the camera viral nucleotide sequence database .v , using tblastx search with an e-value cut off − ; ( ) contigs and singletons clusters were compared against the ncbi nucleotide database ( ) using blastn search with an e value cut off . all blast searches were performed in blast . . + standalone. these files were then analysed by megan v . . [ ] and the lowest common ancestor of known viral sequences were identified (fig ) . finally, all viral contigs and singletons of eukaryotic organisms present in both analyses were aligned and compared with the ncbi reference sequence of the lowest common ancestor given by megan v . . . all alignments were made using sequencher version . . sequence analysis software (gene codes corporation, ann arbor, mi usa) with minimum match percentage of %- % and minimum overlap as assembly parameters, evaluating the percentage of coverage of the genome. all contigs and singletons with no hits were re-evaluated using online blastn with an e value cut off and visualised with megan v . . to evaluate the alignment with its lowest common ancestor. in order to acquire the complete genome of canine astrovirus, multiple sets of primers were selected from the literature or designed based on sequences obtained from illumina reads (s table) . nucleic acids from a single faecal sample from a dog with acute diarrhoea (dd ), which had contigs/singletons of canine astrovirus (after tblastx analysis) was used to determine the complete genome sequence. rna was extracted directly from the centrifuged sample after faecal extraction, previous to enrichment of viral nucleic acids as outlined before. rt-pcr was performed with superscript iii one-step rt-pcr system with platinum taq (invitrogen™). pcr conditions used were: ˚c for min and ˚c for min, cycle of ˚c for sec, ˚c for min and ˚c for min, and a final elongation step of ˚c for min, followed by final hold at ˚c. pcr products were run on a . % agarose tbe gel stained with redsafe™ nucleic acid staining solution (intron biotechnology). all pcr products were excised and cleaned up with wizard sv gel and pcr clean-up system (promega) following manufacturer's protocol and sequenced using sanger sequencing at the agrf. the near complete genome of the canine astrovirus was assembled using sequencher version . . sequence analysis software (gene codes corporation, ann arbor, mi usa) with minimum match percentage and minimum overlap as assembly parameters. phylogenetic analysis of this canine astrovirus was performed aligning protein sequences of the conserved amino acids of the capsid region (orf ) from different species (s fig), using clustal w, from mega version . [ ] with default settings. a phylogenetic tree with bootstrap was generated using the maximum likelihood method based on the jtt matrix-based model [ ] , using mega version . . the percentage of identity was calculated with clustalo . . [ ] supporting information s fig. megan viruses in the faecal microbiota of monozygotic twins and their mothers metagenomic analyses of an uncultured viral community from human feces viral metagenomics. reviews in medical virology viral metagenomics as an emerging and powerful tool in veterinary medicine. veterinary quarterly viral diversity and dynamics in an infant gut viruses in diarrhoeic dogs include novel kobuviruses and sapoviruses bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses the human gut virome: inter-individual variation and dynamic response to diet the fecal virome of pigs on a high-density farm feline fecal virome reveals novel and prevalent enteric viruses viral metagenomic analysis of feces of wild small carnivores metagenomic analyses of viruses in stool samples from children with acute flaccid paralysis rna viral community in human feces: prevalence of plant pathogenic viruses metagenomic analysis of human diarrhea: viral detection and discovery study of the viral and microbial communities associated with crohn's disease: a metagenomic approach characterization of microbial dysbiosis and metabolomic changes in dogs with acute diarrhea common and emerging infectious diseases in the animal shelter. veterinary pathology long-term viremia and fecal shedding in pups after modified-live canine parvovirus vaccination m gene evolution of canine coronavirus in naturally infected dogs. the veterinary record an update on canine coronaviruses: viral evolution and pathobiology canine coronavirus-associated puppy mortality without evidence of concurrent canine parvovirus infection infectious diseases of the dog and cat novel norovirus in dogs with diarrhea fields virology,. . th ed. philadelphia detection and characterization of canine astroviruses isolation and characterization of canine astrovirus in china complete genome sequence of canine astrovirus with molecular and epidemiological characterisation of uk strains prevalence and risk factors of astrovirus infection in puppies from french breeding kennels detection of canine astrovirus in dogs with diarrhea in japan viruses and virus-like particles in the faeces of dogs with and without diarrhoea astrovirus-like, coronavirus-like, and parvovirus-like particles detected in the diarrheal stools of beagle pups genetic characterization of a new astrovirus detected in dogs suffering from diarrhoea enteric disease in dogs naturally infected by a novel canine astrovirus molecular characterisation of calicivirus and astrovirus in puppies with enteritis phylogenetic analysis of astrovirus and kobuvirus in korean dogs a virus discovery method incorporating dnase treatment and its application to the identification of two bovine parvovirus species sequence-independent, single-primer amplification (sispa) of complex dna populations quality control and preprocessing of metagenomic datasets a fast and symmetric dust implementation to mask low-complexity dna sequences introducing mothur: opensource, platform-independent, community-supported software for describing and comparing microbial communities community cyberinfrastructure for advanced microbial ecology research and analysis: the camera resource metavelvet: an extension of velvet assembler to de novo metagenome assembly from short sequence reads cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences mega : molecular evolutionary genetics analysis version . the rapid generation of mutation data matrices from protein sequences. computer applications in the biosciences fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega the authors gratefully acknowledge the helpful assistance of all staff at lost dogs home and university of melbourne u-vet werribee animal hospital with the faecal sample collection. also to dr celeste donato for her valuable scientific input and her assistance with the construction of the astrovirus phylogenetic tree. minot key: cord- -bwxkxvsl authors: mamontov, eugene; cheng, yongqiang; daemen, luke l.; keum, jong k.; kolesnikov, alexander i.; pajerowski, daniel; podlesnyak, andrey; ramirez-cuesta, anibal j.; ryder, matthew r.; stone, matthew b. title: effect of hydration on the molecular dynamics of hydroxychloroquine sulfate date: - - journal: acs omega doi: . /acsomega. c sha: doc_id: cord_uid: bwxkxvsl [image: see text] chloroquine and its derivative hydroxychloroquine are primarily known as antimalaria drugs. here, we investigate the influence of hydration water on the molecular dynamics in hydroxychloroquine sulfate, a commonly used solubilized drug form. when hydration, even at a low level, results in a disordered structure, as opposed to the highly ordered structure of dry hydroxychloroquine sulfate, the activation barriers for the rotation of methyl groups in the drug molecules become randomized and, on average, significantly reduced. the facilitated stochastic motions of the methyl groups may benefit the biomolecular activity due to the more efficient sampling of the energy landscape in the disordered hydration environment experienced by the drug molecules in vivo. chloroquine (cq) and its derivative hydroxychloroquine (hcq) are drug molecules that have been used for malaria treatment and also as immunosuppressants in the treatment of autoimmune disorders such as rheumatoid arthritis and lupus. the drugs have also been suggested as promising agents for antitumor therapy. , however, they have recently attracted attention and debate as potential antiviral drugs, coming to prominence from the coronavirus disease (covid- ) caused by sars-cov- . given the conflicting evidence of cq and hcq efficacy for the treatment of covid- and the potential adverse side effects, − their use and further trials are currently restricted to clinical settings under strict medical supervision due to safety considerations. the therapeutic action of cq and hcq tends to be closely linked to their protonation and deprotonation. these compounds preferentially accumulate in the lysosomes and endosomes of cells, increasing the ph of the environment. this inhibits the processes such as autophagy or virus release from the endosome or lysosome since these processes require acidic conditions. in antiviral therapies, this inhibition may impede the ability of the virus to release its genetic material into the cell. the accumulation of cq and hcq in lysosomes, which is due to their basic properties, may result in a -to -fold increase in the concentration of these drugs compared to the plasma or cytosol concentration. , thus, these drugs may be present in the organism in aqueous environments at vastly different levels. the influence of aqueous hydration on the molecular dynamics and, ultimately, the function of biomolecules has been widely recognized. such influence can be very broad, as hydration-dependent enabling of the general enzymatic activity by proteins, or specific and crucial, as control of kinetic proofreading in the aminoacylation of rna. importantly, even the small-amplitude molecular motions can have a critical influence on the biological function. for example, a static picture of a transmembrane protein cannot adequately describe the function of gramicidin a and kcsa potassium channels without taking into consideration their thermal atomic motions with sub-angstrom amplitude that play a decisive role in facilitating or blocking ion transmission. such thermal fluctuations with sub-angstrom amplitude can be probed efficiently by inelastic (ins) and quasielastic (qens) neutron scattering. while the crystallographic structure of chloroquine-based compounds has been studied extensively, the molecular dynamics have yet to be investigated. in this work, we employ neutron scattering techniques to probe the influence of hydration water on the molecular dynamics in hcq sulfate (hcqs), a commonly used drug of the chloroquine family. the structure of hcq and hcqs units is presented schematically in figure . the protons of h so attach to two of the nitrogen atoms of hydroxychloroquine, resulting in the formation of a hydroxychloroquine cation and a sulfate anion. thus, unlike the free base hcq, hcqs is readily solvable in water. while free base hydroxychloroquine (c for the preparation of hydrated solid hcqs samples, various approaches have been attempted. it should be noted that, despite a high solubility in water, the hcqs powder was not found to be hygroscopic and did not show any weight uptake when exposed to ambient humidity for days. this is in remarkable contrast with the hydration behavior of powders of inorganic hydrophilic compounds and lyophilized biomolecules, , which readily adsorb water from ambient vapor in amounts sufficient for the adsorbed hydration water to exhibit bulk-like traits of microscopic dynamics, albeit without crystallization on cooling. hcqs powder was dissolved in water at mg/ml in an open glass petri dish. the water was then allowed to evaporate in a chemical fume hood for h. the resulting residue was transparent and had a hardened epoxy appearance, which could be scraped from the dish with a steel spatula. the weight uptake was ∼ . %; this value was repeatedly achieved when preparing several samples and , with four water molecules per hcqs molecular unit. we hypothesize that this composition represents a stable low-hydration state of hcqs. interestingly, when the hcqs powder was initially dissolved in water, before obtaining a fully mixed solution, dense regions with filamentary structures with the appearance of the hydrated epoxy-like sample were observed. for the main hydrated solid sample, . g of the c h cln o h so (h o) was used. before the scattering measurements occurred, this main hydrated solid sample was kept inside the sealed sample holder at k for h. a second, less ordered, hydrated solid sample with the same composition was then prepared similarly to the main sample, except that it was not annealed inside the sealed sample holder at k before the measurements. a third, more ordered, hydrated solid sample was prepared by mixing the hcqs powder with a stoichiometric amount of water to achieve a targeted composition of c h cln o h so (h o) and then loaded into a similar sealed sample holder. this approach was previously used to achieve the desired hydration level and behavior in inorganic powders similar to those obtained using vapor. besides the hydrated solid samples, . g dry hcqs was loaded into a similarly sealed sample holder. for a subset of measurements, a . g sample of free base hydroxychloroquine, c h cln o (hcq), was also loaded into a similarly sealed sample holder. neutron scattering measurements were performed at several spectrometers at the spallation neutron source (sns) at the oak ridge national laboratory (ornl): basis, sequoia, vision, and cncs. diffusion in aqueous solutions of hcqs was analyzed using qens data from basis ( figures s and s ). the resulting diffusivity values are displayed in figure and exhibit similar activation energies for the and mg/ml solutions of . and . kj/mol (linear fits in figure ), respectively. since the data that we have fitted following subtraction of the d o buffer signal did not support the presence of more than one dynamic component, the measured qens dynamics in solutions were either similar between the cations and anions or completely dominated by the motion of one species, more likely the cations. even if the hydrated so anions were strongly dissociated from the hcq cations (also hydrated) and their diffusivities were substantially different, the relatively much larger number of hydrogens associated with the hydrated hcq cations would seem to suggest the cation dominance in the measured scattering signal. furthermore, the diffusivity values reported herein are likely related to global (combined translational and rotational) motion of the solute species, whereas the actual translation diffusivity values are somewhat lower. for example, for the particles that could be approximated by the dense, hard spheres, the global diffusion coefficient measured by qens would exceed the actual translational diffusion coefficient by ca. %. the internal dynamics of hcqs molecules were probed using the solid samples. from the neutron diffraction patterns measured at sequoia and cncs ( figure s ), the structures of dry hcqs and more ordered hydrated hcqs look similar, as their features almost overlap after subtraction of a constant from the latter to account for the higher incoherent scattering background from the hydrated sample. the structures of the main hydrated hcqs and less ordered hydrated hcqs are similar to one another (although the latter exhibits somewhat less prominent diffraction peaks) but differ from the dry and more ordered hydrated hcqs. the cncs data demonstrates larger mean-squared displacements (msd) at k in the main hydrated sample compared to the dry, as evidenced by the relatively faster decrease with q of the data baseline that represents the incoherent scattering signal from the protons. the diffraction data in figure s suggest that the hydration of the main and the less ordered samples resulted in the formation of a different structure. the ins spectra of dry hcqs, measured at vision and calculated using density functional theory (dft), are presented in figure . the dft results agree well with the experiment and thus allow for a reliable assignment of the vibrational modes. figure shows the measured spectra of dry and main hydrated hcqs as well as more ordered hcqs and free base hcq. while the difference between dry hcqs and hcq, introduced by the protonation of hcq and the presence of the sulfate anion, appears limited, the effect of hydration (at a similar level) on the vibrational spectra differs dramatically between the main hydrated hcqs and more ordered hydrated hcqs. the latter system, which retains the structure of dry hcqs ( figure s ), exhibits a vibrational pattern similar to hcq and dry hcqs, albeit with a higher background due to the water. on the contrary, the main hydrated hcqs exhibits a very different hydration pattern in the low-energy range, below ∼ mev, indicating significant disorder within the sample. note that figure is intended to make a comparison among the overall vibrational patterns and their relative intensities, whereas the low-energy range will be plotted separately over an expanded energy range and discussed in more detail below. the data in figure suggest that the main hydrated hcqs sample not only has a different crystallographic structure, as evidenced by the diffraction data in figure s , but indeed becomes less ordered. the ins data collected at sequoia also supports this conclusion. qens measurements on basis were used to probe the relaxation dynamics in dry and main hydrated hcqs at physiological (body) temperature. the results for the meansquared displacements ( figure s ) are in agreement with the trend evident from figure s , showing that the displacements for the main hydrated sample exhibit a much stronger temperature dependence. while both dry and the main hydrated hcqs samples show a sustained increase in the mean-squared displacements above k, the latter also exhibits an additional, faster increase with temperature above k. therefore, a relatively more complex, possibly multicomponent scenario of dynamic processes in the main hydrated hcqs sample at physiological temperatures could be inferred. this is indeed confirmed by a comparison of the qens spectra for the dry and main hydrated samples, as presented in figure . the spectra feature a prominent elastic line and quasielastic wings. the dry hcqs spectra can be adequately fitted with the expression where the fraction of the elastic scattering signal, x(q), represents the elastic incoherent structure factor (eisf), Γ(q) is the half-width at half maximum (hwhm) of the lorentzian quasielastic component, r(q, e) is the experimentally measured resolution function numerically convolved with a superposition of the delta-function (elastic) and lorentzian (quasielastic) components, and the term in the parentheses represents a fitted linear background. on the other hand, the spectra collected from the main hydrated hcqs sample, in the bottom panel of figure , require two quasielastic components, broad and narrow, as follows to better illustrate this, in figure we present the same data as plotted in figure , but with the scattering and fitted intensities renormalized as i(q, e)/(n bose (e) + ), where n bose (e) = (exp(e/k b t) − ) − is bose population factor and k b is boltzmann's constant. at higher-energy transfers, where the influence of the spectrometer resolution is relatively weak, such renormalized data approximates the imaginary part of the dynamic susceptibility, χ″(q, e). the maxima of dynamic susceptibility correspond to the characteristic relaxation frequencies in the system, thus enabling intuitive visualization. one can see that for the main hydrated hcqs sample, an additional fit component (cyan solid line) is indeed necessitated by the deviation of the low-energy data points (symbols) from the elastic signal (deep blue solid line). addition of the second fit component results in a good overall fit to the data (red solid line). the hwhms of the qens components ( figure s ) in the energy range of − μev exhibit only a relatively weak qdependence, indicative of localized, as opposed to translational, dynamic processes, whereas the additional narrow ( − μev) component in the main hydrated hcqs sample has no significant q-dependence. this narrow component could be ascribed to the dynamics of the water molecules. the width of the broad component is similar for the dry and hydrated samples at k. however, the temperature dependence is quite different, as it is much more pronounced and systematic in the dry sample. the corresponding arrhenius plot, with relaxation times calculated from the q-averaged qens broadening values as τ = ℏ/⟨hwhm(q)⟩, is presented in figure . also shown are the relaxation times for the more ordered and less ordered hydrated samples obtained in the same way as for the main hydrated sample. it is immediately evident that much smaller activation energies are observed for the main hydrated and less ordered hydrated samples (blue and cyan symbols and lines). at first, we discuss the principal dynamic component, which is represented by the symbols other than squares (whereas the squares represent the dynamic component associated with the hydration water). there is only a relatively small difference in the activation energy between the dry hcqs and free base hcq. the latter is also characterized by a single-component qens signal, just as dry hcqs. furthermore, the more ordered hydrated hcqs sample also exhibits similar activation energy. on the other hand, hydration in the main and less ordered hcqs samples reduces the activation energy by about a factor of . − . . for the main hydrated sample, this reduced activation energy value becomes comparable with the low activation energy exhibited by its hydration water (longer relaxation times, square symbols, in figure ). at the same time, . arrhenius plot of the relaxation times (symbols) and fits (lines) for the dynamic process in dry hcqs and two dynamic processes in the main hydrated hcqs sample. also presented are the data for the free base hcq (one dynamic process) and the more ordered and less ordered hydrated hcqs samples (two dynamic processes). the square symbols (at longer relaxation times) represent the hydration water. when not visible, the error bars are within the size of the symbol. the activation energy exhibited by the hydration water in the less ordered hydrated sample is even lower. interestingly, the activation energy exhibited by the water in the more ordered hydrated sample is essentially the same as that of the principal dynamic component in this sample, suggesting that the motion of the hydration water molecules is highly correlated with the motion of other entities in this sample. this is corroborated by the ins data measured up to mev at sequoia (figure s ) , which exhibit very similar features for the dry and more ordered hydrated sample, in contrast with the typical behavior of surface/interfacial water. while the dynamic component associated with longer relaxation times (square symbols in figure ) can be ascribed to the hydration water molecules, the origin of the main dynamic component warrants further discussion. to this end, the eisf(q) at k is fitted (figure ) with an expression for -fold jumps on a circle of radius r modified to allow for an additional parameter, c, describing the fraction of the immobile particles where j is a spherical bessel function of zeroth order. using a known value of . Å for the proton−proton distance in methyl groups, which corresponds to r = Å in eq , the fits shown with the solid lines in figure were obtained with a single fit parameter, c, whose value is represented by a solid horizontal line. the fitted curve oscillates about the value of c + ( − c)/ since for -fold jumps on a circle, the eisf(q) should asymptotically approach a value of / at high q values. it should be noted that fitting the eisf(q) with any generic model, such as motion on a sphere or within a spherical volume, shows the same deviation at the lower q values between the fits approaching unity and the data points positioned below the fit curve. this indicates not the inadequacy of the -fold jump model, but rather a systematic error in the eisf(q) data in the low-q region, which is known to originate from multiple scattering in the sample. indeed, the discrepancy becomes more pronounced for the main hydrated hcqs sample, as one would expect for the stronger scattering sample with more pronounced multiple scattering effects. it should be noted that some multiple scattering, especially in the main hydrated hcqs sample, was unavoidable due to the sample morphology (hardened epoxy), which precluded the use of a sample holder less than . mm in thickness. with the flat-plate sample oriented perpendicular to the incident neutron beam, the first five q values are measured in transmission geometry and are especially susceptible to multiple scattering effects. nevertheless, there are several compelling reasons to ascribe the broad dynamic component to the rotation of methyl groups in hcqs. first, the fitted values of the "immobile fraction" parameter c of . ± . and . ± . for the dry and main hydrated sample are in excellent agreement with the values one would expect from the two rotating methyl groups with six hydrogens per molecule, ( − )/ = . for the dry hcqs (with a total of hydrogens per molecule) and ( − − )/ = . for the hydrated hcsq (with a total of hydrogens per molecule, out of which the eight hydrogens associated with the four h o units are mobile). second, the activation energy of . kj/mol, or mev, determined from the arrhenius plot for the broad dynamic component in dry hcqs in figure predicts the first rotational excitation energy of mev for the quantum methyl rotor, which is in excellent agreement with the measured value of . mev ( figure ) observed with the vision spectrometer. the free base hcq and more ordered hydrated hcqs also show the methyl rotation peak at the same position, in agreement with their similar activation energies (for the broad dynamic component) presented in figure . however, there are no rotational peaks visible in figure for the main hydrated hcqs at . mev, as one would predict for the quantum methyl rotor from the activation energy of . kj/mol, or mev, determined from the arrhenius plot for the broad dynamic component in figure . the rotational peak at mev, characteristic of hcq and dry hcqs, is also not present in the main hydrated hcqs spectrum. this suggests that, unlike in the more ordered hydrated hcqs, the hydration water likely randomizes the barriers for the methyl group rotations in the main hydrated hcqs, which thus can no longer be described in the framework of the methyl quantum rotations. distribution of the barriers for the methyl group rotations does not give rise to a well-defined peak in the vibrational spectra. yet, the effective average activation energies for the stochastic methyl rotation processes can still be measured by qens, as presented in figure . the data in figure show reduced activation energy (slope) for the main and less ordered hydration samples. in contrast, the corresponding prefactor (intercept) concurrently increases, suggesting that barrier randomization could have increased the average rate of methyl rotation in the higher-temperature range, above physiological temperatures. neither the introduction to hcq of the sulfate groups to produce hcqs, nor the hydration of hcqs in the more ordered hydrated sample, results in a similar smearing effect on the methyl group rotations, as the rotational peak at mev remains unchanged among hcq, dry hcqs, and the more ordered hydrated hcqs, and the corresponding measured activation energies change only slightly (figure ). this suggests that the effect of hydration on the activation energy for the methyl group rotation may be due to the disorder introduced by the water molecules in the main hydrated and less ordered hydrated hcqs samples. it is thus instructive to compare the temperature dependence of the eisf for the dry and main hydrated hcqs samples ( figure s ). the eisf for the dry hcqs is temperature-independent, demonstrating that for the methyl group rotations, the jump rate is temperature-dependent (with an activation energy of . kj/mol). in contrast, the number of methyl groups participating in the rotation is temperature-independent. such behavior is typical for the functional groups in ordered systems. on the other hand, the eisf for the main hydrated hcqs sample is temperaturedependent. thus, while the jump rate of the methyl groups becomes much less temperature-dependent in the main hydrated hcqs (with an activation energy of just . kj/ mol), the number of methyl groups participating in the rotation decreases as the temperature is reduced. the fraction of the immobile hydrogens in the system is defined by the "immobile" parameter c in the eisf(q) fits with eq . when the temperature is decreased to k, the parameter c, that is, the plateau value of the eisf(q) for the main hydrated hcqs, coincides with that for the dry hcsq, indicating that all of the hydration water molecules, which are fully mobile at k, have become immobilized. the temperature dependence of the fraction of the immobilized water molecules in the main hydrated hcqs sample is shown in figure s . therefore, the strong temperature dependence of the ⟨u (t)⟩ exhibited in figure s by the main hydrated hcqs sample above ∼ k (in the absence of bulklike water freezing at k) is driven primarily by changing with the temperature fraction of the immobilized water molecules. such behavior is typical for the disordered hydration water. to further describe the water in the main hydrated hcqs, we performed temperature-dependent measurements of the ins spectra using the sequoia spectrometer. while the upper panels of figures and show the measured spectra, the lower panels present a comparison of the difference spectra between the main hydrated and dry hcqs samples to the spectra of h o ice-ih ( k) and liquid water ( k) as well as the structural h o in wo ·h o data. in figure , the peaks at ∼ mev are mainly due to c−h and n−h stretching modes, and the shoulder at ∼ mev in the main hydrated hcqs sample spectra is due to o−h stretching modes of the hydration water (this peak is prominent in the difference spectrum at k). at k, the peak at ∼ mev can be fitted with two gaussians, centered at and mev for the dry hcqs sample and and mev for the main hydrated hcqs sample. due to the mismatch in the second peak position, the difference (hydrateddry) hcqs spectrum shows a peak at mev, whereas another peak in the difference spectrum ( mev) is due to o−h stretching modes of the hydration water. the lower panel demonstrates that at k, the o−h stretching peak in the difference spectrum is broad, as it covers the peak in ice-ih at its low-energy side and the peak of the structural water at its high-energy side. therefore, the water in the main hydrated hcqs sample exhibits a distribution of hydrogen bonds, which are spread in strength from those observed in ice-ih to the weaker bonds. the spectra for the hydrated sample at and k almost coincide. the o−h stretching modes in the difference spectra decrease in intensity (due to the debye−waller factor) and do not shift ( mev) or broaden, whereas the peak in liquid water at k ( mev) moves to much higher energy. this suggests that the water molecules at temperatures of − k are still confined and not in the bulk-like liquid state. the spectra presented in figure show a sharp peak at about mev due to c−h bending modes. the spectrum of the main hydrated hcqs sample shows a slight shift in the peak to higher energy and a new peak at ∼ mev. due to the change in the mev peak, the difference hcqs spectrum (hydrateddry) shows a minimum at mev and a maximum at mev. the peak at ∼ mev is due to the intramolecular water h−o−h bending (or scissor) mode. at higher temperatures ( k), the intensity of the bending h−o−h mode peak sharply decreased (due to the debye−waller factor). in addition, the difference spectrum clearly shows a band between and mev, which can be assigned to librational vibrations of water. the lower panel of figure shows that at k, this band is similar to the band in ice-ih, and its characteristic sharp low-energy cutoff is shifted to lower energy by about mev compared to that in ice-ih. therefore, the average hydrogen bonds in the confined water are weaker than in ice-ih, which agrees with the stretching modes. at k, the librational peak in the difference spectrum softens (compared to k), but it is located at higher energy than in liquid water. thus, the hydrogen bonds acting on the confined water in the main hcqs sample are larger in size than those in liquid water. the structural water (as in the wo ·h o data, also shown in the figure) exhibits sharper peaks in this range. therefore, the appearance of the broad librational band is indicative of a disordered water network around the hcqs molecules. hydration water molecules may exert a profound influence on the dynamics of methyl group rotations in hcqs, in particular, by randomizing the potential barriers in a more disordered hydration structure. when the hydration results in a highly ordered structure similar to that of dry hcqs, the water molecules move in correlation with the other structural units, such as methyl groups, and do not alter the activation energy associated with the methyl group rotations. however, this changes when the hydration leads to a more disordered structure. such a hydration state, which is more relevant to the state of hcqs in aqueous environments, randomizes and, on average, lowers the activation barriers for the methyl group rotations, even at minimal hydration levels. the modification of the potential barriers experienced by the hcqs methyl groups in the more disordered hydrated state could have implications for the function of the drug. it has been known that the introduction of methyl groups can dramatically increase the potency of drug molecules, sometimes by orders of magnitude. − a boost in the drug efficiency by methyl groups is typically attributed to the altered binding affinity, solubility, or metabolism. on the other hand, it has been argued that a critical advantage of methyl groups in biochemical processes is due to the ease of the thermally activated dynamics that increases the configurational entropy of the molecule and allows for more efficient sampling of the energy landscape, which is crucial for biochemical activity. in this paradigm, further reduction of the activation energy for methyl group rotation would be beneficial to the drug function and efficiency. in contrast to the dry hcqs, or the hcqs in the ordered hydration state, the hcqs in the disordered hydration state experiences further plasticization of the methyl group dynamics via a randomized energy landscape and lowered, on average, potential barriers for rotations, analogous to the plasticizing effect of hydration water in proteins. this effect is remarkably pronounced already at minimal hydration levels of just four water molecules per hcqs structural unit. therefore, the potential barriers and the associated stochastic dynamics of the methyl side groups could be significantly reduced in a disordered hydration environment experienced by the drug molecules in vivo. hcqs powder was purchased from sigma-aldrich and used as received for the preparation of liquid and solid samples. aqueous d o solutions of hcqs at and mg/ml were loaded in flat-plate aluminum gold-coated sample holders of mm height, mm width, and . mm thickness, which were sealed hermetically using indium wire. the use of gold-coated sample holders was necessitated by the mildly corrosive character of the liquid samples. for the solid samples, hermetically sealed with indium wire sample holders of . mm thickness were used. the backscattering basis spectrometer provided a range of neutron energy transfers suitable for data analysis between − μev and + μev, with an energy resolution (averaged over all scattering angles) of . μev full width at half maximum (fwhm). routine data reduction procedures were used, including background subtraction and normalization to a vanadium standard. the quasielastic neutron scattering (qens) measurements were performed at , , , and k. in addition, continuous data collection occurred during a controlled cooling of . k/min. the sample-dependent resolution spectra were collected at a baseline temperature of k. the sequoia spectrometer was used to measure inelastic neutron scattering (ins) data at incident neutron energies (e i ) of and mev, to provide an energy resolution of − % for ins data below mev and a momentum transfer (q) between and Å − . the collected neutron scattering data were transformed from time-of-flight (tof) and instrument coordinates to the dynamical structure factor s(q, e). ins data was also collected for the empty container under the same conditions and subtracted. the vision spectrometer was used to collect ins data over a broad energy-transfer range with a high-energy resolution (Δe/ e < . %). additional measurements were performed using the cold neutron chopper spectrometer (cncs) with an incident energy of . mev. density functional theory (dft) was used to calculate the normal vibrational modes and ins spectra of dry hcqs using vasp. the initial structure was obtained from the ccdc ( ). the calculations used the projector augmented wave (paw) approach , to describe the effects of the core electrons using the pbe exchange-correlation functional and an optb b-vdw functional for dispersion correction. an energy cutoff of ev was used for the plane-wave basis of the valence electrons. the electronic structure was calculated on a × × Γ-centered grid. given the relatively large unit cell (a = . Å, b = . Å, c = . Å), a large number of k points was not required to achieve convergence. we tested × × and × × grids, and the difference in the final energy was rather small (< . mev). the vibrational spectra were also confirmed to be almost identical. the maximum interatomic force after optimization was below . ev/Å using a total energy tolerance of − ev for the electronic energy minimization and − ev for the structure optimization. the vibrational eigenfrequencies and normal modes were then calculated by solving the force constants and dynamical matrix using phonopy. the oclimax software was used to convert the dft phonon results to the simulated ins spectra appropriate for comparison with each experimental measurement. to directly calculate the energy barrier associated with individual methyl group rotations, the climbing image nudged elastic band (cneb) method was used. seven intermediate images were introduced, with the starting and the ending images being the two equilibrium positions of the methyl rotor separated by a relative rotation of °. we note that there are two nonequivalent methyl groups in the unit cell, and the energy barriers are close, with the lower one being mev. the excitation energies for the quantum methyl rotor were solved using the data analysis and visualization environment (dave). ■ associated content the supporting information is available free of charge at https://pubs.acs.org/doi/ . /acsomega. c . diffusivity of hcqs in aqueous solutions; neutron diffraction patterns from the solid samples; mean-squared displacements (msd) in the solid samples; qens broadening in the dry and main hydrated solid samples; hydration water dynamics in the more ordered hydrated solid sample; temperature dependence of the eisf(q) measured at basis for the dry and main hydrated solid samples; temperature dependence of the fraction of immobilized water molecules in the main hydrated sample (pdf) de-ac − ch , for access to supercomputing resources. computing resources were also made available through the virtues and the iceman projects, funded by laboratory directed research and development program at ornl. we thank r. moody for the dedication to obtaining sample materials. the authors also thank dr. jessica v. lamb for assistance with the structural representations and dr. mark d. lumsden for valuable discussion. this manuscript has been authored by ut-battelle, llc, under contract de-ac − or with the us department of energy (doe). the us government retains and the publisher, by accepting the article for publication, acknowledges that the us government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript or allow others to do so, for us government purposes. doe will provide public access to these results of federally sponsored research in accordance with the doe public access plan (http://energy.gov/downloads/doe-public-access-plan). delivery by smart liposomes for optimal autophagy inhibition and improved antitumor efficiency with liposomal doxorubicin in situ autophagy disruption generator for cancer theranostics targeting endosomal acidification by chloroquine analogs as a promising strategy for the treatment of emerging viral diseases safety conciderations with chloroquine, hydroxychloroquine and azithromycin in the management of sars-cov- infection rethinking the role of hydroxychloroquine in the treatment of covid- a systematic review on the efficacy and safety of chloroquine for the treatment of covid- role of lysosomes in hepatic accumulation of chloroquine possible mechanisms of action of antimalarials in rheumatic disease protein hydration and function mechanistic insights into cognate substrate discrimination during proofreading in translation on the importance of atomic fluctuations, protein flexibility, and solvent in ion permeation intermolecular interactions in crystalline hydroxychloroquine sulfate in comparison with those in selected antimalarial drugs observation of fragile-to-strong liquid transition in surface water in ceo dynamics of biological macromolecules: not a simple slaving by hydration water suppression of the dynamic transition in surface water at low hydration levels: a study of water on rutile the new cold neutron chopper spectrometer at the spallation neutron source: design and performance vibrational density of states of strongly h-bonded interfacial water: insights from inelastic neutron scattering and theory quasielastic neutron scattering multiple-scattering effects on smooth neutronscattering spectra dave: a comprehensive software suite for the reduction, visualization, and analysis of low energy neutron spectroscopic data confined interlayer water promotes structural stability for high-rate electrochemical proton intercalation in tungsten oxide hydrates rational design of orthogonal receptor-ligand combinations recent advances in chemical approaches to the study of biological systems the methylation effect in medicinal chemistry a potent and selective s p( ) antagonist with efficacy in experimental autoimmune encephalomyelitis methyl effects on protein-ligand binding profound methyl effects in drug discovery and a call for new c-h methylation reactions discovery of -{ -[ -fluoro- -(( s, r)− -methyl- , -dioxo- -phenyl-[ , ]thiazinan- -ylmethyl)-phenyl]-piperazin- -yl}-ethanone (gne- ): a potent, selective, and orally bioavailable retinoic acid receptor-related orphan receptor c (rorc or ror gamma) inverse agonist the medicinal chemist's toolbox for late stage functionalization of drug-like molecules late-stage oxidative c(sp( ))-h methylation role of methyl groups in dynamics and evolution of biomolecules a unified model of protein dynamics efficient iterative schemes for ab initio total-energy calculations using a plane-wave basis set projector augmented-wave method from ultrasoft pseudopotentials to the projector augmented-wave method generalized gradient approximation made simple ) togo, a.; tanaka, i. first principles phonon calculations in materials science simulation of inelastic neutron scattering spectra using oclimax a climbing image nudged elastic band method for finding saddle points and minimum energy paths key: cord- - arxiu authors: nan title: september new in review date: - - journal: j acad nutr diet doi: . /j.jand. . . sha: doc_id: cord_uid: arxiu nan the authors hypothesized that video game use would be positively associated with body mass index (bmi) while controlling for television use, and that sugar-sweetened beverage and snack consumption, physical activity, and bedtime regularity would be influencers. a cohort study was designed to test this hypothesis using a sample of , children. the sample was derived from the millennium cohort study, a nationally representative prospective birth cohort study of children in the united kingdom between september , and january , . information was included if the participating children had data collected at the ages of , , , and years during the study. the sample was % male, with . % classified as white. the primary exposure variable was weekday video game use, which was measured on a six-point scale ranging from none to more than hours per day and then collapsed into four categories: none, less than hour, to hours, and or more hours per day. television use was measured on a similar scale. physical activity was measured based on days per week participating in sports clubs or physical classes, with bed time quantified with a binary scale as to whether the child did or did not go to bed at the same time each night. consumption of sugar-sweetened beverages was measured in terms of frequency and location, as was high-calorie snack food. the outcome variable was bmi standard deviation score. during home visits, researchers measured both height and weight for its calculation. covariates included sex, race/ethnicity, and socioeconomic status. statistical analyses were performed using spss statistics version . (ibm, ). the authors report that for every standard deviation increase in the number of video game hours at age years, a higher bmi score was observed at age . clinical significance of nutritional risk screening for older adult patients with covid- . liu g, zhang s, mao z, et al. eur j clin nutr. ; https://doi.org/ . /s - - - . researchers assessed nutritional risks among older patients diagnosed with covid- along with their associated clinical outcomes. a retrospective cohort analysis was designed using a sample of participants. patients admitted to the department of infectious diseases at a research hospital between january and march , , diagnosed with covid- , older than years, and hospitalized with a length of stay of over hours were enrolled. the sample was % female, with a mean age of . years. four nutritional risk screening tools were used for the study: the nutritional risk screening (nrs ) , malnutrition universal screening tool (must), mini nutrition assessment shortcut (mna-sf), and the nutrition risk index (nri). the nrs was designed to predict clinical effects of nutritional treatment in hospital settings with two levels: level and level contained factors of bmi status, weight loss history, nutritional intake, and disease severity. the mna-sf was designed to detect undernourishment of the elderly in home care programs, nursing homes, and hospitals and to ascertain the risk of undernourishment progressing. the must was designed to identify the need for nutritional treatment as well as establishing nutritional risk on the basis of the association between impaired nutritional status and impaired function, also containing a bmi score, weight loss score, and acute disease score. the nri was calculated with an equation using serum albumin and recent body weight loss: ( .  serum albumin, g/l), plus . , multiplied by present weight/usual weight  . statistical analysis was performed using spss . (ibm corp., ). the authors report that after accounting for confounding variables through multivariate analysis, patients in the higher-risk groups had significantly longer length of stay, higher hospital expenses, poor appetite, heavier disease severity, and more weight changes. association of work requirements with supplemental nutrition assistance program participation by race/ ethnicity and disability status, - . brantley e, pillai d, ku l. jama netw open. ; ( ):e . the investigators considered the impact of work requirements for supplemental nutrition assistance program (snap) participants against rates of participation, while considering race, ethnicity, and disability status. a cross-sectional study with , participants was designed to consider this issue. the sample was drawn from the to american community survey in conjunction with the us census bureau. the investigators excluded those adults who would be exempt from work requirements for able-bodied adults without dependents, as well as those facing specific snap barriers: noncitizens, students, individuals living with the elderly; and individuals living in group quarters. the sample included individuals without household incomes below % of the federal poverty level. the sample was . % male, with a mean age of . years, and was . % non-hispanic white, . % non-hispanic black, . % hispanic, and . % asian. the outcome sought was snap participation based on responses indicating whether anyone in the household received vouchers over the prior months. the primary exposure was residency in geographic areas in which snap work requirements apply. in addition to race and ethnicity, covariates included sex, educational attainment, marital status, home ownership, household size, and age. statistical analysis was performed using stata mp version (statacorp, ). the investigators report that work requirements resulted in a . percentage point decrease in participation, most acutely felt amongst non-hispanic black adults. association of modifiable lifestyle factors with cortical amyloid burden and cerebral glucose metabolism in older adults with mild cognitive impairment. kimura n, aso y, yabuuchi k, et al. jama network. ; ( ):e . the investigators assessed whether modifiable lifestyle factors are associated with cortical amyloid burden and cerebral glucose metabolism in communitydwelling older adults with mild cognitive impairment. a prospective cohort study using participants was designed to address this issue. inclusion criteria were aged years or older; living in usuki, japan; generally healthy and without dementia; and able to function independently. the sample had a mean age of . years, with . % being male, and a median education level of years. participants wore a wristband sensor (silmee w , tdk corporation, tokyo, japan) day and night except while bathing, and had measured lifestyle parameters to include walking steps, conversation time, and sleep parameters. the studied lifestyle factors included walking steps, conversation time, total sleep time, wake after sleep onset, sleep efficiency, waking time count, and nap time. static carbon- elabeled pittsburgh compound b positron emission tomography (pib-pet) and fluorine- fluorodeoxyglucose positron emission tomography (fdg-pet) findings were acquired using the biograph mct in threedimensional scanning mode. apolipoprotein e phenotype determination was performed using human apolipoprotein e / pan-apoe enzyme-linked immunosorbent assay kit. the association between neuroimaging variables and seven lifestyle factors was examined using multiple regression models. statistical analyses were performed using spss . (ibm corp, ). the investigators report that total sleep time was inversely associated with fluorodeoxyglucose uptake after adjusting for covariates. proteomic and metabolomics correlates of healthy dietary patterns: the framingham heart study. walker m, song r, xu x, et al. nutrients. ; ( ): . the researchers investigated the association of three dietary patterns with , plasma proteins and circulating metabolites in middle-aged adults. a crosssectional study using data from the framingham offspring study was developed using , participants. the framingham offspring study is a longitudinal study investigating coronary heart disease in families. participants were included in this study if in attendance of the examination cycle spanning to and having complete data needed. the sample was % male, with a mean age of years. proteomics profiling was conducted by way of blood samples analyzed using a somascan platform (somalogic inc), with which a total of , proteins were quantified using single-stranded dna-based aptamers. measurements of metabolites were taken with positively charged polar, negatively charged polar and lipid metabolites quantified using liquid chromatography with tandem mass spectrometry. dietary assessments used the harvard semiquantitative food frequency questionnaire. dietary patterns studied included the alternative healthy eating index, dietary approaches to stop hypertension diet score, and mediterranean-style, adherence to which was determined via the food frequency questionnaire. covariates included age, total caloric intake, current smoking status, physical activity index, use of lipid-lowering medication, use of antihypertensive medication, and bmi. statistical analysis was performed using sas version . (sas institute, ). the researchers report that, among other findings, proteins were associated with at least one dietary pattern: with alternative healthy eating index, with dietary approaches to stop hypertension, and eight with mediterranean-style. association between lifestyle factors, vitamin and garlic supplementation, and gastric cancer outcomes: a secondary analysis of a randomized clinical trial. guo y, li zx, zhang jy, et al. jama netw open. ; ( ):e . the researchers evaluated the effects of vitamin supplementation and garlic on gastric cancer (gc) incidence and mortality within subgroups based on lifestyle factors. a secondary analysis of a randomized controlled trial was performed to examine this issue using a sample of , participants. participants for the larger trial were recruited from shandong province in china between and . participants from that study were excluded from this analysis if they were lacking complete relevant data or had prior gc or other cancer diagnoses. the sample was . % female and had a mean age of . years. the primary study outcomes were gc incidence and mortality. the sample was randomized for . % to receive active vitamin supplementation, with . % receiving placebo. the intervention included regular supplementation of garlic extract and distilled garlic oil and a supplementation of vitamin c, vitamin e, and selenium between and . sociodemographic characteristics and anthropometric measurements were taken at baseline and followed up throughout the study. gastric cancer incidence was observed through scheduled gastroscopies, cancer registries, or autopsy reports, with scheduled endoscopies performed and lesions examined while progressing at regular checkups. lifestyle factors documented included smoking status, alcohol intake, and diet by way of a food frequency questionnaire. statistical analysis was performed using sas . (sas institute, ). the researcher report that not drinking alcohol was associated with a stronger beneficial effect of garlic supplementation on gc prevention. adherence to vitamin d intake guidelines in the united states. simon a, ahrens k, et al. pediatrics. ; ( ) :e . the researchers examined trends in meeting vitamin d guidelines among infants in the united states beginning in . data from a cross-sectional study were used to establish a sample of , participants for the study. the sample was drawn from the - national health and nutrition examination survey (nhanes) study. inclusion criteria for study were being an infant aged to months with dietary recall information and breastfeeding status available. the sample was . % male, . % non-hispanic white, . % non-hispanic black, . % hispanic, and . % non-hispanic other. in-person interviews were conducted in nhanes mobile examination centers, and dietary recalls were taken for the participants, with parents and guardians serving as proxies for the infants. researchers estimated the percentage of infants meeting the vitamin d guidelines on a given day, defined as either consuming at least unit of infant formula or receiving a supplement of at least iu vitamin d or both. analyses were conducted across all infants to months of age as well as stratified by breastfeeding status as defined by an infant consuming breast milk on either of the possible days of dietary recall. demographic variables taken included age, sex, race/ethnicity, participation in the supplemental nutrition assistance program, family income level, education of parents, and health insurance status. all analyses were conducted in sas version . (sas institute, ). the researchers estimate that . % of us infants in - met vitamin d intake guidelines, with non-breastfeeding infants more likely to meet them than their peers. association of bariatric surgery with risk of fracture in patients with severe obesity. khalid s, omotosho p, spagnoli a, et al. jama netw open. ; ( ):e . the authors explored the absolute risk of fracture in patients with severe obesity who underwent various forms of bariatric surgery. a retrospective multicenter cohort study using a sample of , participants was designed to assess this issue. the sample was pulled from the longitudinal medicare standard analytic files containing records billed to medicare between january and december . patients who were billed at time during their available history for cancer transplant, end-stage kidney disease, prior gastric operations, gastric banding procedures, or fractures before the bariatric surgery were excluded. the sample was . % female, with % at or under years of age. the primary outcome measured was the odds of fracture based on exposure to bariatric surgery over a -year period. comorbidities considered included hypertension, smoking status, nonalcoholic fatty liver disease, hyperlipidemia, type diabetes, osteoporosis, osteoarthritis, postmenopausal status, and obstructive sleep apnea. of the , participants, . % did not undergo weight loss surgery, . % underwent roux-en-y gastric bypass, and . % sleeve gastrectomy. statistical analysis was performed using r statistical software version . (the r foundation, ). the authors report that bariatric surgery was associated with a reduced risk of fracture in bariatric surgeryeeligible patients. the assessment of eating pleasure among older adults: development and preliminary validation of the anticipatory and consummatory eating pleasure (aceps). bailly n, wymelbeke v, maitre l, sulmont-rosse c. j nutr health & aging. ; https:// doi.org/ . /s - - - . effect of sit-to-stand exercises combined with protein-rich oral supplementation in older persons: the older person's exercise and nutrition study. protein requirements in critical illness: do we really know why to give so much? leyderman i, yaroshetskiy a, klek s. j parenter enteral nutr. ; https://doi.org/ . / jpen. . clinical nutrition evaluating the effects of dietary interventions on disease progression and symptoms of adults with multiple sclerosis: an umbrella review long-term dietary intervention reveals resilience of the gut microbiota despite changes in diet and weight challenges and outcomes for bariatric surgery in patients with paraplegia: case series and systematic review nutrients and porphyria: an intriguing crosstalk salicylate toxicity a checklist to assess adequacy of vitamin d intake assessment of calcium and vitamin d intake in an outpatient gastroenterology and hepatology clinic tutorial: nutrition therapy in eosinophilic esophagitis-outcomes and deficiencies systematic review with meta-analysis of patient-centered outcomes, comparing international guidelinesrecommended enteral protein delivery with usual care oncology consumption of fish and u- fatty acids and cancer risk: an umbrella review of meta-analyses of observational studies dietary supplement use among adult cancer survivors in the united states pediatric modified low-protein infant formula supports adequate growth in healthy assessment of evidence about common infant symptoms and cow's milk allergy current practices using pediatric malnutrition indicators: a survey of dietitians working in pediatrics health supervision for people with achondroplasia ):e . eating vegetables first at start of meal and food intake among preschool children in japan morality from covid- increases with unsaturated fat, and may be reduced by early calcium and albumin supplementation trajectories of picky eating in lowincome us children ethical framework for nutrition support resource allocation during shortages: lessons from covid- inadequacy of immune health nutrients: intakes in us adults ethical framework for nutrition support resource allocation during shortages covid- : a personalized cardiometabolic approach for reducing complications and costs. the role of aging beyond topics trends in the prevalence of metabolic syndrome in the united states management of hyperphosphatemia in end-stage renal disease: a new paradigm revising dietary phosphorus advice in chronic kidney disease the role of protein intake and its timing on body composition and muscle function in healthy adults: a systematic review and meta-analysis of randomized controlled trial weight management impact of covid- stay-at-home orders on weight-related behaviors among patients with obesity spices in a high-saturated-fat, highcarbohydrate meal reduce postprandial proinflammatory cytokine secretion in men with overweight or obesity: a -period, crossover, randomized controlled trial mindful eating and active living: development and implementation of a multidisciplinary pediatric weight management intervention associations of bariatric interventions with micronutrient and endocrine disturbances global lifestyle medicine: for people who need it the most but have it the least association between healthy eating patterns and risk of cardiovascular disease a telehealth lifestyle intervention to reduce excess gestational weight gain in pregnant women with overweight or obesity (glow): a randomized, parallel-group continuous glucose monitoring in pregnancy: importance of analyzing temporal profiles to understand clinical outcomes key: cord- -neqj xf authors: lojkić, ivana; biđin, marina; prpić, jelena; Šimić, ivana; krešić, nina; bedeković, tomislav title: faecal virome of red foxes from peri-urban areas date: - - journal: comp immunol microbiol infect dis doi: . /j.cimid. . . sha: doc_id: cord_uid: neqj xf red foxes (vulpes vulpes) are the most abundant carnivore species in the northern hemisphere. since their populations are well established in peri-urban and urban areas, they represent a potential reservoir of viruses that transmit from wildlife to humans or domestic animals. in this study, we evaluated the faecal virome of juvenile and adult foxes from peri-urban areas in central croatia. the dominating mammalian viruses were fox picobirnavirus and parvovirus. the highest number of viral reads (n = ) was attributed to a new fox circovirus and complete viral genome was de novo assembled from the high-throughput sequencing data. fox circovirus is highly similar to dog circoviruses identified in diseased dogs in usa and italy, and to a recently discovered circovirus of foxes with neurologic disease from the united kingdom. our fox picobirnavirus was more closely related to the porcine and human picobirnaviruses than to known fox picobirnaviruses. red foxes (vulpes vulpes) are the most abundant and widespread carnivore species in the northern hemisphere. populations of the red fox are well established in peri-urban and urban areas, so they represent a potential reservoir of viruses that transmit from wildlife to humans or domestic animals. foxes and dogs (canis lupus familiaris) often share the same viral pathogens such as canine parvovirus , canine enteric coronavirus, rotavirus and canine distemper [ ] . the most important is rabies, which was endemic in croatia since , but today almost eradicated thanks to the ongoing vaccination campaigns using oral rabies vaccine to target the red fox population in croatia [ ] . fox carcasses, collected regularly to access the effectiveness or vaccination, can also be used for various laboratory investigations. today, high-throughput sequencing followed by viral metagenomic analysis has proved to be powerful tool for exploring and analysing new and existing viruses from variety of human and animal sample types and faeces are commonly studied. in the present study we evaluated the faecal virome of asymptomatic juvenile and adult red foxes from peri-urban areas in central croatia using random cdna synthesis followed by high-throughput illumina sequencing. considering the fact that composition of the faecal virome of foxes has been studied recently [ ] , our results showed different viral profile. we detected a new fox circovirus and other viruses including fox parvovirus and fox picobirnavirus but not the astroviruses and hepevirus. twenty four fox carcasses were collected during a regular fox shooting period associated with the nationwide oral rabies vaccination (orv) of foxes approved by croatian ministry of agriculture. all foxes were collected in central croatia in two counties: zagrebačka and bjelovarsko-bilogorska (peri-urban area). fox jaws were subjected to age and tetracycline determination, the key techniques in the evaluation of oral vaccination effectiveness [ ] and according to result classified as adult or juvenile. carcasses were frozen at − • c for week and after defrosting, faecal materials were sampled from the rectum of the foxes and frozen at − • c until processing. samples from juvenile foxes were pooled into four samples, two samples per county. the same criterion was applied to the samples from adult foxes. a % (wt/vol) mixture of faeces in phosphate-buffered saline (pbs) was prepared and centrifuged. the supernatants were then http://dx.doi.org/ . /j.cimid. . . - /© elsevier ltd. all rights reserved. filtered using . m filters (millipore, usa) to remove remaining cell fragments and bacteria. the resulting filtrates were subsequently subjected to nuclease treatment with u of dnase i (new england biolabs, uk) at • c for h. the resulting virion-enriched samples were used for simultaneous viral rna and dna automatic extraction using iprep viral kit and iprep instrument (invitrogen, usa). ribosomal rna was depleted from the genomic dna-depleted samples using l of rna, l of reaction buffer a, . l riboguard rnase inhibitor ( - u/l, epicentre biotechnologies, usa) and l ( u/l)terminator tm -phosphate-dependent exonuclease (epicentre). the mixture was incubated at • c for min. thereafter the samples were subjected to a subsequent round of purification using rna clean xp (beckman coulter, usa) magnetic beads and then used as template for double-stranded cdna synthesis using random hexamers, random primer fr rv-n ( gcc gga gct ctg cag ata tcn nnn nn ) at a concentration of . m and fr rv-t primer ( gcc gga gct ctg cag ata tc (t) ) at . m [ ] with cdna synthesis system kit (roche diagnostic gmbh) according to the manufacturer's instructions. the resulting dsdna was quantified using qubit fluorimeter (life technologies, usa), and diluted to a final concentration at . ng/ul ( ng total of each sample). sequencing libraries were then prepared using nextera xt sample preparation and nextera index kits (illumina, usa) using ul of diluted dsdna according to manufacturer's instructions and then sequenced using the miseq nano cycles kit on miseq platform (illumina). an option for automatic trimming for quality and primer was used. the resulting fastq files for each paired read were subjected to de novo contig assembly (cap ), with criteria of % minimum overlap identity and a minimum overlap length of nucleotides; contigs < bp in length were not analysed further. both, reads and contigs were compared to the genbank non-redundant protein database using blastx with an e-value cut-off of - and search was filtered to be restricted to the sequences in the database that correspond to subset viruses (taxid: ). the blast output was used to create a taxonomic classification of the reads and contigs with megan . . . [ ] . the reference sequences were downloaded from the ncbi (http://www.ncbi.nlm.nih.gov/) for further assemblies using geneious . . . the raw sequence data have been submitted to the sequence read archive (sra) at genbank with sra accession number srp . the nucleotide sequences obtained from our study were deposited in genbank with accession numbers kp -kp . recombination analysis of multiple sequence alignments was conducted with rat [ ] . jmodeltest v. . . . [ ] was used to estimate best-fit model by hierarchical likelihood ratio tests (hlrts) and approximate akaike information criterion (aic). global alignments, neighbour-joining (nj) and maximum likelihood (ml) phylogenetic analyses were generated using mega [ ] . reliabilities of phylogenetic relationships were evaluated using nonparametric bootstrap analysis with replicates for nj and ml analysis. bayesian inference (bi) analysis [ ] was performed with mrbayes v . b . [ ] . the genbank accession numbers of the viral sequences used in the phylogenetic analyses are shown on tree figures. in this study, pooled fox faecal samples of juvenile (marked as , , , ) and adult ( , , , ) animals were used for metagenomic analysis by random cdna synthesis followed by high-throughput sequencing. from the total of , obtained reads, , reads showed significant sequence identities to known viruses. among the viral contigs, . % were bacterial viruses and . % were not assigned to any virus family. the pie charts of assigned viral reads obtained by megan for all samples are shown in fig. . the ratio between assigned eukaryotic virus sequences vs. bacteriophage sequences were . %: . % in the adult foxes and . %: . % in juvenile. the dominating mammalian virus in adult foxes was picobirnavirus. fox circovirus. circovirus sequences were found in three samples, two from juvenile foxes with relatively low number of reads ( and ), respectively. from sample (adult foxes; fig. , table ) a single de novo assembled genome-sized contig was generated from even specific reads with mean coverage = (genbank accession no. kp ). phylogenetic analysis of complete nucleotide (nt) sequence ( fig. ) showed that our sequence clustered with recently identified canine circoviruses ( , h , bari/ , ucd - , ucd - ) and newly described circovirus from fox sera (vs ). the genetic relatedness with circoviruses sequences of other species was low (< % nt identity with porcine circoviruses). our fox circovirus genome comprises nt with a gc content of . %. the genome contains two putative open reading frames (orfs), on complementary strands in opposite orientation that encode the viral replicase (rep) ( amino acids (aa)) and capsid (cap) protein ( aa). it has two intergenic noncoding regions that are and nt long. in the rep gene, the identity to canine circoviruses was generally high with the highest identity to strain ucd and fox isolate vs ( % aa) and the lowest to the bari/ - ( % aa). in contrast to rep, in the cap protein the highest identity was observed to fox isolate vs ( %), dog strain ha ( . %), while identity to strain ucd was lower ( . %). there is no proof for recombination event. fox picobirnavirus. we detected sequences with homology to picobirnaviruses in out of samples (fig. ). all pooled samples of adult foxes had relatively high number of picobirnaviral sequence reads (n = ); there were only two juvenile samples that had picobirnaviral reads (n = ) ( table ). sample (adult foxes) had contigs matching members of picobirnaviridae assembled from reads (max coverage = ×), so the whole-size rna-dependent rnapolymerase (rdrp) sequence has been generated mapping to picobirnavirus consensus sequence. the complete coding sequence of picobirnaviral rdrp gene from the sample (genbank accession no. kp ) consisted of nt ( aa) and was longer than recently described fox picobirnavirus f - (genbank accession no. kf ; nt). phylogenetic analysis of our picobirnavirus nt sequence (fig. ) with other rdrp gene sequences of picobirnaviruses of similar length showed that the obtained sequence of the rdrp gene was more closely related to the porcine picobirnavirus than to fox picobirnavirus. aminoacid sequence alignment and comparison revealed the greatest similarity to porcine picobirnavirus kf ( %), human ab ( . %) and to fox kf ( %). fox parvovirus. parvovirus were present in all four adult and one juvenile sample (fig. , table ) with the highest number of reads (n = ) and contigs (n = ) in sample (adult foxes). the well covered regions of fox parvovirus, including a partial ns ( -bp; kp ) and a partial vp ( -bp; kp ) were acquired and showed an nt identity of . and . %, respectively, to fox parvovirus (kc ). other viruses. sixty-two adenoviral reads assembled to contigs with the largest contig having nt were present in a sample from an adult fox- (fig. , table ) with - % similarity to simian adenoviruses. eighty-two reads - % identical to ias virus were found among adult foxes and only three reads in juvenile. the largest number of ias contigs (n = ) and the longest contig of nt were present in sample (fig. , table ). there were several short contigs grouped with ss-rna viruses that were most similar to ngari virus sequence. a total of reads similar to rodent stool-associated circular virus were present in three pooled samples of adult foxes (fig. , table ). among contigs that were grouped with ss + rna the largest number showed identity with tombusviridae-plant viruses. all samples contained at least one plant virus and most contained sequences from several plant viruses. retroviral contigs (n = ) were present in all sequences and were - % identical to avian leucosis virus (avl), which resulted from contamination of the reagents for reverse transcription. bacteriophages. among the viral reads and contigs, - % were bacterial viruses and those contigs were the largest ones. the most abundant bacteriophages detected were escherichia phage phapec , k , crassphage and enterobacterial phages. sufficient reads were present in sample to assembly escherichia phage phapec genome-sized contig although a proper analysis of this genome is beyond the scope of this study. this study describes the composition of the viral communities in the faeces of two groups of red foxes from peri-urban areas in central croatia and with no clinical signs of disease. from our metagenomic study, the full-length fox circovirus genomic sequence was de novo assembled from the clean reads into a single contig. two other viral genomes (picobirnavirus and parvovirus) were partially assembled from the reads and mapped to the reference genomes. based on the number of viral reads, circovirus was the most represented virus in juvenile and picobirnavirus in adult foxes. juvenile foxes showed the smaller overall number of eukaryotic virus reads in comparison to bacteriophage reads while pooled samples from adults had greater number and diversity of eukaryotic viruses. it might be the case that the adult foxes have been exposed to a variety of viruses and other microorganisms over their lifetime, and therefore have developed specific intestinal viral flora in comparison to juvenile ones with obviously dominant normal intestinal bacterial flora. compared to results of bodewes et al. [ ] other prevalent fox faecal viruses (fox hepevirus and fox astroviruses) were not discovered. preparation of samples from frozen carcasses cannot be the cause since it was identical as in study of bodewes et al. [ ] . to corroborate this fact, we also detected + ss-rna viruses but they were mainly attributed to plant viruses. one of the reasons might be the difference in the library preparation and the other one simply the geographical origin. circoviruses are confirmed pathogens worldwide associated with a wide range of illnesses in birds [ ] , pigs [ ] and recently dogs [ ] [ ] [ ] . recently, there were many new circoviruses identified in animal and human faeces and environmental samples but their natural hosts are mainly unidentified [ , ] . circovirus sequences were found in three samples but the highest number of reads was detected in a sample from adult foxes. from that sample ( ) a single de novo assembled genome-sized contig was generated from even specific reads. some previous results show that full-length viral genomic sequences cannot be assembled from the clean reads due to the low quantity of the virus within samples, the short reads, and the lack of known reference sequences [ , ] . phylogenetic analysis of circoviral nt sequences (fig. ) and sequence analysis of the rep and cap protein showed that our sequence clustered with recently identified canine circoviruses [ ] [ ] [ ] and newly described circoviruses from sera of foxes with neurologic disease [ ] . our results demonstrate that new canine circovirus that has been proved to be pathogenic for foxes [ ] and dogs [ ] [ ] [ ] can be found in asymptomatic foxes too. forty-seven per cent of all viral reads in the sample discussed here were assigned with canine circovirus. such a high number of viral reads obtained by viral metagenomic approach using random primers for cdna synthesis could be attributed to unknown subclinical or clinical disease. the first available data on clinicopathological signs and prevalence of circoviruses in foxes published recently suggested that the prevalence of circoviruses in foxes is relatively high [ ] . future studies should investigate whether a new circovirus is main pathogen or act in co-infection with an unknown agent. we did not detected other prevalent canine viruses such as canine parvovirus , canine enteric coronavirus, canine rotavirus and canine distemper which is reported in both domestic and wild carnivores in many european countries [ ] . we detected sequences with homology to picobirnaviruses in out of pooled samples in this study. it is a similar or slightly higher prevalence than that identified in other host species [ ] [ ] [ ] [ ] but we expected such result since picobirnaviruses have been normally detected in faecal samples of human and various animals with and without clinical disease [ ] . from the same sample where complete new circovirus was assembled (sample ) the complete rdrp sequence was generated by mapping to reference genomes. phylogenetic analysis of our picobirnavirus nt (fig. ) and aa (not shown) rdrp gene sequence showed that obtained sequence was more closely related to the porcine and human picobirnaviruses than to recently described fox picobirnavirus [ ] . the same group of authors identified that picobirnaviruses from different foxes were not identical. the divergence of picobirnaviruses within species has been proved previously in humans [ ] , pigs [ ] and dromedary camels [ ] . parvoviruses cause a variety of mild to severe symptoms in mammalian and avian hosts [ ] . members of the parvoviridae subfamily have recently undergone a large expansion in the number of know genera and species [ , ] . in the analyzed faecal samples of foxes, sequences were detected with homology to recently reported novel parvovirus of red fox [ ] . the newly discovered and other detected viruses in the present study indicate potential exchange of viruses among foxes, domestic animals and possible humans. our viral metagenomic study identified a circovirus that was close relative to dog virus and a picobirnavirus that was closely related to porcine and human picobirnaviruses, suggesting that these viruses may have potential to be transmitted among species. rabies and canine distemper virus epidemics in the red fox population of northern italy diversity of currently circulating rabies virus strains in croatia identification of multiple novel viruses, including a parvovirus and a hepevirus, in faeces of red foxes detection of the tetracycline in red fox teeth and age determination, eurl for rabies viral genome sequencing by random priming methods integrative analysis of environmental sequences using megan recombination analysis tool (rat): a program for the high-throughput detection of recombination jmodeltest: phylogenetic model averaging mega : molecular evolutionary genetics analysis version . markov chain monte carlo algorithms for the bayesian analysis of phylogenetic trees mrbayes: bayesian inference of phylogeny quantification of pigeon circovirus in serum, blood, semen and different tissues of naturally infected pigeons using a real-time polymerase chain reaction molecular biology of porcine circovirus: analyses of gene expression and viral replication complete genome sequence of the first canine circovirus circovirus in tissues of dogs with vasculitis and hemorrhage genomic characterization of a circovirus associated with fatal hemorrhagic enteritis in dog rapidly expanding genetic diversity and host range of the circoviridae viral family and other rep encoding small circular ssdna genomes multiple diverse circoviruses infect farm animals and are commonly found in human and chimpanzee faeces metagenomic analysis of the viromes of three north american bat species: viral diversity among different bat species that share a common habitat metagenomic analysis of viruses from bat fecal samples reveals many novel viruses in insectivorous bats in china detection of circovirus in foxes with meningoencephalitis metagenomic analysis of the viral flora of pine marten and european badger faeces the fecal viral flora of california sea lions human picobirnaviruses identified by molecular screening of diarrhea samples the fecal virome of pigs on a high-density farm genogroup i picobirnaviruses in pigs: evidence for genetic diversity and relatedness to human strains divergent picobirnaviruses in human faeces genome sequencing identifies genetic and antigenic divergence of porcine picobirnaviruses metagenomic analysis of viromes of dromedary camel fecal samples reveals large number and high diversity of circoviruses and picobirnaviruses fields virology acute diarrhea in west african children: diverse enteric viruses and a novel parvovirus genus two novel parvoviruses in frugivorous new and old world bats this research was supported by grant no. - - from the ministry of science, education and sports, republic of croatia. the authors declare that they have no conflict of interest. key: cord- -vm d mj authors: bradt, david a.; drummond, christina m. title: technical annexes date: - - journal: reference manual for humanitarian health professionals doi: . / - - - - _ sha: doc_id: cord_uid: vm d mj this chapter provides guidance on technical issues in the health sector. the annexes contain selective compilations of frequently used reference information. material ( ) financial f. identification of deliverables and timetables g. consolidated reporting and dissemination ( ) group terms of reference ( ) meeting minutes ( ) epidemiology updates ( ) health sitreps ( ) component analysis ( ) field documentation (toolkit) for new arrivals . capacity building of host authorities . civil society partnership and support a. organize community leaders b. encourage gender mainstreaming c. encourage privatization d. discourage entitlements . advocacy . transition to early recovery d. strategy for livelihood/economic relief . restore productive assets (supply side interventions) a. in-kind donations (e.g. food, seeds, tools, fishing nets, etc.) b. types of community projects in food-for-assets programs ( ) natural resources development (a) water harvesting (b) soil conservation ( ) restoration of agri(aqua)culture potential (a) irrigation systems (b) seed systems ( ) infrastructure rehabilitation (a) schools (b) market places (c) community granaries (d) warehouses (e) roads (f) bridges ( ) diversification of livelihoods (a) training and experience sharing . increase individual purchasing power a. cash distribution b. cash for work (cash for assets) c. vouchers d. micro-credit e. job fairs f. artisanal production g. livelihoods/income generation . support market resumption a. market rehabilitation b. infrastructure rehabilitation c. micro-finance institutions e. goals-protect what's left ( month), restore the system ( months), improve the system ( ( ) non-food items c. financial assistance ( ) cash grants ( ) cash for work ( ) microfinance (loans) ( ) livelihood/income generation . ensure responsible resource management a. human resources management ( ) incident management command and control ( ) team structure and function ( ) staff selection (a) internationals (b) homologues ( ) field activities (a) briefing (b) meetings and reports (c) debriefing ( ) operations support (a) comms (b) transport (c) office (d) food and lodging ( ) personal health maintenance and morale b. material resources management c. financial resources management d. supervision e. monitoring and evaluation . scale up coverage of priority health interventions . address bottlenecks of the disrupted health system (otherwise temporary solutions become permanent) . protect essential public health infrastructures . build capacity of local authorities with focus on sustainable systems a. technical oversight-hiring of local experts b. material assistance-production of key commodities c. financial assistance . provide incentives for host government . support host country non-beneficiary population . find new partners in the development community . use health sustainable development goals as targets for recovery activities . seek opportunities and develop mechanisms for transition and phase out f. . programmatic constraints a. staff ( ) western trained ( ) hospital-based ( ) resource intensive ( ) technology dependent ( ) procedurally oriented ( ) invasive ( ) monolingual ( ) hazard naïve b. supervision ( ) limited responsibility ( ) limited authority ( ) limited accountability c. projects ( ) acute ( ) curative ( ) short-term ( ) intermittent d. systems ( ) inadequate security ( ) weak rule of law ( ) limited accountability framework ( ) uncoordinated . international cooperation to protect lives and health . timely and sustained high-level political leadership to the disease . transparency in reporting of cases of disease in humans and in animals caused by strains that have pandemic potential to increase understanding, enhance preparedness, and ensure rapid and timely response to potential outbreaks . immediate sharing of epidemiological data and clinical samples with the world health organization (who) and the international community to characterize the nature and evolution of any outbreaks as quickly as possible . prevention and containment of an incipient epidemic through capacity building and in-country collaboration with international partners . rapid response to the first signs of accelerated disease transmission . work in a manner supportive of key multilateral organizations (who, fao, oie) . timely coordination of bilateral and multilateral resource allocations; dedication of domestic resources (human and financial); improvements in public awareness; and development of economic and trade contingency plans . increased coordination and harmonization of preparedness, prevention, response and containment activities among nations . actions based on the best available science d. program innovations at community level . genocide (article )-acts committed with intent to destroy, in whole or in part, a national, ethnic, racial, or religious group a. killing members of the group b. causing serious bodily or mental harm to members of the group c. inflicting on the group conditions of life calculated to bring about its physical destruction in whole or in part d. imposing measures intended to prevent births within the group e. forcibly transferring children of the group to another group . crimes against humanity (article )-acts committed as part of a widespread or systematic attack against any civilian population, with knowledge of the attack a. murder b. extermination c. enslavement d. deportation e. imprisonment in violation of international law f. torture g. rape, sexual slavery, enforced prostitution, forced pregnancy, enforced sterilization, or other comparable form of sexual violence h. persecution on political, racial, national, ethnic, cultural, religious, gender, or other grounds universally recognized as impermissible under international law i. enforced disappearance j. apartheid k. other inhumane acts intentionally causing great suffering or serious injury to body or to mental or physical health . war crimes (article ) a. grave breaches of the geneva conventions of aug ( ) willful killing ( ) torture or inhumane treatment including biological experiments ( ) willfully causing great suffering ( ) extensive destruction and appropriation of property ( ) compelling a pow to serve in the armed forces of a hostile power ( ) willfully depriving a pow of the right to a fair trial ( ) unlawful deportation ( ) taking of hostages b. serious violations of laws and customs applicable in international armed conflict ( ) intentionally directing attacks against the civilian population or against civilians not taking direct part in hostilities ( ) intentionally directing attacks against civilian objects ( ) intentionally directing attacks against personnel, installations, material, units, or vehicles involved in humanitarian assistance or peacekeeping mission ( ) intentionally launching an attack in the knowledge that it will cause incidental civilian loss of life or severe damage to the natural environment ( ) attacking undefended towns, villages, dwellings, or buildings which are not military targets ( ) killing or wounding a combatant who has surrendered ( ) improper use of a flag of truce, flag or insignia or uniform of the enemy or of the un, or emblems of the geneva conventions resulting in death or serious personal injury ( ) transfer by the occupying power of parts of its own civilian population into the territory it occupies, or the deportation or transfer of all or parts of the population of the occupied territory within or outside the territory ( ) intentionally directing attacks against buildings dedicated to religion, education, art, science, charitable purposes, historic monuments, hospitals, and places where sick are collected, provided they are not military objectives ( ) subjecting persons to physical mutilation or to medical or scientific experiments which are not justified by the medical treatment nor carried out in his/her interest ( ) killing or wounding treacherously individuals belonging to the hostile nation or army ( ) declaring that no quarter will be given ( ) destroying or seizing the enemy's property unless such be imperatively demanded by the necessities of war ( ) declaring abolished, suspended, or inadmissible in a court of law the rights and actions of the nationals of the hostile party ( ) compelling the nationals of the hostile party to take part in the operations of war directed against their own country ( ) pillaging a town or place, even when taken by assault ( ) employing poison or poison weapons ( ) employing asphyxiating, poisonous or other gases, and all analogous liquids, materials, or devices ( ) employing bullets which expand or flatten easily in the human body ( ) employing weapons, projectiles, material and methods of warfare which cause superfluous injury or unnecessary suffering ( ) committing outrages upon personal dignity, in particular humiliating and degrading treatment ( ) committing rape, sexual slavery, enforced prostitution, forced pregnancy, enforced sterilization, or other comparable form of sexual violence ( ) utilizing a civilian or other protected person to render certain areas or military forces immune from military operations ( ) intentionally directing attacks against buildings, material, medical units, transport, and personnel using the emblems of the geneva conventions in conformity with international law ( ) conscripting or enlisting children under the age of years c. serious violations of common article applicable in non-international armed conflict, i.e. acts vs. persons taking no active part in the hostilities, including armed forces placed hors de combat by sickness, wounds, detention, or other cause ( ) violence to life and person ( ) outrages upon personal dignity ( ) taking of hostages ( ) passing of sentences and carrying out of executions d. non-applicability of c (above) to internal disturbances (riots, sporadic violence, etc.) e. other serious violations of laws and customs applicable in non-international armed conflict c. degradation of health system a. ppm = mg/kg (solids) = mg/l (liquids) = μg/ml (liquids) = basic unit of measure for chloroscopes :. , ppm = % a range of generic prevention measures should be considered for its impact on diseases in a biological "all-hazards" environment. overall, excreta disposal, water quantity, personal hygiene, and food hygiene commonly contribute more to environmental health than do other listed measures. epidemic threats will oblige heightened consideration of disease-specific strategies for prevention and control. note: in u , length is the preferred term over height . . this last includes kwashiorkor and marasmatic kwashiorkor in the wellcome classification. • sam = severe wasting cases or bilateral pitting edema cases (where due to malnutrition) • sam = whz < − , muac < . cm, or bilateral pitting edema (who). whm not in definition. • sam prevalence worldwide ≈ , , . • sam mortality ≈ × mortality of normally nourished child and its cfr can be - %. • gam = mam + sam • gam = moderate wasting cases, severe wasting cases, or bilateral pitting edema cases (where due to malnutrition) underweight • underweight is not used for screening or surveys in nutritional emergencies. it reflects past (chronic) and present (acute) undernutrition and is unable to distinguish between them. it encompasses children who are wasted and/or stunted. however, weight gain over time can be a sensitive indicator of growth faltering which is easily tracked on road to health charts. • stunting generally occurs before age . it is irreversible. • stunting prevalence worldwide ≈ , , . • stunting is not a good predictor of mortality, but the cfr from ids in cases of severe stunting ≈ × the cfr from ids in cases without stunting. reference standards can be absolute muac, centile, % of median reference, or z scores: • muac • easy to understand. an excellent predictor of mortality. permits comparisons between age groups insofar as the low growth velocity of muac in the u age group makes data roughly comparable. may be used alone in "quick-and-dirty" convenience samples to estimate local prevalence of wasting. however, not used alone in authoritative anthropometric surveys, and is commonly part of a two stage screening process to determine eligibility for feeding programs. easy to understand. permits comparisons between age groups and outliers. however, data are not convenient to convert. e.g. z - . = . nd percentile • % of median of reference population whm is the preferred indicator to determine eligibility for feeding programs (sphere). calculations are easy and are used in the who road to health charts. however, median reference data are not comparable between ages. eg % wt-for-age = severe malnutrition in infants = moderate malnutrition in school age kids moreover, median reference data are not comparable between indicators. eg % wt-for-age = severe malnutrition in infants % wt-for-ht = death • z scores preferred indicator (sphere, who) for reporting anthropometry survey results because it permits comparisons between age groups and nutritional indices. however, data may be difficult to understand. eg z score wt-for-age for y/o: . . kg kg -= − sd below median for his age overall: whz gives higher prevalence of malnutrition than whm for the same population. this is most marked where there is low baseline prevalence of disease, and especially for adolescents (who get subsequently over-referred). whz is more statistically valid, but whm is better predictor of mortality and is used for admission to tfcs. weight-for-age is influenced by weight-for-height and height-for-age. it can be difficult to interpret. blanket-all hh in geographically targeted catchment area (e.g. where ipc + and gam > % or - % with aggravating factors) targeted-some hh in catchment area (e.g. where gam - % or - % with aggravating factors); u and pregnant or lactating women vs. u alone vs. u alone depending on resources available and challenges with case finding) overall programmatic target- % coverage for sam in rural areas (sphere); % coverage for mam in rural areas admission criteria: pedes: age - mo, muac < . cm, with appetite, discharged from otp, no severe medical complications pregnant & lactating: muac < . cm, and nd- rd trimester or with infant < mo treatment: rusf as dry rations e.g. plumpy'sup ® , csb, csb + (supercereal), csb ++ (supercereal +) nb csb may also be cooked on-site as in emergency school feeding. discharge criteria (pedes): weight gain, muac > . cm, time in program > months community outreach with mobile brigades admission criteria for u : sam (whz < − , muac < . cm, or bilateral pitting edema) discharge criteria for u : whz >− . , no edema, and clinically well (generally takes - weeks) treatment protocol (who, icddr) shock severe dehydration: rl + d , ½ strength darrow's + d , or ½ ns + d dose: cc/kg iv death (~ % sam will die with good care, and % will die with mediocre care) c. measures of association quantify the strength or magnitude of the association between the exposure and the health problem of interest. they are independent of the size of the study and may be thought of as best guess of the true degree of association in the source population. however, they give no indication of the association's reliability. • cohort study-relative risk (rr) = riskexposed/riskunexposed • in acute outbreaks, risk is represented by the attack rate (ar) • case-control study-odds ratio (or) • no denominator with which to calculate an attack rate • cross-sectional-prevalence ratio or prevalence odds ratio c. survey designs (see r magnani [ ] , and f checchi [ ] ) . census-complete enumeration of the entire population . sample a. probability sampling ( ) simple random sampling (srs) it requires a complete enumeration of population n-names and locations of all persons or households (hh)-and sample size n nb much effort is necessary to conform to requirements of random sampling. it is easier to sample less often but take more specimens as a cluster. unfortunately, it is recognized that individuals from a cluster often share characteristics which < the precision of the method. ( ) systematic random sampling it requires a complete enumeration of population n, and sample size n, to calculate the skip interval k = n/n. ( ) stratified random sampling it requires a population size n divided into groups or strata l, then srs within each stratum. the method ensures over-sampling in under-represented groups. it yields separate estimates for each stratum at less cost. however, it requires extra info and has complicated analysis. ( ) cluster sampling, cluster sample survey (css) it is used when you don't have a complete enumeration n of all people in the area, and thus can't do random sampling; or when the area is too big to cover, and thus can't do systematic random sampling. • what should be done to compensate for the bias induced when one samples clusters rather than individuals? use n. empiric data on association within clusters in smallpox immunization suggests doubling n. if n = , n = . • what is the minimum number of clusters that can be selected and still fulfill requirements of the theory on which binomial sampling is based? . statistical theory demonstrates that > clusters help ensure cluster means have a normal distribution. the larger the number of clusters, the smaller the design effect (i.e. study efficiency improves, and the total number of study subjects needed will decrease). e.g. × (n = ) will prove more accurate and efficient than × (n = ). clusters × households will be more precise, but clusters × households may be more logistically feasible. choice of cluster should be driven by what one team can complete in a day. × css leaves . min/hh/team, but × css leaves min/hh/team. if a team can only measure kids/day (which is common), then it's best to increase the number of smaller clusters. • to permit an equal number of children to be selected from each of clusters, children would not achieve the necessary n. therefore, children are selected per cluster ( × = ). b. non-probability sampling ( ) convenience ( ) purposeful/judgment (most affected area, hhs, etc.) ( ) quota bias (see r magnani [ ] , f checchi [ ] , and smart [ ] ) systematic, non-sampling error which lowers accuracy of findings. it is usually not appreciated by the survey team. it is usually not apparent from the survey results. it cannot be arithmetically calculated or corrected. its extent cannot be judged by readers of the report. methods and materials must be explicit. report authors must discuss possible sources of bias as limitations to their study. accuracy depends on validity of findings. it is more important than precision (section e), and bias should be prevented at all costs. awareness of sources of bias is the first step in minimizing its impact on any study. as sample size increases, it is more difficult to control quality. more teams to train and supervise create higher risk of bias. it is better to have smaller sample size with less attendant precision but much less risk of bias. . selection bias-respondents are not representative of the population a. project bias-assessors work where a project may be conceptually familiar to them b. spatial/access bias-assessors work where access is easiest (roadside or "windshield" bias) c. refusal or non-response bias (self-selection) bias-subject nonparticipation may undermine representativeness of the sample d. survivor bias-assessments are conducted where households have disappeared due to family death or migration. mortality rate is thereby underestimated. this bias is most likely where hh size is low, recall period is long, mortality is high, and clustering is present. e. class/ethnic bias-different social classes or ethnic groups are inadequately included if not excluded from the assessment. local assessors may have ethnic bias, or the key informants may be drawn from one particular social class or ethnic group. f. season bias-assessments are conducted during harvest season or periods of weather when segments of the population may be under-represented g. time of day/schedule bias-assessments are conducted at a time of day when segments of the population may be under-represented nb items - below may also be grouped as information/measurement bias. . interview bias a. interviewer bias ( ) cultural bias-assessors cultural norms lead to incorrect assumptions about the interview subjects ( ) mandate or specialty bias-assessors mandate or specialty blinds them to needs outside of that mandate or specialty. e.g., a shelter specialist may only assess shelter needs while neglecting livelihood or nutrition needs. gender bias-assessors interview only one gender ( ) language bias-assessors may have a limited spectrum of people with whom they can communicate ( ) key informant bias-assessors may be partial to key informants who appear credible in ways meaningful to the assessors ( ) information/political bias-assessors focus on information that confirms preconceived notions rather than pursue evidence of alternate beliefs ( ) mistranslation ( ) interviewer error-assessors write down answers incorrectly b. subject (response) bias ( ) event recall bias-retrospective surveys only, esp. with recall periods > yr (a) informants underreport remote events (e.g. neonatal deaths) (b) calendar bias-informants over report events within the recall period ( ) event reporting bias (a) taboos-informants underreport taboo subjects (e.g. neonatal deaths) (b) lies-informants misinterpret surveys as registration activities and over report family members or underreport deaths to maintain assistance (c) political bias-informants present information that conforms to their political agenda ( ) age heaping/digit preference-informants exhibit digit preference . instrument/measurement bias-errors in design or use of instrument (e.g. questionnaire, lab equipment, etc.) a. random errors in measurement random errors in weight measurement, even if yielding equal numbers of high and low measurements, widen the distribution curve without altering the mean. hence, the prevalence of malnutrition is overestimated. the effect is greater for severe malnutrition than for moderate malnutrition, and greater when prevalence is low than when it is high. the data distribution should be checked for normal distribution with an sd between . and . z scores. improving the data quality thus appears to reduce the prevalence of malnutrition. b. systematic errors in measurement systematic errors in weight measurement, even if small (e.g. g error in presence of clothing), may alter the mean, but also widen the distribution curve. hence, the prevalence of malnutrition is overestimated. systematic errors in height measurement, such as erroneous lengthboard, may alter the mean without altering the sd. if the measurement is too short, there will be > stunting, albeit < wasting. if the measurement is too long, there will be < stunting, albeit > wasting. a standardization test is routine before undertaking anthropometric surveys. nb some scholars prefers terms "counted" and "calculated" to "measured" and "derived" . data entry bias . analytic bias a. anchoring bias-focusing on one major piece of information b. confirmation bias-favoring data which confirm underlying beliefs c. familiarity bias-weighing familiar/understandable events and spokespersons more than unfamiliar ones d. recency bias-weighing recent events more than remote ones e. salience bias-weighing vivid events more than mundane ones f. "time will tell" bias-collecting more data or letting time pass instead of making a hard decision e. imprecision (see r magnani [ ] , f checchi [ ] , and smart [ ] ) sampling, non-systematic error which lowers precision of findings and affects the level of certainty in extrapolating sampling estimates to the population's true value. it is always present, unavoidable, and a function of chance. its magnitude depends on sample size, sampling statistics, prevalence of condition, and length of recall period. precision refers to consistency of results obtained from repeated measurements. what is the sample size n of a random sample of binomial variables needed to yield a result of specified accuracy and precision? n = [(z pq)/d ] × design effect e.g. n = first estimate of sample size z = confidence limits (accuracy), or normal deviate. usually set at % :. z "score" = . p = proportion of the target population with attribute p q = proportion of the population without attribute p = -p. usually set at . to maximize the n of a study having a result of specified accuracy and precision. if you knew p and q, you would not need to do a survey. d = confidence interval (precision). usually set at +/− % :. d = . design effect (see e below) . . . once n is calculated, compare it to the size of the target population (n). if n < % of n, then use n as final sample size. if n > % of n, then recalculate the final sample size (n f ) by the following correction (a smaller sample size may be used). n n n n f = + / n f = / . n f = nb n to calculate the mean weight may be much smaller than n to calculate the prevalence of malnourished outliers ( vs. ). . sampling statistics and error measurement a. malnutrition prevalence or death rate the higher the prevalence (or death rate), the lesser the precision (higher d) available through a fixed sample size. (this is a consequence of the formula.) % gam is a common trigger for intervention. but, smart discourages use of this because high survey precision is needed (narrow ci). :. choose highest expected prevalence or rate-tends to > n. nb at levels of malnutrition and mortality generally found in emergencies, precision has much greater effect on sample size than suspected prevalence of malnutrition or death rate. n is related to d . e.g., if the malnutrition rate estimate is %, and assuming a design effect of : • survey statistic with a ci of +/− % requires n = • survey statistic with a ci of +/− % requires n = as rule of thumb, prevalence (%)/ approximates the range of appropriate ci. e.g. malnutrition prevalence of % calls for a precision of +/− % (range of %). it's generally unfeasible to achieve precision greater than +/− %. b. standard deviation (sd, σ). the degree to which individuals within the sample differ from the sample mean (μ); unaffected by sample size c. standard error (se = sd/√n) standard deviation of the sampling distribution of a statistic; decreases with larger sample sizes as estimate of the population mean improves, thus a lower se is more precise ( ) standard error of the mean (sem) is standard deviation of a sample mean's estimate of a population's true mean; an estimate of how close to the population's true mean the sample mean appears to be. ( ) relative standard error (rse)-sem/μ expressed as % • se of g on weight mean of kg = rse of % • se of g on weight mean of kg = rse of % d. confidence interval (ci = μ + z (se)) the margin of error around a point estimate. for normally distributed data, the ci yields the range in which a parameter is % likely to be found. a convention for reporting such data would be: "the most probable estimate of the parameter is x, and we are % confident the parameter lies somewhere between y and z [bounds of the ci]" (paraphrased from checchi, ) . nb in general, the lower the prevalence (or death rate), the greater the precision (lower d) needed to detect it and any subsequent changes in it. (this is intuitive.) overall, there is no benchmark for precision. increasing precision (decreasing d) slightly can dramatically increase n. +/− . deaths/ , /d is a practical limit in precision of mortality surveys. :. choose widest acceptable ci-tends to < n. e. design effect (d eff = variance study design /variance simple random sample ) a measure of the (in)efficiency of a cluster sample survey compared to that of a simple random sample. if d eff > , but the analysis treats it as a srs, then the confidence interval is inappropriately narrowed, and a test for differences is more likely to produce a positive result (type error). • if each child in a cluster had an unrelated probability of immunization, the precision of the sample estimate would match that of a simple random sample in which children were chosen. d eff = . however, this is generally not the case. • if each child in a cluster had an identical probability of immunization, the precision of the sample estimate would match that of a simple random sample in which children were chosen. d eff = cluster size of . nb focal phenomena create clustering of findings which increase the d eff . :. choose largest d eff -tends to > n. . length of recall period a. the shorter the recall period, the more accurate the mortality estimate (more distant events are more likely to be forgotten). b. the longer the recall period, the more precise the mortality estimate for a fixed sample size. the "sample" is effectively the number of person-days. for a fixed level of precision, the length of the recall period is inversely related to number of study subjects needed. if you cannot increase the sample size, you must increase the recall period. confounders are extraneous variables that correlate with both dependent and independent variables of interest (e.g. both the exposure of interest and the outcome of interest), are unevenly distributed across the levels of exposure, but are not causally linked to exposure and outcome. age and sex are the most common confounders. hence, the importance of matching in intervention and control groups. g. validity . study validity a. internal-capacity of the study to yield sound conclusions for the study population after considering bias, imprecision, and confounding (see d-f above) b. external-generalizability beyond the study population (ill-advised) . measurement validity a. criterion validity ( ) concurrent-sensitivity/specificity or correlation with a gold standard ( ) predictive-ability to predict an event b. face validity-common sense c. content validity-all relevant elements of a composite variable are included d. construct validity (usually for a new measure)-extent to which the measure corresponds to theoretical concepts (constructs) e. consensual validity-extent to which experts agree the measure is valid :. strength of evidence: face validity, criterion validity > content, construct, consensual validity. in absence of validity, a measurement may be embraced for its reliability (below). . death rates-calculated incidence of death expressed per , p/d or per p/mo; data collected by retrospective surveys (e.g. month period) to gauge severity of public health emergency particularly where sudden events lead to spike in mortality a. cdr-crude death rate b. asdr-age-specific death rate (e.g. u dr or death rate of children - yr) during a studied time interval (written as m or - dr); age of study cohort, e.g. - yr, should not be confused with study time intervals . mortality rates-calculated probability of dying before a specified age expressed per live births; data collected by national health authorities in periodic (annual) demographic surveys to reflect ongoing health status a. cmr-calculated probability of mortality in given population for specific time b. imr-calculated probability of a live borne child dying before yr c. u mr-calculated probability of a live borne child dying before yr nb mr ≠ dr. e.g. cmr ≠ cdr, u mr ≠ u dr. different rates measure different things and are not directly comparable. however, mrs may be converted into drs by the following: cdr or u dr (deaths/ , /d) = -ln( -p/ ) × . where p = cmr or u mr (deaths/ live births). however, this has little field utility. nb mmr-maternal mortality ratio has different units in numerators (maternal deaths) and denominators (live births), thus is a ratio, not a rate i. . stability-inter/intra-observer variation a. discrete variables-kappa coefficient b. continuous variables-correlation coefficient . internal consistency-correlation among all items in the measure . tests of reliability-cronbach's alpha, kuder-richardson, split halves j. conclusions the application of study findings to an entire population from which the sample was drawn. if the survey was well-conducted, the results may be considered representative of the entire population. this is scientifically justified. however a ci should accompany any parameter estimate of that population. the extension of study findings to a population or period which was not represented in the sample. it works by association-if populations appear to be experiencing similar conditions, the morbidity/mortality experience of one may be imputed to the other. this is not scientifically justified, but is often done where data are insufficient or impossible to collect. .k. • holo-endemic areas (e.g. congo) have an intense level of malaria transmission year-round. epidemics don't occur unless displacement brings in nonimmune populations. infection may be asymptomatic. effective partial immunity develops in adults which enables clinical tolerance of infection and protects against serious episodes. mortality is highest in pedes u and pregnant women. • hyper-endemic areas (e.g. w. africa) have an intense but unstable level of transmission in seasonal peaks when the climatic conditions are favorable. epidemics occur. infection is generally symptomatic. partial immunity fails to develop. mortality occurs across all age groups. • hypo-endemic areas (e.g. thai-burmese border) have a low level of transmission year-round. epidemics occur. infection is generally symptomatic. partial immunity fails to develop. mortality occurs across all age groups. think differential diagnosis (below). know the golden rules of infectious diseases (abstracted from a yung [ ] and used with permission). rigors are always important-serious bacterial infections are the most likely cause. severe muscle pain may be a symptom of sepsis even without fever. elderly patients with sepsis may be afebrile. in elderly patients, fever is rarely caused by a viral infection. septic patients who are hypothermic have a worse prognosis than those with high fever. treat as a medical emergency. fever in a postoperative patient is usually related to the surgical procedure (e.g. pneumonia, uti, wound, or deep infection). fever with jaundice is rarely due to viral hepatitis. think liver abscess, cholangitis, etc. the rash of early meningococcal infection may resemble a viral rash. generalized rashes involving the palms and soles may be due to drugs, viral infections, rickettsial infections, or syphilis. all febrile travelers in or returned from a malaria infected area must have malaria excluded. . disseminated tb must be suspected in all elderly patients with fever and multisystem disease who have been in an area with endemic tb. . septic arthritis may be present even in a joint which is mobile. ddx failure to thrive without f in infants is worked up like f without localizing signs. watch for clinical mimics-malaria presenting as pneumonia or diarrhea in pedes; vl presenting as malaria in adults; lepto presenting as mild df (esp in df endemic areas where the pt has mild onset of illness, worsening course, and no rash but jaundice after a week). do basic things well, use equipment you understand, teach others, delegate. h. count the number of fresh graves or bodies at health facilities and inquire as to cause. . orient the descriptive data-person, place, and time. a. tabulate data on affected patients. b. make a spot map. ( ) when and where was/were the first reported case(s) seen indicating an outbreak? c. plot an epidemic curve. ( ) what is the present # of patients/day or week? ( ) what is the usual # of patients/day or week? ( ) is this an increase? ( ) what is the present # of deaths/week or month? ( ) what is the usual # of deaths/week or month? ( ) is this an increase? d. calculate attack rates and case fatality ratios for total patients, u , o , and gender. . develop hypothesis. a. postulate sources of disease and mechanism of spread. b. estimate the population at risk of contracting disease and of dying from it. consider especially: those with limited access to health services pregnant and lactating ( ) infants not breast fed, children unvaccinated ( ) elderly . initiate control measures considering agent, host, and environment. a. what action has the community taken? b. identify local response capacity. ( ) what number and type of staff are locally available? ( ) what drugs and supplies are locally available? c. determine immediate unmet needs. ( ) specimen collection and lab diagnosis ( ) logistics ( ) support for clinical care-staff, drugs, and supplies ( ) support for environmental health d. undertake further necessary actions. ( ) case management with secondary prevention ( ) patient isolation ( ) health education ( ) agent and reservoir identification ( ) environmental decontamination ( ) primary prevention ( ) public information . inform authorities with investigation report. . initiate ongoing disease surveillance. during epidemic there is no clinical difference between them. other serogroups may cause disease in individuals, but not epidemics. when a suspected cholera serotype (strain) is isolated in the lab, one of the first tests performed is bacterial agglutination with o and o antisera. strains are thereby identified as v. cholerae o , o , or non-o /non-o . • if (+) agglutination to o antisera, then the strain is further tested for agglutination to antiserum of ogawa and inaba serotypes. • if (+) agglutination to o antisera, then the strain is not further subdivided (except as producer or nonproducer of ct as noted below). • if (−) agglutination to o and o antisera, then the strain is known as non-o , non-o v. cholerae. a strain is further identified as a producer or non-producer of cholera toxin (ct). ct production is a major determinant of disease development. strains lacking ct do not produce epidemics even if from the o or o serogroup. • serogroup o exists as main biotypes-classical and el tor-though hybrids also exist. each biotype occurs as two serotypes-ogawa and inaba. classic biotype caused the th and th pandemics but little epidemic disease since the s though it still causes cases in india. el tor biotype caused the th (current) pandemic and almost all recent outbreaks. el tor was first isolated in in el tor, egypt after importation by indonesian pilgrims travelling to mecca. it survives longer in the environment and produces ct similar to the classical biotype. presumably because of ct pathogenicity, the % of cholera patients with severe disease has doubled over the past yrs. these patients tend to require iv fluid therapy. • serogroup o may have evolved from strains of o el tor as they share many properties though not agglutination. in spring of in dhaka, o cases exceeded o el tor cases for the first time, and it was postulated that o may become the cause of an th pandemic. however, since then, o has again become dominant. infective dose depends on individual susceptibility. relevant host factors include immunity produced by prior infection with serogroup o as well as stomach acidity. id may be , orgs, so personal hygiene plays a lesser role than in shigellosis where the id is much lower. shigella has species. • s. dysenteriae type (sd or shiga bacillus) causes the severest disease of all shigella sp because of its neurotoxin (shiga toxin), longer duration of illness, higher abx resistance, higher cfr thru invasive complications, and great epidemic potential. • s. flexneri is the most common, and is generally endemic, in developing countries • s. sonnei is the most common in industrial countries • s. boydii and s. sonnei give mild disease. id may be orgs, so personal hygiene plays a greater role than in cholera. some kinds of e. coli produce a shiga toxin. shiga toxin genes reside in bacteriophage genome integrated into the bacterial chromosome. some abx, e.g. fluoroquinolones, induce expression of phage genes. the bacteria that make these toxins are variously called "shiga toxin-producing e. coli" (stec), "enterohemorrhagic e. coli" (ehec), or "verocytotoxic e. coli" (vtec). all terms refer to the same group of bacteria. • e. coli o :h (often called "e. coli o " or "o ") is the most commonly identified stec in north america, and it causes most e. coli outbreaks. approximately - % of ehec infections result in hus. • non-o stec serogroups also cause disease. in the usa, serogroups o , o , and o are the most commonly identified e. coli pathogens overall. diarrhea epidemiology is seasonally dependent. environmental temperature directly influences biologic activity-∆ °c is proportional to × risk of disease • temperate climates: bacterial diarrhea in warmer, humid season; rotavirus diarrhea in cooler, dry season • tropical climates: bacterial diarrhea in rainy season; rotavirus diarrhea year round with increased incidence in cooler season • most common pathogens for watery diarrhea-rotavirus, etec, v. cholerae; most important pathogen for epidemic watery diarrhea-v. cholerae • most common pathogens for dysentery-shigella species, salmonella species, campylobacter jejuni, clostridium difficile, eiec, ehec, e. coli o :h , entamoeba histolytica, yersinia enterocolitica; most important pathogens for epidemic dysentery-s. dysenteriae serotype (developing countries), e. coli o :h (developed countries) bangladesh has two seasonal cholera peaks: pre-monsoon with hot, humid weather (esp weeks - in apr-may) creating increased biological activity; post-monsoon (esp weeks - in aug-sep) with contamination of water sources. premonsoon epidemics are generally worse than post-monsoon ones. dysentery has low level year-round incidence, but epidemics occur roughly each decade. epidemic strains display new, additive antibiotic resistance which probably triggers the epidemic. once resistant strains have become endemic, antibiotic susceptibility rarely reappears. sd acquires resistance quickly. sf acquires it more slowly, and that resistance may wane with decreasing abx pressure. at icddr, annual proportional incidence approximates the following: • e. coli overall = % of cases, but etec = %. • e. coli tends to dominate before monsoon season and flooding. • cholera tends to dominate after monsoon season and flooding. • overall, - % of diarrhea cases may be vaccine-preventable. • % of pts have no pathogen identified. clean water and waste management for cholera. personal hygiene (hand washing with soap and clean towels) for shigella. water safe drinking water (boiled, chlorinated) nb sphere standards are not enough-you need increased quantities of chlorinated water at household level. san clean latrines for safe disposal of excreta hand washing with soap food safe food (cooked, stored) breast feeding fomites safe disposal of dead bodies with disinfection of clothing nb after outbreak of a fecal-oral pathogen, food hygiene and funereal practices may influence human-tohuman transmission more then water quality. health education to affected population wash hands with soap: after using toilets/latrines. after disposing of children's feces. before preparing food. before eating. before feeding children. is identical for all patients, and thus can't be given to pedes < yr because of volume loading. dukoral has been the main vaccine considered for use in high-risk populations. • morc-vax and shanchol-similar to dukoral except they do not contain the rbs, hence do not require a buffer, and are / the cost to produce. morc-vax, produced in vietnam, is derived from a vaccine administered to millions of people since , but is not who pre-qualified, and is not expected to have international distribution. shanchol, produced in india, has international distribution (e.g. used in the haiti cholera vaccination campaign of ), and is now the agent of choice for who. it confers immunity d p nd dose, effectiveness > % at mo, and protection > % at yr. also confers short-term protection vs etec. dose: . cc vaccine followed by water ingestion but no fasting needed; doses, wks apart; cold chain required except for day of use. orochol-bivalent formulation as in dukoral without rbs of ct. dose: single dose. no longer manufactured. who recommendations: "vaccination should not disrupt the provision of other high-priority health interventions to control or prevent cholera outbreaks. vaccines provide a short-term effect that can be implemented to bring about an immediate response while the longer term interventions of improving water and sanitation, which involve large investments, are put into place" [ ] . icddr recommendations: "because of limitations in terms of transport, formulation, and cost of the current dukoral vaccine, the cots program does not require the utilization of the vaccine during an outbreak; it is not necessary to vaccinate to overcome an outbreak. however, if dukoral is readily available and staff are properly trained in its use according to the guidelines that come with the vaccine, the cots program permits dukoral's use (ideally before an outbreak) in the following high-risk populations: refugee populations in which cholera is present, health care workers managing cholera cases, and communities in which the incidence rate is greater than in annually" [ ] . if undertaken, the following will apply: vaccination campaign requires numerous staff. community mobilizers are key. clinical staff should not be poached from their clinical duties. supervisors must be free to move at will. logistics is key-if the st day goes bad, the campaign goes bad. mark the domiciles which are done. hold after-action meetings each day. last day, use mobilizers with mobile broadcasting to find those missed. second phase vaccination should include chws with multi-purpose messages on water and sanitation. key lessons in epidemic response avoid: press exaggeration abx prophylaxis reliance on ivf and insufficient ors lab investigation of cases once epidemic etiology is ascertained prolonged hospitalization hospital discharge criteria requiring multiple negative stool cultures enthusiasm for ocv during epidemic exaggerated water purification objectives concentration of technical competencies in moh at expense of districts failure to share information with district stakeholders influenza viruses comprise genera-influenza types a, b, and c-each with species. • influenza type a is divided into subtypes based upon serological response to hemagglutinin (ha) and neuraminidase (na) glycoproteins. there are different ha subtypes and different na subtypes. h n , h n , and h n are responsible for the major human pandemics in the last century. h n virus circulated between and but currently does not. only influenza a subtypes infect birds, and all subtypes can do so. bird flu viruses do not usually infect humans. but, in , an outbreak of h n avian influenza in poultry in hong kong marked the first known direct human transmission of avian influenza virus from birds to humans. since then, h , h , and h avian influenza subtypes have been shown to infect humans. • influenza type b is morphologically similar to a and also creates seasonal and epidemic disease. • influenza type c is rare but can cause local epidemics. seasonal human influenza vaccine currently has strains-h n /h n /b. influenza disease in humans has a short incubation period ( - d) . early symptoms are non-specific. it is highly infectious, especially early in the course of the disease, with a large # of asymptomatic carriers. transmission potential (r ) is a function of infectivity, period of contagiousness, daily contact rate, and host immunity. in general, the faster the transmission, the less feasible is interrupting transmission thru usual disease control tools of case finding, isolation, contact tracing, and ring vaccination. case definitions may change and become more specific as epidemic evolves case management guidelines for communicable diseases with epidemic potential outbreak management protocol rapid response teams to investigate case reports epidemic investigation kits to mobilize specimens to collect labs to verify diagnosis and share specimens with peer labs pts to identify, isolate, and treat (ipd and opd settings) contacts to trace and ? quarantine hotline use and rumor investigation secondary prevention specific groups of exposed or at risk in the community-most likely to work when there is limited disease transmission in the area, most cases can be traced to a specific contact or setting, and intervention is considered likely to slow the spread of disease eg quara ntine of groups of people at known common source exposure (e.g. airplane, school, workplace, hospital, public gathering; ensure delivery of medical care, food, and social services to persons in quarantine with special attention to vulnerable groups) (useless once there is community-based spread) eg containment measures at specific sites or buildings of disease exposure (focused measures to > social distance) cancel public events (concerts, sports, movies) close buildings (recreational facilities, youth clubs) restrict access to certain sites or buildings community-wide measures (affecting exposed and non-exposed)-most likely to work where there is moderate to extensive disease transmission in the area, many cases cannot be traced, cases are increasing, and there is delay between sx onset and case isolation. eg infection control measures ari etiquette-cover nose/mouth during cough or sneeze, use tissues, wash hands avoidance of public gatherings by persons at high risk of complications nb use of masks by well persons is not recommended eg "snow" (stay-at-home) days and self-shielding (reverse quarantine) for initial d period of community outbreak-may reduce transmission without explicit activity restrictions eg closure of schools, offices, large group gatherings, public transport (pedes more likely to transmit disease than adults) nb community quarantine (cordon sanitaire)-restriction of travel in and out of an area is unlikely to prevent introduction or spread of disease international travel nb travel advisories to restrict international travel are generally useless in slowing epidemic spread nb health screening for fever and respiratory sx at ports of entry is also generally useless in slowing epidemic spread meningitis is a disease with significant mortality. meningococcus (neisseria meningitides) is renown for its rapid onset, rapid progression (death sometimes within hours), and high mortality ( % untreated). there are serogroups of neisseria meningitides but only (a, b, c, w, x, y) are known to cause epidemics. the bacteria spread from person to person via respiratory and nasal secretions. kissing, sharing eating and drinking utensils, cigarettes, coughing, and sneezing are recognized methods of transmission. close contacts over a period of time, as between household or dormitory residents, are most commonly affected. population movements (e.g. pilgrimages, displacement, military recruitment), poor living conditions, and overcrowding are epidemic risk factors. large, recurring epidemics of meningitis occur in the "meningitis belt" of sub-saharan africa where over million people live. this belt encompasses countries from senegal in the west to ethiopia in the east and as far south as tanzania and the democratic republic of congo. sub-saharan arica has epidemic seasonality. dry seasons and droughts favor epidemics. rains stop them. large regional epidemics, as well as epidemics in displaced populations and refugee camps, have mainly been due to meningococcus serogroup a. since , extensive use of meningococcal type a conjugate vaccine in the meningitis belt has reduced the incidence and case load of type a epidemics by nearly %. in , the most common lab confirmed meningitis isolate was streptococcus pneumoniae. in non-epidemic settings, neisseria.meningitidis, streptococcus pneumoniae, and haemophilus influenzae account for % of all cases of bacterial meningitis. prior to the availability of conjugate vaccines, h. influenza type b (hib) was the most common cause of childhood bacterial meningitis outside of epidemics. where hib vaccines are in the routine infant immunization schedule, hib meningitis has nearly disappeared. polysaccharide vaccines are available with serotypes (a and c), serotypes (a, c and w) or serotypes (a,c, w, and y). duration of immunity is approximately years. meningococcal protein conjugate vaccines confer longer immunity but at higher cost than polysaccharide vaccines. monovalent conjugate vaccine against group c dates from , and tetravalent (a, c, w and y) conjugate vaccine dates from . a group b vaccine made from bacterial proteins has been licensed since but is not readily available. meningococcal vaccines have a very low incidence of side effects. regular disease surveillance is necessary to detect outbreaks. the epidemic threshold is suspected cases/ , population in any given week. two suspected cases of meningitis in the same settlement should trigger an outbreak investigation. nasopharyngeal carriage rates do not predict epidemics. - % of meningococcal disease presents with meningitis. % of cases occur in patients < y/o. peak incidence in meningitis belt is ages - yrs. diagnosis is straightforward when patient presents with signs of meningitis-fever, headache, vomiting, changes in mental status. however, most patients have non-specific illness - days before onset of meningitis. cfr of untreated meningococcal meningitis can be %. cfr of properly treated meningococcal meningitis is < %. - % of meningococcal disease presents with septicemia unaccompanied by meningitis or other focal features. it is a dramatic illness which affects previously healthy children and young adults. it presents with acute fever leading to purpura fulminans (hemorrhagic or purpuric rash), shock, and waterhouse-friderichsen syndrome (acute adrenal failure). etiologic diagnosis can be easily missed. cfr of meningococcal septicemia is % and may be % even with proper treatment. diagnosis may be confirmed by agglutination tests, polymerase chain reaction, culture and sensitivity testing of spinal fluid and blood. in many situations, these tests are not available. throat swabs may be helpful on occasions. do not delay treatment for tests or test results. minutes count. it is more important to have a live patient without a confirmed diagnosis than a dead one with a diagnosis. differential diagnosis in a tropical patient with fever and altered mental status, but without purpura or shock, includes cerebral malaria. co-infection may occur. standardized case management of bacterial meningitis in developed countries involves - days of parenteral antibiotic therapy. drug of choice in adults and older children is ceftriaxone which also rapidly eliminates the carrier state. alternate drugs include ampicillin and benzylpenicillin which do not eliminate the carrier state. in developing countries, days of parenteral antibiotic therapy are empirically shown to be effective. in large epidemics in resource-poor settings, a single im dose of chloramphenicol in oil is the drug of choice. for patients who do not improve in h, a repeat dose may be given. viral meningitis is rarely serious and requires only supportive care, recovery is usually complete. patient isolation and disinfection of the room, clothing, or bedding are not necessary. respiratory precautions are advised particularly early in the course of treatment. chemoprophylaxis of contacts is available in some settings but rarely in the disaster setting. vigilance and education of close contacts is mandatory. epidemic preparedness and early detection of outbreaks are key. vaccines against n. meningitides serogroups a, c, y and w are very effective in controlling epidemics. in epidemic settings, children - are the priority target with serogroups a and c typically the priority antigens. rapid mass vaccination campaigns can contain outbreaks in - weeks. for immunocompetent patients over years, vaccine efficacy rate is % one week after injection. however, duration of immunity may be as little as years in younger children. in some countries, vaccine may also be used with close contacts of sporadic disease cases to prevent secondary cases. chemoprophylaxis of contacts is not recommended in epidemics, but community education and ready access to health care are essential. source control/reduction/elimination avoid unnecessary contact with suspected reservoir animals and known disease carrier species (e.g. primates). avoid direct or close contact with symptomatic patients. undertake quarantine and culling of sick reservoir animals and known disease carrier species. avoid unnecessary contact with or consumption of dead reservoir animals or known disease carrier species. establish appropriate communicable disease controls for burial of the dead. administrative controls environmental and engineering controls avoid needle stick exposure to blood specimens thru automated machine handling ppe use standard precautions-gloves, masks, and protective clothing-if handling infected animals or patients. wash hands after visiting sick patients. active surveillance and contact tracing (enhanced surveillance) through community-based mobile teams active case finding (screening and triage) and contact tracing dedicated isolation facility food provision to isolated patients so they are not dependent on family case definition treatment protocols emphasizing supportive care and treatment of complications essential drugs referral guidelines secondary prevention barrier nursing strictly enforced family and community education ministerial task force to address policy local health authority task force to address procedures national level task forces to comprise guidance note on using the cluster approach to strengthen humanitarian response panel on humanitarian financing report to the united nations secretary-general. too important to fail-addressing the humanitarian financing gap belgian development corporation, government of bulgaria, government of canada, et al. the grand bargain-a shared commitment to better serve people in need available from usaid's development experience clearinghouse gender equality and female empowerment policy national strategy for pandemic influenza selective primary health care-an interim strategy for disease control in developing countries ten great public health achievements-united states ten great public health achievements-united states water and excreta-related diseases: unitary environmental classification infections related to water and excreta: the health dimension of the decade addendum to ipc technical manual version . . tools and procedures for classification of acute malnutrition. rome: ipc global partnership integrated food security phase classification technical manual version . . evidence and standards for better food security decisions nbc domestic preparedness training hospital provider course. undated. curriculum available from the center for domestic preparedness sampling guide interpreting and using mortality data in humanitarian emergencies-a primer for non-epidemiologists. humanitarian practice network, network paper no . london: overseas development institute measuring mortality, nutritional status, and food security in crisis situations: smart methodology. v infectious diseases-a clinical approach cholera vaccines: who position paper cholera outbreak training and shigellosis (cots) program [cd-rom version . , undated history and epidemiology of global smallpox eradication retrieved from us department of health and human services geneva: world health organization. laboratory available? . what tests does it perform? . is there transport to and from the laboratory? . who prepares transport media? . who provides specimen collection material and supplies? . how can these supplies be obtained? . who provides cool packs, transport boxes, car, driver …? . what forms/information must be sent with the specimens? how does the epidemiologist obtain results? if a lab is not available, then you need a sampling strategy that addresses specimen acquisition, preparation, and transportation in compliance with international regulations on the transport of infetious substances. reference . world health organization department of communicable disease surveillance and response. highlights of specimen collection in emergency situations. undated. available from who laboratory and epidemiology capacity strengthening office regulation ) . leak-proof specimen container wrapped with enough absorbent material to absorb the entire content of the st container . leak-proof secondary container usually plastic or metal . outer shipping container whose smallest dimension is mm diagnostic specimens use iata packing instruction without biohazard label. infectious materials use iata packing instruction with biohazard label. what to send with the sample? lab request form with: • sender's name and contact info • patient name, age, sex • sample date, time • suspected clinical diagnosis with main signs and symptoms • sample macroscopic description • context-outbreak confirmation, ongoing verification, outbreak end, etc. • epidemiological or demographic data where to send the sample? a. prior to seasonal epidemic . establish a national coordinating committee (ncc). . designate a lead agency and lead official in the ncc. . establish a local coordinating committee (lcc). . designate a lead official in the lcc. . anticipate roles for partner agencies (e.g. inter-agency and team coordination, disease surveillance, field epidemiological investigation, laboratory identification, case management guideline development, outbreak logistics, public information, and social mobilization). . identify sources of funds. . intensify disease surveillance. . identify reference lab(s) for communicable diseases of epidemic potential. . ensure mechanism for specimen transport. initial response to suspected outbreak . form an emergency team to investigate and manage the outbreak a. identify key roles on the outbreak investigation team(s) ( ) epidemiology and surveillance ( ) case management ( ) water and sanitation ( ) laboratory services ( ) communication b. staff those roles ( ) epidemiologist-to monitor proper data collection and surveillance procedures ( ) physician-to confirm clinical s/sx and train health workers in case management ( ) water and sanitation expert-to develop a plan for reducing sources of contamination ( ) microbiologist-to take environmental/biological samples for laboratory confirmation, train health workers in proper sampling techniques, and confirm use of appropriate methods in the diagnostic laboratory ( ) behavior change communication (bcc) specialist-to assess the population's reaction to the outbreak, create, and disseminate appropriate health messages outbreak investigation protocol in place rapid response teams to investigate case reports epidemic investigation kits to mobilize specimens to collect labs to confirm dx of v. cholerae, s. dysenteriae, other shigella, and e. coli o :h dipstick identification on representative sample of specs is useful for cholera, but c&s is essential because dipsticks are not available for shigella, etec. vibrio are hardy if kept moist and cool. they can survive a week in cary blair media. shigella are fragile and difficult to recover if transport time > d. - isolates initially to confirm outbreak - isolates initially to create abx use policy (bacterial resistance renders cotrimoxazole, amp/amox, nalidixic acid, and tetracycline unusable) - isolates monthly from ipd and opd before abx therapy to assess evolving abx resistance - isolates periodically to reference laboratory to confirm abx resistance patterns and undertake molecular studies isolates at end of the outbreak to confirm that new diarrheas are not epidemic pathogens nb systematic sampling is most representative-e.g. every th pt or all pts q weeks adjusted as needed to collect the necessary specs. sensitivity >> important than specificity in rdt screening during an epidemic. pts from one geographic area are more likely to constitute a cluster involving a new pathogen. an area may be considered cholera-free after incubation periods (total of d) have passed without cholera disease. however, hospital monitoring should continue for a year due to tendency of enteric pathogens to re-emerge long after they are declared gone. cholera may be viable but nonculturable from the environment; environmental monitoring has many false negatives. consider improvements to existing diagnostic labs hotlines set up for reporting of rumor health reference and educational materials in place case definitions case management and referral guidelines for communicable diseases with epidemic potential pt, provider, and community educational materials specimen handling protocols epidemic command & control center established under local health authorities using principles and practices of incident mgmt unified command of multi-disciplinary specialists information channel to government and stakeholders support by government for technical actions coordination with technical sectors-particularly wash (cfr is a function of case mgmt, but ar overall is a function of wash) water supply, purification, and distribution systems bucket chlorination is low tech but reasonable way to reach individual hh or small communities water treatment units need ca hypochlorite, chlorimetric, and colimetric monitoring devices chlorinators worth considering at water sources of high public demand and epidemic activity hygiene promoters with environmental health assessors to address hand and food hygiene in communities around the outbreak area (think ring vaccination with knowledge) safe disposal of medical waste and infectious sludge from treatment facilities medical logistics-resource prepositioning and stockpiles cots (take one and have carpenter make copies) plastic sheets with defecation hole or sleeve buckets ( white color for stool-enables recognition of diarrhea color; different color for emesis; different color for domestic waste) ivf, iv sets, iv poles or suspension cords (cholera kits) key: cord- -uphdzj l authors: sahajpal, nikhil shri; mondal, ashis k.; njau, allan; ananth, sudha; jones, kimya; ahluwalia, pankaj k.; ahluwalia, meenakshi; jilani, yasmeen; chaubey, alka; hegde, madhuri; kota, vamsi; rojiani, amyn; kolhe, ravindra title: proposal of reverse transcription-pcr–based mass population screening for sars-cov- (covid- ) date: - - journal: j mol diagn doi: . /j.jmoldx. . . sha: doc_id: cord_uid: uphdzj l testing for sars-cov- has lagged behind in many countries due to lack of adequate test kits and bottlenecks in the analytical process. the aim of this study was to investigate the feasibility and accuracy of a sample pooling approach for wide-scale population screening for covid- . a total of nasopharyngeal-swab samples ( negative and positive) previously tested for sars-cov- were de-identified and assigned random numbers for analysis. from this, pools of samples each were created. automated rna extraction followed by rt-pcr was carried out in a well plate. positive pools were identified and the individual samples were re-analyzed. the outbreak of covid- (caused by sars-cov- ) is now a pandemic that has caused mass disruption of the world order, impacting public health care systems, social lifestyle, governance and economics. since its identification in the region of wuhan, china, over . m confirmed cases with over , covid- related deaths have been reported globally (https://coronavirus.jhu.edu/map.html, last accessed june , ). the incidence of disease is highly varied across the globe, with the incidence rates ranging from per m in us, per m in spain and ~ per m in africa, compared to the global incidence rate of per m (https://www.worldometers.info/coronavirus/#countries, last accessed june , ). in an attempt to contain the spread of disease, multidisciplinary strategies have been launched in different regions of the world, including implementing social distancing, maintaining personal hygiene, contact tracing, quarantine, travel restrictions and lockdowns. a widely accepted method, though not effectively implemented as a measure to control its spread is testing for sars-cov- , typically utilizing nasopharyngeal swab specimens. patients tested positive require appropriate clinical management by either effective isolation or quarantine at home for mild symptoms or within healthcare facilities for moderate to severe symptoms. wide-scale testing approaches such as those implemented in south korea have resulted in great success at reducing community spread and lowering mortality rates. in addition, wide scale testing provides more informative epidemiological data for drafting policies on disease monitoring and control. currently, at least manufacturers of diagnostic assays have received emergency use authorization (eua) from the federal drug and food administration (fda) for covid- testing (https://www.fda.gov/medical-devices/emergency-situations-medical-devices/emergencyuse-authorizations#covid ivd, last accessed june , ). however, testing has lagged behind in many countries due to various factors, most significant of which being supply chain issues with lack of reagents and adequate test kits. therefore, many patients (both symptomatic and asymptomatic) remain untested and hence are potentially contributing to community spread of the virus. furthermore, many countries including high income countries, have logically resorted to prioritize testing for the hospitalized, symptomatic and high risk population. with this approach, absence of testing or long turnaround times among exposed but asymptomatic individuals and patients exhibiting mild symptoms have been observed; a factor that likely contributes to exponential community spread. herein, we propose a mass population screening approach, based on sample pooling strategy for rapid and wide-scale population screening that may be adopted by laboratories currently using rt-pcr based methods to test for sars-cov- . the strategy we propose leverages on existing high throughput systems that employ high analytically sensitive [limit of detection (lod) - copies/ml] real-time pcr chemistries, coupled with pooling of samples based on current covid- incidence rates. pooling of samples compared to individual testing has been investigated previously such as in screening blood donations, infectious and genetic diseases. , , pooling when carefully executed, has been found to be useful and more cost effective for estimating incidence rates in specific cases. the advantages of this approach include the potential to catch up with huge testing deficits, reducing turnaround times and most importantly ensuring enormous savings through the most efficient use of rna extraction and/or testing kits, which even today are in significant short supply. the rt-pcr based methods have two primary components, first: rna extraction from clinical specimens and second: rt-pcr based detection of sars-cov- nucleic acid region(s the assay is based on rna extraction followed by taqman table s ). table s ). identifying the positive sample: step pool(s) that resulted positive were identified and the samples comprising each positive pool were retrieved and processed for downstream extraction and rt-pcr analysis for the identification of the positive sample(s). the positive sample(s) were identified based on the ct value specified by the manufacturer. in addition, one negative pool was selected randomly and each sample was re-analyzed individually as a qc measure. in step , the qc of negative and positive sample was observed to be within the range table s ). therefore, samples resulted in an overall . % positive (ppa) and % negative percent agreement (npa) compared to individual testing approach. the covid- pandemic has resulted in an overwhelming number of infected patients, leading to a tremendous burden on health care resources to the extent of outstripping the current production capabilities for supplies. innovative ideas in specimen collection, isolation, respiratory support and other patient care plans have been implemented. scaling up testing has been identified as a key component to manage the pandemic. laboratories and manufactures of test kits across the world have also responded by ramping up testing. however, more innovative approaches are needed for wide-scale population testing and to side step the foreseeable shortages in test kits. in this study we demonstrate that pooling patient samples and testing them on high sensitivity, high throughput systems is both practical and accurate. in our proof-of-concept study with samples, pools were formed with samples table s ). another challenge to this approach is inaccurate designation of individual and pooled samples within the well plate, resulting in sample mix-up. to minimize this, use of a minimum of two identifiers and well documented workflows to ensure traceability of all pooled samples is essential. in addition, use of bar code readers and automated sample processing would minimize the chances of such a mix-up. further, selected samples from a randomly selected negative pool(s) should also be analyzed individually as a quality control monitor. in our study, all samples in one negative result pool/well were retested and were found to be in agreement with individually tested samples. in terms of cost analysis, million individuals can be tested for $ . m with the proposed mass population screening approach compared to $ m with routine screening. more important however, is the potential to massively increase the number of people tested using the same quantity of reagents/test kits. this is a critical advantage given the short supply of test kits, a fact with the disparity that ensues especially in low and middle income countries (lmic). the direct saving on reagent and test kits are complemented by indirect savings on laboratory supplies including personal protective equipment that are needed to perform testing on these infectious clinical samples. these savings will enhance sustainable laboratory operations throughout the pandemic or can be deployed to laboratories that are facing dire constraints in supplies. in conclusion, we surmise that this unprecedented crisis requires innovative solutions at all levels. the strategy we propose leverages existing high throughput systems which employ analytically high sensitive rt-pcr chemistries, coupled with pooling of samples based on current covid- incidence rates. in this study, we analyzed samples in a pooled approach using only two-extraction and -pcr runs and achieved . % ppa and % npa. in order to optimize the number of pooled samples, real time region specific data in websites such that hosted by the center for systems science and engineering (csse) at johns hopkins university, baltimore, md, is helpful. in addition, robust validation and knowledge of the analytical performance of the assay to be adopted as well as regional/laboratory positivity rates are critical in this approach. the number of samples pooled are inversely proportional the analytical sensitivity of the assay and the local positivity rate. , the advantages of this innovative approach include potential of catching up with testing, clearing backlogged samples, reducing turnaround times and ensuring enormous savings on rna extraction and/or testing kits and laboratory supplies that are in short supply. this would relieve the pressure mounting on laboratories for increased testing, hopefully making a significant contribution to control of this pandemic. in addition, this strategy may come in handy for effective and consistent disease surveillance as many states and countries begin to reopen businesses, airports, public gatherings and work environments. monitoring spikes in the number of cases in groups of individuals in the same environment will facilitate rapid and early containment. covid- : towards controlling of a pandemic estimating the reproductive number and the outbreak size of novel coronavirus disease (covid- ) using mathematical model in republic of korea impact of pooling on accuracy of hepatitis b virus surface antigen screening of blood donations sensitivity evaluation of the gen-probe amp-ct assay by pooling urine samples for the screening of chlamydia trachomatis urogenital infection a pooling strategy for heterozygote screening of the delta f cystic fibrosis mutation feasibility of pooling sera for hiv- viral rna to diagnose acute primary hiv- infection and estimate hiv incidence sample pooling as a strategy to detect community transmission of sars-cov- assessment of specimen pooling to conserve sars cov- testing resources the steps needed to end the covid- pandemic: bold public health leadership, rapid innovations, and courageous political will. jmir public health and surveillance optimization of group size in pool testing strategy for sars-cov- : a simple mathematical model test in multi-sample pools the authors thank lisa middleton for help with manuscript review. key: cord- - c zwhdh authors: bal, a.; destras, g.; gaymard, a.; bouscambert-duchamp, m.; valette, m.; escuret, v.; frobert, e.; billaud, g.; trouillet-assant, s.; cheynet, v.; brengel-pesce, k.; morfin, f.; lina, b.; josset, l. title: molecular characterization of sars-cov- in the first covid- cluster in france reveals an amino acid deletion in nsp (asp del) date: - - journal: clin microbiol infect doi: . /j.cmi. . . sha: doc_id: cord_uid: c zwhdh nan in december , a novel coronavirus emerged in china, causing outbreaks of pneumonia [ ] . the virus was subsequently identified as a betacoronavirus and named severe acute respiratory syndrome coronavirus (sars-cov- ). sars-cov- is responsible for the coronavirus disease (covid- ) pandemic which includes asymptomatic upper and lower respiratory tract infections. among the first european cases of covid- , six were associated with a cluster of transmissions in the french alps in late january [ ] . the index case of this cluster travelled from singapore to france and went back to the united kingdom (uk) where he tested positive for sars-cov- on february th. here, we aimed to investigate the french cases related to this cluster using metagenomic next-generation sequencing (mngs) analysis. of the six contact patients who tested positive for sars-cov- , the three samples with the highest viral loads (assessed by rt-pcr targeting the rdrp gene) were selected for mngs analysis [ ] . one nasopharyngeal swab was collected from a patient with an upper respiratory tract infection on february th (sample # , ct ¼ . ). the other two samples were collected from the same asymptomatic patient on february th (sample # , nasopharyngeal swab, ct ¼ . ) and th (sample # , nasopharyngeal aspirate, ct ¼ . ). a previously described mngs protocol was used, but dnase treatment was performed after nucleic acid extraction in order to increase the sensitivity for the detection of rna viruses [ ] . lowquality and human reads were filtered out, and remaining reads were aligned to the sars-cov- reference genome (isolate wuhan-hu- , epi_isl_ ) using the bwa-mem algorithm. a mean of reads per sample were generated, of which a mean of reads per sample were mapped to the sars-cov- reference genome. the percentage of genome covered at a minimum depth of coverage of x was . % for sample # , . % for sample # and . % for sample # . the whole-genome sequence (wgs) generated from sample # was deposited on gisaid (global initiative on sharing all influenza data) (epi_isl_ ). the phylogenetic analysis using the wgs of sars-cov- publicly available (as of march th ) found that this sequence clustered with a sequence (epi_-isl_ ) collected in jiangsu, china, on january th, suggesting a separate introduction from asia (fig. ) . compared to the reference sars-cov- sequence, a three-nucleotide deletion in open reading frame a (orf a) at positions e was identified. this deletion was found in % of the reads covering this position with a sequencing depth of x around the deletion. importantly, this deletion was also identified in % of the reads of sample # and sample # with a depth of x and x, respectively. using the cov-glue resource, we found that this mutation leads to a deletion of amino acid in non-structural protein (nsp ) [ ] . this deletion in nsp (asp del) was also characterized in / ( . %) of the wgss available on march th (england n ¼ ; the netherlands n ¼ ). wgs-based phylogenetic analysis found that viruses containing this specific deletion were close to viruses collected in china between december and early february , while viruses with asp del collected in the netherlands have slightly diverged (fig. ). the analysis included wgs of sars-cov- (> bp) collected in humans and available on gisaid (global initiative on sharing all influenza data) from march th, . the following sequences were excluded from the analysisdepi_isl_ , epi_-isl_ , epi_isl_ , epi_isl_ , epi_isl_ and epi_isl_ because they were outliers, and epi_isl_ , epi_isl_ dbecause of incomplete sequences in orf ab. the hcov /wuhan/ipbcamswh / strain was used as an outgroup virus. genetic distances were calculated using the kimura's two-parameter model (k ) and pairwise deletion. the tree was constructed by the neighbour-joining method using r seqinr and ggtree packages and validated using bootstrap pseudo-replicates. sequence from sample # (epi_isl_ ) is indicated by the black arrow. nucleotide alignment ( e ) is depicted as a heatmap on the right panel with the threenucleotide deletion shown in black. corresponding amino acid sequence (nsp : - ) for the reference sequence is indicated below the heatmap. letter to the editor / clinical microbiology and infection xxx (xxxx) xxx sars-cov- sequences were not further compared between the two patients due to largely incomplete coverage of the sars-cov- genome in sample # . nonetheless, the longitudinal samples from the asymptomatic patient (sample # versus sample # ) were compared using a minimum depth of coverage of x in order to make a preliminary assessment of intra-host genetic variability. three snvs were noticed between the two samples: c a (nsp : s y), a g (synonymous mutation in nsp ), and t a (protein s: l h), suggesting intra-host evolution of the virus. for all three positions, nucleotides from sample # were still detected in sample # , but as minor variants. in this short report, we present the first genetic characterization of a covid- cluster in europe. despite low viral loads, the mngs workflow used herein allowed us to characterize the wholegenome sequences of sars-cov- isolated from an asymptomatic patient in two clinical samples collected day apart. comparison of these sequences suggests viral evolution with development of quasispecies. specific studies using high depth of coverage are needed to explore potential intra-host adaptation. in addition, the present workflow identified a new deletion in nsp (asp del) which was found in all three samples originating from this cluster. the analysis of wgs identified this deletion in other viruses collected in england (february) and in the netherlands (march), suggesting the spread of this deletion in europe. the impact of asp del on sars-cov- transmission and pathogenicity, as well as on pcr performances and antiviral strategies, should be rapidly evaluated in further studies. investigations complied with the general data protection regulation (regulation (eu) / and directive / /ec) and the french data protection law (law n e on / / and d ecret n - on / / ). informed consent concerning the disclosure of information relevant to this publication was obtained from the confirmed cases in france. ab and gd have contributed equally to this work. a novel coronavirus from patients with pneumonia in china first cases of coronavirus disease (covid- ) in the who european region detection of novel coronavirus ( -ncov) by real-time rt-pcr quality control implementation for universal characterization of dna and rna viruses in clinical respiratory samples using single metagenomic next-generation sequencing workflow amino acid analysis for the sars-cov- outbreak letter to the editor / clinical microbiology and infection xxx (xxxx) xxx we would like to thank all the patients, clinicians, laboratory technicians and informatics department who contributed to this investigation. we are also grateful to v er ena landel and philip robinson (drci, hospices civils de lyon) for help in manuscript preparation. we thank the authors, the originating and submitting laboratories for their sequence and metadata shared through gisaid on which this research is based. we gratefully acknowledge all the members of cov-glue, nextstrain.org, and virological.org for sharing their analysis in real time. key: cord- -byxuruk authors: fritsch, annemarie; schweiger, brunhilde; biere, barbara title: influenza c virus in pre-school children with respiratory infections: retrospective analysis of data from the national influenza surveillance system in germany, to date: - - journal: euro surveill doi: . / - .es. . . . sha: doc_id: cord_uid: byxuruk introduction: recent data on influenza c virus indicate a possible higher clinical impact in specified patient populations than previously thought. aim: we aimed to investigate influenza c virus circulation in germany. methods: a total of , samples from to year-old children presenting as outpatients with influenza-like illness (ili) or acute respiratory infection were analysed retrospectively. the samples represented a subset of all samples from the german national surveillance system for influenza in this age group in – . the presence of influenza c virus was investigated by real-time pcr. for positive samples, information on symptoms as well as other respiratory virus co-infections was considered. retrieved influenza c viral sequences were phylogenetically characterised. results: influenza c viral rna was detected in ( . % of) samples, including during the / season. the majority ( / ) of influenza c-positive patients had ili according to the european union definition, one patient had pneumonia. viruses belonged to the c/sao paulo and c/kanagawa lineages. most ( / ) samples were co-infected with other respiratory viruses. conclusion: our data are the first on influenza c virus circulation in germany and notably from a european national surveillance system. the low detection frequency and the identified virus variants confirm earlier observations outside a surveillance system. more virus detections during the / season indicate a variable circulation intensity in the different years studied. influenza c virus can be considered for ili patients. future studies addressing its clinical impact, especially in patients with severe disease are needed. influenza viruses are a major threat to human health and are therefore in the focus of national and international health authorities. among these, influenza virus types a and b are the main considered, as they cause annual epidemics with high morbidity and considerable mortality [ ] . in contrast, influenza c virus has been regarded as a pathogen of minor relevance, causing mild or clinically unapparent disease [ , ] . nevertheless, in recent years, detections of influenza c in hospitalised young children with (severe) lower respiratory tract disease were reported [ ] [ ] [ ] [ ] [ ] [ ] . thus, the clinical and epidemiological significance of this virus species might have been underestimated and needs to be reassessed. in europe, the burden of influenza c virus infection in children and adults is largely unknown, as no systematic surveillance data are available. the few studies published mainly focus on clinical data, mostly from hospitalised children [ , , ] . in germany, no surveillance data and no sequence information on circulating influenza c viruses have ever been reported. therefore, we decided to search for influenza c in our outpatient sample collection assembled for the purpose of influenza virus surveillance in germany. as young children are described to have the highest infection rates [ , , ] , we confined our study to the - year-old age group. we furthermore sequenced the haemagglutinin esterase (he) gene from influenza c-positive samples to phylogenetically characterise the detected viruses. all samples were collected from practitioners participating in the national influenza surveillance, who are distributed over the complete german territory and represent a statistically valid proportion of the german population [ ] . these practitioners continuously collect nasal or throat swabs from non-hospitalised patients presenting with symptoms of influenza-like illness (ili) according to the european union (eu) definition or an acute respiratory infection (ari). an ili case is defined by a sudden disease onset with at least one of four systemic symptoms (fever or feverishness, malaise, headache, myalgia) and at least one of three respiratory symptoms (cough, sore throat, shortness of breath) [ ] , while ari is an acute respiratory disease with at least one of the four following symptoms: fever, cough, rhinorrhoea or sore throat. the samples are sent to the german national influenza centre, accompanied by a completed questionnaire on patient characteristics, sampling date, disease symptoms, influenza vaccination status and therapeutic intervention i.e. antiviral treatment. they are routinely analysed for influenza virus types a and b, human respiratory syncytial virus (rsv) as well as -since april -human adenovirus (adv), metapneumovirus (hmpv) and rhinovirus (hrv). all samples are stored at - °c afterwards. for this study, a subset of , samples ( . %) was selected from a total number of , samples taken from children ≤ years of age in the years - as described in the supplementary file (supplement s ). briefly, at least every second sample in a chronological order was retrospectively analysed for influenza c virus. to extend the basis for the co-infection data, all influenza c-positive samples were additionally tested for human parainfluenza viruses types - and coronaviruses oc , nl , hku and e. positive samples taken before april were furthermore retrospectively examined for adv, hmpv and hrv. the conduct of a sentinel surveillance is covered by german legislation ( § , § , protection against infection act). the german national surveillance of influenza and other respiratory viruses was furthermore approved by an ethical committee of the charitè berlin (application number ea / / ). additionally, for all samples a written consent was given for their inclusion in research studies. all analyses were done with pseudonymised data. after their arrival in the laboratory, ml of cell culture medium (minimum essential medium (mem) with for sequence analyses, cdna was synthesised with the accuscript hi-fi reverse transcriptase (agilent, santa clara, us) and a primer that binds to the conserved ' end of the rna gene segments (uni , see table ). assay validation was performed with synthetic double stranded dna strings (gblocks; idt, skokie, us) in singleplex as well as duplex format (including the internal control fcv) on -well as well as -well plates. for the determination of the linear detection range and the correlation (r ) of quantification cycle (c q ) values, a -fold dilution series ( - copies per reaction) was examined in duplicates. pcr efficiency was calculated by inserting the slope value of the standard curve into the formula e = (- /slope)- . the limit of detection (lod) was established as % detection probability, calculated by probit analyses of the results of a -fold examination of low copy numbers ( - . genome equivalents per reaction) applying the ibm spss statistics software. for intraassay precision, gblocks were examined sixfold in a single run, while for interassay precision the intraassay data were extended by two additional runs with double reactions. all reproducibility runs were performed on consecutive days in independent experiments, and precision was described as standard deviation of the observed c q values. conventional pcr for sequence determination of the he gene was carried out in a total reaction volume of µl. the reaction contained x extaq buffer, . mmol/l dntp (thermo fisher scientific, waltham, us) with dutp (ge healthcare, chicago, us), . u extaq polymerase (takara, kusatsu, japan), nm primers (metabion, planegg, germany) as listed in table , and µl of the prediluted cdna. alternatively, the superscript monthly distribution in - year-old children of (a) the number of samples tested for influenza c, as well as testing coverage among samples received by table ) were used for amplicon sequencing in cases where the nested pcr primers did not yield a sequence spanning the complete amplicon. all he sequences were processed and assembled in the geneious software before their deposition at the global initiative on sharing all influenza data (gisaid; www. gisaid.org) database (epi -epi ). the applied amino-acid numbering includes the signal peptide. all he sequence analyses were performed with geneious version . . . multiple sequence alignments were compiled on the basis of the mafft algorithm. the n-terminal sequences including the signal peptide sequence (mffslllmlgltea [ ] ) as well as the c-terminal region with incomplete sequence information (last nt including the stop codon) were excluded. the alignment for phylogenetic analyses thus covered the nt to , of the complete coding sequence and was calculated including reference sequences downloaded from the gisaid database (see supplement s ). maximum likelihood trees were constructed applying the hky model and the spr tree topology search. branching reliability was estimated by performing , bootstrap replicates. trees were manually edited in corel draw x . a qpcr assay for the detection of influenza c viruses was established as singleplex as well as duplex qpcr including our routine internal control fcv. the assay proved to be a robust and sensitive tool and furthermore did not show any cross-reactivity to a variety of viral respiratory pathogens and to human genomic dna (validation results summarised in table ). the duplex qpcr approach was applied to retrospectively examine , throat or nasal swabs, of which , samples gave valid qpcr results, i.e. yielded either an influenza c or a fcv signal (or both) in duplex qpcr runs. twenty samples ( . %) were found positive for influenza c virus rna, with c q values ranging from to . the positive samples predominantly were taken between october and april , reaching an average positivity rate of . % ( / ) in these months ( . - . % per month, figure ). outside of this particular winter season, viruses were identified only sporadically with detection rates of . % ( / , january-april ) or . % ( / , october -april . no virus detection was achieved from may to september of any year studied. also, no particular age distribution could be observed ( figure ). all influenza c-positive samples were additionally examined by qpcr to identify other respiratory viruses. more than half of the influenza c-positive patients ( / ; %) proved to be co-infected with diverse other respiratory pathogens (table ) , with influenza c c q values covering the complete range of to . all patients with influenza c virus infection reported fever and cough. fifteen patients reported a maximum temperature between . °c and . °c, while for the remaining five patients the maximum temperature was not provided. additionally, a sudden disease onset ( / ) , rhinitis ( / ) , sore throat ( / ) and muscular pain and/or headache ( / ) were predominant symptoms. clinical signs of pneumonia were reported for one patient with an influenza c c q value of , but also low amounts of influenza a(h n ) were detected in this sample. in patients with a sole influenza c virus infection, the sudden disease onset ( / ), the maximum fever ( . °c - . °c in patients), rhinitis ( / ), sore throat ( / ) and muscular pain and/or headache ( / ) were reported in similar proportions. the sequencing of the he gene was achieved for samples, of which three yielded only partial sequences. two of the incomplete sequences covered a consecutive stretch of , and , nt, respectively, while the three incomplete sequences were only characterised based on the nt homologies to other sequences. the two fragments of sample - ( nt, nt) are % identical to our sample sequence - , which belongs to the c/victoria/ / subgroup of the c/sao paulo clade. similarly, sample sequence - ( , nt) is % identical to the sequence of c/sao paulo samples - and - , which group into the c/miyagi/ / subgroup of the c/aichi/ / subclade. sample sequence - has a similarity of > . % to the same sao paulo lineage cluster, while the similarity to c/victoria/ / ( . %) and the prototype strain sequences for the other five he lineages is lower (≤ . %). although influenza c virus was discovered years ago, there is only little knowledge on the biology and epidemiology of this virus type. some studies indicated a low clinical impact with only mild symptoms [ , , ] , and in spite of a high seroprevalence in the population, virus detections were rare [ , , ] . these findings led to the conclusion that influenza c infection is common, but clinically inapparent or too mild to require a visit to a doctor [ ] . additionally, the low detection rate may be in part due to the fact that in earlier times virus diagnostics were mainly based on virus culture, which is difficult for influenza c [ , ] and necessitates conditions that differ from influenza a and b virus cultivation [ ] . as a consequence, influenza c virus diagnostics were restricted to specialised laboratories and correspondingly rare [ ] . with the introduction of molecular methods, influenza c has been increasingly included into studies on respiratory pathogens and clinical diagnostics. thereby, the low detection rates in the general population were confirmed, but a higher clinical impact for paediatric patients was indicated, as influenza c was described to also cause lower respiratory tract disease [ ] [ ] [ ] [ ] [ ] . in a -month prospective study (december -may including japanese children with communityacquired pneumonia, bronchiolitis or bronchitis, influenza c infection was identified even with a prevalence approximating those of influenza a or hmpv [ ] . further studies, mostly in children, described the symptoms of influenza c infection to be indistinguishable from influenza a and b infections [ , ] , although the maximum body temperature may be lower and the fever shorter compared with influenza a [ , , ] . in finnish military recruits, influenza c virus caused common cold-like symptoms, but occasionally resulted in pneumonia and bronchitis [ ] . for europe, only little information on influenza c circulation has been published. in adults, a seroprevalence of ca % and more was found in france [ ] , finland [ ] , and united kingdom [ ] . applying pcr on samples from all age groups, a virus detection rate of ≤ % was reported for normandy/france [ ] , scotland [ ] and spain [ ] , but higher detection rates of . - . % were found in two adult studies from finland [ , ] . outside europe, a similar seroprevalence as well as comparable detection rates have been described for australia, canada, japan, nigeria, the philippines, peru and the us [ ] [ ] [ ] , , , , , [ ] [ ] [ ] . in view of the lack of knowledge on influenza c virus circulation in germany, we decided to generate the first systematic data on the basis of our national influenza virus surveillance. we chose to examine the age group of - years, as young children have been shown to have the highest infection rates [ , , , ] . we analysed a representative subset of the , samples received in this age group between and ( . - % of all samples in the corresponding month). first, we validated a previously published qpcr [ ] and duplexed it with our routine internal control, fcv. in an extensive validation effort according to international standards [ ] , we found the singleplex as well as the duplex format to perform with high sensitivity, specificity and precision. we therefore applied it to our sample compilation and identified influenza c rna in of , samples with valid qpcr results ( . %). the vast majority of virus detections ( of ) was found in samples that were collected between october and april , signalling a more pronounced virus circulation during these months with positivity rates of up to . % ( / ) in november . as these samples were collected in of german federal states, virus circulation was not confined to a region, but widespread, maybe even nationwide. the virus prevalence however was markedly lower during the other winter seasons observed in this study, and no virus could be detected during the summer months. this absence of summer circulation is in congruence with reports from japan, france, finland and spain [ , , , , ] , but is in contradiction to a report from catalonia in spain, in which the majority of positive samples were taken in august and september of the observed time span [ ] . an upsurge of influenza c virus circulation in the spring of was also observed in the philippines [ ] , but did not occur in japan, from where virus circulation in even numbered years was reported, including the years and [ , ] . however, a biennial pattern of virus circulation with increased or time-shifted profile has also been described for other respiratory viruses in germany [ , ] and therefore is conceivable, but remains open in our study due to the short study period, which presents a limitation. in total, the proportion of influenza c-positive patients was small, but within the expected range. it needs to be emphasised though, that the obtained overall positivity rate is largely based on only few months during the winter season / with substantial virus circulation in our study population. because of our limited access to clinical data, only few conclusions can be drawn with regard to the clinical relevance of influenza c virus. the vast majority of patients ( / ) carrying the virus fulfilled the eu ili definition. although, due to our study design, there may be a bias to ili cases during periods of influenza a and b virus circulation, our findings are in concordance with other studies, in which ili was described for the majority or all of influenza c infected patients [ , ] . bronchitis or bronchiolitis was not reported for any patient, but one child (infected also with an influenza a virus) presented with symptoms of pneumonia. however, the proportion of pneumonia in our influenza c-positive samples does not differ considerably from that of our complete sample collection of this age group spanning the years to (data not shown). in our ambulant setting, we thus do not see an indication for an accumulation of lower respiratory tract disease in influenza c infected patients, but an influenza-like clinical presentation is common. interestingly, a substantial share of influenza c-positive samples showed co-infection with other pathogens, as reported also in other studies [ , , , ] . in these cases, the cause for ili symptoms cannot clearly be attributed to influenza c. yet, we used qpcr assays with comparable performance characteristics (lod and efficiency), so that a comparison of the obtained c q values can be semiquantitatively interpreted for the different pathogens within one sample. in our study, the majority of co-infected samples ( / ; . %) exhibited the highest viral load for influenza c, including the pneumonia case for whom it was ca , -fold higher than that of influenza a at the time point and the sample site examined. in four samples, the influenza c c q was close to the detection limit (≥ ) and thus influenza c was presumably of minor relevance. repetitive sampling from the same patients and continuous parallel assessment of the patients clinical presentation could clarify the role of the single pathogens in the disease course, but is not included in our routine influenza surveillance system. therefore, we cannot judge on the temporal dynamics of virus replication and the clinical impact of each virus detected. due to the slow evolution and thus high antigenic homology of influenza c virus [ ] , we decided to characterise the german sequences only on the basis of the he gene sequences. the he glycoprotein has a variety of functions in the viral replication cycle and greatly determines the antigenicity of the virus [ ] . based on antigenic and phylogenetic characteristics of this protein, distinct virus lineages have been described that were named after their prototype strains c/taylor/ / , c/kanagawa/ / , c/mississippi/ , c/aichi/ / , c/ yamagata/ / and c/sao paulo/ / [ , ] . all influenza c lineage clusters are comprised of isolates from a multitude of continents, indicating a global circulation of virus lineages [ ] . however, four lineages seemingly disappeared (c/taylor, c/aichi, c/ mississippi, c/yamagata), and only the c/kanagawa and c/sao paulo lineage have been detected within the last decade [ , , , , , , ] . from our positive samples, a total of partial and near full-length he sequences could be generated. we almost exclusively detected c/sao paulo lineage viruses, and only two c/kanagawa viruses were identified. our c/ sao paulo sequences add to both lineage subclades described by matsuzaki et al. and represented by c/ aichi/ / and c/victoria/ / [ ] . a total of sequences ( complete, incomplete) group into the c/aichi/ / subclade and were sampled between march and april , while three additional sequences ( complete, incomplete) group into the c/ victoria/ / subclade and were sampled between november and march . thus, viruses of both subclades co-circulated during the / winter season. in contrast, the two c/kanagawa lineage viruses were sampled in march and april , thus outside of the period with increased infection rates. they form a distinct cluster within the c/kanagawa clade, most closely related to c/miyagi/ / . both c/ kanagawa viruses are almost identical to each other showing a nt homology of . %, although they were sampled with a -year distance. their closest neighbour, c/miyagi/ / even has a homology of . % and . % on the nt level. this further supports the described genetic stability of this virus type compared with influenza a and b viruses [ ] , possibly also reflecting their antigenic properties. to summarise, our study is the first report on influenza c circulation in the context of a nationwide outpatient influenza surveillance system in europe. we found influenza c in a proportion of samples that was in accordance with previous reports. an increased and widespread virus circulation was observed during the winter and spring months of / , with viruses predominantly belonging to c/aichi/ / subclade of c/ sao paulo lineage viruses. infected patients showed symptoms of ili including upper as well as lower respiratory tract infection, although its association to the observed clinical symptoms remain uncertain in the majority of cases due to the identified co-infections. further knowledge is needed about the virus epidemiology, its transmission patterns, its role in sole and mixed infections as well as the associated disease burden, especially in young children and patients with lower respiratory tract disease. bootstrap analyses were performed applying the maximum likelihood algorithm with , replicates as described in the methods section. the tree is rooted at c/taylor/ / . only bootstrap values greater than are displayed at the branch nodes. prototypes of influenza c virus lineages are in bold font and blue colour, subclade representatives are bold and underlined. the german sequences are marked in bold italic letters influenza (seasonal) fact sheet. geneva: who production of common colds in human volunteers by influenza c virus influenza c virus infection in military recruits--symptoms and clinical manifestation prospective study of influenza c in hospitalized children influenza c virus and human metapneumovirus infections in hospitalized children with lower respiratory 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of medically attended respiratory illness in adults minimum information necessary for quantitative real-time pcr experiments genetic lineage and reassortment of influenza c viruses circulating between and human metapneumovirus: insights from a ten-year molecular and epidemiological analysis in germany genetic variability of group a human respiratory syncytial virus strains circulating in germany from to hemagglutinin-esterase-fusion (hef) protein of influenza c virus analyses of evolutionary characteristics of the hemagglutinin-esterase gene of influenza c virus during a period of years reveals evolutionary patterns different from influenza a and b viruses we thank susi hafemann, nathalie tollard and uwe kozian for excellent technical assistance. we also acknowledge all laboratories that contributed influenza c virus sequences to the gisaid database and thereby enabled our analyses. none declared. af: screening and sequencing of patient samples, manuscript preparation; bs: design of study, manuscript preparation; bb: design of study, data analyses, manuscript preparation. this is an open-access article distributed under the terms of the creative commons attribution (cc by . ) licence. you may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence and indicate if changes were made.any supplementary material referenced in the article can be found in the online version. key: cord- -pelz ygf authors: nan title: october new in review date: - - journal: j acad nutr diet doi: . /j.jand. . . sha: doc_id: cord_uid: pelz ygf nan researchers analyzed how experiences with weight-related stigma and discrimination over the course of a lifetime are connected to physical activity among people with obesity. using a social constructivist perspective for analysis, the researchers designed a retrospective biographical study, using a sample of adults. the sample was drawn from southwest germany by way of advertisements via the university hospital help centre. participants had to have a minimum body mass index (bmi) of to be considered. the sample was % female, with a mean age of . years, spanning to years. mean bmi was . . led by the researchers, participants were asked to reconstruct and assess past experiences with weight stigma and physical activity over the course of their life, so as to proceed from a (re)constructivist perspective. a mixed-methods approach was used, combining the individual narrative interviews with a rating-scaled graphic elicitation tool whereby the x-axis represented a chronological sequence of the events, and the y-axis the intensity on a scale of to . data collection was performed by specifically trained research assistants. participants were actively engaged in the data input as well as the biographical narrative to generate insights into a specific generative mechanism of physical (in)activity. all data of the drawn graphs and curves were transferred into spss version (ibm corp, ) for statistical analysis. the quantitative analysis was supplemented with the qualitative data afforded by way of the interviews. the researchers' findings suggest a continuous decrease in physical activity from childhood to mid-adulthood, with weight-related discrimination in both sport and non-sport settings influencing this behavior. the association of a sweetened beverage tax with changes in beverages prices and purchases at independent stores. bleich s, lawman h, levasseur m, et al. health aff. ; https://doi.org/ . /hlthaff. . . investigators evaluated the influence of a beverage tax on prices and purchases among independent stores and customers. a natural experiment was designed to address this, using a sample of stores and , customer purchases. as intervention, the investigators used the january , implementation of a . -cent-perounce excise tax on sugar-sweetened beverages in philadelphia, pa, with baltimore, md, serving as the untaxed control. stores were included if independently owned and selling at least three of the beverages assessed by the tax. at baseline, customer demographics for the sample were . % and . % male in philadelphia and baltimore, respectively. demographically, the customer bases of these stores were . % and . % white, . % and . % black, . % and . % hispanic, and . % and . % termed "other" in philadelphia and baltimore, respectively, at baseline, before the tax implementation. the primary outcome sought was the change in mean beverage price in cents per ounce of the taxed and nontaxed beverages. the taxed beverages numbered , with a comparative list of seven nontaxed. the study was conducted from october to december . the investigators conducted objective purchase assessments at both philadelphia and baltimore stores. researchers stood outside participating stores on weekdays at three intervals: : to : am, : am to : pm, and : to : pm, for days and queried customers on exit. researchers recorded volumes, quantities, and prices for each item purchased, as well as demographic information and shopping frequency of the customers. objective price data were obtained at the participating stores. statistical analysis was performed using sas . (sas institute, ). the investigators report significant declines in purchases of the taxed beverages after the tax implementation. researchers examined the shape of sexspecific associations of dietary protein intake with -and -year changes in muscle mass and gait speed, as well as mobility limitation in older adults. a prospective cohort study was designed, using a sample of , . the sample was drawn from participants in the health abc study, which began in . participants were recruited from a random sample of medicare-eligible residents of memphis, tn, and pittsburgh, pa. participants were eligible if free of difficulty walking one quarter mile and climbing steps. participants were excluded if missing relevant data for the study or if they dropped out during the -year project. the sample was . % male, . % female, . % white, . % black, and had a mean age of . years. body composition was measured annually by dual x-ray absorptiometry scans (hologic a, version . a), with usual gait speed used as an objective measure of physical function. participants were asked to walk a -minute course at their normal pace. absolute -and -year changes in gait speed were calculated by subtracting baseline speed from follow-up measurements. mobility limitation was used as a subjective indicator of physical function and defined as two consecutive reports of difficulty walking the quarter mile or climbing steps. dietary intake was assessed using a -item, interviewer-administered version of the block food frequency questionnaire. dietary protein intake was expressed in grams per kilogram of adjusted body weight daily. demographic variables were collected via a questionnaire. depressive symptoms were measured using the center for epidemiological studies depression scale and the modified mini-mental state examination. statistical analysis was conducted using spss version (ibm corp., ) and r version . . (r foundation for statistical computing, ). researchers report that in women, higher protein intake was associated with less appendicular lean mass loss over years, but not over , or with gait speed decline. prompting consumers to make healthier food choices in hospitals: a cluster randomized controlled trial. allan j, powell d. int j behav nutr phys act. ; https://doi.org/ . /s - - -z. researchers tested the effects of point-ofpurchase prompts (ppps) specifically designed to facilitate product comparison by displaying food products in order from highest to lowest energy content. a twoarm cluster, randomized controlled trial was designed to test this, using a sample of hospital food shops in scotland. the sample included food shops owned by one national retailer, the royal voluntary service. shops were eligible if selling snack food, located in a hospital, and accessible to staff, patients, and visitors. the targeted behavior was the purchase of different single-serve snack items deemed unhealthy, including confectionary, fruit, dried fruit snacks, crisps, savory snacks, cereal bars, pre-portioned cakes, muffins, and tray-bakes. the -week intervention was a ppp in the form of a display sign near shop shelves. the sign displayed the available single-serve snacks in order from lowest calorie to highest, with a picture of the item and number of calories. the ppp was designed by a multidisciplinary team of psychologists and dietitians. the ppp also had in print: "if you are trying to eat less, then choose a snack from the left." the shops in the sample were classified by annual revenue in three levels: low, medium, and high. fifteen of the shops were randomized to the intervention and to the control. the primary outcome was the average energy content of products consumed each day, with secondary outcomes including average fat and sugar content of products purchased per day, as well as the average cost of each product purchased and the number of products purchased. statistical analysis was performed using spss version (ibm, ). the researchers report that snacks purchased from the intervention sites were on average lower in calories and sugar at follow-up, but there was no effect on fat content or number of units sold. enhanced long-term dietary change and adherence in a nutrigenomicsguided lifestyle intervention compared to a population-based (glb/ dpp) lifestyle intervention for weight management: results from the now randomized controlled trial. horne j, gilliland j, o'connor c, et al. bmj nutr prevent health. ; https://doi.org/ . / bmjnph- - . hypothesized that a nutrigenetic-based weight management intervention would motivate greater dietary change relative to a populationbased weight management intervention. a randomized controlled trial using a sample of participants was designed to test this question. participants were recruited through health care professional referrals in ontario, canada, between april and september . inclusion criteria were bmi equal to or greater than . ; years or older; english-speaking; willing to undergo genetic testing; access to the internet; not presently working with another health care provider toward a weight loss goal. the sample had a mean age of . years, was . % female, . % white, and had a mean bmi of . the sample was divided into control and intervention groups, both with participants. both treatments lasted months and incorporated elements of the theory of planned behavior. the group lifestyle balance (glb) group served as control and received only the population-based weight loss intervention, whereas the glbþngx group received the same intervention in addition to the modified nutrigenomics component. the glb group received instructions to follow a calorie-controlled, moderately low-fat nutrition plan. the glbþngx group received that in addition to personalized information relating to resting metabolism and specific calorie deficits recommended for weight loss. the intervention group was also advised to focus on the macronutrient recommendations as highlighted in their genetic report. all participants tracked food and beverage consumption by way of food records. genotyping was performed by way of oragene on- saliva collection kits (dna genotek, ottawa, canada). single nucleotide polymorphisms of interest included: ucp (rs ), fto (rs ), tcf l (rs ), apoa (rs ), ppary (rs ), and mc r (rs ). change in dietary intake was a predetermined secondary outcome and was measured using validated multiple-pass methods at baseline and at the -month, -month, and -month points. statistical analysis was performed using ssps version . (ibm, ). the investigators report that only the glbþngx group significantly reduced total fat intake between baseline and the -month follow-up. , ) . the authors report no overall impact of the legislation on obesity, but for children in poverty, the risk of obesity fell substantially each year after its implementation. global incidence of necrotizing enterocolitis: a systematic review and meta-analysis. alsaied a, islam n, thalib l. bmc pediatr. ; https://doi.org/ . /s - - - the authors assessed the global incidence of necrotizing entercolitis (nec) in verylow-birth-weight (vlbw) infants. a systematic review and meta-analysis was designed to study this issue, using cohort studies involving , neonates. the sample included eight studies from the united states; four from china, korea, singapore, and malaysia; three from australia; one from the middle east; one from india; and from europe. eligible studies included cohort-or population-based studies of newborns, including registry data, both prospective and retrospective, studies reporting the number, and studies including the frequency or incidence of confirmed nec in preterm infants or vlbw infants along with appropriate denominator. excluded were studies with unclear case definitions of nec, randomized controlled trials, experimental studies, and case series in which no denominator data are available to compute the incidence. the search was conducted between september and december and included pubmed, medline, embase, the cochrane library, the african index medicus database, latin america and caribbean center of health science international, open grey, indmed: koreamed, virtual health library, national library of australia, and social care online. search terms included enterocolitis, necrotizing, epidemiology, incidence, cohort studies, population-based studies, epidemiological data, prematurity, very low birth weight, clinical study, cohort analysis, and human. analysis was performed using metaxl and comprehensive meta-analysis (comprehensive meta-analysis version , ). the authors report that seven of vlbw infants in neonatal intensive care units are likely to develop nec, although considerable heterogeneity was found between studies. the relationship between overweight and overactive bladder symptoms. hagovska m, svihra j, bukova a, et al. obes facts. ; https://doi.org/ . / . the authors investigate the relationship between body fat percentage (bfp) and the severity of overactive bladder (oab) symptoms by assessing the impact on quality of life in a group of young, overweight women. a cross-sectional study of participants was designed for the project. the sample was % female and recruited from two universities in slovakia. eligibility criteria included being enrolled students, nulliparous female, aged to years, a bmi between and . . exclusionary criteria were infection of the urinary tract, surgical treatment of gynecological and urological illness, and symptoms of stress urinary incontinence. mean age of the sample was . years, with a mean bmi of . . the study was conducted between march and september . all participants completed a screening questionnaire at the point of application to collect demographic data. body composition analysis was performed using a biospace body composition analyzer, with measurements including bfp, visceral fat area, skeletal muscle mass, body fat mass, and waist-to-hip circumference ratio. participants used a voided diary that evaluated voided volume, number of voidings per hours, voided volume during the day, daytime frequency, voided volume during the night, and nocturia. data for days and average values were calculated. the oab questionnaire was used to determine the symptoms of urgency urinary incontinence, the patient perception of intensity of urgency scale to determine severity, and the urinary incontinence quality of life scale for qualitative measures. statistical analysis was performed using spss version . (ibm, ) . the authors report that young women with a bfp greater than % were % more likely to have oab than their peers. can we walk away from cardiovascular disease risk or do we have to "huff and puff"? a cross-sectional compositional accelerometer data analysis among adults and older adults in the copenhagen city heart study. johansson m, sogaard k, prescott e, et al. int j behav nutr phys act. ; https://doi.org/ . /s - - -w. researchers investigated the relationship between sedentary behavior, walking, and high-intensity physical activity (hipa), and risk factors for cardiovascular disease among adults and older adults. a cross-sectional compositional data analysis of data collected as part of the copenhagen city heart study (cchs) was designed to address this question, using a sample of , participants. participants from the fifth examination of the cchs conducted between october and february were invited to participate. eligibility criteria included having more days of measurements with hours or more of accelerometer recordings per -hour day; no use of antihypertensive diuretics or cholesterollowering medications; and no missing variables. the , -participant sample contained adults younger than age years, and adults older than . the cohorts were . % and % female, with a median age of . and . years, respectively. participants completed a questionnaire regarding socioeconomic status, health, diet, and medication use. participants were examined at the cchs test center in the capital region of denmark for height, weight, waist circumference, and lowdensity lipoprotein cholesterol. acceleratorbased measurements of physical behaviors were obtained by way of two tri-axial accelerometers, actigraph gt xþ, (actigraph, pensacola, fl). the software package matlab-software acti (national research centre for the working environment, copenhagen, denmark) was used to derive the time spent lying, sitting, standing, moving, walking, climbing stairs, running, cycling, and rowing. physical behavior was defined as time in minutes per -hour day spent in sedentary behavior (ie, sum of lying and sitting), standing, moving, walking, hipa, running, cycling, and rowing. the outcomes sought were systolic blood pressure, waist circumference, and low-density lipoprotein cholesterol. statistical analysis was performed using rstudio version . . (rstudio, boston, ma, ) and r version . . (r foundation for statistical computing, vienna, austria, ). the researchers report findings that suggest less sedentary behavior and more walking is associated with lower risk of cardiovascular disease among older adults, whereas hipa types are associated with lower risk among all adults. dietary interventions for healthy pregnant women: a systematic review of tools to promote a healthy antenatal dietary intake. beulen y, super s, de vries j, et al. nutrients. ; ( ): . the investigators sought a review of tools to be integrated into antenatal care for promotion of healthy dietary behavior in healthy pregnant women. a systematic review of the literature was designed to address this issue, using a sample of published papers. the sample consisted of randomized controlled trials and five formative evaluations. six of the studies were conducted in australia, five in the united states, and six in europe. eligibility criteria included journal articles describing at least one tool potentially used by health care providers to promote healthy dietary intake in healthy, pregnant women; and articles published in western countries. databases used in the search were pubmed and web of science. data extracted included general characteristics and relevant outcome measures. reference management software mendeley desktop version . . (mendeley, ) was used during screening and microsoft excel in the data extraction. data were analyzed through a narrative synthesis. the investigators report findings suggesting that custom tools that are sensitive to inequalities are needed to support a diversity of women in obtaining and maintaining a healthy diet during pregnancy. comparative effectiveness of glucoselowering drugs for type diabetes: a systematic review and network metaanalysis. tsapas a, avgerines i. ann intern med. ; https://doi.org/ . /m - . influence of cinnamon on glycemic control in subjects with prediabetes: a randomized controlled trial. romeo g, lee j, mulla c, et al. j endocr soc. ; https://doi.org/ . /jendso/bvaa . effects of vitamin d supplementation on prevention of type diabetes in patients with prediabetes: a systematic review and meta-analysis. volunteering and subsequent health and well-being in older adults: an outcome-wide longitudinal approach. kim e, whillians a, lee m, et al. am j prevent med. ; ( ) : - . effect of probiotic use on antibiotic administration among care home residents: a randomized clinical trial. economic benefit of dieteticnutritional treatment in the multidisciplinary primary care team. casas-agustench p, megias-rangil i, babio n. nutr hosp. ; https://doi.org/ . /nh. . long-term development effect of withholding parenteral nutrition in pediatric intensive care units: a -year follow-up of the pepanic randomized controlled trial. jacobs a, dulfer k, eveleens r, et al. lancet child adolesc health. ; ( ): - . home enteral nutrition in adults: nationwide multicenter survey oncology the role of diet in cancer prevention and chemotherapy efficacy pediatric obesity treatment among adolescents: a review of current evidence and future directions tummy time," and screen time at and months of child age: a -group randomized clinical trial veganism and pediatric food allergy: two increasingly prevalent dietary issues that are challenging when co-occurring change, predictors and correlates of weight-and health-related quality of life in adolescents -years following bariatric surgery the dynamic relationship between asthma and obesity in school-children identification of temporal condition patterns associated with pediatric obesity incidence using sequence mining and big data becoming your healthiest self: an eatwell, get-fit, feel-great guide for teens pediatric to adult transitions of ketogenic dietary therapy for epilepsy maternal long-chain polyunsaturated fatty acid status, methylmercury exposure, and birth outcomes in a high-fish-eating mother-child cohort policy & advocacy cardiorespiratory fitness in youth: an important marker of health: a scientific statement from the position statement-european association for the study of obesity (easo): obesity and covid- : the two sides of the coin dietitians australia position statement on telehealth public health nutrition regulates innate immunity in health and disease health impact and cost-effectiveness of volume, tiered, and absolute sugar content sugar sweetened beverage tax policies in the united states: a microsimulation study dietary micronutrients in the wake of covid- : an appraisal of evidence with a focus on high-risk groups and preventative healthcare observational study of the associations in high school football with self-rated health, obesity, and pain in adulthood the united states: differences by region and rurality renal nutrition effect of dietary potassium restriction on serum potassium, disease progression, and mortality in chronic kidney disease: a systematic review and meta-analysis keeping the diet simple and natural in chronic kidney disease: a south african-based dietary infographic the efficacy of prebiotic, probiotic, and symbiotic supplementation in modulating gut-derived circulatory particles associated with cardiovascular disease in individuals receiving dialysis: a systematic review and meta-analysis of randomized controlled trials research association between adult acne and dietary behaviors: findings from the nutrinet-sante prospective cohort study human milk oligosaccharide profiles and allergic disease up to years the effect of vitamin d supplementation on insulin sensitivity: a systematic review and meta-analysis body mass index and risk for intubation or death in sars-cov- infection: a retrospective cohort study effects of vitamin d supplementation on general and central obesity: results from randomized controlled trials involving apparently healthy populations association between chocolate consumption and risk of coronary artery disease: a systematic review and meta-analysis physical activity associations with bone mineral density and modification by metabolic traits school nutrition association of the healthy, hunger-free kids act with dietary quality among children in the us ): fat mass (rfm): association with mortality in nhanes covid- and the role of chronic inflammation in patients with obesity dietary intake and biomarkers of linoleic acid and mortality: systematic review and meta-analysis of prospective cohort studies quality improvement pilot study of the living your best weight program: a health at every size approach association between plant and animal protein intake and overall and causespecific mortality vitamin d supplements for prevention of tuberculosis infection and disease zinc as nutritional intervention and prevention measure for covid- disease women's health obesity and cardiovascular disease in women promoting cardiovascular health for african american women: an integrative review of interventions fertility and pregnancy outcomes in women with polycystic ovary syndrome following bariatric surgery key: cord- - aj ozpn authors: mutzel, p.; bertram, a.; jünger, p.; krieger, h.; schmitz, s.; jünger, m. title: increasing virus test capacity via recursive pool testing with an application to sars-cov- testing date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: aj ozpn in the context of adequate reactions to the current covid- pandemic, seifried, ciesek et al. [ , ] have proposed the application of sars-cov- pool testing in the pursuit of increasing testing capacity. we show how this method can be substantially improved in realistic scenarios, and we point out a possible impact on the ongoing discussion concerning the need of increased testing as a complementary measure to relaxed restrictions. seifried, ciesek, et al. [ , ] have proposed the application of sars-cov- pool testing in april , see also [ , ] . the described procedure uses two aliquots of each sample. the first set of aliquots is partitioned into pools of a given size and these pools are tested. a negative test result means that all corresponding samples in the pool are negative, a positive test result requires testing the corresponding samples in the pool individually using the corresponding second set of aliquots. the authors state: "dabei wird der abstrichtupfer zunächst in ein archivröhrchen gegeben und anschließend in ein poolgefäß. da sich bei dieser poolmethode das volumen im poolgefäß nicht vermehrt, wird auch keine verdünnung und damit keine abnahme der empfindlichkeit (sensitivität) beobachtet." according to the article, this pool test procedure was developed and patented by the goethe university and the drk blood donation service and leads to the fact that a constantly demanded extension of testing is made possible. the procedure was tested on samples, of which were sars-cov- positive. these samples were divided into pools of samples each. in a newer article, lohse et al. [ ] report on testing , samples, of which are positive, they used pools of size that were again subdivided into subpools of size , and they needed only tests. this strategy requires (rather than ) aliquots of each sample. the authors state that borderline samples might escape detection in pools of size . however, they argue that those stem from almost recovered persons. both research groups emphasize a tremendously increased testing capacity when the infection rate is low and consequently, many pool tests will have a negative result. we review the method in [ , ] , henceforth called "the frankfurt method", also in view of the variant in [ ] , henceforth called "the saarbrücken variant", propose a new variant based on recursive pool testing, demonstrate its potential in comparison to the frankfurt method as well as the saarbrücken variant, and discuss the possible impact on strategies that accompany the current relaxation of restrictions due to the covid- pandemic. the idea is to combine many samples into one combined sample ("pool") and apply the test to the combined sample. if the result is negative, all samples in the combined sample are negative. if the result is positive, further testing is necessary in order to determine negativity/positivity for all samples in the combined sample. this idea has already been applied for syphilis testing of soldiers in the s, and is popular for hiv testing since . we are given n samples {s , s , . . . , s n } for testing. the frankfurt pool testing method proceeds as follows: . prepare to divide each sample s i into up to aliquots s i, and s i, . . partition the set of all n samples into subsets of a given size p < n. (in [ ] we have n = and p = , in [ ] we have n = , and p = .) . make a pool test for each subset using the aliquots s i, . (these tests can be performed simultaneously.) . declare all samples within each negatively tested subset negative. . test all samples within each positively tested subset individually using the aliquots s i, . rather than testing all samples in a positive pool, the idea is to again split this pool subset into (or more) smaller subsets for pool testing, and keep splitting as long as possible. in detail, our enhancement concerns steps and of the frankfurt pool testing method. for ease of exposition, we assume p to be a power of , i.e., p = k for some k. (the method can be easily adapted to arbitrary values of p.) in step , we moderately increase the number of aliquots: '. prepare to divide each sample s i into up to a = + log p aliquots {s i, , s i, , . . . , s i,a }. step is replaced by an application of the divide&conquer principle that is well-known in computer science: '. for each positively tested subset apply the divide&conquer method decribed next. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint for ease of exposition, we assume the pool subset to consist of the sample aliquots s b, , s b+ , , s b+ , , . . . , s b+p− , . these are the p consecutive aliquots starting at index b. e.g., for n = and p = , the pool subsets are {s , , s , , s , , s , } {s , , s , , s , , s , } {s , , s , , s , , s , } {s , , s , , s , , s , }. then, with e = b + p − , dc test(b, e, d) stands for "test the pool of the samples s b , s b+ , s b+ , . . . , s b+p− using aliquots d". so our task is dc test(b, e, ). here is dc test(b, e, d) in pseudocode: we have n = samples s , s , . . . , s all of which are negative but two, namely s and s , which are positive. the pool size is p = . both methods apply pool tests to the subsets the first and the third will have a negative result, the second and the fourth a positive result. up to this point, both methods will have applied tests. the frankfurt pool testing method will now apply individual tests to the sample aliquots s , , s , , s , , s , , s , , s , , s , , and s , ( individual tests) so that a total of tests is performed. the recursive pool testing method will replace {s , , s , , s , , s , } by {s , , s , } and {s , , s , } and make pool tests for both. it will get a positive result for the first and therefore test s , and s , individually. then it will replace {s , , s , , s , , s , } by {s , , s , } and {s , , s , }, make pool tests for both and get a negative result for the first and a positive result for the second. therefore it will test s , and s , individually. this involves another pool tests plus individual tests, so, again, a total of tests, no improvement in terms of the number of tests. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . here is an illustration of the actions of both methods (boxes correspond to tests, blue means "negative", red means "positive". both methods initially divide the entire set of samples so far we have split each pool into subpools. of course, we can just as well split into b > subpools. again, for ease of exposition, we assume that the pool size p is a power of b, i.e., p = b k for some k. in practice, we expect pools in which there is no or only one positive sample. in the former case, no further test is necessary. in the latter case, the positive sample is found after + b log b p tests. e.g., let p = and b = . then the pool of size is positive (first test). one of the two subpools of size (second and third test) is positive and requires another two individual tests (fourth and fifth test), i.e., a total of + log = tests. the situation is illustrated in figure . the case p = and b = with a total of + log = tests is illustrated in figure . is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint the analysis for two or more positive samples in a pool is more involved. if the pool contains two positive samples, then we can show that the function gives the expected number of tests as a function of b and p. the picture on the right of figure shows a plot of the function f (b, p). again, it turns out that b = is the best choice as long as p ≥ . indeed, for pool sizes from to , it would be optimal to split the pools into pieces, for pool size , the value b = would be ideal, and smaller pool sizes should be tested individually. three or more positive samples in a pool of size up to are quite unrealistic. our computational tests confirm that b = is the best choice in realistic scenarios with pool size . so we restrict our attention in the following to the -split version of recursive pool testing. according to the robert koch institute (rki), the number of tests per week from calendar weeks to varied between , and , for about laboratories, which is a total of about , tests per laboratory week on average, i.e., per day. the proportion of positive tests in germany has been between . % and %. in the st calendar week, , laboratory tests for the coronavirus (sars-cov- ) were carried out in germany, , ( . %) have been positive. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . currently, a typical laboratory seems to test about samples per day. accordingly, we have simulated tests for n = samples with pool size p = for varying infection rates. the minima/maxima/averages/medians have been taken over , , samples, in which the infected samples are uniformly randomly distributed. the results for the frankfurt method in comparison to the -split version of the recursive method are presented in table and, for the medians only, as a bar chart in figure . the results show that, with small infection rates up to about %, the new approach outperforms the frankfurt approach by more than %. for example, for an infection rate of . %, our method needs about tests compared to the frankfurt method with tests (which is a reduction of %). for larger rates, the advantage is less significant, and for rates from about %, the frankfurt approach is better. compared to current individual testing methods without pools, our experiments show that with a positive sample rate of . % the number of tests could be reduced by . %. cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . the frankfurt pool testing method increases the test capacity significantly and our simulations indicate that recursive pool testing has the potential to increase the test capacity even further by a factor of about in realistic scenarios. however, pool testing in general and the recursive method in particular have drawbacks that limit their applicability. we recall that we refer to the pool size as p and to the required number of aliquots as a. as lohse et al. [ ] state, borderline samples might escape detection in pools of size . however, they expect those samples might stem from almost recovered persons. with increased p, sensitivity, i.e., the chance to detect a positive sample in a pool of negative samples, diminishes. in pcr technology, ideal amplification follows a c pattern where c is the cycle number. assuming a -cycle pcr, one can estimate the amount of amplified dna by amplified dna = initial dna × . typically, a realtime pcr reliably detects positive samples as long as the target reaches the threshold value by cycle or . with each : dilution ( % virus concentration) the ct-value is increased by . so, for a :p dilution, ct values increase by log p. with a -size pool, one can assume an increase in the ct value from a single sample to a pool of log = . this means that samples with single-sample ct values or above might escape detection. with this in mind, the pool size can be determined by counterbalancing cost and sensitivity. as a working hypothesis for the following, we assume that pools should not be larger than , on the other hand, it is desirable to stay close to . individual testing requires no sample splitting into aliquots, in our notation a = , the frankfurt method requires up to a = , the saarbrücken variant up to a = , and our recursive method with -splits up to a = + log p aliquots of each sample, at pool size p = the latter amounts to a = aliquots like the saarbrücken variant, at pool size p = the latter amounts to a = aliquots. it is unclear, see also below, if a = is tolerable in laboratory practice. we have pointed out the superiority of the -split recursive method, but we have not yet taken a limitation of the number of required aliquots into account. if a ≤ is required, the -split method would have to set the pool size to only which is undesirable. likewise, the -split recursive method (b = ) with "natural" pool size p = = is undesirable due to small pool size. on the other hand, -split with "natural" pool size p = = is undesirable, because the pools get too big. an interesting alternative is to consider -splits (i.e., take b = ) and set the pool size to p = = . we have repeated the experiment reported in table and figure with this alternative in order to see if the number of tests deteriorates in comparison to the previous table for which up to aliquots were needed. we also ran the saarbrücken variant on the same instances in order to see how well it performs in comparison. the results are given in table and figure . we observe that recursive -split consistently outperforms the saarbrücken variant and its performance only moderately deteriorates in comparison to the -split recursive method. we also compared the same two methods as well as the -split recursive method on , , random instances of the original saarbrücken experiment, i.e., n = , samples, of which positive, randomly distributed, , , instances. the pool sizes are for the saarbrücken variant, for the -split, and for the -split recursive method, see table . the -split recursive method significantly outperforms the saarbrücken method (both require up to aliquots) even though it uses pools of size rather than , and its performance is is pretty close to the one of the recursive -split method that requires up to aliquots. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . however, with the / pooling, a lab can use the same pooling device with identical settings to produce -size pools by just using the previously generated size- -pools as input and still keep the original (aliquot ) as well as the size- -pools (aliquot ) and obtain the rd aliquot by making up the -size-pool. no re-programming is needed, and lab personnel does not need to keep in mind whether to pool , or samples as it would always boil down to in . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint with current technology, a batch with up to samples takes a little over hours to be analysed. any time to pool samples would have to be performed beforehand and laboratory experience indicates that this takes about another roughly hours for samples (pooled into pools of size , thus pools). assuming no invalid results and only valid batches, as well as neglecting setup-times and time taken to produce the pools, individual results are available after a hours, i.e., hours for individual testing (up to samples), hours with the frankfurt method, hours with the saarbrücken variant as well as the -split recursive method at pool size , and hours with the recursive -split method at pool size . while it takes hours to test , samples using a roche cobas analyzer, the authors have calculated that with pool size it takes a little more than hours for the same samples ( % positives), and, with recursive pool testing at size and , the turnaround-time would go up to hours. while the extended waiting times are intolerable in many scenarios like testing due to individual doctoral prescription or testing in known hot spots, they might well be tolerable in mass testing without previous indications, e.g., when opening a school with , students and teachers and testing at fortnightly intervals for possible early detection of infections, mainly in settings with very few or no positive samples to be expected. of course, in order to convince laboratories to undertake changes in their workflow that are prone to prolonged turnaround times, the course of action must be provided in an easily digestible form. the question is: how could something like this be realized in practice? first and foremost, the lis (lab information system) needs to be able to distinguish between samples which can be pooled (let's call them "screening samples") and samples which must not be pooled ("diagnostic samples"). those screening samples need to be entered into the system with a specific request, different from the diagnostic samples' entries. also, the user (not necessarily the samples' originators) need to be able to see, at any time, at which point in the prospective pooling step a sample might be just now. therefore, it might be prudent to allow for different analyses if working with different pools: "number analyses = number aliquots". we start with aliquots s i, of the n samples {s , s , . . . , s n } and add p samples into one pool which is then analysed using analysis corona pool . if corona pool turns out negative, the lab order for those patients is fulfilled and the negative result is reported. all those with either a positive or invalid result appear with "not negative" (and hidden to the sender) on the lab report and make the next step (request) appear (open) on the lab report corona pool . same here, any negative pools end up with the negative result behind this analysis while any positive or invalid results appear as "not negative" internally within the lab and make the third analysis appear, visibly, on the report corona screen. all analyses might have the same friendly name appear on the lab report, visible to the samples' sender. the strict consecutive pattern of dividing positive pools into subpools described in section . has been useful for a concise description of the algorithm, but it is not necessary for the correctness of the recursive method. rather, different aliquots of the samples in a positive pool can be arbitrarily assigned to the subpools. within the lab, samples will be archived and can be retrieved if any pool is positive. in order for the lab personnel to know whether to pool the samples with or or or whatever number of other samples, samples should be stored in different archives and marked with different colours, depending on their status within the workflow chain. an easier setup would be a pooling device that inherently can pool samples and use this to undergo a -step pooling. step : make pools of of all screening samples. step : make pools of of all pools from step . this way, using the same pipetting robot and the same programming, the complete chain of barcode numbers can be passed on to the lis after step . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint is complete. this way, also, all containing pools of which might be needed if the -size-pool comes back positive, are already available and ready for second stage testing. the downside is: with low prevalence, a significant number of size- pools are created unnecessarily, costing time. however, already having the lis know the next smaller pools barcode makes it easier to work with residual lists, i.e., which samples still have no result and where do i find them. these days we face the situation that some restrictions imposed due to the covid- pandemic are reduced stepwise and another shutdown would be disastrous for the economy. we regularly test the players in the german soccer leagues but have no plan how to detect new emerging hotspots in, say, re-opened kindergartens, schools, and retirement homes as early as possible. it seems that a substantially increased testing capability might help in developing an enhanced strategy in dealing with this situation. this view is shared, e.g., by the german federal minister of health jens spahn [ ] : "es ist viel teurer, zu wenig zu testen, als zu viel zu testen." currently (end of june ), the german federal state of bavaria offers preventive sars-cov- mass testing to all (roughly , , ) state inhabitants. we believe that pool testing in general and the recursive method in particular may be the method of choice. however, one must not assume that sample pooling reduces the cost of testing by a factor of the pool size. it is clear that laboratories save on reagents with the reduced number of tests. with recursive pooling, we have shown that reagents use can be massively reduced. for positive sample rates of . %, the reduction is about % with respect to current individual testing and % with respect to the frankfurt method, respectively. however, one must consider the downsides, namely the increased personnel and other consumable costs (like secondary tubes and pipette tips) due to longer hands-on times and more procedural steps. a major problem could also be the significantly prolonged turnaround times. however, this setup is certainly feasible for laboratories with limited pcr-capacity or countries with limited reagent supplies as well as those having to deal with sample numbers exceeding the laboratory capacities by a large factor over a prolonged period of time. we definitely think that recursive pool testing may be useful in mass testing when only few infections are to be expected. we can suggest to labs involved in sars-cov- testing to consider evaluating this setup with their own equipment and calculate the "sweet spot" for their circumstances based on an arbitrary number of samples with varying positivity rates. while it is also possible to transfer this idea to antibody-testing, one needs to keep in mind that antibody tests mostly follow a linear dilution formula while the exponential increase in dna-fragments in pcr technology allows for greater dilution factors with relatively smaller impact on possible false-negatives. pooling samples could accelerate new coronavirus testing corona pool testing increases worldwide capacities many times over pooling of samples for testing for sars-cov- in asymptomatic people fact -frankfurt adjusted covid- testing-a novel method enables high-throughput sars-cov- screening without loss of sensitivity. medrxiv pool-testen von sars-cov- proben erhöht die testkapazität weltweit um ein vielfaches we gratefully acknowledge the advice of ulrich jürgens, andrea krieger, dirk schmidt, and norbert schöngen. key: cord- -f ibgn i authors: jawed, shayan; grabocka, josif; schmidt-thieme, lars title: self-supervised learning for semi-supervised time series classification date: - - journal: advances in knowledge discovery and data mining doi: . / - - - - _ sha: doc_id: cord_uid: f ibgn i self-supervised learning is a promising new technique for learning representative features in the absence of manual annotations. it is particularly efficient in cases where labeling the training data is expensive and tedious, naturally linking it to the semi-supervised learning paradigm. in this work, we propose a new semi-supervised time series classification model that leverages features learned from the self-supervised task on unlabeled data. the idea is to exploit the unlabeled training data with a forecasting task which provides a strong surrogate supervision signal for feature learning. we draw from established multi-task learning approaches and model forecasting as an auxiliary task to be optimized jointly with the main task of classification. we evaluate our proposed method on benchmark time series classification datasets in semi-supervised setting and are able to show that it significantly outperforms the state-of-the-art baselines. modern deep learning architectures have taken the fields of computer vision, natural language processing and recommender systems by storm. time series classification is no stranger to recurrent neural networks and convolutional neural networks (convnets) too [ , ] . although proven to learn high level features across a broad domain of time series classification problems, the success of convnets hinges on the availability of large amounts of labeled training data. in reality, however, there is a high cost associated in acquiring such labeled data. as a result, there have been efforts to utilize semi-supervised learning algorithms catered especially for time series classification [ , , , , , ] . the idea behind semi-supervised learning is to exploit unlabeled data for training purpose in the presence of only few labeled instances. the applicability of this learning paradigm naturally extends to time series data as plentiful of it can be acquired trivially. for example, a single polysomnography (sleep study) can generate up to , heartbeats but it takes the time and expertise of a cardiologist to annotate individual heartbeats [ ] . hence, effective methods for semi-supervised learning can lead to mining vast amounts of time series data for which only comparatively few labels might be available. a related stream of works has been dedicated to learning high level convnets based representations that do not require any manual annotation of data. selfsupervised learning has emerged as a prominent learning paradigm among such, where the idea is to define an annotation-free pretext task that is inherent in the data itself. the task stands to provide a surrogate supervision signal for feature learning. example tasks include classifying image rotations [ ] , colorizing images [ ] solving jigsaw puzzles [ ] to learn transferable representations for high-level tasks such as object detection and semantic segmentation. until so far, applications have been limited to the computer vision domain. in the same spirit of learning generalizable representations, we now introduce multi-task learning. multi-task learning is an important paradigm in machine learning which builds upon the idea of sharing knowledge between different tasks [ ] . a set of tasks is learned in parallel, aiming to improve performance over each task compared with learning one of these tasks in isolation. a multi-task learning problem can also be formulated with respect to main and auxiliary tasks. auxiliary tasks are motivated by the intuition that for most problem settings, performance over one particular task is of primary importance. however, in order to still reap the benefits of multi-task learning, related tasks could be modeled as auxiliary tasks [ ] . these exist solely for the purpose of learning an enriched representation that could increase prediction accuracy over the main tasks. in our work, we bring together ideas from these high-impact research ideas of self-supervised learning and multi-task learning to propose an auxiliary forecasting task that is inherent in labeled and unlabeled time series data both. this auxiliary task stands to provide a strong surrogate supervision signal for feature learning which when learned in parallel with the main task of classification of time series boosts the performance of the classifier especially in semi-supervised setting. more specifically, we first define a sliding window function parametrized by hyper-parameters of stride and horizon to be forecasted. next, we augment the training set with generated samples for the forecasting task by providing labeled and unlabeled samples as input to this function. the convnet model is trained jointly to classify the labeled samples and forecast future series values. this exploitation of the unlabeled samples leads to learning representations that help boost the classification accuracy. the intuition is that these unlabeled samples come from the same distribution and if the model learns the complex task of forecasting series values accurately, then the same latent representations could be leveraged for classification. in our experiments we show that is indeed the case and our proposed method excels in semi-supervised setting where only a few labeled instances might be available for the model to learn from. to recap, our contributions are: • a novel self-supervised task that is intuitive, requires close to no changes in the base network structure and provides a strong surrogate supervisory signal for feature learning in the realm of time series classification. • a multi-task network which enables the forecasting and classification task to share latent representations and learns high-order interactions automatically. • extensive experimental evaluation of our self-supervised method in the domain of semi-supervised learning for time series classification and show that it outperforms state-of-the-art baselines. the problem of learning with both labeled and unlabeled data is of central importance in machine learning [ ] . we specifically review works that have focused on time series classification. we note the seminal work in the field from wei et al. [ ] . they proposed a self-training approach based on a nearest neighbor classifier. the work from [ ] later improved the method significantly by proposing a new meta-feature based distance. in [ ] a clustering approach was proposed combined with self-training. another ssl algorithm in [ ] also is in essence a clustering based method. the authors of [ ] proposed a graph theoretic ssl algorithm that constructs graphs relating all samples based on different distance functions and consequently propagates labels. the current state-of-the-art method in the field [ ] is based on shapelet learning [ ] on both labeled and unlabeled time series data. on the other hand, we note recent works [ , , , , ] which showed that strong supervision could be leveraged by describing a task that is inherent in the data itself (requires no manual annotation). we consider the pioneering work by [ ] which leveraged spatial context in an image for self-supervised learning by predicting relative location of one sampled patch to another. similar selfsupervised tasks were image colorization [ ] , solving jigsaw puzzles [ ] and classifying image rotations [ ] . more closely related to our work is a multi-task self-supervised network [ ] . the work firstly tries to compare how the representations learned from recent proposed self-supervised approaches like above compare with each other, and then shows that combining these tasks even in a bare-bones multi-task network without catering for any controlled parameter sharing lifted the accuracy compared with the single-task networks compared before. moreover, we also note the works that cater for temporal structure. such temporal structure is inherent in video data, work in [ ] proposed a sequential verification task to determine whether a sequence of frames was in correct order. it was shown that with this simple but intuitive task, the convnet captures temporally varying information such as human poses and ultimately lifted the accuracy on benchmark action recognition datasets. another closely related example is [ ] where the task was to recognize whether the video is playing forwards or backwards. with motivations behind our method set from the literature review, we now draw the following insights: firstly there exist semi-supervised learning approaches similar to ours that learn from unlabeled data, most notably current state-of-the-art shapelet learning approach [ ] if we consider shapelets to be similar to convolutional filters. however, with our work we exploit deep learning based methods which solve an auxiliary self-supervised task of forecasting which forces the network to learn filters to solve this particular complex task. secondly, there have been a plethora of works that proposed novel self-supervised tasks, however to the best of our knowledge, there are no examples for the time series domain and neither that cast a self-supervised task as an auxiliary task. our aim is to learn a convnet model that can estimate a forecasting function f (.) and a classification function g(.) jointly. to put it in concrete terms, we have a set of univariate time series samples .., x u k } a set of unlabeled samples, and a labeled set comprising of note that k + l = n and total series length is t . furthermore, we define a sliding window function w(.) which is parametrized by a stride s and horizon h. this function takes as input time series from x, and segments each in forecasting samples for e.g., .., y ,t=p+ h } which denote the first sample i.e x 's first window. the next sample is chosen with regard to p = p + s and complete set of forecasting samples is given by it is worth noting that these windows have a total length of h < t of which the later half consists of targets to be forecasted. and, the total number of forecasting samples, m = n × ( × h + )/s where s > . to fix ideas, our objectives are y f = f (x f ) and y l = g(x l ). the loss function for the objective with respect to f (.): specifically, we wish to learn the set of parameters θ f that minimize the loss with respect to predictions,Ŷ f . the model does multi-step predictions for the horizon h and the loss stated above captures this with the outer sum. moreover, for the classification task, the model outputs a probability distribution over all possible classes, c. in contrast to the forecasting samples, the input corresponds to the complete length, t . we also emphasize here the difference in parameters by denoting θ c as the classification task parameters. the loss to be minimized with respect to predicting classes c for samples x l : we adopt this architecture from [ ] where it was shown to outperform variety of baselines on a majority of datasets. we reuse the same parameters for f (.) up-to the last convolutional block, from where a dedicated linearly fully connected layer denoted by fc outputs for the horizon. the core intuition to model forecasting as an auxiliary task is to force the con-vnet model to learn a set of rich hidden state representations from unlabeled but structured data. in the case of only few labeled instances being available as in semi-supervised setting, a fully-supervised approach can overfit on the training instances by learning a poor set of features which can hardly distinguish different classes. however, since training proceeds, by using the same set of features repeatedly the model can be more assuming of its predictions which would decrease the training loss in turn. in order to avoid this, a self-supervised task on unlabeled data could be leveraged that can learn comparatively more discriminative features for training and ultimately lead to a significant lift in accuracy on unseen data. additionally, forecasting is well-studied and easily formulated, but at the same time is complex enough which does not open any doors for cheating, as there are no trivial shortcuts for the model to exploit for solving the task [ ] . moreover, the task allows us flexibility in terms of data generation. by configuring the different values of the horizon and stride, h and s respectively, one could control the number of samples needed to configure an optimal balance between the classification and forecasting task samples. central to the theory of multi-task learning is the leveraging of hidden state representations from multiple tasks simultaneously in order to create a more robust model. this begs the question as to whether there exist tasks that could mutually benefit each other by sharing parameters between. naturally, forecasting fits well with a classification task in a multi-task model as both tasks share the same input space. moreover, the learned feature spaces are expected to be correlated in turn also [ ] . however, designing a multi-task learning network poses two key challenges. firstly, how to divide the feature space in shared and task-specific feature sets. secondly, how to balance the weights between the different loss functions so as to distinguish between the main and auxiliary tasks. we rely on the hard-parameter sharing scheme, in which the learning parameters are all shared between the tasks up to the final fully connected layer in a layered architecture. from thereon, task-specific final layers output predictions for each task. this is illustrated in the fig. where we indicate shared parameters between the two tasks with same colored space. on the other hand, by adopting task specific weights we aim to cast the forecasting as an auxiliary task. we formulate the multi-task learning approach as an optimization process over the weighted sum of the two loss functions. λ is a hyper-parameter that controls parameter updates of the network relative to forecasting loss. it is thus crucial to tune for λ as too high of a value could bias the network weights for the forecasting task. on the other hand, if it is set too low, then the network would not learn for the forecasting task at all [ , ] . so far we have not drawn a link between the two feature sets of the tasks. in order to do so, consider that in a multi-task setting, the model is able to accurately forecast an unlabeled sample. the intuition is, if this unlabeled sample belongs to the same class as the very labeled sample the model is now trying to classify, and hence both are similar, then the latent features that were activated for the unlabeled sample could be leveraged to classify. additionally, since the model is trained end-to-end, we also hypothesize that the model automatically learns to share latent representations between tasks and their corresponding high-order interactions based on this latent space. we compare our proposed multi-task model to multiple baselines on realworld public time series datasets [ ] . since the data generating processes are completely different , the proposed method's performance can be judged without bias to similar data generating processes. previously proposed methods were compared on the same in [ ] . a summary of the datasets is given in table . all experiments were run with pytorch and code is published online to encourage reproducibility. wei's method [ ] is based on self-training through which the classifier iteratively augments the labeled set by adding a sample from the unlabeled set. the choice as to which sample to add is based on the (nearest neighbour) classifier's prediction of which sample was the closest to any of its labeled counterpart in euclidean distance. the newly added sample is then given the same class as its closest neighbour. is a meta-feature based distance. this distance was defined as the ratio of dtw to the euclidean distance. the intuition is to exploit the difference between the two distance's performance mainly the benefit of choosing dtw over the euclidean distance. self-training is then carried out based on this distance. success [ ] does constrained hierarchical clustering of the complete set of training samples, irrespective of labels. the distance metric utilized is dtw and all unlabeled samples are given the top-level seed's label. xu's method [ ] is a graph theoretic ssl algorithm that constructs graphs relating all samples based on different distance functions such as dtw or wavelet transform. a probabilistic method optimally combines these various graphs after which a well studied harmonic gaussian field based method [ ] is adopted for label propagation. bag-of-words [ ] leverages a sliding window procedure to generate local segments from time series data. these local segments are used to create histograms to train an svm model for classification. it is worth noting that this method differs from above as it uses only labeled samples. sssl [ ] is the current state-of-the-art method in the field. it uses shapelets to classify unlabeled samples thereby producing pseudo-labels. a coordinate descent solver wraps the optimization process by iteratively solving for the classification of labeled samples, pseudo-labels and shapelets respectively. base [ ] is a single-task variant of our proposed method that is only trained on the labeled samples to do classification. Π-model [ ] is well-known semi-supervised learning method for image classification task. the basic idea is rooted in incorporating stronger regularization via ensembling. the method relies on dropout and asks the network being trained to output consistent labels for the same input. the input albeit goes through different dropout conditions leading to stochastic outputs. this makes it a welldefined task to exploit especially for unlabeled data. as the training proceeds, it is expected for the network's self-ensembled predictions to converge to the same labels for both labeled and unlabeled data. we sandwiched dropout layers after the batch-normalization layers and trained with dropout values of % and %. transfer learning is common in the regime of self-supervised learning based methods [ , ] . following these works, we train a non-linear classifier on top of each of a network's layers trained particularly for forecasting the datasets under consideration. the forecasting network in question is composed of stacking convolutional layers in successive order with filter numbers , , , , , respectively. moreover, we sandwich maxpooling layers between halving the input in temporal dimension after each convolutional layer. next, flattening and training non-linear fully connected layers with dimensions and . the very final layer's dimensionality corresponds to the horizon. this network is the result of an extensive grid search over multiple forecasting tasks from concurrent work. as we motivated, this network is geared towards forecasting in sharp contrast to the network in fig. adopted for the classification task. we trained this network with a grid search in s × h where, s ∈ { . , . , . } and h ∈ { . , . } , and used the configuration resulting with the least loss in eq. . this baseline serves to evaluate the self-supervised learned features from the forecasting task, by measuring classification accuracy that they achieve when we train a classifier on top of them without any fine-tuning [ ] . this classifier has two non-linear layers corresponding to dimensions of and respectively. we hypothesize that if the features do correlate between the classification and forecasting task, then this non-linear classifier is expected to perform well. we begin this section by shedding light on the evaluation protocol. we randomly split each dataset into train and test splits with % and % of the samples. secondly, we split the train split further into labeled and unlabeled sets by randomly discarding % of the labels. this evaluation protocol is kept in line with the previous published methods, so as to report a direct fair comparison. the metric of evaluation is classification accuracy throughout the experiments. given that the initial splits can bias the maximum achievable accuracy, similar to the works before ours, we report the maximum achieved accuracy on the test split by running the experiments times with different hyper-parameters altogether. table shows the comparison of accuracies for the proposed method and the baselines. the results are also stated for the best performing transfer learning scheme and the Π-model. a number of interesting observations can be drawn from these results. firstly, we observe that our proposed method is able to outperform all other methods by a wide margin across almost all benchmark datasets considered. this is only made possible because of the exploitation of the unlabeled data better than other methods. given that we consider here only % labeled samples, the difference between the performance of the methods boils down to how these cater for the unlabeled samples. by leveraging the forecasting task, the model is able to pick up useful representations that directly effect the final accuracy. we observe that the proposed model fails to correctly model the underlying distribution of the wordsynonyms dataset. firstly, we observe that in contrast to other datasets, the number of classes is the highest and hence we hypothesize that due to intra-class variances the model simply needs more labeled samples to model the underlying distribution. moreover, we plot samples from the dataset together with our forecasts for last window of each of these samples in fig. . our second intuition is that given the dataset is based on handwriting, it can be a difficult task to forecast it correctly. the mean squared error that we achieve also hints in this direction, though exceptionally for these handwriting samples it does not convey explicit meaning. perhaps surprisingly, the base method, is able to come at a close second. with exception to wordsynonyms and the lightning datasets, it is able to beat the current state-of-the-art by a considerable margin. we posit that this relates to the powerful non-linear modeling of the convnet in contrast to other algorithms. also worth considering is that this architecture is the result of an extensive search over a wide variety of benchmark datasets as evident in [ ] . hence because of these reasons, even with few labeled samples available it is able to lead over rest of the baselines. additionally, we notice that the Π-model does not lead to fruitful results. although the underlying architecture is the same as in base model, we observed that the model was unfortunately not able to properly cater for the consistency regularization term for the unlabeled data. as a result, the multi-task loss it optimizes for diverges resulting in poor performance over the test splits not outperforming the base model. despite initially considering to orthogonally integrate the Π-model with our proposed multi-task model, we refrained because of poor performance for this standard configuration. we also summarize the results for the transfer learning based approach which serves to quantify the usefulness of features learned purely for the self-supervised forecasting task. we can see that without any fine-tuning, by learning a nonlinear classifier on top of the layers provides useful results. although, results reported here are generalized over the layers, by reporting only the maximum possible accuracy regardless of the layer, it still serves as a validation of our initial hypothesis that feature spaces correlate heavily among the forecasting and classification tasks. to point out the effect of hyper-parameters on our proposed multi-task model, we state the results with respect to different stride and horizon values in table . it can be noted that for a subset of datasets the search was fruitful. indeed, we find out that performance varies considerably with respect to the size of forecasting samples generated with particular stride and horizon, as it has direct effect on the learned representations. we also wish to highlight further observations here though briefly. we observed that the final forecasting loss (eq. ) was consistent across s and h configurations albeit least at the extremities of stride and horizon values i.e those that lead to maximum possible forecasting samples. also, we observed that network was robust to the different values of λ altogether. more importantly, as we posited earlier, the network performance was indeed biased to labeled samples resulted from random splits. we plot here the qualitative results for the forecasting task. we observe that the network is able to model the underlying distribution, albeit not perfectly. we proposed a novel semi-supervised learning algorithm for time series classification based on a self-supervised feature learning task. we trained a con-vnet model that jointly classified and did auxiliary forecasting by sharing latent representations and learning high-order interactions end-to-end. as a result of exploiting the unlabeled data more effectively, our method was able to outperform state-of-the-art baselines. future work includes extending our method to multivariate time series and researching additional ways to incorporate consistency regularization, which might yield better performance. multitask learning dtw-d: time series semi-supervised learning from a single example the ucr time series classification archive unsupervised visual representation learning by context prediction multi-task self-supervised visual learning deep learning for time series classification: a review unsupervised representation learning by predicting image rotations learning time-series shapelets lars: invariant time-series factorization multi-task learning using uncertainty to weigh losses for scene geometry and semantics temporal ensembling for semi-supervised learning success: a new approach for semi-supervised classification of time-series shuffle and learn: unsupervised learning using temporal order verification positive unlabeled learning for time series classification unsupervised learning of visual representations by solving jigsaw puzzles an overview of multi-task learning in deep neural networks time series feature learning with labeled and unlabeled data bag-of-words representation for biomedical time series classification time series classification from scratch with deep neural networks: a strong baseline learning and using the arrow of time semi-supervised time series classification time series analysis with graph-based semi-supervised learning colorful image colorization semi-supervised learning using gaussian fields and harmonic functions semi-supervised learning literature survey acknowledgements. this work was co-funded by volkswagen financial services through the data-driven mobility services project. key: cord- -dtukacm authors: xu, y.; lewandowski, k.; downs, l.; kavanagh, j.; hender, t.; lumley, s.; jeffery, k.; foster, d.; sanderson, n.; vaughan, a.; morgan, m.; vipond, r.; carroll, m.; peto, t.; crook, d.; walker, s.; matthews, p.; pullan, s. title: nanopore metagenomic sequencing of influenza virus directly from respiratory samples: diagnosis, drug resistance and nosocomial transmission date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: dtukacm background: influenza virus presents a significant challenge to public health by causing seasonal epidemics and occasional pandemics. nanopore metagenomic sequencing has the potential to be deployed for near-patient testing, providing rapid diagnosis of infection, rationalising antimicrobial therapy, and supporting interventions for infection control. this study aimed to evaluate the applicability of this sequencing approach as a routine laboratory test for influenza in clinical settings. methods: we conducted nanopore metagenomic sequencing for respiratory samples from a uk hospital during the / influenza season, and compared results to routine molecular diagnostic testing. we investigated drug resistance, genetic diversity, and nosocomial transmission using influenza sequence data. results: metagenomic sequencing was % ( / ) sensitive and % ( / ) specific for detecting influenza a viruses compared with the diagnostic standard (cepheid xpress/biofire filmarray respiratory panel). we identified a h n genome with the oseltamivir resistant s r mutation in the na protein, potentially associated with the emergence of a distinct intra-subtype reassortant. whole genome phylogeny refuted suspicions of a transmission cluster in the infectious diseases ward, but identified two other clusters that likely reflected nosocomial transmission, associated with a predominant strain circulating in the community. we also detected a range of other potentially pathogenic viruses and bacteria from the metagenome. conclusion: nanopore metagenomic sequencing can detect the emergence of novel variants and drug resistance, providing timely insights into antimicrobial stewardship and vaccine design. generation of full genomes can contribute to the investigation and management of nosocomial outbreaks. and vaccine design. generation of full genomes can contribute to the investigation and management of nosocomial outbreaks. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint influenza a viruses (iav) are enveloped viruses of the orthomyxoviridae family, with a segmented, ~ kb rna genome [ , ] . iav can cause both seasonal epidemics and occasional pandemics, presenting a significant challenge to public health [ ] . seasonal epidemics are estimated to cause half a million deaths globally each year, primarily among young children and the elderly [ ] . estimates suggest a future pandemic could infect % to % of the world population and cause over million deaths within six months [ , ] . tracking and characterization of circulating influenza viruses, in both human and animal populations, is critical to provide early warning of the emergence of novel variants with high virulence and to inform vaccine design. direct-from-sample metagenomic sequencing can potentially identify all viral and bacterial pathogens within an individual clinical sample. the genomic information generated can comprehensively characterize the pathogens and enable investigation of epidemiology and transmission. oxford nanopore technology (ont) is a third generation sequencing technology that can generate long-read data in real-time, which has been successfully applied in the real-time surveillance of ebola, zika, and lassa outbreaks [ ] [ ] [ ] . ont metagenomic sequencing has the potential to be deployed for near-patient testing, providing rapid and accurate diagnosis of infection [ ] , informing antimicrobial therapy [ ] [ ] [ ] , and supporting interventions for infection prevention and control [ ] . we have recently demonstrated proof-of-principle for a direct-from-sample nanopore metagenomic sequencing protocol for influenza viruses with % sensitivity and % specificity compared to routine clinical diagnostic testing [ ] . all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint here we describe nanopore metagenomic sequencing directly from clinical respiratory samples at a uk hospital during the / influenza season, evaluating the applicability of this approach in a routine laboratory as a test for influenza, and investigating where further optimisation is still required before the assay can be deployed in clinical practice. we assessed the performance of this experimental protocol head-to-head with routine clinical laboratory tests, and used the influenza sequence data to investigate drug resistance, genetic diversity, and nosocomial transmission events, demonstrating the diverse benefits that can be gained from a metagenomic approach to diagnostics. residual material was collected from anonymised throat swabs, nasal swabs, and nasopharyngeal aspirates that had been submitted to the clinical diagnostic laboratory at the oxford university hospitals nhs foundation trust during the / influenza season. prior to metagenomic sequencing, samples had been tested in the diagnostic laboratory based on a standard operating protocol using either xpert xpress flu/rsv assay (cepheid, sunnyvale, ca, usa, that detects influenza a/b and respiratory syncytial virus), or biofire® filmarray® respiratory panel assay (biofire diagnostics, salt lake city, ut, usa, that detects a panel of viral and bacterial respiratory pathogens). xpert reports a quantitative diagnostic result (ct value) for the detected pathogen, while biofire® rp reports a binary result (pathogen detected or not all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the first laboratory diagnosis of influenza in our hospital laboratory in the / season was made on th october , and our sample collection ran until th (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. methods for sample processing (sequence independent single primer amplification as described in [ ] ), nanopore sequencing, and genomic and phylogenetic analyses are described in supplementary material. for the nanopore metagenomic sequencing, the sample processing and library preparation time in our protocol was eight hours, the sequencing time was hours, and thus total turnaround time for each sample was < hours. with a team of three members, we prepared the sequencing libraries for samples within days and completed the sequencing runs for the influenza-positive samples within days of commencing sequencing ( figure a ). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint nanopore sequencing generated between . x and . x (mean . x ) total reads per sample (table s ). we retrieved hazara virus reads (spiked as an internal control at genome copies/ml) from / ( %) samples. the samples in which hazara virus reads were not identified were all influenza negative and had comparatively low total cdna concentrations following amplification. therefore, we repeated sequencing of the / samples that had sufficient remaining material with the addition of linear polyacrylamide as a carrier, which produced hazara virus reads in / samples. taken together, we therefore retrieved hazard internal control in / ( %) samples ( were not possible to re-test with carrier). the xpert xpress influenza-positive samples (sensitivity %), ranging from to , reads ( figure a ). iav reads were present in all samples with ct ≤ , and up to a maximum ct value of . (sample , iav reads). there was a strong correlation between ct value and both iav read numbers (r = . , p< . ; figure a ) and the ratio of iav:hazara virus reads (r = . , p< . ; figure b ). the remaining influenzapositive samples for which iav reads were not generated by nanopore sequencing had lower viral titres, reflected by higher ct values (range . - . ). iav reads were not present in / influenza-negative samples (specificity %). all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. among the samples for which we generated iav reads, we could determine the ha subtype of / ( %) samples; were h and were h (designated as blue vs red dots in figure ). we could determine ha subtype for all samples with ct ≤ , and up to a maximum of ct . (sample ) ( figure a ). we retrieved / ( %) consensus sequences with genome coverage ≥ %, among which were h and were h subtype ( figure c ). the genome coverage for samples with ct value between and showed substantial variation, which was not associated with any sample attributes that we were able to measure, including sample type, or percentage of human or bacterial reads (data not shown). from consensus sequences covering drug-resistant positions, we identified the s n amino acid mutation in the m protein in / h n and / h n sequences, which is known to be widespread, conferring reduced inhibition by amantadine [ ] . / h n sequences (sample ) carried the s r amino acid mutation in the na protein, which has been reported to confer reduced inhibition by oseltamivir [ ] . analysing mapping data for sample , / ( %) reads carried the s r mutation. other drug resistance mutations, such as h y in the na protein associated with oseltamivir resistance [ ] , were not present in our dataset. the majority of our h sequences were clustered within clade c. a b, with one sequence in clade c. a ( figure a ). comparison of the h and n phylogenies all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint showed that ha and na segments of each individual sample were clustered within the same clade, except sample had a distinct genotype with the h segment clustered within clade c. a b and the na segment within clade c. a (denoted subsequently as 'r-genotype'), suggesting intra-subtype reassortment ( figure a and b) . interestingly, the s r mutation occurred in the same sample (sample ), motivating us to further investigate the prevalence of this mutation in seasonal iav using all published h n sequences from the last two influenza seasons ( / and / ). in the / dataset, / ( . %) sequences carried the s r mutation, with ha and na segments from clade c. a or c. a . in / , the proportion of sequences with the s r mutation increased to / ( . %), and all belonged to the r-genotype. these results suggest a potential association between the increase in prevalence of the s r mutation and the emergence of this distinct rgenotype. we included a putative clinical cluster of eight influenza-positive samples (group ) collected from patients on the infectious diseases ward over a day period, aiming to investigate potential nosocomial transmission events ( figure a ). we could determine the ha subtype of six samples, three being h and three h . among these, two h n (samples and ) and one h n consensus sequences had > % full genome coverage. a minimum spanning tree (mst) of our h n sequences showed that samples and differed by snps (figure b ), despite being collected on the same day from patients on the ward. these results refuted the suspicion that these all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. figure b ) also demonstrated: • three sequences were identical (cluster ), from one patient on the infectious diseases ward (sample ), one who had been recently on the infectious diseases ward and then under the care of emergency assessment unit (eau) (sample ), and one who had been on the eau for a couple of days and then in the complex medicine unit until discharged (sample ). • two identical sequences (cluster ) differed from cluster by snps, and were from patients on the respiratory ward, taken two days apart. • one sequence (sample ) differed from cluster by snps, and was from an acutely admitted patient in the eau three weeks later. • the remaining four sequences, including sample from the refuted cluster and three from patients elsewhere in the hospital, were separated from cluster , cluster , and each other by ≥ snps. these results suggested that cluster patients on the infectious diseases ward and cluster patients on the chest ward likely reflected nosocomial transmission. there was no clear link between cluster patients, cluster patients, and the acutely admitted eau patient (sample ). one patient in cluster (sample ) and this eau patient were positive for influenza on the first day of their admission to the hospital, suggesting these samples may be associated with a predominant strain circulating in the community. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. phylogenetic analysis of the h segment showed that our sequences clustered within clade b. (figure s a ). at the full genome level, we found no evidence of phylogenetic clustering of ph n iavs recovered from our hospital, suggesting these represent independent introductions. rather, our ph n genomes were closely related to other genomes recovered during the uk / season ( figure s b ). of our ph n genomes had their most closely related sequence within < snps, of these most closely related sequences were from the uk. among the influenza-negative respiratory samples we sequenced, had tested positive for another virus in the clinical diagnostic laboratory. from this small dataset, our metagenomic sequencing dataset was > % sensitive for hmpv, rsv, and piv, but only % sensitive for coronavirus and enterovirus; specificity was high at > % for all five viruses ( table ) . in five influenza-positive samples for which iav reads were generated by sequencing, we also retrieved reads for other viruses, including human coronavirus (table s ). for the influenza-negative all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (table s ). while these organisms may represent agents of respiratory infection, they can also be commensal or colonising flora. in the absence of detailed clinical metadata, we were unable to explore their likely contribution to pathology. in this study, we conducted nanopore metagenomic sequencing of iav directly from clinical respiratory samples at a uk hospital during the / influenza season, reporting a head-to-head comparison with routine clinical diagnostic tests. the total turnaround time for metagenomic sequencing of each sample was < hours. we processed clinical samples and generated sequencing data for clinical samples over a day period. while the turnaround time is still slower than other laboratory diagnostic tests, and the approach requires an investment in labour and sequencing/analysis time, there is potential to reduce this further, through simplification of the wet laboratory protocol and implementation of real-time bioinformatic analysis of reads as they are generated. timeliness is crucial for the deployment of international vaccine strategies. each february, who determines influenza vaccines for use in the following northern all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (figure ), due to the substantial increase of c. a viruses in several regions since november associated with low vaccine effectiveness ( %) [ ] . this one-month delay raised concerns about the timeliness of vaccine manufacturing and distribution for the upcoming influenza season. within our cohort, a clade c. a h n sample was collected on th january , and if we had conducted rapid-turn-around sequencing as a routine assay then the complete genome sequence could be available in < hours, while the first strain from this clade in the country was not officially reported by public health england until th march. this timeline illustrates how routine laboratory sequencing would allow earlier genetic characterization, providing translational advantages in influenza surveillance, monitoring change in the proportion of genetically diverse strains, and contributing to timely insights into seansonal epidemiology vaccine design. our sequencing data showed % sensitivity for iav compared with existing laboratory diagnostic tests, consistent with our previous study with a smaller dataset [ ] . further optimization is needed to improve the sensitivity of our protocol for clinical samples with lower viral titres (ct values > ). potential methods include depletion of host and bacterial rna to reduce the amount of non-target nucleic acid present, and enrichment of the target via probes or primer amplification. our data show that addition of a carrier can improve the detection of internal spiked control in samples with low total cdna, which is likely due to the improved purification and reduced degradation of lower all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint concentration rna, thus we intend to incorporate this approach as a routine part of the protocol in future. the s r na mutation in h n iav has been associated with reduced susceptibility to oseltamivir since the / influenza season [ , , ] . among , h n iavs tested globally during the / season, one strain from south korea showed reduced susceptibility to oseltamivir due to this mutation [ ] . our analysis demonstrates that iavs carrying this mutation from the / season belong to a distinct genotype generated through intra-subtype reassortment between clades c. a b and c. a . a previous study reported a similar observation that the emergence and rapid global spread of adamantane resistant h n iavs (conferred by a s n mutation in the m protein) was associated with a single genotype generated through intra-subtype reassortment [ , ] . s n now occurs in almost all circulating iav globally, causing the cessation of use of adamantane to treat influenza [ ] . the genesis, prevalence, distribution and clinical impact of the s r mutation merits additional study to evaluate potential implications for the clinical usefulness of oseltamivir, which is widely used as a first-line agent when treatment is indicated [ ] . whole genome sequencing can provide high resolution characterization of the spatiotemporal spread of viral outbreaks [ , ] . previous studies have used targeted enrichment combined with next generation sequencing to investigate nosocomial transmission of influenza [ , ] , and our study demonstrates the application of nanopore metagenomic sequencing for this purpose. our sequencing data allow us to all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint refute the suspicion of a single transmission cluster on the infectious diseases ward, although the small number of whole genomes generated limits the extent to which we could draw conclusions about transmission among this specific patient group. furthermore, our dataset reveals two clinical clusters that likely represent nosocomial transmission on the infectious diseases ward and the chest ward, showing proof of concept that nanopore metagenomic sequencing can identify nosocomial transmission with the potential to inform infection prevention and control practices. based on a small exploratory dataset, our protocol shows > % sensitivity for the detection of human metapneumovirus, parainfluenza, and respiratory syncytial virus compared to routine clinical diagnostic testing. the lower sensitivity for enterovirus and coronavirus could be due to low viral titres in these samples, although we are not able to confirm this as the biofire® rp assay is a non-quantitative test. another possibility is that the sispa method is less sensitive for certain viruses [ ] . moreover, no influenza b virus reads are present in our influenza-positive samples, congruent with the global low level of influenza b virus during the / season. further work is needed to determine the limits of detection and optimize the laboratory and bioinformatic protocol to improve the sensitivity for a wider range of potential pathogenic organisms. this study included a limited cohort, with samples stratified by clinical diagnostic results, collection time, and the observation of a putative clinical cluster. we were not able to systematically sequence all influenza-positive samples from the clinical all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint diagnostic laboratory due to limited manpower and laboratory resources. generalisability is limited by this sampling approach, as well as by other confounding influences which we were unable to control, including both laboratory and clinical influences (e.g. diverse sample types, sample exposure to freeze/thawing, underlying immunocompromise, symptom duration prior to sample collection). while metagenomic data holds the promise for simultaneous detection of all pathogens from an individual clinical sample, it poses general challenges to analyze and distinguish between pathogens, commensal flora and potential contaminants. accurate interpretation is based upon the clinical context of the patient, type and quality of the sample, the absolute and relative abundance of the organism in the metagenome, genome coverage and mapping depth, and the occurrence of the organism in samples on the same run (if multiplexed) and historical runs in the same laboratory. expert caseby-case appraisal is currently required if the data are to be used for clinical decisionmaking. in summary, we demonstrate the feasibility of applying nanopore sequencing in clinical settings to simultaneously detect influenza and other respiratory viruses, identify drug resistance mutations, characterize genetic diversity, and investigate potential nosocomial transmission events. while work is still needed to refine and streamline the sequencing protocol and bioinformatic analysis, nanopore metagenomic sequencing has the potential to become an applicable point-of-care testing for infectious diseases in clinical settings. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the study of anonymised discarded clinical samples was approved by the london -queen square research ethics committee ( /lo/ ). following removal of human reads, our sequencing data have been uploaded to the european bioinformatics institute https://www.ebi.ac.uk/, project reference prjeb….. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. hazara virus was spiked as an internal control at genome copies/ml. c) coverage of the iav consensus sequence against ct value. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. distance between each pair of sequence is denoted by number adjacent to the branch. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. all rights reserved. no reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint evolution and ecology of influenza a viruses the biology of influenza viruses are we ready for pandemic influenza? science estimates of global seasonal influenza-associated respiratory mortality: a modelling study the origin of the pandemic influenza virus: a continuing enigma the lancet infectious diseases. how to be ready for the next influenza pandemic real-time, portable genome sequencing for ebola surveillance metagenomic sequencing at the epicenter of the nigeria lassa fever outbreak multiplex pcr method for minion and illumina sequencing of zika and other virus genomes directly from clinical samples rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis diagnostic tests 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on the susceptibility of human influenza viruses to neuraminidase inhibitors recommended composition of influenza virus vaccines for use in the - northern hemisphere influenza season the genesis and spread of reassortment human influenza a/h n viruses conferring adamantane resistance the origin and global emergence of adamantane resistant a/h n influenza viruses whole-genome sequencing provides data for stratifying infection prevention and control management of nosocomial influenza a single primer isothermal amplification (spia) combined with next generation sequencing provides complete bovine coronavirus genome coverage and higher sequence depth compared to sequence-independent single primer amplification (sispa) key: cord- - hkoeca authors: furstenau, tara n.; cocking, jill h.; hepp, crystal m.; fofanov, viacheslav y. title: sample pooling methods for efficient pathogen screening: practical implications date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: hkoeca due to the large number of negative tests, individually screening large populations for rare pathogens can be wasteful and expensive. sample pooling methods improve the efficiency of large-scale pathogen screening campaigns by reducing the number of tests and reagents required to accurately categorize positive and negative individuals. such methods rely on group testing theory which mainly focuses on minimizing the total number of tests; however, many other practical concerns and tradeoffs must be considered when choosing an appropriate method for a given set of circumstances. here we use computational simulations to determine how several theoretical approaches compare in terms of (a) the number of tests, to minimize costs and save reagents, (b) the number of sequential steps, to reduce the time it takes to complete the assay, (c) the number of samples per pool, to avoid the limits of detection, (d) simplicity, to reduce the risk of human error, and (e) robustness, to poor estimates of the number of positive samples. we found that established methods often perform very well in one area but very poorly in others. therefore, we introduce and validate a new method which performs fairly well across each of the above criteria making it a good general use approach. for targeted surveillance of rare pathogens, screenings must be performed on a large number of individuals from the host population to obtain a representative sample. for pathogens present at low carriage rates of % or less, a typical detection scenario involves testing hundreds to thousands of samples before a single positive is identified. although advances in molecular biology and genomic testing techniques have greatly lowered the cost of testing, the large number of negative results still renders any of specimens in order to detect just a few thousand cases. the large number of negative tests struck dorfman as being extremely wasteful and expensive and he proposed that more information could be gained per test if many samples were pooled together and tested as a group [ ] . if the test performed on the pooled samples was negative (which was very likely), then all individuals in the group could be cleared using a single test. if the pooled sample was positive, it would mean that at least one individual in the sample was positive and further testing could be performed to isolate the positive samples. this procedure had the potential to dramatically reduce the number of tests required to accurately screen a large population and it sparked an entirely new field of applied mathematics called group testing. due to practical concerns, dorfman's group testing approach was never applied to syphilis screening because the large number of negative samples had a tendency to dilute the antigen in positive samples below the level of detection [ ] . despite this, sample pooling has proven to be highly effective when using a sufficiently sensitive, often pcr-based, diagnostic assay. in fact, ad hoc pooling strategies have long been used to mitigate the costs of pathogen detection in disease surveillance programs. for example, surveillance of mosquito vector populations in the u.s. involves combining multiple mosquitoes of the same species (typically - ) into a single pool, prior to testing for the presence of viral pathogens [ ] [ ] [ ] [ ] . elsewhere, such pooling techniques have been successful in reducing the total number of tests in systems ranging from birds [ ] , to cows [ ] , to humans [ ] [ ] [ ] . in many wildlife/livestock surveillance programs, sample pooling is used to simply determine a collective positive or negative status of a population (e.g. a herd or flock) without identifying individual positive samples. while this is often appropriate and sufficient for small-to-medium scale research experiments or surveillance programs, a well designed pooling scheme can easily provide this valuable information with little additional cost. for the purposes of this paper, we will focus on pooling methods that provide accurate classification of each sample so that infected individuals can be identified. group testing theory primarily focuses on minimizing the number of tests required to identify positive samples and many nearly-optimal strategies for sample pooling have been described. from a combinatorial perspective, a testing scheme begins by examining a sample space which includes all possible arrangements of exactly k positive samples in n total samples. because the positive samples are indistinguishable from negative samples, a test must be performed on a sample or a group of samples in order to determine their status. the test is typically assumed to always be accurate, even when many samples are tested together (in practice, this is often not the case and approaches that consider test error and constraints on the number of samples per pool have been examined [ , ] ). in the worst case, all of the samples would need to be tested individually requiring n tests. the goal of group testing is to devise a strategy which tests groups of samples together in order identify the positive samples in fewer than n tests. group testing methods are generally more efficient when positive samples are sparse. as the number of positive samples increases, the number of tests will eventually exceed individual testing for all of the methods. this point has been previously estimated to be roughly when the number of positives is greater than n for sufficiently large n [ , ] . in order to establish the most optimal testing procedure, non-adaptive and adaptive pooling approaches and, in each case, we assume that the test applied to the pools is noiseless (the test will always be positive if a positive sample is present in the pool and negative otherwise) and it produces only a binary or two-state outcome (e.g. positive/negative or biallelic snp typing). number of times any two samples are included in the same pool [ ] . this is achieved by staggering the samples that are added to each pool in different sized windows or intervals ( fig ) ; importantly, the size of the windows must be greater than √ n and co-prime to minimize the intersections between samples. the number of different pooling windows (the weight) should be one greater than the expected number (upper bound) of positive samples, w =k + , to ensure accurate results. fig . dna sudoku pooling example. in this example, there are a total of n = samples. the -well plates show which samples are combined into each pool for the two different window sizes (w = and w = which are greater than √ n and co-prime). by using two different window sizes, the weight of this pooling design is w = meaning that k = w − = positive sample can be unambiguously identified in a single step using t = w + w = tests. the positive samples are decoded by finding the samples that appear most often in the positive pools. for example, if g is the only positive sample, we can detect this from the pooling results by noticing that g was added to both of the positive (red) pools while other samples in those pools were added to only one or the other. alternatively, if both g and d are positive, four samples occur with equal frequency (d , g , e , and f ) in the positive pools (red and purple) and it is impossible to determine which are the true positive samples. this ambiguity is introduced because the test was designed to handle only one positive sample. multidimensional pooling is another non-adaptive approach that is generally easier to perform than dna sudoku but can be more prone to producing ambiguous results. as the name implies, this procedure can be extended to many dimensions [ , ] , however it becomes more difficult to perform without robotics when more than two dimensions are used. in the two dimensional ( d) case, n samples are arranged in a perfectly square d grid or in several smaller but still square sub-grids [ ] . for example, when testing samples (as in fig ) , this could be achieved through a single x grid or dorfman's original pooling design for syphilis screening was an adaptive two-stage test. following this method, samples are partitioned and tested in g groups of size n. all of the samples in groups with negative results are considered to be negative and all of the samples in groups with positive results are tested individually. ignoring the constraints of the actual assay, the optimal group size that minimizes the number of tests depends on the number of positive samples, k. specifically, there should be roughly √ n k groups of size n k [ , ] . dorfman's two-stage approach was later generalized to any number of stages using li's s-stage algorithm [ ] , which can reduce the number of tests sobel and groll [ , ] , introduced several adaptive group testing algorithms based on recursively splitting samples into groups and maximizing the information from each test result. they demonstrated that this class of algorithm is robust to inaccurate estimates of k, particularly in the case of the binary splitting by halving algorithm which can be performed without any knowledge of the number of positive samples. binary splitting by halving (fig ) begins by testing all of the samples in a single pool. if the test is negative, all of the samples are negative and testing is complete, if the test is positive, the samples are split into two roughly equal groups and only one of the groups is tested in each step. if the tested half is negative, we know that all of the samples in the tested group are negative and testing is now complete for those samples. we also know that testing [ , ] . this is the only approach discussed here that does not rely on an step ). if the tested half is negative, then all of the samples in the tested half are considered to be negative and at least one negative sample is known to be present in the other non-tested half of the samples. if the tested half is positive, then it contains at least one positive sample and no information is gained about the other untested half. in either case, the method continues by halving and testing whichever group is known to contain a positive sample until a single positive sample is identified (either by individual testing, as seen in step , or by elimination, as seen in step ) . once a single positive sample is identified, the remaining unresolved samples (non-grey wells) are pooled and tested to determine if any positive samples remain and the process continues until all positive samples are identified. only one test is required per round, and in this example, it takes sequential rounds to recover both positive samples. fewer tests [ ] . as the ratio of samples to positive samples ( n k ) increases, the number of tests required to identify k positive samples approaches k log n k which is nearly optimal; however, like binary splitting by halving, the generalized binary splitting approach requires many sequential steps to complete testing. here we are introducing a new approach that we developed with the goal of finding a good balance between the number of tests, the number of steps, simplicity, and robustness. we found that many of the methods described previously focus on optimizing only one of these features usually to the detriment of the others. instead of attempting to perform the best in a single area, we wanted to take a more balanced approach and find tradeoffs that allow good performance across each of these areas. our modified -stage approach (fig ) is based on the s-stage approach but it is modified so that the number of steps is constrained to a maximum of three. at three steps, this approach requires only one additional step than ambiguous non-adaptive approaches that require two steps for complete validation. because the s-stage algorithm is already fairly robust, constraining the number of steps does not have a large impact on the number of tests required. we also modified our method to be simpler and easier to perform by borrowing the recursive subdividing used in the binary splitting approaches. in the s-stage approach, the remaining samples in each step are arbitrarily redivided into pools. not only does this make it difficult to keep track of the remaining samples spread across the plate, it can also make it more difficult to collect the samples for a pool using a multichannel pipette (e.g. step in fig ) . instead, we opted to recursively subdivide the samples from positive pools. this makes it easier to keep track of the samples that should be pooled at each stage and, because the samples are always in close proximity, they are easier to collect using a multichannel pipette (compare and ). is the number of groups tested at each step. the number of pipettings for a single channel pipette was equal to the number of samples in each of the pools that were tested. for multichannel pipettes, the number of samples in each pool was divided by the number of channels and rounded up. in cases where the samples in the pool were not in adjacent wells, additional pipettings were required. experimental validation of modified -stage approach we set up rare pathogen detection experiments in complex microbiome backgrounds to test our modified -stage approach. we used a total of samples (eight -well plates) that contained a background of µl of dna extraction from cow's milk and µl of molecular grade water. these samples originated from distinct cow milk samples and were replicated ( replicates each) to fill eight -well plates -a total of unique microbiome backgrounds. c. burnetti dna ( µl) was added to randomly chosen background samples (∼ . % carriage rate) as we verified that the spike-in was successful using a highly sensitive taqman assay designed to target the is repetitive element in coxiella burnetti [ ] . using the same taqman assay, we also verified that the target pathogen was not present in any of the unique microbiome backgrounds prior to the spike-in. to ensure a consistent amount of background dna, the milk extractions were tested to determine the amount of bacteria with a real-time pcr assay that detects the s gene and compares it to a known standard [ ] . the pooling procedure was carried out by a typical researcher looking to identify the number of sequential steps is one of the major factors that differentiates pooling methods. the major benefit of non-adaptive pooling methods is that, in some cases, all of the tests can be run at the same time which means that testing can be completed faster. clearly, the nonadaptive tests required the fewest number of steps even when the results were ambiguous, necessitating a second round of validation . for samples, the highest weight that we tested was which meant that any simulation with or the number of samples that are combined in a single pool is a very important practical concern because it can determine whether the assay can produce accurate results. in most methods, using a multichannel pipette reduced the number of pipettings by an order of magnitude in some cases. compared to the -channel pipette, the likely because the method requires many samples to be pooled at each step for many steps. the performance is slightly improved when multichannel pipettes are used but it is still the least efficient in many cases. using a single pipette, dna sudoku was not the most inefficient compared to the other methods. however, because the samples that are combined in each pool are spaced out in different intervals instead of in consecutive groups, the number of pipettings did not improve by using multichannel pipettes. this means that, in the best case, a laboratory technician would need to correctly pipette table which shows that method is more sensitive to overestimation than to underestimation. of the methods that depended on an estimate of the number of positive samples, the s-stage (fig , left) and our modified -stage approach (fig , second from left) were the most robust to misestimations of k. the number of steps was more robust in the modified -stage approach than the s-stage due to the -step constraint; however, the modified -stage was more sensitive in the number of tests in some cases (table ) . dna sudoku (fig , left) was the most sensitive method overall. overestimating of the number of positive samples caused the weight of the pooling design (w =k + ) to be set higher than it needed to be. when this happened, all of the positive samples were still unambiguously identified but each unnecessary increase in the weight required more than √ n additional tests. when the number of positive samples was underestimated, fewer tests were performed but the pooling scheme was no longer able to unambiguously identify the positive samples in a single step and a second round of verification was required. a similar pattern occurred in the d pooling simulations (fig , right) . while the grid dimensions did not directly depend on k, generally larger grids were more efficient when the number of positive samples was low and smaller grids reduced ambiguous results when the number of positive samples was high but at the cost of many more tests. however, because d pooling was constrained to two dimensions, the number of tests did not vary as drastically as dna sudoku. table . although the expected number of positive samples per plate was ∼ given the . % carriage rate, the actual number of positives ranged from to and none of the plates had exactly positive samples ( table ). the taqman assay was able to accurately identify the positive pools without any false positives or false negatives even during the first step when the number of samples per pool was the largest at . using an -channel pipette where appropriate, a total of pipettings was required to pool the samples. a total of taqman assays were performed which is ∼ % fewer than would be required to individually test samples. picking the right pooling approach for a given pathogen surveillance campaign can be a complicated decision, which is often driven by a set of conflicting constraints and complexity. dna sudoku, however, is far from optimal for monitoring rapidly changing pandemics due to its extreme sensitivity to misestimation of the carriage rate of the pathogen in population. a good middle ground between the adaptive and non-adaptive pooling approaches is the modified -stage approach -our preference in our own surveillance applications. while it is never the absolute best in any one category, it is always nearly optimal in terms of number of serial steps ( nd best), complexity ( nd best), number of tests ( th best), and extremely resilient to misestimation of the carriage rate ( nd best). the latter is particularly important, as it allows this approach to be useful for surveillance in situations with rapidly changing pathogen carriage rates (e.g. in pandemic or seasonal outbreaks), while keeping number of serial steps as low as possible for an adaptive method. testing pooled sputum with xpert mtb/rif for diagnosis of pulmonary tuberculosis to increase affordability in low-income countries estimating community prevalence of ocular chlamydia trachomatis infection using pooled polymerase chain reaction testing impediments to wildlife disease surveillance, research, and diagnostics evaluating and testing persons for coronavirus disease (covid- ) the detection of defective members of large populations kwang-ming hwang f. combinatorial group testing and its applications searching for the proverbial needle in a haystack: advances in mosquito-borne arbovirus surveillance phylogenetic analysis of west nile virus in maricopa county, arizona: evidence for dynamic behavior of strains in two major lineages in the american southwest detection of west nile virus in large pools of mosquitoes west nile virus in the united states: guidelines for surveillance, prevention, and control active surveillance for avian influenza virus infection in wild birds by analysis of avian fecal samples from the environment pooled-sample testing as a herd-screening tool for detection of bovine viral diarrhea virus persistently infected cattle sample pooling as a strategy to detect community transmission of sars-cov- high-throughput pooling and real-time pcr-based strategy for malaria detection real-time, universal screening for acute hiv infection in a routine hiv counseling and testing population group testing: an information theory perspective. foundations and trends® in communications and information theory to pool or not to pool? guidelines for pooling samples for use in surveillance testing of infectious diseases in aquatic animals a boundary problem for group testing sharper bounds in adaptive group testing dna sudoku-harnessing high-throughput sequencing for multiplexed specimen analysis discovery of rare mutations in extensively pooled dna samples using multiple target enrichment screening of a brassica napus bacterial artificial chromosome library using highly parallel single nucleotide polymorphism assays a two-dimensional pooling strategy for rare variant detection on next-generation sequencing platforms a sequential method for screening experimental variables group testing to eliminate efficiently all defectives in a binomial sample binomial group-testing with an unknown proportion of defectives a method for detecting all defective members in a population by group testing rickettsial agents in egyptian ticks collected from domestic animals bactquant: an enhanced broad-coverage bacterial quantitative real-time pcr assay key: cord- -wrzzb cy authors: brunner, jesse l. title: pooled samples and edna-based detection can facilitate the “clean trade” of aquatic animals date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: wrzzb cy the regional and international trade of live animals facilitates the movement, spillover, and emergence of zoonotic and epizootic pathogens around the world. detecting pathogens in trade is critical for preventing their continued movement and introduction, but screening a sufficient fraction to ensure rare infections are detected is simply infeasible for many taxa and settings because of the vast numbers of animals involved—hundreds of millions of live animals are imported into the u.s.a. alone every year. batch processing pools of individual samples or using environmental dna (edna)—the genetic material shed into an organism’s environment—collected from whole consignments of animals may substantially reduce the time and cost associated with pathogen surveillance. both approaches, however, lack a framework with which to determine sampling requirements and interpret results. here i present formulae for pooled individual samples (e.g,. swabs) and edna samples collected from finite populations and discuss key assumptions and considerations for their use with a focus on detecting batrachochytrium salamandrivorans, an emerging pathogen that threatens global salamander diversity. while empirical validation is key, these formulae illustrate the potential for edna-based detection in particular to reduce sample sizes and help bring clean trade into reach for a greater number of taxa, places, and contexts. www.nature.com/scientificreports www.nature.com/scientificreports/ context of screening u.s. service members for syphilis, pooling samples to reduce the number of tests required to detect rare infections is common in numerous contexts , including surveys for disease freedom. however, the formulae for inference from pooled samples generally assume sampling with replacement or, equivalently, very large populations, which does not align well with the problem of detecting infections in consignments or captive populations. recently a probability formula was developed for pooled samples collected from finite populations without replacement -but it does not accommodate imperfect tests. second, one might screen environmental dna (edna)-the genetic material shed into an organism's environment-for the pathogen or parasite of interest . the edna approach seems especially relevant to aquatic and semi-aquatic taxa, which make up a large portion of the wildlife trade , as edna can be simply filtered from water housing animals; they need not even be handled. (semi-aquatic species or life stages are often shipped in moist sphagnum moss or paper, but can then be placed in water to collect edna during a quarantine period .) environmental dna sampling is being used to detect pathogens in a growing number of settings, from natural environments [ ] [ ] [ ] [ ] to ballast water and even in trade [ ] [ ] [ ] . however, while the statistical framework for making proper inference from edna results is well-developed in certain contexts (e.g., the presence or absence of a target species in ponds using occupancy models , ), these approaches do not translate well to sampling small captive populations or consignments. while both approaches have the potential to reduce sample sizes, we lack the formal framework with which to determine sampling requirements and interpret results. in this paper i therefore develop formulae for imperfect tests of pooled samples in closed populations and edna, discuss the key assumptions and considerations in their application, and illustrate how edna may be especially useful for detecting infections in the live animal trade. i present these results in the context of emerging fungal pathogens that threaten amphibian diversity, for which trade appears to play a key role. the amphibian chytrid fungus, batrachochytrium dendrobatidis (bd), is the most devastating emerging pathogen on record, responsible for declines in over species, including that are likely extinct . it likely owes its global distribution in large measure to the international trade of african clawed frogs (xenopus laevis) used for pregnancy tests and research , american bullfrogs (lithobates catesbeianus) sold for food , , and myriad species involved in the pet trade , [ ] [ ] [ ] [ ] . more recently, the international pet trade aided the emergence of a novel chytrid fungus, b. salamandrivorans (bsal), that is particularly lethal to salamanders [ ] [ ] [ ] [ ] . bsal was introduced into northern europe via the pet trade from southeast asia , [ ] [ ] [ ] . while it has not yet been detected in the wild in much of europe - , it is already prevalent in and appears to have spread among private collections in europe [ ] [ ] [ ] . in north america, a hot-spot of salamander diversity, bsal is apparently absent from both wild and captive amphibians , - . however, the risk and potentially devastating consequences of its introduction via trade [ ] [ ] [ ] led to prohibitions on the importation of species of salamanders into the u.s.a. and all salamanders in canada . while laudable, such bans are inevitably crude. these restrictions were necessarily based on experiments with small samples sizes of few species, extended to others by taxonomic affiliation . moreover, it was quickly out of date since several other taxa, including frogs, are now known to carry bsal , . such bans may also promote black-market trade, which already occurs on a global scale similar to the black-market trade of narcotics . in response to these challenges, as well as the economic interests of the pet trade, there have been calls for a "clean trade" of amphibians [ ] [ ] [ ] . indeed, the european union recently mandated a program requiring screening, using a quantitative real-time pcr (qpcr) assay of skin swabs , or prophylactically treating all consignments of live salamanders into and between member nations of the eu . again, the scale of the international trade of live amphibians makes routine screening a formidable challenge. while the number of consignments of amphibians into or between eu member nations is not available in the literature, an estimated - million live amphibians are imported per year into the usa, alone , and , annually into the u.k. . since bsal is thought to occur at low prevalence in traded animals , the eu regulations require screening all or a substantial fraction of individuals in a consignment to ensure a reasonable chance of detection (fig. ) . while shipments of certain taxa or from certain sources might be excluded, even conservative assumptions about the volume of trade imply screening an enormous number of samples if this program were extended worldwide (e.g., consignments of salamanders each would require testing some , swabs). this scale of sampling is likely to place clean trade beyond the reach of many nations or interested parties (e.g., constituents in the pet trade industry). similar problems arise when considering other amphibian pathogens (e.g., ranavirus ) or those of fishes. the question in this paper is therefore: can pooling swab samples or using edna place routine surveillance in reach? the formulae presented below can account for false positives (i.e., less than perfect specificity, sp). it will be important to consider the sources and likelihood of false positives (e.g., from carry over of target dna from the water rather than infected hosts , vs. from contamination in a laboratory) as well as the consequences (e.g., slowing shipments, economic costs, loss of trust , ). in the case of bsal detection, i would hope that any positive test would be investigated further with additional, independent diagnostic tests. moreover, setting aside the issue of carry over contamination , which might be avoided by transferring animals to clean water prior to sampling, there is no evidence that false positive rates are higher in pools of samples or edna than typical individual samples. in fact, to the extent that fewer tests are conducted with pooling or edna one would expect fewer false positives at a given level of surveillance sensitivity. for these reasons i set aside the issue of specificity in the following discussion and focus on sensitivity. www.nature.com/scientificreports www.nature.com/scientificreports/ pooling individual samples. i extended the "pooled hypergeometric" distribution of theobald and davie to account for imperfect diagnostic tests (eq. ( )). the results are largely intuitive: when n individual-level samples are divided into m = n/k pools of size k, the number of pools that need be screened is reduced up to k-fold, assuming low prevalence of infection and high diagnostic sensitivity , . the gains in efficiency from pooling are reduced somewhat for less sensitive tests when infections are more common, but not markedly so. a key assumption of this formula is that diagnostic sensitivity is not affected by pooling. this implies on the one hand that the combined analyte (e.g., target dna) from multiple infected individuals does not increase diagnostic sensitivity. the probability of detecting a single infection in a pool of samples (i.e., swabs) is at most the diagnostic sensitivity of individual swabs. on the other hand, the formula assumes that the analyte is not overwhelmed or inhibited by non-target materials (e.g., pcr inhibitors in skin secretions, microbial dna) nor diluted below a detection threshold (e.g., as was observed with pools of fish screened for a megalocytivirus ). this later assumption is unlikely to hold with very large pools of samples (see laurin et al. for a review), but may be reasonable for small pools. it is an important assumption to test under realistic conditions, but one could simply use lower values of diagnostic sensitivity (se in eq. ( )) to account for target swamping or dilution. also, it is worth remembering that the same number of samples must be collected with or without pooling; efficiencies are only gained in the processing and screening steps. environmental dna. i developed a new formula for edna-based detection in small, closed populations (eq. ( )). the formula makes two key assumptions. first, it assumes that the edna shed into the water is homogeneously distributed in the water. while pathogen edna is likely clumped (e.g., bsal may be found principally in skin sheds), one could homogenize the water (e.g., with a blender) prior to taking samples to meet this assumption, at least in the smaller volumes used to house animals in shipments or captive populations. thus, replicate edna samples are essentially technical replicates in this framework. second, as with the formula for pooled samples, it assumes that test sensitivity is unaffected by the number of hosts in the water at the same time. that is, the target pathogen edna is not swamped by non-target host and microbial edna or other waste material leading to inhibition of the pcr reaction. this assumption needs to be empirically verified, but it may be possible to minimize pcr inhibition with dedicated kits or by diluting the eluted dna prior to pcr, although such methods risk reducing sensitivity. in any case, the formula i present could be extended to account for this swamping effect if needed. these assumptions have two important consequences for edna-based pathogen detection. first, every sample collects edna from the entire population. environmental dna avoids the issue of whether rare infections are included in any random sample of individuals-they are all sampled because the edna from all individuals is distributed homogeneously in the water. the question is only whether there is sufficient target edna in a sample to ensure a reasonable probability of a positive test. as a consequence, edna sampling can detect rare infections with many fewer samples than individual swabs, or pools of swabs, even when diagnostic sensitivity is very low (figs. and ). (i consider various factors that are likely to affect sensitivity in the next section.) this also means that the probability of detecting an infection is, all else being equal, independent of population size (fig. ) . thus, while individual-level swabbing can be more efficient than edna in small populations, in larger populations edna outperformed swabbing, even when pooled, over a wide range of parameter values (fig. ) . similarly, samples sizes for edna-based detection decrease with population size when prevalence is held constant because there are more infected individuals shedding pathogen edna, whereas they must increase with population size when using individual or pooled sampling to ensure infected individuals are included in any sample (fig. ). once again, it is essential to establish the actual performance of edna-based detection in real world contexts and test how it scales with population size and other variables. www.nature.com/scientificreports www.nature.com/scientificreports/ quantitative nature of detection. diagnostic sensitivity is usually defined in the context of a classification problem with binary infection status determined by a gold standard of infection (e.g., histopathology) or assigned in experiments. as such, sensitivity is generally assumed to be fixed; that is, all infections within a population or experiment are equally detectable. however, any test that reacts directly with the pathogen (e.g., pathogen dna or antigens) or host responses (e.g., antibodies) is necessarily dose-dependent ( fig. ; eq. ( )). the variation in the amount of the analyte between infected individuals or among laboratories, protocols, etc., may be small enough to ignore (but see e.g., rimmer et al. ), or the concentrations generally large enough that detection is assured (e.g., with clearly diseased animals), in which case it may be reasonable to assume diagnostic sensitivity is constant. at the other extreme, recently infected individuals, sub-clinical carriers , or otherwise inapparent infections tend to produce or shed little analyte and are thus much less easily detected than clinically infected animals. quantitative real time pcr of swabs, for instance, often fails to detect low-level bd infections )) of the per target copy detection rate, φ and the number of copies of the target dna or analyte, c. the arrows and dotted vertical lines show the consequences of increasing the volume of water in which animals are held ten-fold, thus diluting the analyte (blue) or increasing the number of infected animals from one to two, three, or four. note, however, that the concrete effect of such changes on the probability of a positive test depends on where one starts along the φc axis. www.nature.com/scientificreports www.nature.com/scientificreports/ have large effects on the pool-wide sensitivity . however, the the concentration-dependent nature of detection will be especially pronounced with edna sampling. the concentration of target dna in an edna sample co-varies with the type and intensity of infection and details of the sampling method (here volume sampled, pore size, storage conditions ), as with individual samples , but also depends on the conditions in which animals are held (e.g., temperature, volume, time in the water; fig. ). thus, one would expect a great deal more variability in sensitivity of edna between settings and studies, not to mention between taxa, than with traditional sampling directly from animals. empirical validation of edna-based pathogen detection should thus focus on understanding the causes and consequences of this variability across consignment types, holding conditions, and sampling approaches, and work to develop standards. equations ( )- ( ) are an initial attempt to explore these influences (fig. ) . they emphasize that equivalent changes in the number of infected animals, their shedding rates, the proportion of the environment sampled, or rates of degradation all have equivalent effects on the amount of analyte in a sample, and thus detection probability. it is worth noting, however, that researchers may have control over certain key variables, such as the duration and volume in which animals are held before collecting samples, allowing them to maximize sensitivity. for instance, if rates of edna shedding and degradation are fairly constant and fast relative to changes in the underlying infection intensity, one would expect edna concentrations in water to approach an equilibrium over time, at least as a first approximation (eq. ( )). the rate at which it asymptotes depends on the rate of degradation, and so water quality, temperature, ph, etc. are likely to be important , . however, unless rates of degradation are exceedingly rapid, one would expect concentrations of edna to accumulate over several days and so investigators might increase sensitivity by holding animals longer prior to sampling. while efficiency is not the only important criteria for a testing scheme (e.g., the expertise, specialized equipment, and costs involved must also be considered), routine pathogen surveillance and thus a "clean trade" becomes more feasible as the number of samples that must be analyzed are reduced. pooling swabs and sampling edna are both, in principle, viable approaches to reducing sample sizes, although to much different extents and with different drawbacks in their use. pooling and batch-processing samples collected from individual animals (e.g., swabs) can reduce several fold the number of diagnostic tests (e.g., dna extractions and pcr reactions) required to detect rare infections. while some n samples must still be collected, pooling them into groups of size k means that only n/k pools need be processed, which may substantially reduce the time and costs of surveillance. the critical question for pooling is the degree to which sensitivity, the probability of correctly identifying infections if present, declines as samples are pooled. laurin et al. found that sensitivity generally declined with pooling in aquatic pathogens, in part because moderate concentrations of analyte (e.g., pathogen dna) may be diluted below reliable detection limits in pools , but there are few studies that properly evaluate this. however, given the quantitative nature of detection (fig. ) and the large amount of variation in infection intensities and analyte in a sample, it may not be possible to provide simple, general estimates of sensitivity when pooling . in the case of bsal, sabino-pinto et al. found that diagnostic sensitivity of the typical qpcr assay was unaffected when dna was extracted from pools of up to four skin swabs. while the authors specifically recommend against pooling in trade and quarantine settings given the risks associated with the failure to detect a single infected individual , their results suggest pooling could reduce several-fold the number of dna extractions and qpcr reactions required to achieve the eu's requisite detection probability in a shipment ( figs. and ) . again, empirical tests in real-world settings, especially with low-level bsal infections, are needed to evaluate the actual sensitivity when pooling samples. hyatt et al. , for instance, found that diagnostic sensitivity of their qpcr assay for bd remained high in pools of five swabs, so long as "equivocal" results (one or two of three wells positive) were scored as positive-likely because of a dilution of target dna-but even here, one low-level infection went undetected in a pool. still, losses in sensitivity may be offset by increases in the fraction of a population or consignment that can be screened when samples are pooled, essentially increasing the chances that a rare infected individual is included in a sample. for instance, even if sensitivity is reduced from a probability of one with individual swabs to . with a pool of four swabs, many fewer assays need be run on the pools than the individual swabs to attain the same confidence of detecting an infection (fig. ) . equation ( ) and the included r code can be used to evaluate this trade-off. environmental dna offers a different trade-off. the samples collect genetic material shed into the water (or environment more broadly), so whatever material is shed, which may be minimal, is diluted in a larger volume and might be degraded. all of which means that fairly little target dna may end up in any given edna sample. thus, one would expect the diagnostic sensitivity of edna samples to be quite low relative to individual-based samples , (fig. ) . however, edna samples from the entire population, at least in the context of small captive populations or consignments of animals in water, perhaps with the aid of mechanical homogenization. this allows edna to circumvent the problem of including rare infected individuals in sample that is intrinsic to individual-level sampling (and pooling). as a result, even with low sensitivity, edna sampling can be much more efficient at detecting infections in large consignments than perfectly sensitive individual-based samples or pools of samples. moreover, sample sizes required to detect a rare infection do not increase with population or consignment size (fig. ) . sampling edna thus offers the promise of dramatically reducing the amount of sampling required to ensure disease freedom in the trade of live aquatic and semi-aquatic animals. the actual performance of edna-based detection likely depends on the nature of the consignment (e.g., volume, time animals have spent in water), collection and processing (e.g., volume filtered, presence and removal of pcr inhibitors), and biological realities (e.g., rates of shedding and degradation). it is important to evaluate these factors empirically so that the results can be properly interpreted. the simple model presented in eqs. ( )-( ) provides a useful starting point, illustrating how edna concentrations, and thus per sample detection www.nature.com/scientificreports www.nature.com/scientificreports/ probabilities, vary with volume, time, and rates of shedding and degradation (fig. ) . the proportion of holding water sampled and time animals spend in the water, shedding pathogen edna, are likely key parameters, but may vary substantially among taxa, life stages, and settings. a clever investigator, however, could use these factors to increase the sensitivity of edna-based samples, for instance by holding animals in small volumes of clean water for longer periods of time to ensure that target edna accumulates to readily detectable levels before collecting edna samples. perhaps the most fundamental question is whether pathogen edna is swamped by non target host or microbial dna or pcr inhibition increases with population size. while not unique to edna-pcr inhibition is common with swab-based detection of chytrid fungi-the issue maybe be magnified for environmental samples. if swamping or inhibition are a problem then the number of edna samples required to attain a particular detection probability would need to increase with population size and eq. ( ) adjusted accordingly. i must stress that it is only with a clear understanding of how these manifold conditions influence detection probabilities can edna-based detection be used reliably in the variety of settings and types of consignments that comprise the live animal trade. the goal of developing a "clean trade" may be the most realistic strategy for preventing the movement and introduction of pathogens such as bsal into new areas and naive hosts. but it is a daunting task given the immense number of animals involved in international and regional trade. the formulae developed here place sample pooling and edna in closed populations on a firm theoretical foundation. they suggest that pooling individual-level samples and, especially, collecting edna can substantially reduce sample sizes required to ensure rare, but important infections are found. moreover, sampling at key nodes in the distribution networks or crucial segments of the live animal trade (e.g., particular species, sources) can make surveillance even more tractable . it is my hope that these approaches can help bring clean trade into reach for a greater number of taxa, places, and contexts. however, diagnostic tests are rarely perfect. infected individuals are correctly detected with probability se (diagnostic sensitivity) and uninfected animals correctly test negative with probability sp (diagnostic specificity); false negatives and false positives occur with probabilities − se and − sp, respectively. cameron and baldock extended the hypergeometric model to include imperfect tests, such that the probability of observing x positive tests (t + ) is: ( ) often our goal is to determine the probability of at least one positive sample, given particular parameter values. consider a consignment of n = salamanders, some % (d = ) of which are infected, screened with individual swabs with a diagnostic sensitivity of se = . , matching the assumption in the eu regulations , and perfect specificity (sp = ). we see that the probability of detecting at least one of these infections is, using the equivalence p(t + ≥ x) = − p(t + = ): note that the part involving specificity simplifies to when se = . because x = , j is always zero and so this simplifies to: www.nature.com/scientificreports www.nature.com/scientificreports/ one can then try values of n such that p(t + ≥ ) ≥ . or any other threshold surveillance sensitivity. in this scenario the threshold is met when n ≥ . while this calculation can be done by hand, it becomes quite tedious. using the r code found below the same calculation can be achieved with pooledhyp_atleast (d= , n= , m= , k= , se= . , sp= ). note that that this function is written for pooled samples (see next subsection), but we can use it for individual samples with m = n "pools" (=samples) of size k = . pooling individual-level samples. when the n samples are divided into m groups of size k = n/m, which are then tested as pools, the probability that x of the m pools contain an infected individual in them (pool + ) is described by the "pooled hypergeometric" of theobald and davies : it is important to note that this formulation assumes diagnostic sensitivity and specificity are not influenced by the composition of a pool. a pool is equally likely to test positive if one or all of the individuals within it are positive, or if the pool is comprised of few or many samples. this assumption may be questionable for large pools, but may be reasonable with small k. returning to the example of a consignment of n = with se = . and sp = , but now pooling swabs into groups of size k = and assuming that the m pools is greater than the d = infections, we can find the probability of at least one pool testing positive as: , we obtain the following expression for the probably of observing x positive samples: note that because each edna sample collects material from all d infected individuals in the population, success is defined in the binomial as the probability of a positive edna sample [ − ( − se) d ], ignoring false positives, rather than the probability a sample includes an infected individual, as is the case when using the binomial to describe detection probabilities with individual samples taken with replacement. multiple infected individuals in a population simply increase the amount of target edna to be detected and thus the effective sensitivity of the edna test. both sensitivity and specificity are assumed to be constant and independent of the n − d uninfected animals in the population. finding the number of replicate edna samples to ensure ≥ ≥ . notice that both the binomial terms and the first term in brackets simplify to one since x = ; in other words, we only need to subtract the probability that all n samples test negative from one. ≥ ≥ . + p t ( ) edna when n ≥ . while this is simple to calculate by hand, the code, edna_atleast (d= , m= , se= . , sp= ) produces the same result. the quantitative nature of detection. any test that reacts directly with a pathogen (e.g., pathogen dna or antigens) or host responses (e.g., antibodies) is necessarily dose-dependent. focusing on dna-dependent detection methods (e.g., pcr), let us assume that every copy, c, of the target dna sequence in a sample has a small, fixed probability of causing a positive result in a reaction, φ. the probability of a positive test result can then be described by a "single-hit" model , : c which results in a typical dose-response relationship (fig. ) . (a logistic relationship between p(t + ) and log(c), which is commonly used in studies of analytic sensitivity, yields similar results, but requires an extra parameter.) if the c copies come from a single infected individual, then this function describes diagnostic sensitivity. the magnitude of c in a sample can therefore have a strong influence on sensitivity. as noted in the main text, one would expect greater variation in c with edna sample than with individual-level samples because edna is not collected directly from an animal, but from the environment, which can also play a role (although external swabs are also influenced by the environment). a simple model is useful in clarifying the factors that likely determine the number of target copies in an edna sample. in this model, the number of copies of target edna in a sample increase as the d infected individuals shed into the water at rate ψ, some portion, α, of which ends up in each edna sample, and decrease as edna degrades at rate δ: the solution is: where c is the initial concentration in the water (the first term on the right-hand side is zero when c = ). the solution asymptotes to c * = ψdα/δ over time at a rate dependent upon δ. substituting the equilibrium solution into the single-hit model (eq. ( )), one can see that detection probability depends on these rates in a straightforward way: while this model has several terms, they have clear biological meaning and could be estimated from experimental data [ ] [ ] [ ] . moreover, they enter the model as a product, with equivalent effects, at least at equilibrium. that is, doubling the volume of water collected in an edna sample is the same as doubling the number of infected individuals or halving the decay rate. this model also provides a way to link the many factors that can affect edna-based detection via their influence on target copy number, c (e.g., degradation, δ, is likely temperature-dependent ), or pcr efficiency (e.g., φ might be a function of ph and environmental inhibitors ). can be implemented in r using the "big integer" versions of multiplication and the binomial expansion found in the gmp package for greater precision: www.nature.com/scientificreports www.nature.com/scientificreports/ if(m*k>n) stop("individuals sampled (m*k) is greater than population size (n)") stopifnot(se<= , se>= , sp<= , sp>= , length(x)== ) innersums <− for(y in :m){ j<− :min(y,x) innersums[y+ ] <-pooledhyper(x=y, d=d, n=n, m=m, k=k) * sum(dbinom(j, size=y, prob=se) * dbinom(x-j, size=m-y, prob=( -sp))) } sum(innersums) # return sum of inner summations } note that when k= the "pools" are individual samples, as in eq. 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degradation in aquatic microcosms multiple precision arithmetic r package version . - i would like to thank kaare graesbøll and ming-wen an for interesting conversations as i was conceiving these ideas and jeb owen, christian yarber and other members of the brunner lab for helpful comments and discussion on earlier drafts, and suggestions from three anonymous reviewers. this work was supported by cgf j.l.b. conceived, developed, and and wrote the manuscript. the author declares no competing interests. correspondence and requests for materials should be addressed to j.l.b.reprints and permissions information is available at www.nature.com/reprints.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -wsmnlnlk authors: grädel, carole; terrazos miani, miguel a.; baumann, christian; barbani, maria teresa; neuenschwander, stefan; leib, stephen l.; suter-riniker, franziska; ramette, alban title: whole-genome sequencing of human enteroviruses from clinical samples by nanopore direct rna sequencing date: - - journal: viruses doi: . /v sha: doc_id: cord_uid: wsmnlnlk enteroviruses are small rna viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. in routine diagnostic laboratories, enteroviruses are identified by pcr-based methods, often combined with partial sequencing for genotyping. in this proof-of-principle study, we assessed direct rna sequencing (drs) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. the approach was complemented by sequencing the corresponding viral cdna via illumina miseq sequencing. drs of total rna extracted from three different enterovirus-positive stool samples produced long rna fragments, covering between % and . % of the most similar reference genome sequences. the identification of the enterovirus sequences in the samples was confirmed by short-read cdna sequencing. sequence identity between drs and illumina miseq enterovirus consensus sequences ranged between % and %. here, we show that nanopore drs can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains. enteroviruses (evs) present a major burden for human health and health care systems worldwide, with large outbreaks consisting of hundreds of thousands of hospitalized cases occurring periodically [ ] . evs are associated with a wide variety of symptoms, ranging from mild respiratory diseases to severe neurological infections, leading potentially to death [ ] . these single-stranded rna viruses that belong to the picornaviridae family possess a relatively small genome size ranging from . to . kb. for routine diagnostics, ev presence is generally determined by real-time, reverse-transcription pcr (qrt-pcr) assays, complemented by partial sequencing of the vp -vp coding regions for genotyping [ , ] . due to the lack of proofreading mechanism in rna replication, ev genomes are highly variable and the likely subject of within-and between-genome recombinations [ , ] . therefore, when diagnostic tests solely rely on pcr using conserved primer sequences, false-negative results cannot be excluded. there is thus a need to generalize the assessment of ev diversity and evolution via a whole-genome approach, rather than from the limited information gained from sequencing single genomic regions. the main limitation towards a general adoption of whole-genome sequencing (wgs) for better ev characterization and better understanding of their evolution and epidemiology is mostly of a technological nature: wgs approaches are more expensive, technically demanding and hence time consuming than standard molecular assays. as such, they are generally not routinely used in diagnostic laboratories. in the case of rna viruses, the challenge is even more exacerbated because various molecular steps (rna purification, extraction, cdna synthesis, and optionally amplification) add to the turnaround time and final cost of the assays. clinical wgs applications are mostly based on sanger sequencing and on second-generation sequencing platforms, with illumina miseq/hiseq (illumina, san diego, ca, usa) and ion torrent machines leading the market, as benchtop sequencing technologies enable high-throughput sequencing at an affordable price per sample when samples are multiplexed [ ] . third-generation sequencers, i.e., smrt technology (pacific biosciences, menlo park, ca, usa) and nanopore sequencing (oxford nanopore technologies (ont), oxford, uk), have recently emerged as complement to or replacement of second-generation sequencers, by enabling the sequencing of single, long dna molecules, a feature that is particularly attractive in the context of sequencing full-length viral genomes [ ] . nanopore sequencing technology has been successfully applied to genome sequencing of rna viruses. based on viruses propagated on cell lines, viral transcriptomes have been examined by nanopore sequencing, for instance for porcine circovirus [ ] , herpes simplex virus [ ] , hepatitis c virus [ ] , cultured influenza virus a, and human cytomegalovirus (hcmv) [ ] . additionally, nanopore sequencing has been used successfully to sequence whole ev genomes from viral cdna extracted from cell cultures: in their proof-of-concept study [ ] , the authors demonstrated that nanopore sequencing could be applied for rapid routine whole-genome sequencing of ev with sufficient accuracy compared to sanger sequencing. metagenomic nanopore sequencing of influenza virus was performed directly from randomly amplified viral cdna obtained from clinical respiratory samples [ ] . furthermore, ont has presented a new, potentially revolutionary nanopore sequencing application, which allows sequencing of rna molecules directly, i.e., without the pre-requirement of cdna synthesis or pcr amplification [ ] . this method, direct rna sequencing (drs), yields full-length, strand-specific rna sequences and enables the direct detection of nucleotide modifications in native rna molecules. drs has been used in several studies, including human poly(a) transcriptome [ ] and dna virus transcriptome, such as hsv herpes simplex virus type (hsv- ) during productive infection of primary cell [ ] . drs was also used for sequencing rna genomes of, e.g., pseudorabies virus propagated on immortalized porcine kidney epithelial cell line [ ] , influenza a virus (h n ) from infected chicken eggs [ ] , human coronaviruses viral rnas produced in cell cultures [ ] , and many other examples of complete sequencing of multiple, single-stranded rna (ssrna) viruses obtained with or without poly(a)-tailing of rna viruses obtained from cell cultures [ ] . here, in a proof-of-concept study, we apply nanopore drs to ev-positive stool samples and show that whole rna genomes of enteroviruses can be retrieved with enough genomic information for the characterization of the infectious agents. we further analyze the metatranscriptomic data provided by the drs approach and compare it with that obtained by illumina miseq sequencing of the same samples. in this study, we used three independent patient stool samples (e , e , e ), which were sent for enterovirus diagnostics to the clinical diagnostic laboratory of the institute for infection diseases, bern, switzerland. the samples were collected in (e , e ) and (e ) and had a cycle threshold (ct) value of . (e ), . (e ) and . (e ) via real-time pcr using enterovirus-specific primers (see description below). ethics approval was granted on march by the swiss ethics committee on research involving humans to conduct sequencing of enteroviruses in clinical samples stored in the ifik biobank (basec-nr: req- - ). after homogenization with a sterile pipette, approximately . g of stool sample was added to ml of transport medium [ ] containing - glass beads (diameter mm; merck ag, zug, switzerland). after s of vortexing, the suspension was centrifuged for min at g and the resulting supernatant was filtered through a pore size of . µm with % penicillin/streptomycin (biochrom, berlin, germany). this preparation was further extracted on the easymag platform (biomérieux, geneva, switzerland) for real-time pcr and with trizol ls reagent (thermo fisher scientific, reinach, switzerland) for illumina miseq sequencing ( figure ). after homogenization with a sterile pipette, approximately . g of stool sample was added to ml of transport medium [ ] containing - glass beads (diameter mm; merck ag, zug, switzerland). after s of vortexing, the suspension was centrifuged for min at g and the resulting supernatant was filtered through a pore size of . µm with % penicillin/streptomycin (biochrom, berlin, germany). this preparation was further extracted on the easymag platform (biomérieux, geneva, switzerland) for real-time pcr and with trizol ls reagent (thermo fisher scientific, reinach, switzerland) for illumina miseq sequencing ( figure ). figure . analytical workflow. the workflow used for diagnostic assays is indicated by greyed boxes and that followed for ngs techniques by white boxes. a total of µl of trizol ls reagent (thermo fisher scientific) was added per µl of stool suspension in pbs. following homogenization by pipetting, the sample was transferred to phasemaker tubes (thermo fisher scientific). after incubation for min, µl of chloroform was added, and the tubes were shaken vigorously by hand for s. after min incubation, the sample was centrifuged for min at , g, at °c. the aqueous phase was transferred to a new tube and µg of carrier rnase-free glycogen (thermo fisher scientific) was added, followed by µl isopropanol. after incubation for min, the sample was centrifuged for min at , g, at °c, and the supernatant was discarded. the total rna precipitate was resuspended in ml of % ethanol. the sample was vortexed briefly, and then centrifuged for min at × g, at °c. the supernatant was discarded and the rna pellet was air-dried for min, before being resuspended in µl of rna storage solution (thermo fisher scientific). after incubation at °c for min, the rna sample was either used directly in downstream applications, or stored at − °c. alternatively, total nucleic acid extraction was performed with nuclisens easymag from µl of the routine diagnostic stool preparation, to which . µl carrier rna (qiagen ag, hombrechtikon, switzerland) was added and eluted in µl. this extract was used for real-time pcr and sanger genotyping. we used the who-recommended protocol for pre-treating stool samples for enterovirus rna isolation (enterovirus surveillance guidelines, [ ] ) adapted from nix et al. [ ] as follows: a low amount ( . to g) of stool sample was added to pbs (thermo fisher scientific) up to ml volume, to which . g of glass beads ( mm diameter, merck) and . ml of chloroform (applichem, aesch, switzerland) were added. the mixture was shaken vigorously using a tissuelyser (qiagen ag) for min at maximum speed. the suspension was centrifuged at × g for min at °c, and approximately ml of the supernatant was transferred to a new . ml tube and continued with rna extraction. for drs of sample e , rna was extracted using easymag, while for e and e , the trizol ls method was used (see supplementary material table s ). a total of µl of trizol ls reagent (thermo fisher scientific) was added per µl of stool suspension in pbs. following homogenization by pipetting, the sample was transferred to phasemaker tubes (thermo fisher scientific). after incubation for min, µl of chloroform was added, and the tubes were shaken vigorously by hand for s. after min incubation, the sample was centrifuged for min at , × g, at • c. the aqueous phase was transferred to a new tube and µg of carrier rnase-free glycogen (thermo fisher scientific) was added, followed by µl isopropanol. after incubation for min, the sample was centrifuged for min at , × g, at • c, and the supernatant was discarded. the total rna precipitate was resuspended in ml of % ethanol. the sample was vortexed briefly, and then centrifuged for min at × g, at • c. the supernatant was discarded and the rna pellet was air-dried for min, before being resuspended in µl of rna storage solution (thermo fisher scientific). after incubation at • c for min, the rna sample was either used directly in downstream applications, or stored at − • c. alternatively, total nucleic acid extraction was performed with nuclisens easymag from µl of the routine diagnostic stool preparation, to which . µl carrier rna (qiagen ag, hombrechtikon, switzerland) was added and eluted in µl. this extract was used for real-time pcr and sanger genotyping. we used the who-recommended protocol for pre-treating stool samples for enterovirus rna isolation (enterovirus surveillance guidelines, [ ] ) adapted from nix et al. [ ] as follows: a low amount ( . to g) of stool sample was added to pbs (thermo fisher scientific) up to ml volume, to which . g of glass beads ( mm diameter, merck) and . ml of chloroform (applichem, aesch, switzerland) were added. the mixture was shaken vigorously using a tissuelyser (qiagen ag) for min at maximum speed. the suspension was centrifuged at × g for min at • c, and approximately ml of the supernatant was transferred to a new . ml tube and continued with rna extraction. for drs of sample e , rna was extracted using easymag, while for e and e , the trizol ls method was used (see supplementary material table s ). one-step rt-pcr was performed with the agpath-id one-step kit (ambion, reinach, switzerland) using published primers and probes [ ] , following the protocol described previously [ ] . primers and probes were synthesized at microsynth ag, balgach, switzerland. genotyping of the samples was performed by vp amplicon sanger sequencing as described previously [ , ] and carried out at microsynth. for nanopore sequencing, we followed the manufacturer's instructions of the protocol for kit sqk-rna (version drs_ _v _revn_ dec ), but substituted superscript iii with superscript iv (thermo fisher scientific). the input rna and amounts loaded on flow cells for each experiment are specified in table s . quantification was performed using a qubit rna hs assay kit (rna) or dna hs assay (cdna) kit on a qubit fluorometer . (thermo fisher scientific). the reverse-transcribed and adapted rna was loaded onto r . . flowcells and sequenced on minion sequencer. each drs run was conducted with a new, previously unused flowcell. the following three specific primers for coxsackievirus a were designed for this project to hybridize to coxsackievirus a sequence kj : r_a : -cccgtttctgccgctt- adapted from primer by oberste et al. [ ] ; r_a : -atatctctgaatttctcatt- adapted from primer hev. c.d by bessaud et al. [ ] ; ev- utr _a _rc: -catattcacgaccagattcctggtg- (this study). all primers were synthesized at microsynth. first-strand synthesis was performed with the superscript iv first-strand synthesis system as follows: for reverse transcription, a reaction mixture was prepared containing . µl of the specific primers ev- utr _a _rc, r_a , r_a ( µm), or µl of random hexamers ( µm) or µl of oligo(dt) ( µm) together with µl of mm dntp mix and filled with rna extract and depc-treated water to a total volume of µl. after heating at • c for min and snap cooling, a mixture of µl ssiv buffer, µl mm dtt, µl ribonuclease inhibitor and µl superscript iv reverse transcriptase ( u/µl) were added. the samples were incubated for min at • c and min at • c (preceded by min at • c when containing random hexamer primers). subsequently, µl of e. coli rnase h ( u/µl) was added and incubated at • c for min. to this mixture, µl of second-strand synthesis reaction buffer nebnext (new england biolabs (neb), ipswich, ma, usa), µl nebnext second-strand synthesis enzyme mix and µl of nuclease-free water were added. the reaction mixture was incubated at • c for min. clean up with µl of agencourt ampure xp magnetic beads (beckman coulter, nyon, switzerland) was performed according to the manufacturer's instructions, with an elution volume of µl for samples e and e and µl for sample e . for the first sample e , an additional end-prep step was performed, but that step was later removed in the protocol as it was deemed unnecessary: to µl of the elution, µl of ultra ii end-prep buffer (neb), µl of ultra ii end-prep enzyme mix (neb), and µl of nuclease-free water were added and the mixture was incubated for min at • c and min at • c. after an additional purification step using µl of agencourt ampure xp magnetic beads, the cdna was eluted in µl of nuclease-free water. libraries were prepared from unamplified cdna using nextera xt dna library prep kit (e ) and nextera dna flex library prep kit (e , e ) (illumina), and sequenced using an illumina miseq benchtop sequencer generating × bp paired-end reads (v ), according to the manufacturer's protocols. sequencing was performed at the next-generation sequencing platform of the inselspital, bern, switzerland. raw fast files produced by minion sequencing were basecalled under the high accuracy mode using the ont basecaller guppy version . . with the parameter: "guppy_basecaller --input_path path --recursive --save_path path --qscore_filtering --min_qscore --flowcell flo-min --kit sqk-rna --cpu_threads_per_caller -num_callers ". statistics for nanopore sequencing output are summarized in table s . we did not perform any adapter trimming due to the inaccuracy of the rna basecaller when calling dna adapter sequences, which results in unreliable identification [ ] , but do not expect it to impair our downstream analysis, as basecalled nanopore reads were classified using blastn against the ncbi's nucleotide (nt) database (downloaded . . ), using blast version . . . blast results were classified using megan (v. . . , [ ] ), with the blast rma parameters "c false --m --ms --me . --mpi --top --supp . --sup --alg naive --mrc --mrefc --ram readcount --a t nucl_acc tax-jul .abin". rna sequences were mapped to the best-scoring reference genome (whole-genome reference with highest bit-score in blastn output) using minimap (version . -r -dirty, [ ] ). for illumina miseq data (table s ) , adapter trimming was performed with bbduk.sh from the bbmap package (v . , [ ] ), and human and rrna reads were removed by mapping reads against the corresponding databases (grch genome, cdna and ncrna, and silva lsu ssu https://www.arb-silva.de/documentation/release- /) using minimap [ ] . non-human, non-rrna reads were then assembled using spades (parameters: -k --rna --only-assembler; version . . ; [ ] ). the resulting contigs were subsequently analyzed using blastn and megan as described for nanopore data. sequence identity between illumina miseq and nanopore drs enterovirus genome consensus sequences was calculated using legacy blast . . . coverage information was generated using bbmap and plots created using r statistical computing environment (version . . ). after removal of any human reads, all illumina miseq sequencing data (fastq), raw and basecalled ont data (fast and fastq, respectively), and sanger sequences were deposited in the european nucleotide archive (ena) under the project reference prjeb . drs was performed with enterovirus-positive stool samples with similar viral load (ct values between and ) in three independent experiments, consisting of samples from three different patients. the samples were prepared using the who-recommended protocol for viral enrichment using chloroform/bead treatment, followed by rna extraction using trizol or easymag (table s ) . we sequenced the total polyadenylated rna using drs on a minion nanopore sequencer. the runs were continued until there was only negligible sequencing output or until the maximum recommended duration of the run was reached. for validation purposes, all three samples were also subjected to cdna sequencing using illumina miseq from samples prepared by the routine diagnostic procedure (figure ). the cdna was produced with either genotype specific primers (e ), or with both oligo-dt primers and random hexamers (e , e ). drs reads from extracted rna were obtained up to h after the beginning of the sequencing run, and sequencing was stopped after h. out of a total output of , raw nanopore reads, reads were successfully basecalled into rna sequences, with an average length of bases (range - bases). more than % of basecalled rna reads ( reads) were taxonomically classified using blastn against the ncbi's nucleotide (nt) database. the large majority (> %) of those hits matched eukaryotic sequences, and only . % bacterial ( reads) and . % viral sequences ( reads) ( table ). the majority of eukaryotic reads were assigned to the yeast species saccharomyces cerevisiae ( reads; %). all other eukaryotic species had less than five reads assigned. within the reads assigned to bacteria, the only species with notable number of reads were escherichia coli ( reads), faecalibacterium prausnitzii ( reads) or bacteroides vulgatus ( reads), all of which are known to be commensal species in the human gut. as far as viral sequences were concerned, all ( . % of ) reads matched the enterovirus genus, species ev-a ( table ). they were on average bases long (range - bases). alignment of the rna sequences to the best-scoring reference genome (coxsackievirus a kj ; figure ) demonstrated that the long reads covered almost the entire genome of coxsackievirus a , with one single read covering alone . % of the reference genome (with only bases missing at the end). the top two longest rna sequences ( and bases long) were obtained within the first hour of sequencing, and most of the larger sequences (> kb) were obtained within the first h of sequencing. after h of sequencing, sequences matching enterovirus were all < bases long. classical amplicon sequencing of the vp region was attempted for sample e , but it did not yield good results. in order to confirm genotype identification obtained via drs and to obtain a highly accurate whole-genome enterovirus sequence, we also subjected sample e to cdna sequencing using illumina miseq. miseq sequencing library preparation was performed with stool material and following the routine diagnostic pre-treatment (as opposed to chloroform/bead treatment) given that low amount of patient stool sample was available (figure ). for this sample, an enterovirus-targeted approach was chosen to produce the cdna by using genotype-specific primers given that low number of reads was obtained by drs. after removal of reads mapping to human genome and bacterial rrna, metatranscriptomic reads were assembled into contigs and further subjected to blastn similarity analysis against sequences in the nt database, and the top hits were taxonomically summarized using megan. most contigs of sample e were classified as bacteria ( ) or viruses ( ) ( table ). such differences in species composition when compared to the results of the drs run may be explained by the genotype-specific approach used for cdna synthesis and different pre-treatment of the sample. based on the promising results obtained for sample e , the drs transcriptomic approach was repeated on two other clinical samples: for the drs of rna extracted from sample e , the total yield of the sequencing run was , reads ( . mb) after h of sequencing. a total of , reads passed basecalling, with an average length of . bases (range of - , ). only % ( , ) of the basecalled sequences were taxonomically classified using blastn. interestingly, there was a drastic difference in read composition as compared to the first sample (e ), and the majority of reads classified as of bacterial origin ( . %, , ), and only very few reads matched to eukaryotic ( , . %) or archaeal ( , . %) species. for bacteria, drs reads were assigned to a large variety of species. the most prominent phyla were bacteroidetes ( , reads) and proteobacteria ( reads). ) . for e , the coverage plot was produced by using the assembled contig sequence of the illumina sequencing run (oligo-dt primers) as reads did not map well to any reference ev genome sequence. for samples e and e , illumina miseq reads based on cdna produced with oligo-dt and random hexamers are indicated by light and dark grey lines, respectively. all viral sequences matched again uniquely to ev sequences. however, megan analyses revealed the presence of two enterovirus species consisting of ev-a ( contigs; contigs lengths ranging from to bases) and ev-b ( contigs; contigs lengths ranging from to bases) in the cdna sample, of which the majority of contigs were assigned to coxsackievirus a and echovirus ( table ). mapping of the viral sequences to a vp database confirmed the presence of these two genotypes and the identification was verified by using the online rivm bioinformatic platform [ ] . alignment of the reads to the closest reference genome sequences revealed that the coverage of cv-a was % with an average depth of . (± . ), while for echovirus , only % of the reference genome was covered (figure ) . the consensus sequence for cv-a obtained by illumina miseq was . % identical ( / ), with % gaps ( / ), to the consensus sequence obtained by drs sequencing. based on the promising results obtained for sample e , the drs transcriptomic approach was repeated on two other clinical samples: for the drs of rna extracted from sample e , the total yield of the sequencing run was , reads ( . mb) after h of sequencing. a total of , reads passed basecalling, with an average length of . bases (range of - ). only % ( , ) of the basecalled sequences were taxonomically classified using blastn. interestingly, there was a drastic difference in read composition as compared to the first sample (e ), and the majority of reads classified as of bacterial origin ( . %, , ), and only very few reads matched to eukaryotic ( , . %) or archaeal ( , . %) species. for bacteria, drs reads were assigned to a large variety of species. the most prominent phyla were bacteroidetes ( reads) and proteobacteria ( reads). a total of viral reads, corresponding to . % of classified reads, were found. as seen for the previous sample, only matches to the enterovirus genus were found. although many more viral reads were found in comparison to sample e , they were, with an average of bases, much shorter, and with a range of - . no reads spanned the complete genome sequence of any known ev species or genotype, and altogether could cover % of the reference genome sequence (figure ). short fragment lengths might result from increased fragmentation of the viral rna during extraction or library preparation. due to the directional sequencing used in the drs process of nanopore sequencing, higher coverage at the ' end of the sequence may be obtained, and fragments without poly-a tail cannot be sequenced. therefore fragmented, incomplete rna molecules may hinder the recovery of the end of the rna genome. nevertheless, although the covered region did not span the vp region, we were able to identify the enterovirus as echovirus by genomic similarity to the best-scoring reference genome sequence. this species identification was independently confirmed by a standard genotyping approach that uses sanger-based sequencing of the partial vp amplicon. the cdna from sample e was also sequenced by illumina miseq, although in this case with non-specific primers for cdna synthesis for a more unbiased approach towards ev detection and not to miss possible ev co-infections. both oligo-dt and random hexamer primers were used to ensure a better chance of obtaining the full genome, with the idea that oligo-dt may lead to results more comparable to those of drs due to polyadenylation requirements, and random hexamers may offer better chance of obtaining a well-covered ' end of the enterovirus genome. the distribution of contigs assigned on the domain level did not vary much between random hexamer and oligo-dt produced cdna: most contigs were assigned to bacteria ( . % or . %, respectively), with only a small minority mapping to eukaryotes ( . %, . %) and viruses ( . %, . %). within bacteria, most contigs were assigned to the phyla proteobacteria ( , , , ), followed by bacteriodetes ( , ) and actinobacteria ( , ). taking a closer look at all the detected viral genotypes (table ) , we found that, besides echovirus , there were also contigs mapping to other virus genotypes, such as rhinovirus a, and a variety of other bacterial or plant viruses, albeit only with few short contigs. the echovirus genome, however, was well covered with either approach (figure ) . comparison of the drs nanopore sequencing consensus to the one obtained from illumina miseq showed . % identity ( / ), with . % gaps ( / ). the drs run of the rna extract from sample e was stopped after the maximal run duration of h. notably, this run had much higher output compared to the other two samples, resulting in a total of . m sequenced reads ( . gb), from which . m reads passed basecalling. the average length was also considerably higher than in previous runs with bases (range - bases). although the rna input material used for library preparation was measured to be of a similar amount for all three samples ( ng), cdna measurement ( ng) before loading on the flow cell indicated that the initial rna concentration might not have been the same. overall, % of basecalled reads ( . m reads) were taxonomically classified using blastn. of those, . % ( , , ) mapped to eukaryotic sequences. as seen for sample e , the vast majority of eukaryotic reads mapped to saccharomyces cerevisiae ( . %), while . % ( , ) were classified as of bacterial origin, and only a negligible amount as archaea ( , . %). viral sequences comprised . % ( ) of the reads, the majority of which were classified as plant viral pathogens (table ) , mostly belonging to the tomato mosaic virus ( reads, % of viral reads). other, more rarely detected species, included melon necrotic spot virus ( ) , and cactus virus x ( ) . in total, reads mapped to enteroviruses. although sequences were mapped to several genotypes, mapping the reads to the best-scoring reference genome and to a vp database identified genotype echovirus as the most likely and only ev genotype present in the sample. although there were relatively few hits to enteroviruses considering the high total output of the run, long sequences (average . , range - bases) were observed, which covered up to . % of the reference genome sequence (figure ). illumina sequencing of the cdna from sample e produced using oligo-dt primers and random hexamers showed similar distributions on the domain level: the majority of contigs belonged to bacterial species ( . % and . %, respectively, for oligo-dt and random hexamer approaches), with a minority of reads mapping to eukaryotes ( . %, . %) and viruses ( . %, . %). this major shift from yeast to bacteria as compared to the drs approach, which was also observed for sample e , could be explained by the different pre-treatment using filtration with a pore size of . µm. additionally, comparison of number of contigs and nanopore reads are of limited value when not considering coverage depth. for e , the genome of echovirus genotype, whose presence was also confirmed by sanger sequencing of the vp gene, was well covered with both types of cdna synthesis ( figure ). some contigs also mapped to another genotype, echovirus , but these were only short contigs, which may have been wrongly taxonomically assigned due to the short sizes of the illumina reads. otherwise, we also found a variety of viral sequences, with the most prominent being the tomato mosaic viruses and other plant pathogens, reflecting the results of the drs run. the consensus sequence identity for echovirus sequenced by drs to illumina miseq was calculated as . % ( / ), with % gaps ( / ). using rna extracted from clinical samples, we were able to repeatedly identify human enteroviruses in stool samples from three independent patients by nanopore-based drs in this proof-of-concept study. beyond identifying ev species and genotypes correctly, we showed that the approach may also provide rich metatranscriptomic information on sample composition for all life domains. clear differences in overall species compositions, with either yeast or bacteria dominating the majority of obtained reads, were observed between samples. viral reads constituted between . % and . % of total passed reads. by complementing drs data with illumina miseq sequencing data for the same samples, we were able to validate the obtained enterovirus sequences and added further metatranscriptomic information. illumina miseq sequencing revealed a higher diversity in viral species in these samples (table ) , which was not captured by the drs approach. in all samples, % to . % of the ev reference genome sequences were covered. the average identity of the drs consensus was - % compared to the illumina miseq consensus sequence. other studies using drs have achieved similar values with - % identity when sequencing viruses from cell culture [ , ] , or up to % consensus identity for influenza a [ ] . for applications with samples with low target concentration such as patient samples, getting enough sequencing depth to obtain accurate consensus sequences constitutes a challenge for the method. the resulting consensus sequences might therefore be of limited use for applications which require high accuracy, such as phylogenomic analysis. however, for the purpose of genotype identification, the approach is sufficiently accurate, especially as it can provide long reads, which can facilitate mapping to the correct reference, and expedite downstream bioinformatic analyses. furthermore, different ev genotypes have < % nucleotide identity in the vp region [ , ] , which makes vp -based genotype identification possible even with the current high error rate of drs (modal read accuracy of > % [ ] ) when considering read length above few hundred bases. our wet laboratory approaches were refined during the course of the study while analyzing further samples, and the experiments had to be adapted to limited patient material availability. the good overall agreement in ev detection between the two ngs approaches suggests that the variability observed in number of reads and composition between samples may be attributable to the natural biological variability that may exist between the sampled patients. yet, finer differences in composition between samples may be explained by the sequencing technology and by the different pre-treatments of the samples: on the one hand, smaller cdna molecules produced by the wet laboratory procedure (cdna synthesis, bead cleaning), followed by short-read sequencing, may be easier to detect, than larger, intact rna molecules via drs. on the other hand, unreliable mapping and poor taxonomical identification may be produced by short reads aligning to genomic regions that are conserved among genotypes, but also by long, error-prone nanopore reads [ ] aligning incorrectly to reference sequences. therefore, further large-scale comparisons of the two ngs approaches would be needed to confirm or reject the hypothesis about the effect of read length on organism detectability in metagenomic or metatranscriptomic studies. while enteroviruses were detected in all tested samples using drs, it should be noted that these were all samples with relatively high viral load, i.e., low ct values. with a range of only - total reads mapping to enteroviruses for the three clinical samples, the approach was close to miss ev detection in those samples. in the case of sample e , this sensitivity issue could explain why co-infection by another enterovirus was not detected by drs, but only by the short-read sequencing-based approach. in a recent study on the use of drs for identification of porcine reproductive and respiratory syndrome virus, viruses could be detected in spike-in samples with . × viral copies and in clinical samples with . × viral copies [ ] . another study has previously reported low sensitivity of nanopore sequencing in a viral metagenomic approach in patient samples with low viral titers, even when sequencing viral genomes via sispa-based amplification [ ] . an improvement in sensitivity would be necessary before attempting to sequence samples with low viral load in general. a limiting factor of the drs approach was the amount of polyadenylated rna used for library preparation, as in two of the sequencing runs, the full flowcell sequencing capacity was not used and the sequencing was stopped before maximal run duration, because low amounts of rna library were loaded on the flowcell. if a sufficient amount of patient samples is available to obtain enough rna, increasing the input concentration to fulfill the recommended input of at least ng polyadenylated (as opposed to total) rna might have provided better coverage of the target genome sequences. furthermore, drs sequencing is expensive if only one sample is loaded per flowcell. recent progress with barcoding for drs [ ] might, however, reduce this limitation in the future. additionally, results might be improved with adaptations in viral enrichment steps. we used a chloroform/bead pre-treatment in drs samples for enrichment of viral capsid and although it might be efficient in enriching viral reads, it could also reduce the overall amount of rna extracted. as the maximum sequencing yield of the flowcell was not always reached, perhaps milder enrichment methods might be preferable, especially if the broader diversity of pathogens is to be studied. currently, the drs method is restricted to sequencing of polyadenylated rna. however, performing polyadenylation of the total rna fraction could expand its applications. another characteristic of the drs method is the expected sequencing bias towards the ' end of the polyadenylated molecules [ ] . hence, coverage of the ' region of the ev genomes was generally low, which may be problematic given that the vp region is the main region used for ev genotyping (e.g., located at position - in echovirus kt . ). thus, correct genotyping would require around bases long rna reads from the ' towards the ' end of the genome sequence. we encountered lesser coverage of the ' region of the genome with sample e , although in that instance, we were still able to identify the same genotype as found by the gold-standard vp pcr-based approach. however, unambiguous identification relies on the capsid region and its presence is crucial in case of recombinations or co-infections. therefore, great care must be applied during extraction and library preparation to avoid shearing or degrading extracted rna. the known high variability of ev genome sequences makes it necessary to have suitable alternatives for genotype identification available if pcr-based approaches fail. in such cases, drs of patient samples could be a valuable alternative, as the sequencing is primer independent and the sequences of long, native rna molecules are recovered. additionally, as is the case with all nanopore sequencing, the data is available in real time, which can provide faster identification and a sequencing-on-demand approach to molecular assays. overall, our proof-of-concept study demonstrated the possibilities offered by the drs technique for genotype identification of human enteroviruses directly from clinical samples. the method requires further optimization to improve the overall sensitivity and to lower costs for possible applications in routine diagnostics. however, with the rapid advancement of the technologies, those issues will likely improve in the near future. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , table s : summary of laboratory experiments; table s : summary of nanopore sequencing data; table s : summary of illumina sequencing 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minimap : pairwise alignment for nucleotide sequences bbmap: a fast, accurate, splice-aware aligner spades: a new genome assembly algorithm and its applications to single-cell sequencing an automated genotyping tool for enteroviruses and noroviruses unbiased prrsv strain detection using minion direct rna sequencing and bioinformatics tools typing of human enteroviruses by partial sequencing of vp resolving ambiguities in genetic typing of human enterovirus species c clinical isolates and identification of enterovirus , and barcoding and demultiplexing oxford nanopore native rna sequencing reads with deep residual learning. biorxiv , . © by the authors key: cord- -lzzi rnz authors: chorna, nataliya; godoy-vitorino, filipa title: a protocol for the multi-omic integration of cervical microbiota and urine metabolomics to understand human papillomavirus (hpv)-driven dysbiosis date: - - journal: biomedicines doi: . /biomedicines sha: doc_id: cord_uid: lzzi rnz the multi-omic integration of microbiota data with metabolomics has gained popularity. this protocol is based on a human multi-omics study, integrating cervicovaginal microbiota, hpv status and neoplasia, with urinary metabolites. indeed, to understand the biology of the infections and to develop adequate interventions for cervical cancer prevention, studies are needed to characterize in detail the cervical microbiota and understand the systemic metabolome. this article is a detailed protocol for the multi-omic integration of cervical microbiota and urine metabolome to shed light on the systemic effects of cervical dysbioses associated with human papillomavirus (hpv) infections. this methods article suggests detailed sample collection and laboratory processes of metabolomics, dna extraction for microbiota, hpv typing, and the bioinformatic analyses of the data, both to characterize the metabolome, the microbiota, and joint multi-omic analyses, useful for the development of new point-of-care diagnostic tests based on these approaches. a growing body of evidence, especially over the last twelve years, suggests that the composition and function of the microbiota in different human body habitats play vital roles in human development and immunity [ ] [ ] [ ] . the vagina and cervix have the lowest biodiversity, with a colonized epithelium dominated by lactobacilli [ ] , thus being an interface that plays a protective role between the host and the environment. the cervix serves a pivotal role as a gatekeeper to protect the upper genital tract from microbial invasion and subsequent pathology. recent studies have indicated that changes of the cervicovaginal microbiome [ ] [ ] [ ] , such as bacterial vaginosis [ , ] , cervical inflammation [ , ] and vaginal ph [ , ] , play a role in the susceptibility to cervical human papillomavirus (hpv) infection and the development of cervical intraepithelial neoplasia due to the profound shifts in the relative abundances of protective bacteria [ ] . although cervical cancer is preventable mainly by vaccination, detection, and treatment of lesions, it remains an important public health problem, being the fourth and the second most common cancer in women globally and in developing countries, respectively ( ). in latin america and the caribbean, it remains the number one cause of mortality due to malignant neoplasms among -to -year-old women ( ) , three times higher in latin america than in the united states (us) ( ) . according to guidelines established by healthy people [ ] , women that have had a pap test in three subsequent years need to be at~ %, however, in some areas of the world such as latin america, this does not happen, with women often lost to follow-up even before treatment occurs. guidelines for genital care include coming to clinics for a pap smear for hpv typing and cervical cytology, and for those who meet the guidelines, a return for a second visit to receive colposcopy and biopsy. these are invasive and expensive methods, and the approaches explained in this article are innovative and have the potential to be useful in advancing the field. a persistent infection with high-risk human papillomavirus (hr-hpv) types is a common, but not sufficient to cause cervical cancer [ , ] . high risk (hr) hpv infection often requires lifestyle factors for cancer development, such as smoking or oral contraceptive use [ ] , however, the reasons for hr hpv promotion and persistence are not completely understood. a gap in our understanding of hpv infection and carcinogenesis resides in the insufficient understanding of the true microbial community structure, preventing our ability to control the microbiota for therapeutic purposes. as widely known, the cervicovaginal microbiome also plays a role in disease pathogenesis in combination with hpv, namely genital dysbiosis, a modifiable risk factor for cervical disease [ ] [ ] [ ] [ ] . a comparison of the vaginal microbiome in women of different ethnicities within the u.s. mainland indicates different relative abundances of the four main groups of lactobacillus and anaerobic bacteria [ ] . different studies have described that infections with hpv are associated with an increase in atopobium vaginae and g. vaginallis [ , ] . other latino populations, like mexicans, have shown sneathia spp. as a cervical biomarker for neoplasia [ ] . not only is bacterial biota associated with hpv, but studies have also found an important role of fungal signatures such as that of malassezia for hr hpv infections and an increase in sporidiobolaceae and sacharomyces in atypical squamous cells of undetermined significance (ascus) lesions [ , ] . thus, non-lactobacillus dominant communities, with a concomitant increase in ph and related to high-grade dysplasia, perturb amino acid and nucleotide metabolisms and drive inflammation and hpv infections. the combination of the microbiome with metabolome can become even more informative than classical hpv typing. recently, microbial inventories via sequencing of s rrna genes, have been complemented with metabolomics, adding a layer of information regarding the metabolic activities of the microbiome that helps elucidate the etiology of the microbiome-mediated disease [ , ] . metabolites produced by commensal microorganisms may affect host metabolic processes, with both pathogenic and protective consequences. multi-kingdom colonization (bacteria, archaea, fungi, and viruses) associated with hpv infections in the vagina and cervix can produce metabolites which translocate through the host's barriers, that could influence systemic health effects in women at risk of developing cervical cancer. indeed, recent findings suggest that cervical microbial metabolic changes may influence systemic health effects in women at risk of developing cervical cancer [ ] . recent data has been emerging on the usefulness of coupling microbiota inventories with systemic metabolomics in biofluids such as urine (that can be self-collected), thus easy to obtain and minimally invasive. indeed, biomarkers for cervical cancer may be exfoliated as debris in urine, a kind of liquid biopsy-which could facilitate diagnostics [ ] . it is, therefore, important to develop protocols to help integrate information between the microbiome, viral pathogenesis, and immunity affecting cancer progression. additional to microbiota analyses, one could develop shotgun metagenomic methods to understand the full diversity more accurately, including archaea and viral composition affecting the dysbiotic or disease status. indeed, shotgun approaches are useful methods that can non-specifically detect hpvs and other highly divergent viruses (specifically dna viruses), using rolling circle amplification (rca) [ ] , or even using meta-total rna sequencing (metrs) methods based on shotgun sequencing of total rna, which could reveal the extent of rna viruses [ ] (even the novel coronavirus-sars-cov- ), which could lead to new methodologies to reveal the impact of the new virus on the microbiome and the immune response responsible for carcinogenesis. it is crucial to develop more microbiome/metabolome studies to understand cancer progression. the early detection and treatment of genital dysbiosis may facilitate better management of the disease [ ] . as such, new quick tests that could monitor genital health based on the use of vaginal secretions and even urine could be useful. dysbioses of the genital microbiome result in high ph due to the decrease in fermenting lactobacillus, and create a genital microbiome/metabolome fingerprint, which in the future could be detected with different applications [ , ] . these metabolomic profiles require advanced diagnostics and point-of-care applications for which big data from microbiome and metabolome are needed. here, we provide a unique and detailed guide for the development of multi-omic integration studies of the bacterial microbiota and urine metabolomics to understand possible biological associations with the host's urinary metabolites. this article discloses detailed materials, methods, sample processing, and data analyses. the coupling of two omic methods is a useful approach to understand the association of the human microbiome with health status and risk of disease severity, and for the development of new point-of-care testing technologies. in the second step (solution c ), what occurs is the precipitation of non-dna organic and inorganic material, including cell debris and proteins. given that cervical swabs are low biomass samples, this step will be joined with inhibitor removal (solution c ). . add µl of c and µl of c . vortex for s. . incubate at − • c for min. . centrifuge at room temperature for min at , rpm. . avoiding the pellet, transfer up to, but no more than µl of supernatant to a clean ml tube. . the next step with solution (rich in salt), is aimed at binding the dna to spin filter. . add µl of c (previously shaken) to the former supernatant and vortex for s. . place the spin filters in the vacuum manifold and load approximately µl onto a spin filter. turn on the vacuum until all the supernatant flows through. add an additional µl of the supernatant to the spin filter and vacuum. . load the remaining of the supernatant and repeat it. a total of three loads for each sample processed are required. . the next step includes washing the dna bound to the membrane and remove contaminants. . add µl of cold % ethanol (− • c) to all the tubes and turn on the vacuum. . add µl of solution c to all tubes and turn on the vacuum. carefully place the spin filter in a clean ml collection tube. avoid splashing any solution c onto the spin filter. it is critical to remove all traces of solution c as it can interfere with many downstream dna applications. . the final step is dna elution from the membrane using sterile solution c ( mm tris) or sterile pcr water. . add - µl of c ( • c) to the center of the white filter membrane and let sit for min. volume range depends on how diluted you wish the sample to be, if low biomass, lower volumes of c are suggested. . centrifuge for min at room temperature at , rpm. . discard the spin filter and store dna at − • c ( figure ). . in the second step (solution c ), what occurs is the precipitation of non-dna organic and inorganic material, including cell debris and proteins. given that cervical swabs are low biomass samples, this step will be joined with inhibitor removal (solution c ). . add μl of c and μl of c . vortex for s. . incubate at − °c for min. . centrifuge at room temperature for min at , rpm. . avoiding the pellet, transfer up to, but no more than μl of supernatant to a clean ml tube. . the next step with solution (rich in salt), is aimed at binding the dna to spin filter. . add μl of c (previously shaken) to the former supernatant and vortex for s. we suggest the use of a highly sensitive short-polymerase chain reaction-fragment assay (labo biomedical products, rijswijk, the netherlands, licensed innogenetics technology). this assay has been used for epidemiological and vaccination studies because it has high analytical sensitivity [ , ] and other cervicovaginal microbiota related studies [ , ] . it starts with a pcr step, followed by a reverse hybridization protocol. thaw the dna previously extracted from clinical samples, mix by vortexing and spin down the tubes to collect all liquid at the bottom of the tube. the assay uses spf primers to amplify a -bp fragment of the l open reading frame of hpv genotypes, followed by a reverse-hybridization step. the -bp pcr fragment assay amplifies the following common mucosal hpv genotypes: prepare the master mix for the pcr. at first use of the pcr master mix (vial a ), it is recommended to mix the contents of the vial on a vortex mixer and aliquot the contents in ready-to-use portions of µl. this further reduces the risk of contamination. after aliquoting, store at − • c . prepare the tubes/ -well plates (depending on the number of samples) considering the number of the samples to process and include negative and positive controls provided in the kit. mark each pcr reaction vial/pcr plate. . spin down the liquid in the tubes with the processed specimen for s at , rpm in a minicentrifuge. . add (one by one) µl of the supernatant of the processed sample to the appropriate vials with pcr master mix. the vials intended for the hpv negative and positive pcr controls remain closed. cap each vial after the addition of dna before proceeding with the next. . mix hpv positive pcr control (vial b ) on a vortex mixer, add µl to the appropriate vial, and close the vial. . use clean filter tips for each sample and mix each sample with the pcr mix by pipetting up and down a few times. in the second step, the amplified fragments undergo a line probe assay by reverse-hybridization assay performed by using the portion of the kit named rha kit hpv spf -lipa . in this second step, the biotinylated amplicons are denatured and then hybridized with specific oligonucleotide probes immobilized as parallel lines in membrane strips. after immobilization of the oligonucleotides on the strips, these were washed, and streptavidin alkaline phosphatase (sap) is added to bind to the biotinylated hybrid formed, yielding a purple precipitate in the strip that visually determines the specific hpv type compared to the kit-provided controls. the lipa strips are visually inspected and interpreted following the standardized reference guide provided by the kit's manual. . prewarm the vials with the hybridization solution (hs) and the stringent wash solution (sw) to at least • c but must not exceed the hybridization temperature of • c (all crystals should be dissolved before the opening of the vial). currently, it is cheaper to extract good quality dna and send directly to any company for outsourced sequencing of s rdna genes. to characterize the microbiota, one sequences the v hypervariable region of the s ribosomal rna using the universal bacterial primers: f ( gtgccagcmgccgcggtaa ) and r ( ggactachvgggtwtctaat ) as described in the earth microbiome project (emp; http://www.earthmicrobiome.org/emp-standard-protocols/ s/;) [ ] . once the facility sends the data, raw fastq reads can be uploaded to qiita https://qiita.ucsd.edu/, an entirely open-source microbial study management platform [ ] . it allows users to keep track of multiple studies with multiple omics data, and use stringent quality criteria phred scores (quality score of the nucleotides generated by automated dna sequencing) with an ascii offset of , and the user can choose the length of the reads for trimming, demultiplexing and binning using a given database (greengenes, silva, or the ribosomal database project-rdp). the usefulness of this platform is that it allows data to be directly made public via ebi (the european bioinformatics institute), a great service offered by the qiita team at the university of california san diego (ucsd). after the otu table is prepared in qiita, an alternative is to use qiime for analyses or r packages such as bioconductor [ ] or the quick tool microbiomeanalyst (https://www.microbiomeanalyst.ca) for preliminary analyses [ ] . reads matching chloroplast, mitochondria, and unassigned sequences should be removed as well as rare singletons. . downstream taxonomy plots, alpha and beta analyses should follow a given rarefaction level considering a minimum number of reads shared by most samples. . taxonomic barplots, alpha richness (number of otus/operational taxonomic units) and geometric diversity boxplots (shannon index of equitability [ ] ), can be built using r's ggplot package [ ] . . heatmaps can be built using the plot_heatmap function from the heatmap library [ ] . . additional analyses can be plotted using a microbiomeanalyst that integrates multiple r programs [ ] and also includes lefse (linear discriminant analysis effect size) analyses [ ] . . for beta diversity, samples can be visualized using pairwise bray-curtis distance between samples using the r package [ ] with vegdist function in vegan [ ] . the global differences in bacteria can be visualized with principal coordinates analysis (pcoa) or non-metric multidimensional scaling (nmds). alternatively, beta diversity can be visualized using unifrac [ ] . all reagents must be an analytical grade (hplc). all aqueous solutions used throughout this protocol should be prepared with milli-q or deionized water ( . mΩ-cm, at • ). when possible, all procedures should be performed using glass appliances. the whole extraction needs to be performed at • c since the metabolites can degrade as little as a few seconds. mix with µl of the cold methanol-water mixture ( : v/v) . vortex mixture for min. keep the sample at • c or ice for min. centrifuge at rpm for min at • c. . de-assemble the screw cap and remove silicone septum. perforate it using scissors and mount vials onto a rotary vacuum evaporator. (see note ). . remove µl of the supernatant from the top phase and transfer to a glass vials, thread - . . prepare reference pool quality control samples (see note ). . attach vials to a rotary vacuum evaporator (see note ). . evaporate supernatants to dryness. . replace caps with solid tops. . put vials in desiccator and store at − • c for at least four weeks. prepare the derivatization solution : mix mg methoxyamine hydrochloride in ml pyridine using ml glass vials with plastic caps solid-top, thread - (see note ) . briefly, vortex the obtained solution at rt until methoxyamine hydrochloride is fully dissolved. take out dried samples from storage and allow them to warm up to room temperature for at least min before derivatization (see note ) and add µl derivatization solution , close tightly the vials with closed caps and incubate at • c for two hours. remove samples, add µl of the derivatization solution (see note ) directly to the reaction mixture in the vial, close tightly the vials with closed caps and incubate for an additional h at • c. transfer the reaction mixture to labeled eppendorf vial and centrifuge at , rpm for min at rt. transfer the supernatant in the new glass vial and immediately close each sample with a solid plastic cap, thread - . store for a long time at − • c and for a short time at • c (see note ). . for the analysis, place a glass insert in the new glass vial and dilute each sample : with hexane prior to gc/ms analysis (see note ). the mass spectrometer must be tuned according to the manufacturer's manuals for optimal parameters for ion lenses, detector voltage, and other settings. usually, this can be performed in autotune operation. inject µl of each sample including quality control samples in the gc-ms. separate metabolites using a gc temperature ramping program. the gc oven can be programmed from • c to • c at a rate of • c/min. the injection port temperature will be • c, and helium is used as the carrier gas at a constant linear velocity of cm/s. injection mode-split ( : ). detect metabolites by setting the ion source filament energy to ev, the ion source temperature − • c, and the scan range (mass-to-charge, m/z) - da. metabolomic data acquisition and analyses obtain the total ion chromatogram and mass chromatograms for each detected metabolite gc/ms instruments generate a single file per sample, a list of mass spectra together with their corresponding retention times (rt). these spectra are commonly shown on a chromatogram represented by rt on the horizontal axis and signal intensity on the vertical axis ( figure ). identify metabolites based on the fragmentation pattern of molecular ions (characteristic fragment ions) using the gc/ms manufacturer's software equipped with nist/epa/nih mass spectral library (nist ) (see note ) . gc/ms instruments generate a single file per sample, a list of mass spectra together with their corresponding retention times (rt). these spectra are commonly shown on a chromatogram represented by rt on the horizontal axis and signal intensity on the vertical axis ( figure ). . identify metabolites based on the fragmentation pattern of molecular ions (characteristic fragment ions) using the gc/ms manufacturer's software equipped with nist/epa/nih mass spectral library (nist ) (see note ). statistical tests on the beta diversity can be completed via community-level differences between sample groups, assessed using the permanova test, which allows sample-sample distance to be applied to an analyses of variance (anova)-like framework [ ] . this permanova test is determined through permutations and provides strength and statistical significance on sample groupings using a bray-curtis distance matrix as the primary input. analyses of variance tests using the aov function in r (team, ) must be used on the rarefied richness (alpha) values as well as for the shannon diversity values, to find significant differences related to a given category. p-values should be considered using an fdr of . to be considered significant. . the differential abundance test for otus, to identify possible signatures significantly associated with the metadata categories (biomarkers), can be completed with non-parametric wilcoxon rank sum tests that have lower false positive rates and are more robust to outliers, without needing a normal distribution assumption. biomarker analyses can be identified also with lefse [ ] . quantify each peak using the maximum peak intensity value and organize data as a "peak intensity table" using ms excel or similar software (see note ) . the table must include metabolite identities and peak intensity values and saved in comma separated values (.csv) or tab delimited text (.txt). perform statistical analyses using free online tool metaboanalyst.ca [ , ] (see note ). . open metaboanalyst.ca, choose "statistical analysis", upload the "peak intensity table" and submit. if the table was organized correctly and saved in the required format, the "data integrity check" will pass the information for further analysis. click the "skip" button to accept the default practice (see note ) . in "data filtering" click "none". . perform data normalization to remove unwanted variations between the samples. first, perform "sample-specific normalization. click "proceed" (see note ). for two groups (control and experimental) perform univariate analysis (fold change analysis, t-tests, volcano plot, correlation analysis). . for more groups, perform chemometrics analysis (principal component analysis (pca), partial least squares-discriminant analysis (pls-da), feature identification and cluster analysis. detect outliers to improve downstream results (see note ). the integration of metagenomics and metabolomics data could be performed using model-based integration of metabolite observations and species abundances (mimosa ) freely available at borensteinlab.com/software_mimosa .html [ , ] . mimosa summarizes paired microbiome-metabolome datasets to support mechanistic interpretation and hypothesis generation. mimosa also characterizes the relative capacity of community members to produce or consume metabolites based on a priori metabolic information of the activity of metabolic enzymes for each species from the kegg database, describes how well each metabolite can be predicted by metabolic potential, and estimates how much each taxon can explain each metabolite. prepare the output data files and save in txt format for each sample in either control or experimental groups separately. for "microbiome": from qiita or qiime, export the species table (biom) with greengenes id as otu identifiers [ ] . this will allow for picrust [ ] to use the otu table and result in a functional-gene-count matrix, telling the count of each functional-gene in each of the samples surveyed, based on the reference genome. for "metabolome"-use kegg [ ] ids for each identified metabolite and it's identified concentration. log in to borensteinlab.com/software_mimosa .html. the following options will be used for the analysis: . mimosa will calculate cmp scores for each metabolite and generate a tab-delimited file for each bacterial species in each condition. additionally, a contribution table will be generated as a tab-delimited file which will relate taxa, to kegg metabolic pathways with its associated p-value, model slope and varshare (represent the fraction of the variation) for each metabolite explained by the taxon in question. varshare values could be used to build heatmaps in r, in the same format as those for microbial taxa. finally, mimosa will identify microbial features that may underlie differences in microbial metabolite concentrations between similar communities. it answers questions such as: ( ) do the metabolites in a given dataset appear to vary depending on microbiome composition? which ones? ( ) can differences in microbiome metabolic capabilities explain metabolite variation? ( ) which taxa, genes, and reactions appear to be playing a role in metabolite differences? [ ] . methoxyamine hydrochloride is very sensitive to moisture. storage is recommended under the vacuum at rt. glass inserts decrease the surface area inside the vial allowing to achieve maximum sample recovery and easier sample removal. de-assemble the screw cap and remove silicone septum. perforate it using scissors and mount the vials onto a rotary vacuum evaporator. this step is required to minimize sample cross-contamination during evaporation. preparation of the reference pool quality control samples. prepare a large pool sample during the preparation of sample extracts by aliquoting µl from each ml authentic subject sample. after a collection of a large pool sample, aliquot all for ml in separate vials (thread - ), label all as reference quality controls and process similar to authentic subject samples. continue as instructed in section . . . the derivatization solution without the sample only, and empty vials, must also be included as quality controls in the analysis to ensure that identified metabolites are derived from the sample [ ] . attach the glass vials to the rotary evaporator [ ] . . derivatization solution must be freshly prepared. . during derivatization, avoid contact with moisture, which will result in metabolite degradation. if samples are not dried, it is necessary to proceed to additional evaporation as said in sections . . - . . . . derivatization solution must be used from freshly opened vials. . if an analysis is performed after samples preparation, wait two hours before injecting the first sample into the gc-ms. . the sample concentration must be adequately adjusted to give sufficient chromatogram, then it needs to be concentrated before injecting the sample into the gas chromatograph. the dilution of a sample ( : ) was found to be sufficient for the analysis. however, if the signal is too low, the concentration could be increased. derivatization solution without the sample only, and empty vials, must also be included as quality controls in the analysis to ensure that identified metabolites . ms detects the mass of the molecular ions and the masses of the fragment ions. the database contains extensive information on many compounds including the masses of fragment ions that help to perform successful metabolite identification. in addition, to ensure a valid peak identification, data can be also processed using a deconvolution method, which is a signal processing technique that estimates the relative area corresponding to each peak when multiple peaks overlap within the same spectral region. this is especially important for low abundant metabolites that might co-elute with abundant major peaks. for general quadrupole mass spectrometers, data deconvolution by the freely available software amdis is recommended (http://chemdata.nist.gov/mass-spc/amdis/). . some of the metabolite's so-called "missing values" will not be identified in chromatograms. therefore, when composing the table for the analysis, missing values should be presented as empty values or na without quotes. . metaboanalyst is a web server designed to permit comprehensive metabolomics data analysis, visualization, and interpretation [ ] . the web interfaces of metaboanalyst are designed to be self-explanatory. therefore, most steps are documented on top of the corresponding pages. available tutorials and sample data sets complement the information by providing step-by-step instructions for several most common tasks. . this step is strongly recommended for metabolomics datasets with a large number of variables (more than metabolites) since many of them are from baseline noises. usually, the gc/ms analysis of brain tissues could identify less than variables. . the normalization procedures are grouped into three categories. the sample normalization allows general-purpose adjustment for differences among your sample, which can include the protein content in the sample or intensity value of internal standard spiked into the derivatization mixture before gc/ms analysis. data can be further transformed by "log" or "cube root" and/or scaled via "mean centering, autoscaling, pareto scaling, and range scaling" (please see the description of each in the metaboanalyst program). according to [ ] autoscaling and range scaling performed better than the other pretreatment methods and showed biologically sensible results in chemometrics analysis. data transformation and scaling are two different approaches to make individual features more comparable that can be used individually or in combination. scaling is a useful procedure when variables are of very different orders of magnitude [ ] . . detect outliers. this can be carried out by visual inspection of the pca plot to identify samples-outliers outside the hotelling t % confidence ellipse [ ] . many outliers could be corrected by normalization or excluded from the analysis. in many cases, outliers are the result of operational errors during the analytical process. if those values cannot be corrected, they should be removed from the analysis, but always justified [ ] . please refer to metaboanalyst.ca tutorials, or faq to select the statistical analysis method. . well-predicted metabolites were identified by the community metabolic potential (cmp) scores model in groups by examining of the total pool of metabolites with positive model slope and a model p-value < . . given that mimosa is prone to false negatives because its model is approximate, it requires a weak statistical threshold (p-value < . ) to capture more relationships of possible interest [ , , ] . additional information could be found at borensteinlab.com/software_mimosa .html helper. we are assisting a new stage of microbiome research, which moves beyond the typical s profiles listing bacteria associated to a given body site or disease, and offers the novel integration of other omics approaches, towards a better understanding of community functions in the disease, and interactions with the host. emphasis is now given to the causality of microbiome research and the development of mechanistic understandings of the influence of microbiota, including their metabolic activities. thus, the integration of metagenomics and metabolomics is becoming a very promising approach in the exploration of associations between the host microbiome and circulating metabolites with the aim of understanding the complex bacterial interactions with the host and their contributions to systemic health. using urine as a liquid biopsy is gaining more popularity. indeed, evidence suggests that biomarkers for cervical cancer may be exfoliated as debris in urine, which could facilitate diagnostics [ ] . since circulating metabolites are finally excreted through urine [ ] , recent studies suggest that exploring the association between urine metabolome and host microbiome (gut or even cervicovaginal) may complement sequencing-based approaches with a functional readout of the host microbiome and its interaction with host [ , ] . indeed, a recent study coupling gut microbiome and urine metabolomics suggests that gut bacterial compositional changes could eventually be monitored and probed using urine metabolomics [ ] . in addition, the application of the current protocol for functional characterization of vaginal and cervical hr hpv microbiota and urinary metabolome has identified species-contributors to the pool of several circulating urinary metabolites associated with cervical cancer development [ ] . the high throughput analyses of these two datasets could be key for the development of new tools (like portable devices) to give women feasible self-testing, and to allow them to be informed of their cervicovaginal dysbiosis via vaginal or urine metabolomic screening, which could benefit low-income countries, and those with a lack of access to cervical surveillance. taken together, this report is a simple and easy-to follow protocol, aimed to be used as a guide for those taking on microbiota and metabolic analyses using human biospecimens, such as cervicovaginal microbiota and urine metabolomics [ , , ] . data integration via the use of freely available mimosa software [ ] facilitates analyses providing easy export files that can be plotted in any other widely used packages such as r [ ] . generating data that combines both the microbiome and metabolomics analyses will guide the development of tools aimed at detecting dysbiosis via changes of the microbial profiles and metabolomic signatures, which will be useful for new point-of-care diagnostics. the authors declare no conflict of interest. an ecological and evolutionary perspective on human-microbe mutualism and disease human microbial ecology and the rising new medicine role of the microbiome in human development vaginal microbiome of reproductive-age women cervicovaginal fungi and bacteria associated with cervical intraepithelial neoplasia and high-risk human papillomavirus infections in a hispanic population the 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detection and identification of anogenital human papillomavirus reactivation of latent hpv infections after renal transplantation high rate of infection by only oncogenic human papillomavirus in amerindians ultra-high-throughput microbial community analysis on the illumina hiseq and miseq platforms qiita: rapid, web-enabled microbiome meta-analysis phyloseq: an r package for reproducible interactive analysis and graphics of microbiome census data microbiomeanalyst: a web-based tool for comprehensive statistical, visual and meta-analysis of microbiome data the mathematical theory of communication elegant graphics for data analysis pretty heatmaps metagenomic biomarker discovery and explanation r: a language and environment for statistical computing; foundation for statistical computing community ecology package. r package version . - . r development core team. r: a language and environment for statistical computing; r foundation for statistical computing unifrac-an online tool for comparing microbial community diversity in a phylogenetic context a new method for non-parametric multivariate analysis of variance metaboanalyst . -making metabolomics more meaningful web-based inference of biological patterns, functions and pathways from metabolomic data using metaboanalyst defining and evaluating microbial contributions to metabolite variation in microbiome-metabolome association studies metabolic model-based integration of microbiome taxonomic and metabolomic profiles elucidates mechanistic links between ecological and metabolic variation. msystems greengenes, a chimera-checked s rrna gene database and workbench compatible with arb predictive functional profiling of microbial communities using s rrna marker gene sequences discussion - metabolomics by gas chromatography-mass spectrometry: combined targeted and untargeted profiling metabolomics analysis of glutamate receptor function centering, scaling, and transformations: improving the biological information content of metabolomics data standardizing gc-ms metabolomics urine and bladder washing cytology for detection of urothelial carcinoma: standard test with new possibilities uremic solutes from colon microbes an untargeted fecal and urine metabolomics analysis of the interplay between the gut microbiome, diet and human metabolism in indian and chinese adults chapter -handing on health to the next generation: early life exposures discriminating high-risk cervical human papilloma virus infections with urinary biomarkers via non-targeted gc-ms-based metabolomics this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -v qdizwx authors: chang, jia jin marc; ip, yin cheong aden; ng, chin soon lionel; huang, danwei title: takeaways from mobile dna barcoding with bentolab and minion date: - - journal: genes (basel) doi: . /genes sha: doc_id: cord_uid: v qdizwx since the release of the minion sequencer in , it has been applied to great effect in the remotest and harshest of environments, and even in space. one of the most common applications of minion is for nanopore-based dna barcoding in situ for species identification and discovery, yet the existing sample capability is limited (n ≤ ). here, we assembled a portable sequencing setup comprising the bentolab and minion and developed a workflow capable of processing samples simultaneously. we demonstrated this enhanced capability out at sea, where we collected samples and barcoded them onboard a dive vessel moored off sisters’ islands marine park, singapore. in under h, we generated minion barcodes, of which belonged to fresh metazoans processed immediately after collection. our setup is thus viable and would greatly fortify existing portable dna barcoding capabilities. we also tested the performance of the newly released r . nanopore flow cell for dna barcoding, and showed that the barcodes generated were ~ . % accurate when compared to illumina references. a total of % of the r . nanopore barcodes also had zero base ambiguities, compared to – % for r . . , suggesting an improved homopolymer resolution and making the use of r . highly recommended. the practice of dna barcoding-involving the generation of standardized genetic markers that, when matched to databases, allow for species identification-was first popularized by [ ] . since then, the field of dna barcoding has evolved and expanded considerably beyond just species identification [ ] [ ] [ ] [ ] to include species discovery, population genetics, and phylogenetics [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this rapid growth in dna barcoding capabilities has occurred as a result of advancements in sequencing technologies. for instance, the rise of second-generation sequencers (e.g., illumina) has greatly enhanced our ability to produce dna barcodes in larger volumes (vis-à-vis sanger sequencing) while maintaining high accuracy and low costs [ ] [ ] [ ] [ ] . however, one of the limitations of second-generation sequencing technologies is that the dna barcoding process and its associated technologies largely remain spatially confined to specialized laboratory settings. the development of the minion sequencer by oxford nanopore technologies (ont) was thus significant for nucleic acid sequencing as it quickly materialized the concept of portable sequencing. its release was game-changing for several reasons, though most notably for its compact size and portability, as well as its ability to generate data in real time [ ] . since then, the minion sequencer has we amplified the -bp region of the mitochondrial cytochrome oxidase subunit i (coi) locus, using the mlcoiintf: ′-ggw acw ggw tga acw gtw tay ccy cc- ′ [ ] and lobor : ′-taa acy tcw ggr tgw ccr aar aay ca- ′ [ ] primer combination. the primer pair was chosen due to the high amplification success of marine fauna [ ] , and was also comparatively cheaper [ , ] than the conventional metabarcoding primer pair jghco [ ] and mlcoiintf metazoan specimens were collected opportunistically from ten coral reef sites across singapore from to , either via intertidal surveys or subtidally via scuba. collections were authorized by the national parks board (permit number np/rp - ), and samples were carefully treated according to nus institutional animal care and use committee (iacuc) guidelines (iacuc protocol b - ) during the collection and vouchering process. during the vouchering phase, samples were grouped into phylum/class based on morphology. this was to facilitate downstream amino acid correction (see section . .), as well as morphology-barcode identity congruence checks. voucher specimens were imaged using the canon ef mm f . /l is usm macro lens on an eos d. genomic dna extractions were either carried out using phenol:chloroform:isoamyl-alcohol ( : : ) phase separation [ ] , or via the abgenix tm automated dna and rna extraction system (aitbiotech pte ltd., singapore) with animal tissue genomic dna extraction kits according to the manufacturer's protocols. all samples were processed individually regardless of extraction method. we amplified the -bp region of the mitochondrial cytochrome oxidase subunit i (coi) locus, using the mlcoiintf: -ggw acw ggw tga acw gtw tay ccy cc- [ ] and lobor : -taa acy tcw ggr tgw ccr aar aay ca- [ ] primer combination. the primer pair was chosen due to the high amplification success of marine fauna [ ] , and was also comparatively cheaper [ , ] than the conventional metabarcoding primer pair jghco [ ] and mlcoiintf (supplementary table s ). pcr primers were each tagged with unique -bp barcode tags on the end to allow for convenient downstream demultiplexing [ ] , and we ensured that forward and reverse tag combinations were unique to each specimen. each pcr reaction mix comprised µl of template dna, µl each of µm -bp tagged primer, µl of bovine serum albumin (bsa; mg/ml), µl of magnesium chloride, and . µl of gotaq ® green master mix (promega), and was topped up to µl with nuclease-free water. a step-up thermal cycling profile was used: • c for s; cycles of • c for s, • c for s, • c for s, followed by cycles of • c for s, • c for s, • c for s, and a final extension for min at • c. amplification success was verified on % gels stained with gelred (cambridge bioscience). pcr amplicons were pooled based on gel band intensity and cleaned using . × sera-mag tm magnetic speedbeads tm (ge healthcare life sciences) in % polyethylene glycol- (peg- ) buffer ( m nacl, nm tris-hcl, nm edta, ph ). we then prepared pcr-free libraries using the nebnext ® ultra tm ii dna library prep kit (new england biolabs), but with truseq dna single indexes (set b, illumina), following the manufacturer's instructions up to the adapter ligation step. libraries were cleaned using the same . × sera-mag peg suspension, and sequenced in batches over two illumina miseq lanes ( × -bp) at the genome institute of singapore. note that each batch utilized only~ % of each sequencing lane. we followed the modified bioinformatic pipeline based on sze et al. ( ) [ ] and leveque et al. ( ) [ ] , where we used pear v . . [ ] to merge paired-end reads, and obitools v . . [ ] for demultiplexing and further downstream processing of assembled reads. we considered illumina barcodes valid if ( ) the dominant read sequence for the sample had a minimum × read coverage, and ( ) if the dominant read sequence was at least five times more abundant than the next most dominant read sequence assigned to that sample [ , ] . finally, we performed a translation check of illumina barcodes on geneious r v . . [ ] to ensure there were no internal stop codons. in preparation for the field sequencing phase, we first tested extractions and gene amplification with the bentolab in the laboratory. we used quickextract tm (lucigen; heron referred to as "qe"), a dna extraction solution which requires only incubation with a heat source to produce pcr-ready genomic dna. this can be easily supplied by the thermocycling component of the bentolab, thus making it a potentially convenient method of dna extraction in situ. the qe solution has been used extensively on insects [ , , [ ] [ ] [ ] as well as zooplankton [ ] but only on a handful of marine macrofauna [ ] . we tested the qe-based protocol on the bentolab for the same group of samples prior to field sequencing. tissue subsamples were immersed in µl of qe solution, and reactions were incubated at • c for min, followed by • c for min [ ] . the qe products were then diluted × prior to pcr with nuclease-free water, following the manufacturer's recommendation, to reduce pcr inhibition. gene amplification was performed using the same primer pair described above. for minion-based barcoding, however, the primers were tagged with -bp tag sequences (instead of the -bp tagged primers used previously for illumina sequencing) to account for the higher sequencing error rate in nanopore sequencing [ ] , while still allowing for accurate sample demultiplexing downstream [ ] . our µl pcr reaction mix was altered to: µl of template dna, µl each of µm -bp tagged primer, µl of bsa ( mg/ml), and . µl of gotaq ® green master mix (promega), and topped up with nuclease-free water. we replaced magnesium chloride with more bsa to better neutralize potential pcr inhibitors that might be present in the extracts [ ] . we also took this opportunity to test if a shortened cycling profile would be feasible. the thermal cycling profile used was • c for s; cycles of • c for s, • c for s, • c for s, followed by cycles of • c for s, • c for s, • c for s, and a final extension for min at • c. gene amplification success was likewise verified on % agarose gels. we pooled the amplicons by gel band intensity, taking and µl for bright and faint to no observed gel bands, respectively. the amplicon pool was cleaned with . × ampure xp magnetic beads (beckman coulter) and stored at − • c till the field sequencing phase. we performed field extraction, pcr, and sequencing as a proof-of-concept demonstration that the entire workflow was field-ready. here, we assembled an in situ barcoding workflow involving the bentolab, minion sequencer, and a laptop computer (intel ® core i - h; figure ). we tested the system out at sea onboard a dive vessel moored off sisters' islands marine park, singapore on july , and documented the process from sample to sequence ( figure ). during the field trip, thirty-one fresh invertebrate metazoan samples were collected via scuba. collections were authorized by the national parks board (permit number np/rp - ). samples were subsampled onboard the diving vessel. all samples, including one negative control, were extracted and gene-amplified using the bentolab with the same methods described above (see section . .), but with minor adjustments. we increased the volume of qe per reaction to µl, and decreased the total number of pcr cycles to . we ensured that the tag combinations used in the field pcr step did not overlap with the tagged amplicons generated at the home laboratory. liquids were mixed by flicking the tubes or pipetting by hand. we also did not check for amplification success on agarose gel, and proceeded to pool the pcr products (taking µl each) together with the amplicons generated ex situ for the bead clean-up using . × ampure xp magnetic beads (beckman coulter). drying of the magnetic pellets was performed using a phone-powered mini fan. the final amplicon pool was quantified using a qubit . fluorometer with the qubit dsdna br assay kit (thermofisher scientific, waltham, ma, usa). we prepared a minion library onboard using the ligation sequencing kit (sqk-lsk ), with the following modifications: ( ) end repair and da-tailing reactions were incubated in the bentolab at • c for min, followed by • c for min, and ( ) ligation reactions were similarly incubated for min at • c. this undoubtedly increased the library preparation time, but we noted improved library success with the protocol changes [ ] . bead clean-ups were performed after end repair and adapter ligation. the library was sequenced on a fresh r . . flow cell, and left to run on a laptop (minknow v. . . ) for~ min. during the field trip, thirty-one fresh invertebrate metazoan samples were collected via scuba. collections were authorized by the national parks board (permit number np/rp - ). samples were subsampled onboard the diving vessel. all samples, including one negative control, were extracted and gene-amplified using the bentolab with the same methods described above (see section . .), but with minor adjustments. we increased the volume of qe per reaction to µl, and decreased the total number of pcr cycles to . we ensured that the tag combinations used in the as we had exhausted the amplicon pool during library preparation for the first flow cell, we re-pooled the amplicons and prepared a second library for sequencing on a fresh r . flow cell on the same laptop back at the laboratory. no changes were made to the reaction conditions. we monitored the sequencing progress and ended the run when an approximately same number of reads was generated as the r . . dataset. run time for r . lasted h min. for both sets of minion raw reads, we performed gpu basecalling via guppy v . . + d e. for the r . . flow cell, we generated two datasets, one produced using the fast basecalling model ("fast"), and the other via the high-accuracy ("hac") model. the latter basecalling model produces higher single read accuracy, but is computationally more intensive than the former, and hence slower. we sought to investigate if the basecalling model had an impact on minion barcodes generated from an error correction pipeline like minibarcoder. for the r . dataset, we started with two raw datasets. the first dataset was subsampled to the same run time as on r . . ( min; hereon referred to as "st"), while the second dataset had approximately the amount of reads generated (~ million) as the r . . flow cell (heron known as "sr"). for both r . read sets, we likewise performed basecalling using the fast and hac models. all six instances of basecalling were performed using the same settings, and we also noted the time taken for each instance (supplementary table s ) . we then performed minion barcode calling using the minibarcoder pipeline [ ] . first, we used the minibarcoder.py script to generate preliminary mafft barcodes via an alignment consensus approach. briefly, the python script employed glsearch [ ] to search for primer sequences in order to retrieve flanking tag sequences (supplementary file s ), which were then used to bin reads into respective samples, before mafft v . [ ] was applied at the sample level for alignment of binned reads to call a majority consensus, or the "mafft barcode" [ ] . any resulting mafft barcodes that had < × read coverage and > % ambiguous bases called as ns were discarded. we then applied racon_consensus.py to map the raw reads back to the mafft barcode using graphmap v . . [ ] before generating consensus sequences using racon v . . [ ] to yield "racon barcodes" [ ] . we subsequently used publicly available genbank sequences (nt database updated july ) for amino acid correction [ ] of the mafft and racon barcodes to yield "mafft + aa" and "racon + aa" barcodes, respectively. as our sample set consisted of fauna from various phyla, the appropriate genetic code (option -g) was applied in the correction process [ ] ; we used code for actinopterygii, code for cnidaria and porifera, code for echinodermata, hemichordata, and platyhelminthes, code for ascidiacea, and code for the remaining invertebrates. we also varied the namino parameters from to [ ] . the final step was to align the corrected mafft+aa and racon + aa barcodes and call a strict consensus (using consolidate.py) to produce "consolidated barcodes" [ ] . we used seqkit v . . [ ] and gnu parallel [ ] to accelerate barcode calling (see supplementary file s for unix script for automating minibarcoder). all minion barcode calling steps were executed locally on the dedicated field sequencing laptop. the entire minibarcoder pipeline took~ - min for each dataset totaling amplicons ( samples + negatives). we first subjected the illumina and minion barcodes to a contamination check. for the minion barcodes, we used the mafft barcode dataset as it was the largest, and correspondingly filtered the other types of minion barcodes of detected contaminants [ , ] . we performed a blastn search (ncbi blast+ v . . ; [ ] ) on the same offline nt database (-evalue e − , -max_target_seqs , -perc_identity ), and blast results were parsed through readsidentifier v . (≥ % identity and -bp overlap [ ] ) to obtain taxonomic identities. we only accepted species-level identities for barcode matches ≥ % [ , , ] . the taxonomic identities from readsidentifier were then compared against morphological classifications made during the sample vouchering process, and any incongruence was flagged for further voucher examination to preclude misidentifications. if a pre-sorting error was deemed unlikely, the barcodes were subsequently removed from the dataset. any barcode that matched any non-metazoan sequence was also excluded from downstream analyses. we then evaluated the minion barcode datasets based on two criteria: ( ) sequencing accuracy, and ( ) barcode ambiguity. sequencing accuracy is defined as the proportion of perfectly matched bases to the total number of bases compared, while barcode ambiguity refers to the proportion of ambiguous bases called as ns that persists after amino acid correction [ ] . these ns were introduced to preserve the reading frame [ , ] , and served to correct the sequencing errors in homopolymeric regions [ ] [ ] [ ] . as a point of reference for sequencing accuracy, we used the barcodes generated via illumina (supplementary file s ) as the sequencing technology has already been proven to be highly accurate [ , ] . our goal was to find the flow cell chemistry and basecalling model that scored high and low on sequencing accuracy and barcode ambiguity, respectively. we used the supplied assess_uncorrbarcodes_wref.py and assess_corrbarcodes_wref.py scripts [ ] ; the former utilized dnadiff v . [ ] to compare uncorrected barcodes against illumina references, while the latter utilized mafft for alignment and pairwise comparisons of the corrected barcodes with illumina ones [ , ] . any minion barcode that differed from its illumina reference by > % was deemed erroneous and flagged for removal. barcode ambiguity was assessed using the measure_ambs.py script [ ] , and visualized as boxplots on r . . (r core team, ) using ggplot [ ] . we then compared results across all six datasets to select the best performing minion barcode dataset. with the chosen minion barcode dataset, we examined samples that failed the ns-filtering step-these usually have a high number of ns in the mafft barcode sequence, which in turn suggested the presence of contaminant reads [ ] -and determined if they could be rescued. we approached these failed samples in a manner analogous to ho et al. ( ) [ ] , which was to treat these samples as small-scale metabarcoding pools, except in this case, the sample sequences were mixed with contaminant reads. we took all the binned reads in each failed sample and subjected them to blastn against the same nt database and parsed the matches through readsidentifier v . [ ] to obtain their taxonomic identities. barcode calling for the sample was repeated using only the reads that matched the morphological assignment of the voucher, and only if the retained read count was still ≥ . only four samples (hs - , hs , and hs ) were re-examined this way. finally, we performed objective clustering to collapse the dna barcodes into molecular operational taxonomic units (motus), i.e., putative species units, based on uncorrected p-distances [ , ] . we performed the clustering at - % to check for motu stability. a final blastn was conducted, and taxonomic identities were obtained by parsing the best matches through readsidentifier. we collected samples between august and january from ten coral reef sites across singapore, representing phyla (figure ). we also included seven samples from a previous study [ ] , for which we were unable to obtain dna barcodes. the sample size for the laboratory trial was . together with samples collected on the field sequencing day, the total sample size for this study was . samples for which whole vouchers were collected have been deposited in the zoological reference collection at the lee kong chian natural history museum, singapore (supplementary file s ). for the laboratory-based bentolab trial, there were samples for which we had sufficient tissue subsamples to re-extract with qe solution, followed by pcr. gel bands were observed for samples ( %). we also repeated the minion-based pcr for the remaining sample extracts with insufficient tissue and obtained gel bands for of them (~ %). three samples (hs , hs , and ip ) did not have a tissue subsample for qe re-extraction or genomic dna for re-pcr (total for laboratory phase = + + = samples). amplification success for the laboratory trial was % on average ( + = bands, out of samples). for the field sequencing phase, an additional samples were collected and subjected to qe-based dna extraction on the bentolab. while we did not run the gel check in situ to save time, a postliminary amplification check on agarose back at the laboratory revealed observable gel bands. for the laboratory-based bentolab trial, there were samples for which we had sufficient tissue subsamples to re-extract with qe solution, followed by pcr. gel bands were observed for samples ( %). we also repeated the minion-based pcr for the remaining sample extracts with insufficient tissue and obtained gel bands for of them (~ %). three samples (hs , hs , and ip ) did not have a tissue subsample for qe re-extraction or genomic dna for re-pcr (total for laboratory phase = + + = samples). amplification success for the laboratory trial was ~ % on average ( + = bands, out of samples). for the field sequencing phase, an additional samples were collected and subjected to qe-based dna extraction on the bentolab. while we did not run the gel check in situ to save time, a postliminary amplification check on agarose back at the laboratory revealed observable gel bands. table s ). we piped three datasets through guppy for gpu basecalling on the laptop: one for the r . . dataset, and two from the r . dataset, one "sr" for the same amount of reads generated as r . . (~ million reads), and the other "st" for the same length of sequencing time as r . . (~ , reads). we ran fast and hac basecalling for each of the three datasets on guppy to obtain six basecalled datasets in total. we observed a - min difference between the fast and hac basecalling models, but did not observe any ostensible difference in basecalling times between r . . and r . _sr table s ). all nanopore fast read sets and corresponding basecalled fastq files have been deposited at the ncbi sequence read archive under bioproject prjna (srr -srr , and srr -srr ). while the guppy results were fairly similar across the six datasets, we noted a more pronounced effect of the basecalling model on the number of minion barcodes obtained. in general, datasets that were called using the fast basecalling model resulted in a lower percentage of successfully demultiplexed reads ( - % for fast vs. - % for hac models). low demultiplexing success rates were expected due to the intrinsically high raw read error rate [ ] , and our values were consistent with past studies [ , ] . the fast datasets also consistently obtained a lower number of consolidated minion barcodes than the hac datasets ( - vs. - ; table ). the r . dataset performed marginally better than r . . with respect to the final number of consolidated barcodes obtained ( - vs. ). remarkably, even with only half the read size of the r . . _hac dataset, the r . _hac_st dataset obtained even more barcodes than the former. we also noted only an increase in one more barcode in the r . _hac_sr dataset, despite doubling the reads sequenced and increasing the run time three-fold. referenced against illumina barcodes, minion barcodes generated from the minibarcoder pipeline scored high on accuracy (≥ %) regardless of the flow cell or basecalling model used ( table ) . while uncorrected barcodes (i.e., mafft and racon barcodes) generated from the fast basecalling model resulted in more gaps compared to the hac model, the minibarcoder pipeline was able to correct this disparity, such that all three types of error-corrected barcodes (mafft + aa, racon + aa, and consolidated barcodes) have zero gaps across all flow cell and basecalling model datasets ( table ) . we did, however, note differences in barcode ambiguities remaining after error correction. in particular, we found that the basecalling model applied greatly influenced the proportion of remaining ambiguities in error-corrected minion barcodes more so than the flow cell type. the hac model was the superior model, and the resultant minion barcodes consistently had fewer remaining ambiguities compared to the fast basecalling barcodes (figure ). in fact,~ % of the consolidated minion barcodes from the r . _hac datasets (st and sr included) had % ambiguous bases. we eventually selected the consolidated (namino ) barcodes from the r . _hac_sr dataset as our primary minion barcode set for two reasons. first, it was the dataset that yielded the highest number of minion barcodes following contamination checks (n = ). second, it was also the dataset that did not have any remaining gaps and scored % sequencing accuracy when matched against illumina references ( table ). the namino dataset performed similarly well, but had a higher number of total ambiguous bases compared to the namino set ( vs. ). in addition, we further rescued two additional minion barcodes (hs and hs ; see section . .) for the final dataset to yield a total of minion barcodes for this study ( % success). combining datasets of illumina and minion barcodes, including overlapping barcodes, we obtained a total of unique dna barcodes from both sequencing platforms ( % success overall out of samples). we derived motus at the % threshold, of which were singletons. motu richness was stable across the - % thresholds. when compared to the existing local singapore barcode database [ ] , we found at least novel motus from this study. dna barcodes generated in this study have been deposited at genbank under accession numbers mt -mt (supplementary file s ). in this study, we assembled an in situ sequencing setup that comprised three main components: the suitcase-sized laboratory in the form of the bentolab, the minion handheld sequencer, and a laptop computer (figure ). our proposed in situ sequencing workflow employed qe solution for thermal-based dna extraction and tagged pcr on the bentolab, before sequencing on the minion and laptop. the laptop computer also served as an analysis terminal for basecalling and minion barcode calling via minibarcoder. we first tested all the protocols back at the laboratory, before conducting an in situ demonstration onboard a diving vessel moored at the sisters' islands marine park, singapore, on july (figure ). we obtained minion barcodes, of which were from samples obtained in the field. to our knowledge, the samples and dna barcodes generated here represent one of the highest throughputs from published studies to date [ , , , ] , with the entire sample-to-sequence workflow completed in under h. in the following, we discuss our experiences with portable sequencing on the bentolab and minion in the form of three takeaways learnt from the entire process. while the minion sequencer has undoubtedly been instrumental in making portable sequencing possible, the field-ready hardware has hitherto not co-evolved to keep pace with the sequencing technology. there thus remain certain logistical and operational limitations to carrying out dna barcoding in situ as discussed recently [ , , , ] . one of the most consequential constraints is the sample throughput of portable laboratory equipment. barcode amplification remains the most crucial yet time-limiting step in any dna barcoding workflow, but only a handful of samples can be processed at any one given time due to the low capacity of existing portable laboratory equipment. this low scalability potentially limits its applicability and buy-in to portable sequencing. as such, we strongly advise new users to carefully consider the sequencing targets and objectives, so as to better plan around the field equipment and conditions to suit their own needs. in our case, we successfully expanded the processing capacity to samples at any one time by using a one-step, heat-based dna extraction method on the bentolab. it is worth noting that the use of spin-column kits, as past studies have done [ , , ] , was impractical for our study given the -well configuration of the bentolab centrifuge. the overall higher throughput here would most certainly fortify existing barcoding capacities for species identification on expeditions or field courses [ , , , , ] , though unlikely to the extent where a "reverse workflow" of sorting specimens with dna barcodes can be fully realized [ , ] given the relatively small number of samples that can be processed each time. specifically, this increased barcoding capacity would be helpful for small-scale operations involving randomly selected samples, or where on-site testing is preferred but laboratory capabilities are not available, particularly in biosecurity, wildlife conservation genetics, and even food safety [ , [ ] [ ] [ ] [ ] . while the bentolab is slightly bulkier compared to the minipcr, our entire sequencing system would still fit in a backpack and not require much space to set up and deploy. one of the strengths of second-and third-generation sequencers is the ability to reduce sequencing costs via sample multiplexing onto a flow cell [ ] ; the greater the number of samples, the lower the resultant cost of each barcode. minion barcodes can cost as low as~usd . each when multiplexing samples per minion flow cell [ ] , though such a volume is unlikely to be achieved in a field setting [ , ] . users thus need to bear in mind that there are financial trade-offs with the lower throughput for portable sequencing. for our entire sample set ( amplicons), we estimated each minion barcode to cost~usd . , whereas if we barcoded only the field collections ( amplicons), minion barcoding would have cost usd . per barcode (supplementary table s ). the latter is nearly double the cost of usd per regular sanger barcode [ ] . our workflow has sought to keep molecular costs low by using qe-based dna extraction, which we estimated to be usd . a sample. it is slightly more costly than the chelex resin (usd . per sample), but still considerably cheaper than other proprietary extraction kits sold by qiagen or biomeme (usd and usd , respectively, per sample) [ ] . the thermal-based dna extraction method complemented the -well capacity of the bentolab and was instrumental in increasing our throughput. for gene amplification, it was cheaper to use tagged primers [ ] which allowed for more samples to be multiplexed onto the flow cell, compared to ont's native barcoding expansion kit for a maximum of samples. the former also saved us an additional barcode ligation step during library preparation. one other fruitful way to reduce sequencing costs is to use the lower-throughput flongle flow cells [ , ] , which are estimated to cost usd per dna barcode on a flongle multiplexed with samples [ ] . we did not test the flongle for this study as the r . chemistry is presently limited to minion flow cells. nevertheless, the field of on-site nanopore barcoding is rapidly growing, and researchers are increasingly finding creative ways to reduce costs, such as d-printing of centrifuges to complement spin-column kit extractions [ ] [ ] [ ] . we expect that as novel techniques emerge and technologies are refined, the cost of in situ nanopore barcoding is likely to fall even more in the near future. this study also investigated how flow cell chemistry and basecalling models affected sequencing accuracy and barcode quality by analyzing six different datasets. we observed that the default hac (high-accuracy) basecalling model was superior. hac datasets attained higher demultiplexing success compared to fast datasets (table ) due to the improved basecalling accuracy in the hac model. in all instances, datasets that employed the hac model resulted in more barcodes overall than the fast datasets (table ) . we also noted ostensible differences in barcode quality, where hac-produced barcodes had fewer persisting ambiguities across all three types of error-corrected barcodes ( figure ) . moreover, we did not observe significant time savings from applying the fast basecalling model (supplementary table s ). there appears to be no compelling reason to adopt the fast model and we recommend that users adhere to the default hac model for basecalling. there was a final difference of just one barcode between the st (r . with the same sequencing time as r . . ) and sr (r . with the same number of reads as r . . ) datasets. prolonging the sequencing time improved coverage but not the final barcode tally (table ) . indeed, a run time of min on r . was sufficient to capture the full range of sample diversity with amplicons, and there was no evidence to suggest that the raw read count impacted the final barcode tally in any way. in fact, the r . _hac_st dataset resulted in more consolidated barcodes than the r . . _hac dataset ( vs. ) with only half the number of raw reads generated. fewer raw reads processed translated to faster guppy basecalling times (~ × faster; supplementary table s ) and would be especially important for field sequencing workflows like ours where rapid turnover is key. the pairing of the hac model with the r . flow cell chemistry resulted in the highest quality of minion barcodes for this study. this finding is evident in how all corrected barcodes had no internal gaps, scored near perfect sequencing accuracy (≥ . %; table ), and a large majority (~ %) had zero ambiguities post-correction ( figure ). in contrast, only~ % and~ % of r . . _hac barcodes from this study and an earlier study [ ] , respectively, were free of ambiguities. n-coded bases are typically inserted during amino acid correction to resolve frameshifts caused by sequencing errors in homopolymeric regions [ ] . the observed increase in samples having zero ambiguous bases points to an improved homopolymer resolution with the r . chemistry. this marked improvement in r . sequencing chemistry is a welcome development and paves the way for furthering nanopore sequencing applications such as dna metabarcoding [ ] [ ] [ ] [ ] . error-prone reads from the r chemistry make it challenging to assign taxonomy [ ] , and previous studies have resorted to complex laboratory procedures [ ] or reference-based polishing [ ] to negate these sequencing errors. one study tested the r . chemistry for nanopore metabarcoding, but the only comparisons made to r . . chemistry were in terms of read coverage and read size distribution; no assessments were made on sequencing accuracy [ ] . given the improved dna barcode performance noted in this study, we believe similar positive knock-on effects for nanopore metabarcoding are to be expected. major advancements in sequencing technology, such as the release of ont's handheld minion sequencer, have made portable sequencing possible, and numerous studies have since emerged to advance this field. however, field-based barcoding capacity remains limited to small sample sizes. here, we expand upon existing capabilities by combining the use of bentolab with the minion. the bentolab boasts a -well thermocycling capacity, and is suited for a thermal-based dna extraction method like qe. our proof-of-principle demonstration out at sea generated minion barcodes, of which were from samples processed immediately after collection. to date, our field collection of specimens represents one of the largest sets of samples processed in situ. we also took the opportunity to test the newly released r . flow cell for dna barcoding, and report that the error-corrected barcodes scored high on sequencing accuracy, had no gaps, and showed an improved homopolymer resolution compared to the existing r . . chemistry. collectively, the illumina and minion sequencing runs here have contributed more barcodes toward efforts to grow the local biodiversity knowledge database. our in situ sequencing workflow is thus viable and joins a growing myriad of related developments aimed at advancing portable dna barcoding capabilities, raising throughput and lowering costs as the field progresses. biological identifications through dna barcodes barcoding animal life: cytochrome c oxidase subunit divergences among closely related species dna barcoding australia's fish species identification of birds through dna barcodes new evidence shows that pocillopora "damicornis-like" corals in singapore are actually pocillopora acuta (scleractinia: pocilloporidae) ten species in one: dna barcoding reveals cryptic species in the neotropical skipper butterfly astraptes fulgerator dna barcoding of marine metazoa dna barcoding: how it complements taxonomy, molecular phylogenetics and population genetics cryptic 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conservation and biodiversity research conservation of endangered wild harvested medicinal plants: use of dna barcoding dna barcoding of traded shark fins, meat and mobulid gill plates in singapore uncovers numerous threatened species barcoding and border biosecurity: identifying cyprinid fishes in the aquarium trade toward on-site food authentication using nanopore sequencing hand-powered ultralow-cost paper centrifuge open source completely -d printable centrifuge. instruments , , a d-printed hand-powered centrifuge for molecular biology speeding up the detection of invasive aquatic species using environmental dna and nanopore sequencing enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with nanopore or pacbio sequencing a workflow for accurate metabarcoding using nanopore minion sequencing nanoclust: a species-level analysis of s rrna nanopore sequencing data acknowledgments: we are extremely grateful to mei lin neo, ria tan, chay hoon toh, lynette s. m. ying, shu qin sam, sudhanshi s. jain, clara l. x. yong, and crew of summit marine system for fieldwork assistance, as well as darren yeo and arina adom for laboratory support. we also thank samuel y. k. chan for it recommendations, and also acknowledge national supercomputing centre, singapore (https://www.nscc.sg), for permitting use of its computational resources. finally, we thank nicholas w. l. yap and daisuke taira for help with specimen identification, and the curators of lee kong chian natural history museum, especially iffah binte iesa, for their help with specimen deposition. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -r bmtloy authors: jendrny, paula; schulz, claudia; twele, friederike; meller, sebastian; von köckritz-blickwede, maren; osterhaus, albertus dominicus marcellinus erasmus; ebbers, janek; pilchová, veronika; pink, isabell; welte, tobias; manns, michael peter; fathi, anahita; ernst, christiane; addo, marylyn martina; schalke, esther; volk, holger andreas title: scent dog identification of samples from covid- patients – a pilot study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: r bmtloy background: as the covid- pandemic continues to spread, early, ideally real-time, identification of sars-cov- infected individuals is pivotal in interrupting infection chains. volatile organic compounds produced during respiratory infections can cause specific scent imprints, which can be detected by trained dogs with a high rate of precision. methods: eight detection dogs were trained for week to detect saliva or tracheobronchial secretions of sars-cov- infected patients in a randomised, double-blinded and controlled study. results: the dogs were able to discriminate between samples of infected (positive) and non-infected (negative) individuals with average diagnostic sensitivity of . % ( % confidence interval [ci]: . – . %) and specificity of . % ( % ci: . – . %). during the presentation of randomised samples, the dogs achieved an overall average detection rate of % (± . %) with correct indications of positive, correct rejections of negative, incorrect indications of negative or incorrect rejections of positive sample presentations. conclusions: these preliminary findings indicate that trained detection dogs can identify respiratory secretion samples from hospitalised and clinically diseased sars-cov- infected individuals by discriminating between samples from sars-cov- infected patients and negative controls. this data may form the basis for the reliable screening method of sars-cov- infected people. the ongoing covid- pandemic highlights the importance of fast and reliable testing for accurate identification of symptomatic and asymptomatic carriers to reduce spread of infection effectively [ ] . current testing regimens usually require nasopharyngeal swabs applied by a trained person and a reverse transcription polymerase chain reaction test (rt-pcr) for pathogen identification. obtaining rt-pcr results is time consuming and can be cost-prohibitive, especially for developing countries, and is therefore currently often used in a targeted fashion, testing predominantly patients with covid- specific symptoms [ ] . there is therefore a need for an additional faster, reliable, noninvasive, and versatile screening tool, especially to identify asymptomatic and pre-symptomatic individuals. several studies have proven the canines' extraordinary olfactory acuity to detect persons with infectious and non-infectious diseases like different types of cancer [ ] , malaria [ ] , bacterial, and viral infections [ ] [ ] [ ] , with usually high rates of sensitivity and specificity [ ] . a pathogen-specific odour that can be detected by dogs may be composed of specific patterns of volatile organic compounds (vocs). compared to bacteria, viruses have no own metabolism, and therefore vocs are released by infected body cells as a result of metabolic host processes [ ] . different technical approaches have used the detection of vocs to discriminate infectious diseases successfully, but none is being used routinely in clinical practice [ ] . as dogs can be trained quickly, the aim of the present study was to test the concept of using dogs reliably and in real-time to discriminate between samples of sars-cov- infected patients and non-infected controls. this method could be employed in public areas such as airports, sport events, borders or other mass gatherings as an alternative or addition to laboratory testing, thus helping to prevent further spreading of the virus or further outbreaks. saliva samples and tracheobronchial secretion samples were collected from hospitalised covid- patients that showed clinical symptoms and were diagnosed as sars-cov- positive using nasopharyngeal swab samples. negative control samples were obtained from sars-cov- rt-pcr negative people with no previous history of covid- , nor had the individuals any history of a recent cold or infection. none of the samples were screened for different human coronaviruses like beta coronavirus hcov-oc or alpha coronavirus hcov- e. after the sample acquisition, the anonymised samples were transported to the university of veterinary medicine hannover. all collected samples were confirmed as positive or negative using the rt-pcr sars-cov- -ip assay from institut pasteur (recommended by the world health organization [ , ] , including an internal control system and protocol as described [ , ] . samples from covid- patients (irrespective of the final rt-pcr result) were further subjected to virus quantification (end point dilution assay) and virus isolation analysis using vero e cells under biosafety level conditions. the cell layers were assessed for cytopathic effects and final results were obtained days after cell infection. since dogs are susceptible to sars-cov- [ ] all samples from covid- patients were inactivated using beta propiolactone (bpl) in order to protect the dogs and their handlers from infection during training. briefly, samples and reagents were kept at °c, μl/ml nahco ( . %) was added, and samples were incubated for min at °c. after addition of μl/ml of % bpl, samples were incubated at °c for to h. hydrolysis of bpl was conducted at °c for to h. samples that showed a cytopathic effect before bpl inactivation using virus isolation or end point dilution assay were tested again after bpl inactivation and were confirmed to be inactivated. only bpl inactivated samples from covid- patients were used for the dog training. furthermore, detection dogs were provided both negative control samples with and without previous bpl treatment to exclude hydrolysed bpl as a potential distracting reagent. for the dog training, a volume of μl per sample was pipetted onto a cotton pad, which was placed into a ml glass tube. the presentation of the samples to the dogs was conducted via a device called detection dog training system (ddts; kynoscience ug, germany), which can present samples in a randomised automated manner without trainer interference. for a short video sequence, see additional file . ddts was utilised for training and testing. the device is composed of seven scent holes. behind each hole two tubes are leading to two metal containers. in the study, the first container enclosed the target sample and the second one carried the control sample. only one container is presented in each sniffing hole at any given time as the pairs of containers are situated on movable slides inside the device. the metal containers were covered with grids, which allowed the odour to escape and reach the sniffing hole. each tube extension was identical and lshaped, which prevented dogs from physical contact with the samples and excluded any visual cues that may have enabled further detection capabilities. for each trial run, only one hole presented a sars-cov- positive sample at a time while the other six holes presented negative samples. after the indication of the hole with the positive sample, the dog was automatically rewarded by the device with food or ball. the indication time was changed during successful training from s to s. while the reward was eaten, the device's software randomly and automatically assigned new positions to the slides for the following session with again only one hole presenting the positive odour sample. the dog, its handler and a person observing the study were blinded during the double-blinded study. all personnel stood behind the dog during the test runs to avoid distraction. the device recorded automatically the number and time length of each nose dip into the scent holes and the location of the positive and negative samples. this was verified by manual time-stamped video analysis. after a weeks habituation process to the ddts, the eight dogs needed days of training in total until the detection rate was above chance. an additional spreadsheet provides background information of the dogs used in the study (see additional file ). the controlled doubleblinded detection study was then conducted after days of training and in total , sample presentations ( table ) . on each training day, unknown and known positive samples and negative control samples were presented to the canines. the response to the new sample was used in order to evaluate if the generalisation process has been achieved. while the dogs had only achieved an average detection rate of % on the second day of training, the values increased to % on day five and even % on day seven indicating a successful generalisation process. after completion of the training process, the detection accuracy of the eight trained dogs was evaluated in a randomised, double-blinded, and controlled study ( table ) . samples from seven infected and seven healthy individuals were used in this study. two of the positive samples were tracheobronchial secretion, the other samples consisted of saliva. within randomised and automated sample presentations, dogs achieved an overall average detection rate of % (± . %) with correct indications of positive, correct rejections of negative, false positive and false negative indications. the canines discriminated between infected and non-infected individuals with an overall diagnostic sensitivity of . % ( % confidence interval [ci]: . - . %) and specificity of . % ( % ci: . - . %). sensitivity ranged from . to . % and specificity from . to . % (fig. ) . there was no notable difference in detection ability between saliva and tracheal secretion (average hit rates . and . %, respectively). timely and accurate detection of sars-cov- infected individuals is of uttermost importance for a society to control the pandemic. our data indicate that detection dogs can be trained in just about a week to discriminate between samples of people infected and non-infected by sars-cov- . the average detection rate was %. analysis for accuracy and precision revealed a diagnostic sensitivity of . % ( % ci: . - . %) and a high diagnostic specificity of . % ( % ci: . - . %) for all dogs. all dogs had a high diagnostic specificity with a small range in variation, which could be important for population screening to avoid false positive results. however, there was quite a range in variation of sensitivity for the individual dog and inbetween dogs. this can in part be explained with the dogs' variable training background (see additional file ), signalment, personality traits and short training period of days. to avoid a bias concerning hospital specific smells, positive samples were obtained from two different hospitals to include a variation in a covariate factor and this appears to have not influenced the current results. understanding better why there is this range in sensitivity and how to potentially improve it would be important prior to considering the use of detection dogs in the field. in comparison, the current gold standard diagnostic rt-pcr test of a nasopharyngeal swab can, in trained hands, have a false detection rate of % and a false positive rate of . - . % [ ] . a new, not yet published study indicated a clear, nearly % voc specific pattern of sars-cov- infected individuals compared to negative controls and individuals infected by the influenza virus using multicapillary column coupled ion mobility spectrometry of breath [ ] . this provides further indications that unique voc imprints exist and can be used for the development of diagnostic procedures. the current study results are promising, although they should be regarded as preliminary and suitability for this detection method in the field can only be acquired after further research has been conducted. our work provides the very first steps of the development of a new sars-cov- screening method. our inclusion criteria for the samples collected were rather non-specific and not stratified by severity of symptoms, disease status or virus load. future studies are needed to address this including a higher number of different samples to evaluate the analytical sensitivity (e.g. dilution of samples/detection level, different disease phenotypes and stages) and analytical specificity (differentiation to other lung diseases or pathogens such as cancer or infection with other seasonal respiratory virus infections, e.g. influenza, respiratory syncitial virus, adenovirus, other than sars-cov- coronaviruses, rhinovirus). in the current study negative control samples were acquired from healthy individuals without clinical signs of respiratory disease. the individuals were only tested for sars-cov- virus and therefore one cannot exclude that a former infection, especially with another human coronavirus like hcov-oc resulted in false positive indications of the dogs and that cross detection occurred. on the other hand, samples included in the current study were from severely affected, hospitalised covid- patients, but one of the main challenges in controlling the current pandemic is to identify pre-symptomatic covid- patients and asymptomatic carriers, which may constitute most covid- cases [ ] . the sensitivity of detection by dogs may also vary across the course of the disease. future research should therefore focus on the ability of dogs to identify the different covid- disease phenotypes and phases of disease expression, such as asymptomatic, pre-symptomatic, mild and severe clinical cases as well as to test samples of the same individuals at different timepoints across the course of the disease. one of the most important requirements regarding handling of infectious samples is infection prevention and control. initially, it was assumed that dogs cannot get infected by sars-cov- , but recent single cases have been reported showing that dogs can get infected by sars-cov- and could potentially play a role in viral spread [ , ] . there is evidence of human-to-animal transmission with a subsequent infection of dogs. it is still unclear whether dogs can function as spreaders of the virus by infecting other animals or humans [ , ] . nevertheless, this needs to be considered when using dogs for detection of infected material or people. it is also unclear how an infection in the dog will alter its sense of smell. in the current study we chose to use an inactivation procedure which should not affect vocs. however, this is not practical for testing in the field and we are currently developing new strategies for a secure presentation of non-inactivated samples. this would eliminate potential risks of virus transmission by detection dogs when used in a non-laboratory setting. detection dogs were able to discriminate respiratory secretions of infected sars-cov- individuals from those of healthy controls with high rates of sensitivity and specificity. the current pilot study had major limitations which needs to be elucidated in future studies. sars-cov- detection dogs may then provide an effective and reliable infection detection technology in various settings like public facilities and function as an alternative or addition to regular rt-pcr screening. in countries with limited access to diagnostic tests, detection dogs could then have the potential to be used for mass detection of infected people. further work is necessary to better understand the potential and limitation of using scent dogs for the detection of viral respiratory diseases. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file : additional video. detection dog working with ddts. the video (additional file ) shows the labrador retriever "seven" can be seen at the bottom of the video. the scent hole with a sample of an sars-cov- infected individual is marked in green on the video (please note the green mark was not seen by the dog and was only used in the video as a visualisation tool for the viewer to demonstrate the dog's search and detection behaviour). at each detection trial run only one hole is presenting the target scent with the other six holes presenting saliva samples from sars-cov- negative tested individuals. when the dog detects the target scent, the nose will be left within the hole for ≥ s to indicate the detection. this will be recorded by the device. a beeping sound announces the food or ball reward, which is automatically ejected by the device, distracting the dog for a short time period. in the meantime, the device rearranges the sample presentation in an automatic and random fashion, presenting one other scent hole with a sample of a sars-cov- positive tested individual and six control scent holes with negative control samples. pathophysiology, transmission, diagnosis, and treatment of coronavirus disease (covid- ): a review diagnostic accuracy of canine scent detection in early-and late-stage lung and breast cancers trained dogs identify people with malaria parasites by their odour using dog scent detection as a point-of-care tool to identify toxigenic clostridium difficile in stool canine detection of the volatilome: a review of implications for pathogen and disease detection realtime detection of a virus using detection dogs biomedical scent detection dogs: would they pass as a health technology? the human volatilome: volatile organic compounds (vocs) in exhaled breath, skin emanations, urine, feces and saliva the scent of disease: volatile organic compounds of the human body related to disease and disorder protocol: real-time rt-pcr assays for the detection of sars-cov- sensitivity assessment of sars-cov- pcr assays developed by who referral laboratories a universal heterologous internal control system for duplex real-time rt-pcr assays used in a detection system for pestiviruses infection of dogs with ars-cov- sensitivity, specificity, and predictive values: foundations, pliabilities, and pitfalls in research and practice. front pub health false positives in reverse transcription pcr testing for sars-cov- . medrxiv rapid detection of sars-cov- infection by multicapillary column coupled ion mobility spectrometry (mcc-ims) of breath. a proof of concept study medrxiv community prevalence of sars-cov- virus in england during may : react study the risk of sars-cov- transmission to pets and other wild and domestic animals strongly mandates a onehealth strategy to control the covid- pandemic. one health susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank the members of the id-uke-covid- study group marylyn m. addo, etienne bartels, thomas t. brehm, christine dahlke, anahita fathi, monika friedrich, svenja hardtke, till koch, ansgar w. lohse, my l. ly, stefan schmiedel, l. marie weskamm, julian schulze zur wiesch at the university medical-center hamburg-eppendorf by helping us with recruitment of patients and sample collection. we further would like to thank stefan hampel, sina knisel and miguel acosta for support at the bundeswehr school of dog, german armed forces, ulmen during dog training and leander buchner from the central institute of medical services, german armed forces in koblenz for the support in getting samples. a special thanks goes to hans ebbers, kynosciences, for providing the ddts free of charge and for the support in dog training. we would like to thank our doctoral student saskia irene peek for her support during sample collection. special thanks go to our "doggy noses" coyote, elli, lotta, donnie, hoss, luigi, jo and seven. heartfelt thanks go to all the people providing us with samples, especially to the sars-cov- infected persons and their relatives with the sincere intention to contribute to the containment of covid- and to scientific progress. we wish you lots of strength and full recovery during the current pandemic. authors' contributions pj participated in the planning of the study, carried out the main practical work, data analyses and drafted the manuscript. cs participated in the planning of the study and carried out the laboratory work including rt-pcr and virus inactivation, as did vp. ft, sm and hav designed and coordinated the study, drafted the manuscript, conducted and coordinated (ft) the sample acquisition and were responsible for data analyses. mvkb and admeo participated in the planning of the laboratory part of the study and were in charge for the legal permission for sample processing. je programmed the ddts software. ip, tw, mpm, af and mma were in charge for the ethical approval, patient recruitment and sample collection (ip, af) at hannover medical school (ip, tw, mpm) and university medical-center hamburg-eppendorf (af, mma). es participated in the planning of the study, was responsible for the dog training and helped with data analyses. all authors have read and approved the final manuscript. not applicable. the datasets used and/or analysed during the current study are available at jendrny, paula, twele, friederike, schulz, claudia, meller, sebastian, von köckritz-blickwede, maren, volk, holger andreas. ( ). sars-cov- detection dogs -a pilot study [data set]. zenodo. https://doi.org/ . /zenodo. ethics approval and consent to participate the study was conducted according to the ethical requirements established by the declaration of helsinki. the local ethics committee of hannover medical school (mhh) and hamburg medical association at the university medical-center hamburg-eppendorf (uke) approved the study (ethic consent number _bo_k_ and pv , respectively). written consent from all people were collected before sample collection. not applicable. the authors declare that they have no competing interests. key: cord- -x zzzw authors: suen, c. y.; leung, h. h.; lam, k. w.; hung, k. p.; chan, m. y.; kwan, j. k. c. title: feasibility of reusing surgical mask under different disinfection treatments date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: x zzzw the possibility to extend the lifespan or even reuse one-off personal protective equipment, especially for n respirator and surgical mask become critical during pandemic. world health organization has confirmed that wearing surgical mask is effective in controlling the spread of respiratory diseases in the community, but the supply may not be able to satisfy all the demands created all over the world in a short period of time. this investigation found that dry heat and uvc irradiance could effectively disinfect the mask material without creating significant damage to surgical mask. uring pandemic, the huge demand of personal protective equipment (ppe) compels countries to investigate the possibility to reuse and extend the lifespan of every one-off ppe, especially for n respirator and surgical mask. world health organization (who) has confirmed that wearing surgical mask is effective in controlling the spread of certain respiratory viral diseases in the community [ ] . however, the supply of surgical masks is still behind the huge demand created all over the world. in order to provide an alternative for the general public to maintain basic protection with limited resource, the possibility of reusing surgical mask after different disinfection methods was investigated. methods of disinfection studied include ℃ dry heat, steaming, boiling, autoclave, % and % ethanol, uvc irradiance and household detergent. all the disinfection treatments used for disinfecting surgical mask were proven to be effective [ ] , except household detergent. however, studies of disinfection efficiency, structural and property changes of surgical mask after various disinfection treatments were very limited. astm-f, en , kf and yy are common standards for testing the quality of surgical masks. although the testing parameters are not completely the same among these standards, they all involve testing of filtration efficiency, breathability and fluid repellency. filtration efficiency typically considers the effectiveness of particles of aerodynamic size from . µm to µm. the cut-off sizes used for the standard tests are classified as particulate filtration efficiency (pfe) and bacteria filtration efficiency (bfe) [ ] . latex sphere is used for the pfe filtration test with the astm standard test [ ] while the kf, yy and niosh use sodium chloride aerosol to perform standard tests for respirator and surgical mask [ ] - [ ] . since droplets will start evaporating when exposed to air, the terminal velocity decreases when the droplet size reduces [ ] . this implies that small-sized droplets can travel a longer distance, resulting in a higher possibility of infection if the droplet nuclei is a pathogen. in assessing the destructive level of different disinfection treatments to surgical masks, filtration efficiency and fluid repellency were the parameters being focused in this study. structural changes of filtration layer were observed and verified after the treatments. the effectiveness of disinfection to surgical mask was thoroughly investigated. medicom astm level surgical mask was chosen as the test subject due to its popularization. this surgical mask model is widely used in hospitals and health care sectors. the samples were labelled with numbers and randomly assigned to various treatment groups. each treatment group (n= ) was disinfected by one of the following methods: a. dry heat -an oven (breville bov bss, w) was used for heating at ℃ for minutes. the temperature was monitored by the lcd display of the oven with reference to an infrared thermometer. b. steaming -samples were placed at the centre of a steamer cooker at ℃ for minutes. c. boiling -samples were placed at ℃ water bath for minutes. d. autoclave -samples were wrapped with aluminum foil individually and placed into the middle basket of an autoclave (hirayama, hve- ). the autoclave sterilization was set at ℃ for minutes (complete cycle time was . hour). e. detergent - . % w/v of household detergent (ultra axion) was prepared by dilution with di water. samples were submerged into the solution for minutes, and then gently rinsed with di water for min to wash away the remaining detergent. f. uvc irradiation -irradiation disinfection was performed by using a biosafety cabinet (nuairs, nu- - s) fitted with nm ( w) tube for minutes. power density at the d . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted may , . . mask level was monitored with uvx radiometer with uvx- nm uv probe. samples were placed at a distance of cm from the tube for irradiation with measured average uvc intensity of µw/cm . the samples were turned over at minutes for even irradiation. g. samples were submerged into % v/v ethanol for minutes. h. samples were submerged into % v/v ethanol for minutes. all samples were air dried in a biosafety cabinet for hours before all measurements. filtration efficiency test % sodium chloride solution (nacl) was aerosolized by particle generator model with a count median diameter at . micrometer (nominal) and a geometric standard deviation of . (nominal). dusttrak™ ii aerosol monitor model was used to measure the particle concentration before and after filter material. an impactor for µm was installed to the aerosol monitor to study the penetration of small droplets. each sample was fitted to the sample holder and fastened by screws and rubber o-rings. the penetration of sample was measured when instruments were operating after minutes for stabilization. measurements were taken at the sampling holes perpendicular to the laminar flow at aerosol upstream and downstream of the sample. the measurement of the concentration of nacl droplet was taken at a face velocity of cm/s. the filtration efficiency was calculated by the following equation: where is filtration efficiency (the background particle count was cancelled before calculation.) bactericidal test staphylococcus aureus (clinical isolate) was inoculated to nutrient broth no. (thermo scientific™ oxoid™ nutrient broth no. (dehydrated)) by an inoculating loop aseptically. the bacteria were then cultivated overnight in an environmental shaker at ℃, rpm. the overnight culture would be re-cultured for a few hours until it reached the concentration of cfu/ml. bacteria culture prepared was diluted to cfu/ml with . % saline and . % tween . mask was cut into inch x inch square and put on supporting glass plate of inch x inch square with external layer of the mask material facing upward. µl of the diluted bacteria suspension was dropped onto each sample and the inoculum was spread evenly to allow soaking into the sample. three samples were picked randomly and treated with the aforementioned disinfection methods. a timer was used to monitor the exposure time of the samples with bacteria. afterwards, the samples with supporting glass plates were transferred to a sterile bottle containing ml of extraction solution ( . % saline, . % tween ). the bottle was vortexed for seconds, allowing sufficient time for dislodgement of microbes into the solution. the suspension was serially diluted with sterilized . % saline solution. µl of the solution was inoculated into tsa agar and cultured at ℃ for hours. the colony forming units (cfus) on agar plates were enumerated. the bactericidal efficiency was calculated by the following equation: where is bactericidal efficiency hydrophobicity test five ul load of di water were added at a distance of cm above the sample at different spots on the outer layer of mask. visual inspection of droplets was carried out for each sample. after wobbling the mask gently, hydrophobicity was recorded if the water droplets hold as beads. hydrophilicity was recorded when water droplet shape was flattened (lowered contact angle) but did not penetrate all three layers. thorough damage of the water repelling layer was recorded when the water droplet was absorbed and penetrated to the bottom. the filtering layer (polypropylene) of each sample after treatments were cut into a size of mm x mm and fixed on copper stage with carbon tapes. the samples were observed with a scanning electron microscope (sem) (hitachi, tm ) at magnification of ×. structural changes such as melting, deformation, entanglement or cracking of polypropylene fibre were recorded. comparison of the control and treatment groups were analyzed by t-test in spss (version ). p values < . were considered significant. sem was used to observe any micro structural change of masks after different treatments. sem images at × revealed that all methods used in the experiment did not cause observable structural change to the filtering layer. no shrinkage, melting, deformation, entanglement or cracking of fibre was noted. all samples were tested for filtration efficiency with nacl droplets. control samples had an average filtration efficiency of . % ± . % at cm/s. the average filtration efficiency of household detergent water treated and ethanol treated samples were significantly lower than the control. samples after boiling, steaming, baking & uvc irradiation were slightly offset from the control but were not significantly different. bead-like droplets were observed in all the samples treated with non-fluid disinfection methods, such as dry heat and uvc irradiation. for the samples underwent other disinfection treatments, the fluid-repelling layers were concluded to be damaged as the water droplets on the mask surface could not retain bead shape. uvc treated sample was scanned using ftir. result showed insignificant difference between control and treated samples. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted may , . . due to the pandemic, limited supply and a sudden surge of need for surgical masks in many places have been reported. surgical mask decontamination methods have been reported from various media. the goal of the study was to evaluate some common surgical mask decontamination methods and to identify the possible methods that cause the least damage to the surgical mask in terms of overall integrity, hydrophobicity and filtration rate. as practical decontamination methods should aim at effectively eliminate harmful microorganisms with high reproducibility while remaining safe to users, certain decontamination methods such as chlorine gas and ozone gas treatment were not included in this experiment. certain drawbacks have been noted in some preliminary tests, for example, aqueous : bleach solution alone cannot penetrate into the mask to perform thorough disinfection. ozone gas is highly hazardous to human being [ ] . it is not recommended to be used in area without proper ventilation. chlorine gas and other chlorinated compounds were known to be incompatible with polypropylene [ ] . treatment with incompatible disinfectants may cause unwanted damages to the surface characteristics and structure of the mask. except household detergent water, all disinfection methods were effective in eliminating s.aureus in the mask material. traditional disinfection methods such as boiling, steaming, submersion in ethanol, dry heat, uvc irradiation have long been used to control microbial growth. s.aureus is a gram-positive bacteria commonly used to demonstrate efficiency of various disinfection methods. moist-heat treatment methods, including boiling, steaming, autoclave, expose the mask material to % r.h. but not in the case of dry heat treatment. disinfection by dry heat has been known to be relatively slower than moist-heat, however, dry heat at ℃ for minutes was still efficient to achieve a -log reduction. ethanol treatment between % to % denatures protein and dehydrate microorganisms [ ] . results from the experiment also showed an efficient -log reduction of s.aureus in minutes. nm uvc irradiation is a physical disinfection method that utilizes electromagnetic wavelength to damage rna, dna, and protein. the samples were turned over after minutes because the irradiation may not be efficient to penetrate the entire mask material and eliminate "dead-corner". experimental result showed the exposure at µw/cm for a total of minutes could effectively eliminate all s. aureus in the mask material. household detergent was not classified as a disinfectant. the detergent used in the experiment did not have bactericidal effect towards s. aureus. reduction demonstrated in table could be a result of the partial suspension of bacteria into the solution. dry heat and uvc treatment did not show observable effect on the hydrophobicity of mask surface. for household detergent, rinsing with water might not completely remove detergent from mask material. all detergent treated masks demonstrated severe water penetration possibly be related to the lowered surface tension [ ] . for treatment by boiling, steaming, autoclaved and ethanol, samples demonstrated flattened droplets. this could possibly be related to altered surface characteristic after treatments. most treatment groups did not demonstrate observable change of the general structure of surgical mask. all the ear loops held unchanged throughout the experiment, except for the samples treated by boiling, steaming and autoclave. treatment by boiling and steaming had generally softened the texture of the mask. uvc did not damage the filtration layer even the exposure time was up to minutes. although uvc is proven to facilitate the degradation of polypropylene, short exposure targeted for disinfection of masks would not cause significant structural damage to the filtration layer [ ] . after one treatment cycle, all treatment methods except uvc demonstrated a drop in filtration efficiency. treatment with household detergent and ethanol resulted in significant decrease of the filtration efficiency. after three treatment cycles, treatment by uvc and dry heat could still maintain high filtration efficiencies (> %). generally, non-fluid treatments perform better than fluid-based treatments in filtration efficiency test. the plausible reason is that the electric charges of polypropylene filtration layer were neutralized after the treatment, especially with the use of organic solvents [ ] . this study had only evaluated a surgical mask in the filtration of . - µm nacl droplets. however, the performance of the surgical mask after treatment has not been tested with more brands, particles of various sizes and different flowrates. as the study result was based on a modified protocol of niosh nacl respirator certification test, it only provides comparisons among different groups of treatments. the study did not determine whether the treated samples could pass any international standards of surgical mask testing. future studies can focus on certain treatments, and investigate the optimal condition of decontamination treatment so as to minimize damage to the mask and to determine detailed disinfection conditions and kinetics that were required for other strains of microorganisms. decontamination and reuse of surgical mask become an alternative strategy to regenerate basic protective equipment so as to control the spread of disease under the current difficult situation. non-fluid contacting disinfection methods such as uvc irradiation and dry heat retained the highest performance regarding filtration efficiency, structural consistence and surface hydrophobicity even after three cycles of treatments. these two disinfection methods for surgical mask would be considered under the severe ppe shortage situation. advice on the use of masks in the context of covid- disinfection, sterilization, and preservation a comparison of facemask and respirator filtration test methods standard test method for determining the initial efficiency of materials used in medical face masks to penetration by particulates using latex spheres comparison of filtration efficiency and pressure drop in anti-yellow sandmasks, quarantine masks, medical masks, general masks, and handkerchiefs surgical mask yy - determination of particulate filter efficiency level for n series filters against solid particulates for non-powered, air-purifying respirators standard testing procedure (stp) how far droplets can move in indoor environments -revisiting the wells evaporationfalling curve review of evidence on health aspects of air pollution -revihaap project polypropylene chemical resistance guide proteomic profiling and analytical chemistry weatherability of polypropylene by accelerated weathering tests and outdoor exposure tests in japan experimental study on charge decay of electret filter due to organic solvent exposure key: cord- - a iztns authors: hayden, randall t.; gu, zhengming; rodriguez, alicia; tanioka, lisa; ying, claire; morgenstern, markus; bankowski, matthew j. title: comparison of two broadly multiplexed pcr systems for viral detection in clinical respiratory tract specimens from immunocompromised children date: - - journal: j clin virol doi: . /j.jcv. . . sha: doc_id: cord_uid: a iztns background: the detection of viral respiratory tract infections has evolved greatly with the development of pcr based commercial systems capable of simultaneously detecting a wide variety of pathogens. objectives: evaluate the relative performance of two commercial broad range systems for the detection of viral agents in clinical respiratory tract specimens from immunocompromised children. study design: a total of patient samples were included in the analysis, representing only the first sample collected for each patient, and excluding failed reactions. samples were de-identified and assayed in parallel using two different, broadly multiplexed pcr systems: resplex™ ii panel v . (resplex), qiagen, hilden, germany and filmarray(®) respiratory panel (filmarray), idaho technology inc., salt lake city, ut. method comparison was based upon pair-wise concordance of results according to patient age, viral target and number of targets detected. results: the two systems showed an overall concordance, by patient, of . % (p = . ). filmarray detected at least one target in . % of samples, while resplex detected at least one target in . %. resplex failed to detect . % of filmarray positives, and filmarray failed to detect % of resplex positives. the relative performance of each system (including which system detected a higher number of positive samples) varied when stratified by target viral pathogen. conclusions: broadly multiplexed pcr is an effective means of detecting large numbers of clinically relevant respiratory viral pathogens. viral respiratory tract infections can cause serious morbidity and increased mortality in immunocompromised pediatric patients. the rapid and sensitive detection of such agents has implications for treatment decisions, clinical care, and infection control practices. as in other areas of diagnostic virology, molecular diagnostic methods have shown promise in markedly improving diagnostic sensitivity when compared to culture or antigen detection assays. while such favorable performance characteristics have made molecular methods appealing, their introduction in the clinical laboratory has been slowed by issues related to cost and technical expertise required to perform this testing. early versions of such tests have focused on a limited number of pathogens, and typically detected only one or two viruses (or groups of viruses) at a time. [ ] [ ] [ ] [ ] [ ] [ ] [ ] thus, detection of all clinically relevant entities has required running an entire panel of tests, compounding costs, and staffing requirements. the advent of broadly multiplexed assays has sought to address some of these issues. , front-end multiplexed amplification (typically pcr) followed by detection is capable of identifying over different targets. thus, a single assay utilizing the sensitivity of pcr can mimic the diagnostic spectrum of culture creating the potential for increasing routine detection beyond cultivable viruses, reducing processing costs, staffing requirements, and turn-around time. this technology also offers the possibility of increased ability to detect multi-viral infection. while the clinical importance of such capabilities is presently uncertain, this may carry important prognostic or infection control related implications, particularly among immunocompromised patients, such as those studied here. a number of different methodologies have now been proposed or marketed that use such an approach; however, limited published studies have compared different broadly multiplexed systems to one-another. in the current study, we compared two such systems to each other and to a panel of real-time pcr assays targeting individual viruses. technologies evaluated included the resplex ii panel v . (resplex), qiagen, hilden, germany and the filmarray respiratory panel (filmarray), idaho technology, inc., salt lake city, ut. both products used for this study were for research use only (ruo). the filmarray product has only recently become available as an fda-cleared assay, and at the time of this submission few studies have been published examining performance of this method using clinical respiratory tract specimens. two broadly multiplexed pcr systems were compared to each other and to a panel of laboratory developed tests for the detection of respiratory viral pathogens in clinical respiratory tract specimens from pediatric immunocompromised children. samples were collected prospectively, as part of routine clinical care at st. jude children's research hospital (sjcrh) between january and may , , from children presenting with signs and symptoms of upper respiratory tract infection. results from patients years of age and above were excluded from the analysis. the sjcrh institutional review board (irb) classified this study as non-human research; the study was exempted from irb approval and informed consent requirements were waived. samples consisted of bronchoalveolar lavage (bal) specimens, nasopharyngeal swabs (nps), nasopharyngeal washes (npw), and tracheal aspirates (ta). unused samples remaining after routine diagnostic testing were de-identified and blinded prior to study inclusion. unique patient and sample identifiers were assigned to each sample in order to match the results obtained from the various methods being compared. samples were divided into two . ml aliquots where one was tested by the laboratory developed test panel(ldtp), which included the pro hmpv assay kit (gen-probe, san diego, ca) for detection of hmpv, at sjrch and the other was tested using the filmarray assay. left over sample from the clinical aliquot was then tested by the resplexii assay at diagnostic laboratory services (dls), aiea, hi. extraction was performed at sjcrh and nucleic acid was sent and stored frozen to dls until testing at this remote site. this collection, transport, and storage method was used to allow preanalytical standardization between the testing methods. a l nucleic acid solution was extracted from l of respiratory sample together with prior addition of rna and dna controls for ldtp test; a second l nucleic acid solution was extracted from l aliquots of the same respiratory sample without addition of the rna and dna controls, for respplexii testing. extractions were otherwise identical and were performed using the nuclisens ® minimag magnetic extraction system (biomérieux inc., durham, nc), per manufacturer's instructions. the rna control was purchased as a ready-to-use lyophilized beadcontaining a kb armored synthetic nucleic acid (cepheid). the dna control was a plasmid constructed by inserting a -bp dna fragment of phocid herpesvirus type gb gene into vector puc (designed in-house at sjcrh and manufactured at genscript corp., piscataway, nj). both controls were detected only in ldtp test. a panel of real-time pcr assays was developed to detect infa and infb, rsv, adenovirus, and parainfluenza viruses , , and from respiratory specimens. the pro hmpv assay kit was also run as part of this panel. the assays utilized taqman chemistry and realtime pcr technology and were carried out on the smartcycler ii platform (cepheid, sunnyvale, ca). the molecular detection targets, primer and probe sequences are listed in table s . the resplex tm ii panel v . uses a multiplex rt-pcr analysis to amplify and detect respiratory viruses ( filmarray utilizes a prefabricated pouch containing lyophilized reagents. the sample is added directly to the pouch wherein specimen preparation, amplification, and detection all take place without further offline sample manipulation. l of original sample was mixed with l of filmarray lysis buffer. approximately ml of hydration solution was added by syringe to the filmarray pouch through hydration solution inlet port and l of sample/lysis buffer mix was added to the pouch through the sample inlet port. the pouch was then loaded on the filmarray instrument, with automated extraction, amplification, and data analysis; total run time, approximately h ( min hands-on time). quality of testing was assured by the inclusion of two rna process controls(pc) in each pouch (proprietary sequences, idaho technology). the rna process controls were carried through all stages of the test process from samples lysis to pcr and dna melt analysis. both controls had to produce positive results for validation of test results. filmarray detected viral targets: adenovirus, bocavirus, coronavirus e, hku , nl , oc , enterovirus, hmpv, human rhinovirus, influenza virus types a and b, parainfluenza viruses , , and , and rsv. the mcnemar test was used to compare the accuracy of all three tests; comparison was made in pair-wise fashion and exact methods were used when discordant cells had less than observations. to reduce bias, only the first sample of each patient with multiple samples was included in the analysis. for purposes of analysis, patient age was divided into four different age groups based on years of age: ≤ , - , - , and - . this was a paired study, pairwise comparisons being made between performance of filmarray and resplex, filmarray and ldtp, and resplex and ldtp. when targets differed between the systems under comparison, results were collapsed into the narrowest taxonomic category that encompassed both systems of a pair. results from individual samples in which the first run failed were excluded from the analysis. however, results from second run from consecutive samples in which the entire run failed were included in the analysis as it was assumed that a systematic or technologist error was most likely the cause for the failure of the entire run for the resplex system, a positive result was determined using a cut-off mean fluorescent intensity (mfi) of . a total of samples were collected from children during the study period. after excluding samples from patients aged and above, samples obtained after the first collection, and failed runs, the total number of samples analyzed was ; only a single sample was tested per patient. while there were no failures with the ldtp, the total number of failed runs by filmarray was and with resplex was . the mean age of children with samples included in the analysis was . years, ranging from months to years. the vast majority of samples were npw with samples ( . %), followed by ta with samples ( . %), bal with samples ( . %) and one nps ( . %). over % of tested samples came from children who were or younger: samples from children two years or less accounted for . % ( ) of samples, between three and five years inclusive were . % ( ), between six and were . % ( ), and . % ( ) of samples came from children aged between and , inclusive. overall difference in detection of targets between filmarray and resplex was found to be significant (p < . ). out of the samples detected positive by filmarray, resplex did not detect a target for . % ( ) of samples, while filmarray missed % ( ) of samples detected positive by resplex ( table ). the total number of samples for which both systems agreed was samples; samples testing positive and samples testing negative. after stratifying by different targets and target groups, differences were observed in the detection of coronavirus e (exact p = . ), infa (exact p = . ), enterovirus (p < . ), and rhinovirus (p < . ). analysis stratified by patient age showed that in ages - , . % of samples were positive by filmarray and of these, resplex missed eight samples ( . %), p = . ; likewise, age group - , . % of samples were positive by filmarray but resplex missed . % ( ), p = . (table s ) . when positive results were stratified by the number of targets detected by filmarray, differences in detection were found to be significant in cases where the filmarray system detected one or two targets (table s ). in the cases where filmarray detected one target, resplex did not detect of those cases, p < . . resplex did not detect any targets in six samples of the samples for which filmarray detected the presence of two targets, p = . . in these six samples, the targets not detected by resplex were rhinovirus ( ), coronavirus hku ( ), rsv ( ), adenovirus ( ), bocavirus ( ), coronavirus oc ( ), and enterovirus ( ). analysis for this comparison included only viruses targeted by both systems. these target groups were: adenovirus, hmpv, infa, parainfluenza types - , and rsv. there were no differences in the number of positives detected by either system over the other, either overall or when stratified by target (table ) . differences in detection rate were not observed when data were stratified by age groups (table s ) . after stratifying by number of targets detected by filmarray, ldtp detected viruses in six samples that filmarray called negative (p = . ); but ldtp did not detect targets in seven samples in which filmarray detected one target, p = . (table s ). the percentage of samples detected positive by resplex ( . %) was lower than that for the ldtp ( . %). among the samples detected positive by resplex, ldtp did not detect . % ( ); resplex did not detect targets in samples ( . %) that were detected positive by ldtp, p = . . the only target group for which significant differences were observed was influenza a; ldtp detected samples as positive for influenza a but resplex did not detect % of these p = . (table ) . when results were stratified by age group, significant differences were observed in samples taken from children aged to (table s ). resplex detected % less positives compared to ldtp in samples from children aged between and ; and % less in the remaining age groups, though these differences were not significant. differences in detection between resplex and ldtp were significant in samples where resplex did not detect any targets (table s ) . the introduction of multiplex molecular amplification assays for the detection of viral respiratory pathogens has improved the sensitivity of routine viral detection methods. the range of agents detected [ ] [ ] [ ] has been expanded to include newly discovered and emerging respiratory viruses such as coronaviruses nl and hku , human metapneumovirus (hmpv), and bocavirus. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the broadly multiplexed molecular approach studied here has the capability to detect and identify more viruses simultaneously than traditional methods (i.e. culture and dfa) and shows an increased detection rate for co-infections. , , [ ] [ ] [ ] [ ] [ ] in the current study, coinfections were detected in the range of - %, which is similar to other investigators. sanghavi et al. investigated both adult and pediatric patients, including organ transplants and found dual infections at . % and triple at . %. in a study, guittet et al. reported a range of - % in hospitalized children. the clinical significance of detecting more than one virus is not clear, but calvo et al. reported multi-viral infections more frequently in hospitalized infants with respiratory tract disease ( . %). these infections were also linked to higher fever, longer hospital stays, and more frequent use of antibiotics than single rsv infections. in another study by semper et al. co-infection with hmpv and rsv revealed a tenfold increase in the relative risk for admission into the pediatric intensive-care unit for mechanical ventilation as a result of severe bronchiolitis. there is little published data beyond this on the clinical significance of multi-viral respiratory tract infection, and no work specific to the importance of detecting > viruses. the findings here suggest that as broadly multiplexed pcr systems become more common, a new body of literature will need to be developed to address the relevance and implications of such findings for prognosis, treatment, and infection control. the current study, to our knowledge, is the first reported that compares the filmarray with the resplex ii v . for the direct detection of viral agents in clinical respiratory tract specimens from immunocompromised children. the viral targets for the two systems were similar with previously noted exceptions. overall even though both systems detected - % more viruses than traditional methods, the filmarray detected significantly more viruses and mixed infections than the xtag rvp. in the current study, resplex detected less rsv, influenza type a, hmpv, and adenovirus than the filmarray. increased rsv detection by the filmarray was also noted by rand et al. compared to the xtag rvp. adenoviruses were not typed in this study, but the reduced rate of detection by resplex may relate to the fact that it detects only subtypes b and e, compared to the filmarray which detects all subtypes. it was noteworthy that the detection rate for enterovirus and rhinovirus were inversely related when the two systems were compared to each other. furthermore, if the filmarray and resplex enterovirus/rhinovirus were added together in each case, they would be approximately equal in the total number of detections ( vs. , respectively). this could be the result of crossover detection related to differences in the nucleic acid sequences between the two assays. rhinoviruses and enteroviruses show extensive genomic similarity, but yet can be very different in their phenotypic characteristics. the self-contained nature of the pouch system used by the filmarray, where sample preparation, amplification, and detection take place in a closed system markedly reduces any risk for carry-over contamination between specimens. both the resplex ii v . and the xtag rvp require external nucleic acid extraction followed by multiplexed amplification and detection using a liquid-phase array technology. this approach may carry a risk of carry-over contamination due to both additional sample manipulation and the lack of a closed system. the difference in nucleic acid extraction prior to amplification and detection, along with other variables, such as nucleic acid sequence variations and chemistry may also contribute to the differences in test performance. specimen collection procedures can also contribute to a difference in the overall test performance for any molecular test. the use of oropharyngeal swabs, nasopharyngeal swabs, or nasopharyngeal washings showed multiplex real-time rt pcr detection rates of . %, . %, and . %, respectively in a study by lieberman et al. other investigators have shown similar differences with the use of different swabs and collection procedures. , the performance of both traditional and molecular-based tests varies according to the age group studied. however, generally the younger children tend to show a higher viral load than older individuals. in general, this observation may account for a more favorable performance between traditional and molecular-based testing in the younger age group. in the present study, both the film-array and the resplex exhibited similar viral detection in the ≤ years age group. however, the filmarray outperformed the resplex in the - year and - year groups. the viral load may have been lower in the > years group, accounting for the overall increase in sensitivity for the filmarray. the material costs for most molecular testing are greater than for dfa and culture, but the overall benefit may depend upon the protocol involved and could result in an overall cost reduction as described by mahony et al. the other aspect of the cost analysis is the actual labor time involved for the filmarray and resplex. in the current study, the hands-on-time for the resplex was approximately h with a total time of h for testing samples. the filmarray involved only min for handson-time with a total time of h/sample. based on the one cartridge per machine design of the filmarray instrument, the throughput for filmarray is integrally related to the number of machines available in a given laboratory. so while laboratories that process less than one sample per hour would be able to take advantage of apparently improved turnaround time with filmarray, resplex is more accommodating of larger specimen loads. it would therefore take six filmarray instruments to meet the -h turnaround time of a full resplex run. likewise, the apparent labor-saving benefits of the film-array system apply primarily to low volume settings. although with practice, processing time might be less than the above stated min per sample, total hands-on time for more than - samples might be expected to meet or exceed that of resplex. this study demonstrates the promise of automated, broadly multiplexed pcr systems for the detection of viral respiratory pathogens. such methods combine the exquisite sensitivity of molecular amplification with a breath of targets previously attainable only through the use of culture-based techniques. these operating characteristics, together with increasingly selfcontained, automated, and easy-to-use methods, bring us closer to the mainstream adaptation of molecular diagnostic testing, no longer limited to academic centers or reference laboratory settings. the widespread use of these powerful techniques will have implications for diagnosis, treatment, infection control, and resource utilization in the healthcare setting. funding for this study was supported in part by the american lebanese syrian associated charities, alsac and the anderson charitable foundation. reagents were kindly supplied by qiagen and idaho technology, inc. instrumentation for the filmarray process was provided by idaho technology, inc. development of three multiplex rt-pcr assays for the detection of respiratory rna viruses multiplex real-time pcr assay for detection of influenza and human respiratory syncytial viruses use of a multiplex real-time pcr to study the incidence of human metapneumovirus and human respiratory syncytial virus infections during two winter seasons in a belgian paediatric hospital multiplex real-time pcr for detection of respiratory tract infections evaluation of the prodesse hexaplex multiplex pcr assay for direct detection of seven respiratory viruses in clinical specimens evaluation of the hexaplex assay for detection of respiratory viruses in children 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multiple simultaneous viral infections in infants with acute respiratory tract infections in spain dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis comparison of two multiplex methods for detection of respiratory viruses: filmarray rp and xtag rvp new complete genome sequences of human rhinoviruses shed light on their phylogeny and genomic features real-world comparison of two molecular methods for detection of respiratory viruses identification of respiratory viruses in adults: nasopharyngeal versus oropharyngeal sampling pooled nasopharyngeal and oropharyngeal samples for the identification of respiratory viruses in adults development and evaluation of a flocked nasal midturbinate swab for selfcollection in respiratory virus infection diagnostic testing performance of diagnostic tests to detect respiratory viruses in older adults cost analysis of multiplex pcr testing for diagnosing respiratory virus infections r. hayden has served as a consultant for idaho technology. other authors have no competing interests to report. the study was classified as non-human research; study was exempted from irb approval and informed consent requirements were waived. supplementary data associated with this article can be found, in the online version, at doi: . /j.jcv. . . . key: cord- - cfenpxm authors: singh, anirudh k.; nema, ram kumar; joshi, ankur; shankar, prem; nema, shashwati; raghuwanshi, arun; patankar, chitra; mathew, bijina j.; shrivas, arti; pandey, ritu; tripathi, ranu; biswas, debasis; singh, sarman title: evaluation of pooled sample analysis strategy in expediting case detection in areas with emerging outbreaks of covid- : a pilot study date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: cfenpxm timely diagnosis of covid- infected individuals and their prompt isolation are essential for controlling the transmission of sars-cov- . though quantitative reverse transcriptase pcr (qrt-pcr) is the method of choice for covid- diagnostics, the resource-intensive and time-consuming nature of the technique impairs its wide applicability in resource-constrained settings and calls for novel strategies to meet the ever-growing demand for more testing. in this context, a pooled sample testing strategy was evaluated in the setting of emerging disease outbreak in central indian districts to assess if the cost of the test and turn-around time could be reduced without compromising its diagnostic characteristics and thus lead to early containment of the outbreak. from nasopharyngeal and oropharyngeal samples received from the three emerging districts, a total of pools were created with consecutive samples in each pool. the diagnostic performance of qrt-pcr on pooled sample was compared with that of individual samples in a blinded manner. while pooling reduced the cost of diagnosis by % and the laboratory processing time by %, of the pools showed discordant results when compared with induvial samples. four pools which tested negative contained positive sample and pool which was positive did not show any positive sample on deconvolution. presence of a single infected sample with ct value of or higher, in a pool of , was likely to be missed in pooled sample analysis. at the reported point prevalence of . % in this study, the negative predictive value of qrt-pcr on pooled samples was around % suggesting that the adoption of this strategy as an effective screening tool for covid- needs to be carefully evaluated. a a a a a the transmission of covid- is difficult to contain owing to the dual factors of high transmissibility of sars-cov- (r = . - . ) and asymptomatic/ mildly symptomatic individuals serving as sources of infection [ ] [ ] [ ] [ ] . extensive testing of suspected cases and asymptomatic direct and high-risk contacts is, therefore, recommended as key to controlling the ongoing pandemic [ ] . however, the testing modality of choice viz. quantitative reverse transcriptase pcr (qrt-pcr), is too technically demanding, time-consuming and resource-intensive to be widely adaptable in low-and middle-income countries as well as remote locations and thus it often fails to inform early identification and quick isolation of the infected patients. innovative methods for expediting the qrt-pcr results, without compromising the diagnostic sensitivity and specificity, are therefore urgently needed. the central indian state of madhya pradesh (mp), with a total of districts, reported the first case of covid- on march th and till th april majority of the reported cases from the state ( . %) were restricted to the districts of indore (district a) and bhopal (district b). from the second week of april , cases started being reported from district dhar (a ), which borders district a, and from districts raisen (b ) and hoshangabad (b ), both of which are adjacent to district b (fig ) . this prompted massive contact tracing in each of these districts in an effort to contain the spread of infection. since the samples collected from these emerging districts converged on a laboratory system that was already over-burdened with unprecedented workload, a need was felt to evaluate strategies for testing additional samples without compromising the diagnostic characteristics and turn-around time of the test. we hypothesized that testing of pooled respiratory samples, collected from potentially infected individuals, could lead to faster laboratory confirmation and quicker containment of the emerging infection in these districts and, thus, undertook this study to evaluate the diagnostic concordance between the strategies of pooled vs. individualized testing and estimate the gain in turn-around time (tat) and resources that could be achieved through pooling. this study was approved by the all india institute of medical sciences, bhopal institutional human ethics committee with the waiver of consent as per the "national ethical guidelines for biomedical and health research" set forth by indian council of medical research, which is an apex government body responsible for making and enforcing policies of medical research in india. as per the guidelines, the institutional ethics committee can grant waiver of consent if the research is done on anonymized biological samples and/or the primary purpose of the research is refinement and improvement of the public health programs. as our study met both these criteria it was approved with the waiver of consent. nasopharyngeal and oropharyngeal swabs were collected by trained healthcare workers from suspected covid- patients belonging to the districts a , b and b in vials containing viral transport medium (vtm) during april and may . samples were transported at - ˚c to the testing laboratory within hours. the relevant clinical and epidemiological details of the patients were entered in a standard form approved by the indian council of medical research, which is spearheading the nationwide laboratory network for covid- testing. aliquots of the original consecutive clinical samples were anonymized and processed in parallel for individualized and pooled analysis in a blinded manner. a pool size of was chosen as per the advisory of the indian council of medical research for pooled sample testing for covid- . for pooled analysis, μl from each of consecutive samples were collected in a single . ml centrifuge tube and processed for rna extraction using qiaamp viral rna mini kit (qiagen, hilden, germany) as per manufacturer's instructions. rna extraction for individualized testing was also performed using the same kit. the extracted rna samples were subjected to diagnosis using real-time fluorescent rt-pcr kit for detecting sars-cov- (bgi, hong kong) as per the manufacturer's protocol on a biorad cfx thermal cycler. the kit targets orf ab for detection of the virus and human β-actin as the internal control. as recommended by the manufacturer, a sigmoidal curve with a ct value � was considered as the criterion for considering a sample as positive for sars-cov- . all the necessary controls namely no-template control, extraction control and positive control were tested in parallel with every batch of samples, as part of the quality control of the procedure. the data was entered in ms excel in wide data format. it was checked for missing values, redundancies and outliers. the descriptive summarization of variables of interest was done by median and iqr for ordinal and interval data (non-parametric distribution) and counts for nominal data. the diagnostic accuracy of pooled strategy, in reference to individual qrt-pcr, for correctly classifying the pool was determined by point and interval estimates of sensitivity, specificity and by likelihood ratios. as positive predictive value (ppv) and negative predictive value (npv) are prevalence-dependent parameters, we ran a simulation to estimate the ppv and npv values with a varying pre-test probability from %- % in addition to group prevalence. further a kappa statistic was calculated between the two strategies in order to detect the agreement beyond chance. we also calculated the nonparametric spearman correlation to check the magnitude and direction of the relationship between pooled ct values and number of positive samples in the same pool. all the analyses were done by base r software which is in open domain and associated epi-r package. the choropleth map was drawn with the aid of gg plot package in r software. the bland altman (ba) plot was drawn and the relevant ba statistic was calculated with 'bland altman leh' and 'ggplot ' r package. a total of samples were collected, with , and of them belonging to districts a , b and b , respectively. both, the individual samples and their pools were processed in parallel for testing. a total of pools were created from consecutive samples received from each district and the diagnostic performance of the strategies were compared. considering the individualized qrt-pcr technique as the gold standard, we observed pcr-positivity of . % ( / ), . % ( / ) and . % ( / ) in the districts a , b and b respectively. the samples that tested positive on individualized testing got sorted into pools and of these pools were detected as positive during pooled sample testing. majority of these pools contained positive sample (n = ). while all pools containing more than one positive sample could be detected on pooling, pools each containing single positive sample were missed by this strategy. although all the pools with more than one positive sample tested positive, we observed a weak negative and non- discordant pools. the statistical significance of this difference could not however be determined by mann whitney u test, due to the statistical constraint of having less than observations in the discordant group (table ) . similarly, pools contained samples that tested negative on individualized rt-pcr and of these pools were also found to be negative on pooled analysis. one of the pools tested positive on pooled analysis with a ct value of . ; though on deconvolution the individual samples tested negative. the prevalence-dependent and prevalence-independent diagnostic characteristics of the pooled sample analysis strategy are depicted in table (this table in a different format is included as s table) , with ppv and npv simulated at % to % background positivity. we observed . % ( . % to . %) sensitivity, . % ( . to %) specificity, . % ( . % to . %) npv and . ( . - . ) negative likelihood ratio values for pooled analysis. the extent of agreement between pooled versus individual sample strategy was estimated further by kappa statistic (κ = . ; % ci = . - .). the pooled strategy expectedly led to a significant gain in turn-around time. the total laboratory processing time required for the analysis of samples through individualized for individual and pooled ct values refer s table. https://doi.org/ . /journal.pone. .t sampling strategy was hours, while the same was found to be hours for pooled sampling including the testing of individual samples after deconvolution of the positive pools. similarly, the pooled testing strategy led to % savings in reagent costs with the total cost of individualized testing being $ whereas the pooled analysis including the deconvoluted samples was $ . in this study we report that the pooled sample analysis strategy, if applied in the scenario of an emerging outbreak of covid- , offers a significant reduction of laboratory turn-around time and reagent requirement at the cost of compromised diagnostic sensitivity. at the reported point prevalence of . % among the individuals tested in this study, the npv of pooled sample strategy was around %; thereby suggesting the possibility of missing infected cases and risking community transmission from undiagnosed individuals. particularly, presence of a single infected individual with relatively low viral load (ct value of � ), in a pool of , was likely to be missed in pooled sample analysis. one of the reasons for this could be the dilution of sample beyond the limit of detection of the assay used. since pcr-positivity is a function of the net viral load in the pooled sample, we understand that the success of the pooling strategy as a sensitive screening tool would be dependent on the number of infected patients in the pool and the viral load in the individual samples. while the latter is likely to be influenced by host-specific factors like immune competence, comorbidities and age, the former is likely to be a reflection of the background prevalence of infection in the community. while pooled sample testing offers the advantage of cost effectiveness and timely reporting, the strategy becomes less useful in communities where the prevalence of the disease is high. with increased prevalence, number of positive pools are likely to increase and more pools are to be deconvoluted for identifying the positive individuals negating the advantages such as cost and time saving. furthermore, with the increase in the prevalence of a disease, npv of the diagnostic test for the disease decreases leading to higher false negative results. based on this premise, national advisories have suggested testing of sample pools for covid- . in communities with prevalence of upto % [ ] . however, our data reflects the lack of a robust relationship between the former factor and the ct value of the pooled sample; thus, implying the greater role of viral titers in individual samples in influencing the pcr results. the relatively low sensitivity and resultant negative predictive value of the pooled sample analysis strategy hint at its weakness as an effective screening tool in reliably "ruling out" the diagnosis of covid- . in our study, pooled sampling led to false negative results in of the patients; all of whom were direct household contacts of known covid- patients and two of them were clinically symptomatic with cough, sore throat and fever. despite being clinically and epidemiologically suggestive, three of these patients had ct values above ; thereby suggesting that pooled analysis is likely to miss the detection of samples with low viral loads. these instances of missed case detection pose the risk of community transmission of infection from undetected sources. earlier studies evaluating the utility of pooled sample testing for surveillance of bovine viral diarrhea virus and porcine reproductive and respiratory syndrome virus demonstrated that sensitivity of rt-pcr tests reduced when run on pooled samples, due to a dilution effect [ , ] . in a recently published study lohse et al., value of were detected as positive in this study as a cut-off value of was used by the authors [ ] . of the pools created from samples testing negative on individualized testing, were negative on pooled analysis and one pool tested positive with ct of . for orf ab gene. however, on deconvolution, each of the constituent samples in that pool tested negative. we observed that this pool comprised of patients with severe acute respiratory infection. we surmise that non-specific inhibitors of pcr amplification, co-habiting the viral genomic target in any of these patients, might have suppressed the amplification of low-level viral target in individual samples beyond the detectable threshold. pooling the samples could potentially dilute these inhibitors and lead to a detectable signal with a relatively delayed ct on poled analysis. in a recent study hogan and co-workers made a similar observation while assessing the utility of pooled sample testing strategy to detect community transmission of sars-cov- in san francisco bay area, ca, usa. one of the pools was positive in their study, however, upon deconvolution all the individual sample tested negative [ ] . our study suffered from several limitations. apart from having a relatively small sample size, as mentioned above, the districts did not have similar background prevalence. hence the diagnostic characteristics like positive and negative predictive values are likely to be dissimilar between the districts. furthermore, pooling of consecutive samples was done in the study. while family members of infected cases were likely to be included in the same pool which may have decreased the possibility of false negative results. adopting a strategy of random pooling could lead to a different proportion of infected individuals in the pools, the effect of which was not explored in the study. we also did not have pools in which multiple infected samples had ct values of � and, hence, the diagnostic performance of such pools could not be assessed. we therefore, conclude that though the pooling strategy could be an alternative in resource-constrained settings with overwhelmed laboratory infrastructure, its adoption could miss the detection of pools having single infected individuals with low viral loads. our study, which is probably the first report on pooled testing in areas experiencing the early phase of emergence of covid- outbreaks, shows that . % of the infected pools could be missed by this strategy. it is therefore recommended that adoption of the pooled sample testing strategy, as a cost effective screening tool for early containment of transmission in emerging outbreaks, has to be tailored to the prevalence of covid- in the geographical area concerned. supporting information s presymptomatic sars-cov- infections and transmission in a skilled nursing facility presumed asymptomatic carrier transmission of covid- delivery of infection from asymptomatic carriers of covid- in a familial cluster the basic reproduction number of novel coronavirus ( -ncov) estimation based on exponential growth in the early outbreak in china from to : a reply to dhungana covid- strategy update advisory on feasibility of using pooled samples for molecular testing of covid- factors affecting sensitivity and specificity of pooled-sample testing for diagnosis of low prevalence infections evaluation of the sensitivity of reverse-transcription polymerase chain reaction to detect porcine reproductive and respiratory syndrome virus on individual and pooled samples from boars pooling of samples for testing for sars-cov- in asymptomatic people sample pooling as a strategy to detect community transmission of sars-cov- key: cord- -bbv f gf authors: greninger, alexander l.; chen, eunice c.; sittler, taylor; scheinerman, alex; roubinian, nareg; yu, guixia; kim, edward; pillai, dylan r.; guyard, cyril; mazzulli, tony; isa, pavel; arias, carlos f.; hackett, john; schochetman, gerald; miller, steve; tang, patrick; chiu, charles y. title: a metagenomic analysis of pandemic influenza a ( h n ) infection in patients from north america date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: bbv f gf although metagenomics has been previously employed for pathogen discovery, its cost and complexity have prevented its use as a practical front-line diagnostic for unknown infectious diseases. here we demonstrate the utility of two metagenomics-based strategies, a pan-viral microarray (virochip) and deep sequencing, for the identification and characterization of pandemic h n influenza a virus. using nasopharyngeal swabs collected during the earliest stages of the pandemic in mexico, canada, and the united states (n = ), the virochip was able to detect a novel virus most closely related to swine influenza viruses without a priori information. deep sequencing yielded reads corresponding to h n influenza in each sample (percentage of aligned sequences corresponding to h n ranging from . % to . %), with up to % coverage of the influenza genome in one sample. detection of h n by deep sequencing was possible even at titers near the limits of detection for specific rt-pcr, and the percentage of sequence reads was linearly correlated with virus titer. deep sequencing also provided insights into the upper respiratory microbiota and host gene expression in response to h n infection. an unbiased analysis combining sequence data from all outbreak samples revealed that % of the h n genome could be assembled de novo without the use of any reference sequence, including assembly of several near full-length genomic segments. these results indicate that a streamlined metagenomics detection strategy can potentially replace the multiple conventional diagnostic tests required to investigate an outbreak of a novel pathogen, and provide a blueprint for comprehensive diagnosis of unexplained acute illnesses or outbreaks in clinical and public health settings. the h n influenza a virus ( h n ) is a novel reassortant virus comprised of genomic segments originating from swine, avian, and human influenza strains [ , ] . after the initial identification of a swine-origin h n by the cdc in april , the novel virus rapidly achieved sustained human-to-human transmission worldwide, prompting a who declaration of a level pandemic and rapid development of a vaccine [ ] . as of august th, , there have been at least million cases and , confirmed deaths worldwide from the h n pandemic (http://www.who.int/csr/don/ _ _ /en/index.html). the emergence of h n was marked by diagnostic difficulties that hampered early efforts at detection. rapid point-of-care diagnostics were insensitive for diagnosing the novel virus, and while existing molecular methods such as rt-pcr were able to detect influenza, they could not differentiate h n from seasonal h n or h n infection [ ] . initial efforts to characterize the virus relied on traditional sanger sequencing and the use of pcr primers to highly conserved regions in the influenza genome, methods that would likely have failed had the virus been more genetically divergent [ , ] . no clinical or laboratory test was initially available to identify h n with high sensitivity and specificity, and the virus may have circulated undetected in the human population for months [ ] . thus, there is a clear need for the introduction of broad-range viral detection methodologies into clinical and public health settings to deal with emerging threats such as h n influenza. metagenomic approaches, including microarrays and deep sequencing, have proven increasingly successful in recent years for the diagnosis of infectious diseases by novel viral pathogens [ ] . the virochip is a pan-viral microarray that is designed to simultaneously detect all known viruses [ , ] . novel viral species or strains can also be detected on the basis of conserved sequence homology to known viruses, as demonstrated by the discovery of the sars coronavirus by virochip in [ , ] . other novel viruses previously identified by virochip include a new clade of rhinoviruses [ ] , a human cardiovirus associated with respiratory and diarrheal illness [ ] , and avian bornavirus, the etiologic agent of outbreaks of proventricular dilation disease (pdd) in birds [ , ] . for detection of known respiratory viruses, the virochip microarray was found to have a sensitivity and specificity comparable to or superior to conventional diagnostic testing [ , ] . deep sequencing is a complementary approach capable of detecting viruses that are too divergent from known viruses to be detected by either pcr or microarray techniques [ , , , , , , ] ; reviewed in [ ] . de novo pyrosequencing has enabled the discovery of two novel arenaviruses, an arenavirus associated with a fatal cluster of cases in solid-organ transplant patients (dandenong virus) [ ] , and a hemorrhagic feverassociated arenavirus from south africa (lujo virus) [ ] . metagenomic approaches are especially attractive in the study of influenza given the constant threat of antigenic drift and shift [ ] . microarrays can rapidly type the origins of different segments to guard against the threat of novel reassortants and to detect coinfections with other pathogens [ , , , , , , ] , while pyrosequencing or the use of resequencing microarrays can monitor the emergence of mutations that confer virulence or resistance to antiviral drugs [ , , ] . deep sequencing strategies have been recently employed to detect seasonal influenza viruses in three clinical samples [ ] , as well as identify quasispecies of h n in a single autopsy lung sample [ ] . here we analyzed a group of early cases from the h n pandemic from the united states (california), canada, and mexico by virochip and deep sequencing. we show that the virochip, in the absence of a priori information, was capable of rapid characterization of h n as an influenza a (h n ) virus most closely related to swine influenza viruses. we also demonstrate the utility of deep sequencing of clinical samples to identify and characterize not only the novel pathogen but also the microbiota and host response to infection. nasopharyngeal swab samples collected from patients in north america from april to june of were included in this study (table s ; fig. a ). study patients were children or young adults, % ( of ) male, with an average age of years (range to years). although the majority of cases ( %, of ) were upper respiratory infections in the outpatient setting, % ( of ) of patients were hospitalized for presumed h n infection. all three study patients from california were hospitalized in the intensive care unit (icu) with h n -associated pneumonia. two of the three california patients were pregnant women, a group previously reported at high risk for severe complications from h n infection [ ] . to determine whether a pan-viral microarray assay was capable of identifying novel h n in the absence of a priori sequence information, we used the virochip to comprehensively screen for viruses in nasopharyngeal swab samples from individuals with influenza-like illness. seventeen samples from mexico (n = ), canada (n = ), and the united states (california) (n = ) were suspected or pcr-confirmed cases of h n infection. the remaining control samples, all from california, were positive for h n seasonal influenza (n = ), h n seasonal influenza (n = ), another respiratory virus (n = ), or negative by all diagnostic testing (n = ). of note, the probes on the virochip were designed prior to the emergence of pandemic h n . virochip hybridization patterns corresponding to of samples suspected or confirmed positive for h n grouped together by hierarchical cluster analysis, and were clearly distinguishable from patterns corresponding to seasonal h n influenza virus ( fig. a) . two of the samples, mex- and mex- , exhibited very weak microarray probe intensities on virochip analysis and were grouped with the negative control samples. the finding of weak virochip intensities for these two samples was reproducible, and likely secondary to low viral titers and/or high background from host nucleic acid which can interfere with the random pcr amplification step of virochip analysis [ , ] . the average normalized microarray intensities for the h n samples were more pronounced with h n probes derived from swine influenza (a/swine/wisconsin/ / ) than with probes derived from human influenza (a/human/puerto rico/ / ); the difference was statistically significant for of samples by t-test analysis (fig. b) . microarrays corresponding to seasonal h n influenza displayed the opposite pattern, with h n probes derived from human influenza stronger in intensity than those derived from swine influenza. minimal cross-hybridization in influenza a probes was observed with samples either negative or harboring other respiratory viruses. the virochip microarray was also analyzed using z-score analysis and e-predict, an automated method for viral species identification in microarrays [ , ] . both methods incorporate information from all of the oligonucleotide probes on the virochip simultaneously, including probes to the full range of influenza strains, and revealed that a virus most similar to swine influenza a (a/swine/wisconsin/ / ) was present in these samples (data not shown). thus, the virochip detected the presence of a virus most closely related to swine influenza a/ h n in samples from individuals infected with h n . to further characterize the metagenomics of h n infection in humans, we labeled the influenza samples positive for h n by virochip with distinct molecular barcodes and analyzed them by paired-end deep sequencing on three lanes of an illumina genome analyzer iix. after trimming reads to remove barcodes and exclude low-complexity or primer sequences, , , high-quality -bp sequence reads were subjected to an iterative blastn analysis pipeline (fig. b) . from the initial set of reads, a total of , , ( . %) reads were alignable (word size = , e-value = or ) to sequences obtained from the ncbi non-redundant nucleotide (nt) database (march build). the distribution of reads aligning to human, bacterial, and viral sequences varied greatly depending upon the laboratory preparation method for the clinical sample prior to sequencing ( figure ). samples from mexico were not treated with dnase post-extraction, resulting in most reads from those samples ( to %) aligning to human genomic dna. in contrast, samples that were treated with dnase post-extraction (united states and canada) contained many reads aligning to abundant, non-coding rnas, including human and bacterial ribosomal rna (rrna). despite dnase treatment, reads aligning to the human mitochondrial genome remained abundant for several samples. the overall percentages of bacterial ( . %), viral ( . %), other ( . %) and nonaligning ( . %) reads were low, with some notable individual exceptions. approximately % of reads from sample bc- aligned to bacterial sequences, mostly corresponding to the streptococcus genus. two samples from british columbia had a disproportionately large number of reads that aligned to viruses due to the addition of ms phage as an internal positive control for the luminex respiratory virus panel (rvp) assay (luminex, austin, tx). to determine if alterations in the upper respiratory tract microbiome were present in samples from patients infected with h n virus, we further analyzed the deep sequencing reads aligning to bacteria. interestingly, all bacterial reads aligned to rrna sequences, likely due to the high relative abundance of rrna transcripts in bacteria [ ] , and no bacterial mrna reads were observed. given the high degree of conservation of rrna sequences in bacteria, each read could only be unambiguously classified at the genus level (or at the family level in the case of enterobactericeae) (fig. ) . in general, a high level of diversity of bacterial families was observed in different samples, reflecting the known diversity of bacterial flora in the nasopharynx [ ] . reads to the top three bacterial families comprised anywhere from to % of all bacterial reads, depending on the sample (fig. a ). moraxellaceae or enterobactericeae were the most common bacterial families found, accounting for one of the three most common bacterial families in of samples (fig. b ). significant numbers of reads aligning to the streptococcus genus were only seen in of samples, while reads to unculturable agents (''environmental samples'') accounted for between to % of sequences aligning to bacteria. the majority of viral reads (n = , , . %) aligned to members of the orthomyxoviridae (influenza) family (table ) . phages comprised the second most common group of viruses, including known controls (e.g. ms control phages), trace reagent contaminants (e.g. bacteriophage m ), and environmental phages. reads aligning to plant viruses were observed in two samples (bc- and mex- ). sixteen reads to murine leukemia viruses (mlvs) were detected in of samples. in general, these mlv reads aligned slightly better to moloney murine leukemia virus (mmlv) than to the recently described human xenotropic murine leukemia-related virus (xmrv) [ , , ] , although, for some reads, the alignments were identical. interestingly, a single overlapping -nt paired-end read from bc- aligned with % identity to a filovirus, ebola-sudan ( % query coverage, e-value = ) and mapped to a gene coding for the ebola viral glycoprotein. this bc- read also aligned to other filoviruses ( - % identity) but did not align to any other viral family. we were able to reproduce detection of this single filovirus read by specific rt-pcr, but, despite the use of multiple sets of primers, we were unable to recover additional filovirus sequence from this sample (data not shown). aside from influenza, the only other respiratory viral pathogen detected was human adenovirus in one canadian sample (bc- , reads). influenza reads aligning to orthomyxoviridae were found in all specimens, ranging from to , reads, or . % to . % of all aligning reads. after stratifying by originating location and corresponding method of sample processing (pre-dnase and/or post-dnase treatment), the percentage of total reads aligning to influenza was linearly correlated with calculated viral titers by realtime quantitative rt-pcr for sites in the united states (california) and canada but not in mexico (fig. a ). treatment with dnase twice yielded the best percentage recovery of however, despite very low viral titers, h n influenza was still detectable by deep sequencing, with and reads aligning to influenza for cal-uc and bc- , respectively (table ) . influenza reads from all segments were found in of samples, while reads from at least two different segments were found in every sample ( fig. ; figs. s and s ). interestingly, reads to the ns (nonstructural) segment were significantly less likely to be found than reads to other segments (p, . by chi-square analysis), with ns reads detected in only of samples. in contrast, reads to pb were found in all samples. samples from mexico had the worst overall coverage, likely due to the high percentage of human genomic background sequences as a result of not treating samples with dnase post-extraction. in the sample with the best overall coverage (bc- ), . % of the h n genome had at least x coverage, while . % of the genome had at least x coverage. we sought to assess the accuracy of deep sequencing relative to traditional sanger sequencing as well as search for single nucleotide polymorphisms (snps) in the assembled deep sequencing reads that may represent heterogeneous populations of influenza. deep sequencing reads from the two samples in the study with the highest coverage of the h n genome, bc- ( . % coverage at x) and cal-uc ( . % coverage at x), were mapped to their full-genome scaffolds obtained by sanger sequencing. overall, there was a high degree of concordance (. . %) with deep sequencing and traditional sanger sequencing for both bc- and cal-uc (table s ) . next, to identify potential mutations at key sites in the genome that mediate drug resistance and virulence, we compared the genomes of bc- and cal-uc with the full-genome reference sequence a/california/ / (h n ) ( table s ). in total, there were nonambiguous nucleotide differences between bc- and the original california strain and nonambiguous nucleotide differences between cal-uc and the original strain, of which % and % were detected by sanger sequencing and % and % by deep sequencing alone, respectively. these nucleotide differences corresponded to and nonsynonymous mutations in the bc- and cal-uc genomes, respectively. the genome segment with the most amino acid changes was the ha gene ( of nucleotide differences, or . %). for both bc- and cal-uc , there were no differences from the reference sequence a/california/ / (h n ) at key sites in the genome corresponding to known mutations for antiviral resistance, enhanced transmission, or increased virulence. database in these samples were considered to arise from mrna transcripts. since no rna-seq data corresponding to nasopharyngeal samples from healthy individuals can be found in the ncbi sequence read archive (http://www.ncbi.nlm.inh.gov/sra), we used a set of nasopharyngeal swab samples collected from kleine-levin syndrome (kls) patients with influenza-like illness but testing negative for influenza as background controls. genes associated with immunity and interferon were significantly upregulated (p, . ) in four of the influenza samples as compared to the control samples, and from two study sites with different specimen processing protocols (fig. a ). the interferon-related genes that were preferentially up-regulated in h n samples relative to controls coded for chemokine ligands, - oligoadenylated synthetases, ifn-inducible proteins, and stat proteins, proteins previously shown to play essential roles in the interferonmediated host immune response to viral infection [ ] . upregulation of interferon and immunity genes was seen in the four samples with the highest x coverage of the influenza genome but not necessarily in samples with a high number of total transcriptome reads (fig. b ). genes associated with cell structure and motility as well as protein metabolism and intracellular protein trafficking were also upregulated in h n samples relative to control samples (fig. a ). we hypothesized that the unprecedented depth of coverage obtainable by deep sequencing could enable detection and even de novo assembly of novel viral pathogens in the absence of any reference genome. to demonstrate the feasibility of this approach in elucidating the cause of outbreaks from hitherto novel or unknown viruses, we generated a modified nt database from which all sequences corresponding to the orthomyxoviridae family had been removed (fig. a) . pure de novo assemblies of paired-end reads pooled from all h n samples which did not align to the modified nt database were then constructed using geneious software (geneious, auckland, nz). of the final contigs (contiguous sequences) of length greater than bp generated from the de novo assembly, contigs mapped onto a reference sequence of h n influenza, with an n contig size of , bp and coverage of . % of the genome (fig. b) . the four longest contigs all corresponded to h n and included the nearly full-length sequences of the pb (polymerase), np (nucleoprotein) and na (neuraminidase) segments (fig. c) . a cursory analysis of the remaining contigs that did not map to the influenza genome revealed that , % of these sequences were low-complexity sequencing artifacts. from a similar analysis performed on the best single sample (bc- ), de novo assembly of . % of the genome was possible. no misassemblies were observed even though the percentage of orthomyxoviridae reads from all h n samples or bc- alone comprised only . % or . % of the corresponding unaligned reads to the modified nt database, respectively. in this study, we demonstrate the utility of a metagenomicsbased strategy combining a rapid, broad-spectrum diagnostic assay (the virochip microarray) with comprehensive deep sequencing to identify and characterize a novel outbreak pathogen, using pandemic h n influenza as a case example. the virochip was capable of differentiating seasonal h n and h n influenza from the novel pandemic strain by the use of multiple probes designed a priori, and accurately characterized the novel virus as closely related to swine influenza viruses. complementary deep sequencing of clinical samples collected early in the course of the h n pandemic enabled detection of reads to the novel strain in every sample, as well as de novo assembly of , % of the genome amidst an enormous background of unaligned sequence reads. as with the sars coronavirus, the lack of a broad-based diagnostic test that was sufficiently sensitive and specific likely contributed to the delayed identification of h n as the cause of an initial outbreak of influenza-like illness in mexico. our approach -using the virochip microarray for rapid screening and deep sequencing for de novo assembly and more detailed characterization -is robust, comprehensive, and directly applicable for the investigation of future outbreaks from novel viral pathogens, especially those which do not grow in culture. nearly all previous studies of metagenomic sequencing of viruses from human clinical material for detection and discovery have employed pyrosequencing [ , , , , , , , , , , ] . the longer reads from pyrosequencing ( - bp) facilitate the assembly of individual reads into contigs, which assists in the classification of reads by homology-based blast alignments. our results suggest, for example, that longer-read sequencing methods such as pyrosequencing may be better suited than illumina sequencing for identification of bacteria. accurate discrimination of bacteria at the species (or even genus) level was not possible with short -bp illumina reads (fig. b) , as rrna sequences corresponding to related bacteria were found to be essentially % identical over a -bp range. we did not de novo assemble the short illumina reads aligning to bacterial rrna into longer, more easily identifiable contigs given the known presence of multiple related bacterial species in the nasopharynx and the very high (and likely) risk of producing misassemblies. on the other hand, a key advantage of illumina sequencing is the significant increase in read depth (, -fold) relative to pyrosequencing. the greater depth of sequencing provides better and more accurate genome coverage as well as increased capability to multiplex clinical samples. in our study of h n influenza by deep sequencing, illumina read lengths of bp were sufficiently long to accurately classify the vast majority of individual reads, thus making de novo contig assembly prior to alignment unnecessary. in addition, we were limited by the computational resources required for de novo assembly of , . million sequences in a reasonable amount of time. our results demonstrate that a computational pipeline consisting of sequential alignments of individual reads using highstringency cutoffs (fig. ) , followed by de novo assembly of the remaining unaligned reads and detailed analysis of the resulting contigs (fig. ) , is an efficient strategy for analyzing the large datasets generated by illumina sequencing of clinical samples and for pathogen discovery. in particular, the final de novo assembly step is critical in identifying divergent sequences that may correspond to novel pathogens. the assembly of longer contigs also eases the burden of computationally intensive amino acid comparison algorithms such as blastx, tblastx and phi-blast by reducing the number of sequences that need to be analyzed. although the costs of deep sequencing (,$ -$ usd/ sample) are still much greater than the reagent costs for diagnostic rt-pcr assays (,$ usd), deep sequencing is able to generate much more useful information including data on the microbiome and host gene expression as well as whole-genome sequencing of pathogens which would require, for example, at least $ , usd per influenza genome via traditional methods. in addition, unbiased deep sequencing allows broad-based detection of novel viruses that may elude diagnosis by specific rt-pcr assays. with paired-end read lengths achievable on the illumina platform now approaching bp, the use of deep sequencing for identification of even highly divergent pathogens at exceedingly low titers becomes feasible. the number of reads corresponding to h n and the ability to map sequences onto the genome were highly dependent on the method of sample preparation. the fewest viral reads were detected in the five samples from mexico that were not treated with dnase after nucleic acid extraction. these samples contained the highest percentage of human genomic sequence, with over % of sequences aligning to the human genome, as well as the fewest reads to bacteria. nakamura et al. also found . % host genomic material in nasopharyngeal aspirates from influenzainfected individuals, and detected a similarly low percentage of reads to influenza, despite the use of centrifugation to remove bacterial and cellular material [ ] . our data indicate that one of the most important steps in viral detection by deep sequencing may be the removal of host genomic dna by post-extraction treatment with dnase -a relatively straightforward procedure for most clinical laboratories. as post-extraction dnase treatment was not % efficient (with the observation of residual dna mitochondrial reads in several samples), it is true that additional pre-processing by filtration, ultracentrifugation, or density-gradient purification would likely have resulted in even better enrichment of viral sequences [ ] . however, for this metagenomics study, we sought to mimic as closely as possible a clinical/public health laboratory setting, and such enrichment strategies may be labor-intensive and neither conducive to the demands of clinical laboratories nor practical given the limited amount of clinical sample available. in addition, an important drawback of directed viral purification methods is that by biasing the sample towards virus detection, they can severely hinder the ability to detect potential non-viral pathogens or investigate patterns of human gene expression in response to viral infection, as we have done here. it has been suggested that co-infections with streptococcus pneumoniae may impact the severity of the clinical presentation in patients infected with h n [ ] . remarkably, very few copathogens, either viral or bacterial, were found among the samples chosen for analysis. deep sequencing revealed that only five samples had a significant number of bacterial reads to the streptococcus genus, which may either represent potential pathogens (e.g. streptococcus pneumoniae), or simply normal human nasopharyngeal viral flora (e.g. streptococcus oralis, sanguis, and/or mitis). one sample from a canadian patient with an upper respiratory infection from h n (bc- ) did contain a small number of reads from adenovirus type (n = ). these results imply that copathogens did not play a significant role in infection by h n influenza in the samples analyzed. further study of a much larger sample set will be needed to fully ascertain the role of co-pathogens in h n infection. although of samples contained reads to murine leukemia viruses, it appears more probable on the basis of sequence alignments that the source of these reads is trace mmlv vector contamination of the reverse transcriptase enzyme used in preparing libraries for deep sequencing rather than infection by xmrv, a gammaretrovirus associated with prostate cancer and chronic fatigue syndrome in humans [ , ] and recently detected in the respiratory tract [ ] . in addition, one sample (bc- ) contained a paired-end read of nt that was a % match to the filovirus ebola-sudan. we considered this most likely a laboratory contaminant, since we did not identify any other reads to filoviruses and were unable to amplify additional filovirus sequence from bc- . however, the origin of this sequence ultimately remains unknown, as we could not subsequently detect it in any sample or reagent by specific rt-pcr, and none of the laboratories involved in the analysis works with ebola virus. we also found reads corresponding to plant viruses in two samples (bc- and mex- ). detection of plant viruses in nasal swab samples is not unexpected given that the nasopharynx is contiguous with the oropharynx, and that an abundance of plant viruses of presumed dietary origin can be found in the human digestive tract [ ] . our discovery of unexpected viral reads corresponding to murine leukemia viruses, filoviruses, and plant viruses in the deep sequencing data underscores the challenges of analyzing rare or unique sequences that may correspond to colonizing flora, reagent/sample contamination, or even true pathogens. using deep sequencing, we were able to recover and assemble near full-length genome sequences of the h n virus from two individual patient specimens. since the molecular determinants of influenza pathogenesis have been well-studied, we were able to analyze the genomic sequence for single nucleotide polymorphisms (snps) that specifically correlate with antiviral resistance, enhanced transmission or increased virulence. for a novel virus, where such genotypic findings would not yet be correlated with phenotypic data, the overall sequence and structure of the genome may still yield valuable insights into viral transmission and mechanisms of pathogenesis. analysis of transcriptome reads demonstrated that immunity and interferon genes were significantly up-regulated in nasopharyngeal swab samples from h n -infected patients as compared to background controls. interestingly, these samples did not necessarily have a high number of transcriptome reads, but did have the highest x coverage of the influenza genome (fig. b) . coverage of the influenza genome may be the best proxy measure for influenza activity and the associated host response in deep sequencing analysis, as it is relatively independent of pcr duplication artifacts that can be introduced during sample preparation for deep sequencing [ ] . our finding of increased expression of interferon-associated host genes in clinical samples from patients with h n infection is intriguing and consistent with very recent data showing that the ns protein of h n lacks the ability to block general host gene expression in both human and swine cells in vitro [ ] . further studies are needed to assess the clinical relevance of increased interferon-associated host gene expression in h n infection in vivo. nevertheless, this finding highlights the power of an unbiased deep sequencing approach by not only facilitating detection of the viral pathogen but also enabling elements of the host response to be simultaneously interrogated, thus generating new hypotheses for analysis of host-pathogen interactions. our study is the first to explore the relationship between viral titer and depth of sequencing required to detect a candidate viral pathogen. in samples that are treated with dnase after nucleic acid extraction (''post-dnase''), there is a linear correlation between percentage of viral reads in the deep sequencing data and viral titers as estimated by quantitative rt-pcr (fig. a ). there is no such observed correlation if samples do not undergo post-dnase treatment, likely secondary to high residual host genomic background, and thus these findings may only apply to rna viral targets. however, it is encouraging that sequence reads corresponding to h n were detectable in all samples, even in those samples with very low viral titers approaching the analytical limits of detection for rt-pcr assays (i.e. cal-uc and bc- ) (fig. b) . more studies are clearly needed on the metagenomics of different viral agents (e.g. rna vs. dna viruses; lysogenic vs. lytic, etc.) across different stages of infection (e.g. pre-symptomatic, acute, or chronic) and in different types of clinical samples (e.g. respiratory secretions, stool, blood, cerebrospinal fluid, tissue etc.). in addition, although the virochip microarray has a to hour turnaround time and can be rapidly deployed as a comprehensive screen for viral pathogens in the setting of outbreaks, the complementary deep sequencing approach is currently limited by the time necessary to perform a full pairedend sequencing run on an illumina genetic analyzer iix (, week) and to analyze the data on a highly parallel computational platform running cores simultaneously (, week). nevertheless, the data that can be obtained in days to weeks by microarrays and deep sequencing would take months to years using conventional methods. furthermore, third-generation sequencing technologies which can generate deep sequencing data within hours are now available [ , ] . the results described here provide a blueprint for the eventual use of microarrays and sequencing as routine diagnostic tools for pathogens in clinical and public health laboratories. cases of influenza-like illness in canada and mexico that were confirmed or suspected positive for h n infection were identified by each individual state public health agency. informed consent was not obtained as the analysis of samples for pathogens is part of the mandate of clinical testing for each individual agency, and samples as well as demographic, clinical, and laboratory data were de-identified prior to analysis. cases in california were enrolled in a viral diagnostics study approved by the university of california, san francisco (ucsf) institutional review board (irb) (protocol #h - ), and written informed consent was obtained from all study participants and/or their legal guardians. for all cases, collected samples were analyzed under protocols approved by the ucsf irb (protocol #h - ). cases of influenza-like illness in canada and mexico were reported by providers and hospitals to the british columbia centre for disease control (bc-cdc), the university of toronto/ ontario agency for health protection and promotion (oahpp), and the veracruz ministry of health. suspected or confirmed h n cases were identified through routine laboratory surveillance under protocols approved by each individual state public health agency. for each case, non-identifying demographic and clinical data were reported on standardized forms. cases in california were identified by infectious disease physicians at university of california at san francisco (ucsf), and demographic/clinical data were abstracted from the medical record. nasopharyngeal swab samples collected in viral transport media from patients with either laboratory-confirmed or suspected cases of h n influenza virus were analyzed. twelve additional nasopharyngeal swab samples from california were included as negative controls for the virochip. rna extractions were performed differently using established protocols specific to individual laboratories. for mexico, ml of sample with linear polyacrylamide (ambion, inc., austin, tx) added as an rna carrier was treated with turbo dnase (ambion, inc., austin, tx)) for minutes at uc (''pre-dnase'' treatment); nucleic acid was then extracted using the purelink rna-dna kit (invitrogen, carlsbad, ca) according to the manufacturer's instructions. for british columbia and ontario, ml and ml, respectively, of sample were extracted using the nuclisens easymag automated extraction system and then treated with turbo dnase (''post-dnase'' treatment). for california, ml of sample was treated with turbo dnase, nucleic acid was extracted with the zymo viral rna kit (zymo research, orange, ca), and then the extracted nucleic acid was treated again with turbo dnase (''pre-/post-dnase'' treatment). total rna was reverse-transcribed to cdna, amplified by a modified round a/b random pcr method, and labeled with cy or cy fluorescent dye as previously described [ , ] . the labeled samples were normalized to pmol of incorporated dye and hybridized overnight using the agilent gene expression hybridization kit according to the manufacturer's protocol (agilent technologies, santa clara, california). the microarray slides used were custom-designed k or k arrays synthesized by agilent technologies, each containing , virochip -mer oligonucleotide probes in common. these , common probes include conserved as well as specific probes representing all viral species in genbank, and were derived from a previous virochip design [ ] . the virochip is not yet commercially available, but is currently implemented as a core diagnostic service of the ucsf viral diagnostics and discovery center (http:// vddc.ucsf.edu). slides were scanned at mm resolution in xdr (extended dynamic range) mode using an agilent dna microarray scanner. virochip microarrays were analyzed with hierarchical cluster analysis, e-predict, and z-score single oligonucleotide analysis as previously described [ , , ] . average normalized microarray intensities were analyzed using a twosample, two-tailed t-test for unequal variances. all virochip microarrays used in this study were submitted to the ncbi geo database (study accession number gse ; microarray accession numbers gsm - ; microarray design accession numbers gpl for the k array and gpl for the k array). sequences corresponding to each influenza segment were obtained by one-step rt-pcr using either a qiagen one-step rt-pcr kit (qiagen, valencia, ca) or an invitrogen super-scriptiii one-step rt-pcr kit with high fidelity platinum taq (invitrogen, carlsbad, ca), with primers designed based on conserved terminal and central regions of each segment (table s ). reactions run with the qiagen kit were done in duplicate to account for potential polymerase errors. pcr products were run on a . % agarose gel, and bands of the correct size were cut and extracted. fragments were cloned into a pcr . vector (invitrogen, carlsbad, ca) and sanger sequenced (elim biopharmaceuticals, hayward, ca) with m f and m r primers as well as the original conserved pcr primers. at least three-fold redundancy was obtained for each segment. the whole-genome sequences of h n samples bc- and cal-uc , including all segments, have been submitted to genbank (genbank accession numbers cy -cy for bc- and cy -cy for cal-uc ). quantitative rt-pcr for influenza was performed with primers based on conserved regions in the ha segment, h n -ha- f ( -gggaaatccagagtgtgaatcact- ) and h n -ha- r (gctctcttagctcctcataatcgatg- ) using a stratagene mx p real-time pcr system. the pcr was performed using a qiagen one-step rt-pcr kit (qiagen, valencia, ca) in a . ml reaction containing . ml h o, . ml x pcr buffer, . ml q solution, . ml dntp, . ml rt/taq enzyme mix, . ml of each primer, . ml sybr green and . ml total rna. conditions for the pcr were uc for min, uc for min; cycles of uc for s, uc for s, and uc for min; and a final extension at uc for min. the viral titer was estimated using a standard curve derived from an in vitro-transcribed and quantified influenza mrna standard (data not shown). for the canadian samples, viral titers as determined through ct values were compared with real-time rt-pcr data obtained independently from reference laboratories in vancouver (bc-cdc) and toronto (oahpp) to check for accuracy (data not shown). libraries for deep sequencing were prepared from amplified cdna libraries using previously published protocols [ ] . briefly, libraries were cleaved with type iis restriction endonucleases (gsui), and truncated adapters containing unique or bp molecular barcodes were ligated on the resulting strand ends. fulllength adapters were subsequently added via an additional to cycles of pcr. libraries were size-selected on a % polyacrylamide gel at approximately bp average length, and then loaded at a final concentration of pm on three lanes of a second-generation genome analyzer iix (illumina, san diego, ca). paired-end reads were sequenced for cycles in each direction. fluorescent images were analyzed using the illumina basecalling pipeline . . to obtain paired-end sequencing data. paired-end reads were subsequently classified by strict barcodes, filtered to eliminate low-complexity sequences with an lzw compression ratio cutoff of . , split into individual reads, and stripped of any remaining primer sequences using blastn alignments (word size = , e-value = ). blastn alignments to publicly available sequence databases were performed using four extralarge high cpu-instance amazon elastic cloud computing (ec ) servers comprising a total of core processors, with an approximate computational time of hours per sequencing lane. sequence reads that aligned to human rrna and mitochondrial genome by blastn (word size = , e-value = ) were initially removed. remaining reads were then classified by successive blastn alignments to the refseq human transcriptome database (word size = , e-value = , september build [release ]) and ncbi non-redundant nucleotide (nt) database (word size = , e-value = , march build). all reads that aligned to nt were sorted by their best hit into their respective taxa for analysis. reads that aligned to nt but that did not align to human, bacterial, or viral sequences were categorized as ''other'', and consisted solely of environmental sequences or plasmids/artificial constructs. the presence of murine leukemia virus and ebola virus in the deep sequencing libraries was independently confirmed by specific rt-pcr and direct sequencing (data not shown). we also separately performed directed blastn alignments of the original set of unclassified deep sequencing reads to an influenza sequence database (word size = , e-value = ) to verify that no influenza reads were incorrectly classified as human or other nonviral sequence (data not shown). all high-quality sequence reads have been submitted to the ncbi sequence read archive (http://www.ncbi.nlm.nih.gov/sra) (accession number sra ). reads corresponding to the human mrna transcriptome were counted by gene and compared to transcriptome reads from nasopharyngeal swab samples from kleine-levin syndrome (kls) patients with influenza-like illness. kls is a neuropsychological disorder marked by hypersomnolence, hyperphagia, and hypersexuality usually found in adolescent boys [ ] . an ongoing metagenomic analysis of samples from patients with kls (''kls samples'') by deep sequencing found only reads to a single rhinovirus a isolate in one sample and no reads to influenza in any sample; in addition, all kls samples were negative for both pandemic and seasonal influenza by specific rt-pcr (data not shown). as such, it was thought that the kls samples could serve as appropriate background controls from individuals with influenza-like illness (but testing negative for influenza) for the purposes of transcriptomic analysis. in particular, the kls samples were derived from a similar demographic as the h n samples (children and young adults) and were processed identically for deep sequencing. the transcriptomic analysis was performed as follows. first, we selected all genes in refseq that were significantly overexpressed in h n nasopharyngeal swab samples relative to kls background samples (p, . , by bonferroni-corrected fisher's exact test). to identify overrepresented categories of genes, we inputted the overexpressed refseq genes into the panther (protein analysis through evolutionary relations) database (http://panther.appliedbiosystems.com) and compared them to a human reference list of transcribed genes [ ] . categories of genes significantly up-regulated in the influenza data relative to the human reference list (p, . , by bonferroni-corrected fisher's exact test) were reported. snp analysis of sequence reads mapping to influenza was performed using the ssaha software package [ ] . both pairedend read information and fastq-formatted quality scores were incorporated in the calling of snps. snp calling was performed at very high stringency to ensure accuracy. specifically, the illumina deep sequencing data had a calculated overall error rate of , % by analysis of the phix control lane (illumina inc., hayward, ca), and at least -fold coverage and a quality score of were required for mapping of deep sequencing reads at any given position in the genome. a minority snp was defined as present if occurring $ % of the time at a given nucleotide position. paired-end de novo assembly of the influenza genome was performed using geneious version . . software [ ] , with strict parameters (word length = ; % mismatch/gap tolerance; only paired-end reads included in assembly; expected paired-end mate distance = ) to avoid misassembly, the de novo assembly algorithm used by geneious is a greedy algorithm similar to that used in multiple sequence alignment programs such as clustalw (matt kearse, geneious inc., personal communication) [ ] . initial generated contigs of size $ bp were further assembled de novo in geneious at high sensitivity with ''fine tuning'' of gaps and -nt trimming of contig ends. final de novo assembled contigs were then mapped to the full-genome reference sequence a/california/ / (h n ) (genbank accession numbers fj -fj and fj -fj ). out of the remaining contigs that did not map to influenza, , % of the contigs corresponded to low-complexity sequence that was identified as such by applying an lzw compression ratio of . , slightly higher than that used in the initial filtering ( . ). emergence of a novel swine-origin influenza a (h n ) virus in humans triplereassortant swine influenza a (h ) in humans in the united states pandemic potential of a strain of influenza a (h n ): early findings rapid-test sensitivity for novel swine-origin influenza a (h n ) virus in humans origins and evolutionary genomics of the swine-origin h n influenza a epidemic metagenomics for the discovery of novel human viruses microarray detection of human parainfluenzavirus infection associated with respiratory failure in an immunocompetent adult microarray-based detection and genotyping of viral pathogens characterization of a novel coronavirus associated with severe acute respiratory syndrome viral discovery and sequence recovery using dna microarrays pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity identification of cardioviruses related to theiler's murine encephalomyelitis virus in human infections achalasia and viral infection: new insights from veterinary medicine recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent utility of dna microarrays for detection of viruses in acute respiratory tract infections in children identification of a novel astrovirus (astrovirus va ) associated with an outbreak of acute gastroenteritis the complete genome of klassevirus -a novel picornavirus in pediatric stool metagenomic analyses of viruses in stool samples from children with acute flaccid paralysis bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses multiple diverse circoviruses infect farm animals and are commonly found in human and chimpanzee feces genetic detection and characterization of lujo virus, a new hemorrhagic feverassociated arenavirus from southern africa a new arenavirus in a cluster of fatal transplant-associated diseases influenza vaccine-outmaneuvering antigenic shift and drift broadspectrum respiratory tract pathogen identification using resequencing dna microarrays robust sequence selection method used to develop the fluchip diagnostic microarray for influenza virus panmicrobial oligonucleotide array for diagnosis of infectious diseases experimental evaluation of the fluchip diagnostic microarray for influenza virus surveillance detection in of the swine origin influenza a (h n ) virus by a subtyping microarray large-scale evolutionary surveillance of the h n influenza a virus using resequencing arrays identifying influenza viruses with resequencing microarrays detection of molecular markers of drug resistance in the pandemic influenza a (h n ) viruses using pyrosequencing direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach characterization of quasispecies of pandemic influenza a virus (a/h n / ) by de novo sequencing using a next-generation dna sequencer california pandemic working g ( ) severe h n influenza in pregnant and postpartum women in california e-predict: a computational strategy for species identification based on observed dna microarray hybridization patterns development and quantitative analyses of a universal rrna-subtraction protocol for microbial metatranscriptomics the nih human microbiome project xenotropic murine leukemia virus-related gammaretrovirus in respiratory tract identification of a novel gammaretrovirus in prostate tumors of patients homozygous for r q rnasel variant detection of an infectious retrovirus, xmrv, in blood cells of patients with chronic fatigue syndrome antiviral actions of interferons clonal integration of a polyomavirus in human merkel cell carcinoma metagenomic analysis of respiratory tract dna viral communities in cystic fibrosis and non-cystic fibrosis individuals streptococcus pneumoniae coinfection is correlated with the severity of h n pandemic influenza rna viral community in human feces: prevalence of plant pathogenic viruses substantial biases in ultra-short read data sets from high-throughput dna sequencing inefficient control of host gene expression by the pandemic h n influenza a virus ns protein real-time dna sequencing from single polymerase molecules the next generation becomes the now generation the long march: a sample preparation technique that enhances contig length and coverage by high-throughput short-read sequencing kleine-levin syndrome: a systematic study of patients panther pathway: an ontology-based pathway database coupled with data analysis tools ssaha: a fast search method for large dna databases multiple sequence alignment using clustalw and clustalx we thank aurora parís at the ministry of health bsl- state laboratory in veracruz for providing h n samples from mexico. we thank david schnurr, sharon messenger, and shigeo yagi at the california department of public health for providing two seasonal h n samples for virochip analysis. we thank emmanuel mignot at stanford university for providing respiratory samples from kls patients as negative controls. we thank catherine liu, peter chin-hong, and brian schwartz for their help in identifying patients infected with h n influenza at ucsf. we also thank clement chu, peter skewes-cox, and maría soto del río for expert technical assistance. key: cord- - jup v authors: gouveia, duarte; miotello, guylaine; gallais, fabrice; gaillard, jean-charles; debroas, stéphanie; bellanger, laurent; lavigne, jean-philippe; sotto, albert; grenga, lucia; pible, olivier; armengaud, jean title: proteotyping sars-cov- virus from nasopharyngeal swabs: a proof-of-concept focused on a min mass spectrometry window date: - - journal: j proteome res doi: . /acs.jproteome. c sha: doc_id: cord_uid: jup v [image: see text] rapid but yet sensitive, specific, and high-throughput detection of the severe acute respiratory syndrome coronavirus (sars-cov- ) in clinical samples is key to diagnose infected people and to better control the spread of the virus. alternative methodologies to pcr and immunodiagnostics that would not require specific reagents are worthy to investigate not only for fighting the covid- pandemic but also to detect other emergent pathogenic threats. here, we propose the use of tandem mass spectrometry to detect sars-cov- marker peptides in nasopharyngeal swabs. we documented that the signal from the microbiota present in such samples is low and can be overlooked when interpreting shotgun proteomic data acquired on a restricted window of the peptidome landscape. in this proof-of-concept study, simili nasopharyngeal swabs spiked with different quantities of purified sars-cov- viral material were used to develop a nanolc–ms/ms acquisition method, which was then successfully applied on covid- clinical samples. we argue that peptides adetqalpqr and gfyaqgsr from the nucleocapsid protein are of utmost interest as their signal is intense and their elution can be obtained within a min window in the tested conditions. these results pave the way for the development of time-efficient viral diagnostic tests based on mass spectrometry. the new severe acute respiratory syndrome-related coronavirus (sars-cov- ) is the causative agent of covid- , the coronavirus disease that was first reported in december in the city of wuhan, china. because of its easy interhuman transmission, sars-cov- has since quickly spread worldwide, causing more than million covid- diagnosed infections and more than thousand deaths officially reported as off mid-july (https://covid .who.int/). the rapid, sensitive, and specific detection of the sars-cov- virus in large cohorts of clinical samples is of utmost importance to identify infected people and control the propagation of the virus by specific containment measures. at the same time, being able to catch the numerous sars-cov- variants represents an opportunity to identify attenuated forms of the virus. however, the occurrence of specific mutations, especially deletions, may challenge current molecular detection methodologies. the research community has been placing great efforts in the development of quick and accurate detection tests. − the gold standard in diagnostics relies on the amplification and measurement of the viral rna by reverse transcription polymerase chain reaction (rt-pcr). rt-pcr is highly specific and achieves a good compromise between speed ( − min) and sensitivity. however, due to the great demand for pcr-based testing, shortage of rna extraction kits and pcr reagents may have limited the testing capacity in some countries at the early stage of the pandemic. besides, rt-pcr testing of clinical samples may be in some case less efficient due to nucleic acid variations in the targeted regions, primers or their close vicinity, that could affect the amplification rate. , for these reasons, alternative detection strategies that address these concerns should be developed to complement conventional tools. immunoassays, whole-genome sequencing and mass spectrometry (ms) technologies are commonly suggested alternatives to pcr-based assays. among these, new generation ms offers a highly sensitive technology that allows the rapid identification of thousands of proteins present in a single sample. the typing of organisms by tandem mass spectrometry (ms/ms), commonly referred to as "proteotyping", is based on the identification of specific peptide sequences that allow the unambiguous identification of organisms. − the uniqueness of the mass to charge ratios and fragmentation patterns measured in ms/ms allows identification of peptides that differentiate organisms at the subspecies level. although classical ms-based identification of pathogens in the clinical setting is based on whole-cell maldi-tof technology, the field has thrived with the increases in speed, sensitivity, and accuracy of new ms instrumentation in the past decade. the coupling of new generation instruments with the separation power of liquid chromatography makes lc−ms/ms a valuable technology to implement in the routine of clinical laboratories. despite their high potential, the application of lc−ms/ms approaches for virus proteotyping is still scarce. among the few examples available in the literature, lc−ms/ms was shown to be able to detect purified influenza virus and human metapneumovirus in clinical samples. because of the considerable damages of the covid- pandemic, the mass spectrometry community quickly proposed to mobilize its efforts at helping to understand the molecular mechanisms of infection − and at improving detection methods. several research groups started investigating msbased quantification of peptides for the detection of sars-cov- in clinical samples, but most of these results are not yet published. , , these preliminary results indicate that targeted ms, in which the mass spectrometer is programmed to precisely detect and quantify a limited number of peptides of interest, can be successfully applied to virus detection. targeted ms is considered as the gold standard for peptide quantification due to its higher sensitivity when compared to shotgun proteomics approaches. nevertheless, this approach has a much lower throughput and is commonly used to test hypotheses on a subset of proteins of interest, in contrast to discovery shotgun proteomics. by being more flexible, the latter provides a more comprehensive picture of the viral peptidome including the detection of variant sequences because of the possibility of detecting peptides without any previous knowledge of their sequences. here we established the proof-of-concept of the use of ms/ ms for the rapid proteotyping of sars-cov- from clinical samples. we recently published a data set from a shotgun lc− ms/ms experiment performed with sars-cov- infected cells and proposed a list of specific viral peptides that could be used for the development of targeted approaches. interestingly, we observed that some sars-cov- specific peptides eluted from the lc column at narrow windows of retention time. here, we used lc−ms/ms with an orbitrap instrument (q exactive hf) for analyzing the peptidome from nasal swabs spiked with different quantities of viral material. by using a short lc gradient focusing on the region of interest identified in our previous study, we tested the detection of the virus in samples containing different quantities of viral peptides, as well as covid- clinical samples, paving the way for the development of time-efficient viral diagnostic tests based on an alternative platform. two nasopharyngeal swabs were collected using a sterile polyester swab with semiflexible polystyrene handle (puritan) from two healthy volunteers (swabs r and r ). each swab was soaked into a tube containing μl of sterile water, incubated for min at room temperature, and then rinsed with μl of sterile water. the biological material from the μl of solution was precipitated with the addition of μl of trichloroacetic acid at % (w/v) and centrifugation at g for min. the supernatant was discarded. the hardly visible pellet was dissolved into μl of lds x containing % beta-mercaptoethanol, heated for min at °c, and centrifuged briefly. for each swab sample, a volume of μl of lds x sample was deposited on a sds-page gel and run for min. after migration, the gel was rinsed with water, stained with simply blue safestain (invitrogen), and destained overnight in water. the two polyacrylamide gel bands corresponding to the whole proteome of each matrix were excised, processed as described, and then subjected to trypsin gold proteolysis (promega) using . % proteasemax surfactant (promega). the nasal matrix peptide fractions were μl for each swab. peptides from the nasal swab matrices were analyzed with a q-exactive hf mass spectrometer (thermo) coupled with an ultimate lc system (dionex-lc) and operated in datadependent mode as previously described. a volume of μl of peptides was injected, desalted onto an acclaim pepmap c precolumn ( μm, Å, μm id × mm), and then resolved onto a nanoscale acclaim pepmap c column ( μm, Å, μm id × cm) with a min gradient at a flow rate of . μl/min. the gradient was developed from to % of ch cn, . % formic acid in min, and then from to % in min, washed, and re-equilibrated. peptides were analyzed with scan cycles initiated by a full scan of peptide ions in the orbitrap analyzer, followed by high-energy collisional dissociation and ms/ms scans on the most abundant precursor ions (top method). full scan mass spectra were acquired from m/z to at a resolution of with internal calibration activated on the m/z . signal. ion selection for ms/ms fragmentation and measurement was performed applying a dynamic exclusion window of s and an intensity threshold of × . only ions with positive charges + and + were considered. vero e (atcc, clr- ) cells were cultured at °c in % co in dulbecco's modified eagle's medium (dmem, gibco, themofisher) supplemented with % fetal calf serum (fcs) and . % penicillin−streptomycin. the sars-cov- strains -ncov/italy-inmi (genbank mt ) was provided by the lazzaro spallanzani national institute of infectious diseases (rome, italy) via the evag network (european virus archive goes global). sars-cov- stocks used in the experiments had undergone two passages on vero e cells and were stored at − °c. virus titer was . × plaque forming units (pfu)/ml, as determined by standard plaque assay (three dilutions in duplicates). all experiments entailing live sars-cov- were performed in our biosafety journal of proteome research pubs.acs.org/jpr article level facility and strictly followed its approved standard operating procedures. vero e cells ( × ) seeded into cm flasks were grown to cell confluence in ml of dmem supplemented with % fcs and . % penicillin−streptomycin for one night at °c under % co . they were infected at multiplicity of infection (moi) of . . cells were harvested at days post infection (dpi), and viral suspension was recovered after centrifugation at rpm for min to remove cell debris. thirty-three milliliters of the viral suspension was laid on ml of % (w/ v) sucrose cushion prepared in nacl . m, edta mm, and mm tris hcl buffer (ph . ) (tne buffer) in ultra-clear ml tubes (beckman coulter). samples were centrifuged at rpm for h at °c. pellets were solubilized in μl of cold tne buffer, and a volume of . ml was laid on a five step − % (w/v) sucrose gradient prepared in ultra-clear ml tubes (beckman coulter). the tubes were centrifuged at rpm for h at °c in a beckman sw rotor. after recovery of the virus band, the viral suspension was inactivated by incubation with betapropiolactone at a final concentration of . % for h at °c. plaque assay titration was used to quantify the purified virus and validate the viral inactivation. the inactivated purified virus sample (equivalent to . × pfu/ml) was quantified in terms of protein concentration ( . mg/ml) by uv spectrophotometry. a volume of μl was mixed with μl of lds x to obtain a protein fraction of . mg/ml. after denaturation at °c for min, a volume of μl ( . μg of proteins) was deposited on a nupage − % gel (invitrogen) and subjected to min electrophoretic migration. the whole proteome was excised as a single polyacrylamide gel band and subjected to trypsin proteolysis as previously described. an aliquot of μl of peptides was extracted. ms/ms analysis was performed to confirm the high content of viral proteins in this sample (gallais and armengaud, unpublished results). sars-cov- viral peptides ( μl) were diluted in μl of h o, . % tfa. after mixing, μl of this tube was removed and diluted with μl of h o, . % tfa. this was repeated several times to obtain a one-third dilution cascade of viral peptides. two series of simili swabs were prepared in parallel. the two peptide fractions obtained from nasopharyngeal swabs ( μl) were diluted with μl of h o, . % tfa. a volume of μl of this diluted matrix was added to each simili swab samples, giving a final volume of μl per sample. thus, each simili swab contained the equivalent of . % of the proteins harvested by a nasal swab. two biological replicates were prepared using each nasal swab matrix. a volume of μl per sample was injected in the q-exactive hf tandem mass spectrometer. they were analyzed in the same conditions as above except that the gradient was developed from to . % of ch cn, . % formic acid for min at a flow rate of . μl/min. the min ms/ms acquisition started min after injection. nasopharyngeal swabs were collected from covid- diagnosed adult patients as routine medical controls to monitor virus clearance after their hospital isolation. they were tested by rt-pcr assay for detecting sars-cov- in a nasopharyngeal sample (swabs t -t ). this study was approved by the institutional review boards of the university hospital of nimes, france ( − − ). patients have been previously be informed that part of these samples could be used for research purpose and agreed. each swab was soaked into a tube containing ml of phosphate buffered saline (ph . ) sterile solution and transferred in the biosafety level facility. the biological material was precipitated with the addition of . ml of trichloroacetic acid at % (w/v). after centrifugation, the supernatant was discarded and the pellet was dissolved into μl of lds x containing % betamercaptoethanol, heated for min at °c, and deposited on a nupage − % gel (invitrogen). the proteins were subjected to min electrophoresis and treated as described here above to obtain tryptic peptides. ms/ms acquisition was done as for the simili swabs. the min ms/ms acquisition started min after injection with an inclusion list comprising m/z values corresponding to viral peptides. ms/ms spectra from the nasopharyngeal swabs were searched against the generalist ncbinr database ( sequences totalling amino acids) with the mascot daemon . . search engine (matrix science). the search parameters were as follows: full-trypsin specificity, maximum of two missed cleavages, mass tolerances of ppm on the parent ion and . da on the ms/ms, carbamidomethylated cysteine (+ . ) as a fixed modification, and oxidized methionine (+ . ) and deamidation of asparagine and glutamine (+ . ) as variable modifications. psms with an fdr < % were selected for peptide inference. peptides were assigned to taxa using the unipept . web interface with default parameters (equate i/l, filter duplicate peptides). ms/ms spectra from the simili sars-cov- contaminated swabs and from the covid- nasopharyngeal swabs were assigned with the mascot daemon . . search engine (matrix science) as follows: the spectra were first queried against the crap_contaminants_ − − .fasta file and then against the swissprot_human_isl_ _ − − database ( sequences totalling amino acids) in follow-up mode and with the decoy option activated. this last database is the merge of the sars-cov- viral proteins and the swissprot human proteome. the mascot search was performed with the same parameters as above. all peptide matches presenting a mascot peptide score with a fdr lower than % were assigned to protein sequences. ms peak areas were evaluated with skyline. briefly, we created spectral libraries based on the dat files from each mascot search (cut-of . ) and uploaded the ms full scan information contained in the raw files. the protein database previously used for the mascot search was used as background proteome. only the viral proteins were added to the target panel. peptide settings were matched to those used in the mascot search. peak peaking was manually checked for all peptides. the mass spectrometry and proteomics data acquired on simili swabs have been deposited to the proteomexchange consortium via the pride partner repository with the data set identifiers pxd and . /pxd . to assess the performance of shotgun ms-based proteomics in detecting sars-cov- peptides in a background matrix consisting of nasopharyngeal swab protein material, we experimentally created tryptic peptidomes from (i) a purified virus solution obtained from vero e cells infected with a sars-cov- reference strain, and (ii) nasopharyngeal swabs obtained from two healthy volunteers ( figure ). we first characterized the nasal peptidomes and searched for the presence of detectable microorganisms by metaproteomic data analysis. then the virus peptidome was serially diluted into nasopharyngeal swab peptidomes to obtain two sets of seven tubes containing from ng (equivalent to infectious particles) to . ng (equivalent to infectious particle) of viral protein material. the samples were subsequently analyzed by lc−ms/ms. a window of min of acquisition within a min lc gradient was adjusted to target the region of elution of five previously identified virus-specific peptides. the rationale for focusing the mass spectrometry measurements on these peptides was their remarkable sequence conservation among the numerous sars-cov- strains sequenced to date or their specificity to the novel coronavirus. these peptides were the following: eitvatsr, gfyaegsr, htpinlvr, iaghhlgr, and adetqalpqr. while known variants exist for the latter, the other four peptides are conserved along the several sars-cov- sequenced genomes. the ms/ms spectra acquired over min on the two nasopharyngeal swabs were analyzed to infer the main microbial components present in these samples as such presence should be taken into account for creating an ad hoc database for ms/ms interpretation. swabs r and r yielded and ms/ms spectra, respectively, from which and were attributed to and peptide sequences from organisms present in the ncbinr database (fdr < %). these peptide sequences were analyzed with the unipept tool to assess the biodiversity present in each sample through their taxon-specificity characteristics based on the lowest common ancestor approach. only a small proportion of the peptide sequences mapped by unipept belonged to microorganisms (table s ) . a rather low number ( and ) of peptides from the r and r swabs, respectively, were attributed to bacteria, archaea, or fungi. these corresponded to . % and . % of the mapped peptide sequences, respectively. to exclude false positive identifications, we applied a threshold of at least three-taxon specific peptides for organism validation at the species level, corresponding to . % of the total number of species-specific peptides ( and in each sample), as suggested by ref . thus, one low-abundant corynebacterium was identified in sample r , namely corynebacterium accolens, with specific peptides. in swab r , corynebacterium propinguum, corynebacterium pseudodiphtheriticum, and dolosigranulum pigrum could be identified at the species taxonomical rank with , , and specific peptide sequences, respectively. simili swabs containing specific quantities of sars-cov- virus and the equivalent of . % of the nasal matrix protein material collected during sampling were analyzed by ms/ms with a short gradient. we first confirmed on the most diluted fraction that the bacterial signal was negligible for both fractions, thus not to consider at the ms/ms attribution search stage. for this, the two data sets were searched against the generalist database ncbinr to check for the presence of nonhuman peptides in the swab peptidomes. the unipept analysis of the detected peptide sequences showed that only and peptides, from replicate and , respectively, were attributed to bacteria and no bacterial species could be confidently identified (table s ). the results from the short gradient ms analysis on the simili swabs against the specific human/virus database yielded ms/ms spectra recorded in the samples. from these, were attributed to peptide sequences with a journal of proteome research pubs.acs.org/jpr article fdr below % (table s ) . these data allowed for the identification of protein groups (table s ) . a small fraction of peptide-to-spectrum matches (psms), corresponding to . % of the total psms, allowed identification of different viral peptide sequences including the five peptides of interest. the peptides report for structural proteins from the virus: peptides from the nucleocapsid protein (n), peptides from the spike protein (s), and peptides from the membrane glycoprotein (m). at least one viral peptide was identified in all samples independently of the concentration of the viral material, from ng ( pfu) to ng ( pfu). however, no peptide from the virus was identified in the sample containing viral peptides corresponding to . ng ( pfu). the heatmap in figure displays the ms peak areas, the number of psms attributed to each peptide in each sample, and the number of viral peptides identified in each sample. the five peptides with the highest ms peak areas across samples were the following: eitvatsr, gfyaegsr, lnqlesk, adetqalpqr, and kadetqalpqr. among them, eitvatsr, gfyaegsr, and adetqalpqr are between the five peptides of interest. peptide htpinlvr was the seventh most abundant. inversely, peptide iaghhlgr was among the peptides with the lowest ms peak areas, along with peptides msecvlgqsk, lddkdpnfk, and eidrlnevak. as expected, the number of identified peptides decreased as the viral load decreased in the sample. while all peptides were identified in the initial dilution containing ng of viral proteins ( pfu), in highly diluted samples containing ng of viral proteins ( pfu) and ng ( pfu), the virus was proteotyped with only and peptides, respectively. in these samples, only peptides from protein n were detected (adetqalpqr, kadetqalpqr, and gfyaegsr). generally, the peptides from protein n were the most consistently detected across samples. despite being among the peptides with higher peak areas in the chromatograms, peptides of interest htpinlvr and eitvatsr were only detected in the simili swabs containing an estimated ng of viral proteins ( pfu). on the other hand, the two other peptides of interest from protein n, gfyaegsr and adetqalpqr, allowed virus proteotyping in the sample containing ng of viral proteins ( pfu). of note, the peptide identified in the condition with ng of viral proteins ( pfu) is a miss-cleaved version of the adetqalpqr peptide: kadetqalpqr. peptide asanlaatk, that had not been previously selected among the "best" candidates for sars-cov- proteotyping, was detected in the dilution with ng of viral proteins ( pfu) and was the most sensitive peptide from protein s. figure represents the retention times of viral peptides from to min of ms acquisition, and their intensities in the nasopharyngeal swabs were sampled from nine covid- diagnosed patients with different clinical manifestations (moderate symptoms and asymptomatic) and at different postdiagnostic stages (table ). these patients were monitored for virus clearance before hospital discharge, thus the viral load in some of these samples was particularly low. the cohort was not established for assessing the performances of the methodology in terms of clinical diagnostic. because of the complexity of the samples, an inclusion list of m/z signals corresponding to the five peptides of interest as well as other sars-cov- peptides detectable in this gradient region was added to the acquisition method to increase the likelihood of their detection. table s reports this inclusion list, which contained m/z values for different precursors from different viral peptide sequences. the short gradient ms analysis on these clinical samples yielded between and ms/ms spectra recorded per sample. sixty-five spectra were attributed to viral peptide sequences with a fdr below % (table s ) . these data allowed for the detection of six peptides reporting for two viral proteins (table s ) : lddkdpnfk, kadetqaipqr, kkadetqaipqr, adetqaipqr, gfyaegsr from protein n, and eitvatsr from protein m. the heatmap in figure displays the ms peak areas, the number of psms attributed to each peptide in each sample, the number of viral peptides identified in each sample, and the result from the pcr testing performed on the same sample. the virus was confidently proteotyped in clinical swab t moderated ct negative swab t asymptomatic negative negative swab t moderated negative negative swab t asymptomatic negative negative swab t asymptomatic ct negative swab t asymptomatic negative negative swab t moderated ct positive swab t asymptomatic ct positive swab t moderated ct negative a moderated severity with radiological visible signs or asymptomatic. b pcr control done within h after control sampling. ct stands for "cycle threshold". to foster the development of alternative detection methods for sars-cov- , we performed a proof-of-concept study to assess the potential of ms/ms for proteotyping sars-cov- : (i) in simulated nasal swabs containing different quantities of viral peptides and (ii) in nasopharyngeal swabs from covid- diagnosed patients. the two nasal peptidomes collected from healthy donors for the first experiment were first analyzed with a gradient of min to check for the presence of detectable microorganisms from the natural microbiota. a search against a generalist database such as ncbinr detected only trace levels of very low abundant bacteria commonly found in the nasal tract, thus confirming the absence of a measurable microbiome in the swab samples. on the basis of this metaproteomic analysis, we used a human-only database as representative of the nasopharyngeal matrices for the subsequent analysis. the simili sars-cov- contaminated swabs contained a fixed amount of swab peptidome, plus a precise amount of viral peptidome corresponding to the expected quantities extracted from ng ( pfu), ng ( pfu), ng ( pfu), ng ( pfu), ng ( pfu), ng ( pfu), and . ng ( pfu) of sars-cov- to cover the range of the viral load kinetics of sars-cov- infection. it is important to note that the virus produced in vero e cells and purified on sucrose gradient is only partially infectious, and thus, the data are also presented in quantities of viral proteins. the real number of viral particles could be much higher in these samples and could be roughly estimated as the molecular weights of each viral protein are known and if the numbers of molecules per virus particle were documented for sars-cov- . here, we refer to the infectious dose as this is the most important parameter in terms of health concern, but the ratio of infectious particles in the nasopharyngeal swabs of patients may drastically differ from the purified virus fraction used here and could even fluctuate during the course of the pathology. the strategy proposed for the analysis of these simili swabs consisted in a shotgun ms analysis based on a short acquisition of min with a short lc gradient. for the clinical samples, we added an inclusion list of viral peptides in the ms method. the inclusion list allowed forcing the fragmentation of candidate viral peptide ions contained in the background matrix, even when they were not included in the top from the data dependent acquisition method. the shotgun strategy resulted in the detection of viral peptides in six out of the seven conditions tested for the simili swab experiment. from the five peptides of interest, gfyaqgsr and adetqalpqr proved to be the most detectable and most sensitive in this background matrix, allowing proteotyping the virus up to the condition of ng of viral material ( pfu). one of the most interesting results was the omnipresence of peptide adetqalpqr and its two misscleaved versions kadetqalpqr and kkadetqalpqr. these peptides were consistently detected in out of identifications in the six most abundant conditions from the simili swab experiment. peptide kadetqalpqr was identified in all simili swabs from figure . these results clearly show that adetqalpqr, despite being prone to missed-cleavages, is one the most abundant and ionizable peptides and should be the main target for proteotyping sars-cov- . this result was confirmed from the analysis of the clinical swab samples since peptides adetqalpqr, kadetqalpqr, and kkadetqalpqr were undoubtedly the most abundant in samples from covid- patients ( figure and table ). in our previous work, we showed that this peptide sequence is also specific to sars-cov- but presented several variants among the available sars-cov- genomes. therefore, when targeting this peptide for viral detection with ms/ms, we can also take into account both its missed-cleaved versions and its different variants. surprisingly, the high intensity peptide eitvatsr was only identified in simili swabs with high concentration of viral proteic material (figure ). by analyzing the ms and ms/ms spectra from these samples, we confirmed that this peptide coeluted with another intense precursor from the background matrix that was fragmented simultaneously. the low mascot ion score attributed to these spectra hindered the confident identification of this peptide. this coelution effect is most likely due to the use of the short chromatographic gradient, and one way to tackle it would be to use smaller isolation windows for fragmentation. this parameter was tested for the analysis of clinical swabs, but little or no improvement was observed. no ms/ms spectra were validated at fdr % for this peptide in swabs t and t , even with the presence of a ms peak corresponding to this peptide in swab t . this peptide is therefore problematic in this type of matrix and probably not suited for tracking sars-cov- in nasal swab samples with our specific experimental setup. the distribution of the peptides along with the chromatogram from figure shows that the two most detectable peptides gfyaqgsr and adetqalpqr eluted in a narrow window of retention time between − min in simili swab samples. for the clinical swab samples, we observed that the retention time for these two peptides was . ± . min for peptide adetqalpqr, and . ± . min for peptide gfyaqgsr as established with skyline (table s ). in light of these new results, we argue that targeting peptides adetqalpqr and gfyaqgsr with an extra short lc gradient of min coupled to the enrichment of these hydrophilic peptides prior the lc injection could be one way to develop quick and robust assays for detection of the virus in clinical samples and gain in signal/noise ratio. besides their high intensity, these peptides provide the needed specificity for a confident assay: peptide gfyaegsr is highly conserved among different sars-cov- genomes, and peptide adetqalpqr is specific to sars-cov- . the simultaneous detection of these two peptides therefore could provide unequivocal evidence for the presence of the virus. interestingly, a recent not yet published study showed the high potential of the same two peptides by using a targeted proteomics assay. the authors report limits of detection in journal of proteome research pubs.acs.org/jpr article the midattomole range corresponding to theoretically sars-cov- particles in their specific experimental setup. besides shortening the lc gradient to less than three min, sample preparation can also be optimized to develop more rapid peptidome preparation assays and remove too hydrophilic and too hydrophobic peptides that could saturate the chromatography column. here, we performed a sds-page gel and in-gel proteolysis with trypsin to denature proteins and to remove any mass spectrometry-chromatography deleterious compounds that could be present in the nasal swab. this procedure is known to not be optimal as only % of the peptide material deposited on the gel is recovered. the literature is becoming rich in alternative sample preparation protocols for ms-based proteomics. for example, we recently proposed a proteotyping assay based on sp magnetic beads for protein purification and digestion in roughly min. being easily adapted to -well plates and robotization, sp based digestion is the method of choice for quick, highthroughput, and highly reproducible proteome digestions, as recently demonstrated. , pathogen proteotyping by mass spectrometry has emerged in recent years as an interesting alternative to molecular biology assays because of its high specificity and speed. numerous strategies based on tandem mass spectrometry have been already proposed for the detection of pathogens from a large source of samples including environmental and clinical samples. as recently highlighted, faster protocols can now be developed for routine mass spectrometry diagnostics for covid- patients. , however, efforts at automatizing sample preparation and diminishing the costs of tandem mass spectrometry are urgently required to deploy this technology in routine diagnostic laboratory if superior performances or throughput can be achieved. a simplified sample preparation protocol, an associated robust instrument, and low operating costs made the success of whole-cell maldi-tof mass spectrometry. here, the clinical setting of the proposed methodology would require a tandem mass spectrometry instrument coupled to chromatography and an automatized sample preparation. we have recently shown that a sp automated preparation of samples for proteotyping can be performed in a -well plate format within half an hour. the high-throughput of such sample preparation protocol is perfectly adapted to the min tandem mass spectrometry measurement established in the present study. in this proof-of-concept study, we did not evaluate extensively the limit of detection and false positive rate of the methodology. however, we could document on medical samples its feasibility. we have shown that nasal swabs with relatively low viral loads (ct and ct ) can be detected positively with the proposed tandem mass spectrometry method. samples with a trace amount of virus (ct ) were negative. as recently demonstrated, patients with ct above are not contagious and thus can be discharged from hospital care or strict confinement for nonhospitalized patients. therefore, the limit of detection of the method should be further documented once sample preparation, chromatography, and mass spectrometry parameters are further optimized. indeed, sample preparation based on sp capture, as well as the use of functionalized swabs, may further significantly increase the sensitivity of the tandem mass spectrometry proteotyping proposed in the present work. furthermore, more sensitive instrument and ms acquisition modes could be tested to gain further sensitivity. importantly, because test sensitivity is considered as secondary to frequency and turnaround time for covid- surveillance, quick approaches such as the mass spectrometry methodology developed in this proof-ofconcept may have direct application in clinical settings. in this proof-of-concept study, we tested the potential of lc− ms/ms based methods for proteotyping sars-cov- in nasopharyngeal swabs. with a min ms-acquisition window, we were able to identify and quantify several virus-specific peptides that allowed proteotyping the virus in simulated swabs and clinical swabs from covid- patients. we argue that peptides adetqalpqr (and its variant forms) and gfyaqgsr from the nucleocapsid protein are of utmost interest to develop quick and robust targeted assays for proteotyping the virus in nasopharyngeal swab samples. a bigger number of clinical specimens must be tested to validate the usefulness and limits of detection of these peptides to calculate the percentage of confirmation rate of positive pcr results and to develop the shortest possible pipeline to ultimately increase the throughput of the method. given the success of the measures adopted to limit the spread of sars-cov- , a large scale test of our strategy is currently not foreseeable in our country. as a result, while further studies in this context will certainly be of great benefit, results described here offer insight into potential opportunities for the development of new types of clinical diagnostics and significantly facilitate future studies. author contributions ¶ dg and gm contributed equally and should both be considered as first coauthor. ja conceived the study with help from dg, gm, lg, and op. gm and jcg performed the mass spectrometry experimental work. jpl and as contributed the medical covid- swab samples. fg, sd, and lb contributed the sars-cov- biological material. dg, lg, gm, op, and ja analyzed the data. dg and ja wrote the manuscript with help from lg. the authors declare no competing financial interest. the mass spectrometry and proteomics data acquired on simili swabs have been deposited to the proteomexchange consortium via the pride partner repository with the data set identifiers pxd and . /pxd . the authors are indebted to dr silvia meschi (national institute for infectious diseases "lazzaro spallanzani" irccs, via portuense , rome, italia) for making the human -ncov strain -ncov/italy-inmi ( n- ) available. this publication was supported by the european virus archive goes global (evag) project that has received funding from the european union's horizon research and innovation programme under grant agreement no. . the authors are also grateful to the french alternative energies and atomic energy commission (cea) and the anr program "phylopeptidomics" (anr- -ce - - ) that supported part of this study. a pneumonia outbreak associated with a new coronavirus of probable bat origin shortlisting sars-cov- peptides for targeted studies from experimental data-dependent acquisition tandem mass spectrometry data mass spectrometric identifcation of sars-cov- proteins from gargle solution samples of covid- patients development and clinical application of a rapid igm-igg combined antibody test for sars-cov- infection diagnosis rapid detection of covid- causative virus (sars-cov- ) in human nasopharyngeal swab specimens using field-effect transistor-based biosensor detection of sars-cov- in different types 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was developed, using studies. of the studies sampled, were conducted in the united states, in europe, six in canada, four in china, and three in australia. an experimental research design was applied in , with using a pretest-posttest design, a factorial design, two a cluster randomization design, one a posttest design, and one a crossover design. a comprehensive search was performed using pubmed, embase, psycinfo, and the cochrane library from inception through december , . studies were eligible for inclusion if they investigated the effect of a choice architecture intervention on physical activity or sedentary behavior as well as the intention to engage in these; studied an adult population older than years; and contained an experimental or quasi-experimental study design. studies were excluded if they were not written in english; the population consisted entirely of individuals with a communicable disease, psychiatric disorder, or cancer; or a combination of choice architecture and other behavioral change techniques was used. high heterogeneity between studies with regard to design, intervention characteristics, type of outcome measured, and outcome measure assessment prevented a meta-analysis. data were extracted narratively by summarizing the characteristics, quality, and findings of the included studies. the synthesis was structured around the choice architecture techniques identified and their effectiveness. the authors report that findings suggest that prompting can effectively encourage stair use in adults. barriers and facilitators to implementation of menu labelling interventions from a food service industry perspective: a mixed methods systematic review. kerins c, mchugh s, mcsharry j, et al. int j behav nutr phys act. ; https://doi.org/ . /s - - - . the investigators synthesized existing research on the perceived barriers and facilitators to implementation of menu labeling interventions from the perspective of the food service industry and mapped these to the consolidated framework for implementation research (cfir) constructs. a systematic review was designed to address this question, using a sample of studies. the sample contained eight studies using quantitative data collection methods, seven using qualitative, and two using mixed methods. two studies used an explicit theory, the diffusion of innovation theory. inclusion criteria included samples representing direct supply-side stakeholders with a role in menu labeling implementation; interventions with no menu labeling format; all primary research studies using qualitative, quantitative, or mixed methods approaches; no restrictions on language or publication year; and outcomes measuring any barrier or facilitator to the implementation of menu labeling. exclusion criteria were samples using demand-side stakeholders and indirect supply-side stakeholders, policy makers, guideline developers, health professionals; and interventions using menu labeling as part of a multicomponent intervention. the investigators searched pubmed, embase, cinahl, pyscinfo, web of science, and scopus electronic databases up through february . data were extracted by two independent reviewers. qualitative analysis was performed by the investigators through the narratives. frequently cited barriers were coded to the cfir constructs "consumer needs & resources," and facilitators were coded to the cfir constructs "relative advantage." the investigators report evidence suggesting that implementation is influenced by multiple interdependent factors, particularly related to the external and internal context of food businesses and features of the menu labeling intervention. effects of resistant starch interventions on circulating inflammatory biomarkers: a systematic review and meta-analysis of randomized controlled trials. vahdat m, hosseini s, mohseni g, et al. nutr j. ; https://doi.org/ . /s - - - . the researchers examined the effect of resistant starch (rs) on several inflammatory biomarkers. a systematic review and meta-analysis was designed to examine this, using a sample of studies. studies included in the sample ranged in size between and , with a total of participants, with six conducted in iran, two in the united states, one in canada, one in china, one in denmark, one in france, and one in brazil. the researchers searched pubmed, scopus, embase, cochrane library, and the world health organization international clinical trial registry platform from through may . combinations of search terms included resistant starch, resistant maltodextrin, resistant dextrin, indigestible dextrin, indigestible starch, high amylose starch, slowly digestible starch, inflammation, c-reactive protein, and tumor necrosis factor. researchers used the jadad scale and the downs and black assessment tools to evaluate quality. funnel plots were used to visually inspect for publication bias along with begg's rank correlation and egger's linear regression tests. all analyses were performed using stata software. the researchers reported evidence suggesting that higher consumption of resistant starch caused a significant reduction in the interleukin and tumor necrosis factor alpha. however, no significant changes were found in the c-reactive protein concentration factors. food groups and the likelihood of nonalcoholic fatty liver disease: a systematic review and meta-analysis. he k, li y, guo x, et al. br j nutr. ; https:// doi.org/ . /s . non-alcoholic fatty liver disease (nafld). a systematic review and meta-analysis was designed, using a sample of studies. the sample contained crosssectional and nine case-controlled studies. seventeen of the studies were conducted in asian nations, five in european nations, and two in the united states. an electronic search was conducted in the medline, embase, and web of science databases without restrictions to time or language, using words and terms surrounding nafld and the targeted food groups: refined grains, whole grains, red meat, fish, vegetables, fruits, dairy products, legumes, eggs, nuts, and soft drinks. inclusion criteria included adult participants; observational studies that investigate food groups in relation to likelihood of nafld; and diagnosis of nafld. excluded were animal studies; adolescents or pregnant women; present hepatitis b surface antigens, antibody against hepatitis c or human immunodeficiency virus; excess consumption of alcohol; and diagnosed malignancy. the authors employed the pico format (population intervention, comparison, outcome) in considering the relationship between the individual food groups and nafld. statistical analysis was performed using stata version . (statacorp, ). the authors report findings that included a positive association between red meat consumption and nafld, as well as with soft drinks. an inverse relationship was found with nut consumption. enteral nutrition protects children undergoing allogeneic hematopoietic stem cell transplantation from blood stream infections. zama d, muratore e, biagi e, et al. nutr j. ; https://doi.org/ . /s - - - . the researchers evaluated the role of enteral nutrition (en) in clinical and nutritional outcomes in pediatric allogeneic hematopoietic stem cell transplantation (allo-hsct) recipients and considered the impact of nutritional support on the main complications. a cross-sectional study was designed to address this question, using a sample of participants. the sample had a mean age of . months and was % male. participants were consecutive patients undergoing allo-hsct for either malignant or nonmalignant disease within a hospital pediatric unit in italy between january and july . the sample was grouped into patients receiving en for more than days (n ¼ ) and those who received it for fewer (n ¼ ). to assess the effect of nutritional support on clinical and nutritional outcomes, data points were recorded from admission to discharge. neutrophil and platelet engraftments, oral mucositis occurrence, and acute graft-vs-host disease were measured by training clinicians. venoocclusive disease, bloodstream infections, and body weight throughout the process were also documented. qualitative variables were compared using fisher's exact test, and means were compared using the t test corrected in unequal variances. variables associated with the incidence of outcomes were studied using univariate and multivariate analyses. the researchers report findings suggesting that en is a feasible and nutritionally adequate method of nutritional support considering the clinical outcomes, although body mass index was no different between the two groups. effectiveness and cost-effectiveness of the daily mile on childhood weight outcomes and wellbeing: a cluster randomized controlled trial. breheny k, passmore s, adab p, et al. int j obes. ; https://doi.org/ . /s - - - . the researchers evaluated the clinical and cost effectiveness of an exercise intervention on obesity prevention in schoolchildren relative to other health and well-being activities within schools. a cluster randomized controlled trial using schools and , children was designed to address this question. the sample was pulled from all state-funded primary and junior high schools located in south birmingham, england, with a minimum of students. children with disabilities preventing them from walking or running minutes were excluded, as were those unable to have height and weight measured at baseline. the sample of students analyzed was . % female, with a mean age of . years. participating schools were randomized to either control or intervention. the intervention contained a total of , participants and assigned the daily mile, which was conducted by way of the daily mile website. participants were taken from the classroom each day and allowed to either run or walk around a predefined route for minutes, estimated to be a distance roughly equal to a mile. participants were empowered to set their own pace. the , control participants received no active intervention. primary outcome measure was body mass index z-score at the -month follow-up, with secondary outcomes being fitness and body fat percentage at the -month, then reported quality of life and teacher-rated academic attainment. statistical analysis was conducted, using stata version (statacorp, ). the researchers reported the daily mile had a small but nonsignificant effect on bmi z, greater in girls than boys. assessing diet in a university student population: a longitudinal food card transaction data approach. the investigators aimed to study the utilization of food purchase transactions from all students living in catered residence halls and examined the differences between demographic characteristics across dietary patterns. a longitudinal study was designed to address this question, using a sample of students who collectively conducted , transactions. the sample was derived from first-year students at the university of leeds in the united kingdom living in oncampus, catered residence halls. students younger than years or older than years were excluded from the study. the sample was . % female, with . % age , . % age , and . % between and . investigators extracted food card data for the first semester of years between september and . food card data were used to derive unique items. to explore demographic differences across dietary patterns, food card records were linked with university-held data on age and sex. dietary patterns were clustered into groups: vegetarian; omnivores; dieters; dish-of-the-day; grab-and-go; carb lovers; and snackers. all data analysis and visualization were carried out using r studio version . . (r studio team, ) and r . . (r foundation, ). the investigators reported that females were significantly more likely to participate in the vegetarian and dieter cluster, with students older than representing a high proportion of omnivores. are obstructive sleep apnea and sleep improved in response to multidisciplinary weight loss interventions in youth with obesity? a systematic review and meta-analysis. roche j, isacco l, masurier j, et al. int j obes. ; https://doi.org/ . /s - - - . the investigators hypothesized that multidisciplinary weight loss interventions will effectively reduce obstructive sleep apnea (osa) indicators such as the apnea-hypopnea index, oxygen desaturation index, and obstructive apnea-hypopnea index, and in improving sleep duration and sleep architecture. a systematic review and meta-analysis was designed to address this hypothesis, using a sample of studies, with all featured in the qualitative analysis and seven in the meta-analysis. the total sample contained participants, youth identified with osa and without osa, and was . % male. all were uncontrolled intervention studies. the investigators used pubmed, central, and embase search engines for studies providing pre-and post-multidisciplinary weight loss interventions results for osa and overall sleep in youth. searches were conducted between february and may , . randomized controlled trials and uncontrolled interventional studies were eligible based on criteria including patients aged between and years; supervised multidisciplinary intervention including both diet and exercise training; no use of continuous positive airway pressure; both pre-and post-intervention results; and at least one of the most common osa parameters. studies published in english or french were accepted. the initial search yielded studies, with two additional records identified through other sources. the pool was narrowed to based on criteria. the cochrane risk of bias tool was used to assess risk of bias. statistical analyses were conducted using stata software. the investigators reported evidence suggesting a decrease in osa prevalence postintervention and significant reductions in apnea-hypopnea index, as well as oxygen desaturation index. comparison of dietary macronutrient patterns of popular named dietary programmes for weight and cardiovascular risk factor reduction in adults: systematic review and network metaanalysis of randomized trials. ge l, sadeghirad b, ball g, et al. bmj. ; https://doi.org/ . /bmj.m . the authors assessed the effectiveness of different dietary programs and macronutrient patterns among adults who are overweight or obese. a systematic review and meta-analysis was performed for this study, using eligible trials with , patients. the sample of participants across the studies had a mean age of . years, a median of mean body mass index (bmi) of . , a median of mean weight of . kg, a median proportion of women at %, and a median intervention duration of weeks. the authors searched medline, embase, cinahl (cumulative index to nursing and allied health literature), amed (allied and complementary medicine database), and the cochrane central register of controlled trials (central) through september . search terms included controlled vocabulary and keyword searches related to randomized controlled trials, diets, weight loss, and cardiovascular risk factors. eligible studies included randomized adults older than age who were overweight (bmi of - ) or obese (bmi over ) to an eligible popular named diet or alternative active or non-active control diet, reported weight loss, changes in lipid profile, blood pressure, or c-reactive protein levels at months' follow-up or longer. reviewers focused on two sets of outcomes: weight loss and related markers of cardiovascular disease risk at the -and -month follow-ups. statistical analysis was performed using stata version . (stata-corp, ). the authors report, among other findings, significant weight and blood pressure reduction among atkins and zone diets. hypothetical lifestyle strategies in middle-aged women and the longterm risk of stroke. jain p, suemoto c, rexrode k, et al. stroke. ; https://doi.org/ . /strokeaha. . . the authors estimated the effects of lifestyle on stroke risk. a longitudinal study was designed to address this issue, using a sample of , participants. the sample was taken from the nurses' health study, a longitudinal study using a food frequency questionnaire, and surveys beginning in with , nurses aged to years, which was followed up every year via questionnaires. the authors excluded participants with missing data. participants were drawn from those participating through june or diagnosis of stroke or death. the sample had a mean age of years at baseline and was % white, % female, and % married. in addition to other medical events, at each questionnaire period, participants were asked whether they had a physician-diagnosed stroke. diet was measured using a validated item food frequency questionnaire. physical activity was measured as hours per week of moderate-to-vigorous activity by way of questionnaire, and self-reported height and weight were used to calculate body mass index at each period. all analyses were conducted using sas . (sas institute, ). the authors report results indicating that sustained lifestyle modifications were estimated to reduce the -year risk of total stroke by % and ischemic stroke by %. encouraging adults to choose healthy now: a hawaii'i convenience store intervention. beckelman t, sinclair- white b, mcgurk m, et al. j nutr educ behav. ; ( ) : - . touch.or can you? dietitians' perceptions of expressive touch in client encounters transforming personalized nutrition practice education pairing feeding content with a nutrition education curriculum: a comparison of online and in-class delivery tutorial: development and implementation of a multidisciplinary preoperative nutrition optimization clinic development and validation of surveys to estimate food additive intake a current gap analysis of education for clinical nutrition managers simulation-based learning experiences in dietetics programs: a systematic review assessment of specifications grading in an undergraduate dietetics course asking young children to "do science" instead of "be scientists" increases science engagement in a randomized field experiment gerontology older adults' physical activity and healthcare costs management/administration how a malnutrition quality improvement initiative furthers malnutrition measurement and care: results from a hospital learning collaborative ):e . nutrigenomics a gene-diet interaction-based score predicts response to dietary fat in the women's health initiative relationship between respiratory muscle strength, handgrip strength, and muscle mass in hospitalized patients oncology safety and feasibility of intermittent fasting during chemotherapy for breast cancer: a review of the literature impact of bariatric surgery on cancer risk reduction the correlation between hand grip strength and nutritional variables in ambulatory cancer patients dietary polyunsaturated fat intake in relation head and neck, esophageal and gastric cancer incidence in the nih-aarp diet and health study chili consumption and risk of gastric cancer: a meta-analysis associations between human milk oligosaccharides and growth in infancy and early childhood dietary fats and atherosclerosis from childhood to adulthood )e . adolescent obesity and dietary decision making e a brain-health perspective lancet child adolesc health evidence-based updates on the first week of exclusive breastfeeding among infants weeks ):e . effects of -year infancy-onset dietary counselling on cardiometabolic risk factors in the special turku coronary risk factor intervention project (strip): -year post-intervention follow-up policy & advocacy regulatory and policy-related aspects of calcium fortification of foods: implications for implementing national strategies of calcium fortification breastfeeding initiation duration, and supplementation among mexicanorigin women in texas diet quality and biomarker profiles related to chronic disease prevention: the multiethnic cohort study cannabis-infused edible products in colorado: food safety and public health implications children's fruit "juice" drinks and fda regulations: opportunities to increase transparency and support public health high-fidelity patient simulation increases saudi dietetics students' self-efficacy in applying the nutrition care process. alkhaldy a, mosli r gender differences in vegetarian identity: how men and women construe meatless dieting goal-setting program improves nutrition and physical activity among supplemental nutrition assistance program eligible adults research a systematic review and meta-analysis of medium-chain triglycerides effects on acute satiety and food intake standard testing fails to identify patients who tolerate baked milk effect of dairy intake with or without energy restriction on body composition of adults: overview of systematic reviews and meta-analyses of ran sports nutrition cherry intake as a dietary strategy in sport and diseases: a review of clinical applicability and mechanisms of action weight management impact of ketosis on appetite regulation: a review emotional eating and obesity in adults: the role of depression, sleep and genes self-weighing and visual feedback facilitates self-directed learning in adults who are overweight and obese translational aspects of body image research for obesity-related quality of life and weight loss maintenance postbariatric surgery wellness/prevention approach and avoidance strategies in health goal pursuits: the moderating role of weight status association between walking pace and stroke incidence: findings from the uk biobank prospective cohort study women's health weight gain during pregnancy and the risk of severe maternal morbidity by prepregnancy bmi exploring breastfeeding knowledge and attitudes and noncaregivers: a narrative review the authors assessed the relationship between food groups and key: cord- -c umbcvn authors: reed, patricia e.; mulangu, sabue; cameron, kenneth n.; ondzie, alain u.; joly, damien; bermejo, magdalena; rouquet, pierre; fabozzi, giulia; bailey, michael; shen, zhimin; keele, brandon f.; hahn, beatrice; karesh, william b.; sullivan, nancy j. title: a new approach for monitoring ebolavirus in wild great apes date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: c umbcvn background: central africa is a “hotspot” for emerging infectious diseases (eids) of global and local importance, and a current outbreak of ebolavirus is affecting multiple countries simultaneously. ebolavirus is suspected to have caused recent declines in resident great apes. while ebolavirus vaccines have been proposed as an intervention to protect apes, their effectiveness would be improved if we could diagnostically confirm ebola virus disease (evd) as the cause of die-offs, establish ebolavirus geographical distribution, identify immunologically naïve populations, and determine whether apes survive virus exposure. methodology/principal findings: here we report the first successful noninvasive detection of antibodies against ebola virus (ebov) from wild ape feces. using this method, we have been able to identify gorillas with antibodies to ebov with an overall prevalence rate reaching % on average, demonstrating that ebov exposure or infection is not uniformly lethal in this species. furthermore, evidence of antibodies was identified in gorillas thought previously to be unexposed to ebov (protected from exposure by rivers as topological barriers of transmission). conclusions/significance: our new approach will contribute to a strategy to protect apes from future ebov infections by early detection of increased incidence of exposure, by identifying immunologically naïve at-risk populations as potential targets for vaccination, and by providing a means to track vaccine efficacy if such intervention is deemed appropriate. finally, since human evd is linked to contact with infected wildlife carcasses, efforts aimed at identifying great ape outbreaks could have a profound impact on public health in local communities, where ebov causes case-fatality rates of up to %. emerging infectious disease (eid) epidemics and pandemics arise without warning, even with global efforts aimed at tracking pathogens early and at the source, a fact most recently evidenced by the swift global spread of influenza h n [ , ] and a current outbreak of ebolavirus affecting multiple west african countries simultaneously [ ] . most major human eids are of zoonotic origin and include viral infections of both global (hiv- , hiv- , h n ) and localized significance (ebolavirus, monkeypox, marburgvirus, nipah virus, severe acute respiratory syndrome [sars]-associated coronavirus) [ ] . systematic monitoring of people and wildlife at hotspots of eid is one strategy for preventing human pathogens of animal origin from reaching a pandemic state [ ] . by detecting animal pathogens before or just as they emerge in humans, it may be possible to mitigate against their worldwide spread [ ] . furthermore, in the case of some diseases such as ebola virus disease (evd), the monitoring of wildlife disease serves as a critical component of early warning systems aimed at preventing the transmission of zoonotic diseases to humans [ , ] . evd has repeatedly passed from infected apes to hunters, leading to multiple epidemics and human deaths ( cases) in gabon and the republic of congo (roc) alone since [ , [ ] [ ] [ ] . more significantly, human epidemics are often preceded by observed animal outbreaks, underlining the human health implications of surveillance and control of epizootics [ , ] . the international union for conservation of nature (iucn) currently lists the western lowland gorilla (g. gorilla gorilla) as critically endangered and cites infectious disease as one of the top two threats to this species [ ] . ebolavirus is lethal in humans and nonhuman primates and has been described as a significant threat to the survival of western lowland gorillas and chimpanzees (pan troglodytes) in central africa [ , , ] . data from ecological surveys in central african ape habitats illustrate declines in ape signs (nests, feces, prints) temporally and spatially linked with confirmed human evd outbreaks [ ] [ ] [ ] . mathematical modeling suggests that, between and , gorilla numbers in gabon dropped by more than %, and it is hypothesized that infectious pathogens, including ebolavirus and bacillus anthracis, may contribute to gorilla mortality in africa [ , , ] . despite the significance to both human and wildlife health, direct evidence of great ape exposure to ebolavirus or other pathogens (either by pathogen or immune response detection) is scant, complicating our ability to monitor epizootics. therefore, to fill this gap, there is a need for prospective epidemiologic studies combining ecological data with laboratory screening. most currently available data regarding primate pathology and immune response comes from experimentally infected laboratory macaques [ , ] . in direct response to the challenges associated with collecting blood or tissue from wildlife, non-invasively collected biological samples such as feces have been used for wildlife disease screening [ , ] . primate feces have been screened for the presence of viral nucleic acids due to shedding of simian immunodeficiency virus (siv), circoviruses, enteroviruses and hepatitis viruses [ ] [ ] [ ] [ ] . for siv, feces have also shown the presence of virus-specific antibodies [ ] . we developed a non-invasive immunological assay to detect ebolavirus antibodies in great ape feces, allowing us more insight into wild ape ebolavirus infections and their surveillance, and leading the way to identifying the best approaches for their protection. in addition, this new assay may prove valuable in the development and employment of prospective epidemiological ebolavirus studies in wild great ape populations. eighty gorilla fecal samples were collected in different habitats in the roc. in zone a, gorilla fecal samples were opportunistically collected while following habituated gorillas roughly and years after ebolavirus infection was confirmed in ape carcasses at that site using a combination of rt-pcr, immunohistochemistry and antigen capture [ , ] . in june , samples were collected in zone b during a reconnaissance walk survey (recces) composed of eight , km linear recces radiating every u from a central point with terminal ends of every other pair connected by km recces. this zone is southwest of the mambili river in the southeastern-most area of odzala-kokoua national park (oknp), and samples were collected two years after two gorilla carcasses found in this area tested positive for ebov using rt-pcr and antigen capture assays [ , ] . for these surveys, two teams operated simultaneously, each averaging . km per day over days, and following pre-determined global positioning system (gps) points. a continuous gps track log was maintained and uploaded to a garmin xl gps (www.garmin.com) with a position recorded every km. three missions occurred in zone b . the first occurred from th august to th september when a km closed loop survey was conducted on the northeast side of the mambili river. this search was for evidence that would indicate that the abovedescribed may epizootic southwest of the mambili river had also affected wildlife on the opposite side of the waterway. ten gorilla fecal samples were collected and a continuous gps track log was maintained and uploaded to a garmin xl unit, with points taken every km. also, in , a large-scale ecological and large mammal survey was conducted throughout oknp under the auspices of the wildlife conservation society and the projet espèces phares of the european union [ ] . from september th to th , , five gorilla fecal samples were collected during these missions by means of reconnaissance walk surveys and of a systematic unbiased line transect design aimed to estimate animal abundance derived from the density of animal sign, multipliers decay rate and production, and the area of the survey zone; both designed with and analyzed by the distance software program [ ] [ ] [ ] . lastly, in june , the original km loop described above was repeated during which gorilla fecal samples were collected. in november-december (zone d) and march-april (zone c), reconnaissance walk surveys, similar to the approach applied in zone b , were conducted in great ape habitats that, by the end of the study period, had no reported disease outbreaks. the purpose of these missions was to estimate ape abundance by recording all ape nests. gps points were taken every km and samples were collected. ebolavirus causes deadly outbreaks in wild great apes, and has been reported as a significant threat to the survival of wild lowland gorillas in central africa. improved knowledge of basic information regarding geographic distribution of ebolavirus in great ape populations, including the identification of immunologically naïve populations and the determination of whether apes survive virus exposure, will be needed in order for protective interventions such as immunization to be effective. however, monitoring ebolavirus infection in wild gorillas by current methods is challenging because of the difficulty in obtaining diagnostic samples from these elusive primates. additionally, there are limitations associated with the available laboratory assays used to document ebolavirus infection. here we report the first successful noninvasive detection of ebov immunity in wild great apes, demonstrating survival in this species. this tool will be useful in a comprehensive strategy aimed at the protection of this endangered species and improved prevention of evd outbreaks in human populations. sample collectors wore disposable latex gloves and surgical masks while collecting feces. approximately g of fresh feces was placed in ml of rnalater (qiagen gmbh, hilden, germany) in a ml plastic screw-top vial (corning incorporated, corning, new york, usa), sealed with parafilm (pechiney, menasha, wi, usa), and placed in zip-closure plastic bags and stored at ambient temperature (, uc or uf). samples collected in zone b were placed in liquid nitrogen vapor in a dry shipper (arctic express dual , thermolyne) at the end of each day and maintained in this state until arrival at the analyzing laboratory. feces were determined to be that of gorillas when recovered under one of the following conditions: post-observation collection (after seeing gorillas) or post-audition collection (after hearing gorillas), in association with gorilla nests or in association with gorilla trails [ , ] . genotype studies have demonstrated that feces collected using these methods are accurately classified as gorilla feces % of the time [ ] . in addition, the presence of long tri-lobed sections, ample fiber, and abundant green leafy material further classified these samples as gorilla dung [ , ] . only feces estimated to be less than hours old using published criteria [ ] were collected. the plasmid encoding ebov np is a p derivative [ ] . to purify the recombinant viral protein, plasmid p np was tagged at the c-terminus by site-directed mutagenesis with the quick-change xl site-directed mutagenesis kit (stratagene, la jolla, ca, usa). p np was provided with the hexa-histidine tag. the tagged plasmids were transfected into human embryonic kidney cells (freestyle -f cells, catalog no. r - ) obtained from invitrogen (carlsbad, ca) and grown in a shaking flask at uc under % co with freestyle expression medium (invitrogen). the ebov his-tagged np was purified by nickel-affinity gel, ni sepharose fast flow (ge healthcare, piscataway, nj), and eluted with mm imidazol. the concentration of purified np protein was measured with quick start bradford protein assay reagent (biorad, hercules, ca, usa) and used in the western blot assay. to screen nonhuman primate fecal samples for ebolavirus antibodies, we adapted an existing enhanced chemiluminescent western blot assay [ ] . feces were vigorously mixed in rnalater (ambion life technologies, grand island, ny, usa), . ml of the mixture diluted in . ml of pbs-tween- ( . %), heated at uc for minutes, centrifuged at g for minutes and dialyzed in pbs x with stir bar at uc for to hours to resuspend fecal immunoglobulins that normally precipitate in rnalater. purified or cell lysate np protein was denatured in sample reducing agent (invitrogen nupage), heated at uc for minutes, separated by - % gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) ( . mg per well) (invitrogen np , carlsbad, california, usa), and followed by transfer to nitrocellulose membrane (invitrogen lc , carlsbad, california, usa) which was blocked with % nonfat milk in pbs-tween ( . %) and bovine albumin ( . %). membranes were then cut into strips and incubated overnight in fecal extract on a rocking plate. specific np-bound antibody was detected with goat-anti-human igg peroxidase conjugate and the blot was visualized using an enhanced chemiluminescence detection system. the films were exposed to the immunoblot strips then scanned using an epson perfection photo scanner. to define a cut-off of positivity we used the image j program (imagej, nih, bethesda, maryland, usa) that allowed us to subtract the background in each strip and to compute the integrated density of the band that is the sum of the values of the pixels in the selection in the blot. specimens which showed no visible specific band in the blots were scored as negative whereas those which showed specific band (reactivity with a protein of approximate molecular mass of kda corresponding to ebov nucleoprotein np) were regarded as positive if their integrated density was in excess of the mean of integrated density plus standard deviations of the negative blots. blots with a weak specific visible band and an integrated density below this cutoff were classified as uncertain. a subset of samples collected in (the year of the last evd outbreak in the region) was also screened for the presence of filovirus rna using a nested rt-pcr. fifty nanograms of total rna isolated from rnalater preserved fecal samples were extracted using the rnaqueous pcr kit (ambion life technologies, grand island, ny, usa) and used in a onestep rt-pcr, followed by a nested pcr step. we used degenerate primer pairs in order to amplify a bp fragment of the l polymerase gene from any filovirus. the one step rt-pcr primers are: -atmgraayttttcyttytcatt- and rytataawartcactracatgcat- ; the nested pcr primers are -ttyccwagyaayatgatggt- and -ggdattrdrwartgcatcca- . to assess the quality of the total rna from the fecal sample, we amplified a housekeeping gene for each sample, the ß-glucuronidase gene (gus, accession number af ) using a nested pcr assay. the gus primers used for the one step rt-pcr were -gcttaccacccagtttgag- and -tgggga-tacctggtttcattg- , whereas the nested primers were -tcagagcgagtatggagc- and -gcactttttggttgtctc- . we generated a bp fragment. positive and negative controls were included to ensure that cdna product could be amplified and that no contamination from cdna or previous pcr products occurred. we compared antibody prevalence between sampling locations using a log-likelihood ratio test (g-test) [ ] . a % confidence interval (ci) was constructed for the prevalence. in order to examine ebolavirus exposure in wild great apes we sought to develop a strategy of detection in samples collected by non-invasive methods that would be sensitive and specific enough to detect multiple ebolavirus species with minimal false positive results. it has been shown previously that an enhanced chemiluminescent western immunoblot assay is able to successfully detect specific antibodies in rnalaterpreserved feces from simian immunodeficiency virus-infected chimpanzees (sivcpz) [ ] . the sensitivity and the specificity of sivcpz antibody detection in fecal samples were estimated to be % and %, respectively. viral sivcpz nucleic acid could be amplified in an immunoblot-positive fecal sample, confirming sivcpz infection [ ] . furthermore, a similar approach was used to diagnose simian foamy virus infection in wild chimpanzees (sfvcpz). the sensitivities of sfvcpz antibody and viral nucleic acid detection in fecal samples from captive chimpanzees were % and % respectively, and assay specificities were % [ ] . these studies show the potential of assessing rnalater-preserved fecal samples to document wild apes' exposure to viruses. given the success of this approach, we developed a fecal western blot assay to detect ebolavirus antibodies. we chose purified ebov np as the antigen for antibody detection since it is one of the most abundant structural proteins produced during infection and a major target of the host immune response. this is supported by previous studies showing that humans who have survived natural ebov infection developed strong antibody responses mostly against np [ ] [ ] [ ] . in addition, the np sequence is well conserved among ebolavirus species (figure s ), making it useful for detection of antibodies against multiple ebolavirus species [ , ] and potentially increasing the breadth of this detection method. we first assessed the ability to detect np antibodies in rnalater-preserved fecal samples from captive cynomolgus macaques. fecal specimens were experimentally spiked with different dilutions of positive serum containing polyclonal immunoglobulin from a monkey that was vaccinated with a genetic vaccine encoding np [ ] . serum from this vaccinated monkey displayed antibody reactivity with np by both elisa and western blot analysis (not shown). extracts from these positive serum-spiked feces were then used to incubate immunoblot strips containing immobilized np. anti-np antibodies were detected by enhanced chemiluminescent western blot immunoassay in fecal samples at seropositive nonhuman primate (nhp) plasma dilutions of up to -fold (figure ) , indicating a high sensitivity of the assay for fecal antibody detection. a similar level of sensitivity was observed for detection of anti-siv and anti-hiv antibodies by western immunoblots using plasma samples from sivsm-infected nhp diluted up to and plasma samples from hiv- infected individual diluted up to [ ] . in contrast, fecal extracts from captive and uninfected nonhuman primates (cynomolgus macaque and western lowland gorilla species) treated in the same way showed no reactivity in the np immunoblot, demonstrating low background for the assay and lack of cross-reactivity with serum antibodies directed against irrelevant proteins. these results demonstrated that np antibodies present in primate fecal samples can be extracted and detected by immunoblotting. to evaluate whether wild apes show evidence of previous ebolavirus exposure, we screened fecal samples from gorillas living in the roc for ebolavirus antibodies. fecal samples were opportunistically collected from great ape habitats using one of two survey methodologies. the first method employed a systematic unbiased line transect design aimed to estimate animal abundance or the density or size of wildlife [ ] [ ] [ ] . the second consisted of reconnaissance walks to provide a general overview of large animal distributions and investigate animal trails where animal dung is likely to be encountered [ ] . fecal samples were collected from two regions within or adjacent to oknp in western roc near the border with gabon ( figure ). the first is an evd diagnostically confirmed outbreak (dco) region where human cases were laboratory confirmed between and [ ] , and ape carcasses collected between and tested positive for ebov [ , , ] . the presence of long-term and functioning wildlife disease surveillance programs and gorilla habituation and research studies in the roc allowed for immediate access to the dco region during and after evd epidemics which facilitated the collection of samples from gorillas with a high likelihood of previous exposure to ebov, and samples were collected at this site within - months of confirmed great ape evd cases being found. the second region is an area with no reported outbreaks at that time (nro). here, there were no reported human cases, observable signs of epidemics, ebov-positive animal samples, or significant losses in ape numbers despite repeated visits up until the end of this study in april . routine and systematic reconnaissance missions for table ) . all human evd outbreaks that had previously occurred in our sampling zone study are thought to be the result of handling infected wild animal carcasses, including gorillas [ ] . samples from carcasses were used to document ebov outbreaks in gorillas by rt-pcr, antigen detection elisa and immunohistochemistry [ ] . to explore whether ebolavirus antibodies could be detected in fecal samples obtained from wild apes we focused initially on the dco region (zones a and b ; figure ) in order to maximize the likelihood of obtaining fecal samples from apes that had been exposed to ebov. among fecal samples collected from the dco region, tested positive for np antibodies by immunoblot ( figure , table ). two ebov antibody positive fecal samples out of ( %, ci: - . %) came from zone a where, in late and early , ebov was laboratory confirmed in great ape carcasses at the lossi sanctuary [ , ] (figure ). of samples collected in zone b in , two samples were uncertain (defined in methods) and three were positive for np antibodies ( . %, ci: - . %). samples were collected from zone b during a mission two years after ebolavirus was detected in ape carcasses at the site [ , ] (figure ). we also tested ape fecal samples obtained from the outbreakfree (nro) region to explore whether np antibody detection can be used as a potential surveillance tool. the nro region contains zones b , c and d. zone b is adjacent to b , yet separated from it by the relatively large mambili river. in the fall of , fifteen ape fecal samples were collected in zone b to determine whether a may epizootic had also affected wildlife on the opposite side of the waterway. two years later, in june , the original km closed loop track was repeated to explore any temporal changes in ape density or np seropositivity. three positive fecal samples out of twenty ( %, ci: - . %) were found in zone b (table ) and one sample was uncertain. twenty-five fecal samples were collected in zone c (march and april ) and zone d (november and december ). the zone c mission followed the discovery of one chimpanzee carcass that later tested negative for ebov (e. leroy, personal communication, april , ) . no antibodies were found in the fecal samples from zones c and d, where no outbreaks had been reported. altogether, eighty fecal samples from wild great apes were analyzed by western blot and eight ( %) were found to be np antibody positive (table ) . three samples (one from zone b and from zone b ) had blots with a weak specific visible band and an integrated density below the cutoff, and were thus classified as uncertain (not shown). the remaining fecal samples showed no detectable np-specific antibodies and were classified as antibody negative. roughly half of the samples were collected in the dco region and . % of these samples were found to be antibody positive, whereas a smaller proportion ( . %) of samples collected in the nro region were positive. the difference between the nro and dco regions is not statistically significant (log likelihood ratio statistic (g) = . , x-squared df = , p-value = . ) ( table ) , but overall the data show that anti-np antibodies are present in fecal samples from wild ape populations even in areas with no prior reports of human or wild great ape outbreaks. these data demonstrate that the screening of wild gorilla feces by western blot for the purpose of monitoring ebolavirus exposure was successful in detecting np antibodies. this study represents the first time that ebolavirus antibodies have been detected in wild great ape fecal samples, and carries important implications for the future management and survival of these primates. this is especially relevant because intervention strategies to protect apes against future evd infections are being actively explored, including vaccination since ebolavirus vaccines have been shown to protect laboratory monkeys from disease [ , ] . there have been no studies or observations involving great apes that have described immune response, clinical signs, precise mortality rates or whether survivorship provides long-term immunity, and little is known regarding the overall ebolavirus serological status of apes in central africa. to date, serum samples from gorillas (n = ) and chimpanzees (n = ) in central africa have been screened for antibodies against ebov [ ] . most of these animals were sampled while living in captive settings (pets, rescue centers, primate centers); four subjects were free-ranging and sampled directly from the wild in oknp but were seronegative. obtaining samples from free-ranging wildlife is needed to improve our understanding of infectious agents circulating in the environment. all other health data related to ebolavirus from free-ranging apes comes from necropsies performed during wildlife die-offs and, in those cases, the vast majority of samples collected are too degraded to have diagnostic value [ ] . as expected, there is also nothing known regarding potential co-infections involved in great ape evd which may modify the host immune response, alter pathogenesis, increase mortality or influence the effectiveness of any future prophylactic plans, such as the administration of a vaccine once available. this is due to the difficulty in acquiring diagnostic samples from wild populations. capture and subsequent blood collection for serological screening is costly, time consuming, and carries some risk to the animals while providing information on only a few individuals. in fact, despite the disappearance of a staggering number of great apes in gabon and the roc and years of sustained and active surveillance in these countries during the course of this study, only carcasses were recovered, with confirmed ebov infection in of those individuals [ ] [ ] [ ] ] . moreover, finding animal carcasses in vast tracts of rain forest is difficult; it requires intensive searching and often results in the acquisition of highly degraded samples, which are not suitable for detection of viral antigens or nucleic acid and are more prone to negative results [ ] . this newly developed approach for non-invasive sampling of great apes has allowed the successful detection of anti-ebov antibodies in fecal samples, yielding a seroprevalence rate of % in gorillas. since genetic identification of individual fecal samples was not performed, we cannot rule out the possibility of resampling, so the prevalence rate is an upper limit for this data set. however, recent genetic analysis of gorilla and chimpanzee samples collected during iron-cross recces (the type of surveillance executed in sites b , c, and d) from - suggest a low resampling rate. of samples, three were identified with genetic identity the same as three previously sampled individuals, yielding a % resampling rate for sites in which the same site was revisited with the shortest interval of seven-months apart; the resampling rate in the currently study could be higher because two sites were sampled one-month apart and two locations, a and b , were not iron-cross recces (personal communication, k.j. lee) in addition to estimating ebolavirus exposure in nhp, this technique of screening feces by western blot is in fact a multipurpose tool. it provides the potential to employ serial fecal collections to detect a temporal change in incidence exposure in a given zone. for instance, we saw a trend toward a decrease in ebolavirus fecal antibodies in zone b /b from % in to % in , which can be tested in the future using formal prospective studies. fecal antibody screening can also be used before and after vaccination to demonstrate vaccine-induced immune responses developed in great ape populations, noting that antibody levels in vaccinated non-human primates are an immune correlate of protection [ ] . finally, this approach will facilitate the identification of immunologically naïve populations for largescale vaccination trials, thereby improving cost-effectiveness by identifying communities that could benefit the most from vaccination efforts. along these lines, it provides us with the first real possibility to investigate patterns of evd emergence in wild apes independent of animal mortality and the role natural barriers, such as rivers, may have in mitigating its spread. this ability to map exposure patterns across central africa may also provide insight into how this virus spreads within and between ape populations, a question that has generated two disparate theories: multiple virus introductions and a single spreading outbreak [ , , , ] . key to pandemic prevention is disease surveillance at the human/wildlife interface, especially considering the fact that the majority of emerging infectious diseases events (over %) are of animal origin and that those caused by wildlife pathogens are increasing [ , ] . the strategy described herein will be valuable in providing zoonotic information of public health concern from regions where resources are poor and help counter the emergence of diseases which have potential to become the next pandemic. monitoring diseases in animals using methods such as those we describe here allows for the identification and surveillance of many pathogens, including those with potential to adapt and spread in humans, like hiv and plasmodium parasites [ , , ] . these findings also illustrate the role in situ conservation organizations can play in disease surveillance programs. adapting these tools for use in other wildlife species may provide information regarding the transmission of ebolavirus and other emerging infectious diseases to human populations. recent concerns surround the role pigs play in the emergence of diseases such reston ebolavirus and h n [ , ] . central africa's forests are home to tens of thousands of wild pigs, including the giant forest hog (hylochoerus meinertzhageni) and the red river hog (potamochoerus porcus), and are characterized as emerging disease hotspots [ ] . although no evidence has emerged supporting speculation of ebolavirus-associated wild pig die-offs in africa, employing this assay in these species may address whether pigs are amplifiers, victims or carriers of the virus [ ] . it is noteworthy that in the case of influenza pigs are considered ''mixing vessels'' for viruses and capable of generating new strains transmissible to humans [ ] . the extensive bush meat trade in africa provides ample opportunity for pathogen transmission from pigs to humans, and underlines the importance of disease surveillance in this species. wildlife managers frequently perform wide scale ecological surveys, simultaneously collecting biological samples and data on the density and distribution of wildlife. with the benefit of the new diagnostic capacity and sampling strategies described herein, different fecal sampling approaches can be integrated into these surveys to provide information that has thus far eluded us concerning the distribution, ecology and epidemiology of ebolavirus. for the first time, both the logistical and diagnostic capacities are available to immunologically screen large popula-tions of wild great apes for previous exposure to ebolavirus and even estimate and monitor prevalence rates. figure s ebolavirus nucleoprotein sequences. sequence alignment of the nucleoprotein np from zaire ebolavirus (ebov, accession no. np_ ), tai forest ebolavirus (tafv, accession no. aci ), reston ebolavirus (restv, accession no. bab ), sudan ebolavirus (sudv, accession no. aad ) and bundibugyo ebolavirus (bdbv, accession no. aci ). the numbering of the amino acids is according to their position in the sequence. ''*'', identical residues; '':'' conserved residues; ''.'', semi-conserved residues. 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recombinant nucleoproteins noninvasive detection of new simian immunodeficiency virus lineages in captive sooty mangabeys: ability to amplify virion rna from fecal samples correlates with viral load in plasma what it will take to monitor forest elephant populations correlates of protective immunity for ebola vaccines: implications for regulatory approval by the animal rule predicting the vulnerability of great apes to disease: the role of superspreaders and their potential vaccination gorilla susceptibility to ebola virus: the cost of sociality global trends in emerging infectious diseases origin of the human malaria parasite plasmodium falciparum in gorillas african origin of the malaria parasite plasmodium vivax scientists puzzle over ebola-reston virus in pigs the pig as a mixing vessel for influenza viruses: human and veterinary implications we thank the congolese authorities for long-term support, especially the key: cord- - w g qk authors: walker, faye m.; hsieh, kuangwen title: advances in directly amplifying nucleic acids from complex samples date: - - journal: biosensors (basel) doi: . /bios sha: doc_id: cord_uid: w g qk advances in nucleic acid amplification technologies have revolutionized diagnostics for systemic, inherited, and infectious diseases. current assays and platforms, however, often require lengthy experimental procedures and multiple instruments to remove contaminants and inhibitors from clinically-relevant, complex samples. this requirement of sample preparation has been a bottleneck for using nucleic acid amplification tests (naats) at the point of care (poc), though advances in “lab-on-chip” platforms that integrate sample preparation and naats have made great strides in this space. alternatively, direct naats—techniques that minimize or even bypass sample preparation—present promising strategies for developing poc diagnostic tools for analyzing real-world samples. in this review, we discuss the current status of direct naats. specifically, we surveyed potential testing systems published from to , and analyzed their performances in terms of robustness, sensitivity, clinical relevance, and suitability for poc diagnostics. we introduce bubble plots to facilitate our analysis, as bubble plots enable effective visualization of the performances of these direct naats. through our review, we hope to initiate an in-depth examination of direct naats and their potential for realizing poc diagnostics, and ultimately transformative technologies that can further enhance healthcare. nucleic acid amplification tests (naats) have become indispensable tools in biology and medicine. for example, for infectious diseases diagnostics, naats are generally faster, more sensitive, and more specific than the current gold standard of culture-based techniques. in fact, a number of dna-and rna-based diagnostics are now recommended by the us food and drug administration (fda) for infectious diseases such as human immunodeficiency virus (hiv) [ , ] . bringing naats to the point of care (poc), and particularly to resource-poor settings, is envisioned to revolutionize healthcare. unfortunately, many naats require access to expensive, specialized equipment and a degree of expertise that is highly unlikely to be found in decentralized laboratories. as an additional challenge, these tests typically require an extraction step to isolate dna or rna from blood, urine or sputum, and a purification step to eliminate contaminants from the sample matrix that can confound the actual detection procedure (figure ). these procedures necessitate expensive instrumentation and can add up to several hours to sample-to-answer results, which further restricts the use of naats within centralized laboratories. direct nucleic acid testing is much more convenient and streamlined than the three-step method with preparatory techniques. in a typical extraction experiment, buffer with lytic agents is added to dilute the sample and homogenized with a mixer. sonication creates pressure waves that burst the cells in mechanical lysis. lysozyme enzymatically destroys cells, and is removed from the reaction with vortexing and centrifugation in a phenol/chloroform phase separation. the dna is precipitated in fresh ethanol and the resulting mixture is washed to remove excess contaminants. excess liquid is removed so that the dna can be resuspended in an appropriate buffer. many groups have attempted to develop portable, integrated, microfluidics-based platforms to increase the functionality of diagnostic sensing and analysis [ ] [ ] [ ] , and some of these have even been commercialized (e.g., biomerieux's nuclisens easyq tests, twistdx's twistamp kits, and enigma diagnostic's minilab). these platforms present breakthrough technologies for rapid, cost-effective, and user-friendly diagnostics. while it remains to be seen whether these systems are simple and error-free enough for developed and developing settings, they demonstrate the feasibility of implementing existing nucleic acid amplification methods for poc use [ ] [ ] [ ] [ ] . an alternative approach to time-consuming and cumbersome sample preparation is performing naats directly from complex samples ( figure ). the advantage of traditional amplification technologies, such as pcr with real-time spectroscopic or mass spectrometry detection, is that the results are highly specific and quantitative. however, these sensing platforms are expensive and require prior extraction of genetic material from the sample. direct naats are advantageous when complicated, costly laboratory apparatuses are not available. they not only reduce the time, labor, and technical constraints of molecular testing, but also bring the additional benefit of standardizing results [ ] . indeed, a growing number of groups are developing such "direct" naats. most notably, the alere i influenza a&b assay became the first fda clinical laboratory improvement amendments (clia)-waived nucleic acid-based test [ ] in january . as the alere i system requires no front-end nucleic acid extraction, and can be used outside of traditional laboratory sites [ ] [ ] [ ] [ ] [ ] , its development and clia-waived status provide strong support for further development of direct assays that can minimize or even bypass sample preparation. thus motivated, we present the current state of direct assays and platforms that achieve nucleic acids detection and analysis from clinically-relevant, complex samples but with either minimal or biosensors , , of even no sample preparation procedures. we surveyed the literature from - and came across a significant number of works that reported naats from bodily samples (e.g., blood-based liquids, oral samples, swabs) without the complex steps generally involved in sample preparation. this meant discarding the works that depended on sophisticated instruments and operations that are labor-, time-, and cost-intensive, such as enzymatic (proteinases), chemical (acids, detergents), or physical (temperature shock, mechanical disruptions) treatments. then, we describe examples whereby data visualization can be used to reveal the connections between the robustness, sensitivity, and efficacy of technologies developed for direct dna-and rna-based tests. it is our hope that in reviewing technologies such as these, and presenting these promising early findings in an information-rich and accessible fashion, we can help to accelerate the development of approaches that make poc nucleic acid testing rapid, accurate, simple, and affordable. in order to find relevant articles with data on naat parameters, we performed literature searches from december to february . we searched google scholar with a combination of search terms. these followed a formula of combining a descriptor (e.g., "point-of-care"), an amplification technology (e.g., "lamp" or "loop-mediated isothermal amplification") and a sample matrix (e.g., "blood"). references of previously published reviews, as well as those included in original studies, were checked for possible candidate articles. articles were initially screened on the title, and secondly on the abstract. any articles that relied on microfluidic platforms or commercialized extraction devices were excluded. publications that required complex pre-processing with enzymatic treatment or chemical purification were not selected. studies were included if they involved direct amplification and detection of genetic material from one of six representative sample types: blood, dried blood spot, serum and plasma, saliva and sputum, swabs, urine, and stool. the full text of appropriate articles was read to extract the necessary information. from each of the published works surveyed, we extracted and recorded data that corresponded to test performance. there are many parameters that cannot be ignored when considering naats: accuracy, specificity, user-friendliness, training requirements, and so on. as such, we provide an extensive examination of nucleic acid template specificity (including single or multiplexed reactions), amplification methodologies (enzymes, operating temperatures, and amplification technology), and user-friendliness (storage considerations, pretreatment requirements, and physical involvement) in supplementary table s . in addition, we have classified assays that can feasibly be completed without extensive training or high-end instrumentation as "direct," whereas those with greater labor or equipment demands (e.g., freezers, high-speed centrifugation, or incubation for multiple hours) are deemed "semi-direct." specifically, the "semi-direct" assays have the following exceptions to a simple laboratory setup: alternating between two or more incubation temperatures (other than room temperature), relying on enzymatic activity, or requiring more than a brief, low-speed (< × g) centrifugation. methods categorized as "semi-direct" face some hurdles to implementation as an on-site service for patient care. what these tests do offer is a way to deliver actionable results that can link diagnosis to treatment. with appropriate conversion from requirements for highly trained staff and sophisticated tools to easy-to-use methods, "semi-direct" procedures will meet the requirements for poc diagnostic devices. we found certain parameters could be distilled into numerical data, yielding particularly useful insights when examining different tests. we have devised three major criteria that are indicative of each platform's robustness, sensitivity, and clinical efficacy: tolerance to the sample of interest-ideally, the assay should be able to detect its target against a high concentration of background contaminants. we note that, although sample dilution sometimes provides a convenient way of permitting amplification, doing so inevitably reduces the limit of detection (lod). most naats analyze only a fraction of the sample volume. sample dilution therefore increases the likelihood of false negative results, especially when the samples already have low target concentrations. . lod-by foregoing sample preparation, one generally sacrifices the opportunity to concentrate bulk samples, reducing the limit of detection and making sensitivity an important consideration. clinical evaluation-recognizing assays that have been validated with clinical samples. finally, we sought to devise a visual strategy that would clearly and quickly communicate the importance of our criteria, compare the wide range of assays, discover trends in the data, and reveal patterns in a single glance. specifically, the essential information of the reviewed publications is presented quantitatively in a single plot. relevant values are standardized and communicated in terms of visual attributes of position, size, shape, and color. we have found it particularly useful to visualize the data as "bubble plots." in a bubble plot, numerical values from three parameters are simultaneously visualized via the two axes and the size of the circular marker. different categories can also be grouped according to the color of the markers. in our case, we can readily display the essential information (e.g., sample tolerance, lod, and instances of clinical testing) of related procedures to discern those that enhance test performance. in our survey, we came across eight dna-and rna-based testing techniques. as expected, pcr (and reverse transcription pcr, or rt-pcr) has been the predominant technique. notably, a number of isothermal amplification techniques have also been used to develop direct naats. herein, we provide brief overviews of these lesser known isothermal amplification techniques. while pcr is the most commonly reported method of amplification, there is an increasing number of isothermal amplification technologies that can be truly used at the poc. the single reaction temperature enables the use of less costly, complicated instruments than for thermal cycling tests. loop-mediated isothermal amplification (lamp) is one such widely researched, developed, and characterized method [ ] . amplification employs a strand-displacing polymerase and two or three pairs of primers: one that is sacrificed to linearize the template, and one or two others that prime the dna synthesis to produce concatenated, cauliflower-like products [ ] . as with pcr, lamp has been modified to target rna as reverse-transcription (rt)-lamp [ ] . lamp has been compared to pcr in other ways as well, including applications with bacterial, viral, fungal, and parasitic assays. not only has the specificity and sensitivity been equivalent to that of pcr, the robustness of lamp to certain preparations of serum, swabs, and blood has shown it to be more tolerant to inhibitors than pcr [ ] . the nucleic acid sequence-based amplification (nasba) method is unique in its ability to amplify single-stranded rna directly [ ] . this is most desirable for targeting rna viruses and for transcriptome analysis [ ] . the continuous, homogeneous, isothermal process relies on rna polymerase, rnase, and reverse transcriptase. first, the reverse transcriptase creates a double stranded rna:dna hybrid from the rna template; next, the original rna is destroyed; a dna duplex is synthesized; then, the polymerase can transcribe rna from the dna. each new rna molecule can repeat the cycle for exponential amplification. nasba has been applied to a wide-ranging set of research problems, including hiv diagnosis during the aids epidemic of the s and automated, real-time, clinical tests in blood with the modern nuclisens (biomerieux, inc., durham, nc, usa) or in urine with the aptima assay (hologic, san diego, ca, usa). nasba is also used outside of the commercial sector with systems to monitor viruses in serum [ ] . the strand displacement amplification (sda) technique is based upon the abilities of a restriction enzyme and a dna polymerase. a primer containing a recognition sequence for the restriction enzyme binds to its complementary, single stranded dna target. after extension by the polymerase, the restriction enzyme nicks the unmodified strand of the double-stranded hemiphosphorothioate recognition site. dna polymerase then extends the end of the nick, displacing the downstream strand. the end result is exponential target amplification from the displaced strands, which serve as targets for new reactions. sda is not complex, but it does suffer from sensitivity issues in the presence of background dna. the best way to overcome off-target amplification, and hence reduce false-positives, is to use simple pretreatment procedures like those that have been developed for detection with the bdprobe-tec (becton dickinson microbiology systems, sparks, md, usa) and in-house systems for urine [ ] . recombinase polymerase amplification (rpa) avoids thermal cycling by using three core proteins that operate optimally between - • c [ ] . the first protein, recombinase, binds to primers that recombine with a duplex target for strand displacement. the second, a single-stranded dna binding protein, attaches to the displaced strand before a strand-displacing polymerase copies the dna from the primer onwards for exponential amplification. one of the requirements for rpa technology is sequence-specific detection. this avoids the problem of primer artifacts that add to background fluorescence with nonspecific, intercalating dyes. with its specific readout and rapidity (< min to results) as two main features, rpa provides an alternative to the time-consuming processes of culturing and bacterial genotyping when testing for pathogens [ ] . strand invasion based amplification (siba) is another amplification process that relies on recombinase activity. in siba, there is a separate recombinase substrate that is inserted between two primer-binding sites. the duplex peripheral to this insertion site is separated, enabling the primers to bind. dna polymerase can then extend the template from the bound primers. this use of an invading substrate, one that is neither consumed nor included in the extension of dna, is advantageous because it abolishes primer artifacts. siba can therefore be used to reliably detect low copy numbers of pathogens-other isothermal methods generate non-specific amplification products in the absence of target dna [ ] . going further, the specificity of siba enables multiplexing for the detection of templates that differ by as little as two bases [ ] . multiple displacement amplification (mda) is a technique that exploits the strand displacement, proofreading, and polymerase activity of the φ bacteriophage dna polymerase [ ] . the highly processive polymerase uses random primers to amplify an entire genome. mda is therefore well-suited for whole genome amplification from crude biological samples, which can be followed by single nucleotide polymorphism (snp) testing and genotyping [ ] . the concept of hybridization chain reaction (hcr) [ ] -an enzyme-free, room-temperature method-relies on a dna trigger to initiate amplification. the initiator interacts with two stable dna hairpins to create nicked double helices. amplification of this initiation event continues until the hairpins are depleted. hcr is a useful assay for detecting short dnas, such as human immunodeficiency virus type (hiv- ) in serum [ ] . time-series plots offer an effective means for showing the growing prevalence of direct naats. specifically, within each sample type, we plotted the number of clinical samples that have been analyzed by direct naats from to ( figure ). here, we also divided the data into two cohorts based on whether samples were subjected to pcr (figure , black) or isothermal amplification techniques ( figure , red). as expected, we saw an overall rise in the number of clinical samples analyzed via naats with minimal or no sample preparation over the analyzed period. across all six sample types, we observed sharp spikes, which indicate studies of high numbers of clinical samples. based on the sample type, swab samples had been most analyzed, while urine and stool samples had been least analyzed. within each sample type, after an initial lag, we saw a notable rise in the use of isothermal amplification techniques. this first became apparent as early as , several years after the advent of lamp in , and twelve years after the introduction of nasba [ ] . pcr-based systems emerged within five years of the technique's inception in [ ] , and pcr largely continues to dominate the realm of nucleic acid testing. the one notable exception is seen in our time-course of blood testing, where isothermal techniques have surpassed pcr and rt-pcr in terms of the number of assays performed on whole blood samples. the concept of hybridization chain reaction (hcr) [ ] -an enzyme-free, room-temperature method-relies on a dna trigger to initiate amplification. the initiator interacts with two stable dna hairpins to create nicked double helices. amplification of this initiation event continues until the hairpins are depleted. hcr is a useful assay for detecting short dnas, such as human immunodeficiency virus type (hiv- ) in serum [ ] . time-series plots offer an effective means for showing the growing prevalence of direct naats. specifically, within each sample type, we plotted the number of clinical samples that have been analyzed by direct naats from to ( figure ). here, we also divided the data into two cohorts based on whether samples were subjected to pcr (figure , black) or isothermal amplification techniques (figure , red). as expected, we saw an overall rise in the number of clinical samples analyzed via naats with minimal or no sample preparation over the analyzed period. across all six sample types, we observed sharp spikes, which indicate studies of high numbers of clinical samples. based on the sample type, swab samples had been most analyzed, while urine and stool samples had been least analyzed. within each sample type, after an initial lag, we saw a notable rise in the use of isothermal amplification techniques. this first became apparent as early as , several years after the advent of lamp in , and twelve years after the introduction of nasba [ ] . pcr-based systems emerged within five years of the technique's inception in [ ] , and pcr largely continues to dominate the realm of nucleic acid testing. the one notable exception is seen in our time-course of blood testing, where isothermal techniques have surpassed pcr and rt-pcr in terms of the number of assays performed on whole blood samples. because blood contains circulating nucleic acids, cells, and over , different proteins, it offers an abundance of biomarkers for disease detection. molecular diagnostics in blood are useful for detecting specific dna or rna sequences from a range of bacterial, toxic, and viral infectious agents. platforms for hepatitis and human immunodeficiency virus [ ] , staphylococcus aureus [ ] , and plasmodium species are just a few of the most-used systems enabling rapid diagnostics in whole blood. blood-based testing generally demands sophisticated detection instruments or extensive preparation to recover inhibitor-free and high-purity dna. not all inhibitory blood components are known [ ] , but heme compounds, anticoagulants, and immunoglobulin g (igg) can all interfere with amplification reactions by inhibiting dna polymerase activity [ ] or chelating necessary cofactors [ , ] . although a wide range of bloodborne viruses, bacteria, and parasites can in principle be detected with nucleic acid testing, extraction-and purification-free means of detecting these pathogens are not currently commercially available. we have visualized the general trends of direct and semi-direct nucleic acid testing in blood as a function of the lods ( figure ). the % (v/v) of blood tolerated in a reaction is plotted against the lod in g of template, with the number of clinical samples encoded as the area of the bubble. we have also assigned colors to indicate the type of amplification technology. it is evident that many studies have achieved high sensitivity in detecting their target in a low concentration of blood. this shows that nucleic acid testing has great potential for blood-based tests in poc situations where collection volumes are small (e.g., finger pricks) and parasite loads may be low. those examples from the literature that were not demonstrated on patient samples are considered separately in the plot, and represented by xs rather than bubble markers. some of these are purported to have very low lods that reach below the fg level (table s )-it remains to be seen whether such tests will perform with the same extreme sensitivity in a clinical context. because blood contains circulating nucleic acids, cells, and over , different proteins, it offers an abundance of biomarkers for disease detection. molecular diagnostics in blood are useful for detecting specific dna or rna sequences from a range of bacterial, toxic, and viral infectious agents. platforms for hepatitis and human immunodeficiency virus [ ] , staphylococcus aureus [ ] , and plasmodium species are just a few of the most-used systems enabling rapid diagnostics in whole blood. blood-based testing generally demands sophisticated detection instruments or extensive preparation to recover inhibitor-free and high-purity dna. not all inhibitory blood components are known [ ] , but heme compounds, anticoagulants, and immunoglobulin g (igg) can all interfere with amplification reactions by inhibiting dna polymerase activity [ ] or chelating necessary cofactors [ , ] . although a wide range of bloodborne viruses, bacteria, and parasites can in principle be detected with nucleic acid testing, extraction-and purification-free means of detecting these pathogens are not currently commercially available. we have visualized the general trends of direct and semi-direct nucleic acid testing in blood as a function of the lods ( figure ). the % (v/v) of blood tolerated in a reaction is plotted against the lod in g of template, with the number of clinical samples encoded as the area of the bubble. we have also assigned colors to indicate the type of amplification technology. it is evident that many studies have achieved high sensitivity in detecting their target in a low concentration of blood. this shows that nucleic acid testing has great potential for blood-based tests in poc situations where collection volumes are small (e.g., finger pricks) and parasite loads may be low. those examples from the literature that were not demonstrated on patient samples are considered separately in the plot, and represented by xs rather than bubble markers. some of these are purported to have very low lods that reach below the fg level (table s )-it remains to be seen whether such tests will perform with the same extreme sensitivity in a clinical context. bubble plot of nucleic acid diagnostics performed in whole blood. percent concentration (v/v) of blood per reaction in a given procedure is displayed as a function of the limit of detection (lod) in g of template. the number of patient samples tested is proportional to the log of the marker area, as shown at top, and the testing methodology is indicated by marker color. cases shown with × instead of bubble markers illustrate that patient testing was not reported. by assessing the pcr-and isothermal-based data, we could obtain some insight into how to optimize these techniques to better tolerate blood as a sample matrix. several of the semi-direct works with pcr have employed over % blood in a reaction after heat-cold shock [ ] . more noteworthy is a truly direct example that relied on the specificity and efficacy of the phusion polymerase (new england biolab, ipswich, ma, usa) to perform pcr in % blood [ ] . pcr typically employs the taq polymerase from thermus aquaticus. chemical additives, whether commercially-available cocktails [ , ] or in-house buffers [ - ], allow the taq family of polymerases to amplify dna from whole blood. pcr can likewise be optimized through the use of more unconventional polymerases [ - ] and physical heating steps [ , [ ] [ ] [ ] [ ] [ ] [ ] to reduce the inhibitory effect of blood components. these referenced works offer expedited methods to obtain amplifiable templates with similar sensitivities to chemical-based extraction kits [ ] . though several authors include the use of a centrifuge in the extraction process, these semi-direct methods of template preparation could likely be completed by relying on careful pipette-based transfer of supernatants rather than high-speed centrifugation [ , ] . most isothermal amplification-based diagnostics in blood make use of lamp [ ] , which offers a highly tolerant means of amplification [ ] . simple treatments with heat [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] or chemicals [ , [ ] [ ] [ ] [ ] [ ] can increase the sensitivity of the lamp or rt-lamp reaction. most impressive are the examples of direct amplification of dna in blood with lamp-based technologies [ , ] and other isothermal amplification methods like mda [ ] . some of these assays employ a post-heating centrifugation step, but since poon et al. have demonstrated that lamp can be performed directly on heat-treated blood without a spin-down process, this step could likely be avoided in semi-direct processes [ ] . even though these successful examples of simple, direct nucleic acid testing methods highlight the promise of dna amplification in whole blood, there is an ongoing need for further improvements. no assay has come close to reaching the capacity of burckhardt et al.'s pcr amplification with taq polymerase in nearly % whole blood, as demonstrated over years ago in [ ] . the associated treatment method is one of the more technically-involved and time-intensive, demanding cycles of heating and cooling. it remains to be seen whether an isothermal amplification method could equal this tolerance. perhaps these techniques will make up for their decreased level of tolerance in their ease of use, as evidenced by suzuki et al. achieving % incorporation of whole blood in lamp with only a five-minute heating [ ] . dried blood spots offer a convenient alternative for screening for genetic disorders, testing for infectious diseases, and profiling drug metabolism in settings with limited laboratory or storage capabilities. such samples are typically prepared by spotting whole blood, either from venous blood or a finger prick, onto filter paper [ ] . sampling time is quick, temperature-controlled storage is unnecessary, and biohazard risks are minimized for health care workers [ ] . the downside of such samples is that the dna in the dried blood must be eluted from the paper-based cellular components before it can be amplifiable. filter paper has been used as medium to test blood for infectious diseases since the s [ ] . from syphilis diagnosis during world war ii [ ] , to infant screening in the s [ ] , to hiv detection and monitoring in the modern day [ , ] , there are important assays with dried blood spots in naats. commercial technologies are even becoming widely available to map, monitor, and survey blood spots from patients infected with malaria or other neglected tropical diseases [ , ] . in a similar manner, the preparation and processing techniques for dried blood samples presented below could open new avenues for disease control and elimination when combined with well-standardized assays for detecting bloodborne pathogens. as shown in figure , all of the tests we surveyed have been validated with actual dried blood spots. in the most-heavily tested example ( clinical samples) by raskin et al., pretreatment with heating-cooling cycles and addition of spermidine to the reaction helped boost the efficiency and yield of the amplification [ ] . blood spot-containing filter paper can typically be directly added into a mixture of reagents, though it necessitates overcoming the high background interference of the filter paper and detecting small amounts of dried blood. biosensors , , x for peer review of as shown in figure , all of the tests we surveyed have been validated with actual dried blood spots. in the most-heavily tested example ( clinical samples) by raskin et al., pretreatment with heating-cooling cycles and addition of spermidine to the reaction helped boost the efficiency and yield of the amplification [ ] . blood spot-containing filter paper can typically be directly added into a mixture of reagents, though it necessitates overcoming the high background interference of the filter paper and detecting small amounts of dried blood. to overcome the background interference from filter paper in directly amplifying dried blood spots via pcr, researchers have bolstered the enzyme's resistance to inhibitors and included various buffer components [ , ] . pretreatments, such as fixing [ , [ ] [ ] [ ] [ ] or heating [ , [ ] [ ] [ ] , also aid in improving sensitivity and specificity. even if the procedures are reported as being too lengthy for poc, there are appropriate ways to scale down the waiting period: for instance, an overnight drying period with methanol while under vacuum [ ] can be streamlined into a five-minute methanol fix [ ] . most of the approaches to amplify dna directly in blood spots use eluants-either from commercial kits [ , ] , in-house buffers [ , , - ], or water [ , ] -to overcome the various difficulties that impede pcr reactions with paper matrices. buffer-based eluants, in addition to being cost-effective, can achieve even higher sensitivities than standard extraction protocols [ ] . similar preparatory approaches are used for lamp, wherein heating in water [ , ] , phosphate buffered saline (pbs) [ ] , or sodium dodecyl sulfate (sds) buffer [ ] enables a fast and easy nucleic acid elution with amplification results that are comparable to the conventional gold standard of microscopy [ , , ] . one of the difficulties in making blood spots suitable for lamp and pcr is the need to resuspend the spots in liquid, then filter out the species of interest. the easiest way to address the former problem is through long elution times; the latter, through centrifugation. this makes taylor et al.'s amplification of plasmodium spp. dna directly from clinical filter paper samples such a remarkable achievement for low-resource settings. the combination of an inhibitor-resistant taq mutant and an enhancer cocktail resulted in a specificity and sensitivity of % for patient to overcome the background interference from filter paper in directly amplifying dried blood spots via pcr, researchers have bolstered the enzyme's resistance to inhibitors and included various buffer components [ , ] . pretreatments, such as fixing [ , [ ] [ ] [ ] [ ] or heating [ , [ ] [ ] [ ] , also aid in improving sensitivity and specificity. even if the procedures are reported as being too lengthy for poc, there are appropriate ways to scale down the waiting period: for instance, an overnight drying period with methanol while under vacuum [ ] can be streamlined into a five-minute methanol fix [ ] . most of the approaches to amplify dna directly in blood spots use eluants-either from commercial kits [ , ] , in-house buffers [ , , - ], or water [ , ] -to overcome the various difficulties that impede pcr reactions with paper matrices. buffer-based eluants, in addition to being cost-effective, can achieve even higher sensitivities than standard extraction protocols [ ] . similar preparatory approaches are used for lamp, wherein heating in water [ , ] , phosphate buffered saline (pbs) [ ] , or sodium dodecyl sulfate (sds) buffer [ ] enables a fast and easy nucleic acid elution with amplification results that are comparable to the conventional gold standard of microscopy [ , , ] . one of the difficulties in making blood spots suitable for lamp and pcr is the need to re-suspend the spots in liquid, then filter out the species of interest. the easiest way to address the former problem is through long elution times; the latter, through centrifugation. this makes taylor et al.'s amplification of plasmodium spp. dna directly from clinical filter paper samples such a remarkable achievement for low-resource settings. the combination of an inhibitor-resistant taq mutant and an enhancer cocktail resulted in a specificity and sensitivity of % for patient samples [ ] . all the approaches have interesting characteristics that make them special, but none achieve the ease in use of this assay for malaria. blood plasma and serum are widely used for quantitative molecular diagnostics in the areas of clinical decision-making and therapeutic management [ ] . plasma is the pale yellowish fluid that normally holds the blood cells of whole blood in suspension, whereas serum is the remnants of blood plasma after the removal of clotting factors [ ] . circulating dna in serum and plasma is a biomarker for a diverse array of systemic, infectious, and genetic diseases. these include particular disorders such as diabetes [ ] and hepatitis b virus [ ] . refining blood into serum or plasma historically requires expensive equipment for centrifugation or sedimentation. recovering dna or rna from blood-based proteins, nutrients, electrolytes, antibodies (particularly igg), antigens, hormones, and exogenous substances becomes even more challenging when considering the low relative levels of cell-free or cell-bound nucleic acids [ ] [ ] [ ] . more recently, however, paper-or card-based devices [ , ] , membrane-based sedimentation [ ] , and microscale devices for cell differentiation and filtration [ ] have made blood separation a single step process at the poc. as such, we include these sample types here. in assessing nucleic acid testing with plasma or serum, we see that most reactions are performed at sample concentrations in the % range ( figure ). however, it is important to note that the sensitivity does not necessarily suffer in much more concentrated samples-in liu et al.'s highly robust two-step amplification process with direct hairpin assembly and hcr-based detection of snp dna sequences in % (v/v) serum, they achieved a very low lod of pg [ ] . these plasma/serum-based tests are especially promising for use in real-world contexts, because their clinical relevance is well-documented- out of the cases examined here included testing with patient samples. samples [ ] . all the approaches have interesting characteristics that make them special, but none achieve the ease in use of this assay for malaria. blood plasma and serum are widely used for quantitative molecular diagnostics in the areas of clinical decision-making and therapeutic management [ ] . plasma is the pale yellowish fluid that normally holds the blood cells of whole blood in suspension, whereas serum is the remnants of blood plasma after the removal of clotting factors [ ] . circulating dna in serum and plasma is a biomarker for a diverse array of systemic, infectious, and genetic diseases. these include particular disorders such as diabetes [ ] and hepatitis b virus [ ] . refining blood into serum or plasma historically requires expensive equipment for centrifugation or sedimentation. recovering dna or rna from blood-based proteins, nutrients, electrolytes, antibodies (particularly igg), antigens, hormones, and exogenous substances becomes even more challenging when considering the low relative levels of cell-free or cell-bound nucleic acids [ ] [ ] [ ] . more recently, however, paper-or card-based devices [ , ] , membrane-based sedimentation [ ] , and microscale devices for cell differentiation and filtration [ ] have made blood separation a single step process at the poc. as such, we include these sample types here. in assessing nucleic acid testing with plasma or serum, we see that most reactions are performed at sample concentrations in the % range ( figure ). however, it is important to note that the sensitivity does not necessarily suffer in much more concentrated samples-in liu et al.'s highly robust two-step amplification process with direct hairpin assembly and hcr-based detection of snp dna sequences in % (v/v) serum, they achieved a very low lod of pg [ ] . these plasma/serum-based tests are especially promising for use in real-world contexts, because their clinical relevance is well-documented- out of the cases examined here included testing with patient samples. researchers have developed convenient pcr assays for both direct and semi-direct testing in blood-based fluids. by using enhanced enzymes, dna [ , , , ] and rna [ , ] targets have been successfully amplified in plasma and serum. additionally, heat-based pretreatment can be used to release nucleic acids prior to carrying out amplification [ , , [ ] [ ] [ ] [ ] . the effect of preheating can be seen in lamp as well. lamp is generally tolerant to the serum or plasma environment [ , ] , but preheating the input sample has been found to have a favorable effect [ ] [ ] [ ] [ ] that produces up to a -fold improvement in sensitivity [ ] . this heating also enabled pardee et al. to detect zika virus rna in serum with high sensitivity using nasba [ ] . hcr performs especially well in serum without any pretreatment [ , , [ ] [ ] [ ] , presumably because the reaction relies on cascaded hybridization events instead of polymerases. because plasma and serum contain very low-abundance analytes, nucleic acid tests need to operate with high sensitivity. fortunately, lamp-based applications are achieving increasingly low limits of detection. nijru et al., for instance, demonstrated that their lod of trypanozoan parasite/l serum in hat diagnosis was -fold more sensitive than pcr testing. such methods could still benefit from user-friendly techniques for large-scale processing. some semi-direct examples presented above include a centrifugation step to collect condensate formed after heating, but could just as easily rely on pipette collection to obviate the need for a high-speed centrifuge. others might benefit from certain stand-alone modules for plasma and serum separation that could be integrated into a poc workflow [ , ] . saliva and sputum are abundant and easy to obtain, and are thus attractive samples for diagnostics. saliva flows into the oral cavities through salivary glands, where blood vessels secrete the same protein and nucleic acid biomarkers as in peripheral blood. in contrast with blood-based samples, saliva sampling does not require trained technicians, presents fewer antigen-associated risks, and can be more easily purified (saliva is % water) [ ] . sputum, a necessary sample for respiratory infections, is mucus from the lower airways. unfortunately, saliva and sputum are very heterogeneous with respect to the distribution of organisms, chemical composition, and the presence of outside contaminants such as toothpaste, cigarette smoke, coffee, or mouthwash. technical extraction kits such as rnaqueous and magmax (life technologies, grand island, ny, usa) are often used to eliminate inhibitors and nucleases from oral samples. the viscosity of sputum requires particularly cumbersome protocols for sample preparation: full processing begins with mucolytic agents such as n-acetyl-l-cysteine (nalc) and dithiothreitol (dtt), disruption of mycobacteria by detergents and proteolytic enzymes, then isolation of target dna by organic solvents or capture reagents [ , ] . the human salivary microbiome has importance as a diagnostic indicator of oral cancer, oral diseases such as periodontitis, and systemic diseases such as pneumonia [ ] . as for sputum, it has become the specimen of choice for detecting tuberculosis [ , ] . naats for mycobacterium tuberculosis have been endorsed by the who (world health organization) and the fda for their high accuracy [ , ] . the systems introduced below extend the practical usage of sputum for poc testing by reducing the requirements for sputum manipulation. most reported nucleic acid testing methods for saliva and sputum show fairly high numbers for patient samples tested, with only two of cases that did not examine clinical specimens ( figure ). the approaches we examined generally employ dilutions of % or less, and achieve low detection limits. this is critical for avoiding false positives, as the target concentrations in sputum and saliva are small. the lowest lod achieved- fg of acinetobacter baumannii bacterial gdna in sputum-required pretreatment with sputazyme (kyokuto, tokyo, japan) and heat before lamp analysis [ ] . in contrast with blood, the high water content of saliva should make it relatively easy to augment the concentration of matrix that can be employed in an amplification reaction. there are several examples of amplification directly on dried sputum collected via filter cards, which is an especially promising direction for direct testing at the poc if samples need to be stored, handled by multiple clinicians, or reevaluated at later dates [ , , ] . amongst the collection of approaches for direct pcr amplification on saliva samples, those that begin with dried saliva swabs fully circumvent dna extraction, purification, and quantification [ ] . hall et al. added punches of saliva stains directly to the reaction mixture in order to perform str analysis. genetic testing in saliva or sputum is often performed directly in liquid samples, and dilution into the reagents is typically sufficient to negate the effect of any inhibitors [ ] [ ] [ ] , although heat [ ] or additives such as polyethylene glycol (peg), hydroxides, or dithiothreitol (dtt) may be added to further process samples [ , , , ] . with the broad range of bacterial species present in the mouth (over inventoried), differing in terms of their contributions to health and disease, pcr is very useful for profiling large numbers of bacteria. some species are recalcitrant to lysing, like streptococcus. so, instead of relying on bead-beating, phenol treatments, or other steps, aas et al. applied proteinase k lysates directly to pcr reagents [ ] . several other groups have followed suit in detecting bacterial taxa in healthy [ , ] and diseased saliva samples [ ] . direct amplification is also possible in the field of lamp-based diagnostics, as evidenced in testing for zika [ ] and malaria [ ] . du et al. went further than simplifying the sample treatment, a ten-minute heating of zaire ebolavirus dna in saliva, by actually providing a lamp-to-glucose transduction that can be read out on a handheld glucometer [ ] . designing direct tests for sputum is inherently difficult because many nucleic acid-based methods process samples analogously to culture-based protocols. in this n-acetyl-l-cysteine (nalc)-naoh method [ ] , the viscous sputum matrix is liquefied through several buffer exchanges and high-speed centrifugation into a more manipulative sample for testing. an additional concern is decontamination. this is necessary for culture, but also useful to protect the operator from biosafety hazards in molecular testing [ ] . several semi-direct examples based on lamp [ ] [ ] [ ] , recombinase polymerase amplification (rpa) [ , ] , or pcr [ ] [ ] [ ] [ ] [ ] have adopted this practice. however, side-by-side comparisons of nucleic acid testing on sputum samples with or without naoh-nalc treatment have indicated that naoh-nalc processing could be removed to enable truly direct protocols for sputum. mitarai et al. reported that the addition of nalc to a sputum specimen prior to extraction had no effect on testing for tb [ ] . furthermore, tarhan et al. showed amongst the collection of approaches for direct pcr amplification on saliva samples, those that begin with dried saliva swabs fully circumvent dna extraction, purification, and quantification [ ] . hall et al. added punches of saliva stains directly to the reaction mixture in order to perform str analysis. genetic testing in saliva or sputum is often performed directly in liquid samples, and dilution into the reagents is typically sufficient to negate the effect of any inhibitors [ ] [ ] [ ] , although heat [ ] or additives such as polyethylene glycol (peg), hydroxides, or dithiothreitol (dtt) may be added to further process samples [ , , , ] . with the broad range of bacterial species present in the mouth (over inventoried), differing in terms of their contributions to health and disease, pcr is very useful for profiling large numbers of bacteria. some species are recalcitrant to lysing, like streptococcus. so, instead of relying on bead-beating, phenol treatments, or other steps, aas et al. applied proteinase k lysates directly to pcr reagents [ ] . several other groups have followed suit in detecting bacterial taxa in healthy [ , ] and diseased saliva samples [ ] . direct amplification is also possible in the field of lamp-based diagnostics, as evidenced in testing for zika [ ] and malaria [ ] . du et al. went further than simplifying the sample treatment, a ten-minute heating of zaire ebolavirus dna in saliva, by actually providing a lamp-to-glucose transduction that can be read out on a handheld glucometer [ ] . designing direct tests for sputum is inherently difficult because many nucleic acid-based methods process samples analogously to culture-based protocols. in this n-acetyl-l-cysteine (nalc)-naoh method [ ] , the viscous sputum matrix is liquefied through several buffer exchanges and high-speed centrifugation into a more manipulative sample for testing. an additional concern is decontamination. this is necessary for culture, but also useful to protect the operator from biosafety hazards in molecular testing [ ] . several semi-direct examples based on lamp [ ] [ ] [ ] , recombinase polymerase amplification (rpa) [ , ] , or pcr [ ] [ ] [ ] [ ] [ ] have adopted this practice. however, side-by-side comparisons of nucleic acid testing on sputum samples with or without naoh-nalc treatment have indicated that naoh-nalc processing could be removed to enable truly direct protocols for sputum. mitarai et al. reported that the addition of nalc to a sputum specimen prior to extraction had no effect on testing for tb [ ] . furthermore, tarhan et al. showed that the sensitivity in tb testing was better for sputum samples that were measured directly, rather than after extraction with naoh-nalc [ ] . in pursuing alternatives to the lengthy naoh-nalc method, sputum has been used in pcr after adding mucolytic agents [ , ] , diluting in buffer [ ] , or bead-beating [ ] to reduce viscosity. a more aggressive pretreatment, relying on chemical, thermal, and mechanical means of disruption, used heating and centrifugation to detect tb in sputum samples with comparable performance to more expensive molecular-based systems [ ] . one particularly noteworthy example of direct testing used lamp to diagnose tuberculosis at three peripheral laboratories [ ] . in the study, lamp had a sensitivity of . %, detecting out of smear-negative, culture-positive sputum samples. the authors canvassed the laboratory personnel after they implemented the heating, washing, and filter-tip capture steps before direct amplification to verify that the assay had significant potential to be adopted for routine use. several early examples of pcr on sputum samples with mycobacterium spp. reported sensitivities in the single-digit copy number range. however, the pretreatment methods were quite divergent. sjobring et al. used long centrifugation and sonication steps, in addition to boiling, to detect down to eight organisms [ ] . sritharan et al. was able to cut down the steps to a min boiling period, with a resulting lod of organism [ ] . since these examples in the s, only one technique with a pre-amplification wash and an rpa reaction has been able to match this performance in detecting a single mycobacterium [ ] . as far as combining specificity and sensitivity without adding technical difficulty, priye et al.'s recent multiplexed rt-lamp detection system for zika, dengue, and chikungunya achieved lods of copies/reaction with no need for lysis or extraction [ ] . these impressive outcomes from isothermal technologies like rpa and lamp illustrate that fancy hardware is not necessary for testing modalities to achieve high specificity directly in human samples. swabs have become a mainstay in testing for viral pathogens. molecular systems that identify respiratory tract infections in nasal swabs [ ] or stis (sexually transmitted infections) in dermal, genital, and conjunctival swabs [ ] are used for rapid, accurate patient diagnosis. dna collection from swabs is attractive because it is simple, minimally invasive, and even enables self-sampling. however, swab-collected specimens are likely to contain polymerase inhibitors such as secreted minerals, electrolytes, hormones, enzymes, immunoglobulins, and cytokines, as well as topical medications [ , [ ] [ ] [ ] [ ] . as a result, many swab tests now on the market remove these inhibitors via extraction methods that are too involved and complex to be suitable for the poc [ ] . all of the swab sample studies we examined employed at least one patient sample, and most achieved high sensitivity at a reasonable level of dilution in the buffer used for dna elution from the solid swab (figure ) . one remarkable study examined patient swabs in direct pcr for str (short tandem repeat) analysis [ ] -unfortunately, the authors did not report the yields of dna obtained or the lowest amounts detected. this is a particularly common problem amongst these references-when the lods are not reported, it is especially difficult to replicate these procedures or compare the manipulations used in sample storage, dna replication, and detection [ , ] . special attention should therefore be paid to reproducibility in future efforts at direct amplification of swab samples. typically, elution either at room temperature [ , , [ ] [ ] [ ] [ ] [ ] or with heating [ , , [ ] [ ] [ ] [ ] [ ] is sufficient to generate pcr-amplifiable template from swabs in solution. these methods of dilution or heating can save over thirty minutes of processing time, as with nihonyanagi et al.'s heating protocol to release mrsa in celleaseii (biocosm inc., hyogo, japan) diluents [ ] . lamp-based testing of swab samples can also be performed at room-temperature [ ] [ ] [ ] [ ] [ ] or while heated [ , [ ] [ ] [ ] [ ] [ ] [ ] , as can mda [ ] . in particular, mahony et al.'s lamp-based test for influenza a and b achieved an analytical sensitivity of one genome equivalent, operating via a novel swab preparation procedure of vortexing and heating [ ] . moving towards instrument-free molecular diagnostics systems, a lateral-flow stranddisplacement amplification (sda) [ ] assay could directly detect mrsa from nasal swabs with a sensitivity of copies/reaction [ ] . lateral flow eliminates the need for expensive detectors. rogdriguez et al. went one step further by combining paper-based extraction and in situ amplification with lateral flow to develop an rt-lamp assay for h n in patient nasopharyngeal specimens. their sensitivity of copies/reaction was well below the mean viral load for h n patients [ ] . pushing to even lower limits of detection, an hda-based assay with a vertical-flow dna strip readout [ ] could directly test clinical genital swabs in transport medium for hsv types and [ ] . the nucleic acid assays had lods of . and . copies/reaction for hsv- and hsv- , respectively, and were able to detect low-viral loads below the sensitivity of culture tests. the most sensitive tests on swabs rely on lamp reactions: for example, fewer than copies/reaction of viral targets were seen after brief heating steps in hank's buffer (whittaker bioproducts, boston, ma, usa) [ ] , m-swab diluent (copan diagnostics inc., murrieta, ca) [ ] , or water [ ] . it is noteworthy is that entire swab samples can be used in amplification reactions, eliminating the loss of starting material that accompanies liquid and hardware transfers. this approach has also been successful in several instances of str testing [ , ] . rodriguez et al. used a similar approach in detecting clinical levels of h n by passing an in-house elution buffer with the sample of interest through a filter paper-based setup, then amplifying on the filter membrane [ ] . future developments could focus on direct reactions with swabs that integrate extraction, figure . swab-based procedures for nucleic acid testing, with data presented as in figure . typically, elution either at room temperature [ , , [ ] [ ] [ ] [ ] [ ] or with heating [ , , [ ] [ ] [ ] [ ] [ ] is sufficient to generate pcr-amplifiable template from swabs in solution. these methods of dilution or heating can save over thirty minutes of processing time, as with nihonyanagi et al.'s heating protocol to release mrsa in celleaseii (biocosm inc., hyogo, japan) diluents [ ] . lamp-based testing of swab samples can also be performed at room-temperature [ ] [ ] [ ] [ ] [ ] or while heated [ , [ ] [ ] [ ] [ ] [ ] [ ] , as can mda [ ] . in particular, mahony et al.'s lamp-based test for influenza a and b achieved an analytical sensitivity of one genome equivalent, operating via a novel swab preparation procedure of vortexing and heating [ ] . moving towards instrument-free molecular diagnostics systems, a lateral-flow strand-displacement amplification (sda) [ ] assay could directly detect mrsa from nasal swabs with a sensitivity of copies/reaction [ ] . lateral flow eliminates the need for expensive detectors. rogdriguez et al. went one step further by combining paper-based extraction and in situ amplification with lateral flow to develop an rt-lamp assay for h n in patient nasopharyngeal specimens. their sensitivity of copies/reaction was well below the mean viral load for h n patients [ ] . pushing to even lower limits of detection, an hda-based assay with a vertical-flow dna strip readout [ ] could directly test clinical genital swabs in transport medium for hsv types and [ ] . the nucleic acid assays had lods of . and . copies/reaction for hsv- and hsv- , respectively, and were able to detect low-viral loads below the sensitivity of culture tests. the most sensitive tests on swabs rely on lamp reactions: for example, fewer than copies/reaction of viral targets were seen after brief heating steps in hank's buffer (whittaker bioproducts, boston, ma, usa) [ ] , m-swab diluent (copan diagnostics inc., murrieta, ca, usa) [ ] , or water [ ] . it is noteworthy is that entire swab samples can be used in amplification reactions, eliminating the loss of starting material that accompanies liquid and hardware transfers. this approach has also been successful in several instances of str testing [ , ] . rodriguez et al. used a similar approach in detecting clinical levels of h n by passing an in-house elution buffer with the sample of interest through a filter paper-based setup, then amplifying on the filter membrane [ ] . future developments could focus on direct reactions with swabs that integrate extraction, amplification, and detection in a single tube for poc usage. this would give low-resource settings alternatives to instrument-dependent assays like the alere i platform for detecting influenza a & b from nasal swabs. diseases of the kidney or genitourinary tract can often be detected from stool or urine. urea destabilizes interactions between primers, template and polymerase, and since urea is typically present in adult urine at concentrations six-fold greater than can be tolerated in pcr reactions, ultracentrifugation, or related procedures are typically used to prepare such samples for nucleic acid testing [ ] [ ] [ ] . molecular tests for the identification of pathogens in stool rely on extraction methods to remove the proteinases, bile salts, polyphenols, and acids that directly inhibit the activity of dna polymerases [ , ] . methods for testing external specimens have been integrated into diagnostic screens for pathogens such as chlamydia trachomatis, neisseria gonorrhoeae, and clostridium difficile with high sensitivity and specificity [ ] [ ] [ ] [ ] [ ] . in surveying direct nucleic acid testing methods (figure ), we noted that the lods are generally lower for urine-based tests than for feces-based. however, there is one example of direct pcr in stool samples, taking advantage of buffer additives and enhanced phire polymerase (new england biolabs, ipswich, ma, usa), which achieved a lod of . pg of bacterial dna. however, there is a clear need for increased testing with patient samples, as nearly half of the referenced works do not include any clinical validation. this issue is especially notable with fecal testing, where patient testing is only reported in one study. diseases of the kidney or genitourinary tract can often be detected from stool or urine. urea destabilizes interactions between primers, template and polymerase, and since urea is typically present in adult urine at concentrations six-fold greater than can be tolerated in pcr reactions, ultracentrifugation, or related procedures are typically used to prepare such samples for nucleic acid testing [ ] [ ] [ ] . molecular tests for the identification of pathogens in stool rely on extraction methods to remove the proteinases, bile salts, polyphenols, and acids that directly inhibit the activity of dna polymerases [ , ] . methods for testing external specimens have been integrated into diagnostic screens for pathogens such as chlamydia trachomatis, neisseria gonorrhoeae, and clostridium difficile with high sensitivity and specificity [ ] [ ] [ ] [ ] [ ] . in surveying direct nucleic acid testing methods (figure ), we noted that the lods are generally lower for urine-based tests than for feces-based. however, there is one example of direct pcr in stool samples, taking advantage of buffer additives and enhanced phire polymerase (new england biolabs, ipswich, ma, usa), which achieved a lod of . pg of bacterial dna. however, there is a clear need for increased testing with patient samples, as nearly half of the referenced works do not include any clinical validation. this issue is especially notable with fecal testing, where patient testing is only reported in one study. though complex extraction methods are recommended prior to pcr in order to remove inhibitory components of urine and feces, changes to the reaction chemistry could effectively relieve the negative effects of the sample matrix. with urine, this alteration takes the form of a hydrogelencased reaction [ ] . the authors developed a pre-assembled, desiccated, gel-based cassette that is rehydrated by the liquid in raw urine samples. the polyacrylamide gel matrix effectively filters though complex extraction methods are recommended prior to pcr in order to remove inhibitory components of urine and feces, changes to the reaction chemistry could effectively relieve the negative effects of the sample matrix. with urine, this alteration takes the form of a hydrogel-encased reaction [ ] . the authors developed a pre-assembled, desiccated, gel-based cassette that is rehydrated biosensors , , of by the liquid in raw urine samples. the polyacrylamide gel matrix effectively filters biological inhibitors out of the amplification reaction, enabling detection of mycoplasma homonis and ureaplasma urealyticum. for feces, the options are to employ inhibitor-resistant polymerases [ ] and buffer additives [ ] . in the case of one large-scale characterization by hall et al., the combination of both phire polymerase (new england biolabs, ipswich, ma, usa) and ampdirect (biomatrica, san diego, ca) gave an lod of nearly one copy/reaction in pcr for francisella tularensis in . % stool [ ] . one can also lyse bacteria in urine [ ] or stool [ , ] through heating to release an amplifiable amount of target dna with minimal levels of inhibitors. moore et al. managed to detect human norovirus repeatedly in out of outbreak stool samples after boiling the diluted feces in pbs [ ] . although centrifugation is employed in some semi-direct methods to create a supernatant from the collected stool, we believe a dilution step could accomplish the same feat by allowing solids to settle at the base of a highly aqueous, non-viscous sample. isothermal amplification techniques like lamp and rpa generally demonstrate a higher tolerance than pcr for urine, as they can be carried out directly. as such, lamp-based assays without any pre-processing steps or chemical enhancements have detected the causative agents of viral infections [ , , ] or stis [ , ] , and pathogenic bacteria such as escherichia coli [ ] . the developers of the recently established isothermal method siba showed the utility of this amplification technique by detecting chlamydia trachomatis and neisseria gonorrhoeae in a low-copy urine sample [ ] . the landscape of molecular diagnostics is constantly advancing, as is the current paradigm of healthcare. the advent of mobile health and telemedicine has decentralized patient care. it has also put a new emphasis on usability and non-invasiveness in disease testing. nucleic acid diagnostics that reduce the difficulties and expenditures of standard multi-step procedures by direct amplification can expedite patient testing in poc, hospital, and laboratory situations [ ] . in this review, we are thus motivated to discuss the current state of the art for direct naats: assays and platforms that require minimal or no sample preparation procedures. we search the literature from to and find published works that we consider direct naats. we first categorize these works based on the type of complex samples. we subsequently employ bubble plots to facilitate the comparison of the amplification method, robustness in complex media, sensitivity to target, and clinical usage. our findings indicate that the majority of direct naats exhibit a tolerance of less than % for their sample of interest, and fewer than patient-based evaluations. still, there are diagnostic procedures that far surpass these averages. sim et al.'s study of direct pcr on buccal swabs [ ] , for instance, is robust and carried out directly on the entire sample for maximal ease. improvements must continue to be made for all sample types in terms of facilitating this level of evaluation with clinical samples. despite significant developments to date, there remain several challenges for realizing direct naats with user-friendliness, consistency, and generalizability. in furthering the development of direct testing, it is important to take a holistic approach and consider the type of sample to be analyzed, the method of sample acquisition, the throughput and volume, and any chemical or mechanical requirements, and the amplification technique. another key point to note is that different matrices will function best in different environments. an improved understanding of the mechanisms behind emerging nucleic acid amplification reactions and mutant polymerases will enable the rationalization of how inhibitory compounds can make or break an amplification system. furthermore, molecular assays have much to learn from diagnostics that are continuously being developed in the commercial pipeline. proprietary technology will always hold knowledge at a cost to the user, but the implementation of new ideas can lead researchers towards better and more successful ways in which to modernize the ever-changing field of disease testing. as we enter the age of electronic, mobile, and personalized medicine, there remains much room for creativity and innovation in the design of naats and poc diagnostics. molecular diagnostics, as the highest-growing segment of all in vitro diagnostic products [ ] , truly have great potential for both developed and low-resource areas. the diagnostic community continues to strive for tests that are reliable against variable electrical resources, water quality, trained staff, or harsh environmental conditions [ , ] . researchers continue to seek approvals such as the fda's clia waivers or the who's assured (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and delivered to end-users) criteria. in this regard, direct naats present a promising approach. through this review, it is our hope to stimulate the discussion on direct naats and their potential as poc diagnostics. ultimately, we seek to help accelerate the development of poc diagnostics that can be clia waived and/or meet the who assured criteria, thereby ushering in the next revolution in healthcare. the following are available online at http://www.mdpi.com/ - / / / /s , table s : detailed properties of amplification assays. point of care diagnostics for sexually transmitted infections: perspectives and advances advances in developing hiv- viral load assays for resource-limited settings commercialization of microfluidic point-of-care diagnostic devices micro total analysis systems for cell biology and biochemical assays microfluidic dna amplification-a review nucleic acid isothermal amplification technologies: a review point-of-care nucleic acid testing for infectious diseases isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings fully automated quantification of cytomegalovirus (cmv) in whole blood with the new sensitive abbott realtime cmv assay in the era of the cmv international standard fda grants first clia waiver for nucleic acid-based flu diagnostic test; food and drug administration focus diagnostics receives fda clearance for moderate complexity simplexa hsv & direct molecular test for aiding the diagnosis of encephalitis performance of the molecular alere i influenza a&b test compared to that of the xpert flu a/b assay detection of influenza a and b with the alere tm i influenza a&b: a novel isothermal nucleic acid amplification assay evaluation of the alere i influenza a&b nucleic acid amplification test by use of respiratory specimens collected in viral transport medium evaluation of alere i influenza a&b for rapid detection of influenza viruses a and b loop-mediated isothermal amplification of dna accelerated reaction by loop-mediated isothermal amplification using loop primers development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus nucleic acid sequence-based amplification low-cost detection of zika virus using programmable biomolecular components reliability of nucleic acid amplification methods for detection of chlamydia trachomatis in urine: results of the first international collaborative quality control study among laboratories reliability of nucleic acid amplification methods for detectio dna detection using recombination proteins invasion based amplification (siba): a novel isothermal dna amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte multiplex strand invasion based amplification (msiba) assay for detection of chlamydia trachomatis and neisseria gonorrhoeae comprehensive human genome amplification using multiple displacement amplification triggered amplification by hybridization chain reaction highly sensitive electrogenerated chemiluminescence biosensor based on hybridization chain reaction and amplification of gold nanoparticles for dna detection specific enzymatic amplification of dna in vitro: the polymerase chain reaction. cold spring harb standardization of nucleic acid tests for clinical measurements of bacteria and viruses highly sensitive detection of staphylococcus aureus directly from patient blood a potent inhibitor of taq polymerase copurifies with human genomic dna identification of the heme compound copurified with deoxyribonucleic acid (dna) from bloodstains, a major inhibitor of polymerase chain reaction (pcr) amplification capacity of nine thermostable dna polymerases to mediate dna amplification in the presence of pcr-inhibiting samples pcr based diagnosis in the presence of % (v/v) blood a reliable and rapid method for molecular detection of malarial parasites using microwave irradiation and loop mediated isothermal amplification detection of toxoplasma gondii dna by pcr following microwave treatment of serum and whole blood comparison between toxoplasma gondii dna and specific immunoglobulins during pregnancy. la rev whole-blood polymerase chain reaction and restriction fragment length polymorphism: a simplified method by microwave irradiation improved method for direct pcr amplification from whole blood direct pcr from whole blood, without dna extraction rapid point-of-care isothermal amplification assay for the detection of malaria without nucleic acid purification rapid and sensitive detection of orientia tsutsugamushi by loop-isothermal dna amplification african trypanosomiasis: sensitive and rapid detection of the sub-genus trypanozoon by loop-mediated isothermal amplification (lamp) of parasite dna sensitive and inexpensive molecular test for falciparum malaria: detecting plasmodium falciparum dna directly from heat-treated blood by loop-mediated isothermal amplification a simple, high-throughput, colourimetric, field applicable loop-mediated isothermal amplification (htlamp) assay for malaria elimination highly sensitive detection of malaria parasitemia in a malaria-endemic setting: performance of a new loop-mediated isothermal amplification kit in a remote clinic in uganda clinical evaluation of a loop-mediated amplification kit for diagnosis of imported malaria short report: evaluation of loop-mediated isothermal amplification (lamp) for malaria diagnosis in a field setting rapid detection of haptoglobin gene deletion in alkaline-denatured blood by loop-mediated isothermal amplification reaction mitochondrial dna targets increase sensitivity of malaria detection using loop-mediated isothermal amplification detection of plasmodium vivax infection in the republic of korea by loop-mediated isothermal amplification (lamp) heat denaturation increases the sensitivity of the cytomegalovirus loop-mediated isothermal amplification method molecular diagnostic field test for point-of-care detection of ebola virus directly from blood direct blood dry lamp: a rapid, stable, and easy diagnostic tool for human african trypanosomiasis direct loop-mediated isothermal amplification from plasmodium chabaudi infected blood samples: inability to discriminate genomic and cdna sequences sequence-specific detection method for reverse transcription, loop-mediated isothermal amplification of hiv- a smartphone-based diagnostic platform for rapid detection of zika, chikungunya, and dengue viruses quenching of unincorporated amplification signal reporters in reverse-transcription loop-mediated isothermal amplification enabling bright, single-step, closed-tube, and multiplexed detection of rna viruses unbiased whole-genome amplification directly from clinical samples dried blood spots in hiv monitoring: applications in resource-limited settings early infant human immunodeficiency virus type detection suitable for resource-limited settings with multiple circulating subtypes by use of nested three-monoplex dna pcr and dried blood spots review article: an overview of the clinical use of filter paper in the diagnosis of tropical diseases the diagnosis of syphilis on a dessicated and defibrinated blood drop a simple phenylalanine method for detecting phenylketonuria in large populations of newborn infants dried blood spots for hiv- drug resistance and viral load testing: a review of current knowledge and who efforts for global hiv drug resistance surveillance a diagnostics platform for the integrated mapping, monitoring, and surveillance of neglected tropical diseases: rationale and target product profiles cystic fibrosis genotyping by direct pcr analysis of guthrle blood spots an evaluation of direct pcr amplification enhanced direct amplification of guthrie card dna following selective elution of pcr inhibitors dna analysis of cystic fibrosis in brazil by direct pcr amplification from guthrie cards screening for cystic fibrosis: feasibility of molecular genetic analysis of dried blood specimens polymerase chain reaction amplification from dried blood spots on guthrie cards rapid, efficient method for multiplex amplification from filter paper gene amplification directly from guthrie blood spots molecular genetic diagnosis of sickle cell disease using dried blood specimens on blotters used for newborn screening dna extraction from small blood volumes and the processing of cellulose blood cards for use in polymerase chain reaction validation of a fast and low-cost alkaline lysis method for gdna extraction in a pharmacogenetic context a reliable and effective method of dna isolation from old human blood paper cards comparison of six commercially-available dna polymerases for direct pcr short report: rapid dna extraction from archive blood spots on filter paper for genotyping of plasmodium falciparum polymerase chain reaction amplification of dna from aged blood stains: quantitative evaluation of the "suitability for purpose" of four filter papers as archival media improved procedure for eluting dna from dried blood spots adaptation of a visualized loop-mediated isothermal amplification technique for field detection of plasmodium vivax infection point-of-care technologies for molecular diagnostics using a drop of blood multilaboratory evaluation of real-time pcr tests for hepatitis b virus dna quantification cell-free nucleic acids as biomarkers in cancer patients characterization of circulating dna in healthy human plasma isolation and characterization of dna from the plasma of cancer patients blood separation on microfluidic paper-based analytical devices simple, miniaturized blood plasma extraction method membrane-based, sedimentation-assisted plasma separator for point-of-care applications micro-scale blood plasma separation: from acoustophoresis to egg-beaters enzyme-free and ultrasensitive electrochemical detection of nucleic acids by target catalyzed hairpin assembly followed with hybridization chain reaction comparative study of sensitivity, linearity, and resistance to inhibition of digital and nondigital polymerase chain reaction and loop mediated isothermal amplification assays for quantification of human cytomegalovirus direct serum assay for cell-free bmi- mrna and its potential diagnostic and prognostic value for colorectal cancer direct serum assay for microrna- concentrations in early and advanced breast cancer circulating dna of hotair in serum is a novel biomarker for breast cancer simple and rapid identification of low level hepatitis b virus dna by the nested polymerase chain reaction microwave treatment of serum facilitates detection of hepatitis b virus dna by the polymerase chain reaction. results of a study in anti-hbe positive chronic hepatitis b improved detection of hbv dna by pcr after microwave treatment of serum tolerance of loop-mediated isothermal amplification to a culture medium and biological substances direct detection of human herpesvirus b by the lamp method using newly developed dry-reagents direct detection of human herpesvirus dna in serum by variant specific loop-mediated isothermal amplification in hematopoietic stem cell transplant recipients rapid detection of hiv- by reverse-transcription, loop-mediated isothermal amplification (rt-lamp) development of a loop-mediated isothermal amplification assay for rapid detection of bk virus direct detection of human herpesvirus dna in serum by the loop-mediated isothermal amplification method enzyme-free and label-free ultrasensitive electrochemical detection of dna and adenosine triphosphate by dendritic dna concatamer-based signal amplification g-quadruplex based two-stage isothermal exponential amplification reaction for label-free dna colorimetric detection pyrene-excimer probes based on the hybridization chain reaction for the detection of nucleic acids in complex biological fluids advances in addressing technical challenges of point-of-care diagnostics in resource-limited settings saliva: physiology and diagnostic potential in health and disease inhibition of the polymerase chain reaction by mucolytic agents direct detection of mycobacterium tuberculosis in sputum by polymerase chain reaction and dna hybridization defining the normal bacterial flora of the oral cavity defining the normal bacterial flora of the oral cavity analytical and clinical evaluation of the epistem genedrive assay for detection of mycobacterium tuberculosis exploring alternative biomaterials for diagnosis of pulmonary tuberculosis in hiv-negative patients by use of the genexpert mtb/rif assay determinants of pcr performance (xpert mtb/rif), including bacterial load and inhibition, for tb diagnosis using specimens from different body compartments rapid molecular detection of tuberculosis and rifampin resistance clinical specimen-direct lamp: a useful tool for the surveillance of blaoxa- -positive carbapenem-resistant acinetobacter baumannii flinders technology associates (fta) filter paper-based dna extraction with polymerase chain reaction (pcr) for detection of pneumocystis jirovecii from respiratory specimens of immunocompromised patients operational feasibility of using loop-mediated isothermal amplification for diagnosis of pulmonary tuberculosis in microscopy centers of developing countries successful use of saliva without dna extraction for detection of macrolide-resistant mycoplasma pneumoniae dna in children using lna probe-based real-time pcr rapid typing of strs in the human genome by hybeacon melting ultra-rapid dna analysis using hybeacon probes and direct pcr amplification from saliva comparison of boiling and robotics automation method in dna extraction for metagenomic sequencing of human oral microbes comparison of dna extraction methods in analysis of salivary bacterial communities study of inter-and intra-individual variations in the salivary microbiota microbiological diversity of generalized aggressive periodontitis by s rrna clonal analysis a sweet spot for molecular diagnostics: coupling isothermal amplification and strand exchange circuits to diagnosis of mycobacterial infections by nucleic acid amplification: -month prospective study pilot study of a rapid and minimally instrumented sputum sample preparation method for molecular diagnosis of tuberculosis efficacy of loop mediated isothermal amplification (lamp) assay for the laboratory identification of mycobacterium tuberculosis isolates in a resource limited setting a novel and more sensitive loop-mediated isothermal amplification assay targeting is for detection of mycobacterium tuberculosis complex development of an in-house loop-mediated isothermal amplification (lamp) assay for detection of mycobacterium tuberculosis and evaluation in sputum samples of nepalese patients rapid detection of mycobacterium tuberculosis by recombinase polymerase amplification polymerase chain reaction for diagnosis of m. tuberculosis: comparison of simple boiling and a conventional method for dna extraction detection of mycobacterium tuberculosis in sputum samples by polymerase chain reaction using a simplified procedure detection of mycobacterium tuberculosis in clinical samples by two-step polymerase chain reaction and nonisotopic hybridization methods rapid, simple method for treating clinical specimens containing mycobacterium tuberculosis to remove dna for polymerase chain reaction an extremely rapid and simple dna-release method for detection of m. tuberculosis from clinical specimens evaluation of a simple loop-mediated isothermal amplification test kit for the diagnosis of tuberculosis evaluation of the efficacy of five dna extraction methods for the detection of mycobacterium tuberculosis dna in direct and processed sputum by an in-house pcr method clinical usefulness of multiplex pcr lateral flow in mrsa detection: a novel, rapid genetic testing method development and evaluation of a rapid multiplex-pcr based system for mycobacterium tuberculosis diagnosis using sputum samples polymerase chain reaction for detection of mycobacterium tuberculosis a simple method for diagnosing m. tuberculosis infection in clinical samples using pcr self-collected versus clinician-collected sampling for chlamydia and gonorrhea screening: a systemic review and meta-analysis validation of real-time pcr for laboratory diagnosis of acanthamoeba keratitis effects of topical anaesthetics and fluorescein on the real-time pcr used for the diagnosis of herpesviruses and acanthamoeba keratitis inhibition of pcr by aqueous and vitreous fluids use of the polymerase chain reaction to detect bordetella pertussis in patients with mild or atypical symptoms of infection diagnostic accuracy of a prototype point-of-care test for ocular chlamydia trachomatis under field conditions in the gambia and senegal high-throughput str analysis for dna database using direct pcr multicenter clinical evaluation of the novel alere i influenza a&b isothermal nucleic acid amplification test collection of buccal cell dna using treated cards a miniaturized and integrated gel post platform for multiparameter pcr detection of herpes simplex viruses from raw genital swabs an enclosed in-gel pcr amplification cassette with multi-target, multi-sample detection for platform molecular diagnostics quick detection of herpes viruses from skin vesicles and exudates without nucleic acid extraction using multiplex pcr real-time pcr for detection of herpes simplex virus without nucleic acid extraction detection of chlamydia trachomatis in endocervical specimens by polymerase chain reaction genotyping single nucleotide polymorphisms in human genomic dna with an automated and self-contained pcr cassette direct pcr amplification of the hvsi region in mitochondrial dna from buccal cell swabs evaluation of pcr for diagnosis of bordetella pertussis and bordetella parapertussis infections these include: evaluation of pcr for diagnosis of bordetella pertussis and bordetella parapertussis infections use of polymerase chain amplification reaction for the detection of adenoviruses in ocular swab specimens comparison of polymerase chain reaction and culture techniques for detection of chlamydia trachomatis rapid detection of diagnostic targets using isothermal amplification and hybeacon probes-a homogenous system for sequence-specific detection clinical utility of loop-mediated isothermal amplification assay for the diagnosis of common alpha herpesvirus skin infections rapid diagnosis of herpes simplex virus infection by a loop-mediated isothermal amplification method rapid detection of varicella-zoster virus infection by a loop-mediated isothermal amplification method rapid and sensitive diagnosis of adenoviral keratoconjunctivitis by loop-mediated isothermal amplification (lamp) method. curr four dna extraction methods used in loop-mediated isothermal amplification for rapid adenovirus detection development of a sensitive loop-mediated isothermal amplification assay that provides specimen-to-result diagnosis of respiratory syncytial virus infection in min multiplex loop-mediated isothermal amplification (m-lamp) assay for the detection of influenza a/h , a/h and influenza b can provide a specimen-to-result diagnosis in min with single genome copy sensitivity evaluation of a direct reverse transcription loop-mediated isothermal amplification method without rna extraction for the detection of human enterovirus subgenotype c in nasopharyngeal swab specimens detection of mycobacterium ulcerans by the loop mediated isothermal amplification method rapid detection of epstein-barr virus dna by loop-mediated isothermal amplification method isothermal in vitro amplification of dna by a restriction enzyme/dna polymerase system a rapid, instrument-free, sample-to-result nucleic acid amplification test paper-based rna extraction, in situ isothermal amplification, and lateral flow detection for low-cost, rapid diagnosis of influenza a (h n ) from clinical specimens nucleic acid assay system for tier ii laboratories and moderately complex clinics to detect hiv in low-resource settings a rapid and simple isothermal nucleic acid amplification test for detection of herpes simplex virus types and evaluation of a strand displacement amplification assay (bd probetec-sda) for detection of neisseria gonorrhoeae in urine specimens nuclisens basic kit for detection of chlamydia trachomatis and neisseria gonorrhoeaein genital tract specimens using nucleic acid sequence-based amplification inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus dna a low complexity rapid molecular method for detection of clostridium difficile in stool effects of amplification facilitators on diagnostic pcr in the presence of blood, feces, and meat effects of amplification facilitators on diagnostic pcr in the presence of blood, feces, and meat performance of clostridium difficile toxin enzyme immunoassay and nucleic acid amplification tests stratified by patient disease severity evaluation of clostridium difficile fecal load and limit of detection during a prospective comparison of two molecular tests, the illumigene c. difficile and xpert c. difficile/epi tests poilane, i. comparison of commercial molecular assays for toxigenic clostridium difficile detection in stools: bd geneohm cdiff, xpert c. difficile and illumigene c. difficile detection of toxigenic clostridium difficile: comparison of the cell culture neutralization, xpert c. difficile, xpert c. difficile/epi, and illumigene c. difficile assays evaluation of the gen-probe chlamydia trachomatis transcription-mediated amplification assay with urine specimens from women sensitive and rapid detection of chlamydia trachomatis by recombinase polymerase amplification directly from urine samples mohd-zain, z. pentaplex pcr assay for detection of hemorrhagic bacteria from stool samples removal of pcr inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to pcr development of a recombinase polymerase amplification assay for detection of epidemic human noroviruses a novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods loop-mediated isothermal amplification test for detection of neisseria gonorrhoeae in urine samples and tolerance of the assay to the presence of urea loop-mediated isothermal amplification assay for rapid detection of common strains of escherichia coli molecular diagnostics of infectious diseases the global molecular diagnostics market report #a point-of-care tests for diagnosing infections in the developing world simple amplification-based assay: a nucleic acid-based point-of-care platform for hiv- testing this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors acknowledge the support of h.t. soh in the initial stages of manuscript preparation. the authors declare no conflict of interest. key: cord- - q iecq authors: tian, wei-chang; finehout, erin title: microfluidic applications in biodefense date: - - journal: microfluidics for biological applications doi: . / - - - - _ sha: doc_id: cord_uid: q iecq nan the growing threat of bioterrorism attacks, combined with repeated outbreaks of emerging infectious diseases, underlines the importance of infrastructure improvement for the detection and diagnosis of biowarfare agents and emerging pathogens. the growing threats posed by terrorists and rogue nations⎯as evidenced by iraq's acknowledgement following the first gulf war that it had loaded biological weapons, and multiple biological "incidents" worldwide⎯has raised serious concerns about bioterrorism attacks directed against the united states and other nations. following the october anthrax attacks in the united states, biodefense has become an area of utmost national and international urgency [ ] [ ] . this incident sparked the testing of tens of thousands of samples for the presence of anthrax, straining the laboratory response network (lrn) system. the new york city experience after the anthrax attacks is telling [ ] . the increase in incoming samples went from one every several months to about , in two weeks, requiring a coordinate growth of analytical staff and laboratories by over twenty-fold. the operational expectation had been that any surge would primarily be composed of human clinical samples; instead most of the samples were environmental. a recent study of acute care facilities in mississippi found that the diagnostic capacity of hospitals would be overwhelmed by a weapon of mass destruction (wmd) attack [ ] ; similar conclusions regarding the lack of diagnostic surge capacity in alternate locales were reached in other studies [ ] [ ] . the destructive potential of genetically engineered bioagents is huge. toxic genes can be hidden in innocuous organisms and expressed at high levels. expression timing and genotypic specificity could be controlled to maximize impact and potentially limit spread to a defined racial pool. the purported accomplishments of the soviet bioweapon program [ ] accentu-ate how real the problem is: sophisticated state-sponsored bioweapons programs have already genetically engineered bacteria and viruses to increase their devastating impact on human populations. "capitalizing" on post- advances in biotechnology such as genetic engineering, the soviet union program researched and produced a range of weapons employing smallpox, anthrax, plague, and other dangerous pathogens [ ] . fortunately, terrorists and rogue states have not yet fully incorporated biological weapons into their arsenals, to our knowledge [ ] . the detection of bioengineered organisms presents even greater challenges than detecting conventional pathogens, and may require multi-tiered screening, including high resolution detection of target genes and dna sequencing. similar to the needs for biodefense of engineered organisms, naturally emerging infectious diseases present another major threat to human health, through the natural spread of these organisms, their rapid evolution to human hosts, and the potential for bioterrorism using these agents. the recent outbreaks of severe acute respiratory syndrome (sars) and bovine spongiform encephalopathy (bse), commonly known as mad-cow disease, have raised global concerns on the need for rapid identification of causative agents and infected individuals before the virus spreads beyond control. hantavirus pulmonary syndrome and west nile virus are examples of additional infectious diseases now emerging in the us. rapid molecular diagnostic methods and monitoring platforms that can adaptively be configured for newly emerging infectious diseases or newly engineered bioagents will be essential for combating these diseases and for biodefense applications. past incidents and the dangers of future bioterrorism attacks highlight the critical need for improved field-and laboratory-based systems to detect, identify, and subtype bioagents [ ] [ ] . as will be seen, the state-of-art of biodefense systems today is operational but rudimentary: all us mail is screened at sorting centers. there is a strong demand from the u.s. government for next-generation systems for civilian and military applications. biodefense monitoring equipment has even more stringent requirements: the size of the current equipment, and the acquisition and operating costs place severe constraints on widespread implementation and deployment. new analytical systems are needed that are scalable, more automated, and capable of rapid deployment in response to surging needs or field operations. the equipment needs to be smaller, use less reagents, be simpler to use, more integrated, and automated-all attibutes of microfluidic systems, making microfluidics an ideal platform to fulfill biodefense needs. this review summarizes approaches to detecting and characterizing biological threat agents for both civilian and military biodefense; describes biodefense programs in place and under development; and delineates some of the approaches where microfluidics is presently being applied to monitor, detect, and characterize biothreat agents. some of the primary needs are outlined, and the challenges to design and build fully integrated microfluidic systems are described. the majority of the review surveys microfluidic technologies that might or could be used in future biodefense systems. sections cover microscale sample preparation methods; immunomagnetic separations and immunoassays; proteomics; polymerase chain reaction (pcr), quantitative pcr (qpcr) and other nucleic acid amplification methods; dna microarrays, microelectrophoresis, and finally integrated lab-on-a-chip systems. there are two basic biodefense detection approaches: detect-to-warn and detect-to-treat. detect-to-warn systems aim to identify biothreats rapidly enough to provide sufficient warning to prevent exposure by the threat. detect-to-treat systems aim to identify the causative agent for diagnostic purposes and thereby to direct healthcare workers to the most effective treatment as quickly as possible. for all systems, low false alarm rates (far) and affordable acquisition and operating costs are essential for widespread adoption. civilian biodefense is based upon surveillance to detect biothreat agents, response networks to warn and direct the treatment of the affected population [ ] , and the development of countermeasures [ ] . bioterrorism incidents, releases of a bioagent in a form that can harm individuals or larger populations, can range from mailing of toxin or bacteria, to release of aerosols in high profile events, to attacks on the food supply. bioshield, a countermeasures program [ ] , is a -year, $ . billion u.s. program for the advanced development and purchase of medical countermeasures. acquisition programs have been announced to counter bacillus anthracis (anthrax), variola virus (smallpox), botulinum toxins, and radiological/nuclear agents. the primary programs that have been implemented for civilian biodefense detection include screening of all postal mail at sorting centers, the bio-watch program, selected localized screening in subways and other undisclosed locations, and the lrn. the currently deployed systems principally use full volume or meso-scale fluidics. despite the need for more advanced detect-to-warn biodefense detection for the general public, these systems have largely remained undeveloped in large part due to the complexity of integrating the complete process. the largest monitoring program is biowatch [ - ], a joint effort by the department of homeland security (dhs), the centers for disease control and prevention (cdc), and the environmental protection agency (epa). biowatch is a 'detect-to-treat' program and monitors the air in at least cities (and as many as cities) [ ] for significant release of bioagents. dhs does not report cities monitored by the biowatch program or the assays used. in and , dhs funding for the biowatch was $ . million for the epa and $ . million for the cdc [ ]. the epa is responsible for continual air sampling by aerosol collectors that trap airborne particles onto filters. the filters are collected for analysis every hours. the cdc is responsible for the analysis of the filters at state and local public health laboratories, and developing new protocols in coordination with both the department of energy (doe) national laboratories and the epa. the assays are generally acknowledged to be pcr amplified detection of specific targets using classified primer sequences. the list of target agents is similarly classified but thought to include at least anthrax, smallpox, plague, and tularemia. the biowatch program has now processed over million samples without a false positive [ ]-an impressive accomplishment. however, the daily sampling frequency and the amount of coverage of the biowatch program still leaves the civilian populace vulnerable. dhs has been funding the biological autonomous networked detectors (band) program as a next-generation system to alleviate some of the deficiencies of the biowatch program. the original goals of the band program were continuous air monitoring with sample analysis every three hours. the detection limits were organisms or ng of toxin per , liters of air processed, with a very low far. the instrument was planned to have an acquisition cost of $ , , an annual operating cost of $ , , and run autonomously for days. additional requirements were for dimensions of ft and the ability to detect up to organisms and tox-ins. the band participants have almost uniformly taken a microfluidics approach. their efforts are described in more technical detail in a later section of this chapter. in addition to detecting bioagents in the primary environment, a major effort has gone into upgrading the response of the medical community to detect unannounced attacks [ ] , since the expectation is that in many scenarios the first alert will be in the form of patients presenting at doctors' offices or hospitals [ ] . biodefense systems are also required to monitor food and water sources [ - ], suspect powders, the exposure of first responders, and test for decontamination after treatment of personnel, equipment, and key environments. the u.s. military biodefense programs aim to detect and identify biological warfare agents that an enemy might use to degrade forces, contaminate bases, and spread confusion throughout command and control systems. various defense programs are delivering technologies that are beginning to counter these vulnerabilities. for example, the portal shield program is designed for facilities protection; the joint biological agent identifiation and diagnostic system (jbaids) is designed for both detection and diagnostics of environmental and clinical samples [ ] , and the joint biological point detection system (jbpds) is designed for detect-to-warn capabilities in the field. portal shield is an array-able sensor system developed to provide early warning of biological attacks for high-value, fixed-site assets, such as air bases and port facilities. portal shield is designed to detect and identify threats simultaneously within min. it is programable to survey continuously as well as perform random or directed sampling. portal shield was deployed in the persian gulf region in february during operation desert thunder, and the current instrumentation is about two thirds the size of a typical office desk. it's fully modularized, selfcontained, and can detect eight different agents. as many as sensor units may be arrayed in a given area and are able to communicate with each other, so there is no reliance on just one of them sounding an alarm. using an array system, the false positive rate diminishes towards zero. jbaids is the dod's first common platform for identification and diagnostic confirmation of biological agent exposure or infection. jbaids block i is currently operational as a real-time pcr instrument with fda approved assays. the current jbaids block i system (and jbtds systems) utilizes manual sample preparation (disrupting the cell or spore and extracting the nucleic acid or protein of interest) which results in: ) complex operator procedures that may result in human error, ) increased operational costs, and ) support and logistical requirements that preclude remote operations. the intent of jbaids block iii, next generation diagnostics (ngd), is to establish a new system incorporating the capabilities of block i and block ii capabilities (table . ) and adding immunoassay capabilities and the ability to identify up to agents including toxins in minutes using automated, miniaturized sample preparation integrated with analysis for nucleic acids and proteins, in a hand held or smaller format. jbpds is planned to detect, identify, and warn against the presence of up to biowarfare agents at discrete points within a given field environment. jbpds is being designed with a sampler, trigger detection, and identification technologies that allow it to rapidly and automatically detect and identify biological threat agents. jbpds was committed to initial limited production and procurement during fy , with planned full production in . future jbpds improvements will reduce size, weight, power consumption, and reagent use while increasing the number of agents recognized, sensitivity, and system reliability. a number of the requirements of the above programs may be met through the implementation of microfluidics. for example, reduced consummable use, weight and size are directly tied to the drastic reduction of reaction volume employed in microfluidic systems and the use of miniaturized microfluidic components. increasing the number of recognized agents can also be accomodated without high order reaction multiplexing by using multi-channel microfluidic devices with single-plex reactions carried out in parallel. in theory, with microfabricated microchips adding more channels is simple a 'cut and paste' exercise once the issues of connections and integation are solved. there are considerable issues in integrating all processing and analysis steps in a 'hands-free' device. however, microfluidics requires a 'hands-free' implementation once samples are loaded since typically there is nothing the user can do to intervene. the detection of biothreat agents today can be segmented into laboratory detection and field detection methods, and by the type of sample matrix processed (reviewed in [ , [ ] [ ] [ ] [ ] ). the detection must be sensitive, specific for the test organism(s), and may require substantial up-front sample preparation before the read-out assay can be performed. there are a number of commercial tests available today to detect biothreats using nucleic acid, immunological, and biochemical methods. the appropriate test may be determined by the level of information required (i.e., phenotypic or genotypic), timeframe, and the consequences. in general, nucleic acid tests are more sensitive, but require a higher order of skilled operators and more sophisticated equipment than immunological tests, and cannot detect toxins. confounding the problem is the need to assess samples for the presence of many possible biothreat agents; the detection of genetically engineered organisms potentially designed to evade standard detection methods; and the vast numbers of people crossing international borders ( million international travelers per year [ ]). sample matrix and sample preparation methods are key variables in biodefense detection. targets may be contained in air, food, water, bodily fluids, powders, swabs, swipes, cloth, or filters, among many other possibilities. significant reductions in sample volume are required and sample preparation methods must remove contaminants such as metal ions, heme, humic acids, and other compounds, which inhibit pcr or other assays. to enable input of samples into microfluidic systems, the target analyte needs to be highly concentrated without concentrating inhibitors. sample preparation issues are further detailed in section . . traditional identification of pathogens is often tedious and prolonged, involving batteries of tests that often take days or even longer to confirm. standard clinical laboratory identification of bacillus anthracis serves as an example [ ] . bacillus anthracis testing by a lrn level a laboratory begins with growth of the organism, a gram stain, capsule observation and routine culturing on sheep blood agar. this is followed by observation of colony morphology, motility, sporulation, and hemolysis. the presumptive identification can take up to hours with additional days for confirmation. confirmatory testing at level b labs (state and federal laboratories) consists of phage lysis and immuno-fluorescence assays of a cell wall and capsule. level c laboratories determine antimicrobial susceptibility and apply more advanced technology including pcr, qpcr, and time-resolved fluorescence measurements. finally, at level d, labs (cdc), in-depth molecular characterization is performed using multiple-locus vntr (variable-number tandem repeat) analysis (mlva), s rdna ribotyping and other methods, including sequencing to provide subtyping information for identification [ ] . full characterization can include complete genomic sequencing to identify the exact strain variant for epidemiological and forensic analysis. similarly the armed forces institute of pathology uses dna extraction, dna quantification, qpcr of unique genetic targets, s rrna gene sequencing, amplified fragment length polymorphism polymerase chain reaction (aflp-pcr), and repetitive element polymerase chain reaction dna fingerprinting to characterize strains [ ]. the results are compared to extensive databases that have been assembled [ - ]. for more rapid detection in the field, current detection methods use immunoassays, pcr, and other molecular typing methods to provide information on suspected biothreat samples. direct fluorescence assay with monoclonal antibodies to cell wall and capsule components have also worked well for b. anthracis [ ] . real-time pcr or qpcr methods are frequently performed [ ] and can yield rapid identification in the field, using the lightcycler (roche), genexpert (cepheid), jbaids (idaho technology) or other systems. dna sequencing of s ribosomal sequences, plasmids, or variable regions can yield the highest resolution identification, but is relatively slow and has a low throughput compared to other methods; sequencing has not yet been adapted to field applications. one of the most advanced field detection concepts is qpcr packaged in portable devices. researchers at lawrence livermore national laboratories (llnl) demonstrated real-time detection of pcr products in a miniaturized silicon reactor with thin film heaters and integrated fluorescence detection [ ] . this work was extended at llnl to a -channel advanced nucleic acid analyzer and a portable version was devised [ ] . cepheid developed commercial versions of this device with integrated (multimicroliter) sample processing for qpcr analysis, now incorporated into the northrop grumman biohazard detection system (bds), used by the postal system for monitoring mail facilities. the bds incorporates upstream air sampling with the genexpert (cepheid), which prepares dna from a fluidized aerosol sample and then performs qpcr. the system is fully integrated, automated, and reports data to a central point. the biowatch program grew in part from the biological aerosol sentry and information system (basis) project developed at the llnl and los alamos national laboratory. basis used filters to collect aerosolized particulate samples at large events such as the olympics. filters were analyzed by pcr in separate laboratory facilities using largely manual protocols [ ] . llnl also developed the autonomous pathogen detection system (apds) [ ] as a stand-alone, autonomous aerosol detection device. apds, a podium-sized system, monitors air for all three biological threat agent types (bacteria, viruses, and toxins) by continuously performing aerosol collection, sample preparation, and multiplexed biological tests. the apds first employs fluorescent bead-based immunoassays to detect more than ten agents. if a positive signal is detected, a second tier confirmation using qpcr is enabled. apds systems have been field tested at major transportation centers and at special events [ ] . a number of companies (e.g., tetracore, alexeter technologies) have developed and market lateral flow immunoassays for field detection of a number of relevant biothreat agents. these are strips of porous materials to which sample is added to one end, and during migration to the other end, encounters a region containing antibodies specific to the target along with a chromophore. while most immunoassays are not particularly sensitive, they are the normally used field screening method and are also used in situations where large numbers of negative samples are anticipated. rapid detection, identification, and subtyping analysis of pathogenic organisms and toxins are critical needs for biodefense and for the management of emerging infectious diseases. autonomous systems that can detect and provide initial identification of bioagents are required for field monitoring and to provide any reasonable degree of protection to civilians. laboratories require automated systems that can rapidly genotype microorganisms from human samples, environmental samples, or food and differentiate at the strain level and better direct treatment. all of these newly developed systems must detect nucleic acids and/or toxins in varying amounts, formats, and in many different matrices. they will need to be completely automated or simple to use; incorporate advanced technologies including sample preparation starting from primary samples (aerosols, blood, etc.), molecular detection, automation, microfluidics, and bioinformatics; reduce reagent consumption and space requirements; and provide cost and performance advantages compared to present systems. analytical techniques such as pcr, vntr, mlva, aflp, and single molecule detection are well suited to analysis on microfluidic systems. in addition, the systems should be capable of accommodating new assays as they become available. the burgeoning field of microfluidics can offer remedies that fulfill many of these needs and is thus becoming an ever increasingly desired component of next-generation systems. microfluidics can provide the fundamental platform technology that reduces the footprint, minimizes reagent consumption, and fully automates monitoring and analytical equipment for operation in the field, monitoring of cities, and detection in the clinic. however, microfluidics faces many challenges before it is ready for wide-spread deployment. the first challenge is interfacing microfluidics with the full scale samples input at the front end. 'real world' samples may be measured in milliliter volumes while microfluidics typically manipulates nanoliter volumes: therefore the sample must be concentrated or many orders-of-magnitude of detection sensitivity will be sacrificed at the front end of the system. in addition, for biodefense and many other applications, a potentially dilute target must be detected from what can be very large volumes. the band program specification for civilian protection is organisms in , liters of air. to work in a microfluidic system, the 'real world' input sample must therefore be reduced in volume by orders-of-magnitude while still achieving the necessary signal-to-noise to maintain sensitivity. this means taking s of microliters of liquids or thousands of liters of air and concentrating the input sample into nanoliters before further microfluidic processing and reactions can take place. this can be achieved by chemically, biochemically, or physically concentrating the sample. paramagnetic beads provide one elegant solution to both the 'macro-tomicro' interface and specificity. the beads, typically about several microns, can specifically or non-specifically capture nucleic acids, cells, viruses, or toxins, from large volumes of solution and move samples from the full volume world of milliliters into a hundreds of nanoliters. when a capture chemistry such as immunomagnetic separations or hydridization is performed with the beads, they can extract the desired target from high backgrounds and clutter. paramagnetic beads simplify sample handling in the microfluidic world by minimizing the positioning demands on the fluidic system since magnetics can be used to recapture beads at any location and potentially eliminate diffusional losses. once the sample has been introduced into the submicroliter realm, the next challenge is integrating the workflow steps in a microfluidic device. as we will see, microfluidics has been applied to the individual processes and proof-of-concept publications on almost any conceivable individual step are a proof of the potential of microfluidic approaches. however, while numerous microfluidic components are well developed in academic or research settings, a key challenge for microfluidics is to either ( ) fully integrate all processes to build complete 'sample-to-answer' microfluidic systems, or ( ) seamlessly interface microfluidic components with each other and with 'full scale' components into a complete system. the later requires interfacing components (or modules)-such as upstream samples from aerosol collectors, swabs, and blood-with sample preparation components and downstream analytical devices, such as real-time pcr or mass spectroscopy. ideally best of breed modules could be interfaced once microfluidic interfaces and connections are standardized. in any case, full process integration within a microfluidic system or module must be accomplished. the integration requires the coupling of different reactions, which may have multiple steps of sample purification, reagent addition, mixing, separation, and detection. microvalves and micropumps are invaluable to isolate processes or reactions as individual steps and to move fluids to integrate different steps into a workflow. the fourth challenge is designing manufacturable biodefense microfluidic systems. the system should be lower in costs to build and operate, preferably by an order-of-magnitue. the 'valley of death' from proof of concept to product must be crossed and scalable manufacturing capabilities must be implemented with only low volumes as early adopters provide the initial orders. the commercial challenge of crossing the valley of death without prior governmental commitment is substantial. the focus of the remainder of this review is on the microfluidic technologies that can provide advanced rapid detection and identification of bioagents. we review microfluidic technologies that may be used with an emphasis on critical battlefield needs and civilian biodefense. in each section, the technology is briefly introduced, how microfluidics is being applied to advance the state of the art is reviewed, and how this is being applied to biodefense is described. in the final sections, fully integrated microfluidics systems are assessed and some of the devices under development for biodefense are presented. other chapters in this book review the general state of microfluidics and many of the different formats having components that might be applicable to biodefense. starting from the sample, microfluidics can be applied to lyse organisms, concentrate, pre-separate, and purify components for further processing. state-of-the-art rapid, automated, miniaturized, modular universal sample preparation systems are required to prepare nucleic acids and proteins from biological samples in order to detect and identify high priority bioagents. this section describes some of the technologies that can be drawn upon to create a front-end that interacts with 'real world, full volume' samples. analysis of intracellular nucleic acids or proteins requires that cells be disrupted by physical (sonication, heating, or bead beating) or chemical means. the most challenging biothreat organisms to disrupt are the gram positive bacterial spores, bacillus anthracis and clostridium botulinum. sonication and bead beating are the most common ways to disrupt spores today. microfluidic-based cell disruption using sonication has been reported. belgrader and coworkers from llnl reported in the development of a mini-sonication device that disrupted spores in s; when coupled with a mini-chip pcr instrument, the complete analysis took min [ ] . this work was further extended at cepheid where spores were lysed by a sonication horn (in conjunction with glass beads) through a flexible interface using a pressurized microfluidics cartridge [ ] then the sonicated lysate was pcr amplified after reagent addition in a disposable cartridge [ ] . marentis et al. developed a piezoelectric microfluidic mini-sonicator and determined that it could lyse eukaryotic cells and spores with an efficiency of % lysis of b. subtilis spores in s [ ] . laser induced disruption in a polydimethylsiloxane (pdms) microchip has been reported for b. atrophaeus spores using a laser absorbing matrix at fluencies below mj/cm and without matrix above that level [ ] . small laser diodes and carboxyl-terminated magnetic beads have been shown to disrupt e. coli, gram-positive vegetative bacteria, and hepatitis b virus mixed with human serum in a microchip with real time detection [ ] . following lysis, the cellular material often requires separation into component fractions. beads, gels, and membranes can also be incorporated to perform pre-separations to remove inhibitors or concentrate samples. they can provide high mass transfer rates and be made from polymer, silica, or other substrates for microchip liquid chromatography and electrochromatography applications. agilent has developed a commercial polymer microfluidic chip for hplc separation using ablation of polyimide to form channels [ ] . a microfluidic technique which is increasingly applied is the use of monoliths as a stationary phase (reviewed in [ ] [ ] [ ] ). the monoliths can be made with a variety of surface chemistries, pore sizes, and functionalized coatings. dielectrophoretic separations can also be performed on microdevices [ ] ; in this regard, the nanogen digital array will be discussed in the microarray section. biodefense samples are derived from a wide variety of substrates and matrices. the matrix may contain complex mixtures including inhibitory compounds (e.g., mold, hemes, indigo, humic and fulvic acids, chelating agents, dnases, rnases, and proteases) that interfere with dna amplification, the gold standard for bioagent identification. the dod critical reagents program now provides standardized test kits composed of commonly encountered inhibitory compounds to aid biodefense development and testing. a number of approaches have been taken to purify nucleic acids before analysis at a full volume. early work showed that dilution of the sample can relieve inhibition contained in soil extracts for pcr amplification reactions [ ]. however, dilution may not be an option if target concentrations are low. low-melting-temperature agarose has been used to extract dna from soil samples [ ] . solid phase extraction that adsorb analytes onto columns, beads, and surfaces and spun separation gels in column format are commonly used to purify dna before analysis. multistep purifications such as organic extracts combined with sephadex columns have also been developed. while these methods are effective, they were best suited for research laboratory environments due to their reliance on supplementary equipment, trained personnel, and time-consuming procedures [ ]. most are not amenable to a microfluidic format. microfluidics and microchips are now being applied to miniaturize dna extractions and concentrate nucleic acids. . silicon microfluidic channels have been modified with amino silanes and dna selectively eluted with alkaline rinses [ ] . immobilized beads in microchannels increased the extraction efficiency from serum -fold compared with free beads [ ] . an integrated microfluidic device was developed to pre-treat whole blood samples using a micro-filter, micro-mixer, micro-pillar array, micro-weir and porous matrix [ ] . sol gels in capillary chromatography have been reviewed [ ] , as have monoliths for preconcentration and sample extraction [ ] . the landers group reported silica bead purification of dna with chaotrophic agents and sol gel immobilization [ ] on microchips, and demonstrated purification of b. anthracis dna [ ] . they have demonstrated a silica-based monolithic column in a fused-silica capillary [ ] and on a glass microchip [ ] with extraction of model dna from complex samples with efficiencies of %. for whole blood and other mixtures they combined a c reverse phase column, to remove proteins and other compounds, with a monolithic column to create a dual phase sample preparation microchip [ ] [ ] . the authors of this chapter have been developing a device, beadstorm™, for automated magnetic bead purification technology on microfluidic handling on microchips ( fig. . ) at microchip biotechnologies inc. the plastic sample processing cube, about in³, with a ul processing chamber is integrated with pneumatically actuated microvalves on a glass microchip to direct pressure-driven flows consisting of fluids, beads, and samples among reagent and reaction reservoirs. microvalves replace both conventional valves and tubing between reservoirs providing a leak-free, self-contained fluid transport. the beadstorm module manipulates input liquid and swabs of biological samples by bead purifying the samples, fully preparing them for downstream analysis such as pcr and immunoassays. immunomagnetic separations have also been performed in this format. the beadstorm device has been successfully used to automate dna extraction from buccal swabs for str amplification. liquid blood samples have also been successfully prepared by the beadstorm module with automated preparation of dna from liquid blood sample in less than minutes. immunological techniques are widely used for rapid purification and detection of cells, viruses, and proteins and are the most widely used diagnostic method in clinical medicine. the primary antibody is typically attached to a solid surface such a microtiter plate or a bead. the secondary antibody is added to generate a 'sandwich' assay that can provide a highly specific and rapid readout. the antibodies can be polyclonal, with wide variation of specificity from batch-to-batch, or monoclonal, which can be produced repeatedly with identical avidity to the corresponding antigen. sandwich assays are well developed for use in laboratories, clinics, and field applications, and are the most common type of assays for toxin testing. there are many immunoassays that are being applied to biodefense and microfluidic immunoassays will be increasingly important in the future. immunomagnetic separation (ims) is a powerful technology that allows targets to be captured and concentrated in a single step using a mechanically simplified format that employs paramagnetic beads and a magnetic field (reviewed in [ ] [ ] [ ] ). ims is used to capture, concentrate, and then purify specific target antigens, proteins, toxins, nucleic acids, cells, and spores in a single step. ims works by binding a specific affinity reagent, typically an antibody, to paramagnetic beads, which are only magnetic in the presence of an external magnetic field. the beads can be added to complex samples such as aerosols, liquids, bodily fluids, or food. after binding of the target to the affinity reagent (which itself is bound to the paramagnetic bead) the bead is captured by application of a magnetic field. unbound or loosely bound material is removed by washing purifing the target from other, unwanted materials in the original sample. a similar approach can purify nucleic acids using a complementary nucleic acid strand attached to a bead. because beads are small and bind high levels of target, when the beads are concentrated by magnetic force, they form bead beds measured in the hundreds of nanoliters (or as low as a single bead), thus concentrating the target at the same time it is purified. the purified and concentrated targets can be conveniently transported, denatured, lysed or analyzed on-bead, or eluted off-bead for further sample preparation and analysis. immunomagnetic separations are commonly used as an upstream purification step before qpcr, electrochemiluminescence, and magnetic force discrimination. as mentioned, paramagnetic beads provide an excellent solution to the macroscale-to-microscale interface: beads are an almost ideal vehicle to purify samples at the macroscale from large volumes and concentrate the specific biomolecules or targets to the nanoscale for introduction into microfluidics devices. immunomagnetic separations are used widely for the detection of microorganisms in food and agriculture. typically, immunomagnetic beads coated with the appropriate antibody are added to material that had been homogenized in a stomacher. the pathogenic strain escherichia coli o :h can be detected directly in ground beef with greater sensitivity than traditional plate enumeration methods by using ims before plating [ ] . ims has also been coupled with qpcr [ ] , fluorescence microscopy, and solid-phase laser scanning cytometry [ ] . key parameters affecting detection sensitivity were shown to include the amount of nanoparticles per assay, immunoreaction incubation time, concentration of the target organism, matrix background, and interferents. generally, interference by non-target microorganisms is less than % [ ] . ims can also be combined with pcr to effectively increase the specificity of the overall process by combining the specificity of antibody-antigen recognition and the sensitivity of pcr. immuno-quantitative pcr for s. aureus enterotoxin b (seb) detection was found to be , times more sensitive than enzyme linked immunosorbent assays (elisa), with little cross-reactivity [ ] . immunomagnetic separations have been adapted to microfluidic devices. in one application, electromagnetic gradients were generated using cu micro-coil arrays embedded in a silicon substrate with magnetic pillars composed of nicop alloy with an integrated sensing coil to produce tunable, localized magnetic forces that were able to trap up to % of applied particles [ ] . a miniature, integrated microfluidic device to separate magnetic particles from laminar flows was developed by the whitesides group and demonstrated to be effective in separating live e. coli on magnetic beads [ ] . clinical applications development includes capture of targeted t cells from blood in bead beds contained in microfluidic channels. these studies resulted in - % t cell capture, but only allowed flow rates of ul/min [ ] . rates of hundreds of microliters per minute are required for most applications that can be achieved by increasing flow rates or by upstream deployment of ims or other technologies that specifically capture and concentrate targets. in addition to performing ims purifications, extensive work has been done on developing and integrating complete immunoassays in microfluidic devices. immunoassays include immunochromatographic lateral flow devices, elisa, ims-electrochemiluminescence, time-resolved fluorescence, and magnetic force assays. in addition to widespread clinical applications, immunoassays are routinely used to detect biosecurity threats [ ] [ ] [ ] . future immunoassays will continue to exploit advances in antibody production and screening, miniaturization, integration, and multiplexing [ ] . lateral flow assays are very simple and compatible with portable applications. a drop of test solution in buffer is added to a pad containing antibodies coupled to colloidal gold or other labels. the antibody and antigen, if present, bind and wick down the pad laterally and intercept detection lines that contain capture antibody-gold complexes. the gold aggregates due to the bivalency of the antibodies and produces a line that is visible by eye. home pregnancy tests are probably the best-known lateral flow devices. lateral flow devices are simple to use, require little training or equipment, and have been devised to detect biothreat agents. simple dipstick immunoassays for e. coli o :h can detect cell per g in ground beef, after outgrowth of the sample [ ] . a comparison of lateral flow assays in a handheld device with elisas and pcr found the sensitivity of the lateral flow assays were approximately one-hundredth of elisas, which were in turn one-tenth the sensitivity of pcr assays [ ] . lateral flow devices are being improved with microfluidic technologies. monolithic beds have been combined with electrophoretic separations to produce a fast (< min), sensitive assay for saliva analysis [ ] . an elisa detected staphylococcal enterotoxin b (seb) in a handheld assay for food at about pg/g of matrix using lateral flow [ ] . an integrated microfluidic device with sample preparation (filtration and mixing) has been described to detect botulinum neurotoxin directly from whole blood [ ] . elisa is the most commonly used form of immunoassay. elisas use an antibody bound to a solid phase support, such as a microtiter plate, to capture analytes from liquids. after washing away unbound material, a secondary antibody with a label or coupled to an enzyme (e.g., horseradish peroxidase) is used to produce a visual or fluorescent readout. elisas are relatively inexpensive, scalable to -and -well technologies, and can be sensitive and specific, depending on the antibody pairs. early work with polyclonal antibodies demonstrated detection of the enterohemorrhagic e. coli o :h in food at about cell/g sensitivity [ ] . anti-s. aureus enterotoxin b (seb) antibody has been immobilized on carboxylated polystyrene microparticles and a competitive assay between fitc-labeled seb was developed to detect . ng/ml of seb in drinking water and . ng/ml in whole milk [ ] . seb has also been assayed by direct labeling of secondary antibody with detection limits of pg/well [ ] [ ] . immunoassays are being adapted to microfluidic devices. microfluidic molded silicone integrated devices have successfully detected botulinum neurotoxin serotype a with results equivalent to full volume assays [ ] . microfluidic immunoassays in plastic devices offer the affordability of plastics, the availability of diverse microfabrication methods, and many well-developed polymer surface modifications [ ] . a heterogeneous immunoassay with antigens immobilized on pdms-coated glass microchips with electrokinetic-control for multiple analyte detection had detection limits for e. coli o :h of ug/ml in an automated prototype [ ] . a poly(methyl methacrylate) (pmma) microfluidic immunoassay device was modified with poly(ethyleneimine) (pei), an amine-bearing polymer, to increase antibody binding ten-fold [ ] ; the authors believe this is due to the spacer effect as well as the addition of amine groups. due to the smaller dimensions, the microchip reactions were ten-fold faster than well plates and had a dynamic range of to ng/ml. an early study using glass capillary tubes as a solid support to assay e. coli o :h employed a competitive-based immunoassay and achieved a detectable limit of cfu per g of ground beef [ ] . capillaries have also been employed to separate complex matrices and detect a model antigen at pm with immunoaffinity chromatography using dual syringe pumps, a silica bead packed bed, and laser-induced fluorescence [ ] . beads have been used on microchips to separate binding steps from the secondary detection to reduce background [ ] and to mix immunoassays on microchips [ ] . microfluidic immunoassays are being adapted for biodefense needs. liu and coworkers [ ] developed multi-stage integrated microfluidics for immunoassays utilizing electrochemical detection. micropumps and circuits were integrated to perform parallel immunoassays for model organisms with enzyme-generated signal detected by active cmos circuitry with resulting sensitivity in the fm range [ ] . a multi-analyte array biosensor (maab) was developed at the naval research laboratory (nrl) to detect multiple target agents in complex samples using a novel fluidics cube module to control the flow of solutions over six different immunoarray sensors in a small portable device with an evanescent wave detector [ ] . the maab could rapidly detect three toxins: ricin, staphylococcal enterotoxin b, and cholera toxin [ ] , and s. typhimurium [ ] . liquid array-based immunoassays with multiplexed detection have also been developed and tested for model organisms [ ] . yang and coworkers at nanogen exploited the unique characteristics of their addressable arrays to develop a device that performed automated electric-fielddriven immunocapture and dna hybridization [ ] . immunomagnetic separations have been combined with electrochemiluminescence (ecl) detection [ ] . ecl-based assays contain ruthenium labels, which emit light when electrochemically reduced. in ecl detection, tripropylamine can be oxided at the surface of an array of electrodes, and in turn it reduces the ruthenium, which then emits light. background noise is reduced since the reaction activation is localized and controlled by the electrode. several commercial systems are available, and over immunoassays are available on one clinically aimed system. ecl has been further developed with microelectrodes manufactured using screen-printing of carbon inks onto microtiter plates. for biodefense, work in showed that the ecl detection of biotoxoids and bacillus anthracis spores in less than one hour [ ] . an ecl assay was compared with fluorogenic chemiluminescence (fcl) for the detection of biological threat agents [ ] . seb at a concentration of pg/ml has been detected in a range of matrices using ecl in a min immunoassay and found to be significantly better than elisa reactions [ ] . clostridium botulinum toxins a, b, e, and f were detected at about pg/ml in clinical samples and food using ims ecl detection, about the same sensitivity as elisa, but with much more rapid time to results [ ] . ecl immunoassays have also been used to detect ricin at . ng/ml [ ] . in the future, ecl detection may well be integrated with microfluidics to produce fully integrated and sensitive laboratory and portable detection systems for biothreat agents. time resolved fluorescence uses lanthanide chelate labels that have very long fluorescent decay times and large stokes shifts [ ] . the secondary antibody can use a lanthanide such as europium that fluoresces in an enhancer solution. the long decay time can lead to a very low background and sensitivity that are an order of magnitude better than traditional eli-sas, but with greater variability [ ] . commercial full scale systems are in use to detect biothreat agents including franciscella tularensis, clostridium botulinum toxin, and seb with detection at a range of low pg/ml [ ] . immunoassays have been integrated with magnetic detectors to produce microfluidic systems that are being applied to biodefense and other fields. using this approach, the strength of intermolecular interactions can be measured by the force required to disrupt a bond when the target is attached to a magnetic bead [ ] [ ] . the magnetic bead serves as the label which can be detected by microfabricated magnetoresistive transducers on microchips. multiple analytes can be measured in less than min in an array format with sensitivity close to elisa [ ] . model spores and viruses can be detected at about cfu/ml and pfu/ml, respectively, while seb was detected at ng/ml. multiple samples can be measured simultaneously and magnetic force is tolerant of different types of analytes. multiplexed femtomolar detection of proteins from complex mixtures has been shown in a format that may be adapted for a handheld platform for both nucleic acid hybridization assays and immunoassays [ ] . proteomic approaches for biodefense rely on identification of proteins and peptides to evaluate and characterize potential biothreat agents. primary proteomic approaches for biodefense include separation of proteins and peptides by mass spectrometry platforms [ ] , two-dimensional gel analysis [ ] , and protein arrays. proteomic approaches are being used to build a cyberinfrastructure of niaid-funded centers that are applying these tools for biodefense to develop vaccines and proteomic targets [ ] . data from mass spectrometry, yeast two-hybrid (y h), gene expression profiles, x-ray and nmr for bacillus, brucella, cryptosporidium, salmonella, sars, toxoplasma, vibrio and yersinia, human tissue libraries, and mouse macrophages have been developed [ ]. microfluidics is being widely adapted to proteomic systems as upstream devices and nozzle systems for mass spectroscopy, as reviewed in [ ] [ ] . in essence, microfluidics is compatible with the low flow rates, small sample volumes, and microseparations that are required for mass spectrometry. both microfabricated nozzles and hplc devices are presently commercially available. the various developments are beyond the scope of this review, but are described in chapter of this book. for biodefense applications, protein profiling has been developed at sandia national laboratory into an autonomous microfluidics system combining microfluidics sample preparation modules with microchip gel electrophoresis [ ] . this system is fully automated, with a total min sample preparation and detection time. sensitivity for b. subtilis spores was agent-containing particles per liter of air. protein arrays [ ] containing either antibodies to different epitopes or to different proteins arrayed on a solid surface, have been applied to characterize and type biothreats. a protein chip for the arraytube platform was developed that uses a microtube-integrated protein chip that accomplishes detection using the classical sandwich assay and horseradish peroxidase colorimetric substrate. [ ] . invitrogen has been developing its highdensity protein microarrays (protoarrays™) for detection of plague, smallpox, anthrax, and a number of hemorrhagic diseases, such as ebola and dengue fever. phage display, where the probes on the array are selected from billions of clones, is a potentially powerful application of protein arrays and may be adopted for biodefense [ ] , as are peptide arrays. for nucleic acids following sample preparation, amplification technologies can be applied that greatly increase the signal. the best known and most used dna amplification method is pcr [ ] . pcr uses thermal cycling to exponentially amplify dna using a thermally stable dna polymerase. each cycle of amplification doubles the amount of template, thereby exponentially increasing the amount of target, and can amplify from as little as a single copy of dna. the specificity of the amplification is determined by the pair of primers that initiate the amplification. pcr has become a standard clinical and research technique for nucleic acid testing (reviewed in [ ] [ ] [ ] ) and for biodefense [ ] . many different variations of the basic pcr reaction have been developed, including qpcr, nested pcr, multiplexed pcr, and single nucleotide polymorphism pcr. qpcr has revolutionized the detection of specific dna sequences in the laboratory, clinic, and field (reviewed in [ ] [ ] ). qpcr quantifies the amount of original target sequence using a fluorescently-labeled probe that acts as a reporter and detection system. in one version, qpcr employs a fluorescently-labeled probe that contains a quencher that suppresses the fluorescent signal. during the replication process, the signal becomes un-quenched, emitting light. as the amplification progresses, the fluorescent signal increases. the presence and amount of target dna in the sample can be determined from the cycle number when the signal increases over a threshold value, commonly called the cycle threshold (c t ). multiplexed reactions make it possible to detect multiple agents in each reaction vessel. the amount of dna produced can also be measured non-specifically using dna intercalating dyes and other (specific) probes such as molecular beacons. qpcr has specificity and sensitivity equivalent to pcr, while simultaneously amplifying, detecting, and quantifying the original dna target in a single, contained reaction. pcr and qpcr are the most widely used methods to detect and identify biowarfare agents by identifying target dna sequences in the laboratory and field [ ] . multiplexed pcr assays for virulence factors on two plasmids, pxo and pxo , in b. anthracis were able to distinguish it from closely related strains [ ] . highly specific assays can identify b. anthracis using pxo , pxo , protective antigen (paga), and capsular protein b (capb) [ ] are commercially available for clinical samples [ ] [ ] . b. anthracis has been detected from soil samples at spores per ml [ ] , in aerosols [ ] , and in food [ ] . qpcr detection assays have been multiplexed to detect four biothreat agents, y. pestis, f. tularensis, b. anthracis and b. mallei, simultaneously using molecular beacons complementary to conserved s rrna targets [ ] , and with minor groove binding probes with a sensitivity of fg of target with no cross reactivity [ ] . melting point curves combined with the multiplex amplification helped distinguish b. anthracis from y. pestis and leishmania [ ] , and between members of the b. cereus group [ ] . the performance of three commonly used qpcr instruments was compared and generally comparable limits of detection, sensitivity, and specificity were found [ ] . the genexpert system (cepheid) is a standard instrument for semi-automated sample preparation and qpcr (using the smartcycler as the qpcr platform). its performance has been evaluated and the incorporation of the nucleic acid purification component of the sample preparation has been credited with its , -fold improved detection over the smartcycler alone for b. anthracis sterne spores due to removal of inhibitors and concentration of the sample [ ] . miniaturized pcr has been an area of intense development with devices developed that use microchips and capillaries to perform thermal cycling in stationary formats and continuous flow regimes. the advantages of miniaturized pcr devices are more rapid assay times, low reagent consumption, and potential integration with upstream sample preparation modules. miniaturization of pcr, including discussion on the types of common designs, issues with surface chemistries that can inhibit pcr, coating procedures, and heating strategies has been reviewed [ ] [ ] . miniaturized pcr was initially performed in glass capillary tubing. capillaries are off-the-shelf items that are suitable for sub-microliter reactions. wittver first demonstrated (in ) pcr amplification of dna in sealed capillary tubes using a hot air cycler [ ] . this basic design became the rapid cycler (idaho technologies), the forerunner to razor, in use today for biodefense applications. sample volumes were reduced to - ul with cycling times of less than min [ ] . a medium-throughput automated capillary sample preparation system that processed , one ul samples per day was developed [ ] ; the acapella- k (u. washington, seattle) utilized a mechanical capillary handling system, air cycling, and a piezoelectric reagent dispenser [ ] . jovanovich and co-workers developed a nl sample preparation system also using an air cycler with a -channel capillary cassette (fig. . ) . this system used um id capillaries without a coating and standard pcr conditions (except for elevated mg + concentrations). dna samples were forced onto the surface of the glass capillary using chaotrophic agents, the dna-coated capillary was then evacuated, and nl of pcr reagents re-filled the tube by capillary action. pcr reaction efficiencies were equivalent to full volume reactions. capillaries have many applications in microfluidics but are currently more difficult to integrate with other functions than microchips. the integration requires robust microfluidic connectors. the first miniaturized pcr reactions in a microchip were performed in an etched silicon wafer by northrup and coworkers, reported in [ ] . wilding et al. reported successful cycling in and ul silicon reactors cycled by an external peltier device in [ ] . pcr reactions have been performed in nl in silicon devices [ ] , which was expanded on by belgrader et al. by addition of capillary electrophoresis on microchip [ ] . belgrader and coworkers further reported pcr amplification in seven minutes [ ] . the first miniaturized thermal cycler with real time detection was from northrup and colleagues at llnl and used silicon based reaction chambers with diode detectors and integrated heaters [ ] , this same group devised a portable battery-powered unit [ ] . many other reports of qpcr devices have followed [ ] [ ] [ ] . some of the challenges of microfluidic qpcr are control of liquid positioning, bubble formation, sealing chambers using microfluidic valves, and surface interactions. liquids and targets can be manipulated by precise positioning by micropumps, by capture onto a surface, or by exploiting beads and solid phase chemistries. microvalves can restrict the liquid solutions to proper chambers. one group has simultaneously sealed the reaction chamber with valves that serve as the macro-to-micro interface [ ] . for poly(cyclic olefin) plastic fabrication, gel valves were used to confine amplicons before integrated on-chip gel electrophoresis [ ] . bubble formation has been found to be primarily due to surface wetting properties of the chamber [ ] . surface interactions can be minimized by improving the surface chemistry or by increasing channel dimensions to over microns. a challenge for microfluidics is improving heating and cooling rates while maintaining good uniformity. one approach has been to microfabricate resistive heaters and sensors directly onto microchips. this has been used to achieve o c/s heating and o c/s cooling rates to detect upper respiratory tract infection microorganisms in min [ ] . a second approach, pioneered by the landers group, used infrared heating to rapidly heat water in fluidic channels [ ] [ ] ; cycle times as low as s were achieved with successful pcr amplification and cycle sequencing. ir driven pcr was integrated on a microchip with upstream solid-phase extraction of dna using silica beads in sol gel to isolate dna from an anthrax spore-spiked nasal swab; amplified target dna sequences were detected on-chip with a total processing time of min [ ] . a third approach is flowthrough devices with fixed-temperature zones. control of temperature and uniformity is simplified, and power consumption is low since the zones stay at a constant temperature. an added advantage is that cycling times can be reduced due to the elimination of temperature ramping. the challenge to this approach is to reduce surface interactions which can degrade pcr reaction performance. this can be accomplished using surface coatings, by controlling surface to volume ratios, or by using emulsions. soper's group has built flowthrough devices combining flowthrough pcr in a polycarbonate chip and a pmma chip using detection with a ligase assay [ ] . reverse transcription and pcr have been integrated in a flow through glass microchip with um channels integrating different temperature zones; cycles of pcr amplification were achieved in min [ ] . a ferrofluidic actuator has also been used to move a pcr sample plug rapidly between temperature zones [ ] . a hybrid chip with silicon and pmma has performed high speed pcr with three heated zones [ ] . pcr amplification in small volumes has also been accomplished in microarray formats. the target dna is spotted onto a glass slide that has covalently attached primers, enabling amplification of bacterial target dna [ ] . this approach has been applied to identify bacteria using amplification of rdna sequences [ ] . the solexa dna sequencer uses a similar process to amplify clusters of pcr products from a single template molecule on a surface. one of the first miniaturized real-time pcr instruments was developed for biodefense applications. the advanced nucleic acid analyzer (anaa), devised at llnl, used an array of silicon reaction chambers with thinfilm resistive heaters and solid-state optics to rapidly test samples for simulants of biothreat agents with detection limits of to , /ml [ ] [ ] . a compact version of a qpcr instrument with a notebook computer, two reaction modules with integrated four-color fluorescence detection was developed and shown to detect bacillus spores [ ] . a handheld device, the handheld advanced nucleic acid analyzer (hanaa), also developed at llnl, used plastic reaction tubes with silicon and platinum-based thermal cycler units and two light emitting diodes to detect bacteria including b. anthracis ames [ ] . the llnl group, working with sandia, went on to develop the apds, which performs continuous monitoring with a multiplexed immunoassay trigger and confirmatory qpcr assays. this has been demonstrated for aerosolized b. anthracis, yersinia pestis, bacillus globigii, and botulinum toxin [ ] . llnl and sandia also developed a "biobriefcase" device with a smaller footprint that has multiplexed, autonomous detection with immunoassays, toxin assays, and qpcr. the biobriefcase incorporated inhibitor removal and concentrated the sample by mixing aerosol-collected liquid with a chaotrophic agent prior to silica bead bed purification [ ] . the llnl group has also developed a -plex pcr amplification with hybridization to beads upstream of a flow cytometry readout; samples were processed in hours [ ] . the lightcycler (idaho technology) has been adapted into a "ruggedized" advanced pathogen identification device, rapid, which has been used for field analysis of bioagents and pathogens [ ] . y. pestis was detected at about genome equivalents in min using this system. the latest innovation from llnl is amplification in emulsion of single copy dna in a lab-on-a-chip format; the system produces pl droplets that can perform qpcr with thresholds exceeded significantly earlier than conventional instruments [ ] . in addition to detecting dna targets, biodefense needs require that rna viruses be detected. detection of two rna-based viruses-dengue virus and enterovirus has been demonstrated with a pdms microchip that integrates reverse transcriptase and pcr amplification [ ] . parallel reverse transcriptase-pcr assays have been performed in pl and shown to detect as little as copies [ ] . a fully integrated handheld device using isothermal nucleic acid sequence-based amplification (nasba) [ ] , which specifically amplifies rna from primers, was built and shown to have comparable results to laboratory instruments. in addition to pcr there are a number of other nucleic acid amplification technologies that are being adapted for biodefense: strand displacement amplification (sda), loop-mediated isothermal amplification (lamp), exponential amplification reaction (expar), and rolling circle amplification (rca). sda uses the primer-directed nicking activity of a restriction enzyme and the strand displacement activity of an exonuclease-deficient polymerase to amplify dna [ ] . sda can achieve to amplification in about min [ ] . fluorescence resonance energy transfer (fret) probes can be incorporated and real time instruments for the clinical laboratory are in commercial use [ ] [ ] . expar is a dna amplification reaction that produces short ( - nucleotides) in a linear or exponential amplification in isothermal homogeneous assays [ ] . expar can be extremely rapid with amplification of greater than . both real time [ ] and end point formats have been developed. expar has been applied to biodefense applications as part of the band project and in other projects as a quick, specific amplification technique. lamp is an isothermal dna amplification method that relies on autocycling strand-displacement dna synthesis. four sets of primers are used with the thermostable bacillus stearothermophilus (bst) dna polymerase that has high strand displacement activity [ ] . the reaction produces a white precipitate, magnesium pyrophosphate, which can be easily detected as an indicator of a positive amplification reaction [ ] . the specificity and sensitivity are competitive with pcr, and in some cases more sensitive than nested pcr [ ] . rca is an isothermal method that uses phi dna polymerase to amplify circular dna [ ] . phi dna polymerase is a single subunit with excellent processivity and can amplify > fold [ ] . rca is widely used for whole genome amplification, for scarce material, and can nonspecifically amplify trace amounts of dna. rca is routinely used at genome centers preparing template for sample preparation for dna sequencing [ ] . rca has also been used for cell free cloning of genomic dna that might be lethal to cells [ ] . rca is a powerful tool for forensic and biodefense applications. dna microarrays have many potential applications in biodefense. dna microarray technology is a widely used powerful technique that uses large arrays of microspots of dna on a solid support or beads to detect complementary dna or rna products from a sample. (protein arrays were briefly discussed in the proteomics section). for rna samples, amplification of rna is commonly done by first performing reverse transcriptase to create dna from rna, and then the resulting dna can be amplified using standard dna amplification methods such as pcr, or whole genome amplification, or by in vitro transcription followed by another cycle of re-verse transcriptase. fluorescent labeling can be introduced into the sample at various steps in the process. typically, tens of thousands of spots, each containing a unique sequence, are interrogated in a single experiment with fluorescent detection. the data can represent a fingerprint of the transcriptional state of an organism (e.g. biothreat agent, or of the response of a human potentially infected with the agent), identify dna sequences present in an organism, or resequence organisms [ ] [ ] . the strength of the microarray platform is the depth of characterization. the , s of analytes measured on a single microarray slide can generate massive amounts of data. in the future, dna microarrays may be displaced or challenged by digital gene expression methods using next-generation dna sequencing to produce , 's or more sequences from a sample or single mrna detection and enumeration strategies (e.g., nanostring). microarrays have been combined with pcr amplification to identify and genetically discriminate b. anthracis from closely related bacterial species from the b. cereus group and determine if the strains harbor plasmids [ ] . dna microarrays can potentially detect multiple pathogens in a single sample. the fda has developed a microarray, fda- , to screen for several food pathogens and virulence factors, including seb [ ] . saic developed its phase i band project based upon a fully automated system that would collect air samples and then analyze them for pathogens using microarrays. after collection and filtration, reverse transcriptase and pcr amplification were used before hybridization to a dna microarray in a cartridge for ten min. the data was then analyzed for fingerprints that indicated the presence of threat agents. microarray sample preparation is a complicated, multistep process that is dependent on the variability of individual operators. microfluidics, with its potential to automate and integrate processes, has been applied to simplify and standardize sample preparation (reviewed in [ ] ). this seminal work was performed by anderson and colleagues at affymetrix, where a miniaturized integrated microdevice was developed to prepare samples by accessing reagents and performing automated operations before performing hybridizations to a microarray. anderson and coworkers elegantly demonstrated a plastic miniaturized sample preparation system for microarray sample preparation and analysis by hybridization, and showed the detection of mutations in the hiv genome from serum samples [ ] . the advantages of microfluidics for microarray biodefense applications are in-tegration of sample preparation steps, potential reductions in reagent cost, ease of use, and decreased hybridization times. the soper group has integrated microarrays with microfluidic technology in plastics. a microarray was fabricated in pmma using uv exposure of the polymer surface, coupling of amine-terminated oligonucleotide probes to the surface, and washing the surface [ ] . the hybridization and allelespecific ligase detection reactions were performed in a polycarbonate flowthrough biochip. the system could screen for mutations in min. work by liu and coworkers at motorola and then combimatrix has developed biochips that integrate sample preparation, pcr, and microarray detection [ ] . electrochemical pumps were used with paraffin-based single-use microvalves to regulate flow. detection of pathogens from whole blood [ ] , identification of influenza virus [ ] , and gene expression from a cell line [ ] were shown. a microelectronic array system was developed by nanogen for microarray testing [ ] . this system employs dielectrophoresis as a sample preparation method and the hybridization of nucleic acid to probes attached to electrodes is accelerated by application of an electrical potential. the electrodes are covered with a hydrogel. detection is performed with fluorescence probes [ ] . electrophoresis is a powerful separation technology with many biodefense applications. the advent of capillary electrophoresis (ce) and the multichannel capillary array electrophoresis (cae) propelled the applications of electrophoresis by increasing separation speeds, automating the analysis, and improving data reliability. most notably, cae technology was partially responsible for the early completion of the human genome project and has become the separation method of choice for most nucleic acid applications. several pcr-based methods for detailed laboratory characterization of microorganisms have been developed that rely on electrophoretic separation. in this section, we first review several of the separation based typing methods for bacteria identification and discrimination, describe the application of microfluidics on microchips to microelectrophoresis, and discuss applications of dna sequencing in a microfluidic platform to biodefense [ ] . vntr loci analysis [ ] is a powerful laboratory tool for identifying bacteria, humans, and other organisms. analogous to microsatellite genotyping, vntr pcr amplification detects regions where genetic drift has created variable numbers of tandem repeats inserted into the genome, plasmids, or other extra-chromosomal elements. vntr has been used to identify y. pestis [ ] , mycobacterium tuberculosis [ ] , and numerous other organisms. mlva [ ] extend vntr to assay multiple alleles and provide a fingerprint, analogous to forensics identification by microsatellite dna analysis. keim and co-workers have identified a set of eight vntr regions that are diagnostic for b. anthracis and characterized b. anthracis isolates into distinct genotypes [ ] . mlva was used to subtype the anthrax strains from the bioterrorist attack in within eight hours of receiving isolates [ ] . mlva has been applied to type closely related bacillus strains but at times required additional information for confirmatory determination of b. anthracis [ ] . mlva types of bacillus anthracis could be further differentiated by single-nucleotide repeats [ ] . aflp is a pcr-based method that can fingerprint microbes [ ] , type organisms, and identify phyllogenetic relationships. aflp is rapid-it employs a relatively simple multi-step workflow with standard reagents regardless of the organism typed. aflp has been applied to differentiate a wide variety of microorganisms at the subspecies level [ ] , including b. anthracis [ ] and is widely used to classify plants, yeasts, and other organisms. the aflp process begins with a two-enzyme restriction digest followed by ligation of fluorescently labeled restriction half-site adapters to reconstruct the restriction site at the end of the fragments and serve as pcr primers. two or three degenerate nucleotides on the end of the adapter reduce the complexity of the amplified products during the high stringency pcr amplification. by selection of proper restriction enzyme pairs, fluorescently labeled fragments in the range of to , bases can be produced, and then are separated and detected by capillary electrophoresis. the resultant pattern is a fingerprint of the test organisms' genomic and extra-chromosomal restriction patterns. a post-labeling fluorescent method using dye-terminator chemistry can visualize both rflp and aflp products [ ] . cdna-aflp can also be performed to visualize the gene expression pattern of an organism [ ] . subtle strain-to-strain variations can be characterized by single nucleotide polymorphisms (snps). snp typing of intragenic spacers in the s- s region has been shown to differentiate closely related bacillus strains including b. cereus from b. anthracis [ ] . the identification of the specific strain of a biothreat organism can help microbial forensics trace the origin and determine whether multiple incidents were caused by release of identical organisms and therefore share a common origin. to determine strain variations, the genomic sequence can be determined by dna sequencing. following the september th attacks, the cdc sequenced the s rrna gene to definitely identify b. anthracis from culture-negative clinical specimens of patients with confirmed anthrax [ ] . in other studies, s rrna and s rrna sequences for all species in the b. cereus group showed disagreement with phenotyping clustering, but by utilizing rrna together with gyrb sequences these workers could discriminate between groups [ ] . ruppitsch and coworkers showed for a wider variety of strains that sequencing of the s rrna gene is not always sufficient, but that additional sequencing of intragenic sequences can increase resolution and thus differentiate bioagents [ ] . cae is based upon separation in a capillary, itself a type of microdevice. in this review, we use microelectrophoresis to connote separations on microchips. the interested reader is referred to reviews [ ] [ ] [ ] for details of chip construction, coatings, operation, and equipment. most commonly, glass microchips are employed but plastic devices have also been developed [ ] . microchips have the potential to simultaneously separate hundreds of samples in minutes. microchips typically consume only picoliters of samples and thus can be well matched to microscale sample preparation volumes. fluorescently labeled amino acids [ ] , dna restriction fragments [ ] [ ] [ ] , pcr products, short oligonucleotides [ ] , and sequencing ladders [ ] have been separated by microchip capillary electrophoresis [ ] . the analyses are extremely rapid, from less than a minute for oligonucleotides to less than minutes for dna sequencing [ ] . we note that next generation microfluidic sequencing-by-synthesis [ ] , microfluidic pyrosequencing [ ] , sequencing by ligation, and nanopores may, in the future, have biodefense applications for detecting genetically modified organisms and digital gene expression. interfacing upstream microfluidic sample preparation with microscale separations is challenging. the classic twin-t injector defines roughly a to picoliter volume in a short sample plug ( to um) in the separation channel with electrokinetic loading [ ] . this is only a small fraction of the sample, even if the prepared sample is only nl: for biodefense applications this means losing orders of magnitude of potential sensitivity during the analysis step. this can be ameliorated with isotachaphoresis, field amplified stacking, dna binding, and other methods that locally increase the sample concentration. isotachaphoresis concentrates samples between leading and trailing electrolytes and can increase detection limits by -fold or more for dna [ ] [ ] , and stack up to a million-fold [ ] . field amplified sample stacking, routinely used in capillary electrophoresis, has been adapted to microchip electrophoresis using pressure driven flows to move low osmotic strength samples into position for stacking on the column. concentration effects range from -fold increase in signals [ ] to -fold for model compounds [ ] . separations on microchips are most developed for dna separations. in , hemochromatosis samples were genotyped in less than min by microelectrophoresis on a microchip [ ] . locus-specific, multiplex pcr products specific for deletions causing duchene/becker muscular dystrophy have been separated on a silicon-glass microchip, and t-cell receptorgamma genes and immunoglobulin heavy chain gene on glass microchips [ ] . fluorescently-labeled cttv pcr samples and short tandem repeats have been analyzed on single-channel ce microchips [ ] . the mathies laboratory genotyped hemochromatosis samples in less than min, with detection by a four-color rotary confocal fluorescence scanner [ ] . dna sequencing on microchips was first performed in in the mathies' lab [ ] ; fluorescently labeled dna sequencing fragment ladders were separated on glass microchips with a denaturing polyacrylamide sieving matrix and readlengths of about bases obtained in min. mathies' lab later reported readlengths of about bases in about min in single channel [ ] . jovanovich's group reported the first multichannel dna sequencing in an array of microchannels with readlengths of bases in min in an automated instrument [ ] . high throughput dna sequencing in channels obtained readlengths of high quality bases [ ] . to achieve longer readlengths, microchips with to cm separation channels were constructed and readlengths of to bases reported with plates containing up to channels [ ] . separations on plastic microchips have also been achieved. one of the first microelectrophoresis devices, in , used a twin t injector on a pdms substrate to separate proteins and dna [ ] . plastic substrates offer the advantage of potential low cost fabrication once high production volumes are achieved, but have been plagued by higher background fluorescence signals than glass or quartz devices and require specialized surface coatings [ ] . while a detailed review is outside the scope of this article, injection molded microelectrophoresis devices have achieved separation of dna fragments [ ] [ ] and dna sequencing [ ] . hot embossed pmma with ir detection was shown to sequence dna out to bases in linear polyacrylamide [ ] . for biodefense, the breakdown products of g-type and v-type nerve agents can be assayed by ce and microelectrophoresis [ ] . wang et al. [ ] combined microchip electrophoresis with derivatization and electrochemical detection of thiol-containing degradation of v-type nerve agents at micromolar concentrations in less than four min. a microchemlab portable device, developed at sandia national laboratory, with a fused silica microfluidic separation chip, a miniature lif detector, and high voltage power supplies [ ] can assay proteins and toxins at nanomolar concentrations [ ] . the achievement of working devices for both microfluidic based sample preparation and analysis has created the possibility for complete system integration to meet biodefense needs. for robust biodefense applications, sample preparation and analysis must be integrated either directly in a monolithic format or by microfluidic connections. as described above, for dna microarrays sample preparation has been integrated with analysis, as first shown by anderson, and brought to commercial product by combimatrix. today, one of the most advanced areas for complete system integration has been the integration of upstream sample preparation reaction steps with microelectrophoresis on microchips. microchip and capillary-based analysis systems have the advantages of high-resolution separations with extremely fast separation times, automation, and nanoliter-scale consumption of reagents. integration of pcr reactions in microfabricated devices with microelectrophoresis was first demonstrated in in collaboration be-tween northrup's llnl group and the mathies laboratory; pcr amplification was performed in a silicon device mounted on a glass separation device and complete analysis was achieved in min for a cloned human gene and min for a bacterial genomic target [ ] . early work exploited the small volumes in capillaries to prepare samples and analyze them by capillary electrophoresis. swerdlow and colleagues developed an automated prototype with a sample loop in an air cycler coupled to a separation capillary [ ] . a fully integrated miniaturized integrated microsystem using capillaries was shown to perform all steps including pcr amplification in nl and separations starting from buccal cells [ ] . integration of pcr reactions in capillaries with separations in capillaries has been shown to be effective for dna typing from blood [ ] and other materials for human and viral targets [ ] . pcr reactions and dna cycle sequencing in capillaries and capillary separations have been fully integrated to create a microvolume system with readlengths of bases in hrs from human dna [ ] . pcr reaction chambers have now been monolithically integrated with microelectrophoresis to create fully integrated microsystems that perform pcr on the same device as the separation. burns et al. integrated sample preparation, gel electrophoresis, and on-chip detection for low resolution separation of dna fragments [ ] . the mathies lab showed pcr amplification and microelectrophoresis separation to determine sex of humans from dna in min [ ] . a portable system with nl pcr reactors, solid state lasers, pneumatically actuated on-chip micropumps, and microelectrophoresis was able to detect - e. coli or s. aureus cells in less than min including determining drug resistance [ ] . the mathies lab has extended their dna sequencing on microchips upstream to include integrated nanovolume sample preparation and purification. first, nanoscale pcr reactions were combined with capillary electrophoresis analysis [ ] . then, sample preparation, cleanup, and dna separations were combined to integrate dna sequencing, as shown in fig. . , [ ] . a nl cycle sequencing reaction was performed on microchip, and then moved by micropumps onto an acrylamide gel capture matrix with an oligonucleotide hybridization probe to capture the target dna in a nl capture chamber. the affinity capture removes template dna, desalts, and pre-concentrates the sample for microchip electrophoresis for higher injection efficiencies. the sample is then electrophoresed into an injector and separated on microchip. readlengths up to bases were obtained from fmol of template. the key to the integration was pneumatically actuated microvalves and micropumps [ ] . the landers laboratory has completely integrated pcr, sample cleanup, and capillary electrophoresis on microchips, as shown in fig. . . a device with pcr reactions, ir mediated heating, pneumatically actuated onchip micropumps, and microelectrophoresis has achieved amplification and good separations in less than min [ ] . they have detected of b. anthracis from whole blood of asymptomatic mice and bordetella pertussis from nasal aspirate of a patient using on-chip nucleic acid purification with a nl pcr reactor coupled to microelectrophoresis analysis with control by pneumatic microvalves [ ] . fully integrated 'industrial strength' microfluidics is being applied to homeland security and biodefense in the band program by several groups including microfluidic systems inc, iquum, and us genomics. iquum (www.iquum.com) is developing a liat™ "detect-to-treat" system. the system has a disposable cassette containing reagents and equipment that contains air sampling, sample preparation, and real time detection using pcr in a "lab-in-a-tube" [ ] . the sample moves through segmented tube sections containing the reagents using peristaltic pumps and moves back and forth between temperature zones to amplify the dna. us genomics (www.usgenomics.com) has taken a very different approach. single stranded dna is prepared from aerosol samples and a set of fluorescently labeled probes added. the probes bind to their homologous targets, if present. the labeled dna is then moved through a narrow channel one molecule at a time where it is interrogated by laser-induced fluorescence. the resulting pattern of binding sites is a fingerprint that can identify known and unknown agents from a database [ ] . one advantage of the approach is the analysis of individual molecules is done without apriori knowledge of the sequence. this potentially enables detection of genetically engineered or previously unknown pathogens. in the future, next generation dna sequencers, mass spectroscopy, and dna microarrays will provide additional solutions to begin to address genetically engineered organisms. microfluidics will become an increasing important element of the future biodefense portfolio. as shown in this and other chapters in this volume, microfluidic components are available in many different shapes and formats: proof-of-concept of just about any imaginable type of sample extraction, lysis, pre-separations, sample preparation, assay, separation, and detection component has been demonstrated in research settings, at universities, research facilities, national laboratories, and in industry. the missing component is the integration of the many parts into complete working systems that are robust, manufacturable, and maintainable. complete integration of complex workflows has proved elusive in full volume devices and in microfluidics. to fully integrate a microfluidic system for biodefense requires taking a sample of several hundred microliters or larger and processing it completely to yield an answer. the chemical and biochemical workflow usually has many steps that all have to be developed, tested, and integrated. in addition, of course, surface chemistries, temperature, and other variables must be controlled. the work described in this review has shown the promise for microfluidics for many types of genomic and proteomic devices. fully microfluidic integrated devices that can input samples and perform sample preparation and analysis are just beginning to appear, as are field portable microfluidic genetic analyzers. while there are some examples of success at the research and prototype levels, there are only a few fully integrated solutions and fewer commercial successes. two main microfluidic elements are needed for full integration. the first element is an appropriate level of control of the microfluidic volumes. this typically requires microfluidic valves, a pumping mechanism, and routers to move, mix, and split, and aliquot liquids. single use valves enable many applications in a disposable cartridge, while programmable valves that enable multiple uses are required for environmental monitoring and reusable applictions. pneumatically driven programmable microvalves are only now becoming more mature and are in use in academia and industry to control fluid flows and mixing. these microvalves [ ] have proved invaluable in the integration by the mathies and landers groups, and in industry to integrate processes onto monolithic microdevices. the second enabling element will be to connect different microfluidic devices together using standard connections. in microelectronics, usb, firewire, and other standard connectors allow devices to interconnect by defining the interface and the protocols. microfluidics now needs to establish standards for connections that will enable 'best in class' microfluidic devices to work together in a 'plug and play' manner. a symposium of industry and academia with government representation, including dod, dhs, and nist, to discuss achievable initial standards would be invaluable and may serve to begin the path towards interconnectivity for microfluidics for biodefense and other applications. in this regard, the physical dimension of spacing for connectors needs to be standardized, and standard protocols for transfer of materials developed. the future for microfluidics will be bright as individual steps are optimized and integrated. future microfluidic systems will connect directly to 'real world' samples and fully integrate upstream sample concentration and analysis in a single autonomous device. the full integration of micro-fluidic processes will enable man-portable and then hand-held biodefense devices. eventually, if biothreats become pervasive, microfluidic home and business security devices akin to smoke detectors may provide the massive sampling capability needed to detect to warn the public. the public and the biodefense community await the transformation that complete microfluidic integration of biodefense detection can bring to increase the biosecurity of the world. fear of terrorism in new york after the september terrorist attacks: implications for emergency mental health and preparedness bioterrorism-a new challenge for public health laboratory response to anthrax bioterrorism chemical or biological terrorist attacks: an analysis of the preparedness of hospitals for managing victims affected by chemical or biological weapons of mass destruction hospital infectious disease emergency preparedness: a survey of infection control professionals physicians' preparedness for bioterrorism and other public health priorities the knowledge report to congressional requesters, biological weapons: effort to reduce former soviet threat offers benefits, poses new risks the looming threat of bioterrorism biological threat characterization research: a critical component of national biodefense multiplex diagnostic platforms for detection of biothreat agents bioterrorism: implications for the clinical microbiologist medical countermeasures to wmds: defense research for civilian and military use project bioshield: what it is, why it is needed, and its accomplishments so far comparative genomics tools applied to bioterrorism defense a two-component direct fluorescent-antibody assay for rapid identification of bacillus anthracis report to congressional requesters: bioterrorism information technology strategy could strengthen federal agencies' abilities to respond to public health emergencies apds: the autonomous pathogen detection system a minisonicator to rapidly disrupt bacterial spores for dna analysis lysing bacterial spores by sonication through a flexible interface in a microfluidic system a microfluidic cartridge to prepare spores for pcr analysis microfluidic sonicator for q-disruption of eukaryotic cells and bacterial spores for dna analysis laser induced disruption of bacterial spores on a microchip microchip-based one step dna extraction and qpcr in one chamber for rapid pathogen identification the fundamental aspects and applications of agilent hplc-chip monolithic continuous beds as a new generation of stationary phase for chromatographic and electro-driven separations recent development of monolithic stationary phases with emphasis on microscale chromatographic separation a plastic microchip for nucleic acid purification fabrication of amino silane-coated microchip for dna extraction from whole blood microfluidic chip for high efficiency dna extraction continuous flow microfluidic device for cell separation, cell lysis and dna purification advances in sol-gel based columns for capillary electrochromatography: sol-gel open-tubular columns less common applications of monoliths: preconcentration and solid-phase extraction microchip-based purification of dna from biological samples evaluation of silica resins for direct and efficient extraction of dna from complex biological matrices in a miniaturized format dna extraction using a tetramethyl orthosilicate-grafted photopolymerized monolithic solid phase microchip-based macroporous silica sol-gel monolith for efficient isolation of dna from clinical samples microfluidic-based dna purification in a two-stage, dual-phase microchip containing a reversed-phase and a photopolymerized monolith microfluidic chip-based protein capture from human whole blood using octadecyl (c ) silica beads for nucleic acid analysis from large volume samples microfluidic system integration in sample preparation chip-sets -a summary microfluidic immunoaffinity separations for bioanalysis bacterial separation and concentration from complex sample matrices: a review detection and recovery rates achieved using direct plate and enrichment/immunomagnetic separation methods for escherichia coli o :h in minced beef and on bovine hide rapid detection of escherichia coli o :h by immunomagnetic separation and qpcr sensitive detection of escherichia coli o :h in food and water by immunomagnetic separation and solid-phase laser cytometry magnetic nanoparticle-antibody conjugates for the separation of escherichia coli o :h in ground beef immunoquantitative qpcr for detection and quantification of staphylococcus aureus enterotoxin b in foods magnetic-based microfluidic platform for biomolecular separation combined microfluidic-micromagnetic separation of living cells in continuous flow immunomagnetic t cell capture from blood for pcr analysis using microfluidic systems immunological methods for detection and identification of infectious disease and biological warfare agents immunoassay of infectious agents heightened sense for sensing: recent advances in pathogen immunoassay sensing platforms microfluidic immunosensor systems dipstick immunoassay to detect enterohemorrhagic escherichia coli o :h in retail ground beef detection of francisella tularensis in biological specimens using a capture enzyme-linked immunosorbent assay, an immunochromatographic handheld assay, and a pcr microfluidic immunoassays as rapid saliva-based clinical diagnostics detection of staphylococcal enterotoxin b (seb) by enzyme-linked immunosorbent assay and by a rapid hand-held assay microfluidic tectonics platform: a colorimetric, disposable botulinum toxin enzyme-linked immunosorbent assay system rapid procedure for detecting enterohemorrhagic escherichia coli o :h in food development of a fluorescent latex microparticle immunoassay for the detection of staphylococcal enterotoxin b (seb) a simple and rapid fluorescence-based immunoassay for the detection of staphylococcal enterotoxin b a rapid and sensitive magnetic bead-based immunoassay for the detection of staphylococcal enterotoxin b for high-through put screening integrated bioassays in microfluidic devices: botulinum toxin assays bead-based microfluidic immunoassays: the next generation an electrokineticallycontrolled immunoassay for simultaneous detection of multiple microbial antigens surface modification for enhancing antibody binding on polymer-based microfluidic device for enzyme-linked immunosorbent assay a solid phase fluorescent capillary immunoassay for the detection of escherichia coli o :h in ground beef and apple cider a capillary-based microfluidic instrument suitable for immunoaffinity chromatography microfluidic elisa on non-passivated pdms chip using magnetic bead transfer inside dual networks of channels enzymatically-generated fluorescent detection in micro-channels with internal magnetic mixing for the development of parallel microfluidic elisa integrated microfluidic biochips for immunoassay and dna bioassays a portable array biosensor for detecting multiple analytes in complex samples array biosensor for detection of toxins detection of salmonella enterica serovar typhimurium by using a rapid, array-based immunosensor multiplexed liquid arrays for simultaneous detection of simulants of biological warfare agents an integrated, stacked microlaboratory for biological agent detection with dna and immunoassays sensitive detection of biotoxoids and bacterial spores using an immunomagnetic electrochemiluminescence sensor detection of biological threat agents by immunomagnetic microsphere-based solid phase fluorogenic-and electro-chemiluminescence rapid and sensitive immunomagnetic-electrochemiluminescent detection of staphyloccocal enterotoxin b rapid detection of clostridium botulinum toxins a, b, e, and f in clinical samples, selected food matrices, and buffer using paramagnetic bead-based electrochemiluminescence detection an activity-dependent assay for ricin and related rna n-glycosidases based on electrochemiluminescence comparison of dissociation-enhanced lanthanide fluorescent immunoassays to enzyme-linked immunosorbent assays for detection of staphylococcal enterotoxin b, yersinia pestis-specific f antigen, and venezuelan equine encephalitis virus rapid and sensitive detection of biological warfare agents using time-resolved fluorescence assays a biosensor based on magnetoresistance technology giant magnetoresistive sensors and superparamagnetic nanoparticles: a chip-scale detection strategy for immunosorbent assays array biosensor for simultaneous identification of bacterial, viral, and protein analytes rapid, femtomolar bioassays in complex matrices combining microfluidics and magnetoelectronics proteomics for biodefense applications: progress and opportunities a prototype two-dimensional capillary electrophoresis system fabricated in poly(dimethylsiloxane) an emerging cyberinfrastructure for biodefense pathogen and pathogen host data microfluidic systems in proteomics microfabricated devices: a new sample introduction approach to mass spectrometry autonomous microfluidic sample preparation system for protein profile-based detection of aerosolized bacterial cells and spores biomarker discovery using protein microarray technology platforms: antibody-antigen complex profiling a simple and rapid protein array based method for the simultaneous detection of biowarfare agents phage display for detection of biological threat agents enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle-cell anemia molecular diagnosis of medical viruses nucleic acid-based methods for the detection of bacterial pathogens: present and future considerations for the clinical laboratory advances in nucleic acid-based diagnostics of bacterial infections nucleic acid approaches for detection and identification of biological warfare and infectious disease agents qpcr in the microbiology laboratory cockerill fr rd, smith tf ( ) qpcr in clinical microbiology: applications for routine laboratory testing multiplexing for the detection of multiple biowarfare agents shows promise in the field identification and characterization of bacillus anthracis by multiplex pcr analysis of sequences on plasmids pxo and pxo and chromosomal dna fluorescent detection techniques for q-multiplex strand specific detection of bacillus anthracis using rapid pcr detection of bacillus anthracis dna by lightcycler pcr application of rapid-cycle q-polymerase chain reaction for the detection of microbial pathogens: the mayo-roche rapid anthrax test sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by qpcr application of the qpcr for the detection of airborne microbial pathogens in reference to the anthrax spores real-time nucleic acid-based detection methods for pathogenic bacteria in food molecular beacons for multiplex detection of four bacterial bioterrorism agents simultaneous qpcr detection of bacillus anthracis, francisella tularensis and yersinia pestis a novel semiquantitative fluorescencebased multiplex polymerase chain reaction assay for rapid simultaneous detection of bacterial and parasitic pathogens from blood rapid genotypic detection of bacillus anthracis and the bacillus cereus group by multiplex qpcr melting curve analysis detection of biological threat agents by qpcr: comparison of assay performance on the r.a.p.i.d., the lightcycler, and the smart cycler platforms evaluation of the cepheid genexpert system for detecting bacillus anthracis miniaturized pcr chips for nucleic acid amplification and analysis: latest advances and future trends microchip pcr automated polymerase chain reaction in capillary tubes with hot air minimizing the time required for dna amplification by efficient heat transfer to small samples capillary tube resistive thermal cycling acapella- k, a capillary-based submicroliter automated fluid handling system for genome analysis dna amplification with a microfabricated reaction chamber pcr in a silicon microstructure development of a microchamber array for picoliter pcr functional integration of pcr amplification and capillary electrophoresis in a microfabricated dna analysis device pcr detection of bacteria in seven minutes a miniature analytical instrument for nucleic acids based on micromachined silicon reaction chambers a battery-powered notebook thermal cycler for rapid multiplex real-time pcr analysis disposable q-micropcr device: labon-a-chip at a low cost ultra fast miniaturized qpcr: cycles in less than six minutes towards a portable microchip system with integrated thermal control and polymer waveguides for qpcr world-to-chip microfluidic interface with built-in valves for multichamber chip-based pcr assays integrating polymerase chain reaction, valving, and electrophoresis in a plastic device for bacterial detection microfluidic handling of pcr solution and dna amplification on a reaction chamber array biochip micromachined polymerase chain reaction system for multiple dna amplification of upper respiratory tract infectious diseases infrared-mediated thermocycling for ultrafast polymerase chain reaction amplification of dna infrared temperature control system for a completely noncontact polymerase chain reaction in microfluidic chips a simple, valveless microfluidic sample preparation device for extraction and amplification of dna from nanoliter-volume samples polymerase chain reaction/ligase detection reaction/hybridization assays using flow-through microfluidic devices for the detection of low-abundant dna point mutations microfabricated device for dna and rna amplification by continuous-flow polymerase chain reaction and reverse transcription-polymerase chain reaction with cycle number selection automated chip-based device for simple and fast nucleic acid amplification high-throughput pcr in silicon based microchamber array microarray-based detection of bacteria by on-chip pcr microarray-based identification of bacteria in clinical samples by solid-phase pcr amplification of s ribosomal dna sequences rapid pathogen detection using a microchip pcr array instrument a miniature analytical instrument for nucleic acids based on micromachined silicon reaction chambers a handheld real time thermal cycler for bacterial pathogen detection autonomous detection of aerosolized bacillus anthracis and yersinia pestis flow through pcr module of biobriefcase. smart medical and biomedical sensor technology iii a multiplexed pcr-coupled liquid bead array for the simultaneous detection of four biothreat agents a q-fluorescence polymerase chain reaction assay for the identification of yersinia pestis using a field-deployable thermocycler on-chip, q-, single-copy polymerase chain reaction in picoliter droplets miniature qpcr system for diagnosis of rna-based viruses parallel picoliter qpcr assays using microfluidics characteristics and applications of nucleic acid sequence-based amplification (nasba) detection of m. tuberculosis dna using thermophilic strand displacement amplification strand displacement amplification: a versatile tool for molecular diagnostics homogeneous q-detection of single-nucleotide polymorphisms by strand displacement amplification on the bd probetec et system isothermal reactions for the amplification of oligonucleotides isothermal dna amplification coupled with dna nanosphere-based colorimetric detection loop-mediated isothermal amplification of dna sensitive and inexpensive molecular test for falciparum malaria: detecting plasmodium falciparum dna directly from heat-treated blood by loop-mediated isothermal amplification use of loop-mediated isothermal amplification of the is sequence for rapid detection of cultured mycobacterium avium subsp. paratuberculosis rapid amplification of plasmid and phage dna using phi dna polymerase and multiply-primed rolling circle amplification tem-pliphi, phi dna polymerase based rolling circle amplification of templates for dna sequencing templiphi: a sequencing template preparation procedure that eliminates overnight cultures and dna purification cell-free cloning using phi dna polymerase potential of dna microarrays for developing parallel detection tools (pdts) for microorganisms relevant to biodefense and related research needs potential applications of dna microarrays in biodefense-related diagnostics identification of bacillus anthracis by multiprobe microarray hybridization multipathogen oligonucleotide microarray for environmental and biodefense applications merging microfluidics with microarray-based bioassays a miniature integrated device for automated multistep genetic assays fabrication of dna microarrays onto polymer substrates using uv modification protocols with integration into microfluidic platforms for the sensing of low-abundant dna point mutations self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and dna microarray detection validation of a fully integrated microfluidic array device for influenza a subtype identification and sequencing fully integrated miniature device for automated gene expression dna microarray processing microelectronic array system for molecular diagnostic genotyping: nanogen nanochip and molecular biology workstation active microelectronic array system for dna hybridization, genotyping and pharmacogenomic applications chemical and biological threat-agent detection using electrophoresis-based lab-on-a-chip devices variable number of tandem repeat (vntr) markers for human gene mapping diversity in a variable-number tandem repeat from yersinia pestis discrimination of m. tuberculosis complex bacteria using novel vntr-pcr targets multiple-locus variable-number tandem repeat analysis reveals genetic relationships within bacillus anthracis molecular subtyping of bacillus anthracis and the bioterrorism-associated anthrax outbreak, united states comparison of minisatellite polymorphisms in the bacillus cereus complex: a simple assay for large-scale screening and identification of strains most closely related to bacillus anthracis singlenucleotide repeat analysis for subtyping bacillus anthracis isolates aflp: a new technique for dna fingerprinting evaluation of the dna fingerprinting method aflp as an new tool in bacterial taxonomy genetic comparison of bacillus anthracis and its close relatives using amplified fragment length polymorphism and polymerase chain reaction analysis frflp and faflp: medium-throughput genotyping by fluorescently post-labeling restriction digestion visualization of differential gene expression using a novel method of rna fingerprinting based on aflp: analysis of gene expression during potato tuber development strategy for identification of bacillus cereus and bacillus thuringiensis strains closely related to bacillus anthracis sequencing of s rrna gene: a rapid tool for identification of bacillus anthracis use of s rrna, s rrna, and gyrb gene sequence analysis to determine phylogenetic relationships of bacillus cereus group microorganisms suitability of partial s ribosomal rna gene sequence analysis for the identification of dangerous bacterial pathogens capillary electrophoresis on microchips (review) microfabricated electrophoresis systems for dna sequencing and genotyping applications: current technology and future directions high resolution dna separations using microchip electrophoresis plastic microchip electrophoresis for analysis of pcr products of hepatitis c virus micromachining a miniaturized capillary electrophoresis-based chemical analysis system on a chip ultra-high-speed dna fragment separations using microfabricated capillary array electrophoresis chips high-speed separations on a microchip high-speed dna genotyping using microfabricated capillary array electrophoresis chips high-speed separation of antisense oligonucleotides on a micromachined capillary electrophoresis device optimization of high-speed dna sequencing on microfabricated capillary electrophoresis channels automated parallel dna sequencing on multiple channel chips microfluidic device reads up to four consecutive base pairs in dna sequencing-by-synthesis pyrosequencing in a microfluidic flow-through device micromachining a miniaturized capillary electrophoresis-based chemical analysis system on a chip tandem isotachophoresis-zone electrophoresis via base-mediated destacking for increased detection sensitivity in microfluidic systems preconcentration and separation of double-stranded dna fragments by electrophoresis in plastic microfluidic devices on-chip millionfold sample stacking using transient isotachophoresis sample preconcentration by field amplification stacking for microchip-based capillary electrophoresis online sample preconcentration using field-amplified stacking injection in microchip capillary electrophoresis high-throughput genetic analysis using microfabricated -sample capillary array electrophoresis microplates degenerate oligonucleotide primed-polymerase chain reaction and capillary electrophoretic analysis of human dna on microchipbased devices dna typing in thirty seconds with a microfabricated device microfabricated -lane capillary array electrophoresis bioanalyzer for ultrahigh-throughput genetic analysis ultra-high-speed dna fragment separations using microfabricated capillary array electrophoresis chips microfabricated capillary array electrophoresis dna analysis systems high throughput dna sequencing with a microfabricated -lane capillary array electrophoresis bioprocessor optimization of high-performance dna sequencing on short microfabricated electrophoretic devices modification of poly(methyl methacrylate) microchannels for highly efficient and reproducible electrophoretic separations of double-stranded dna high-performance genetic analysis on microfabricated capillary array electrophoresis plastic chips fabricated by injection molding modification of a poly(methyl methacrylate) injection-molded microchip and its use for high performance analysis of dna application of disposable plastic microfluidic device arrays with customized chemistries to multiplexed biochemical assays near-infrared time-resolved fluorescence lifetime determinations in poly(methylmethacrylate) microchip electrophoresis devices analysis of nerve agents using capillary electrophoresis and laboratory-on-a-chip technology microchip capillary electrophoresis with electrochemical detection of thiol-containing degradation products of v-type nerve agents microchip separations of protein biotoxins using an integrated hand-held device hand-held microanalytical instrument for chip-based electrophoretic separations of proteins functional integration of pcr amplification and capillary electrophoresis in a microfabricated dna analysis device fully automated dna reaction and analysis in a fluidic capillary instrument capillary-based fully integrated and automated system for nanoliter polymerase chain reaction analysis directly from cheek cells high-throughput polymerase chain reaction analysis of clinical samples by capillary electrophoresis with uv detection on-line coupling of polymerase chain reaction and capillary electrophoresis for automatic dna typing and hiv- diagnosis on-line integration of pcr and cycle sequencing in capillaries: from human genomic dna directly to called bases an integrated nanoliter dna analysis device fully integrated pcr-capillary electrophoresis microsystem for dna analysis integrated portable genetic analysis microsystem for pathogen/infectious disease detection single-molecule dna amplification and analysis in an integrated microfluidic device microfabricated bioprocessor for integrated nanoliter-scale sanger dna sequencing on-chip pressure injection for integration of infrared-mediated dna amplification with electrophoretic separation a fully integrated microfluidic genetic analysis system with sample-in-answerout capability nucleic acid-based technologies ramp up alternative methods for amplifying and detecting specific dna sequences dna mapping using microfluidic stretching and single-molecule detection of fluorescent site-specific tags an integrated microfluidic processor for single nucleotide polymorphism-based dna computing miniaturized electrophoresis in planar foramtas produced by semiconductor fabrication techniques key: cord- -r r bc v authors: morel, noelia; lassabe, gabriel; elola, susana; bondad, mauricio; herrera, silvia; marí, carlos; last, jerold a.; jensen, oscar; gonzalez-sapienza, gualberto title: a monoclonal antibody-based copro-elisa kit for canine echinococcosis to support the paho effort for hydatid disease control in south america date: - - journal: plos negl trop dis doi: . /journal.pntd. sha: doc_id: cord_uid: r r bc v cystic echinococcosis is still a major concern in south america. while some regions show advances in the control of the disease, others have among the highest incidence in the world. to reverse this situation the pan american health organization (paho) has launched a regional project on cystic echinococcosis control and surveillance. an early concern of the program was the lack of a standardized diagnostic tool to monitor infection in dogs, a key target of control programs. under this premise, we have developed a new copro-elisa test after extensive screening of a large panel of monoclonal antibodies (mabs) and polyclonal sera, which performs with high standards of sensitivity ( . %) and specificity ( . %) as established by necropsy diagnosis of dogs. the key component of the test, mabeg has a convenient igg isotype and reacts with a periodate-resistant epitope found in high molecular weight components of the worm. time-course analysis of experimentally infected dogs showed that even animals with a very low number of parasites could be detected as early as day post infection. the test was formulated in a ready-to-use kit format with proven stability of each component for a minimum of months at room temperature. this characteristic facilitates its standardized use and shipping to other laboratories, which was demonstrated by the identical results obtained by two different laboratories in peru and our own laboratory when a large number of field samples were analyzed independently in a blind fashion. cystic echinococcosis, caused by infections with echinococcus granulosus, is one of the main zoonoses related to dogs and is affecting most parts of the globe [ ] . different control programs aimed to decrease the impact of the disease have been instrumented in many countries. although the rate of success has been highly variable, it has become evident that a tight control of dog infections is the key element to arrest the life cycle of the parasite. this has created a demand for reliable diagnostic tests for canine echinococcosis as a tool to obtain epidemiological baseline information and help in the surveillance of hydatid control [ ] [ ] [ ] [ ] . however, accurate diagnosis of dog infection is complex and challenging, and other than careful necropsy of dogs, there is no perfect gold standard [ ] . parasitological examination of eggs is unsafe and not very useful because e. granulosus are morphologically difficult to distinguish from other taeniae eggs, and appear late (after the first month) of infection. traditionally, screening of dogs for e. granulosus has been done by arecoline purgation, followed by examination of the purge for parasites by trained personnel. the method is highly specific, but it is tedious, biohazardous, unpopular among dog owners, and its sensitivity is modest, particularly when the parasite burden is low and bowel evacuation is incomplete [ , ] . as an alternative, different laboratory tests for ante-mortem diagnosis of canine echinococcosis have been developed, including detection of antibodies in serum, polymerase chain reaction (pcr) amplification of parasite dna and immunological detection of antigens (coproantigens) in fecal samples. different studies have been carried out to explore the potential use of dog serology to diagnose the disease. however, the systemic immune response to the parasite is poor and consequently the sensitivity attained is also low [ ] . parasite dna excreted with eggs, proglottids or cells has been detected in fecal specimens by pcr amplification using specific primers derived from mitochondrial dna [ , ] . the method has provided the high specificity of pcr, but due to the presence of inhibitors the dna extraction procedure is cumbersome and the technique requires expensive reagents and specialized laboratories [ ] . in addition, the pre-patent period of the infection, when there is no egg production, is a critical time-window that challenges the sensitivity of the test. introduced at the beginning of the 's, the detection of parasite antigens in fecal samples by immunoassays became a widely accepted diagnostic test [ , ] . the major attractive features of the method include its simplicity, the possibility of detecting parasite components even in the prepatent period, and the fact that the target antigens (coproantigens) are highly stable. samples can be collected in % of % formaldehyde and kept at room temperature for extended periods of time, facilitating the logistics of large-scale studies in remote areas [ , [ ] [ ] [ ] . the study of a large group of infected dogs necropsied at the end of the pre-patent period demonstrated a superior sensitivity for the copro-elisa ( %), when compared to copro-pcr ( %) or arecoline purgation ( and % after one or two doses, respectively). most failures to detect positive infected dogs occurred when the number of worms was less than one hundred [ ] . despite its proven utility and the numerous reports of copro-elisa developments, the availability of commercial or homemade tests is often a major obstacle to the implementation and evaluation of echinococcosis control programs. the need to overcome this difficulty has been recognized as an early goal of the southern cone sub-regional project on cystic echinococcosis control and surveillance: argentina, brazil, chile and uruguay, and paho (pan american health organization), a joint and collaborative effort to battle the disease in the region. in this framework, we have developed an immunoassay for e. granulosus copro-antigen detection under the premise that in addition to performing with high standards of proven sensitivity and specificity, it had to be robust, standardized and developed in a kit format to be available for its use in regional programs for the control of the disease. all activities involving animals were performed with special care to establish high standards of biosafety and assure animal welfare. all protocols were performed according to the international guiding principles for biomedical research involving animals, (cioms) and were approved by the comisión nacional de zoonosis and the research department of the ministry of health of the province of chubut. nineteen adult dogs, of mixed breeds and sex, were vaccinated against distemper encephalomyelitis, parvovirosis, rabies, leptospirosis, hepatitis and coronavirus (merial, france), treated orally with praziquantel mg/kg, pyrantel embonate mg/kg and febantel mg/kg (disper, uruguay) to eliminate worm parasites, and topically with a solution of fipronil and methoprene (merial) to arrest the development of ectoparasites. dogs were purchase from local suppliers and maintained on commercial dog food and water ad libitum. nineteen fecal samples (n -n ), corresponding to non-infected dogs, were collected two days after deworming. then, ten dogs (identified as p -p ) were infected orally with , - , viable (. %) protoscoleces from ovine fertile hydatid cysts. two additional dogs (identified as th and th ) were infected orally with and larvae of taenia hydatigena, respectively. parasite material was obtained from sheep slaughter in local abattoirs in paysandú, uruguay. stool samples from each dog were collected daily during the experimental period, at the end which ( - days post-infection (dpi)), the animals infected with e. granulosus were euthanized for necropsy diagnosis by intramuscular injection of acepromazine maleate ( . mg/kg) and ketamine ( mg/kg) (laboratorio holliday, argentina), following by an intravenous overdose of sodium thiopental (laboratorio crusur vet, uruguay). the dogs infected with t. hydatigena were purged at dpi with arecoline bromhydrate (crescent chemical co. inc., new york, usa), following by treatment with anti-parasitic drugs. three additional samples from dogs experimentally infected with e. granulosus ( dpi) and two with t. hydatigena (about dpi) from previous studies [ , ] were obtained as a kind gift from dr. carlos carmona. in addition, unwanted dogs from the province of chubut, argentina, and unwanted dogs from junin, peru, were euthanized following the procedure described above, and the small intestines were removed post mortem, opened longitudinally and examined directly for the presence of parasites following the recommendations of the who/oie [ ] . all activities were performed following the local legislation and the international guiding principles for biomedical research involving animals (http://www.cioms.ch/). stool samples from each dog were collected daily during the experimental infection period, or from the rectum upon necropsy, in % of % formaldehyde, phosphate buffered saline (pbs), in a : v/v ratio. samples were vigorously shaken to obtain homogeneous slurries, and were then boiled for min in a water bath, to eliminate their biological risks. after centrifugation for min at , g the supernatants were aliquoted and kept frozen at uc until used. the parasite antigens were prepared essentially as described by casaravilla et al. [ ] . cystic echinococcosis, caused by infection with the larval stage of the echinococcus granulosus tapeworm, is a lifethreatening zoonosis of worldwide distribution. the adult worm parasitizes the small intestine of dogs, which become infected after eating offal of an animal contaminated with the parasite, and releases eggs into the environment that can be accidentally ingested by domestic animals or humans, maintaining the life cycle of the parasite. deworming of dogs is a major component of control programs, and simple and reliable methods are needed to monitor the base-line infection in the canine population. the lack of these tests was recognized as a major obstacle to the paho effort to control the disease in south america. this paper describes the development of a diagnostic assay that detects parasite antigens in dog feces. the key component is a monoclonal antibody carefully selected to attain high levels of sensitivity and specificity, which were established with a large panel of field fecal samples obtained from animals diagnosed by necropsy. several aspects of the long-term stability of the test were optimized to facilitate its shelf-life and transference to other laboratories. unit with an ym- membrane (millipore, usa), followed by dialysis against pbs. e. granulosus somatic antigen (sm) was obtained by sonication of adult parasites in pbs supplemented with ultra protease inhibitor tablets (roche, indianapolis) on ice, centrifugation and filtration ( . mm). antigens from t. hydatigena were similarly prepared. protein content was determined using a bca kit (pierce, rockford, illinois). to prepare polyclonal antibodies, mg of sm or e/s antigens were dissolved in ml of pbs and vigorously mixed with ml of freund's complete adjuvant (pierce, illinois) to form a thick emulsion. this emulsion was then injected subcutaneously into several points on the back of new zealand white rabbits. after and weeks, the animals were immunized intramuscularly with additional doses of mg of antigen emulsified in freund's incomplete adjuvant. ten days after the final booster the animals were bled. the igg fraction was prepared by precipitation of the sera with ammonium sulfate, affinity purified on protein g agarose (amersham, uppsala, sweden), and kept at uc until used. for monoclonal antibody (mab) preparation, balb/c mice were primed intraperitonally with mg of e. granulosus sm or e/s antigen in freund's complete adjuvant, and boosted after and weeks with the corresponding antigen using freund's incomplete adjuvant. three days after the last booster mice were sacrificed and the splenocytes fused with sp / cells using standard procedures. cultures producing mabs reactive with the corresponding antigen were selected by elisa. we initially performed a fusion experiment using mice immunized with alternate doses of e/s and sm antigens, and additional selection in a sandwich format using fecal samples. mab cpr was selected due to its convenient isotype and performance. several fusion experiments were performed to produce a large panel of monoclonal antibodies. the screening of the monoclonal antibodies was initially performed as described before, and then each of the double-positive supernatants ( clones) was tested in a sandwich format in combination with the different polyclonal antibodies. however, this time instead of using fecal samples from heavily infected dogs, each supernatant was tested against the following: i) a negative sample from a non-infected dog, ii) a sample collected from a low-burden dog at dpi (dog p , worms), iii) a sample from a heavily infected dog taken at an early stage ( dpi) of the pre-patent period (dog p , worms), and iv) a sample from dog th taken at dpi that was positive in the cpr copro-elisa. out of this complex screening clone mabeg , in combination with polyclonal antibody pabc (prepared from a rabbit immunized with e/s), was chosen because this antibody pair produced high signals with samples (ii) and (iii), and low readings with samples (i) and (iv). parasite preparations were resolved by sds-page % under reducing conditions and then transferred onto nitrocellulose sheets (bio-rad, hercules, california, usa). the nitrocellulose was blocked with % non-fat milk powder in pbs h at uc and was incubated for h at uc with a : dilution of the rabbit antisera in pbs containing . % of tween (pbs-t), % non-fat milk, or directly with the culture supernatants of the mabs antibodies. the membranes were then incubated for h at uc with alkaline phosphatase-conjugated to anti-rabbit igg or antimouse igg (pierce) (diluted / ). after extensive washing, a substrate solution containing -bromo- -chloro- -indolylphosphate p-toluidine salt (bcip) and nitro-blue tetrazolium chloride (nbt) was added according to the manufacturer's instructions (sigma). five mg/ml of polyclonal igg ( ml/well) (pab cg or pabc for the mab cpr or mabeg copro-elisa, respectively) was dispensed into microtitration plates (greiner, germany) and incubated overnight at uc. the plates were then blocked with % non-fat milk (pbs) and washed with pbs-t. alternatively, the plates were further treated with pbs, . % bovine serum albumin (bsa), % sucrose, . % sodium azide, flapped repeatedly against adsorbent paper, dried in a % relative humidity chamber for h, and kept in sealed aluminum foil bags containing adsorbent packets (sigma) until used. samples were analyzed in triplicates, after a : dilution in pbs; ml/well were incubated for h at room temperature, the wells washed and loaded ( ml/well) with a : dilution of the mab supernatants and incubated for h at room temperature. finally, the wells were incubated ( h upon necropsy at - dpi, of the experimentally infected dogs p -p dogs harbored e. granulosus worms. the number of worms recovered from each dog varied widely, from to , , and showed no correlation with the initial number of protoescoleces used for infection. no other parasites were observed in addition to the e. granulosus worms. adult worms (typically less than m long) were recovered from dogs th and th infected with t. hydatigena. after careful washing, the recovered parasites were kept in culture to prepare excretion/secretion antigens, or were sonicated as described to obtain somatic antigens. the sds-page analysis of these antigens is shown in figure a ; a protoescoleces extract was also included as a reference antigen. all preparations possessed a complex composition and the sds-page profile did not reveal any obvious similarities among the individual components of the different preparation. initially, the reactivity of polyclonal antibody pabcgb raised against the sm antigen was characterized, figure b . there was a strong response to a large number of components of the immunizing antigen, as well as to high molecular weight components of ege/s and t hydatigena. destruction of sugar epitopes by periodate treatment significantly decreased the reactivity of this antibody with the somatic antigen, as has been observed before [ , ] , but had little effect in the case of the e/s preparation. the marked cross-reactivity of the polyclonal antibodies with e/s components of t. hydatigena evidences how difficult it is to obtain a specific assay using these antibodies as reagents. for that reason we concentrated our efforts in the preparation and selection of monoclonal antibodies after initial screening against e/s and sm and further selection using pab cgb for capture and the hybridoma supernatants for detection, four clones providing the best signal/noise ratios with fecal samples were selected. among these, clone mabcpr , secreting an igg b, was chosen. this elisa was used to examine fecal samples obtained at the end of the infection, days - for e. granulosus or at selected dpi (highest cross-reactivity) for t. hydatigena ( figure ) . the assay performed with negligible reactivity with samples from noninfected dogs and moderate cross-reactivity with t. hydatigena infected animals. all e. granulosus infected dogs were positive, however, the readouts of the samples corresponding to dogs with small parasite burdens were significantly higher, but too close to the mean value of the negative dogs to give unequivocal results. the assay was then used to study the time course of coproantigen excretion during the experimental infection period ( figure ) . using a cut-off derived from the analysis of field samples (see below), we found that dogs with high parasite burdens became positive after two weeks, and in spite of some fluctuations remained so thereafter. on the other hand, dogs with less than worms at necropsy, presented low readouts throughout the experiment, becoming positives only at the end of the experimental infection. along the time course of the examined period, the dogs harboring t. hydatigena worms presented very low values (below the cut-off), showing positive values only in very few days after dpi (not shown). due to the limited sensitivity of the cpr copro-elisa, new antibodies were prepared. the best combination resulted when pab c was used as capture antibody in combination with mab eg . the reactivity of pabc with different parasite antigens in western blot was similar to that of pabcgb (figure b) and it is not shown. mabeg is an igg; its reactivity with different parasite extracts is displayed in figure c . the antibody reacted with a large number of the egsm bands, mostly in the middle to high molecular weight range. two components of and kda are the most prominent immunoreactive bands of the e/s antigens, which appear to be also present in the sm preparation. the crossreactivity with th e/s antigens was negligible (as was also the case with protoscoleces antigens, not shown). treatment with periodate had no effect upon the reactivity of mabeg with any of the antigens, which was unexpected because previous studies have shown that the sugar epitopes have a major role in the immune response against e. granulosus coproantigens [ , ] . to rule out technical artifacts, the efficacy of the periodate treatment was tested in parallel using the monoclonal antibody e /g that defines a sugar epitope highly expressed in e. granulosus protoscoleces preparations [ ] , figure d . when the experimentally-infected dog samples were analyzed with the new assay, it was evident that the eg copro-elisa produced stronger signals with samples from dogs with small numbers of worms, allowing a better discrimination between noninfected and infected dogs ( figure ) . this is an important improvement because most of the reported tests failed to detect these kind of samples. cross-reactivity with t. hydatigena coproanti-gens did not seem to be a major problem when the time course of coproantigen excretion was study with the eg copro-elisa ( figure ). using the cut-off estimated by the receiver operating characteristic (roc) curves from the analysis of field samples (see below), only on very few occasions and after dpi were some of the samples from t. hydatigena infected dogs positive. the eg copro-elisa allowed an earlier detection of infected dogs. animals with or more parasites became positive between - dpi and by dpi all infected dogs were positives and remained so thereafter. curiously, coproantigens in dogs p and p samples could be detected very early. this is more remarkable in the case of dog p that harbored a rather small number of worms ( ) at necropsy, and may be the result of the spontaneous expulsion of worms a few days after infection. the cut-off and cross-reactivity of the assays were evaluated with field samples collected from unwanted dogs in the provinces of chubut, argentina and junin, peru. upon necropsy, of dogs from chubut, and of dogs from junin were positive for e. granulosus, were infected with other parasites and were found negative, table . fecal samples from these dogs were tested using the cpr and eg copro-elisas and the cutoff selected from receiver operating characteristic (roc) curves [ ] to have a convenient balance between sensitivity and specificity as follows: cpr elisa = . au and eg elisa = . au. using these values, the sensitivity of the tests were . and . % respectively, which is in agreement with the results obtained with the experimental infection of dogs, figure . the specificity of the eg test was also superior . % versus . % for the cpr elisa. in addition, the cpr test produced readings close to the cut-off for an important number of samples, figure , which may be problematic for the use of a cutoff internal calibrator, because small inter-assay variations in the reading of the calibrator may have an important effect upon the number of samples that are classified in either category. actually, if samples that fall between % of the cut-off are considered as undetermined (a common practice), none of the samples analyzed with the eg test would fall in this category, while four of the samples analyzed with the cpr would. while it was not possible to obtain an exact counting of parasites in the dogs from the province of chubut, this was feasible in the case of the animals necropsied in junin. although most dogs had a large number of worms, two of them did not, and these two dogs were exaustively examined. we found only e. granulosus and ascaris lumbricoides, and e. granulosus and very few dipylidium caninum worms in the first and second dogs, respectively. interestingly, the average absorbance readings of the fecal samples obtained from these animal were . and . au, which represent strong positive results, indicating the sensitivity of the test and confirming the capacity of the assay to detect small numbers of parasites that had been observed in the experimentally infected dogs. in this regard, when the e. granulosus positive dogs from the experimental infection are included, the overall sensitivity of the test rises to . %. despite the fact that mabeg was selected for its reduced cross-reactivity with the thsm antigen, most of its cross-reactivity was against dogs infected with t. spp (mostly hydatigena) alone or together with other parasites. only one dog infected with t. canis ( . au) and one of the non-infected dogs ( . au) from the province of chubut were classified as positive with the eg elisa, figure . the relatively high readings of these two samples are unexpected. based on the results of the experimental infection of dogs and the fact that all other samples ( ) from dogs harboring t. canis alone or together with dipylidium caninum showed very low readings, a misdiagnosis at necropsy can not be discarded. it is worth noting that cross-reactivity was essentially against taenia. despite the fact that we could not identify to species all field samples, the great majority of samples from chubut and all from junin corresponded to t. hydatigena; hence most false positive results will be related to dogs eating offal. development of the elisa test in a kit format and interlaboratory analysis of blind samples the eg test was then formulated in a kit format to facilitate its use and transference to other laboratories. initially, we studied different conditions for coating and drying the plates and found that using sugars as additives during drying have the best effect on the stability of the capture antibody. trehalose and sucrose showed the best results and the latter was chosen because of its much lower cost. figure a shows that dried plates showed similar capacity as fresh ones to discriminate between weak positive and negative samples over a one year period of storage at uc. similar results were obtained after years of storage (not shown). to set up the value of the cut-off an internal standard was prepared by adjusting the dilution of the e. granulosus sm antigen in order to produce a readout equal to the value of the cut-off. the stability of this cut-off calibrator solution and other components of the assay (in their ready-to-use dilution) were tested using different diluent buffers at uc, room temperature, and uc, and some representative results are shown in figure b-d. different formulations of the calibrator solution were stable over a month period, even at uc; only the results for the sm antigen diluted in pbs, % glycerol, . % kathon (dow chemicals, midland, michigan) are shown. the reactivity of mab eg started to diminish after a few days at uc, but was highly stable over the month period at uc (not shown) or at room temperature, figure c . the most critical component was the secondary antibody. for several different formulations (only two shown in figure d ) all of them rapidly lost the enzymatic activity at uc, but using pbs, . % bsa, . % kathon, . mm tmb, the conjugate remained stable for up to days at room temperature (or uc, not shown). overall, we can conclude that even at room temperature the kit has a safe shelf life of months, which can surely be extended upon refrigeration, which is a convenient advantage for the shipment and use of the kit in remote places. due to the availability of commercial ready-to-use tmb substrate solutions, the preparation of this kit component was not pursued in this study. in case of high demand of the kit, it will be necessary to prepare new pab. in our experience, this is a critical component of the test, but we have selected similarly performing pabs from a panel of to immunized rabbits, after initial selection with fecal samples from dogs harboring low number of e. granulosus parasites and counter selection with samples from t. hydatigena infected dogs. the reproducibility of the results obtained with the copro-elisa kit and the feasibility of its transference were demonstrated by the analysis of blind samples. to this end, fecal samples collected in the province of junin, peru were processed as described, and aliquots were distributed to two laboratories (ins and digesa) in lima, peru, where the copro-elisa kit had been transferred, and to our laboratory in uruguay (facultad de química). the samples were independently analyzed by the three laboratories and classified as positive, negative or uncertain if their readout felt within the calibrator absorbance reading % range. twenty four samples were positive, indicating a prevalence of . % in the studied areas (canchayllo and jauja). the kit performed with excellent reproducibility and all individual samples were equally classified by the three laboratories with the exception of two of the samples with readings close to the cut-off, one of them categorized as negative by two of the labs and uncertain by the third one, and another sample classified as (weak) positive by two of the labs and uncertain by the remaining one. two tests were developed and the difference in their performance highlights the importance that careful selection of antibody pairs has to attain a high standard of sensitivity and specificity. the eg test performed with high sensitivity ( . %) and good specificity ( . %). these parameters are closely similar to previously reported values for other tests. however, it is very important to keep in mind that the only trustworthy comparison of tests is that obtained with a common panel of samples assayed under identical conditions, which highlights the need of interlaboratory studies to compare the performance of existing test. the test cross-reactivity was low, and in addition, since it was basically restricted to t. hydatigena, most false positive results will still indicate access of dogs to offal. a major contribution of our study is the use of a well-characterized mab that assures availability and batch-to-batch reproducibility, as well as the formulation of the assay in a kit format with extended shelf life. the eg test is being shared with other control programs in the region and we hope that it will help to monitor the progress of control programs and standardize the epidemiological baseline information in the region. checklist s stard checklist for the eg copro antigen test. flowchart s stard flow chart detailing the method used to assess the diagnostic performance of the eg copro antigen test. (docx) partial characterisation of carbohydrate-rich echinococcus granulosus coproantigens control of hydatidosis echinococcosis: disease, detection and transmission screening for echinococcus granulosus in dogs: comparison between arecoline purgation, coproelisa and copropcr with necropsy in pre-patent infections echinococcosis: diagnosis and diagnostic interpretation in population studies coproantigens in taeniasis and echinococcosis detection of echinococcus granulosus coproantigens in australian canids with natural or experimental infection copro-diagnosis of echinococcus granulosus infection in dogs by amplification of a newly identified repeated dna sequence polymerase chain reaction for detection of patent infections of echinococcus granulosus (''sheep strain'') in naturally infected dogs copro-dna tests for diagnosis of animal taeniid cestodes coproantigen detection for immunodiagnosis of echinococcosis and taeniasis in dogs and humans detection of echinococcus coproantigens by enzyme-linked immunosorbent assay in dogs, dingoes and foxes coproantigen detection in dogs experimentally and naturally infected with echinococcus granulosus by a monoclonal antibody-based enzyme-linked immunosorbent assay detection of echinococcus granulosus coproantigens in faeces from naturally infected rural domestic dogs in south eastern australia production and characterization of monoclonal antibodies against excretory/secretory products of adult echinococcus granulosus, and their application to coproantigen detection manual on echinococcosis in humans and animals:a public health problem of global concern the meaning and use of the area under a receiver operating characteristic (roc) curve echinococcus granulosus coproantigens: chromatographic fractionation and characterization modulation of the cellular immune response by a carbohydrate rich fraction from echinococcus granulosus protoscoleces in infected or immunized balb/c mice we thank dr. carmona and dr. cecilia casaravilla for their advice and provision of samples. we are also grateful to the medical veterinarians and staff of the comisión nacional de zoonosis, uruguay, for help with the experimental infection of dogs. the personnel and medical veterinarians from the dirección regional de salud de huancayo, peru, are also acknowledged for their help with the pilot study of dog infection. key: cord- -zvu n f authors: compagnone, d.; van velzen, k.; del carlo, m.; mascini, marcello; visconti, a. title: chapter rapid detection of organophosphates, ochratoxin a, and fusarium sp. in durum wheat via screen printed based electrochemical sensors date: - - journal: comprehensive analytical chemistry doi: . /s - x( ) - sha: doc_id: cord_uid: zvu n f publisher summary most of the inhibition bioassays or biosensors for organophosphate and carbamate pesticides are based on the amperometric detection of the enzymatic product of the reaction. applications of amperometric biosensing strategies for pesticide detection in real or spiked food samples have been recently reported. most of the applications have been developed for vegetable matrices. different formats of biosensors have been used: disposable screenprinted choline oxidase biosensors using acetylcholinesterase (ache) in solution were utilized to detect pesticides. screen-printed sensor developed using photolithographic conducting copper track, graphite–epoxy composite, and either ache or butirrylcholinesterase was also used in the analysis of spiked (paraoxon and carbofuran) samples of tap water and fruit juices at sub-nanomolar concentration. additionally, the developed device, which consists of the hand-held potentiostat, the multiplexer for eight-channel control and a dedicated software, can be used to detect organophosphates pesticides, such as dichlorvos and pirimiphos methyl at contamination level below the maximum residue limit settled by the european union and also amplified dna of f. culmorum. durum wheat safety is affected by different threats comprising both abiotic and biotic agents. in the first class, pesticides are widely represented including neurotoxic molecules such as organophosphates and carbamates. organophosphorus (op) compounds are substances widely used in agricultural practices as pesticides having low environmental persistence and high efficacy. these compounds act by inhibition of acetylcholinesterase (ache) activity as neurotoxic agents via an excessive stimulation of the cholinergic receptors in both insects and mammals, including humans [ ] . this can lead to various clinical implications and high acute toxicity [ ] . among organophosphates dichlorvos ( , -dichlorovinyl dimethyl phosphate) and pirimiphos methyl (o-[ -(diethylamino)- -methyl- -pyrimidinyl]o,o-dimethylphosphorothioate) are important contaminants for the durum wheat industry. dichlorvos is one of the most widely used pesticides worldwide in the storage of many products, such as corn, rice, and durum wheat, finding widespread use in most european countries [ ] . european union regulation foresees a maximum residue limit (mrl) for dichlorvos in durum wheat at . mg/g [ ] . dichlorvos is also classified as a probable human carcinogen on the basis of the effects observed in mice and rats. therefore, the u.s. environmental protection agency (epa) proposed cancellation of most uses of dichlorvos and proposed potato, carrot, and sweet pepper samples spiked with aldicarb, propoxur, carbaryl, carbofuran, and methomyl tested with this ache biosensor, exhibited acceptable recoveries ( - %) [ ] . an interesting application on spiked (aldicarb, carbaryl, carbofuran, methomyl, and propoxur) fruit and vegetable samples was based on screen-printed electrodes (spes) chemically modified with a carbon paste mix of cobalt(ii)phtalocyanine and acetylcellulose [ ] . in this paper, an appealing solvent-free extraction procedure of the spiked samples is reported. this was performed by mixing the fruits and passing the resulting juice through a sieve. no effect of the matrices ph on the biosensor performance is reported. screen-printed sensor developed using photolithographic conducting copper track, graphite-epoxy composite, and either ache or butirrylcholinesterase was also used in the analysis of spiked (paraoxon and carbofuran) samples of tap water and fruit juices at sub-nanomolar concentration [ ] . a recent application of a tetracianoquinodimethane (tcnq)-modified spe for the development of a biosensing device for chlorpyrifos methyl was also reported. this method was demonstrated to detect the active molecule both in standard solution and in commercial products (reldan s ) with comparable sensitivity. the analytical protocol was then applied to grapes and vine leaf samples in order to improve safety in wine-making process [ ] . other food samples that have been recently investigated with electrochemical biosensor based on ache inhibition are infant foods. the european union has set a very low limit ( mg/kg) for pesticides in infant food [ ] . an amperometric biosensor that met these limits both for infant food and orange juice has been reported. the method included an oxidation step of phosphorothionates pastiches to produce the oxygenated derivative, which represent the active pesticide molecule. moreover, the biosensors could be regenerated via the recovery of ache activity through a chemical activation with pyridine- -aldoxime methochloride (pam). the biosensors performed well in solvent extract containing water, though they exhibited a reduced recovery in food with a lower water content [ ] . following work by the same group addressed some of the major problems arising when electrochemical biosensors are in contact with food matrices: ph effect and particle effect. both problems were solved treating the biosensor surface with a tween s /phosphate buffer solution (ph . ) after the incubation with pesticide. the treatment was successful in removing the particulate, the correct ph for enzyme activity measurement was attained and the pesticide enzyme inhibition retained. a large number of samples were analyzed and the results were in agreement with reference standard methods [ ] . to our knowledge, there are only few reports on application of screenprinted cholinesterase-based biosensors to food samples. many other amperometric detection schemes and chemistries have been recently investigated that could enhance the overall analytical performance of screen-printed devices. nevertheless, the possibility to successfully apply these approaches to real food samples is strictly linked to their ability to surmount the matrix effect that leads either to complicated multi-step sample preparation or to poor recovery. carbon nanotubes (cnts) are an emerging class of material due to their exceptional structural and electronic characteristics [ ] . the possibility of the promotion of electron transfer at a lower overpotential and their high surface area provide the basis for improving biosensing systems [ ] . they catalyze the electrochemical reaction of important analytes involved in biosensor development such as nadh [ ] , thiols [ ] , and hydrogen peroxide [ ] . moreover, cnts appear to be an interesting material for the immobilization of biological molecules [ ] [ ] [ ] in biosensor applications [ ] [ ] [ ] . the fabrication and evaluation of cnt-derived screen-printed disposable electrochemical sensors based on a cnt ink has been reported [ ] . cnts have recently been used in the development of a disposable biosensor based on thick filmstrips for the sensitive detection of op pesticides. in this work, the dual role of cnt, electrocatalytic activity toward thiocholine and immobilization matrix for the enzyme, is demonstrated leading to the development of a redox mediator-free, simple, and robust single-enzyme biosensor able to detect . nm of paraoxon in solution [ ] . an interesting and emerging technology that has led to astonishing results in op determination is the use of sonochemical fabrication of spe to obtain micro-electrode array. this technology result in the ablation of non-conductive polymer films that coat, and so insulate, underlying conductive surfaces. sensors were fabricated via the electropolymerization of an insulating film at planar electrode surfaces and then ablated with ultra-sound to expose microdiameter scale areas of underlying conductor. a second polymer of polyaniline with conducting properties, carrying co-entrapped ache, may then be electrodeposited in situ at the micro-electrode cavities forming an enzyme micro-electrode array. with this device the authors obtained a stir-independent response, as expected for micro-electrodes able to detect as low as À m of dichlorvos. the authors claim that this astonishing result is due to the combined effect of enzyme inhibition amplification because of the biosensor geometry and the use of recombinant ache selective for the particular pesticide used [ ] . to complete this overview on the strategies that are pursued to enhance the sensitivity and stability of screen-printed biosensors for the detection of pesticides new immobilization approaches have to be mentioned. spe modified with concanavalin a have been used to bind, via a high affinity interaction, ache. the obtained biosensors, optimized for manufacturing conditions, were able to detect À m of clorpyrifos [ ] . vakurov et al. [ ] evaluated a strategy to improve the covalent binding of ache to screen-printed carbon electrodes modified with polyamines. to improve the extent of dialdehyde modification, electrodes were aminated. initially, this was performed by electrochemical reduction of -nitrobenzenediazonium to a nitroaryl radical permitting attachment to the carbon surface; subsequent reduction of the -nitrobenzene yielded a -aminobenzene-modified carbon surface. the obtained biosensors resulted in very sensitive devices measuring as low as À m of ops. the application of such interesting technologies to real food samples has to overcome the difficulties of extraction strategies and the problems arising from matrix components that can affect the biosensor performance. in the recent years, our group have been involved in the realization of biosensing strategies based on spes for the detection of dichlorvos and pirimiphos methyl in durum wheat. the proposed methods were all based on a choline oxidase biosensor obtained by modification of carbon spes able to determine ache activity in solution. in order to avoid cumbersome regeneration steps, we decide to use free cholinesterases rather than immobilized on the choline oxidase biosensor surface. we successfully applied an ache inhibition assay to the detection of dichlorvos in durum wheat samples using a simplified extraction procedure. the aqueous extraction solvent (phosphate buffer) was used as the assay buffer, thus simplifying the overall procedure and limiting the negative effects of organic solvents on enzyme activity. the total assay time, including the extraction step, was min.the choline oxidase biosensor exhibited an excellent stability: after days from preparation, the blank measurement lost only % of the signal intensity. the calculated limits of detection (lods) in buffer solution were and . ng/ml, respectively, using either electric eel ache (eeache) or a recombinant ache specifically engineered to be inhibited by dichlorvos (rache) [ ] . the developed assay was also applied to milled samples. an optimized extraction protocol using exane as extraction solvent exhibited recoveries of dichlorvos at . - . mg/g from . % to . %, with a lod of . mg/g. an aliquot of the filtered hexane extract was partitioned with phosphate buffer solution, and the organic layer was evaporated prior to electrochemical analysis. an lod of . mg/g of dichlorvos was obtained with mean recoveries of - % at spiking levels of . - . mg/g. a good correlation ( . ) was found between the results obtained with the electrochemical and those obtained with the gas chromatographic methods. the electrochemical method was peer-validated in two laboratories that analyzed blind samples (five duplicates), including a blank and four spiked samples with dichlorvos at levels of . , . , . , and . mg/g. within-laboratory repeatability (rsd r ) and between-laboratory reproducibility (rsd r ) ranged from . % to . % and from . % to . %, respectively [ ] . the possibility of detecting a phosphotionate pesticide in durum wheat has been also investigated. the determination was accomplished via chemical oxidation of the phosphothionate molecule both in buffer and in matrix extract solution optimizing the experimental parameters (reagents concentration and reaction time). the procedure was then applied to standardize the pirimiphos methyl detection obtaining the calibration curves under different conditions. the lod with matrix extract was or ng/ml, depending on the extract % addition, which allowed the detection of samples contaminated at the mrl ¼ mg/kg. the samples mean recovery was . % and no false positive samples were detected [ ] . in the last years, there has been a continuous increase in the use of nucleic acid combined with electrochemical transducers to produce a new kind of affinity biosensor. among the different kind of electrochemical sensor formats available, spe based on thick and thin film technology have played an important role. this is surely due to their recognized advantages in terms of cost that allow their disposable use. as well as other molecular-based biosensors dna biosensors rely on highly specific recognition events to detect their target analytes. the role of the transducer is to provide a suitable platform that facilitates formation of the probe-target complex in such a way that the binding event triggers a usable signal for electronic readout. according to iupac definition, a biosensor includes a molecular recognition layer and a signal transducer that can be coupled to an appropriate readout device. dna is especially well suited for biosensing applications, because the base-pairing interactions between complementary sequences are both specific and robust. different strategies of electrochemical sensing have been used for dna electrochemical detection: ( ) direct dna electrochemistry, ( ) indirect dna electrochemistry, ( ) dna-specific redox indicator detection, ( ) dna-mediated charge transport, and ( ) nanoparticle-based electrochemistry amplification. direct dna electrochemistry allows highly sensitive detection of dna, down to femtomoles of target sequence without a labelling step, which simplifies the overall detection protocol, the main limitation of this approach being the high background signals that often limits a simple readout. this strategy has been applied to different kinds of electrodes in terms of geometry and material ranging from the hanging mercury drop electrode [ ] , carbon paste electrode [ ] , to screen-printed carbon transducers [ ] . methods to oxidize target dna indirectly through the use of electrochemical mediators have also been explored. an especially attractive approach uses polypyridyl complexes of ru(ii) and os(ii) to mediate the electrochemical oxidation of guanine [ ] . this detection approach provide high sensitivity without complex instrumentation through redox-mediated indirect dna oxidation, the main limitation consisting in the electrode preparation that can be difficult to handle. several applications have been described in which target dna sequences are labelled with redox-active reporter molecules. appearance of the characteristic electrochemical response of the redox reporter signals the occurrence of the hybridization event. using physical separation methods to isolate the labelled sequences, lods of the order of $ À molecules have been reported [ , ] . as an alternative to chemical labelling schemes, dna-mediated charge transport electrochemistry has been exploited as detection scheme using redox-active molecules that noncovalently associate with the double helix. in these analyzes, rather than serving as a reactant, the dna is the mediator. these assays can provide high sensitivity and simplicity. nanoparticle-based electrochemistry amplification is another detection scheme that has been exploited. in this case, intercalative probe molecules are used taking advantage of the electronic structure of double-helical dna, using intercalated redox probe molecules to report the perturbations occurring in base stacking [ ] . in this format the intercalator is not used to report the amount of dna or whether it is double stranded versus single stranded; here the dna base pair stack mediates charge transport to the intercalator bound to the dna. the method relies on the switch of inherent characteristic of duplex dna and its ability to mediate charge transport is used for the detection. assays of dna-mediated electrochemistry, using both dnamediated charge transport, and nanoparticle-based electrochemistry amplification, are therefore uniquely suited to sense changes in dna: damage, mistakes, mismatches, and even protein binding [ , ] . dna biosensors, based on the summarized schemes, have been proposed in different area of analysis: environmental, clinical, food control, biotechnology, and pharmaceutical. in this report, we review some recent applications of electrochemical dna screen printed based biosensor for the detection of microorganisms including, viruses, bacteria, and fungi. an electrochemical hybridization biosensor based on the intrinsic oxidation signals of nucleic acids (guanine oxidation) and proteins (tyrosine and tryptophan) has been designed, that makes use of the unique binding event between escherichia coli single-strand binding (ssb) protein and single-stranded dna (ssdna). the voltammetric signal from guanine oxidation significantly decreased upon binding of ssb to single-stranded oligonucleotides (probe), anchored on a single-walled cnt-modified screen-printed carbon electrode. simultaneously, oxidation of the tyrosine and tryptophan residues of the ssb protein increased upon binding of the ssb protein to ssdna and ss-oligonucleotides. the lod of . mg/ml of target dna can be applied to genetic assays [ ] . some work devoted to food safety resulted in the realization of an electrochemical biochip able to specifically detect bacillus cereus. b. cereus constitutes a significant cause of acute food poisoning in humans. the dna biosensor was specifically designed using a capture probe for the toxin-encoding genes. a bead-based sandwich hybridization system was obtained in conjugation with electric chips for detection of both vegetative cells and spores of bacillus strains. the system consisted of a silicon chip-based potentiometric cell, and utilizes paramagnetic beads as solid carriers of the dna probes. the specific signals from amol of bacterial cell or spore dna were achieved in less than h. the method could be also applied directly to unpurified spore and cell extract samples. the developed method can offer a contribution in the rapid identification of bacillus strains without preceding nucleic acid amplification [ ] . a recent advancement in dna biosensor consisted in the realization of an electrochemical microfluidic biosensor with an integrated minipotentiostat for the quantification of rna based on nucleic acid hybridization and liposome signal amplification for dengue virus detection. the detection scheme was ensured by short dna probes that hybridize with the target rna or dna sequence. a reporter probe, coupled to liposomes entrapping the electrochemically active redox couple potassium ferri/ferrohexacyanide, was used to amplify the recognition reaction. the capture probes were bound to superparamagnetic beads that were isolated on a magnet in the biosensor. upon hybridization reaction, the liposomes were lysed to release the electrochemical markers. the detection occurred at interdigitated ultramicroelectrode array. the system was completed by a miniaturized instrumentation (miniec). the functionality of the miniec was successfully demonstrated with the detection of dengue virus rna [ ] . a biosensor for virus detection, particularly for sars (severe acute respiratory syndrome) virus, has been described by abad-valle et al. [ ] . the functioning scheme was a typical hybridization biosensor constructed on a gold thin-film electrode. the selected target, a -mer sequence, was chosen in a lysine-rich region, unique to sars virus and the complementary strand, the probe, was immobilized to the gold surface. hybridization reaction with the biotinconjugated sars strand (at the -end) was obtained and monitored using alkaline phosphatase (ap)-labelled streptavidin that permitted amplified indirect electrochemical detection. the analytical signal is constituted by an electrochemical process of indigo carmine, the soluble product of the enzymatic hydrolysis of -indoxyl phosphate. the use of a sensitive electrochemical technique such as square wave voltammetry allowed an lod of pm to be obtained for this dna sequence [ ] . a multiple pathogen detection system based on hybridization coupled with bioelectronic detection was described by vernon et al. [ ] . the method was developed using papillomaviruses as a model system. in this case, two chips were spotted with capture probes consisting of dna oligonucleotide sequences specific for hpv types and two electrically conductive signal probes were synthesized to be complementary to a distinct region of the amplified hpv target dna. the measuring system was able to successfully detect % of the hpv types contained in clinical samples demonstrating the feasibility of integrated detection of multiple pathogens. recently, our group has devoted some research activity to the realization of dna screen-printed biosensors for the detection of fungi [ ] . to our knowledge there is no evidence in the literature of analysis of fungi via electrochemical detection of dna or rna. in our research, we proposed a post amplification analytical method to detect f. culmorum, a pathogen causing ''foot rot'' and ''head blight'' diseases in cereals, and can produce mycotoxins such as zearalenone, deoxynivalenol, and other trichothecenes that can enter the food chain. the early identification of this fungal pathogen is therefore recommended in order to avoid crop losses and protect consumer health [ ] . the sensing principle of this work is based on the hybridization reaction between a probe and the complementary target sequence. the hybridization is followed by the electrochemical detection using a square wave voltammetry (swv). the direct label-free detection was accomplished by monitoring the guanine oxidation peak of the target sequence relying on the use of inosine-modified (guanine-free) probes [ ] [ ] [ ] . the specificity and selectivity of the methods aroused from the selection of four different probes immobilized on sensor surface, complementary to different regions of the amplified sequence. a sequence was identified that is able to grant selectivity and specificity to the sensing system. immunosensors are analytical devices, which selectively detect analytes and provide a concentration-dependent signal [ ] . electrochemical immunosensors employ either antibodies or their complementary binding partners (antigens) as biorecognition elements in combination with electrochemical transducers [ ] . among electrochemical transducers, spe appears as the most useful due to their characteristics [ ] . spes are mass produced and therefore the single device has a low cost as compared to conventional electrodes, hence they can be used in a disposable manner, avoiding cumbersome regeneration steps following the sample measurement. they have a small size thus reducing the volume of sample that can be analyzed ( - ml). some work has been carried out from prof. palleschi's group in rome leading to the development of disposable immunosensor for the determination of domoic acid (da) in shellfish [ ] . da is neurotoxic amino acid responsible for the human syndrome known as ''amnesic shellfish poisoning'' (asp). the method involves the use of disposable spes for the immunosensor development based on a ''competitive indirect test''. da conjugated to bovine serum albumin (bsa-da) was coated onto the working electrode of the spe, followed by incubation with sample (or standard toxin) and anti-da antibody. an anti-goat igg-alkaline phosphatase (ap) conjugate was used for signal generation via the use of an electrochemical substrate, naphtyl phosphate. the enzyme product, naphtol was detected by differential pulse voltammetry (dpv). the immunosensors allowed the detection of mg/g of da in mussel tissue. moving toward toxins produced by fungi, the same group has devised an electrochemical immunosensor for aflatoxin m . the analyzed matrix was milk [ ] . the immunosensors were fabricated by immobilizing afm antibodies directly on the surface of spes, and allowing the competition to occur between free afm and that conjugated with peroxidase (hrp) enzyme. the electrochemical technique used was the chronoamperometry, performed at À mv. results have shown that using spes aflatoxin m can be measured with an lod of ppt and with a working range between and ppt. moreover, the authors claim that interference problems have been addressed for the direct analysis of aflatoxin m in milk. another micotoxin that is widely present in food commodities is ota, -(l-b-phenylalanylcarbonyl)-carboxyl- -chloro- -hydroxy- , -dihydro- r-methylisocumarin. it is produced by several aspergillus and penicillium species. it is considered as secondary toxic metabolite with nephrotoxic, teratogenic, carcinogenic, and immunotoxic activity in several animal species. immunosensing strategies have been proposed to be able to detect ota in different matrix, such as wine [ ] and durum wheat [ ] . in the former matrix, the assay was carried out on carbon-based spes. the immunosensors were developed using polyclonal antibodies. the assay gave an lod of pg/ml and sensitivity of . . ng/ml. the immunosensors were challenged with wine to assess a matrix effect. recoveries obtained were in the - % range. a more challenging matrix was faced in the latter research [ ] carried out in collaborations with authors' groups. in this case, the immunosensors were realized using monoclonal antibodies, and two formats of immunosensors compared. the immunosensor in the direct format was then used for the determination of ota in wheat. samples were extracted with aqueous acetonitrile and the extract analyzed directly by the assay without clean up. the i in real samples was . mg/ l corresponding to . mg/kg in the wheat sample with a lod of . mg/ kg (calculated as blank signal-- s). within-and between-assay variability were less than % and %, respectively. results obtained on naturally contaminated wheat samples were compared with a reference method with a good correlation (r ¼ . ). in this report, we describe the application of spes to the determination of different contaminants in durum wheat samples. all the reported applications have been developed using palmsens hand-held potentiostat equipped with palmsens pc software for the elaboration of current data (palmsens, amsterdam, the netherlands) (fig. . ). the different biosensors were obtained using thick-film spes produced by biosensor laboratory, university of florence and commercialized by palmsens. the electrochemical cell, consisting of a graphite working electrode and silver counter and pseudo-reference electrodes, was printed on a planar polyester substrate (fig. . ) . the applications (screen-printed electrochemical sensors for the detection of ache inhibitor, screen-printed electrochemical dna sensors for identification of microorganisms, and screen-printed electrochemical immunosensors for the detection of toxins) have been finalized to the assembly of a dedicated instrumentation and software able to handle delocalized analysis of ops, fusarium sp. dna, and ota. the instrumentation consisted of the hand-held potentiostat interfaced with a ch multiplexer (palmsens) (fig. . ) that allow different sensor configuration: a. sensorarrays with eight working and eight combined reference/ counter electrodes b. sensorarrays with eight working electrodes sharing a reference and a counter electrode c. sensorarrays with eight working electrodes sharing a combined reference/counter electrode the software front page reported in fig. . enables the choice of the analytical application among pesticides, fusarium sp., and ochratoxin. each application allows the use of eight independent channels that depending on the procedure can be used to perform individual or duplicate analysis. for instance, in the ota application the default setting of the software permit to dedicate two channels for the blank measurement and two channels for each of the three calibrators or samples whereas in the ops protocol the user can utilize single channel or multiple channels. in each application, a check biosensor option is available to test the correct functioning and positioning of the sensors. in ota and ops protocols calibration and determination windows are present. the user can either create his/her own three-point experimental calibration, which can be saved for further use, or load external data to obtain the calibration curve. the concentration levels of the standard solutions are then used by the software to classify the sample result according to a ''traffic light scheme'' (red light for sample exceeding the legal limit, yellow for samples that need analytical confirmation, and green light for negative samples). the fusarium sp. dna application consists of a check biosensor tool and a pcr sample window. the check biosensor enables the control of the probe immobilization procedure, then in the pcr sample window the amplified samples are measured. four out of the eight channels are used as ''negative'' control and four for sample measurement. the software, using an internal algorithm, allows the discrimination between positive and negative samples. experimental details are reported in procedure (cd accompanying this book). both the determinations reported here rely on the inhibition activity of ops pesticides toward ache combined with the detection of the ache enzyme product choline at the surface of a mediator-modified screen-printed choline oxidase electrochemical biosensor. the biochemical-electrochemical pathway used to determine the inhibition consisted of two enzymatic reactions (eqs. ( . ) and ( . )) generating a chemical oxidation (eq. ( . )) that was determined by cathodic chronoamperometry (eq. ( . ) ). the used iron containing electrochemical mediator was the widely used prussian blue. feðiiiÞ þ e À ! feðiiÞðelectrochemical reductionÞ ( . ) standard and sample extract solutions were analyzed according to the following experimental scheme: first, the current intensity of a blank sample extract was measured, and then the current intensity of either the standard or the sample extract was measured. the current intensity of the blank sample (i ) and of the contaminated sample or standard solution (ii) was used to calculate the percent inhibition according to the following formula: electrochemical experiments were carried out using a hand-held potentiostat equipped with dedicated software for the elaboration of current data (fig. . ). the current was sampled min after the reaction started. figure . shows the chronoamperogram where current versus time is plotted. the current shape was reproducible from time to time and hence the precision assured sampling the current, min after the start of the measurement. after min the current was continuously increasing due to the residual activity of ache in solution. the biosensor fig. . . typical amperogram recorded for durum wheat extract at and mg/kg using the hand-held potentiostat. reprinted with permission from ref. [ ] . used to detect the extent of the ache inhibition was a choline oxidase biosensor assembled on thick-film electrode. here we report experimental data obtained using the proposed electrochemical assay with screen-printed choline oxidase biosensor for the detection of dichlorvos in durum wheat samples. as described in p e , two different extraction approaches have been optimized: the former using whole wheat kernels and aqueous extraction and the latter using grounded samples and hexane extraction. both studies were carried out using free cholinesterases because the aim of the protocol was to devise a rapid procedure that did not include cumbersome regeneration steps, which were unavoidable with immobilized ache. the former approach led to a simplified extraction protocol where the extraction solvent was then used as assay buffer. in this application, the use of wild-type electric eel ache and a recombinant ache, specifically selected as very sensitive to dichlorvos, was compared. the effect of the matrix extract was determined by using various sample: solvent ratios, : . , : , : , and : . the optimal extraction ratio, considering the electrochemical interferences and the effect on enzyme activity and bioavailability of the pesticide, was : . the method was calibrated both in buffer and durum wheat extract. the lods in durum wheat samples were . mg/kg for the wild-type ache and . mg/kg for rache. these characteristics allowed the detection of contaminated samples at the legal mrl, which is mg/kg [ ] . moreover, fortified samples of durum wheat were obtained with both dichlorvos and the commercial product didivane, which contains dichlorvos as active molecule. at all the tested levels, the occurrence of contaminant was detected with an average recovery of %. the total assay time, including the extraction step, was min. because several extractions as well as most of the assay steps can be run simultaneously, the throughput for one operator is determinations per hour. in table . , we summarize the results obtained for the fortified samples. samples (n ¼ ) fortified at different levels ( , , , and . mg/kg) were extracted and analyzed with the proposed electrochemical assay using both eeache and rache. for eeache, the recovery for dichlorvos-spiked samples ranged between and %. the incomplete recovery may be due to concurrent causes, such as the use of whole kernels as sample, water-based extraction solvent, and adsorption exerted by the matrix. although the advantage of using the extraction solvent as measuring buffer has an important influence on the speed of the assay, in order to detect dichlorvos at . mg/kg with better accuracy, it was necessary to use the rache; in that case, an i% of . % was obtained, which is well above the minimum detectable inhibition of the method ( %). moreover, the recovery of the samples spiked with didivane was comparable to that obtained with the pure molecule, dichlorvos. the recovery data suggest that the buffer used in the extraction protocol allowed a repeatable recovery at an applicable contamination level of both dichlorvos in pure form and dichlorvos in commercial formulation (didivane), thus avoiding the use of a polar organic solvent such as methanol or acetonitrile, which may influence enzyme activity and sensor stability. despite aqueous-based extraction using whole wheat kernels has been demonstrated as effective to detect dichlorvos, the procedure might lead to inadequate homogeneity of testing samples. moreover, aqueous solvents cannot be used for extraction of ground wheat samples due to the formation of a slurry, preventing filtration of adequate amounts of extract. to apply the electrochemical method to ground wheat samples, as generally required in food analysis, a non-aqueous extraction solvent was required. we have hence tested a number of extraction solvents that could be used with the grounded samples for dichorvos extraction and then easily coupled, via liquid-liquid partitioning with the assay procedure. to perform electrochemical analysis with the choline oxidase biosensor, dichlorvos needed to be transferred to pbs solution, thus avoiding any electrochemical interference by organic solvents. therefore, the filtered hexane extract was submitted to liquid-liquid partitioning with pbs and the upper organic layer was removed by evaporation. finally, the buffer solution containing dichlorvos was analyzed by the biosensor. dichlorvos was easily measured in ground wheat by electrochemical bioassay at levels as low as . mg/g. the method was peer-validated by two laboratories, and the results of the validation test are reported in table . . as shown in table . , mean recoveries of dichlorvos ranged from % to %, rsd r values ranged from . % to . %, and rsd r values ranged from . % to . %. the dichlorvos mean recovery was calculated for each spiking level as the mean of four measurements, n ¼ (table . ). no false negative or false positive results were obtained by the electrochemical assay. a good correlation between dichlorvos concentrations obtained by electrochemical biosensor and gc analysis was also found as shown in fig. . . the correlation coefficient (r) was . . the electrochemical assay for the detection of ache inhibitors outlined above has been optimized for the detection of pirimiphos methylpirimiphos-methyl in durum wheat. pirimiphos methyl is a phosphotionate insecticide and therefore it requires to be transformed in the corresponding oxo-form to act as an effective ache inhibitor, in fact it did not show inhibition of ache in a concentration range - ng/ ml (data not shown). the procedure for the oxidation of pirymiphos methyl via nbromosuccinimide (nbs) and ache inhibition was optimized in buffer solution for reagents, concentration, and inhibition time (see procedure in cd accompanying this book for more details). as a compromise, between analytical performance and overall assay length, a -min incubation of nbs and ascorbic acid (aa) and -min ache incubation was selected. a statistical analysis, using stepwise general least squares analysis (sglsa), of the data demonstrated that only ache incubation time had a significant and positive influence on the inhibition of ache. the method was calibrated both in buffer solution and in durum wheat extract. the intra-electrode rsd (%) ranged between . and . , whereas the inter-electrode rsd (%) was comprised between . and . the lod was ng/ml, and the i % was ng/ml. the assay conditions were then re-optimized to work with durum wheat extracts and calibrations were obtained under different experimental conditions such as sample pretreatment (milled or whole grains) and extract concentration ( % or %). the calibrations were slightly affected by the sample matrix resulting in an increased lod ( - ng/ml) and i % ( - ng/ml). the lod referred to the sample, determined using the best operational condition, was mg/kg. spiked samples were prepared at the eu regulated level ( mg/kg) and analyzed with the optimized protocol resulting in an average recovery of . %. acetone and methanol were tested for the extraction and the effect on the assay compared (fig. . ) . acetone appeared to strongly affect the enzyme activity and was therefore discarded, whereas methanol did not strongly influence the enzyme and hence was used for sample extraction. once the oxidation of the phosphothionate pesticide was optimized, the assay was applied to nine spiked samples resulting in acceptable recovery (table . ). the spiking concentration was mg/kg for all the samples, which correspond to the legal limit settled by the european union. in this application, the samples were extracted using pure methanol, which was then directly diluted in the assay buffer. the assay scheme used to detect ache inhibitors is not selective, as a great number of molecules inhibit ache. the methods here proposed are ''target oriented''; that is the sought for analyte is known to the analyst. this occurs in the situations where the grower must use a certain production protocol, including pesticide treatments, as stated by contract. in the case where the analyte is not known, the assay can be used as a toxicity test able to detect the presence of a total anticholinesterase activity (taa). competitive electrochemical enzyme-linked immunosorbent assays based on disposable spes have been developed for quantitative determination of ota. indirect and direct formats of immunosensors-based assay were developed. a total of ml of ota-bsa in buffer (indirect format) and ml of goat igg (anti-mouse igg) (direct format) in buffer were dispensed on the graphite-based screen-printed working electrodes and kept overnight at c. another ml of % pva solution was used to block the surface for min at room temperature. also, ml of ota monoclonal antibody were added to the electrode surface for min at room temperature. binding or competition was run with ml of ota-ap conjugate or conjugate+standard for min at room temperature. washing was then carried out. the activity of the label enzyme was measured electrochemically by the addition of ml of substrate solution ( mg/ml -naphthyl phosphate in dea buffer; prepared daily), for min at room temperature. the enzymatic product, -naphthol, was detected by differential pulse voltammetry (dpv) using the following conditions: potential range - mv, pulse width ms, pulse amplitude mv, and scan speed mv/s (fig. . ) . the assays were carried out using monoclonal antibodies in the direct and indirect formats. ota working range, i and lods were . - . mg/l and . - . mg/l, . ( . ) mg/l and . ( . ) mg/l, mg/l and mg/l in the direct and indirect assay formats, respectively. the immunosensor in the direct format was selected for the determination of ota in wheat. samples were extracted with aqueous acetonitrile and the extract analyzed directly by the assay without clean-up. the i in real samples was . mg/l corresponding to . mg/ kg in the wheat sample with an lod of . mg/kg (calculated as blank signal-- s). within-and between-assay variability were less than % and %, respectively. a good correlation (r ¼ . ) was found by comparative analysis of naturally contaminated wheat samples using this assay and an hplc/immunoaffinity clean-up method based on the aoac official method . for the determination of ota in barley as reported in table . . fig. . . differential pulse voltammograms obtained for the samples assayed (one representative voltammogram for each of the spiked levels, from . to mg/kg). competition blank signal is relative to ota level ¼ , blank signal is ota-free wheat extract with no ota-ap conjugate used in the assay. reprinted with permission from ref. [ ] . dna electrochemical sensors were developed for the detection of specific sequences of f. culmorum. the sensing principle of the proposed application was based on the affinity interaction between complementary strand of nucleic acids: the probe was immobilized on the sensor surface and the target analyte was free in solution. the hybridization is followed using an swv. the direct label-free detection was accomplished by monitoring the guanine oxidation peak of the target sequence. this approach rely on the use of inosine-modified (guanine-free) probes. in fact, the nonelectroactive base inosine still forms a specific base pair with the cytosine residue. this results in a flat baseline (in the potential region + . - v versus pseudo ag/agcl reference electrode) for the probe-modified electrode. the duplex formation was thus detected through the appearance of the guanine oxidation peak of the target sequence, following hybridization. the use of such a label-free biosensor for the detection of post pcr samples can improve the safety, since the indicators are usually toxic or carcinogenic compounds, and save time. the sensors were first applied to synthetic complementary oligonucleotides, and then to pcr-amplified samples of f. culmorum. the specificity of the genosensors was tested in the presence of different amounts of non-complementary pcr sample. the crucial part of this work was to understand how to increase the occurrence of the hybridization reaction between the probe and the denatured single strand of pcr product. in this application, the probe selection was carried out using a computational approach. computational tools can greatly improve the rational design of the ligands by exploiting different knowledge about the structure and the chemical properties of the target molecule. the computational run was performed by fixing the folding temperature at c with a concentration of sodium of mm to fit the experimental conditions. the other parameters were left as default. an important purpose of this work was to evaluate the relation between experimental and computational data. therefore the probe selection was focused on the study of the steric hindrance of the simulated secondary structure by emphasizing as primary parameter the intrastrand hydrogen bonds within the pcr single strand. for that reason, we selected the probes complementary to the pcr single-strand regions with different intra-strand hydrogen bonds as reported below (fig. . ): -probe complementary to a region with six intra-strand hydrogen bonds ( g-c; a-t) -probe complementary to a region with five intra-strand hydrogen bonds ( g-c; a-t) -probe complementary to a region with three intra-strand hydrogen bonds ( g-c; a-t) -probe complementary to a region with two intra-strand hydrogen bonds ( g-c; a-t) the different probes were used to obtain four independent sensors that were then tested for their selectivity and specificity toward synthetic complementary sequences and pcr samples. in fig. . we report the response curve obtained for probe dna sensor versus different concentration of the complementary strand and versus three non-complementary sequences. thereafter, the different dna biosensors were used to test pcr samples obtained from f. culmorum. the same dna biosensor was also challenged with completely non-complementary dna (table . ). the results exhibited a dose-dependent response up to mg/ml; for higher concentrations a hook effect was observed. in order to avoid false negatives, a control procedure was introduced. the pcr samples were split into two aliquots, one of which was thermally denatured, for the analysis. it was experimentally evaluated that if the denaturated/nondenaturated signal was higher than , the sample could be considered as positive, while for lower ratio the sample has to be classified as negative. particularly, interesting results were obtained using the probes complementary to the regions and . the spe modified with the probe complementary to region exhibited acceptable repeatability (cv within % on three consecutive measurements) with no measurable analytical signal in the presence of different amounts of noncomplementary pcr sample. the applications described above, coupled with the realization of a dedicated portable instrumentation and software, represent a userfriendly analytical tool dedicated to durum wheat safety. moreover, all the applications are based on the use of one single type of thick-film spe facilitating the overall procedure for the final user that has to store and handle one single type of transducer. the developed device, which consists of the hand-held potentiostat, the multiplexer for eight-channel control and a dedicated software, can be used to detect ops pesticides, such as dichlorvos and pirimiphos methyl at contamination level below the mrl settled by the european union, ota, and also amplified dna of f. culmorum. the software has a user-friendly interface that informs the user on the procedural steps that must be performed (e.g., ''add ml of vial a to vial b, mix and transfer on the sensor surface'') allowing the realization of the measurement even for non-trained personnel. all the internal controls result in a ''pass or fail'' message as well as all the incubation times are controlled by the software. at the end of every protocol the user receives a screen message that contains a self-explanative result via a color code according to a ''traffic light scheme''. for dichlorvos detection, the major achievements were the development of two alternative extraction methods using alternatively the assay buffer or organic solvent as extraction mean. the former procedure allowed a simplified protocol, using whole kernels, that could readily be used in field to measure as low as . mg/kg using wild-type eeache and . mg/kg using a specifically designed engineered rache. the latter procedure performed using solvent extraction allowed a complete recovery of the pesticide from grounded kernels. the procedure that can be used in a laboratory environment as screening method allowed the detection at one order of magnitude lower ( . mg/kg) than in the former protocol using wild-type eeache. the method has been peer-validated in an inter-laboratory experiments. a second challenging pesticide was investigated for method development resulting in a procedure, finally transferred on the developed software, for the analysis of pirimiphos methyl in durum wheat. the phosphotionate pesticide was oxidized and transformed in the active oxo form using a dedicated optimized procedure able to function in durum wheat extract. an extraction protocol carried out in methanol was used as this solvent could then be used in the electrochemical assay protocol. the lod referred to the sample, determined using the best operational condition, was mg/kg. spiked samples were prepared at the eu-regulated level ( mg/kg) and analyzed with the optimized protocol resulting in an average recovery of . %. for ota detection, the optimized immunosensors and the protocol that was implemented on the electrochemical device allowed the detection of . mg/kg, with within-and between-assay variability less than % and %, respectively. the method was evaluated with respect to a reference instrumental method (hplc/immunoaffinity clean-up method based on the aoac official method . ) obtaining good agreement (r ¼ . ). finally, an electrochemical dna biosensor able to discriminate between positive and negative pcr-amplified samples of f. culmorum was shown. in conclusion, the overall device enables a thorough control on durum wheat samples in a user-friendly manner, at detection levels that allow an improvement in the control protocols that have to be carried in field by non-specialized personnel. pesticide monitoring database insecticides and acaricides, european directory of agrochemical products commission directive / /ec of rapid detection of organophosphates, ochratoxin a, and fusarium sp safety evaluation of certain food additives and contaminants. zearalenone safety evaluation of certain mycotoxins in food. deoxynivalenol, ht- and t- toxin. fao food and nutrition paper mycotoxins in food some naturally occurring substances. food items and constituents, heterocyclic aromatic amines and mycotoxins (iarc monographs on the evaluations of carcinogenic risks to humans toxicological evaluation of certain food additives and contaminants, who food additives series , world health organization (who) commission regulation no military chemical and biological agents food and agricultural organization of the united nations product yearbook commission directive / /ec of may amending directive / /eec on infant formulae and follow-on formulae rapid detection of organophosphates, ochratoxin a, and fusarium sp the authors wish to thank the miur (dl / july ), sinsiaf project for funding. key: cord- -jat pttf authors: fan, haoyi; zhang, fengbin; wang, ruidong; xi, liang; li, zuoyong title: correlation-aware deep generative model for unsupervised anomaly detection date: - - journal: advances in knowledge discovery and data mining doi: . / - - - - _ sha: doc_id: cord_uid: jat pttf unsupervised anomaly detection aims to identify anomalous samples from highly complex and unstructured data, which is pervasive in both fundamental research and industrial applications. however, most existing methods neglect the complex correlation among data samples, which is important for capturing normal patterns from which the abnormal ones deviate. in this paper, we propose a method of correlation aware unsupervised anomaly detection via deep gaussian mixture model (cadgmm), which captures the complex correlation among data points for high-quality low-dimensional representation learning. more specifically, the relations among data samples are correlated firstly in forms of a graph structure, in which, the node denotes the sample and the edge denotes the correlation between two samples from the feature space. then, a dual-encoder that consists of a graph encoder and a feature encoder, is employed to encode both the feature and correlation information of samples into the low-dimensional latent space jointly, followed by a decoder for data reconstruction. finally, a separate estimation network as a gaussian mixture model is utilized to estimate the density of the learned latent vector, and the anomalies can be detected by measuring the energy of the samples. extensive experiments on real-world datasets demonstrate the effectiveness of the proposed method. anomaly detection aims at identifying abnormal patterns that deviate significantly from the normal behavior, which is ubiquitous in a multitude of application domains, such as cyber-security [ ] , medical care [ ] , and surveillance video profiling [ ] . formally, anomaly detection problem can be viewed as density estimation from the data distribution [ ] : anomalies tend to reside in the low probability density areas. although anomaly detection has been well-studied in the machine learning community, how to conduct unsupervised anomaly detection from highly complex and unstructured data effectively, is still a challenge. unsupervised anomaly detection aims to detect outliers without labeled data for the scenario that only a small number of labeled anomalous data combined with plenty of unlabeled data are available, which is common in real-world applications. existing methods for unsupervised anomaly detection can be divided into three categories: reconstruction based methods, clustering based methods, and one-class classification based methods. reconstruction based methods, such as pca [ ] based approaches [ , ] and autoencoder based approaches [ ] [ ] [ ] [ ] , assume that outliers cannot be effectively reconstructed from the compressed low-dimensional projections. clustering based methods [ , ] aim at density estimation of data points and usually adopt a two-step strategy [ ] that performs dimensionality reduction firstly and then clustering. different from previously mentioned categories, one-class classification based methods [ , , ] make the effort to learn a discriminative boundary between the normal and abnormal instances. although the above-mentioned methods had their fair share of success in anomaly detection, most of these methods neglect the complex correlation among data samples. as shown in fig. , the conventional methods attempt to conduct feature learning on the original observed feature space of data samples, while the correlation among similar samples is ignored, which can be exploited during feature learning by propagating more representative features from the neighbors to generate high-quality embedding for anomaly detection. however, modeling correlation among samples is far different from those conventional feature learning models, in which highly non-linear structure needs to be captured. therefore, how to effectively incorporate both the original feature and relation structure of samples into an integrated feature learning framework for anomaly detection is still an open problem. to alleviate the above-mentioned problems, in this paper, we propose a method of correlation aware unsupervised anomaly detection via deep gaussian mixture model (cadgmm), which considers both the original feature and the complex correlation among data samples for feature learning. specifically, the relations among data samples are correlated firstly in forms of a graph structure, in which, the node denotes the sample and the edge denotes the correlation between two samples from the feature space. then, a dual-encoder that consists of a graph encoder and a feature encoder, is employed in cadgmm to encode both the feature and correlation of samples into the low-dimensional latent space jointly, followed by a decoder for data reconstruction. finally, a separate estimation network as a gaussian mixture model is utilized to estimate the density of the learned latent embedding. to verify the effectiveness of our algorithms, we conduct experiments on multiple real-world datasets. our experimental results demonstrate that, by considering correlation among data samples, cadgmm significantly outperforms the state-of-the-art on unsupervised anomaly detection tasks. in this section, we formally define the frequently-used notations and the studied problem. problem . anomaly detection: given a set of input samples x = {x i |i = , ..., n }, each of which is associated with a f dimension feature x i ∈ r f , we aim to learn a score function u(x i ) : r f → r, to classify sample x i based on the threshold λ: where y i denotes the label of sample x i , with being the normal class and the anomalous class. in this section, we introduce the proposed cadgmm in detail. cadgmm is an end-to-end joint representation learning framework for unsupervised anomaly detection. as shown in fig. , cadgmm consists of three modules named dualencoder, feature decoder, and estimation network, respectively. specifically, the relations among data samples in the original feature space are correlated firstly in form of the graph structure. in the constructed graph, the node denotes the sample and the edge denotes the correlation between two samples in the feature space. then, a dual-encoder that consists of a graph encoder and a feature encoder, is employed to encode both the feature and correlation information of samples into the low-dimensional latent space jointly, followed by a feature decoder for sample reconstruction. finally, a separate estimation network is utilized to estimate the density of the learned latent embedding in the framework of gaussian mixture model, and the anomalies can be detected by measuring the energy of the samples with respect to a given threshold. to explore the correlation among non-structure data samples for feature learning, we explicitly construct a graph structure to correlate the similar samples from the feature space. more specifically, given a set of input samples x = {x i |i = , ..., n }, we employ k-nn algorithm on sample x i to determine its k nearest neighbors n i = {x i k |k = , ..., k} in the feature space. then, an undirected edge is assigned between x i and its neighbor x i k . finally, an undirected graph being the edge set, and x ∈ r n ×f being the feature matrix of nodes. based on the constructed graph, the feature affinities among samples are captured explicitly, which can be used during feature learning by performing message propagation mechanism on them. in order to obtain sufficient representative high-level sample embedding, dual-encoder consists of a feature encoder and a graph encoder to encode the original feature of samples and the correlation among them respectively. to encode the original sample features x, feature encoder employs a l x layers multi-layer perceptron (mlp) to conduct a non-linear feature transform, which is as follows: where z x(lx− ) , z x(lx) , w x(lx− ) and b x(lx− ) are the input, output, the trainable weight and bias matrix of (l x - )-th layer respectively, l x ∈ { , , ..., l x }, and z x( ) = x is the initial input of the encoder. σ(•) denotes an activation function such as relu or tanh. finally, the final feature embedding z x =z x(lx) is obtained from the output of the last layer in mlp. to encode the correlation among the samples, a graph attention layer [ ] is employed to adaptively aggregate the representation from neighbor nodes, by performing a shared attentional mechanism on the nodes: where w i,j indicates the importance weight of node v i to node v j , attn(•) denotes the neural network parametrized by weights a ∈ r d c and w c ∈ r d c ×f that shared by all nodes and d c is the number of hidden neurons in attn(•), || denotes the concatenate operation. then, the final importance weight α i,j is normalized through the softmax function: where n i denotes the neighbors of node v i , which is provided by adjacency matrix a, and the final node embedding z v = {z v i } can be obtained by the weighted sum based on the learned importance weights as follows: given the learned embedding z x and z v , a fusion module is designed to fuse the embeddings from heterogeneous data source into a shared latent space, followed by a fully connected layer to obtain the final sample embedding where w and b are the trainable weight and bias matrix, and ⊕ indicates the element-wise plus operator of two matrices. feature decoder aims at reconstructing the sample features from the latent embedding z f : where zx (lx− ) , zx (lx) , wx (lx− ) and bx (lx− ) are the input, output, the trainable weight and bias matrix of (lx- )-th layer of decoder respectively, lx ∈ { , , ..., lx}, and zx ( ) = z f is the initial input of the decoder. finally, the reconstructionx is obtained from the last layer of decoder: to estimate the density of the input samples, a gaussian mixture model is leveraged in cadgmm over the learned latent embedding. inspired by dagmm [ ] , a sub-network consists of several fully connected layers is utilized, which takes the reconstruction error preserved low-dimentional embedding as input, to estimate the mixture membership for each sample. the reconstruction error preserved low-dimentional embedding z is obtained as follows: where z r is the reconstruction error embedding and dist(•) denotes the distance metric such as euclidean distance or cosine distance. given the final embedding z as input, estimate network conducts membership prediction as follows: where m ∈ r n ×m is the predicted membership of m mixture components for n samples. with the predicted sample membership, the parameters of gmm can be calculated to facilitate the evaluation of the energy/likelihood of input samples, which is as follows: where μ m and Σ m are the means and covariance of the m-th component distribution respectively, and the energy of samples is as follows: where | • | indicates the determinant of a matrix. the training objective of cadgmm is defined as follows: where the first term is reconstruction error used for feature reconstruction, the second is sample energy, which aims to maximize the likelihood to observed samples, the third is covariance penalization, used for solving singularity problem as in gmm [ ] by penalizing small values on the diagonal entries of covariance matrix, and the last is embedding penalization, which serves as a regularizer to impose the magnitude of normal samples as small as possible in the latent space, to deviate the normal samples from the abnormal ones. λ , λ , and λ are three parameters which control the trade off between different terms. the anomaly score is the sample energy e z , and based on the measured anomaly scores, the threshold λ in eq. can be determined according to the distribution of scores, e.g. the samples of top-k scores are classified as anomalous samples. in this section, we will describe the experimental details including datasets, baseline methods, and parameter settings, respectively. -one class support vector machines (oc-svm) [ ] is a classic kernel method for anomaly detection, which learns a decision boundary between the inliers and outliers. -isolation forests (if) [ ] conducts anomaly detection by building trees using randomly selected split values across sample features, and defining the anomaly score as the average path length from a specific sample to the root. -deep structured energy based models (dsebm) [ ] is a deep energy-based model, which aims to accumulate the energy across the layers. dsebm-r and dsebm-e are utilized in [ ] by taking the energy and reconstruction error as the anomaly score respectively. -deep autoencoding gaussian mixture model (dagmm) [ ] is an autoencoder based method for anomaly detection, which consists of a compression network for dimension reduction, and an estimate network to perform density estimation under the gaussian mixture model. -anogan [ ] is an anomaly detection algorithm based on gan, which trains a dcgan [ ] to recover the representation of each data sample in the latent space during prediction. -alad [ ] is based on bi-directional gans for anomaly detection by deriving adversarially learned features and uses reconstruction errors based on the learned features to determine if a data sample is anomalous. the parameter settings in the experiment for different datasets are as follows: in this section, we will demonstrate the effectiveness of the proposed method by presenting results of our model on anomaly detection task, and provide a comparison with the state-of-the-art methods. as in previous literatures [ , , ] , in this paper, precision, recall and f score are employed as the evaluation metrics. generally, we expect the values of these evaluation metrics as big as possible. the sample with high energy is classified as abnormal and the threshold is determined based on the ratio of anomalies in the dataset. following the settings in [ , ] , the training and test sets are split by : and only normal samples are used for training the model. the experimental results shown in table demonstrate that the proposed cadgmm significantly outperforms all baselines in various datasets. the performance of cadgmm is much higher than traditional anomaly detection methods such as oc-svm and if, because of the limited capability of feature learning or the curse of dimensionality. moreover, cadgmm also significantly outperforms all other deep learning based methods such as dsebm, dagmm, anogan, and alad, which demonstrates that additional correlation among data samples facilitates the feature learning for anomaly detection. for small datasets such as arrhythmia, we can find that traditional methods such as if are competitive compared with conventional deep learning based method such as dsebm, dagmm, anogan, and alad, which might because that the lack of sufficient training data could have resulted in poorer performance of the data hungry deep learning based methods, while cadgmm is capable of leveraging more data power given the limited data source, by considering the correlation among data samples. in this section, we study the impact of noise data for the training of cadgmm. to be specific, % of randomly split data samples are used for testing, while the rest % combined with % to % anomalies are used for training. as shown in table , with the increase of noise data, the performance of all baselines degrade significantly, especially for oc-svm, which tends to be more sensitive to noise data because of its poor ability of feature learning on high-dimensional data. however, cadgmm performs stable with different ratios of noise and achieves state-of-the-art even % anomalies are injected into the training data, which demonstrates the robustness of the proposed method. in this section, we evaluate the impact of different k values during the graph construction on cadgmm. more specifically, we conduct experiments on all three datasets by varying the number of k from to , and the experimental results are illustrated in fig. . during training, the batch sizes are set as , , and for kdd , arrhythmia, and satellite, respectively, the experimental results show that the changing of k value causes only a little fluctuation of performance on all datasets with different settings, which demonstrates that cadgmm is less sensitive to the k value and easy to use. in order to explore the quality of the learned embedding, we make a comparison of the visualization of sample representation for different methods in fig. . specifically, we take the low-dimensional embeddings of samples learned by dagmm and cadgmm, as the inputs to the t-sne tool [ ] . here, we randomly choose data samples from the test set of kdd for visualization, and then we generate visualizations of the sample embedding on a twodimensional space, in which blue colors correspond to the normal class while orange the abnormal class. we can find that cadgmm achieves more compact and separated clusters compared with dagmm. the results can also explain why our approach achieves better performance on anomaly detection task. in this paper, we study the problem of correlation aware unsupervised anomaly detection, which considers the correlation among data samples from the feature space. to cope with this problem, we propose a method named cadgmm to model the complex correlation among data points to generate high-quality lowdimensional embeddings for anomaly detection. extensive experiments on realworld datasets demonstrate the effectiveness of the proposed method. enhancing one-class support vector machines for unsupervised anomaly detection uci machine learning repository anomaly detection: a survey one-class svm for learning in image retrieval principal component analysis robust kernel density estimation improving one-class svm for anomaly detection isolation forest. in: icdm visualizing data using t-sne robust feature selection and robust pca for internet traffic anomaly detection using an ensemble of one-class svm classifiers to harden payload-based anomaly detection systems unsupervised representation learning with deep convolutional generative adversarial networks unsupervised anomaly detection with generative adversarial networks to guide marker discovery real-world anomaly detection in surveillance videos fast anomaly detection for streaming data graph attention networks group anomaly detection using flexible genre models robust pca via outlier pursuit cxnet-m : anomaly detection on chest x-rays with imagebased deep learning adversarially learned anomaly detection deep structured energy based models for anomaly detection anomaly detection with robust deep autoencoders deep autoencoding gaussian mixture model for unsupervised anomaly detection this work was supported in part by national natural science foundation of china (no. , ) . key: cord- -dw qsl s authors: porter, emily; tasker, séverine; day, michael j; harley, ross; kipar, anja; siddell, stuart g; helps, christopher r title: amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis date: - - journal: vet res doi: . / - - - sha: doc_id: cord_uid: dw qsl s recent evidence suggests that a mutation in the spike protein gene of feline coronavirus (fcov), which results in an amino acid change from methionine to leucine at position , may be associated with feline infectious peritonitis (fip). tissue and faecal samples collected post mortem from cats diagnosed with or without fip were subjected to rna extraction and quantitative reverse-transcriptase polymerase chain reaction (qrt-pcr) to detect fcov rna. in cats with fip, % of tissue, and % of faecal samples were pcr-positive, as opposed to % of tissue, and % of faecal samples in cats without fip. relative fcov copy numbers were significantly higher in the cats with fip, both in tissues (p < . ) and faeces (p = . ). pcr-positive samples underwent pyrosequencing encompassing position of the fcov spike protein. this identified a methionine codon at position , consistent with the shedding of an enteric form of fcov, in % of the faecal samples from cats with fip, and in % of the samples from cats without fip. in contrast, % of the tissue samples from cats with fip and % from cats without fip had a leucine codon at position , consistent with a systemic form of fcov. these results suggest that the methionine to leucine substitution at position in the fcov spike protein is indicative of systemic spread of fcov from the intestine, rather than a virus with the potential to cause fip. feline coronavirus (fcov) infection is ubiquitous in domestic cats, particularly in multi-cat households where up to % of animals may be infected [ ] [ ] [ ] . the majority of fcov infections are asymptomatic or are associated with mild enteric disease [ ] . however, approximately - % of infected cats develop the invariably fatal disease, feline infectious peritonitis (fip) [ ] [ ] [ ] . one of the most important questions in fcov research is why some fcov-infected cats develop fip, whereas others remain healthy. one current model of fip pathogenesis proposes that cats are infected with fcov by the faecal-oral route. subsequently, the virus mutates into the virulent form. this form has an enhanced tropism for monocytes/macrophages, and in vitro studies suggest that this is reflected as sustainable replication in, and subsequent activation of, monocytes [ , ] . these activated monocytes carry the virus in the blood and, as a result of complex interactions with endothelial cells, induce the granulomatous phlebitis that is the pathogenic hallmark of fip [ , ] . the age, breed, gender, reproductive status and immune response of individual cats also influence the development of fip [ ] . currently, there is intense interest in determining which mutations alter the virulence of fcovs. a recent paper published by chang et al. [ ] derived full genome sequence data from a collection of fcovs obtained from the faeces of healthy cats and from the tissues of cats diagnosed with fip. they provided evidence of an association between fcov virulence and an amino acid substitution (methionine to leucine at position , m l) within the putative fusion peptide of the fcov spike (s) protein. specifically, the authors concluded that the m l substitution distinguished fip from non-fip associated fcovs in % of cases. a second substitution, two amino acids downstream of m l (serine to alanine at position , s a) distinguished a further % of fip from non-fip associated fcovs. the s protein fusion peptide is a critical element in the fusion of viral and cellular membranes during virus entry [ ] and it is reasonable to think that amino acid substitutions within this peptide may alter the tropism of the virus. in addition, a study by licitra et al. has shown that it is possible to distinguish between fcovs from animals with and without fip on the basis of one or more substitutions in the amino acid sequence that comprises the furin cleavage motif within the s protein [ ] . this furin cleavage site (consensus motif r-x-k/r-r, where r is the basic arginine residue, x is any residue and k is the basic lysine residue) delineates the border of the receptor-binding (s ) and fusion (s ) domains of the s protein and is distinct to the m l substitution site described above. mutation at this site is proposed to alter proteolytic cleavage of the s protein and modify s protein fusogenic properties, which again may relate to the tropism of the virus [ ] . finally, pedersen et al. [ ] concluded that truncating and non-truncating mutations in the c gene occur in a significant proportion of fcovs associated with fip. chang et al. [ ] suggest that functional c protein expression is crucial for fcov replication in the gut but is dispensable for systemic replication. however, they also do not exclude the possibility that the loss or alteration of the c protein may enhance the fitness of the virus in the monocyte/macrophage environment. over the past years, the university of bristol has collected a large number of post-mortem tissue and faecal samples from a cohort of thoroughly examined cats. these include cats with a definite diagnosis of fip, confirmed by the presence of the typical histological fip lesions, in which immunohistochemistry (ihc) demonstrated fcov antigen within macrophages [ ] , and cats with diseases other than fip that completely lacked any histological changes consistent with fip. importantly, this long-term study has enabled both faecal and tissue samples to be collected from fcov-infected cats with and without fip, allowing comparable samples from naturally infected cats to be examined. samples were screened for fcov rna by quantitative reverse transcriptase-polymerase chain reaction (qrt-pcr) [ ] and, if positive, were assessed for the m l substitution by pyrosequencing. post-mortem tissue samples, and faeces whenever possible, were collected from cats that were euthanized with suspected fip, or due to other diseases. fip was then definitively diagnosed or excluded by histopathology and, in the case of fip, the demonstration of fcov antigen in fip lesions by ihc [ ] . tissues of cats without fip were also tested by ihc for the presence of viral antigen. tissue samples were collected into rnalater (life technologies) within h of death for subsequent molecular analysis. the tissue samples were left in rnalater for - h at room temperature or °c before the rnalater was discarded, and the tissue samples stored at − °c. the faecal samples were stored at − °c until use. further samples were collected into % neutral-buffered formalin for histology and ihc. the tissues collected comprised primarily mesenteric lymph node, liver, kidney, spleen and omentum, while other tissues (e.g. intestine, brain, lung, pericardium, pancreas or other lymph nodes) were included based on gross pathological findings or reported clinical signs. the formalin-fixed tissue samples were subjected to standard processing for histopathology. they were embedded in paraffin wax and sections prepared and stained by haematoxylin-eosin. sections were examined by a boardcertified veterinary pathologist (mjd) at the university of bristol for histopathological changes. selected wax blocks were then sent to veterinary laboratory services, university of liverpool for ihc analysis as previously described [ ] . for a cat to be assigned to the "fip group", it needed to have histopathological changes consistent with fip in which fcov antigen was demonstrated within macrophages in lesions [ ] . for a cat to be assigned to the "non-fip group", histopathological changes consistent with fip needed to be completely absent, and fcov antigen within macrophages needed to be absent for all tissues. only individual tissue samples with a positive qrt-pcr result, and lesions consistent with fip on histopathology were used for pyrosequencing. faecal samples were classified on the basis of the diagnosis attributed to the cat from which the sample originated. total rna was extracted from mg of tissue or mg of faeces with a nucleospin rna ii kit (macherey-nagel) using methods based on previous work by dye and siddell [ , ] . reverse transcription was done using a mj mini gradient thermal cycler and improm ii reverse transcriptase (promega). nine microlitres of total rna solution were combined with μl improm ii × reaction buffer, . μl mm mgcl , μl dntps ( mm each), μl random hexamers ( . μg/μl) and μl improm ii reverse transcriptase. the reaction was made up to a total volume of μl with rnase-free water. the following thermal profile was used; °c for min, °c for min, °c for min and °c hold. the resulting μl of cdna was added to μl of rnase-free water and stored at − °c. randomly selected samples were checked for inhibition of the rt reaction using an rna internal amplification control. no inhibition was detected (results not shown). the qpcr was done on an agilent mx p qpcr system (agilent technologies). a qpcr master mix was made for each reaction with . μl × gotaq master mix (promega), . μl of μm forward and reverse primer (p /p ) ( table ) , . μl of μm taqman probe (table ), . μl mm mgcl and made up to μl with rnase-free water. the primers and probe were produced by metabion (metabion international) and were described previously by dye et al. [ ] . one-tenth of the randomly primed cdna ( μl) was added to the pcr master mix. the reaction plate was heat sealed and the following thermal profile was used: °c for min, cycles of °c for s, °c for s and °c for s. fluorescence was detected at nm during the extension phase. feline cov cdna was used as a positive control and rnase-free water as a negative control. reactions that failed to reach the threshold cycle (ct) value by cycle were deemed to be negative. a ct value of was assigned a relative copy number of [ , ] , and the following equation, which takes into account the % efficiency of the qrt-pcr assay [ ] , was used to calculate the relative copy number of each qrt-pcr positive sample: . ( -ct value) . all samples that were positive by fcov qrt-pcr underwent conventional pcr to amplify a base-pair dna fragment encompassing position in the s protein gene. pcr was done using a mj mini gradient thermal cycler. briefly, for each reaction, a pcr mix was made that included . μl × gotaq master mix, . μl of μm forward and reverse primer (f /r ) (table ) , μl of randomly primed cdna reaction products and water to a volume of μl. the following thermal profile was used; °c for min, cycles of °c for s, °c for s and °c for s, before being held at °c. the pcr products were used for the pyrosequencing reaction or stored at - °c until required. samples that failed to produce definitive sequence data were pyrosequenced, following repeat amplification using the same pcr protocol with cycles of amplification. single strand sequencing templates were produced by binding the biotinylated pcr product to streptavidincoated sepharose beads (fisher), followed by chemical denaturation and neutralisation. for each sample, the following mix was prepared; μl streptavidin beads, μl pyromark binding buffer (qiagen) (ph . containing mm tris-hcl, m nacl, mm edta, . % tween ) made up to μl with water. the bead mixture was added to the pcr product and shaken at rpm for min. the sequencing primer mix contained . μl μm sequencing primer (table ) and . μl pyromark annealing buffer (qiagen) ( mm tris-oac, mm mg-oac ph . ) for each sample. the control oligonucleotide (a self-sequencing oligonucleotide) mix contained μl μm oligonucleotide c and μl annealing buffer. twenty five microlitres of the sequencing primer mix was added to the appropriate wells of a pyrosequencing plate, μl of control oligonucleotide was added to one well, and the plate was placed on the pyrosequencing workstation. the pyrosequencing workstation was prepared with trays containing wash buffer ( mm tris-oac ph . ), denaturing buffer ( . m naoh), % ethanol and distilled water. the streptavidin bead bound pcr product was taken up using a vacuum pump and the pyrosequencing bead collector. the bead collector was then placed in the % ethanol for s, in the denaturing buffer for s and in the wash buffer for s. after turning off the vacuum, the bead collector was placed in the pyrosequencing plate and agitated for s to dislodge the beads. the pyrosequencing plate was heated on a plate holder at °c for min, before being placed into the pyromark q (qiagen) and left to cool for min. while the plate was cooling, the pyrosequencing cartridge was prepared. pyromark gold q enzyme, substrate and dntps (qiagen) were added into the appropriate wells of the cartridge. volumes were as outlined by the pyromark q software. during the experimental set up, the dispensation order of the nucleotides was defined as; cgctcatg. the cartridge was placed into the pyromark q instrument and the protocol run. all primers used in the pyrosequencing assay were designed using a combination of pyromark assay design software (qiagen), primer ' software [ ] and mfold [ ] , and were made by eurofins (mwg operon) ( table ). the primer positions were based on those used by chang et al. [ ] , and numbered according to the fcov c je genome [genbank:dq ]. degeneracies were added to the primers, and the location of the primers optimised, based upon a sequence alignment comprised of all available type i fcov genomes (data not shown). conventional pcr to amplify a base-pair dna fragment encompassing position in the s protein gene was carried out as described above, (see pyrosequencing methods) on samples that did not show a m l substitution in the pyrosequencing assay. the pcr primers (f /r ) were then used as sequencing primers in a standard sanger sequencing protocol (eurofins, mwg operon). the fcov relative copy numbers were entered into a database (excel , microsoft) and exported into ibm spss statistics software (version . ). the data sets were evaluated for normal distribution using the kolmogorov-smirnov (k-s) test. non-normally distributed data were described as median and range (minimum and maximum values). data evaluating fcov relative copy numbers in tissue and faecal samples from cats with and without fip were analysed using a multilevel modelling approach (mlwin v . ) [ ] , to account for the repeated measures within cats, and a non-parametric mann-whitney u test. the conclusions drawn from both analyses were in full agreement, so the simpler mann-whitney u test analysis is presented here. relative copy numbers were compared between the fip and non-fip samples for tissue and faecal samples combined, for faecal samples only, and for tissue samples only. significance was assigned at a level of p < . . historical samples were collected with full informed consent from owners that samples could be used for research purposes. the project has been approved under ethical review by the university of bristol animal welfare and ethical review board (vin/ / ). a total of samples were analysed and full details of the samples and results are shown in table . in cats with fip, the diagnosis was confirmed by histopathology and the demonstration of fcov antigen within macrophages in fip lesions by ihc. in cats without fip, the diagnosis was made by histology; neoplasia (e.g. lymphoma, astrocytoma, chemodectoma, or biliary cystadenoma), and inflammatory processes (e.g. chronic lymphoplasmacytic infiltrates of unknown aetiology in liver and kidney, and bronchopneumonia) predominated. a total of faecal samples were analysed by fcov qrt-pcr, and ( %) were positive. these comprised of ( %) faecal samples from cats with fip, and of ( %) faecal samples from cats without fip (table ) . a total of tissue samples were analysed by fcov qrt-pcr, and ( %) were positive. these comprised of ( %) tissue samples from cats with fip, and of ( %) tissue samples from cats without fip (table ) . relative fcov rna copy numbers were not normally distributed (p < . ). the relative copy numbers in pooled faecal and tissue samples in the fip group (median; range: ; - ) were significantly higher (u = . , p < . ) than in the non-fip group ( , - ). when only tissue samples were considered, the relative copy numbers in the fip group ( ; - ) were also significantly higher than those in the non-fip group ( ; - ) (u = , p < . ). finally, analysis of faecal samples alone showed that the relative copy numbers in the fip group ( ; - ) were significantly higher than those in the non-fip group ( ; - ) (u = . , p = . ). the faecal and tissue samples with positive qrt-pcr results were subjected to the pyrosequencing assay. of the faecal samples successfully sequenced, were obtained using the cycle pyrosequencing assay, whereas required the cycle assay. of the tissue samples successfully sequenced, were obtained using the cycle pyrosequencing assay, whereas required the cycle pcr. in of the ( %) faecal samples positive by qrt-pcr, a methionine (aug) codon alone was found at position (table and figure a ). in of the ( %) samples, a leucine codon (uug) alone was found at position (table and figure b) . additionally, ( %) sample (cat , faeces) showed a mixed population of rna coding for either methionine (aug) or leucine (cug) at this position (table and figure c ). importantly, methionine and leucine codons were identified in faecal rna samples from both cats with and without fip (table ) . specifically, a methionine codon was identified in samples from fip cats, and a leucine codon was identified in samples from fip cats. overall, a methionine codon was found in the majority ( / ; %) of faecal samples from cats with fip, but a significant number had a leucine codon ( / ; %). similarly, a methionine codon was identified in samples from cats without fip (including one mixed infection), and a leucine codon was identified in sample from cat without fip (which had the mixed infection). overall, a methionine codon was found in all faecal samples from cats without fip, and sample in cat had a leucine codon ( / ; %, which also had a methionine as a mixed infection). in of the ( %) tissue samples positive by qrt-pcr, a leucine codon ( uug, cug) alone was found at position (table and figures a and b ). in the remaining of the ( %) tissue samples, a methionine codon (aug) was found at this position ( table and tissue types are defined by superscripts: a omentum, b liver, c kidney, d lung, e stomach, f lymph node, g cerebrum, h abdominal lymph node, i heart, j spleen, k colon, l mesenteric lymph node, m brain, n mesentery, o pleura, p pericardium, q medulla, r pons, s small intestine, t intestine. the amino acid coded at position in the s protein of fcov rna from tissue samples without the m l substitution is defined by the superscripts: ucu (ser) and gcu (ala). figure c ). no mixed infections were found in tissue samples. importantly, leucine and methionine codons were identified in rna from tissue samples from both cats with and without fip (table ) . specifically, a leucine codon was identified in samples from fip cats, and a methionine codon was identified in samples from fip cats. overall, a leucine codon was found in the majority ( / ; %) of fip tissue samples, but a significant number had a methionine codon ( / ; %). similarly, a leucine codon was identified in samples from cats without fip, and a methionine codon was identified in sample from cat without fip. overall, a leucine codon was found in the majority ( / ; %) of tissue samples from cats without fip, with a minority ( / ; %) having a methionine codon. in one specific case (cat ), we noted that the sample from one tissue (spleen) contained a leucine codon, whereas samples from three other tissues (liver, lung and mesenteric lymph node) all contained a methionine codon. conventional rt-pcr amplification products of rna from tissue samples that did not contain the m l substitution (from cats , and ) were analysed by sanger sequencing for the s a substitution. one of the samples (cat , pleura) showed an alanine codon at position . the remaining four samples ( from cat and three from cat ) all showed a serine codon at this position (table ). the most important finding in this study is that the m l substitution in the fcov s protein does not correlate with fip disease phenotype, as was proposed by chang et al. [ ] . we reach this conclusion because of two observations. first, although a leucine codon was found in the majority ( %) of tissue samples with fip lesions, a leucine codon was also found in the majority ( %) of non-fip tissue samples. second, a significant number ( %) of fip tissue samples had a methionine codon at this position. tissue samples from naturally fcov infected cats without fip have not been previously evaluated and provide an important insight into fcov infection in the absence of fip. we believe that the m l substitution is more likely to be a marker of systemic fcov infection, as opposed to a marker of fip or the development of disease. as our tissue samples were collected post-mortem, we cannot exclude the possibility that the cats without fip ( samples, from cats , , , , and ) and the leucine codon at position , would have gone on to develop fip if they had not been euthanized due to other reasons. however, histopathological changes consistent with fip were absent in all cats, and ihc did not identify fcov antigen. the finding that the majority of tissue samples from both cats with and without fip have a leucine codon at position does not challenge the idea that systemic spread of fcov is an important step in the development of fip. indeed, the latter is supported by the findings of our study, since fcov rna was found in a far greater proportion of tissue samples with fip lesions ( %) than tissue samples from cats without fip ( %), and, in those samples that were qrt-pcr positive, significantly higher fcov relative copy numbers were found in the fip samples, as has been found in a previous study in naturally infected cats [ ] . however, as sampling in our study took place post-mortem, it could also be argued that the elevated fcov levels in fip tissues were a consequence of the massive immunological dysregulation that results from the disease, rather than a contributing factor towards the development of disease. another viewpoint is that the low levels of fcov rna from the tissues of cats without fip are a result of fcov infecting only fully differentiated macrophages and monocytes, among which are tissue-specific macrophages. this view is supported by our results as ihc, a detection method of low sensitivity, did not detect fcov antigen anywhere in the pcr-positive tissues from cats without fip, providing further evidence of low level viral infection either of tissue macrophages (in persistently infected animals) or in monocytes that were in vessels in the respective organ at the time of sampling [ ] . these findings are also in accordance with recent in vivo and in vitro studies that showed only the virulent form of fcov can effectively and sustainably replicate in monocytes [ , , ] . the m l substitution was not the only s protein substitution that was proposed by chang et al. [ ] to correlate with the fip disease phenotype. they also showed that a second substitution, s a, distinguished a further % of fip from non-fip associated fcovs. we confirmed this result in so far as rna obtained from one of five tissue samples that did not show the m l substitution showed the s a substitution. we believe that, as was proposed by chang et al. [ ] , changes such as the m l and s a substitutions, and potentially others, could be representative of a class of mutations that influence the fusogenic activity of the fcov s protein and, as such, deserve particular attention with regard to the pathogenesis of fip [ ] . it is noteworthy that the substitutions identified by licitra et al. [ ] that are proposed to distinguish between fcovs from animals with and without fip are also suggested to have an effect upon the fusogenic activity of the s protein. with regard to faecal samples, our study found that an unexpectedly high percentage ( %) of faecal samples from cats with fip were fcov qrt-pcr positive and their relative copy numbers were significantly higher than those of faecal samples from cats without fip. moreover, the majority ( %) of fcov rna sequences in faecal samples from cats with fip had a methionine codon at position in the fcov s protein gene, suggesting that these animals were shedding an enteric form of the virus. it seems reasonable to suggest that these cats were infected with an enteric, and a systemic, virulent form of fcov. whether one form was derived from the other following a single infection or whether these cats were infected twice with different fcovs cannot be determined. it has been proposed that the severe immune dysregulation in cats with end-stage fip might create an opportunity for super-infection by enteric fcov circulating in surrounding carriers [ ] . more interestingly, a smaller but significant proportion of faecal samples from cats with fip ( %) provided fcov rna samples that encoded leucine at position . the current model of fip pathogenesis proposes that once the enteric form of the virus has mutated to a virulent form, it is generally no longer horizontally transmitted via the faeces [ , ] . this view has been challenged [ ] , and the current results also suggest that a systemic form of the virus can be found in the faeces of fip cats. however, we accept that this does not mean that excreted virus is necessarily able to infect further cats by the enteric route, as has recently been shown in some experimental studies [ ] . further research is necessary to resolve these issues. in contrast to the pattern shown by the analysis of faecal samples from cats with fip, the analysis of the faecal samples from cats without fip seemed more straight-forward. all "non-fip" faecal samples that were fcov qrt-pcr positive encoded methionine at position , indicative of infection with the enteric form of the virus. also, as found in our study, a shedding proportion of % by cats without fip is not unexpected [ , ] . an interesting faecal sample from a cat without fip (cat ) showed a mixed population of rnas encoding for either methionine or leucine at position . there was no evidence of fcov rna in the single tissue sample taken from cat , and, therefore, one interpretation could be that the m l substitution in the faecal sample was a relatively recent event and the virus had not yet spread systemically. however, this interpretation has to be considered as tentative because we have observed examples of negative qrt-pcr results in tissue samples from cats that were clearly fcov infected due to their fip grouping; cat , liver; cat , liver. as histopathology results and ihc for these two liver samples showed changes consistent with fip, these negative qrt-pcr results are likely to have arisen due to an absence of fcov in the particular samples taken for molecular analysis, as a variable distribution of fcov in affected tissues has been reported [ ] . our study importantly also demonstrated that a pcrbased pyrosequencing [ ] approach is a rapid and accurate method to identify single nucleotide differences at a specific position within a dna fragment, or in our case a viral genome. however, it has some limitations. some samples required , rather than , cycles of pcr amplification to generate adequate amounts of dna for sequencing. this was especially true for pcr products generated from samples that contained low amounts of viral rna, e.g. tissue samples from cats without fip. there were also several samples which, despite containing quantities of viral rna measurable by qrt-pcr, did not produce sufficient pcr products for pyrosequencing, even after amplification cycles. these samples were excluded from the results as they did not contribute any additional sequence data to the study. however, one explanation may be that, despite the degeneracy of the primers used (f /r ), differences in the viral primer binding sites may have precluded efficient amplification in these samples. in summary, we have used a pyrosequencing assay to determine the distribution of a specific m l substitution in the s protein of fcov rna obtained from a large number of post-mortem tissue and faecal samples from cats with and without fip. additionally, sanger sequencing was used to determine whether the s a substitution was present in tissue samples that did not contain the m l substitution. this represents the first study that compares similar samples from cats with and without fip with regard to the viral phenotype. our results contribute to a better understanding of fcov genomic mutations and how they may, or may not, be used as markers of the virus phenotype. the results also make clear that the relationship between the viral genotype and the development of fip is complex. we are currently using an approach that involves the deep sequencing of complete fcov genomes in clinical samples, in order to throw further light on this relationship. feline coronavirus antibodies in uk cats serologic studies of naturally occurring feline infectious peritonitis isolation and characterization of viruses related to the sars coronavirus from animals in southern china feline infectious peritonitis. abcd guidelines on prevention and management clustering of feline coronaviruses in multicat households common virus infections in cats, before and after being placed in shelters, with emphasis on feline enteric coronavirus a study of naturally occurring feline coronavirus infections in kittens activation of p mapk by feline infectious peritonitis virus regulates pro-inflammatory cytokine production in primary blood-derived feline mononuclear cells replication of feline coronaviruses in peripheral blood monocytes morphologic features and development of granulomatous vasculitis in feline infectious peritonitis altered expression of adhesion molecules on peripheral blood leukocytes in feline infectious peritonitis a review of feline infectious peritonitis virus infection: - spike protein fusion peptide and feline coronavirus virulence the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex mutation in spike protein cleavage site and pathogenesis of feline coronavirus feline infectious peritonitis: role of the feline coronavirus c gene in intestinal tropism and pathogenicity based upon isolates from resident and adopted shelter cats feline infectious peritonitis: insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral c gene cellular composition, coronavirus antigen expression and production of specific antibodies in lesions in feline infectious peritonitis evaluation of real-time rt-pcr for the quantification of fcov shedding in the faeces of domestic cats genomic rna sequence of feline coronavirus strain fcov c je development and use of real-time pcr to detect and quantify mycoplasma haemocanis and "candidatus mycoplasma haematoparvum" in dogs use of real-time quantitative pcr to detect chlamydophila felis infection primer on the www for general users and for biologist programmers mfold web server for nucleic acid folding and hybridization prediction mlwin version . . in centre for multilevel modelling natural fcov infection: cats with fip exhibit significantly higher viral loads than healthy infected cats sites of feline coronavirus persistence in healthy cats acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein a mrna pcr for the diagnosis of feline infectious peritonitis an outbreak of feline infectious peritonitis in a taiwanese shelter: epidemiologic and molecular evidence for horizontal transmission of a novel type ii feline coronavirus use of a reverse-transcriptase polymerase chain reaction for monitoring the shedding of feline coronavirus by healthy cats pyrosequencing: an accurate detection platform for single nucleotide polymorphisms amino acid changes in the spike protein of feline coronavirus correlate with systemic spread of virus from the intestine and not with feline infectious peritonitis the authors thank the veterinary practices, cat breeders and rescue centres that helped in the acquisition of samples used in this study. we also thank our colleagues, dr emi barker, dr chris palgrave, louise dawson and debra fews at the feline centre and the veterinary pathology unit, langford veterinary services, university of bristol, who have assisted in obtaining post mortem samples. we would also like to thank members of the histology laboratory, veterinary laboratory services, school of veterinary science, and university of liverpool for technical assistance. professor toby knowles of the school of veterinary sciences, university of bristol, is also thanked for his help with statistical analyses. this research was supported by a project grant from the petplan charitable trust. the authors declare that they have no competing interests. key: cord- - wtv bt authors: yang, wenbo; yang, dianlong; gong, shisong; dong, xiaobing; liu, luyao; yu, shengda; zhang, xiaolei; ge, shengxiang; wang, dong; xia, ningshao; yu, duli; qiu, xianbo title: an immunoassay cassette with a handheld reader for hiv urine testing in point-of-care diagnostics date: - - journal: biomed microdevices doi: . /s - - - sha: doc_id: cord_uid: wtv bt currently, most hiv tests are performed with blood samples, or alternatively saliva samples are used for hiv testing. simple hiv tests need to be performed in hospitals or other medical agencies instead of more invasive hiv blood tests. to enable point-of-care (poc) hiv diagnostics, based on a recently developed lateral flow strip for hiv urine testing, a microfluidic immunoassay cassette with a handheld optical reader is developed. based on lateral flow strip with gold colloid reporter, the integrated immunoassay cassette can perform sample introduction, metering, discharging, applying and detection which simplifies hiv testing. an indicator is incorporated into the cassette to guide sample introduction based on color change, and further, the excess test sample is stored inside the sealed cassette to avoid any contamination. the low-cost handheld optical reader can provide a test result within a few seconds, which is useful for simple, sensitive and affordable hiv onsite detection. instead of using normal white leds, a customized back light module embedded with green leds is adopted to illuminate the lateral flow strip with an appropriate working current to achieve optimal performance. compared to the standard lateral flow strips using a benchtop reader, with the disposable immunoassay cassette assisted by the handheld optical reader, more convenient, easier-to-operate, and more affordable hiv urine testing can be achieved in poc diagnostics. acquired immune deficiency syndrome (aids) is still a serious global disease since it cannot be cured completely. for people infected with hiv, aids is a stage when the level of their cd + t lymphocytes falls below acritical, for example cells/μl (cheng et al. ). by , it was estimated that the global number of people living with hiv was~ . million and more than two-thirds of them reside in developing countries (world health organization ; vos et al. ). there are common methods for hiv detection based on different principles chen et al. ) . using an immunoassay, hiv can be diagnosed by detecting anti-hiv antibody or hiv viral antigen. based on molecular analysis, hiv can be diagnosed by detecting viral nucleic acids. because hiv molecular diagnostic viral testing needs complicated sample processing for nucleic acid extraction and elaborate nucleic acid amplification, it is difficult to perform molecular diagnosis in developing countries where medical infrastructural facilities are lacking (zhao et al. ) . among different detection methods based on hiv immunoassay, lateral flow strips are widely used for on-site screening tests, especially in resource-poor settings at poc because of their ease of use, low-cost, simplicity and rapidity (granade et al. ) . various types of lateral flow strips have been developed for hiv immunoassays (paredes et al. ) . for example, anti-hiv antibody is can be detected in serum samples using lateral flow strips (faulstich et al. ). in principle, the detection result can be observed by eyes when the gold colloid reporter lateral flow strips are used. to avoid the interference from red blood cells, the raw whole blood sample needs to be centrifuged to obtain the cell-free serum sample. alternatively, an additional filtration pad adjacent to the sample pad can be incorporated into the lateral flow strip to block the red blood cell in lateral flow assay. hiv saliva tests can also be performed with lateral flow strip (delaney et al. ). compared to hiv blood testing, hiv detection can be implemented much more conveniently with saliva samples. to improve detection sensitivity, up-converting phosphor (ucp) reporter particles have been used to label the hiv antibody and avoid interfering background ). in this case, a companion optical reader is required to perform hiv saliva test with lateral flow strip when fluorescence labeling is adopted, which may partly limit its application field. urine testing is a routine examination in vitro-diagnosis. in fact, because of the superiority with noninvasive collection of samples, urinalysis is becoming a competitive format in poc diagnosis as more diagnostic biomarkers are identified in urine (mahoney et al. ; hart et al. ). for example, as an emerging application of urine analysis, in situ monitoring of activities of daily living (adl) is able to provide systematical information of the body status with continuous multi-parameter measurements of urine in a home setting for poc testing (taramasco et al. ). traditionally, urinalysis is able to provide not only valuable clinical information for diagnosis of various urologic and renal diseases, but also evidence in asymptomatic patients, for example, with sexually transmitted infections (sti's) (fogazzi and perazella ) . in poc diagnosis, widely used urinalysis devices include dip strips or sticks for pregnancy testing, urinary tract infections (utis), and urine test strips for other types of analytes of urine samples, for example, specific gravity, creatinine, glucose, ions, ketones, lactate, nitrite, ph, protein, and uric acid (kingston et al. ; suresh et al. ) . for high throughput detection, dna microarrays can be used for simultaneous detection of different dna markers in urine (gaber et al. ) . to avoid the inconvenience with urine sample storage and transportation, poc diagnosis devices, which can be easily implemented at the clinic or even at home, are highly desired in urine testing. with simple, convenient and easyto-handle operation, poc urinalysis devices developed based on microfluidic or lab-on-chip technology are able to detect infectious diseases or monitor chronic diseases with only modest resources (lepowsky et al. ; jalal et al. ). compared to blood testing requiring comparatively large sample volumes, poc microfluidics-based urine testing is quite promising and attractive for home use on account of the noninvasive nature of urine sample collection (lin et al. ) . to effectively control hiv epidemics, sensitive, simple, low-cost and easy-to-use detection methods need to be developed for rapid hiv test in poc settings. in this article, we describe a microfluidic immunoassay cassette with a companion handheld optical reader for hiv test based on urine sample analysis. based on a recently developed gold colloid lateral flow strip available for hiv urine testing, a disposable immunoassay cassette is developed to perform self-assisted urine metering, discharging, applying and detection. a low-cost handheld optical reader is developed to detect the test result from the cassette. once the raw, undiluted urine sample is loaded into the cassette, lateral flow immunoassay can be easily performed by the untrained operators themselves. excess sample will be retained inside the cassette to prevent any environmental contamination. the affordable handheld optical reader with a total component cost of seventy dollars improves the detection sensitivity, and logs, displays and transmits the test result if necessary. the performance of both the cassette and the handheld optical reader is systematically evaluated. experimental results show that successful hiv urine testing can be conveniently performed in poc settings with the test reported here. based on the immune specificity of hiv viral surface antibodies, an easy-to-operate, one-step lateral flow immunoassay was developed for hiv- antibody test with urine sample. for each test, the applied volume of urine sample is~ μl. similar to existing lateral flow strips (qian and bau ) , as shown in fig. a , the hiv urine test strip ( mm × mm) is comprised of a backing, sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad. on the conjugate pad, staphylococcal protein a (spa) conjugated to gold colloid nanoparticles is stored in dry form after diluted in a blocking reagent with bovine serum albumin (bsa). witnessing the fact of low concentration of biomarkers with urine sample, instead of using the sandwiched immunoassay which normally adopted in hiv blood testing, indirect immunoassay is adopted in hiv urine testing to ensure the detection sensitivity by capturing hiv antibody from urine sample through spa. moreover, to improve the detection sensitivity, large sample volume, e.g., μl is adopted in hiv urine testing. in the test line area of the nitrocellulose membrane, hiv antigens are immobilized to form the capture line. in the control line area, the second antibodies are immobilized to form the verification line. once the urine sample is in contact with the sample pad of the lateral flow strip, it is wicked along the lateral flow strip by capillary force. at most min later, the result becomes readable by observing the test or control line with red color. as shown in fig. b , in a predefined sequence based on indirect immunoassay, first the hiv antibodies in the test urine sample bind to the gold colloid particles stored on the conjugate pad through spa, and then they move together along the strip until captured by the test line based on the specific immune bioreactivity between the immobilized hiv antigens and the detected antibodies. finally, the remaining unbound free gold colloid particles are captured by the control line area for assay verification through the binding reaction between spa and the second antibody. the lateral flow strip recently developed by beijing wantai biological pharmacy enterprise co (beijing, china) for hiv urine testing has been evaluated thoroughly and systematically using a large sample bank for clinical trials. it has demonstrated . % of accuracy with the hiv positive subjects without antiretroviral therapy (art), and % specificity with hiv negative subjects (beijing wantai ). compared to hiv blood testing, as a noninvasive detection method, hiv urine testing can be performed more easily and conveniently, which is suitable for self-test in poc settings. it is well known that hiv saliva test is a commercialized noninvasive detection method. compared to hiv salvia testing, hiv urine testing can be performed with relatively simpler procedure for sample collection and pre-treatment. however, the unfriendly smell from the urine sample could be one of the disadvantages with hiv urine testing. nevertheless, similar to hiv saliva test, hiv urine testing provides another choice for noninvasive hiv poc test. in poc diagnosis, to allow on-site hiv urine tests with the lateral flow strip performed by laymen instead of trained operators for example for home use testing, an immunoassay cassette with the capability of sample metering, discharging and applying was developed. figure depicts the immunoassay cassette equipped with a lateral flow strip. as shown in fig. a , the immunoassay cassette consists of three major modules, e.g., sample introduction module on the top, sample metering module in the middle, and detection module on the bottom. the sample introduction module consists of a mm × mm × mm poly(methyl methacrylate) (pmma) loading chamber whose top is partly covered with a pmma layer. as shown in fig. a , the urine sample from a normal container can be directly added into the sample introduction module through the loading port. an accessory tape layer on top is used to seal the loading port after sample loading, which reduce the risk of environmental contamination. the sample metering module consists of a mm × mm × mm pmma storage chamber with a vent hole on top. as shown in fig. a , in the center of the storage chamber, there has an independent metering chamber with a size of~ μl. meanwhile, the space around the metering chamber in the storage chamber is used as the overflow chamber to hold the excess urine sample. an absorbent paper layer (nanjing huiyuechi electronic technology company ltd.) is deposited into the overflow chamber to not only absorb any excess urine sample, but also to work as an indicator for sample loading since its color will obviously change from white to red on contact with urine. opposite to the metering chamber, an extruded connection head is fabricated on the bottom of the sample loading chamber to allow urine sample to smoothly enter into the metering chamber. the metering chamber also has an extruded head to establish a flow path to the lateral flow strip once the device is fully assembled. initially, the bottom of the extruded head is sealed with a thin mm × mm paper layer (shenzhen siweituoda technology company ltd.). the specific thin paper layer, which is made from stretched polytetrafluoroethylene (ptfe) with average pore sizes from . to . μm, has an average pore density larger than %. the specific thin paper layer is able to hold the urine sample inside the metering chamber while allowing air to pass through. the detection module consists of a lateral flow strip, a strip holder, and a sharp needle partly penetrating into the strip from the sample pad. to simplify the process of fabrication, different parts of the cassette were all cut with a laser machine (htc- -w , jinan hantong digital control device enterprise co. ltd., jinan), and then laminated with the double-sided tape (type # , m). to reduce the complexity of cassette fabrication and binding, before laser cutting, the pmma sheet was first laminated with the double-sided tape from one side to allow both of them to be cut simultaneously, which was helpful to improve the binding performance. initially, the sample introduction module has been bond with the sample metering module with double-sided tape, and the combined two modules are partly assembled with the detection module through two guiding shafts from both sides (as shown in fig. a) . after sample loading, the two combined modules can be conveniently assembled with the bottom detection module by hand. for safety, two doublesided tape layers on both sides are used to combine and fix three modules together after assembling (as shown in fig. a ). during assembly, the bottom needle (as shown in fig. a ) sitting on the detection module will penetrate through the thin paper layer under the sample metering chamber, which allows the urine sample to be continually imbibed by the sample pad of the lateral flow strip for immunoassay. as shown in fig. b , the top window of the detection module is covered by a transparent pmma layer for optical detection. a handheld optical reader ( mm × mm × mm) with low-cost was developed to read and analyze the test result from the lateral flow strip, which allows the test result to be saved, displayed and transmitted if necessary. in principle, with the handheld optical reader, more sensitive, accurate and consistent test result can be achieved compared to typical eye observation which can be affected by a couple of issues. as shown in fig. a , a small-size cmos camera module (openmv cam m , openmv, llc) equipped with an optical lens was used to collect image from the lateral flow strip when it was illuminated by a back light module from the top. since the gold nanoparticles have a peak wavelength of absorbing light around nm, green light from the back light module consisting of green leds (wavelength: - nm) was adopted to illuminate the lateral flow strip from top to improve the detection sensitivity. a circuit module consisting of a single-chip microcontroller (n e at , nuvoton technology corporation) was used to control the camera module to collect and analyze image, and then the test result was sent to the lcd module for display. a bluetooth module was connected to the single-chip microcontroller for data transmission. to reduce system power-consumption, instead of using an advanced camera, a compact camera module relying on a micro-controller based on arm (stm f , stmicroelectronics) was adopted, which was able to perform image processing with a built-in function library based on micro python after image collection. especially, for the image collection and processing module, a micro-controller with significantly low power consumption was adopted, which was critical to reduce the system total power consumption. the system currents for idle and working states are ma and ma, respectively. powered by two . v batteries, the device is able to continually work up to h. figure c , d, respectively, depict the optical reader with a partly-and fully-inserted immunoassay cassettes. for the hiv urine test, once the urine sample is applied onto the lateral flow strip, the test result becomes detectable after min. with the companion hand-hold optical reader, rapid, on-site, hiv urine testing with lateral flow immunoassay can be conveniently performed at home or in other poc settings. the total cost of components of the hand-hold optical reader is around seventy dollars, which is beneficial to poc diagnosis. a custom algorithm was developed to analyze the test, control lines and the background of the lateral flow strip to achieve the detection result for hiv urine testing. as shown in fig. a , the original image was captured by the cmos camera in the handheld optical reader with the resolution of × pixels. as shown in fig. b , an interest area was extracted from the original image for further analysis, and then it was converted into a gay image (fig. c) . as shown in fig. d , to improve the detection sensitivity, laplacian operator was used to process the gray image to strengthen the boundary between different parts of the lateral flow strip (tian et al. ). there are three major steps for image processing of the lateral flow strip. similar to the test or control line, a background line is adopted here to represent the background area of the lateral flow strip. because of the limited resolution of the captured image, the test, control and background lines are all regarded as a short line (g (x, y)) with a fixed height of one pixel. first is to search the control line within the image processing domain (fig. e) . the location of the control line is identified by analyzing the difference of the mean gray (graymean ) between the control line and its adjacent area. to reduce the time required by image processing, the boundaries of the searching area are defined as y = y c ± n ( < n ≤ ). here, y c is the estimated location of the control line. as shown in fig. f , gray mean at different locations, for example, y = y c or in between y = y c ± n( < n ≤ ) can be calculated by eq. ( ): the control line can be identified by comparing the difference of the mean gray between (y = y c ) and (y = y c ± n( < n ≤ )) with a predefined threshold when the image processing domain is scanned with different y = y c . here, the threshold was set to . within the image processing domain (fig. e) , in the x direction, the searching range of image processing domain is within w ¼ w þ n À Á − w −n À Á , and in the y direction, it is between y ¼ y þ h and y = y + h. in the y direction, the searching range is limited to the lower half part of the domain to reduce the time required for image processing. the second step is to identify the test line. based on the location of the control line, similar algorithm can be used to find the test line within a specific image processing domain. third, the middle area between the control and test lines can be identified as the background line. to compensate the test fluctuation coming from the device or the process of detection, the gray difference between the test and the background lines (relative gray) is adopted as the calibrated result. finally, the detection result for hiv urine testing with lateral flow strip can be achieved by comparing the relative gray of the test line with a predefined threshold. urine samples from healthy donors were collected by national institute of diagnostics and vaccine development in infectious diseases (nidvd) of xiamen university with relevant guidelines and regulations approved by the ethics committee of the nidvd and stored in . ml collection tubes before use. all fresh urine samples were used within min after collection. hiv serum sample with a high concentration was provided by beijing wantai. the original hiv serum sample was diluted with serum to achieve multiple test samples with different concentrations. hiv antibodies from beijing wantai were spiked in healthy urine to simulate hiv positive urine samples. the original hiv urine sample was diluted with urine to achieve multiple test samples with different concentrations. . urine metering, discharging and applying with immunoassay cassette as described above, in each test, the urine sample was first loaded into the metering chamber, and then discharged and applied onto the lateral flow strip for immunoassay. first, water was used as the test sample for performance evaluation of metering. in each experiment, approximately μl of water was loaded into the cassette, and after a couple of seconds, the stored water from the metering chamber was weighted with a precise balance (fa b, shanghai yoke instrument co. ltd.) to determine its volume based on its density ( g/cm ). parallel experiments with urine samples were performed. next, experiments for performance evaluation of discharging and applying were also performed. similarly, both water and urine were used as the test samples. in each experiment, approximately μl of water or urine was loaded into the cassette. first, the detection module of the cassette was weighted with a precise balance before sample discharging. secondly, after the sample was discharged into the detection module within a couple of min, the detection module was weighted again with a precise balance. based on the sample density, the volume of the discharged sample can be estimated by comparing the two weights of the detection module before and after sample discharging. figure a depicts the measured sample volume respectively from metering or discharging in different experiments. as shown in fig. a , it can be seen that either with water or urine, the volume deviation of the metered or discharged sample is within ± μl, which is acceptable for hiv lateral flow immunoassay. as shown in fig. b , the indicative absorbent paper works well since after sample loading, its color changes from white to red, which is helpful to guide proper operation. in principle, the gold nanoparticles have a peak wavelength of absorbing light around nm. therefore, to optimize the system performance, different light sources including white led and green led were used to illuminate the lateral flow strip for comparison. to uniformly illuminate the entire detection area of the lateral flow strip for consistent performance, instead of directly using two independent leds as point light sources, a customized mm × mm back light module (shenzhen baohui photoelectricity company ltd.) was adopted. in the back light module, the light from multiple embedded white or green leds enters into a planar light waveguide from the side wall, which is able to provide uniform illumination. in all experiments, the driving current of the back light module was appropriately set to . ma. standard gold colloid lateral flow strips, instead of the developed cassette, were used to evaluate the performance of the handheld reader itself. hiv serum samples with different concentrations were used in experiments. in each test, μl of hiv serum sample was applied onto the standard lateral flow strip specific for hiv serum detection and incubated for min. figure depicts the experimental results from two back light modules with different colors. error bars come from multiple repeated experiments. the signal amplitude is defined as the signal intensity difference between the test line and the background. as shown in fig. , it can be found that with green leds, optimal performance can be achieved compared to white leds. inserts show photographs of the standard lateral flow strips. as shown in the insert of fig. , a specific number (#) is assigned to each lateral flow strip based on a customized rule to represent the signal intensity of the test line. from # to # , they respectively correspond to low-positive to high-positive. for the described handheld optical reader, low-positive detection close to # is the acceptable limit of detection. in fact, it is difficult for a person to locate the test line from the background for # low-positive detection just by eyes. therefore, compared to traditional eye observation, the handheld optical reader is helpful to improve the detection sensitivity of hiv urine test with lateral flow strip. as shown in fig. , for both cases, instead of # , # lateral flow strip with low-positive can be successfully detected. compared to white leds, significantly higher signal reading with the test line can be achieved with green leds for different sample concentrations (# , # , and # in fig. ), which demonstrate that higher detection sensitivity can be achieved with green leds. therefore, a customized back light module with green light is adopted in the handheld reader. since the detection is performed in an enclosed box where the lateral flow strip is illuminated with a green lighting source, appropriate lighting conditions with a properly set driving current is critical to the detection performance. for comparison, experiments with different driving currents were performed. standard gold colloid lateral flow strips, rather than the cassette, were used to evaluate the performance of the handheld reader itself. hiv serum samples with different concentrations were used in experiments. in each test, μl of hiv serum sample was applied onto the standard lateral flow strip and incubated for min. figure depicts the experimental results for three cases respectively with low ( . ma), medium ( . ma) and high ( . ma) driving currents. error bars come from multiple repeated experiments. as shown in fig. , it can be found that with a medium driving current ( . ma), optimal performance can be achieved compared to others. as shown in fig. , when the driving current is too low or too high, # lateral flow strip with low-positive cannot be successfully detected with acceptable credit. inserts are the recovered photographs of the standard lateral flow strips with medium-positive (# ) respectively with different driving currents, which are taken by the handheld optical reader with green light illumination. it has been demonstrated that optimal performance can be achieved with a medium driving current when the lateral flow strip is properly illuminated without saturation. it was found that comparable performance can be achieved when the driving current was between . ma and . ma. therefore, a medium driving current ( . ma) was applied in the handheld reader. the performance of the hiv immunoassay cassette was evaluated with hiv urine sample. hiv urine samples with different concentrations were used in experiments. in each test, more than μl urine sample was loaded into the described cassette ( μl was used in the test), and the lateral flow strip was detected with the described handheld optical reader after -min incubation, once the cassette was fully assembled. for comparison, parallel tests were performed with standard lateral flow strips. a benchtop immunoassay gold colloid reader (rtr-g , xiamen innovax biotech co., ltd) was used to detect the standard lateral flow strip after -min incubation once μl of urine sample was applied. figure depicts the fig. experimental results with metering and discharging: (a) metering or discharging sample volume respectively with water or urine sample; (b) two photographs of the immunoassay cassette before and after sample loading fig. experimental results of lateral flow strip illumined with green or white light. inset shows photographs of standard lateral flow strips taken by a smartphone camera fig. experimental results detected by the handheld optical reader with different driving currents. inset shows recovered photographs of standard lateral flow strips taken by the handheld optical reader experimental results for both cases respectively with the described cassette and the standard lateral flow strip. error bars come from multiple repeated experiments. as shown in fig. , for both methods, both low (# . ) and medium (# ) positive samples can be successfully detected and differentiated from the negative one. as shown in fig. , acceptable detection sensitive with the described method can be achieved. inserts are the photographs for both cases with detection to negative, the low (# . ) and medium (# ) positive samples. for each insert (negative, # . , and # ), the left photograph is from the described cassette, while the right one is from the standard lateral flow strip. it has been demonstrated that comparable performance between the described method and the standard lateral flow strip with a benchtop reader can be achieved. however, both the cost and the size of the described handheld optical reader are significantly less than those of the benchtop reader, which is more suitable for poc diagnosis at resource-poor settings. we describe a disposable, easy-to-operate microfluidic immunoassay cassette for hiv urine tests with a handheld optical reader for use at the point of care including home use. the hiv immunoassay cassette consists of three different modules respectively for sample introduction, metering and detection. the sample introduction module provides a user-friendly interface for users to conveniently add urine sample into the cassette. the metering module can adequately control the test sample volume with acceptable deviation. the detection module is able to perform hiv urine detection with an integrated lateral flow strip that uses gold colloid reporter. once the cassette is fully assembled, the metering chamber will be fluidically connected from its bottom by a sharp needle from the detection module, which allows the urine sample to be continually aspirated by the sample absorbing pad of the lateral flow strip for immunoassay. the whole operation can be performed just by hand without any instrument or special equipment. after -min incubation, the cassette can be inserted into a handheld optical reader to detect the test result within s. a custom image processing algorithm was developed to search and locate the test and control lines of the lateral flow strip. the total cost of components of the handheld optical reader is around seventy dollars, which is suitable for poc diagnosis or home test. compared to the traditional test by just adding the urine sample to a lateral flow strip, there have a couple of advantages with the developed method. first, with the microfluidic cassette, the allowable test sample volume could be potentially increased for higher sensitivity. second, there has no need for any sample metering tools since there has a metering chamber within the microfluidic cassette. third, the risk of environmental contamination (including hiv sample contamination and even spread of the unfriendly smell from urine sample) can be reduced since both the lateral flow strip and the excess sample are sealed inside the cassette. fourth, the detection sensitivity and accuracy can be improved by combining the cassette with the handheld optical reader compared to direct eye observation which could be unfavorably affected by the operator and the environment. the performance for both the hiv immunoassay cassette and the handheld optical reader has been systematically evaluated. consistent metering, discharging and applying ensure adequate and reproducible test sample volumes. to improve the system performance, green light instead of white light was adopted to illuminate the lateral flow strip. a proper working current for the green back light module was chosen to ensure the system performance. hiv antibody-spiked urine samples were successfully detected by the immunoassay cassette with a handheld optical reader. it has been demonstrated that comparable results can be achieved with the described method comparing to the standard lateral flow strip with a benchtop reader. however, simple, easy-to-operate, low-cost, and integrated hiv urine testing, which is highly desired for poc diagnosis or even home test, can be conveniently achieved with the described method based on a disposable cassette and a handheld, affordable optical reader. user's manual: diagnosis kit for hiv- antibody test with urine sample a rapid, self-confirming assay for hiv: simultaneous detection of anti-hiv antibodies and viral rna enhancing the performance of a point-of-care cd + t-cell counting microchip through monocyte depletion for hiv/aids diagnostics performance of an oral fluid rapid hiv- / test: experience from four cdc studies developing rapid mobile poc systems. part : devices and applications for lateralflow immunodiagnostics the urinary sediment: an integrated view, rd edition comparison between real-time polymerase chain reaction and dna-microarray in detection and identification of mycobacterium species rapid detection and differentiation of antibodies to hiv- and h i v - u point-of-care oral-based diagnostics paper-plastic hybrid microfluidic device for smartphone-based colorimetric analysis of urine shelf life'of trichomonas vaginalis based assays for urine analysis urine analysis in microfluidic devices a timer-actuated immunoassay cassette for detecting molecular markers in oral fluids point-of-care urinalysis with emerging sensing and imaging technologies rapid hiv testing: a review of the literature and implications for the clinician analysis of lateral flow bio-detectors: competitive format finger-actuated, self-contained immunoassay cassettes a portable, integrated analyzer for microfluidic -based molecular analysis non-invasive paper-based microfluidic device for ultra-low detection of urea through enzyme catalysis a novel low-cost sensor prototype for nocturia monitoring in older people medical imaging and diagnosis of subpatellar vertebrae based on improved laplacian image enhancement algorithm global, regional, and national incidence, prevalence, and years lived with disability for diseases and injuries, - : a systematic analysis for the global burden of disease study nucleic acid testing and molecular characterization of hiv infections publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations conflict of interest the authors declare that they have no conflict of interest. key: cord- -fbeb nw authors: sridhar, sushmita; forrest, sally; kean, iain; young, jamie; bartholdson scott, josefin; maes, mailis; pereira-dias, joana; parmar, surendra; routledge, matthew; sparkes, dominic; rivett, lucy; dougan, gordon; weekes, michael; curran, martin; goodfellow, ian; baker, stephen title: a blueprint for the implementation of a validated approach for the detection of sars-cov in clinical samples in academic facilities date: - - journal: wellcome open res doi: . /wellcomeopenres. . sha: doc_id: cord_uid: fbeb nw the covid- pandemic is expanding at an unprecedented rate. as a result, diagnostic services are stretched to their limit, and there is a clear need for the provision of additional diagnostic capacity. academic laboratories, many of which are closed due to governmental lockdowns, may be in a position to support local screening capacity by adapting their current laboratory practices. here, we describe the process of developing a sars-cov diagnostic workflow in a conventional academic containment level laboratory. our outline includes simple sars-cov deactivation upon contact, the method for a quantitative real-time reverse transcriptase pcr detecting sars-cov , a description of process establishment and validation, and some considerations for establishing a similar workflow elsewhere. this was achieved under challenging circumstances through the collaborative efforts of scientists, clinical staff, and diagnostic staff to mitigate to the ongoing crisis. within days, we created a validated covid- diagnostics service for healthcare workers in our local hospital. the described methods are not exhaustive, but we hope may offer support to other academic groups aiming to set up something comparable in a short time frame. sars-cov , the viral agent of covid- , is a recent introduction into the human population, and the disease epidemic is expanding nationally (within the uk) and internationally at an unprecedented rate , . as a result, national diagnostic services are stretched and there is a need for alternative laboratory facilities to provide additional diagnostic capacity. the screening of asymptomatic individuals and healthcare workers (hcws) will be key for controlling the epidemic and also to ensure hcw are a) working in safe conditions with functional personal protective equipment (ppe), b) not transmitting sars-cov to vulnerable patients on wards, and c) can return to work if they are not actively infected . however, in almost all cases, embedding a rapid testing workflow for screening asymptomatic individuals and hcws within the current hospital structure would add additional pressure upon an already overstretched diagnostic service. the uk government and other organisations have recognised the critical role of additional screening . for example, the uk has recently initiated the establishment of national testing centres for hcws; however, there is a need for guidance on how such systems be standardised or scaled. the expected turnaround times from sampling collection to results being reported back to the affected hcw is also a key issue. whilst central screening facilities will ultimately be beneficial in curtailing the epidemic, smaller academic and non-academic laboratories can (and should) contribute to these efforts. critically, local facilities (academic laboratories in proximity to or within healthcare facilities) can frequently provide a quicker turnaround time than larger remote facilities due to simpler sampling and shipping logistics or even overstretched, onsite diagnostic laboratories; current turnaround time is typically > hours from sample being taken to provision of result. such delays can have a negative impact on healthcare provision, for example staffing levels may be strained due to hcws isolating as a result of a respiratory illnesses other than covid- . therefore, there is an urgent unmet need to establish local screening workflows for hcws and those working in essential service industries. one of the key limitations associated with the expansion of diagnostics to tackle the covid- outbreak is the availability of protocols, or a scheme, that can be used in suitable laboratories for detecting sars-cov in relevant clinical samples. in the given circumstances, a robust diagnostic test for covid- should be able to generate a result rapidly, but also maintain a high level of reproducibility, specificity, and sensitivity . the test also needs to be conducted on an easily accessible clinical sample, such as a dual nose and throat swab, urine, or blood. or in those with more severe illness sputum or bronchoalveolar lavage. such tests can be based upon a direct amplification assay for a component of the viral genome, a suitable biomarker or metabolic signature, or the measurement of an indicative acute antibody response. given a paucity of reliable alternatives, a pcr based approach is currently the most suitable and scalable model, whilst providing an acceptable compromise between turnaround time and accuracy. the key issues for rapidly establishing a new diagnostic testing platform are sampling, safety, reagents, cleanliness, methodology, and reporting. early indications for covid- infections is that there are relatively high titres of virus in the respiratory tract, possibly in the gastrointestinal tract, but lower concentrations in blood . consequently, nose and throat swabs are widely accepted as the optimal clinical sample for hcws and others who are likely to have a higher occupational exposure risk and potential upper respiratory tract symptoms to the virus through their work. these swabs need to be handled safely, so the use of a sampling method that inactivates the virus rapidly is essential to protect the sampler and those handling the sample, including couriers, and laboratory staff . the availability and the expense of reagents required for an effective testing programme at a specific scale (e.g. hospital, company, or local community) is critical given the ongoing demand for specific kits. tests that require expensive mainstream reagents, or those in short supply, should be avoided where possible. pcr diagnostics are prone to issues with contamination and appropriate workflow and strict sample/reagent segregation needs to be adopted, which may be problematic in some settings. methodologies and equipment are variable, but every attempt should be made to ensure the tests are performed using a standardised and validated test with appropriate controls. lastly, the resulting data needs to be authenticated by a qualified individual and reported in a timely fashion through an existing and official reporting system, whilst at the same time ensuring patient confidentiality. here we describe our experience in establishing a covid- diagnostics laboratory in an academic containment level (cl ) research facility (uk) in which we validated and established a real-time pcr workflow to detect sars-cov in nose and throat swabs from hcws. we developed an assay and workflow over eight working days (set-up to validation to screening) that can produce a quantitative diagnostic result ~ hours after swabbing. for the swabbing of known covid- patients and hcws we developed a kit that can be easily assembled and provided in bulk. not only does this significantly reduce ppe usage, but also reduces the need for significant interaction in version of this article, we responded to the reviewers' comments and suggestions. specifically, we added a table to accompany figure , to clearly show the results of the concordance testing we performed to validate our assay. this table is " table . assessment of known patient samples". we added additional detail to the section on qrt-pcr validation to more explicitly explain how rigorously we validated our qrt-pcr protocol. we further added a statement clarifying that our workflow used a -well plate setup and the consequent throughput. any further responses from the reviewers can be found at the end of the article revised between those running the testing clinic and potential covid- positive hcws. the kit contained: swabbing instructions, an individually packed sterile swab that can be broken (vwr), a labelled sample tube containing lysis buffer, and gloves (see extended data: protocol ). the instructions indicate the individual to put on the gloves, remove the sterile swab from the packet and swab the back of throat and then the nasal cavity (one swab, two sites). the swab is placed into the labelled sample tube ( ml long necked externally threaded cryovials (nunc ; to avoid aerosols), and the end is submerged in µl lysis buffer ( m guanidine thiocyanate (merck) in mm tris-hcl, . % β-mercaptoethanol (sigma), and carrier rna ( µl of µg/µl stock; qiagen). the swab is snapped carefully to avoid disturbing the buffer, and the cap is placed back onto tube containing the buffer and swab and tightened. the tube is gently agitated to ensure even distribution of lysis buffer and labelled with an ethanol resistant pen. the outside of the tube is sprayed with % ethanol, placed into a zip lock bag (onecall) and sealed. one glove is removed, and the zip lock bag is sprayed with % ethanol while being held in gloved hand and then passed to a clean hand. the sealed bag is placed in a secure biohazard labelled container for dispatch to a certified cl laboratory. the combination of m guanidine thiocyanate and . % β-mercaptoethanol should ensure complete lysis and deactivation of the virus, but to ensure additional safety, the samples are received and unpacked in a class ii microbiological safety cabinet (msc) (see extended data: protocols and ) . notably, whilst this process should be conducted in a sterile and clean environment with routine cleaning sessions, given the nature of the samples this room is isolated as "a dirty room" and all molecular reagents kept elsewhere. those working in this room should not enter the room in which molecular reagents are kept and laboratory clothing remains restricted to this room. the class ii msc should be running as 'safe' prior to work to ensure a stabile airflow. the cabinet is cleaned sequentially with % bleach, % ethanol, and rnasezap (sigma). the class ii msc cabinet should be set up with the required reagents and waste vessels before sample bags are placed directly inside and sprayed with % ethanol. barcodes are scanned for tracking and tubes arranged into batches of ≤ for extraction, dependent on centrifuge rotor capacity. the sample tubes, still containing the swab, are placed into a rack, µl % ethanol (final ethanol concentration %) added to each tube one-by-one and incubated at ambient temperature for minutes. top-up lysis buffer containing the internal extraction and amplification control ( µl of - ms (~ × pfu/ml) per ml of lysis buffer in this case) is next added to each sample ( µl to make % final ethanol concentration) resulting in a total volume of ~ . ml per tube. in total, µl of the media is transferred into a spin column (nbs biologicals) over a ml rnase-free collection tube (thermofisher). to avoid contamination, only one column is open at any one time and filter pipette tips are used for each sample. the tubes are loaded into a microcentrifuge rotor inside the class ii msc, and the aerosol-tight lid closed before returning the rotor to the microcentrifuge. the samples are centrifuged for seconds at , rpm (two spins are required per sample to load the entire volume of lysis buffer). all pass-through liquid is discarded into designated liquid collection containers (do not mix with disinfectants containing bleach). µl of wash buffer ( m guanidine thiocyanate in mm tris-hcl, with % ethanol) is added onto the columns and tubes are centrifuged for seconds at , rpm. the pass-through liquid is discarded, and µl of wash buffer ( mm tris-hcl buffer with % ethanol) is added and again tubes are centrifuged for seconds at , rpm. finally, a second µl of wash buffer is added, and the tube is centrifuged for minutes at , rpm. the silica spin column is transferred to a new collection tube and centrifuged at , rpm for minute to remove residual ethanol. the silica spin column is transferred to a new rnase free tube with the appropriate sample label. µl of nuclease free water is added to each column and left to stand for minute before centrifugation for minute at , rpm. the spin columns are discarded, and the eluted samples are either directly taken for qrt-pcr or an aliquot is frozen at - °c for subsequent amplification. the remaining nucleic acid extracts are frozen at - °c with the location recorded on the 'sample record' form. once the nucleic acid (viral rna) has been extracted, it can be amplified to detect sars-cov (see extended data: protocol ). notably, this work should be done in a "clean room," and the operators should wear laboratory clothing that is restricted to this room. movement to other working areas where biological or molecular contamination may be an issue should be restricted, and there should be no access to the dirty room. per reaction, the master mix is made up of: . µl x luna universal probe one-step reaction mix, . µl of pmoles/µl wu forward primer (atgggttgggattatcct aaatgtga), . µl of pmoles/µl wu reverse primer (gcagttgtggcatctcctgatgag), . µl of pmoles/µl mgb probe fam (atgcttagaattatggcctcac), . µl of pmoles/µl of internal control forward primer (ms ) (supplied by eurogentec), . µl of pmoles/µl internal control reverse primer (ms ), . µl of pmoles/µl internal probe (ms rox), µl of luna warmstart rt enzyme mix (new england biolabs) and . µl water. once the master mix is prepared, it can be stored at °c short term or - °c longer term. if using immediately, µl can be aliquoted into each well of a -well plate in a clean class ii cabinet and then combined with µl of each rna extract, using a different pipette tip for each well. ideally, the master mix preparation and addition of rna to each well should be done in separate class ii cabinets to minimize contamination. the ms internal extraction and amplification control that underwent the full extraction protocol is included as the negative extraction control in a minimum of two wells on the qrt-pcr plate. to check for contamination in the qrt-pcr process, µl nuclease-free water (minimum wells) is included as the qrt-pcr negative control. µl of spiked sars-cov template plasmid is included in a single well as the qrt-pcr positive control. after adding µl of each sample to its designated well, the plate is sealed carefully with an optically clear plastic seal. the plate is centrifuged for minute at , rpm at °c and then inserted in the qrt-pcr machine (quantstudio, thermofisher scientific) and the run is parametrised. fam and rox are acquired; rox is used to detect the internal control; fam is used to detect sars. the assay is run for minutes at °c, minutes at °c (for the reverse-transcriptase), minutes at °c, before cycles of °c for seconds followed by °c for seconds. establishing and validating the workflow in our setting establishing a workflow for sars-cov qrt-pcr upon the decision to rapidly establish the qrt-pcr assay we identified several challenges, and these included: a) establishment and validation of a method suitable for diagnostic reporting, b) safe extraction of nucleic acid from a highly transmissible virus, c) accessing reagents required for performing extractions and amplifications, d) establishing a "clean" diagnostic workflow to minimise the risk of contamination, and e) creating a system in which hcws could be swabbed and the data reported confidentially within a specified timeframe. setting up a diagnostic qrt-pcr in our setting, diagnosis of infections for the hospital is normally performed in the region public health england (phe) diagnostic laboratory, which is co-manned by hospital and phe staff, serving our and local hospitals. upon agreement with senior diagnostic staff we sought their approval to duplicate their in-house generated, validated assay on our equipment. the diagnostic laboratory provided access to their in-house method (designed by martin curran and surendra parmar) and provided a collection of anonymised sars-cov positive extractions (as determined by the same pcr method) and a cloned positive control. the required reagents were ordered and the quantstudio machine calibrated to run the qrt-pcr. qrt-pcr was initially performed using existing positive samples and ten-fold dilutions of the cloned target gene (a conserved region with the orf polyprotein). upon amplification, we were able to replicate the positive signals from known positive samples (with comparable ct values of between and ) and generate a reproducible standard curve that could be used for all following amplifications and validations (figure a) . additionally, during this process we validated the amplification process by the addition of a positive control; ms nucleic acid was added to all samples with the exception of the positive sars-cov control and the negative controls ( figure b) . notably, these assays were run a minimum of three occasions over differing days to assess the degree of experimental variation. through this procedure of testing, troubleshooting, and assay development, we were able to show reproducible amplifications and have an assay ready for downstream validation. it should be noted that at the time of starting the scheme, samples with the potential to harbour sars-cov virus were classified by the uk health and safety executive (hse) as requiring a containment level (cl ) laboratory. this level of security was required due to the infectious nature of the virus and potential for airborne transmission. existing sampling procedures exploited a viral transport media containing ingredients to preserve the virus and restrict the growth of non-viral pathogens. the extraction of nucleic acid (or viral inactivation prior to downstream processes) in a cl facility was deemed a major bottleneck that could be circumvented. consequently, we considered it essential to inactivate the sample safely at source so as to minimise risk. a protocol was established that was risk assessed by the university health and safety committee to inactivate combined nose and throat swabs immediately after they are taken from the individual being sampled. the protocol is outlined in methods (for the full protocol see extended data: protocol ) and was established from existing methods known to chemically inactivate viruses. we utilised existing data regarding coronavirus and other highly infectious viral pathogens. several methods, including heat inactivation were considered, but the selected method using a mixture of guanidine thiocyanate and β-mercaptoethanol was considered to be the most suitable for validation. whilst existing data demonstrated that the designated approach was safe for viral extraction it had not been tested on covid- patients. a recent publication has highlighted that traditional avl lysis buffer, on which our home-made equivalent is based, does not lead to % inactivation of live virus when mixed at a ratio of : and left with a contact time of minutes. however, our protocol relies on the use of dry swabs, therefore any dilution effect of the lysis buffer is negated . in addition, because of the locality of the testing laboratory, the minimum contact time between the swab and the lysis buffer is typically > hr. this was followed by the addition of ethanol to a final concentration of % in an msc. sample workflow a critical step in establishing a diagnostics facility is the segregation of workspace and staff, preventing the cross contamination of samples, equipment, and reagents. the mode of operation is not typical for many research laboratories where communal facilities are used according to the requirements of the specific project. the research laboratory was reorganised to create "dirty", "clean", and amplification areas (figure a) . these were strategically located in separate rooms and a strict regime was created where equipment, staff, ppe, and samples were restricted to these specific rooms. all laboratory staff were trained in the new containment structure and in the assays being performed. this component involved the transfer of materials, knowledge, and protocols between phe cambridge and the research laboratory. laboratory staff were given specific roles and were restricted to the "clean" or "dirty" work areas for a single working day. ultimately, we had developed a workflow that could be tested for screening sample from covid- patients (figure b ). final validation of qrt-pcr assay from known covid- patients the next stage in validating the process was to run the full extraction protocol and assays on samples from patients that had previously tested positive for sars-cov in the assay performed by the hospital diagnostic laboratory. buffers and extraction kits were constructed in the "clean" rooms and provided for patient sampling. in agreement with the hospital, for the purposes of developing a diagnostic test, we approved that a group of known covid- patients and a group of individuals assumed not to be infected with sars-cov would be screened. consequently, swabs were taken from these individuals according to the protocol; these were dispatched to the laboratory for processing and analysis. the samples were anonymised, and research workers were blinded from knowing which samples were positive or negative. additionally, instead of a precise / split in the provided samples, were from known sars-cov patients and from uninfected patients. again, this was not revealed to the staff performing the assay until after the tests results were known. data from this experiment are shown in figure a and table . there was % correlation between the test results initially generated by the diagnostic laboratory and the research laboratory, with generating ct values of between and , and generating no detectable signal. all controls were as expected. at this point the assay was repeated several times to be further validated for reproducibility. specifically, the initial extracted rna samples provided by the hospital were assessed by qrt-pcr on our system three times to determine reproducibility of the qrt-pcr signal. in addition, further testing rna samples were provided by the hospital as known positives and negatives for use. a total of ~ of these samples were analysed by qrt-pcr to check for robustness and concordance of our qrt-pcr assay. upon completion of these validation samples, the assay was offered to the hospital for the screening of hcws. within two weeks of the start of the process, the screening procedures were approved by the hospital and made available for hospital staff through occupational health. a firewall figure . establishing a diagnostic workflow for qrt-pcr for sars-cov . (a) diagram displaying the segregation of the "dirty", "clean" and "amplification" rooms. note the use of separate cabinets for the preparation of reagents in the "clean" room. individuals working in the "dirty" or "amplification" rooms are unable to enter the "clean" room on the same working day. (b) diagram showing a suitable workflow of samples from swabbing to amplification to reporting. was built between the hospital and the research laboratory to protect confidential data without losing track of samples. a system was created where samples and data could be managed within a single system through use of a unique identifier number and barcode, hence there was a logged transfer of the samples to the research laboratory, where samples could be tested, and data reported within the research laboratory. the hospital established a swabbing pod and offered structured screen-ing to staff, and a depository for samples was established at a single point within the hospital. samples were transported securely to the research laboratory in a risk assessed, spill proof container by selected courier, which was a member of clinical staff involved in the project. data from the first screening run is shown in figure b and permitted the detection of several positives. the ct values from these ranged from - and the turnaround time was ~ hours from sample arrival to result being available for reporting. however, given the scheduled sampling, we aimed to report the data within hours. using our -well plate setup and allowing for multiple negative and positive controls per plate, we were able to assay and report up to patient samples per qrt-pcr run. data were checked and validated prior to reporting by a senior member of laboratory staff. all ct values and curves were checked, and the presence of amplification in the controls was verified. all data were entered into the official hospital database and verified by a clinical virologist prior to reporting back to occupational health. residual rna samples were suitable for downstream analysis and we were able to contribute to ongoing covid- genome sequencing projects affiliated to cog-uk. clearly establishing an assay rapidly in difficult circumstances requires frequent validation, reappraisal, and troubleshooting. as the project developed, more levels of management, oversight, and communication were brought in. for example, within the hospital, links had to be established between those working within the wards and occupational health to determine hcw populations for priority and routine screening. thus ethical, logistical, and practical barriers had to be identified and managed. within the laboratory setting, potential for the contamination of materials was a key consideration that had to be managed. for example, at one stage, background levels of amplification on negative samples were elevated above acceptable levels, which was assessed to be contamination. based on the controls used in the specific plate (having negative and positive extraction controls, swabs extracted using two different kit batches, qrt-pcr negative and positive controls), we hypothesized that the contamination was occurring in the quantstudio equipment. this was potentially due to sars-cov dna that was being amplified inside the machine and causing all samples to have ct values ~ . consistency in ct value suggested that the issue was at the amplification stage and not at the extraction or qrt-pcr preparation stage. the quantstudio comes with a background calibration plate as part of its calibration system. this plate was checked to assess whether the background profile had changed substantially. if the machine "passed" the calibration, we assessed whether the profile of the background fluorescence differed from when it was previously calibrated. we performed a background calibration plate run and then the bottom plate of the machine was cleaned sequentially with % bleach, % ethanol, and milliq water. the baseplate and optical plate were removed from the machine for deep cleaning. the baseplate was rinsed in % bleach, followed by milliq water, % ethanol, then water again. liquid was aspirated from the wells, which were then wiped with a lens cleaner tissue. the upper optical plate was wiped with cotton swabs containing % ethanol in case any dust particles were occluding the surface. a further background calibration plate was run after cleaning and several wells were found to have high readings. to revalidate the machine, a plate of µl mastermix plus water (negative controls) in any "problem" wells (to rule out the possibility of well-specific amplification/contamination) and additional wells scattered around the plate were assessed. based on the location of the problem wells, contamination was often more severe at the edges of the plate, so these were also checked. the rest of the wells of the -well plate were filled with µl of water only. the location of the "problem" wells suggested that there might have been a failed plate seal at some point, which may have released some dna into the machine to amplify. this plate was found to give low background; several positive samples and positive and negative controls were run, and the contamination issues were found to be resolved. in the continuing public health crisis, we need as much capacity as possible for supporting diagnostic services to ensure key workers and hcw are screened frequently. this places an enormous pressure on an already saturated system. the introduction of large screening services will play a huge role in tackling the epidemic in the uk and elsewhere but lacks some of the speed and flexibility that small on-site diagnostic laboratories can provide. we recognised the need to repurpose our laboratory for covid- screening; this was initiated without request to provide some additional local capacity. many academic facilities may be in a similar position but may be unsure about how to start proceedings and what regulations are in place. we suggest that groups establish the assay and processes so they can be prepared as the need arises. the route we describe here is not a scalable solution to the international lack of diagnostic testing, but a blueprint for what can be established in a standard academic research laboratory within days. at the time of writing we have a full sample workflow from swabbing to diagnostic testing of hcws at our healthcare facility, with the capacity of approximately tests a day with a result provided within hours. this number of tests can be expanded, but is dependent on maintaining enough extraction rooms, key staff, and of course key reagents. we sought to develop a test that works independently of kits from major suppliers, but there will potentially be issues with other resources as the crisis develops. the theoretical turnaround time is hours, but this is dependent on integration with occupational health facilities and the diagnostic laboratory and ensuring there is a sustainable communication and enough staff to maintain the process. in setting up this process there are many challenges and pitfalls, especially given the time constraints of providing a functional service that can be rapidly deployed, and we recognise that everything described here is not exhaustive. many laboratories differ in equipment, facilities, capacity, expertise and staffing; additionally, being in close proximity to a major infectious disease centre with an excellent diagnostic facility is a major advantage. however, the methods and stages of laboratory repurposing described will, we hope, be of value to other academic laboratories in the uk and internationally that are aiming to make a useful contribution. particularly, with some simple modifications and training we feel that this could be developed and rolled out in low and middle-income countries, providing vital molecular capacity for this and future epidemics. in summary, the key problems to solve are safety, reagents, cleanliness, methodology, validation, and reporting. here, we tackled new challenges on an almost daily basis, but inactivating the virus on contact improved the process and ensured the swabs could be extracted safely in a cl laboratory. access to reagents is key, and we suggest that groups become less reliant on kits from major manufacturers, unless essential. this step reduces costs and puts less pressure on existing supply chains of key kits and equipment . cleanliness is paramount, and sample flow, room segregation, and dedicated staff are essential. having a reliable diagnostic facility that you can partner with will reduce many of the initial issues. these groups, such as the phe laboratory here, provided excellent advice, reagents, methodology, and support for set up. having access to good clinical facilities for validating the assay is essential; the whole process (from swab to report) needs to be comprehensively tested before being rolled out. lastly, reporting needs to be conducted with the provision of experienced staff, again a link with clinical diagnostic facility is essential. here we provide a brief outline of our experience in establishing a covid- diagnostic laboratory in a standard molecular bacteriology laboratory, which we hope is useful to other groups in a similar position. it was achieved under challenging circumstances through the collaborative efforts of scientists, clinical, and diagnostic staff with the ability to generate something constructive that we hope will contribute to the ongoing crisis. underlying data all data underlying the results are available as part of the article and no additional source data are required. development. i confirm that i have read this submission and believe that i have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reviewer report september https://doi.org/ . /wellcomeopenres. .r © wren b. this is an open access peer review report distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. department of infection biology, london school of hygiene and tropical medicine, london, uk the article is a true and necessary blueprint for the rapid set up of facilities for the diagnosis of covid- infection. the article is most timely and provides all the necessary details that many university/research centre laboratories with pcr thermal cycling facilities could adopt. this should prove very useful, for example enabling universities to offer regular testing of students and staff, relieving the pressure on national health care systems. the protocols (including sampling, safety, reagents, cleanliness, methodology and reporting) are very well written, carried out to the highest of standards and are of sufficient detail to be widely adopted. a useful section was on contamination with background levels of nucleic acid amplification on negative samples. this was traced to the quantstudio equipment which was subsequently stripped and cleaned. an alternative proof of the source of contamination would have been to use a different machine. the conclusions fully supported the findings presented. a statement on the capacity and throughput of the set up would have been useful, presumable this is dependent on the -well plate format. given the current pandemic and capacity difficulties encountered by many countries in testing for the virus, it is imperative that this article should be indexed as soon as possible. is the description of the method technically sound? yes pubmed abstract | publisher full text | free full text . eurosurveillance editorial team: updated rapid risk assessment from ecdc on the novel coronavirus disease (covid- ) pandemic: increased transmission in the eu/eea and the uk challenges for nhs hospitals during covid- epidemic covid- : towards controlling of a pandemic pubmed abstract | publisher full text | free full text the laboratory diagnosis of covid- : current issues and challenges virological assessment of hospitalized patients with covid- rt-qpcr detection of sars-cov- rna from patient nasopharyngeal swab using qiagen rneasy kits or directly via omission of an rna extraction step rapid and simple method for purification of nucleic acids the use of control charts in the clinical laboratory performance characteristics of rules for internal quality control: probabilities for false rejection and error detection countries: covid- and risks to the supply and quality of tests, drugs, and vaccines pubmed abstract | publisher full text | free full text a blueprint for the implementation of a validated approach for the detection of sars-cov in clinical samples in academic facilities: extended data we wish to acknowledge all involved from the onset in giving their time, effort, and knowledge in getting this going. we additionally acknowledge the healthcare workers at addenbrooke's hospital, cambridge.a previous version of this article is available on biorxiv: https://doi.org/ . / . . . . reviewer report october https://doi.org/ . /wellcomeopenres. .r © jambo k. this is an open access peer review report distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the authors have made appropriate changes and the paper should be indexed as soon as possible. competing interests: no competing interests were disclosed. if any results are presented, are all the source data underlying the results available to ensure full reproducibility? yesare the conclusions about the method and its performance adequately supported by the findings presented in the article? yes competing interests: no competing interests were disclosed.reviewer expertise: micobial pathogenesis and application to diagnostics and vaccine development. reviewer report september https://doi.org/ . /wellcomeopenres. .r what are the key recommendations that would help minimise the quantstudio contamination? can the authors comment on the potential for incorporating sample pooling into their workflow? . the authors should consider presenting the validation data in figure in the form of a table, in addition to the amplification plots. the authors should provide the data to support this statement "at this point, the assay was repeated several times to be further validated for reproducibility before being offered to the hospital for the screening of hcws". this would help qa and qc for those who want to adopt the setup. is the description of the method technically sound? yes if any results are presented, are all the source data underlying the results available to ensure full reproducibility? partlyare the conclusions about the method and its performance adequately supported by the findings presented in the article? yescompeting interests: no competing interests were disclosed.reviewer expertise: immunology, biomedical laboratory science i confirm that i have read this submission and believe that i have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however i have significant reservations, as outlined above. key: cord- -pyg vwb authors: tabak, tom; purver, matthew title: temporal mental health dynamics on social media date: - - journal: nan doi: nan sha: doc_id: cord_uid: pyg vwb we describe a set of experiments for building a temporal mental health dynamics system. we utilise a pre-existing methodology for distant-supervision of mental health data mining from social media platforms and deploy the system during the global covid- pandemic as a case study. despite the challenging nature of the task, we produce encouraging results, both explicit to the global pandemic and implicit to a global phenomenon, christmas depression, supported by the literature. we propose a methodology for providing insight into temporal mental health dynamics to be utilised for strategic decision-making. mental health issues pose a significant threat to the general population. quantifiable data sources pertaining to mental health are scarce in comparison to physical health data (coppersmith et al. ). this scarcity contributes to the complexity of development of reliable diagnoses and effective treatment of mental health issues as is the norm in physical health (righetti-veltema et al. ). the scarcity is partially due to complexity and variation in underlying causes of mental illness. furthermore, the traditional method for gathering population-level mental health data, behavioral surveys, is costly and often delayed (de choudhury, counts & horvitz b) . whilst widespread adoption and engagement in social media platforms has provided researchers with a plentiful data source for a variety of tasks, including mental health diagnosis; it has not, yet, yielded a concrete solution to mental health diagnosis (ayers et al. ) . conducting mental health diagnosis tasks on social media data presents its own set of challenges: the users' option of conveying a particular public persona posts that may not be genuine; sampling from a sub-population that is either technologically savvy, which may lend to a generational bias, or those that can afford the financial cost of the technology, which may lead to a demographic bias. however, the richness and diversity of the available data's content make it an attractive data source. quantifiable data from social media platforms is by nature social and crucially (in the context of our cases study) virtual. quantifiable social media data enables researchers to develop methodologies for distant mental health diagnosis and analyse different mental illnesses (de choudhury, counts & horvitz a) . distant detection and analysis enables researchers to monitor relationships of temporal mental health dynamics to adverse conditions such as war, economic crisis or a pandemic such as the coronavirus (covid- ) pandemic. covid- , a novel virus, proved to be fatal in many cases during the global pandemic that started in . governments reacted to the pandemic by placing measures restricting the movement of people on and within their borders in an attempt to slow the spread of the virus. the restrictions came in the form of many consecutive temporary policies that varied across countries in their execution. we focus on arguably the most disruptive measure: the national lockdown. this required individuals, other than essential workers (e.g. healthcare professionals) to remain in their own homes. the lockdown enforcement varied across countries but the premise was that individuals were only permitted to leave their homes briefly for essential shopping (food and medicine). this policy had far reaching social and economic impacts: growing concern towards individuals' own and their families' health, economic well-being and financial uncertainty as certain industries (such as hospitality, retail and travel) suspended operations. as a result, many individuals became redundant and unemployed which constrained their financial resources as well as being confined to their homes, resulted in excess leisure time. these experiences along with the uncertainty of the measures' duration reflected a unique period where the general public would be experiencing a similar stressful and anxious period, which are both feelings associated with clinical depression (hecht et al. , rickels & schweizer . in this paper, we investigate the task of detecting whether a user is diagnosis-worthy over a given period of time and explore what might this appropriate time period be. we investigate the role of balance of classes in datsets by experimenting with a variety of training regimes. finally, we examine the temporal mental health dynamics in relations to the respective national lockdowns and investigate how these temporal mental health dynamics varied across countries highly-disrupted by the pandemic. our main contributions in this paper are: ) we demonstrate an improvement in mental health detection performance with increasingly enriched sample representations. ) we highlight the importance of the balance in classes of the training dataset whilst remaining aware of an approximated expected balance of classes in the unsupervised (test) dataset. ) we analyse empirically proven relationships between populations' temporal mental health dynamics and respective national lockdowns that can be used for strategic decision-making purposes. unlike physical health conditions that often show physical symptoms, mental health is often reflected by more subtle symptoms (chung & pennebaker , de choudhury, counts & horvitz a . this yielded a body of work that focused on linguistic analysis of lexical and semantic uses in speech, such as diagnosing a patient with depression and paranoia (oxman et al. ) . furthermore, an examination of college students essays, found an increased use of negative emotional lexical content in the group of students that had high scores on depression scales (rude et al. ) . such findings confirmed that language can be an indicator of an individuals psychological state (bucci & freedman ) which lead to the development of linguistic enquiry and word count (liwc) software (pennebaker et al. , tausczik & pennebaker which allows users to evaluate texts based on word counts in a variety of categories. more recent and larger scale computational linguistics have been applied in conversational counselling by utilising data from an sms service where vulnerable users can engage in therapeutic discussion with counsellors (althoff et al. ) . for a more in-depth review of uses of natural language processing (nlp) techniques applied in mental health the reader is referred to trotzek et al. ( ) . the widespread engagement in social media platforms by users coupled with the availability of platforms data enables researchers to extract population-level health information that make it possible to track diseases, medications and symptoms (paul & dredze ) . the use of social media data is attractive to researchers not only due to its vast domain coverage but also due to the cheap methodologies by which data can be collected in comparison to previously available methodologies (coppersmith et al. ) . a plethora of mental health monitoring literature have utilised this cheap and efficient data mining methodologies from a variety of social media platforms such as: reddit (losada & crestani ) , facebook (guntuku et al. ) and twitter (de choudhury, gamon, counts, & horvitz ). twitter user's engagement in the popular social media platform give way for the creation of social patterns that can be analysed by researchers, making this platform a widely used data source for data mining. additionally, the customisable parameters querying available in the application programmable interface (api) allows researchers to monitor specific populations and/or domains (de choudhury, counts & horvitz b). in the context of the covid- pandemic, we found a handful of projects with similar intentions as our own, to mon-itor depression during the pandemic. li et al. ( ) gather large scale, pandemic-related twitter data and infers depression based on emotional characteristics and sentiment analysis of tweets. zhou et al. ( ) focus on detecting community level depression in australia during the pandemic. they use the distant-supervision methodologies of shen et al. ( ) to gather a balanced dataset, they utilise the methodology of coppersmith et al. ( ) to model the rates of depression and observing the relationship with the number of covid- infections in the community. our work differs from this in three main areas: ) we investigate the implication of different sample representations to provide more context to our classifier. ) we retain an imbalance in our development dataset. ) we investigate european countries (france, germany, italy, spain and the united kingdom) that experienced a relatively high number of covid- infections. in this section we describe the data mining methodology used to build a distantly supervised dataset and the classifier experiments conducted on this dataset. to conduct the proposed experiments, we firstly construct a distantly supervised development dataset for each country, to be used in training and validation of the classifier. the data mining methods follow the novel distant-supervision methodology proposed in coppersmith et al. ( ) as it is relatively cheap but also well-structured for clinical experiments. we follow the widely-accepted methodology proposed by watson ( ) where diagnosed (diagnosed) and nondiagnosed (control), groups are created. in this paper we will only be exploring depression as a mental health condition, accordingly we will have a single diagnosed group for each country's development dataset. however, if multiple mental issues were to be explored, then the same number of different diagnosed groups would be required for each country's dataset. ) diagnosed group: we gather public tweets with a geolocation inside the country of interest, posted during a two-week period during . as we are searching for a depression diagnosed tweets, this two-week period needs to be chosen strategically, as we want to capture users that have been diagnosed with depression rather than seasonal affect disorder (sad), a separate albeit a condition with similar symptoms. tweets collected via twitters api , were retrieved based on lexical content indicating that the user has history/is currently dealing with a clinical case, e.g. i was diagnosed with depression, rather than expressing depression in a colloquial context. human annotators were then instructed to remove tweets that are perceived to have made a nongenuine statement regarding the users' own diagnosis, most of these were referring to a third party. examples of genuine and non-genuine tweets encountered can be seen in table i . we then collect all (up to , most recent) tweets made public by the remaining users between the start of and october . further filtering includes removal of all users with less than tweets during this period or those whose tweets do not meet our major language of instruction benchmark. this benchmark requires % of the tweets collected to be written in the major language of instruction of the country of interest (i.e. united kingdom is english, italy is italian etc.). following this filtering process and some preprocessing on the tweet level, which includes medial capital splitting, mention white-space removal (i.e. if another user was mentioned this will be shown as a unique mention token), the same has been done with urls, all uppercase and non-emoticon related punctuation were removed. ) control group: we gather , public tweets with a geolocation in the country of interest, posted during the same two-week period as the diagnosed in and remove any tweets made by diagnosed group users. we then follow a similar process to that of the diagnosed collection methodology by collecting up to , most recent tweets for each user from the period mentioned above. as can be seen in table ii , we construct imbalanced datasets. world health organisation (who) claim million people suffer from depression worldwide . whilst, at the time of writing, the global population is approximately . billion . this would suggest that in individuals suffer from depression. however, these figures are approximations. therefore, the extent to which our datasets are imbalanced is not an attempt to create datasets that are representative of the expected balance of classes, as these are unverifiable. nevertheless, our datasets present ratios of control:diagnosed samples between . : and . : , which came about from the data mining methods previously described. we accept these ratios to retain imbalanced datasets in a similar order of magnitude as the expected balance whilst achieving reasonable classifier performance. we inherit the caveats to the distantsupervision approach of coppersmith et al. ( ) : (a) when sampling a population we always run the risk of only capturing a subpopulation of the control or diagnosed that is not fully representative of the population, especially considering that diagnosed samples are identified based on the fact that they publicly speak out about what is a deeply personal subject this attribute may not generalise well to the entire population. (b) we do not implement a verification of the method used to identify users in diagnosed but rather rely on the social stigma around mental illness whereas it could be regarded as unusual for a user to tweet about a diagnosis of a mental health illness that is fictitious. (c) control is likely contaminated with users that are diagnosed with a variety of conditions, perhaps mental health related, whether they explicitly mention this or not. we have made no attempt to remove such users. (d) depression is often comorbid with other mental health issues (aina & susman ) . as such, it is plausible that the users forming diagnosed are suffering from other mental health conditions. this could suggest that the classifier will be trained to pick up these hidden meaning representations of other mental health issues and classify them as depression. we have made no attempt to further investigate nor remove such users from diagnosed as having a complex diagnosed group is a realistic representation of the task. in this section we describe the experiments conducted in training our classifier to diagnose depression. the trained classifier is deployed in section iv for classifying samples from an unsupervised experiment dataset which is then used in analysing temporal mental health dynamics. we investigate the most appropriate sample representation of our distantly supervised dataset. we are posed with these considerations: (a) symptoms' temporal dependencies: as the tweets gathered come from a variety of days, weeks, months and even years, symptoms may only be present in specific timedependant samples. however, when represented by overwhelming tweet-enriched samples the classifier perfor-mance is traded-off with retaining the symptoms' temporal dependencies. (b) as our final task will be to monitor and analyse the temporal mental health dynamics, we are interested in modelling the rate of depression as fine-grained as possible. therefore, the ability to accurately identify diagnosed samples and correctly discriminate between control and diagnosed with the least tweet-enriched samples will be vital in modelling a fine-grained rate of depression in the deployment stage of the final task where conclusions could be drawn in the context of the national lockdowns. the sample representations we examined: • individual each sample constitutes of a single tweet. • u ser day -each sample constitutes of all tweets by a unique user made public during a given day. • u ser week each sample constitutes of all tweets by a unique user made public during a given week. • all user -each sample constitutes of all tweets made public by a unique user. we examine the performance of a benchmark, support vector machine (svm) with a linear kernel function (peng et al. ) , on the different sample representations datasets where the benchmark classifier inputs are sparse many-hot encoding representations of the samples' lexical content. as we are working with imbalanced datasets we need to think about the metrics we use to assess the classifiers' performance. the accuracy metric is insufficient for imbalanced datasets and is best illustrated with an example. if we have a dataset with : ratio split between the samples of each class, the classifier could achieve % accuracy by classifying every sample as the majority class. the classifier is clearly not discriminating between the distributions of the two classes but yet achieving high performance. as such, we will be assessing the performance of the classifier on the individual classes' precision (p), recall (r) and f score measures as well as the macro f score for this and the remainder of the experiments in this paper. the precision measure will tell us: of all the samples the classifier labelled as a particular class, what fraction are correct. the recall measure will tell us: of all the samples that actually belong to that particular class, how many did the classifier correctly identify. whilst the f score is a harmonic mean between the two and the macro f score takes the f scores of all classes and calculated a non-weighted mean between them. by having a more classspecific breakdown of the classifiers' performance we can better understand the strengths and limitations of our classifiers and hence make a more informed decision when choosing the highest performing classifier. the results in table iii suggest that our benchmark classifier improved in identifying diagnosed, with increasingly tweet-enriched, samples. interestingly however, when presented with the u ser day sample representations a sharp decreased in performance in control samples causing a decrease in macro f score when compared with the f scores of both individual and u ser week sample representations. barring this decrease in macro f score, we can say that we are able to achieve improved performance when using increasingly tweet-enriched samples. however, the end task would benefit from fine-grained modelling of the rate of depression, providing us with more detailed relationships between the temporal mental health dynamics and noteworthy dates. as such, our task is bias towards the two fine-grained sample representations, individual and u ser day. as our benchmark classifier achieves superior performance on the individual sample representation we will adopt this representation, as denoted by the asterisk in table iii . ) classifier experiments on u.k. development dataset: once we have chosen the sample representation that balances out or fine-grained sample requirements with the benchmark classifier performance, we must now build a classifier that best discriminates between our two classes. the higher the performance of the classifier, the more accurate the temporal mental health dynamics will in section iv. we outline the classifier architectures included in our experimentation: • sv m : linear kernel svm as used in section iii-b . this classifier will serve as our benchmark. • av ep l ef c : average pooling layer. we set hyper-parameters where an adam optimiser (kingma & ba ) is used with a learning rate of . , batch size of , . all classifiers were trained for a single epoch with a dataset training:validation split of : and weighting the samples of diagnosed as times more valuable than those of control. training was done on a single tesla p -pcie with gb of ram available through google's colaboratory . table iv shows that all classifiers achieve significantly higher performance on control than diagnosed. as we are trying to correctly detect diagnosed samples and discriminate between the two classes, we prioritise the diagnosed precision and macro f score metrics. based on these chosen metrics to guide our classifier selection process candidates emerge: av ep l, bilst m and bilst m -self a achieving {diagnosed precision, macro f } scores of: { . , . }; { . , . } and { . , . } respectively. whilst the performance of these classifiers is similar the performance of bilst m -self a is the highest performance combination of the desired metrics (indicated by the asterisk) and as such we will be adopting this classifier in further experiments. in this section we investigate the distribution of our datasets in training and validation of our classifier. by conducting this experiment we intend to gather an in-depth understanding of our task from a linguistic standpoint. we train and validate the classifier on datasets with varying balances to investigate the role of our imbalanced dataset in the depression diagnosis task. this experiment analyses the performance of the bilst m -self a classifier on a number of different training regimes: • balanced: a dataset containing all diagnosed samples and downsampling from control. • imbalanced: a dataset of the development dataset's distribution (see table ii) . furthermore, we explore the effects of sample weighting of the classes by weighting diagnosed samples as times more valuable than control samples as mentioned in the previous experiment. the performance of the bilst m -self a classifier on the different training regimes can be seen in table v the balanced-balanced (t raining-v alidation) training regime achieves encouraging results in terms of its precision-recall trade-off, for both classes, as well as the macro f score. this shows that the problem is reasonably linguistically achievable, when the imbalance challenge is removed from the equation. the imbalanced-imbalanced training regime shows that adjusting the sample weighting is a successful measure we can implement to adjust the precision-recall trade-off in our class of interest (diagnosed). our classifier performs significantly worse in the balanced-imbalanced regime when compared to the performance on the imbalanced-imbalanced regime, this performance is reduced by the introduction of sample weighting. this means that when training on a balanced dataset our classifier is less robust to an imbalanced dataset at validation. finally, whilst our classifier experiences a significant improvement in performance on the imbalanced-balanced training regime when sample weighting is introduced due to our final depression diagnosis task in which we expect an imbalanced unsupervised dataset (discussed in section iii-a ) the training regimes implementing balanced validation datasets are not suitable approximations of our classifier's depression diagnosis performance. therefore, we conclude that imbalanced training, with suitable sample weighting, yields more desirable and robust depression diagnosis performance as it's able to see a broader range of data examples in training (i.e. no sub-sampling). we train separate bilst m -self a classifiers for each of the countries' imbalanced development datasets following the individual sample representation. the test performance of these classifiers can be seen in table vi . we observe that the bilst m -self a classifier architecture achieved similar performance on the remaining countries' datasets as was acheived on u.k. dataset. this shows that the bilst m -self a architecture is able to generalise well to different languages and cultural differences after training. hence, producing an encouraging set of results and increase our confidence in its classification ability. whilst the bilst m -self a classifier architecture achieved the highest performance of all our classifier architectures, a combination of . diagnosed precision and . macro f score leaves much to be desired. as such, we perform an error analysis and examine the significance of its results. table vii shows the input samples, text, the prediction type as well as the sigmoid output which is the output layer of the classifier and is responsible for the final classification of the samples. the sigmoid output is normalised in the range of [ , ] ∈ r, where an output of . represent the decision boundary, as such it can be interpreted as complete uncertainty by the classifier as to how the sample should be classified. a sigmoid output of is complete certainty that the sample should be classified as positive (diagnosed) and an output of is complete certainty the sample should be classified as negative (control). we observe that the true positive example mentions having "overcoming depression" which implies that the user has recovered from depression, as one overcomes other health issues. the sigmoid output for this sample is . which is extremely high certainty by the classifier that this sample follows the distribution of diagnosed. whilst on the other end of the scale, the true negative sample discusses a topic that is completely unrelated to nor implies that the individual suffers from depression, as such it is classified as part of control with a sigmoid output of . . however, we find the texts of the two samples misclassified by the classifier are rather similar. they both use words rooted from the word 'depress' in rather colloquial contexts, with no indication of a past diagnosis or clinical appropriation of depression -which is rather a desirable ability for our classifier to be able to discriminate between. it is also noteworthy that the sigmoid outputs of these two samples are much less polarised than the correctly classified samples, with the sigmoid output of the false positive sample just about falls within the diagnosed classification region. however, these two incorrectly classified samples reflect the complexity of depression diagnosis from distantly supervised tweets. ) significance of results: we investigate the significance of our classifiers' results by performing a χ significance test. our null hypothesis, h , states that both sets of data, our classifiers' predictions (d p ) and the distribution it is being tested against (d t ), have been drawn from the same distribution (d). we compare the distribution of the classifiers' predictions against a random uniformly distributed set (denoted by uniform) and against a random distributed set following the distribution of the development datasets (denoted by weighted). all classifier results in table vi are statistically significant from the random baselines, according to the χ significance testsee table viii in appendix a. therefore, we can reject h and conclude that the predictions of the classifier and those of the respective randomly distributed benchmarks have not been drawn from the same distribution. we prepare the unsupervised dataset and deploy the previously trained bilst m -self a classifier to annotate this dataset. we analyse the relationships between the temporal mental health dynamics to respective national lockdown dates. in this section we discuss the procedure for constructing the unsupervised experiment dataset, to be used for monitoring the temporal mental health dynamics during the respective pandemic-inflicted national lockdowns. the experiment dataset is used for the deployment of the classifier, which is trained and validated on the development set, to analyse the temporal mental health dynamics of a country. we start by gathering tweets made public by users during the first two weeks of with a geolocation within the country of interest. we then follow the same methodology for data mining as control outlined in section iii-a, for the periods starting from december until may , where we apply similar user-level and tweet-level preprocessing and filtering on these dataset. the composition of these experiment datasets can be seen ( table ix in appendix b) along with key dates. the key dates specified observe the official date announcing the commencement of and the announcement of first step towards easing of the national lockdowns, rather than the first official data implementing these measures as we anticipate that the announcement would provoke users to express their opinion more than the implementation of the measures. we acknowledge some caveats to the methodology with relations to the temporal mental health dynamics during the respective national lockdowns: (a) the activity-level of users whose lifestyles have been highly disrupted by the national lockdowns may be overstated during this period, due to increased leisure time. (b) the language-based filtering component may exclude certain users of the population such as stranded expatriates that use a non-majority language to communicate their thoughts. such samples may contain a bias towards a higher rate of depression. to monitor and analyse temporal mental health dynamics we must firstly deploy our trained bilst m -self a on the respective countries' unsupervised experiment datasets. once we have the classifier's predictions, we must model the rate of depression by calculating the rate of depression at any given day, r t , using the following equation: where Φ represents our trained classifier, x i is the input, n t is the total number of samples on day t. the output of the classifier, Φ(x i ) takes the form [ , ] ∈ n. r t is a normalised continuous value between and , interpreted as the proportion of tweets at t that classify as diagnosed: meaning all samples belong to control and meaning all samples belong to diagnosed. figures and (see appendix c) display the temporal mental health dynamics for the countries under investigation. it is noteworthy that r t across the different countries is a function of the ratio of control:diagnosed samples in the country specific datasets on which the classifier was trained. as such, the rates across countries are not directly comparable but are rather analysed by the momentum of how r t in a country changed over time and how it differed from r t of other countries. foremost, we note that we categorically cannot, nor do we, state that the rates of depression discussed in this section are caused by the imposition of respective national lockdowns or any other measures of any type, taken by governments to combat the spread of the virus. in this section, we merely offer interpretations of the rates of depression in line with explicit relationships that we discover between the rates of depression and key events that occurred during the time-period included in this case study. upon examination of the u.k. rate of depression (r u k ), the first distinct observation we make is not related to any pandemic-related measures but rather the sharp, non-sustained, increase of r u k of over % on christmas day, before decreasing back to the status quo the next day. upon further investigation we find that this phenomenon is well-documented (hillard & buckman ) and seeing that our classifier was able to identify this phenomenon, without explicitly being aware of its existence, is encouraging. we continue observing r u k chronologically, on march th , italy national lockdown begins. from this point on we observe a sharp, sustained increase in r u k approximately until march rd , u.k. national lockdown begins (the last country to impose such a restriction in this study), where r u k somewhat plateaus. we interpret this as an increase in anxiety, a symptom of depression, amongst the u.k. population as neighbouring countries take decisive measures to slow the spread. a key theme in the build up to the u.k. national lockdown implementation was the intentional delay so that to ensure maximum utility from the policy . however, a report published on the th of march by the imperial college covid- response team estimated that the the current combative approach taken up by the u.k. government would result in , deaths. the report was well-publicized by the british media and was arguably a factor in the change of combative approach by the u.k. government. this is somewhat supported by the change in r u k during the u.k. national lockdown where we see a sustained decrease for the majority of the period. finally, we observe a slight increase in r u k towards the end of and in the aftermath of the national lockdown that could perhaps be interpreted as anxiety and concern from the population at the uncertainty with which they are faced both from a social and an economic perspective. the rates of depression of france (r f r ), germany (r de ), italy (r it ) and spain (r es ) behave rather differently from r u k . firstly, r it increases sharply by over % over the initial days of the italian national lockdown. this can be interpreted as anxiety and concern in response to the quickly imposed stringent measures by the italian government in response to the outbreak of the virus in italy, which at this point was largely believed to be the epicentre of the pandemic. this was coupled with economic turmoil and great concern over the capacity of hospitals and their ability to handle the high requirement for intensive care units that would ensue . a similar pattern emerges in r f r , a sharp increase over the initial days of the french national lockdown period, afterwhich r f r continues to rise throughout the lockdown period at a lower and inconsistent rate. a similar story could be tailored to r es . whereas, the major increase in r de occurs fig. . u.k. rate of depression before and during the national lockdown. noise in the rate of depression has been smoothed with a -day moving average. in the build-up month, whilst during the german national lockdown, r de increases in the initial days, albeit at a lower rate. r de then plateaus and decreases -creating a turning point in r de during the german national lockdown. furthermore, we can observe the r of the respective countries following the easing of respective lockdowns and interpret them as the countries' outlook on the easing of restrictions. from the time period that we have available it seems that the french and spanish general populations experienced a reduction in symptoms of depression, such as anxiety, as is evidenced by the clear reduction in r f r and r es respectively. we can therefore conclude by, tentatively, stating that the easing of restrictions were received by an improvement in the mental state of the general populations of france and spain, the mental state of the italian and german general populations deteriorated, whilst the general mental state of the u.k. was rather agnostic to the easing of restriction. we are hesitant to state the changes in the rates of depression had been caused by the imposition/easing of national lockdowns. to make such a claim we would be required to undertake a more fine-grained causality study which is beyond the scope of this paper, however we note this for future work. we can however claim to have discovered clear relationships between the drastic changes in the behaviour of rates of depression during the periods of the build-up to, during and in the aftermath of national lockdowns v. conclusion our set of experiments have been conducted with the aim of providing organisations with a methodology for monitoring and analysing temporal mental health dynamics using social media data. we examine sample representations and their ability to impact classifier performance. we investigate the role of including an imbalanced dataset in the classifier training regime. our classifier provides encouraging performance on two fronts: the first being that it is able to discriminate with high certainty between clear diagnosed and control samples and secondly, it was able to identify the christmas depression phenomenon supported by the literature. finally, we present an analysis and discussion of the rates of depression and their relationships with key events during the covid- pandemic. we reiterate that through the analysis conducted in this paper, we cannot state that the measures imposed caused the changes in rates of depression during the pandemic and leave this causality analysis for future work. mental health monitoring methodologies such as the one proposed in this paper can be adopted by governments, to identify relationships between the general population's mental health state to imposed measures, mental health authorities, to assist in planning and targeting individual locations in which to dynamically concentrate their resources, as well corporations involved in producing or disseminating drugs, such as pharmaceuticals, to combat mental health issues for a more commercial use case. coronavirus: boris johnson announces uk government's plan to tackle virus spread, youtube impact of non-pharmaceutical interventions (npis) to reduce covid- mortality and healthcare demand doctors: covid- pushing italian icus toward collapse understanding comorbidity with depression and anxiety disorders large-scale analysis of counselling conversations: an application of natural language processing to mental health could behavioral medicine lead the web data revolution? the language of depression the psychological functions of function words quantifying mental health signals in twitter predicting postpartum changes in behavior and mood via social media social media as a measurement tool of depression in populations predicting depression via social media bert: pre-training of deep bidirectional transformers for language understanding detecting depression and mental illness on social media: an integrative review anxiety and depression: comorbidity, psychopathology, and social functioning christmas depression long short-term memory adam: a method for stochastic optimization what are we depressed about when we talk about covid : mental health analysis on tweets using natural language processing a test collection for research on depression and language use the language of paranoia you are what you tweet: analyzing twitter for public health multi-kernel svm based depression recognition using social media data psychological aspects of natural language use: our words, ourselves the treatment of generalized anxiety disorder in patients with depressive symptomatology' risk factors and predictive signs of postpartum depression language use of depressed and depression vulnerable college students depression detection via harvesting social media: a multimodal dictionary learning solution the psychological meaning of words: liwc and computerized text analysis methods utilizing neural networks and linguistic metadata for early detection of depression indications in text sequences attention is all you need an account of a series of experiments, instituted with a view of ascertaining the most successful method of inoculating the small-pox detecting community depression dynamics due to covid- pandemic in australia france imposes -day lockdown and mobilises , police to enforce coronavirus restrictions france eases lockdown after eight weeks germany bans groups of more than two to curb virus germany says football can resume and shops reopen all of italy is in lockdown as coronavirus cases rise coronavirus: italy's pm outlines lockdown easing measures spain orders nationwide lockdown to battle coronavirus spain plans return to 'new normal' by end of coronavirus updates: 'you must stay at home' uk public told -bbc news uk past the peak of coronavirus, says pm purver is partially supported by the epsrc under grant ep/s / , and by the european unions horizon pro-gramme under grant agreements (saam, supporting active ageing through multimodal coaching) and (embeddia, cross-lingual embeddings for less-represented languages in european news media). the results of this publication reflect only the authors views and the commission is not responsible for any use that may be made of the information it contains. we express our thanks to all of our data annotators: l. achour, l. del zombo, n. fiore, m. hechler and r. medivil zamudio. key: cord- -j cecioe authors: fager, edward w. title: determination and analysis of recurrent groups date: - - journal: ecology doi: . / sha: doc_id: cord_uid: j cecioe nan the primary information available from an ecological survey usually consists of a mass of sampling results involving many localities and many species. in order to investigate the interspecific processes which may be involved in controlling the abundance and distribution of the species found, it is useful to be able to group together those species which frequently occurred together in the samples, which were in this sense a nearly constant part of each other's biological environment. the grouping will be most valuable as a classificatory device if it consists of defined units obtained by a procedure which can be repeated. whatever the method of grouping employed, the sampling procedure is certain to introduce subjective elements; the size of sample, the number and selection of sampling sites and the sampling methods used will always be influenced by the judgment of the person doing the study and they will in turn influence the composition of the groups. if, however, the sampling conditions are explicitly given, the probable extent and character of these influences can be taken into account and a meaning given to the groupings in this context. some of the methods of grouping which have been suggested will be briefly reviewed (see also macfadyen, ) and a new method will be outlined which overcomes some of the difficulties review of some methods of grouping animals have often been grouped on the basis of vegetation or of various physical or chemical factors in the environment, but many difficulties are involved in vegetational classification or in deciding what is a critical limit in respect to a certain physical or chemical factor. furthermore, animal groupings do not always conform to vegetation or other factors ; a group may extend over several recognizable vegetation or soil divisions or there may be several distinct assemblages of animal species within an environmental complex which is uniform in many other respects. the characterization of the habitat in which an animal group is often found must include the associated plants and other factors, but it seems better to use the animals as the primary basis for their grouping. gisin ( gisin ( , gisin ( , has suggested a procedure using what he has called "differential" species. in the hands of an experienced worker this method can lead to useful groupings, but the choice of the species which will be designated as "differential" is so subjective that comparison between groupings by different workers, even if based on the same material, may be impossible. moreover, using this method groups may be suggested the constituent species of which so seldom occur together that it seems unlikely that they could have any relation to each other. for example, gisin ( ) in his rearrangement of agrell's ( ) swedish lapland collembola suggested four groups. members of three of these were found together moderately often. the fourth group contained four species, representatives of which were recorded in a total of thirty-one samples ; no sample contained all four, one sample contained three, five samples contained different pairs and twenty-five samples contained only one of the species. the subjective element can be reduced if the grouping is based on a defined index of affinity. perhaps the simplest expressions which might be used for this purpose are those proposed by jaccard ( ) and sprenson ( ) as measures of the similarity between floras. as given, however, neither differentiates between a coefficient based on ten samples and one based on one thousand samples, though the latter would surely be a more reliable estimate. the expressions also fail to take account of changes in the relative number of total occurrences of the two species; as long as the sum of the total occurrences remains constant, the proportions can change without affecting the value of the coefficient. a method based on correlation between pairs of species is precisely definable but, as cole ( ) has pointed out, product moment correlation cannot be used directly because sampling results for most species are not normally distributed in regard to numbers of individuals per sample; there are qsually a few outsize samples which will have a disproportionate effect on the calculations. transformation of the data or the use of rank correlation could overcome this difficulty, but any method which involves a measure of abundance may, in certain cases, not lead to the desired results : two species may always occur together and never separately and yet, unless there is a nearly constant relation between the relative numbers of individuals of the two species, a correlation coefficient will indicate no relationship even though they are a constant part of each other's biological environment. for this reason it seems best to base species groupings upon presence and absence alone and to consider abundance relations within such groupings after they have been determined. in his paper, cole ( ) discussed the coefficients of association which had been proposed up to that time and indicated the deficiencies of each. he then proposed a new coefficient using only presence and absence and based on a x con-ti~ig-ency table for which a chi-square test of sigmficance can be calculated. a basic assumption of his expression is that all samples taken should be included; i.e., the probabilities of occurrence of the two species being tested for association are assumed to be uniform throughout the area sampled. under this assumption, the chi-square indicates the significance of the departure of the observed number of joint occurrences from the number expected on the hypothesis of independent distribution of the two species over all the samples. although cole did not use his coefficient for grouping species, it has been so used several times since. the following example shows that it is not a satisfactory criterion for grouping if the groups are to be composed of species which form a nearly constant part of each other's biological environment: species and show no evidence of association when examined by cole's method and yet they nearly always occur together-over % of the occurrences of each are in company with the other -and should be considered together in any grouping based on this set of samples. on the other hand, species and are significantly associated by cole's criterion even though their affinity is rather low-less than % of the occurrences of each are in company with the other. in general, two species which occur in most of the samples taken will show little or no evidence of association and two species with a low percentage of joint occurrences can show significant evidence of association if they are rare enough or if a large enough number of samples in which they could not occur are included. bray ( s ) has recently noted these difficulties connected with the use of cole's index and has attempted to overcome them by considering only quadrats within plots in which both species occurred. macan ( s ) using the method without modification found a very large number of positive associations in his analysis of collections of corixidae, a finding which, as he indicated, was unsatisfactory because the objective analysis produced a less well-defined picture than that which could be obtained from a subjective technique based on a knowledge of habitat features. an expression of the sort suggested by cole has certain desirable properties; in particular, it is sensitive to changes in the relative frequency of occurrence of the two species and a "significance a t-test is preferred to chi square because it can be used as a one-tailed test [ . s ,....., p (.os) ] for positive affinity; negative affinity, being based on the failure to find a species, seems potentially subject to too many unavoidable errors. there are both mathematical and biological reasons for setting an upper limit on the value of nn which will be considered with a given na: the closer the ratio nnjna is to , the closer the normal approximation will be to the true value; keeping the ratio close to ensures that whenever evidence of affinity is obtained the number of joint occurrences will be a considerable proportion of the total number of occurrences of both of the species. this is a desirable property for an index to be used as a basis for grouping if the purpose is to put together organisms which are a nearly const·mt part of each other's biological environment. an upper limit of for the ratio nn/na is therefore sug-level" can be defined for use as an objective criterion of the presence of affinity. it can be adapted for use as an index of affinity if it is assumed that the probability of finding species a is najna + nb and of finding species b is nbjna + nb, where na is the total number of occurrences of species a and nb is the total number of occurrences of species b; i.e., the probalilities of occurrence of the two species are related to the sum of their occurrences (na + nb) rather than to the total number of samples taken. the use of this assumption removes the premium on rarity and also makes it possible to find evidence of affinity between two species which occur in most of the samples taken. it seems neither more nor less arbitrary than the use of the total number of samples for the latter is usually determined arbitrarily by the person doing the work, often mainly on grounds of practicality or subjective judgment that the number taken is sufficient. if the preceding assumption is used and the species are assigned to the letters so that na is less than or equal to ne, the number of joint occurrences (j), on the hypothesis of independent distribution, will have a hypergeometric distribution with expected j equal to nanejna + ne and variance equal to (nanb) j(na + ne) (na + ne- ). for values of na greater than and p and q not too different from j , a normal approximation can be used to test the significance of the deviation of the observed number of joint occurrences from the expected number. for this purpose, the following expression can be taken as a normal deviate with mean and unit variance (pearson ): gested. the use of such a limit has the disadvantage of preventing the finding of affinity between such a pair of species as a rare parasite and its relatively abundant host, but the existence of a relationship of this kind can, in any case, probably be satisfactorily established only by field observation and breeding studies. minimum values of j which are significant at the .os level have been calculated for values of na from s to using exact probabilities obtained by leslie's method ( ss) . similar values have been calculated for each tenth value of na from to using the normal approximation. both sets are given in table in the appendix. these values and intermediate ones which can be estimated by interpolation will make it possible to quickly sort pairs of species into those which certainly show evidence of affinity, those which cer-tainly do not and those which are doubtful and require calculation. when applied to the examples used in the discussion of cole's coefficient, the index proposed above indicated significant evidence of affinity between species and but none between species and . it is, therefore, a better index of the pro-portion of occurrences which are joint occurrences and should provide a more satisfactory basis for grouping. it may be repeated that cole did not develop his index as a basis for grouping and that the two indices measure different things: cole's index measures the departure of the two species from independence of distribution on the assumption that the probability of occurrence of each is constant over all the samples taken ; the index proposed in this paper provides an objective measure for the word "frequently" in the statement "these species frequently occurred together." the procedure to be outlined for the determination of recurrent groups may appear complicated but with a little practice it can be gone through at least as rapidly as the "fitting by eye" of a trellis diagram. it has the virtue of repeatability ; given the same primary information, two workers will always arrive at the same groups. this means that if several workers using similar sampling methods make studies in different localities and find different recurrent groups, there is some assurance that the differences are real and not the result of differences in judgment or emphasis. any dichotomous index of relationship between species can be used as a basis for grouping by this procedure; the meaning of the groups will be determined by the nature of the attributes measured by the index. in this paper, a recurrent group is defined as one which satisfies the following requirements : ( ) the evidence for affinity is significant at the . level for all pairs of species within the group. ( ) the group includes the greatest possible number of species. ( ) if several groups with the same number of members are possible, those are selected which will give the greatest number of groups without members in common. ( ) if two or more groups with the same number of species and with members in common are possible, the one which occurs as a unit in the greater number of samples is chosen. these requirements are to be taken in order; i.e., the group or groups which fulfill requirements and are determined and then, if there are several possible combinations, a choice is made on the basis of , followed, if necessary, by . such groups will represent the largest, most frequent, separate units within which all the species formed a nearly constant part of each other's biological environment. they would be a likely choice if one wished to make an intensive study of interspecific processes which are quantitatively important in the habitats sampled. the following procedure will give a recurrent group or groups as defined above. the affinity information for the species concerned may be set out in a trellis diagram as shown for the example (table i) . if there are many species, it has been found more convenient to record the information for each species on a separate card. . =non-affinity the species are put in order in terms of the number of other species with which they have affinity. in the example this order is a, b, c, . . . p, q. starting with the species with the largest number of affinities, species are counted in the direction of decreasing number of affinities until the number of species counted (x) exceeds the number of affinities ( y) of the last species counted. in the example this occurs at species h where x = and y = . two possible relationships between x and y must now be considered: if this is true, as it is in the example, the largest potential group will contain y + species ; species in the example. it cannot contain more because species h has affinities with only other species and can, therefore, only form a group of species satisfying requirement ( ). if species h is omitted, only species are left. because the species were put ecology, vol. , no. in order in terms of the number of other species with which they had affinity, the species beyond h will either have the same number of affinities as h, in which case they are also potential members of a group of species, or fewer affinities than h, in which case they cannot be included in a group of species satisfying requirement ( ). if species h had had affinities with other species, the largest potential group would have contained the first species, a to h. [x > y + ] if this is true, the largest potential group will contain the x - species preceding the last one counted. for example, if species h had had affinities with or fewer other species, the largest potential group would have been composed of the species, a to g, for h and any species beyond it could only form a group containing or fewer species. the counting procedure thus selects those species which are potentially members of a group satisfying requirements ( ) and ( ). these must be further examined to determine whether such a group can be formed. two preliminary tests of the possibility of its formation can be applied. the first test can be stated as follows : in order to form a group of z members from v potential members, there must be at least z of these with more than z- affinities with others of the potential group. for this test the affinities of each of the selected species with all others of the potential group are tabulated. this tabulation for the example is shown below : a b c d e f g h v = ;z = . there are only species which have more than affinities and, therefore, a group of species cannot be formed. passing the preceding test is a necessary but not sufficient condition for the formation of a group as can be seen by consideration of the following : if the interrelationships among the potential members had been such that there was affinity between all species except the four pairs, a-b, c-d, e-f, and g-h, the tabulated affinities would have all been and yet no group containing species and satisfying requirement ( ) could have been formed. on the other hand, if the species a to g had had affinity with all others except h, the tabulated affinities would have been seven 's and one and a recurrent group of species could have been formed. in general, the affinities lacked by the species needed to form the group must be accounted for by species which are not necessary for its formation. the second test determines whether this condi-tion is met. it can be expressed algebraically as follows : in order to form a group of z members from v potential members the following inequality must hold: (v- ) ( z-v) < + ~ z largest affinities -~ the rest of the affinities. applied to the two cases discussed in the preceding paragraph, it gives the following results: incorrect, a group of cannot be formed. ( - ) ( [ ] - ) < + ( )- ( ); correct, a group of can be formed. this is a necessary and sufficient condition for the formation of a recurrent group when v = z or z + ; it is necessary but not sufficient when v = z + or more for in this case the two sums on the right hand side of the expression can vary independently to some extent, the more so as there are more possible pairs of species within the category "rest." although passing these tests does not make the potential group a certainty in all cases, the two tests will prevent unnecessary work in many instances in which no group can be formed. returning now to the example, no group of species could be formed so the possibility of the next smaller group, species, is investigated. two additional species, j and k, must now be considered for, having affinities with other species, they could be members of a group of . the affinities within the group of species are shown in the first line of the tabulation below. both tests indicate that it may be possible to form a recurrent group of species : there are over species with more than affinities; ( - )( [ ] - ) is less than + ( + + + + + ) -( + + + ). the affinity interrelationships must now be examined in detail in order to make the final determination. perhaps the easiest way to do this is to eliminate those species which do not have more than z - affinities, g and k in the example ; repeat the tabulation as shown in the second line below, eliminate species which now do not have more than z - affinities and repeat until no more species can be eliminated. if at the end of this process v is less than z + , the second test can be applied as a both necessary and sufficient condition for the formation of a recurrent group. v=lo z= v= z= v= z= v= z= in the last line ( - ) ( [ ] - ) is less than + ( ) - and as v = z this is a necessary and sufficient condition for the formation of a recurrent group. therefore, the species a-e, j constitute the group. the presence of j in the group emphasizes the necessity of considering all species which, on the basis of the number of other species with which they have affinities, might be part of the group. the initial counting procedure takes care of this automatically for the largest potential group, but the appropriate species must be added if this group cannot be formed and the next smaller must be considered. as there were no alternative groups of satisfying requirements ( ) and ( ), requirements ( ) and ( ) do not have to be considered. the species not included in the group are now examined to see if any have affinities only with members of the recurrent group. this is true of l and q. such species are considered associates of the recurrent group. the procedure is repeated on the remaining species, omitting their affinities with members of the first recurrent group (table ii) . the largest potential group contains species; x = and y = at species n. when the affinities within this group of are tabulated, however, p and n have only and a group of species cannot be formed. all of the species must be included when the next smaller group, species, is considered. none can be eliminated on the basis of the first test and as ( - ) ( [ ] - ) is less than + ( + + ) -( + + + ) a group may be possible. when the interrelationships are examined in detail, the following groups are found to satisfy requirements ( ) and ( ) : fgn, fgp, fhp, fkp, gmn. requirement ( ) will be satisfied if gmn and either fhp or fkp are selected. in order to decide between the latter two, the original sampling results would have to be investigated on the basis of requirement ( ) . it will be assumed here that fhp occurred as a unit more often than did fkp. the relation- ships between the species might be set out as shown in figure . this method has been applied to several studies reported in the literature and to an unpublished survey of the invertebrate fauna of decaying wood done by the author. in all cases the groups determined were consistent with what was known of the species' requirements, preferences, etc. the invertebrates from decaying wood were grouped using code numbers for the species in order to eliminate the possibility of bias ; the groups found were in general agreement with impressions gained during sorting and counting. recurrent groups based on the index of affinity proposed in this paper and determined by the procedure outlined above are composed of species which very frequently form a part of each other's biological environment. such interspecific concepts as dominance, concordance and correlation should, therefore, be particularly meaningful when examined within these groups. the several methods of analysis suggested below use ranking procedures which, as kendall ( ) has shown, are very useful for detecting general trends in material which includes some extreme values, as sampling results from natural populations nearly always do. before the analytical methods can be applied, samples representative of each recurrent group must be selected. for groups containing only a few species, selection can be based on the requirement that all species in the group are present in the sample. for groups containing many species, this may too greatly reduce the number of samples and some compromise must be accepted. the requirement should be set at a high percentage present but the exact value to be used will depend on the sampling results which are available. it should be explicitly stated in any case. table iii presents some sampling results, related to actual observations but somewhat modified in order to show the different results which dominance numerical dominance is often expressed as a mean percentage of total individuals, obtained from the summed percentages of the samples considered separately. such an expression, using percentages calculated for samples with different total numbers of individuals, is hard to interpret and gives no information about the constancy of the relation. the difficulties of interpretation are increased if the species are dissimilar in size, activity, etc. it is, therefore, suggested that determinations of dominance be restricted to species within recurrent groups which are, or appear to be, similar in regard to size, activity, food requirements, etc. and that the determination be based on ranks within samples, summed over the set of samples. this is equivalent to n things (species) ranked m times (number of samples in the set). the concordance among the rankings can be tested by the statistic w (kendall , pp. - ) , which can range from to ; from no agreement to perfect agreement among the rankings. details of the calculation of w and of an f-test of its significance are given in the appendix. the use of ranks within samples gives every sample an equal voice in the decision and eliminates the bias which one or two samples with exceptionally large or small percentages may impose on the per cent method. rankings within samples for the species a, b and c and d and e are shown in the second part of table iii. for the value of w is significant at the . level showing that there is concordance among the samples; i.e., the dominance relations between the species tend to be constant over the set of samples. the sum of ranks is for each species the best estimate, in the sense of least squares, of the over-all rank of that species and if there is significant evidence of concordance the species may be ordered by these sums. the results may be stated as c >a, b; i.e., c is constantly dominant to a and b, and a is probably dominant to b but their sums of ranks are so nearly equal that the relative positions are less certain. the value of w[w = . ; f(n = . ; n = . ) = . (p > . )] is not significant for d and e indicating that dominance relation between these two species is not constant. relative abundance over all the samples is related to but not the same as numerical dominance. for example, assume that the numbers of individuals of two species found in samples were as follows: species x would be found to be constantly dominant to species y and yet their relative abundances would not be significantly different. the difference between the relative abundances could be tested by the usual t-test of the difference between the means, but if there were any outsize samples they would exert a disproportionate influence. as these are often present among samples taken from natural populations, it is probably better to use the distribution-free statistic u and a t-test based on it (hoel , pp. - ). this tests the hypothesis that the distributions of the two populations of values are the same against the alternative that one distribution is situated to the left (lower valued) of the other. it is not affected by outsize samples. details of the calculation will be found in the appendix. in the case of species a and b the observed value of u is not significantly different from the expected value. there is, therefore, no evidence that the distribution of abundances of a was different from that of b. as is apparent by inspection, c was significantly more abundant than either a or b. the observed value of u is significant at the .os level, indicating that e was significantly more abundant than d even though determination of dominance had shown that it was not constantly dominant to d. the concordance among the species of a recurrent group on what constitutes a good habitat can be tested by the same methods ( w) as were used in examining numerical dominance except that in this case ranking is done within species and the meaning of n (number of samples in the set) and n (number of species) are the reverse of those used in the previous test. if the observed number of individuals of a species is taken as an estimate of the goodness of a sample for that species, the samples can be ranked in regard to each species ; this eliminates difficulties due to differences between species in average abundance, efficiency of collection, etc. the sum of these ranks over all species of the group is for each sample the best estimate, in the sense of least squares, of the rank of that sample among the samples selected as representative of the group. if there is significant evidence of concordance, the samples can be put in order. rankings within species are shown in the third part of table iii. for the recurrent group considered as a unit; showing that there is agreement among the species on what constitutes a good or bad habitat. the samples can be put in order of decreasing goodness as follows: > , > , , , , , > . an examination of the other characteristics of these samples, especially the extremes, might reveal a good deal concerning the preferences and requirements of the group as a whole. rank correlations between species should also be calculated because the preceding generalization may miss significant relations: if, in a group consisting of four species two rankings are alike and the other two are exactly the opposite, the concordance will be zero although the six possible pairs of species will give four perfect negative correlations and two perfect positive correlations. because of the possibility of parent correlations, nonsense correlations, etc., the interpretation of correlation analysis must always be viewed with reservations. if, however, correlation coefficients are calculated only for those pairs of species for which there are other reasons for expecting interrelationships, they may serve as useful guides to the mature and constancy of the relations. kendall's ( , pp. - , ) the nearly significant positive correlation between the two species of poduromorph collembola, ac, might be due to chance, might indicate that the species have a favourable effect on each other, or might result from their liking the same conditions and not interfering. the significant positive correlation shown by the presumed prey-predator pair, cj, indicates that the predator did tend to congregate in those places where its prey was abundant. the absence of significant negative correlation between similar species means that in none of the pairs of species was an increase in the number of individuals of one always accompanied by a proportionate decrease in the number of individuals of the other. more evidence than the presence or absence of correlation is necessary before biological relationships can be claimed, but such an analysis will suggest those which might best repay further investigation. this paper presents a defined, repeatable method for grouping together species which are frequent components of each other's biological environment. such a method makes it possible to compare groups found in different habitats or at different times or localities. as the composition of these recurrent groups is influenced by the method of sampling used and by the "level of significance" required of the index of affinity, the groups are abstractions. it has, however, been found that if sampling is related to the size, activity, etc., of the organisms and the significance requirement is made stringent, the groups formed have ecological unity in the sense of significant intragroup agreement on what constitutes a good or bad habitat. it seems reasonable, therefore, to consider them as natural assemblages, somewhat artificially delimited but nonetheless real. because the recurrent groups are composed of species which very often occur together, they represent particularly appropriate units within which to examine interspecific relationships. the analytical procedures suggested for this examination have two purposes; the provision of a summary description of relationships and the suggestion of working hypotheses upon which to base further field and laboratory work. the use of methods employing ranking gives each species an equal voice in the analyses, removes many of the difficulties due to non-normality of distributions and makes it possible to determine the constancy of general trends. a new index of affinity between species, based on presence and absence, is proposed and a table is provided from which the significance of an observed number of joint occurrences can be estimated. using this index and a four-part definition of a recurrent group, a procedure is outlined which leads to the largest, most frequent, separate groups within which all species formed a nearly constant part of each other's biological environment. ranking methods are suggested for the examination of interspecific concepts such as numerical dominance, relative abundance, concordance and correlation within these groups. the essentials of the methods of testing for con-cordance ( w) and correlation ( -rb) and of the u test are given below for the convenience of those to whom the texts may not be readily available. the expressions given for the first two tests include the corrections required when tied ranks are present. in both cases, t or u represent the extent of ties present; e.g., if a ranking has members with one rank and others with another rank, the summation is ( - ) + ( - ) in the case of wand ( - ) + ( - ) in the case of "b· for the calculation of w all ties are considered together ; for the calculation of 'rb the extent of ties in one ranking is represented by t and that in the other by u. when tied ranks are absent, the summations are and the expressions are considerably simplified. concordance (kendall ) : if n things are ranked m times, the ranks for each of the n things are summecl and the sum of the squares of the deviations of these sums from their mean is represented by s, the concordance between the rankings ( w) is given by the following expression : w- s m n(n - )ml'jt(t - ). this does not have to be modified for tied ranks unless the number and extent of ties is large. to determine significance the usual table of the variance ratio (f) is entered with degrees of freedom n and n • correlation (kendall ) : the rankings are arranged so that one is in the natural order- , , , ... n. following this arrangement, scoring is based entirely on the other ranking. starting with the lefthand member, + is scored for every larger rank and - for every smaller rank to the right of it. after this has been done for each member of this ranking, -rb is calculated from the sum of the scores ( s) as follmvs : s 'rb = y'[n(n- )l'jt(t- ) ][n(n- )l'ju(u- )] . t is the sum of ranks of the y's when the x's andy's are ranked together. these expressions can be used in a one-tailed t-test of the hypothesis that the distributions of the two populations are the same against the alternative that the x population is situated to the left (lower valued) of the y population. for m and n equal to or more, the following expression may be taken as a normal deviate with mean and unit variance : this test is nearly as efficient as the usual t-test when applied to normally distributed populations ; as it is distribution-free, it is applicable to nonnormal distributions. example: if na = and nb= , a j of at least is necessary for significant evidence of affinity; if na= and nb= (nb/na= . ), minimum significant j can be zur okologie der collembolen: untersuchungen im schwedischen lappland a study of mutual occurrence of plant species the measurement of interspecific association vegetationsforschung auf soziationsanalytischer grundlage analyses et syntheses biocenotiques . l'ecologie introduction to mathematical statistics nouvelle recherches sur ia distribution florale rank correlation methods a simple method of calculating the exact probability in x contingency tables with small marginal totals a contribution to the study of the ecology of the corixidae (hemipt the invertebrate fauna of the choice of statistical tests illustrated on the interpretation of data classed in a x table a method of stabilizing groups of equivalent amplitude in plant sociology based on the similarity of species content and its application to analyses of the vegetation on danish commons key: cord- -istz iql authors: nan title: procedures to investigate waterborne illness date: - - journal: procedures to investigate waterborne illness doi: . / - - - - _ sha: doc_id: cord_uid: istz iql humanity could not survive without a reliably clean, safe, and steady flow of drinking water. since the early s when typhoid fever and cholera were frequently causes of waterborne illness in developed countries, drinking water supplies have been protected and treated to ensure water safety, quality, and quantity. having access to safe drinking water has always been one of the cornerstones of good public health. not only safe water is limited to drinking water, but recreational water can also be a source for waterborne illness—both from treated waters such as in swimming pools, whirlpools, or splash pads and from non-treated surface waters such as lakes, rivers, streams and ponds. recreational waters may cause illness not only from ingestion of pathogens, but also when in contact with eyes, ears, or skin. some pathogens in water can be acquired by inhalation of aerosols from water that is agitated or sprayed such as in humidifiers, fountains, or misting of produce. this poses a potential risk to those exposed, particularly if they are immunocompromised. humanity could not survive without a reliably clean, safe, and steady flow of drinking water. since the early s when typhoid fever and cholera were frequently causes of waterborne illness in developed countries, drinking water supplies have been protected and treated to ensure water safety, quality, and quantity. having access to safe drinking water has always been one of the cornerstones of good public health. safe water is not limited to drinking water, since recreational water and aerosolized water can also be sources for waterborne illness, from treated waters such as in swimming pools, whirlpools, or splash pads and from non-treated surface waters such as lakes, rivers, streams and ponds. recreational waters may cause illness not only from ingestion of pathogens, but also when in contact with eyes, ears, or skin. some pathogens in water can be acquired by inhalation of aerosols from water that is agitated or sprayed such as in humidifiers, fountains, or misting of produce. this poses a potential risk to those exposed, particularly if they are immunocompromised. often when an outbreak is first suspected, the source is not clear, i.e., food, water, animal contact. investigation is usually needed to find the common source. in some outbreaks the food may first be identified as the source, such as with produce, but the ultimate source could be contaminated irrigation water. investigators have to keep an open mind until laboratory and/or epidemiologic evidence links cases to the primary source. although we frequently think of waterborne illness originating from a microbiological agent, we should be aware that water may also be contaminated by pesticides, fertilizers, and other chemicals which may enter through industrial discharge, agriculture runoff, or deliberate contamination. waterborne illness acquired from microorganisms may be classified as: • toxin-mediated infections caused by bacteria that produce enterotoxins or emetic toxins that affect water, glucose, and electrolyte transfer during their colonization and growth in the intestinal tract; • infections caused when microorganisms invade and multiply in the intestinal mucosa, eyes, ears, or respiratory tract, or contact the skin; • intoxications caused by ingestion of water containing poisonous chemicals or toxins produced by other microorganisms manifestations range from slight discomfort to acute illness to severe reactions that may terminate in death or chronic sequelae, depending on the nature of the causative agent, number of pathogenic microorganism or concentration of poisonous substances ingested, and host susceptibility and reaction. the public relies on public health regulators to investigate and mitigate waterborne illness. mitigation depends upon rapid detection of outbreaks and a thorough knowledge of the agents and factors responsible for waterborne illness. public health and law enforcement agency officials should always be alert to the rare possibility of an intentional contamination of water supplies by disgruntled employees or terrorists. the purposes of a waterborne illness investigation are to stop the outbreak or prevent further exposure by: • identifying illness associated with an exposure and verifying that the causative agent is waterborne • detecting all cases, the causative agent, and the place of exposure • determining the water source, mode of contamination, processes, or practices by which proliferation and/or survival of the etiological agent occurred • implementing emergency measures to control the spread of the outbreak • gathering information on the epidemiology of waterborne diseases and the etiology of the causative agents that can be used for education, training, and program planning, thereby impacting on the prevention of waterborne illness • determining if the outbreak under investigation is part of a larger outbreak by immediately reporting to state/provincial/national epidemiologists in the instance of a bottled water outbreak, halting of distribution and sale of product and recall of product, some of which may already be in consumers' homes, are necessary to prevent further illness. as epidemiologic data accumulate, information will indicate the source of the problem, whether a municipal water treatment plant, bottled water manufacturing plant, or recreational water exposure, and suggest methods for controlling and preventing waterborne illness. this information will guide administrators in making informed decisions to provide the highest degree of waterborne safety. a flowchart, sequence of events in investigating a typical outbreak of waterborne illness (fig. ) shows the sequential steps, as presented in this manual, in investigating a typical outbreak of waterborne illness and illustrates their relationships. a description of each step is presented in this manual. can be recognized at any point during the outbreak investigation. if intentional contamination is suspected follow your notification scheme in emergency response plans (this could include law enforcement, emergency management and other government agencies) the primary purpose of a waterborne disease surveillance system is to systematically gather accurate information on the occurrence of water-related illnesses in a community, thus allowing development of a rational approach for the detection, control and prevention of waterborne illness. other purposes are to (a) determine trends in the incidence of waterborne diseases, (b) characterize the epidemiology of waterborne diseases, (c) gather and disseminate information on waterborne diseases, and (d) develop a basis for evaluating control efforts. it may be useful to coordinate this system with, or integrate it into a foodborne disease surveillance system. however, while the procedures are quite similar from an epidemiologic viewpoint, they may differ with respect to personnel or agencies involved. an effective disease surveillance system is essential for detection of disease caused by either unintentional or intentional contamination of food. an effective waterborne disease surveillance system consists of: • early reports of enteric and other illnesses that may be related to water exposure or consumption • coordinated effort between local and state public health partners, water utility and water recreation staff • systematic organization and interpretation of data • timely investigation of identified outbreaks or clusters of illness • dissemination of outbreak reports and surveillance summaries to all appropriate stakeholders many types of reporting systems may already exist at the local or state/provincial level, and these should be incorporated into a waterborne disease surveillance program. these include (a) mandatory (or voluntary) laboratory-or physician-based reporting of specific infectious diseases, (b) national-based surveillance systems such as calicinet (cdc ) or nors (cdc in the us, (c) physician office, hospital emergency, and urgent-care clinic medical records, (d) public complaints made to health agencies and/or local water utilities, (e) school illness and absentee records, (f) absentee records of major employers, (g) water treatment records kept by water utilities (e.g., turbidity, disinfection levels, occurrence of coliforms), (h) increased sales of antidiarrheal drugs and anti-nausea medications, and (i) source water quality data kept by environmental agencies (e.g., departments of natural resources and geological survey agencies). another type of surveillance mechanism that may supplement or enhance existing reporting systems is a daily log of illness and water quality complaints. agency contemplates initiation or development of a waterborne illness surveillance program, give top priority to identification of appropriate financial, political, strategic, and administrative support. then, identify a key person to create, implement, and manage the system. this person takes responsibility for: • reviewing the types of reporting systems that already exist in your agency or in other agencies that could be incorporated into a waterborne illness surveillance system • identifying the types of information that cannot be obtained from existing reporting systems but that need to be collected or addressed by the waterborne illness surveillance system • identifying ways to merge or integrate the data collected by existing systems with data gathered in the waterborne illness surveillance system • identifying collaborating agencies and staff • develop a mechanism to communicate and update all stakeholders (may be by blast e-mail or periodic conference calls) • providing training in surveillance methods for agency staff and other partners to enhance cooperation • assembling materials that will be required during an outbreak investigation • evaluating the effectiveness of the system. develop procedures to seek and record complaints about waterborne illnesses, water supplies, and water recreational sites. for example, list the telephone number of the waterborne illness investigation unit prominently on local and state public health and water utility websites. to be most effective, have this number monitored / by staff or an answering service. if possible, the utilization of social media such as facebook or twitter should be considered and monitored as many large municipalities (including drinking water utilities) and recreational facilities have an internet presence. if your agency has social media accounts, consider using this vehicle to further disseminate information regarding waterborne illness clusters or outbreaks. identify medical care facilities and practitioners and seek their participation. direct educational activities, such as newsletters and talks at meetings, to stimulate participation in the program. encourage water treatment utilities and operators of recreational water sites to report suspected complaints of waterborne illness to the appropriate local agencies. also, encourage private and hospital laboratories to report isolations of parasitic agents (e.g., giardia, cryptosporidium), viruses (e.g., norovirus and hepatitis a virus), bacteria (e.g., e. coli (pathogenic), salmonella, shigella, vibrio cholerae), and other agents that may be waterborne. develop a protocol for notification and coordination with agencies that might cooperate in investigational activities, including -h-a-day, -days-a-week contacts. a comprehensive contact list should be constructed and updated at least twice a year as individuals may change. notify and coordinate with state/provincial or district agencies, national agencies that have surveillance and water regulatory responsibilities, and other national and international health agencies, as appropriate. for example, it may be useful to find out the level of participation within a certain jurisdiction in national-level outbreak surveillance programs such as nors (cdc, ) or other national surveillance system. delegate responsibility to a professionally trained person who is familiar with epidemiologic methods and with the principles of water treatment and recreational water protection. this person will (a) direct the surveillance program, (b) take charge if waterborne and enteric outbreaks are suspected, and (c) handle publicity during outbreaks. delegate responsibility to others who will carry out specific epidemiologic, laboratory and on-site investigations. if an intentional contamination event is suspected, local and national law enforcement agencies will likely become the lead agency responsible for the investigation. with this in mind, it is critical to identify appropriate individuals and include them in communication and any practice drills that may occur. if a relationship has been established prior to any event, the investigation may run more smoothly. enlist help from a team of epidemiologists, microbiologists, sanitarians/environmental health officers/public health inspectors, engineers, chemists, nurses, physicians, public information specialists, and other (e.g., toxicologists) as needed. free flow of information and coordination among those participating in waterborne disease surveillance and investigation are essential, particularly when several different agencies are involved. water-related complaints are equally likely to be directed at health departments or water utilities but also perhaps to different jurisdictions. therefore, it is essential that these complaints be registered by an agency and that the information is rapidly shared within and perhaps outside of a particular jurisdiction as part of an integrated surveillance system. whenever possible, share the information with participating parties by rapid means such as e-mail and by calling / contact phone numbers. if an intentional contamination event is suspected, contact emergency response and law enforcement for their early involvement. select staff members who will participate in the waterborne disease surveillance program on the basis of interest and ability. inform them of the objectives and protocol of the program. emphasize not only the value of disease surveillance, but also the value of monitoring water quality and treatment performance. if possible, provide printed learning material that can be referenced later. encourage the use of epidemiologic information and approaches in routine disease surveillance and prevention activities. develop their skills so that they can carry out their role effectively during an investigation, and teach them how to interpret data collected during investigations. conduct seminars routinely and during or after investigations to update staff and keep agency personnel informed. train office workers who will receive calls concerning waterborne illnesses to give appropriate instructions. those who participate in the investigation will learn from the experience and often are in a position to implement improvements after completion of the investigation. assemble and have readily available kits with forms and equipment as specified in table a (equipment useful for investigations). restock and maintain kits on a schedule recommended by, and in cooperation with, laboratory staff to ensure their stability and sterility. verify expiration dates, and use kits before this date or discard and reorder. assemble a reference library on waterborne illnesses, investigation techniques, and control measures from reference books, scientific journal articles, manuals, and reputable internet sources (e.g., www.cdc.gov, www.who.int/en/, www.hc-sc.gc.ca/ index-eng.php, www.gov.uk/topic/health-protection/infectious-diseases); make it available to the staff in an easy-to-access format. (see further reading for suggestions). organize a multiagency team with representatives from public health agencies, regulatory agencies, and water utilities and with local political officials to review and exercise existing emergency response plans in the event of a large scale waterborne disease outbreak or other disaster likely to result in waterborne illnesses. local public health agencies have the primary responsibility for the restoration and protection of health of a community following an outbreak event or other emergency. emergency operational procedures should include the following: • an emergency notification list that includes phone numbers and e-mail addresses of key persons/agencies that need to be informed about possible outbreaks and that should receive emergency press releases. every state/province has an emergency management agency and depending upon the scale of the event, it may be useful to coordinate efforts. • clear guidelines for household water consumption following an event. for example, boil-water advisories or instructions to drink only bottled water. statements should be reviewed to ensure current relevance and updated to reflect the most current knowledge. • a plan for dissemination of information to the public; select a coordination point to which all news media and outside agencies will be directed, and designate one person or one telephone number as the contact. (more than one contact person can create confusion). • alternative drinking water sources to be used in cases of emergency and plans for the distribution of this water, if necessary. these include alternative munici-pal systems, bottled water supplies, portable filtration and/or disinfection devices and home treatment units. (special attention should be given to backup supplies for hospitals, nursing homes and other places where lack of safe water would be immediately life-threatening). • identification of specialty laboratories at the state/provincial and national level that are capable of performing (and willing to perform) procedures not routinely done at local laboratories (e.g., large volume water sampling and processing for pathogenic parasites and viruses, serotyping, electron microscopic examination of stool samples, molecular and immunodiagnostic tests for pathogens in environmental and clinical samples). one or more of these tests may be necessary to identify the causative agents in an outbreak and confirm their transmission route. • a plan to exercise procedure with tabletop exercises involving all pertinent partners on a regular basis and implement any necessary adjustments based upon review of after-action findings. notification of a suspected outbreak is often received by health authorities from a laboratory report or a family physician and can be documented on a log such as form a (foodborne, waterborne, enteric illness complaint report). public health investigators will then interview cases and persons at risk who are well (controls) to make epidemiologic associations to find a common source. from here a hypothesis can be formed. further investigation will involve: • collecting clinical samples and water samples • conducting an on-site investigation at the facility alleged to be responsible to determine the mode of contamination or process failure, e.g., low disinfectant level • characterizing the etiologic agents by laboratory analysis using various typing schemes. dna profiling or pulsed-field gel electrophoresis (pfge), of isolates from clinical and water samples to "fingerprint" and link strains of the etiologic agent among cases and to the source prompt handling and referral of water-related complaints, rapid recognition of the problem, and prevention of further illnesses are the foundations of a successful investigation. complaints of water problems are as likely to be reported to a water utility as to a health department. communication is essential between these agencies. this first contact with the public is a vital aspect of an investigation of a potential outbreak and needs to begin by public health professionals as quickly as possible, usually within hours, sometimes by putting less urgent activities aside. as indicated earlier, any action respecting a potential deliberate contamination of water will generate a specific approach to further action. upon receiving a complaint or an alert about a water supply or water exposure or illness potentially attributed to these sources, record the information on form a. alerts may be initiated by reports from physicians, laboratories, or from hospital emergency rooms. alerts may also include an increase in a particular pfge pattern from clinical isolates. an investigation may be initiated to determine if there is a common water exposure among patients with the pfge pattern. the pattern may be compared with similar pfge patterns in pulsenet databases to determine if there are similar occurrences of the pattern in water and clinical isolates nationwide or internationally, e.g., for food that might have been contaminated with water, bottled water. the form provides information upon which to decide whether an incident should be investigated. form a is not difficult to fill out and can be completed by a public health professional, a trained water utility staff member, or trained office worker. assign a sequential number to each complaint. if additional space is needed to record information, use the reverse side or attach additional sheets. always ask the complainant to provide names of other persons at the event, or using the water supply or recreational water under suspicion, whether or not ill, and names of any other persons who are known to be ill with the same symptoms. follow up by contacting these additional persons. emphasize to the persons making alerts or complaints the need to retain a sample of the suspect water and to save clinical specimens (vomitus and stool) from ill persons using a clean utensil in a clean jar or plastic bag and to seal tightly and label clearly with the name of the person and date, and store in a refrigerator (do not freeze). also consider family members not ill for case-control studies. advise complainants to collect a liter (quart) of water immediately, preferably in sterile containers but otherwise in jars that have been boiled or in plastic bags, or if this is not feasible, in other clean containers. tell the complainant to save any ice cubes or refrigerated water, either in their present containers or in unused plastic bags, in the refrigerator or freezer (if already frozen) where they are normally kept. instruct the ill person to hold all clinical specimens and water samples until the health agency evaluates the epidemiological evidence and arranges, if necessary, to collect them. if it is determined that the specimen or sample is not necessary, notify the complainant and advise on proper disposal of the material. unfortunately, the specific etiologic agent cannot be identified in a large proportion of waterborne outbreaks because water samples and clinical specimens (a) were not collected in an appropriate time-frame (not early enough during illness), investigate outbreaks (b) are too old, (c) are too small in volume, especially for giardia and viruses which require liters, (d) have not been examined for the appropriate agent. contaminants may be in the water system for only a short time, and concentrations of toxic substances and numbers of microorganisms may decrease significantly because of dilution or disinfection. if there is a cluster of cases, monitor reports from physicians, complaints about water, or records of laboratory isolation of enteric pathogens that may suggest outbreaks of disease or contributory situations. collect clinical specimens and water as soon as practicable according to the section obtain clinical specimens in this book. extract key information (see* and † entries) from form a and enter it onto form b (foodborne, waterborne, enteric illness and complaint log). record time of onset of the first symptom or sign of illness, number of persons who became ill, predominant symptoms and signs, whether ice or water was ingested, and the name of the water supply or recreational water alleged to have caused the illness, and whether a physician had been consulted, and/or had taken feces or emesis samples, and/or prescribed antibiotics. also, enter on form b names of the places or common gatherings (other than home) at which the stricken persons ingested water during the weeks before onset of illness (see table for an example). enter a code for the water source (e.g., community, non-community, individual well, bottled, stream/ lake, vended, or untreated) . under "history of exposures" column indicate whether the afflicted person had recent domestic or international travel, attended a child care facility, or had recent contact with ill persons or animals. under "comment" column, enter notations of type of agent isolated, results of specimen tests, places where water was consumed during travel, names and locations of restaurants or other foodservice facilities, and other pertinent information including hospitalization, occupation, or place of employment. at this phase of the investigation it will probably not be known whether the illness is waterborne, foodborne, or person-toperson spread. this log can be kept either in hardcopy or in electronic format. see table (below) as an example of a log. interpretation of table . entry -get further details on the patient's symptoms and seek other cases. the report of foreign travel suggests an infection that may have been acquired outside the country. follow-up of such cases may identify an outbreak of international scope. if so, inform state/provincial and national authorities concerned with surveillance of waterborne disease about the situation. entry -possibly food associated; alert food safety officials. entry -initiate investigation; the two cases of conjunctivitis suggest the possibility of a common-source outbreak associated with the motel pool. entry -initiate investigation; cases indicate an outbreak that has a common time-place association. entry -this could be related to entry , because this person reported swimming in the same pool. exposure history: dt domestic travel (out of town, within country); it international travel; cc child care; ci contact with ill person outside household or visitor to household; a an exposure to ill animal; c contact with ill person within household responsible for the investigation, if this was not done when the surveillance protocol was established. delegate sufficient authority and provide resources to the head investigator so that the investigation tasks can be accomplished effectively and efficiently. inform all outbreak investigative team members that any findings are to be reported to this delegated authority. a list of all team members and additional contacts such as administrative contacts, sanitarians/environmental health officers/ public health inspectors, local and regional contacts, physicians, clinical laboratories, or other persons who may become involved in outbreak investigations should be assembled. before beginning the investigation, check the supply of forms and the availability of equipment suggested in table a (equipment useful for investigations) and obtain any needed materials or additional equipment. general resource materials describing signs and symptoms, incubation times, and specifics regarding specimen collection and appropriate kits to be used should be maintained and readily available to those processing the initial calls, which may help to formulate the initial hypothesis. if the alert or complaint suggests a possible outbreak, inform laboratory personnel of the type of outbreak and estimated quantity and arrival time of clinical specimens and water samples collected. this information will give laboratory managers time to prepare laboratory culture media, prepare reagents, and allocate personnel. at a minimum, the laboratory should have six to eight stool culture kits on hand or readily available, since in many cases, stool specimens must be collected within h of onset of illness to isolate and identify certain pathogens (e.g., campylobacter spp., and salmonella spp.). consult laboratory personnel about proper methods for collecting, preserving, and shipping environmental samples and clinical specimens if such information is needed. obtain appropriate specimen containers and sample submission (chain of custody forms) from them. once the investigation is underway, the proper clinical specimens should be collected as soon as possible before patients recover and become less likely to submit specimens. all suspected waterborne outbreaks should be examined and a determination made regarding the feasibility of conducting a thorough investigation even if the time to collect proper clinical specimens has passed. an ill person or family member, physician, hospital staff member, or operator of a water utility or recreational site may report suspected cases of waterborne illness. whatever the source of the report, verify the diagnosis by taking a thorough case history and, if possible, by reviewing clinical information and laboratory findings. (this analysis can be further substantiated by detecting suspected etiologic agents in water). verification of the diagnosis is done in consultation with medical professionals. when a complaint involves illness, complete forms c - (case history: clinical data and case history: food/water history and common sources) either at the time of initial notification, during a personal visit, or during a telephone call to the person reported to be ill. use this same detailed interview approach with every person who has been identified in the initial complaint or alert, even though some may not have been ill. be aware that potential cultural and language barriers can make interviews difficult. a different interviewer may be needed to accommodate these barriers. continue this until sufficient information is obtained to decide whether there is, indeed, an outbreak of waterborne illness. from persons who are at risk of illness but who remained well, also obtain water and -hour food histories, inquire about recreational water exposure in past weeks, and information about their activities in common with the ill persons. information from these persons is as important to make epidemiologic associations as it is from the cases. when it is apparent that an outbreak has occurred and a specific event has come under suspicion, substitute forms d - (case history summaries: clinical data and case history summaries: water/laboratory data) for form c. form d can be used initially in many routine waterborne illness outbreak investigations where it is obvious that a common-source outbreak has occurred or when all of the ill persons consumed water together (e.g., drank from the same public system, consumed ice at an event) or recreated at the same place (e.g., swam in the same lake or used the same hot tubs). this will simplify recording, because most affected persons will give similar information. at this time, notify the district, state, or provincial epidemiologist about the outbreak. if a specific pathogen (e.g., norovirus, e. coli o :h , cryptosporidium spp.) has been identified as the etiologic agent, consider developing a form for recording relevant information. many state/provincial or national public health agencies have standard forms tailored to specific pathogens. include signs and symptoms of the illness and other clinical information, the etiology of the agent, and usual methods of transmission. computer programs (e.g., epi info™) can aid in the design of such standard forms. upon contact with the affected person, identify yourself and your agency and explain the purpose of the visit or call. a professional attitude, appropriate attire, friendly manner, and confidence in discussing epidemiology and control of waterborne illnesses are essential for developing rapport with affected persons or their families and in projecting a good image of the investigating agency. keep in mind that you are not interviewing someone you inspect or regulate, but that you are providing a service to the affected person. exhibit genuine concern for persons affected and be sincere when requesting personal and confidential information. communicate a sense of the urgency of the investigation, and emphasize that their participation will make a positive contribution for the control and prevention of waterborne illness. parental consent must be obtained before interviewing children under years of age. in some locations, consent from the affected person's physician may also be required. after asking open-ended questions about the person's food exposures and illness history, follow up with more specific questions to fill in the details and better ensure a thorough recall. base your level of communication on a general impression of the person being interviewed, considering information about age, occupation, education, or socioeconomic status. tact is essential. use either form c or form d, as appropriate, as a guide. state questions so that the persons who are being interviewed will describe their illnesses and associated events in their own words. try not to suggest answers by the way you phrase questions. fill in form c - (if appropriate) and take additional notes during the interview. ask specific questions to clarify the patient's comments. think questions through before conducting the interview. realize that people are sometimes sensitive to questions about age, sex, special dietary habits, ethnic group, excreta disposal, and housing conditions. nevertheless, any or all information of this type can be relevant. word questions thoughtfully when discussing these characteristics and habits. such information can often be deduced from observations. if doubt remains, confirm your guesses by asking indirect questions. information on recent travel, gatherings, or visitors may provide a clue to common sources or events that would otherwise be difficult to pinpoint. review known allergies, recent immunizations, recent changes in the patient's medical status, and similar information. remember that the agents associated with waterborne disease can also be spread by other means such as consuming food, person-to-person, visiting child care centers, animal-to-person in petting zoos, through walk-in-spray fountains, and pools for young children. as persons describe their illnesses, check boxes next to appropriate symptoms or signs on form c . do not ask about all symptoms or signs listed; however, ask about those marked with an asterisk if the ill person does not mention them. if there are questions, explain symptoms to the patient in understandable terms. the symptoms and signs in the first two columns of form c are usually associated with poisoning or intoxication, although some occur during infections. those in the third, fourth, and fifth columns are usually associated with enteric infections, generalized infections, and localized infections, respectively. those in the last column are usually associated with disturbance of the central nervous system. diseases in any category will sometimes be characterized by a few symptoms and signs listed in the other columns, and not all signs and symptoms occur for any one ailment or for all persons reporting illness. if an illness seems to fall into one of these categories, mention other symptoms in the category and record the patient's response. whenever possible, use physician and hospital records to verify signs and symptoms reported by patients. clinical data may strengthen or dismiss the possibility of waterborne illness. before contacting a physician or a hospital, become familiar with laws and codes relating to medical records to ensure that you have legal access to these records. legal release forms may be necessary to obtain some records. do not distribute names of patients, their other personal identities (e.g., address, phone number), or their clinical information to unauthorized persons. the entries begin with the day of illness, followed by the previous days. if the illness, however, began early in the day or before any of the listed meals, modify the entries on the form so that the -hour history can be completed in the space pro-vided on the form. if the incubation period is days to a week in duration, use additional copies of form c and modify day or day before subtitles. signs and symptoms will sometimes give clues to the transmission route by indicating the organ systems affected. if the early and predominant symptoms are nausea and vomiting, ask about the most recently ingested water or beverage within the past h. in these situations, suspect high-acid water supplies, carbonated beverages and fruit drinks, because these tend to leach metallic ions from water pipes and containers. if diarrhea, chills, and fever predominate, be suspicious of water and beverages ingested - hours before onset of illness for salmonellosis, shigellosis, and norovirus related gastroenteritis. if the incubation period averages - weeks, consider typhoid fever, cryptosporidiosis or giardiasis. diseases with incubation periods exceeding weeks (e.g., hepatitis a and e, amebic dysentery, or schistosomiasis) can be handled as special cases for which longer histories would be sought. others, such as chronic lead and arsenic poisoning, have incubation periods of variable durations and onsets so gradual as to be indeterminable. see table b (illness acquired by ingestion of contaminated water: a condensed classification by symptoms, incubation periods, and types of agents) for details on specific pathogens, table c (illnesses acquired by contact with water: a condensed classification by, symptoms, incubation period, and types of agents), and table d (illnesses acquired by inhalation of microorganisms aerosolized from water. a classification by symptoms, incubation period, and type of agent). other microorganisms not listed in tables b, c, and d that can be potentially spread by water include the bacteria klebsiella pneumoniae, mycobacterium avium complex, acinetobacter calcoaceticus, elizabethkingia meningoseptica, stenotrophomonas maltophilia, pseudomonas putida, serratia marcescens, protozoa isospora, microsporidium, algae schizothrix calcicola. these microorganisms are less frequently identified with waterborne illness, but they may become opportunistic pathogens, particularly for highly susceptible and immunosuppressed persons. further investigation is needed to confirm their role in the spread of waterborne diseases. gather information about all sources of water to which the patient(s) may have been exposed weeks before onset of illness. the water supply and the event that precipitated the illness might not be obvious. persons often have difficulty recalling exposure to all water sources including; ice or water ingested; aerosols and recreational water contact. therefore, if the person does not remember specific exposures to water, ask about the water consumed in usual or routine daily habits and the amounts ingested; exposure to recreational waters; and unusual exposures or events attended during this interval. this may stimulate recall of away-from-home water consumption or contact that was unusual. ask about other risk factors for enteric illness, such as contact with young children and child care centers, animal contact, ingestion of raw foods of animal origin, and usual food preference habits. for persons who have been traveling, ask them where (both cities and rural areas) they have traveled during the incubation period of suspected agents. determine if they drank water from any taps or pumps in rural areas they visited. ask whether unheated (or untreated) tap water or beverages containing unheated (or untreated) water or ice was ingested at restaurants, in hotels or at events in the places they visited. also, ask whether they ingested bottled water and, if so, the brand name. find out whether they drank water from streams, ponds, springs, or other natural water sources. if they did, ask if they observed any abnormal condition of the water such as algal growth, high turbidity or discoloration. ask if domestic or wild animals had access to the water. if they have skin or eye infections or generalized infections, ask them to name all swimming pools, water slides, beaches, lakes, ponds, or other chlorinated and nonchlorinated water courses where they swam, waded or bathed during their trip. also ask them whether they used any hot tubs, spas, whirlpools, or similar devices. this information sometimes provides clues to common sources or to events that otherwise would be difficult to discover. record the information on form c . in a protracted outbreak, or when investigating an outbreak of a disease with a long incubation period, expect recall to be poor. in this situation, obtain from ill persons and others at risk a listing of their water, ice, and beverage preferences and amounts usually ingested, or their purchases of these items within the range of the incubation period of the suspected disease. as a guide, draw up a list of either water, ice, and beverages that are commonly consumed by the affected group or those waters, ice, and beverages previously identified as vehicles of the suspected disease under investigation. summarize data from all copies of forms c - on form d. form d allows rapid review of all exposed persons (ill or not ill) and serves as a basis for analyzing the data. diagnosis of most diseases can be confirmed only if etiologic agents are isolated and identified from specimens obtained from ill persons. get specimens from the ill persons to confirm an etiologic agent. • in large outbreaks, obtain fecal specimens from at least ten persons who manifest illness typical of the outbreak • in smaller outbreaks, obtain specimens from as many of those ill and those at risk as practicable, but from at least two, and preferably ten, ill persons • try to collect specimens before the patient takes any medication. if medication has already been taken, collect specimens anyway, and find out the kinds and amounts of medicine taken and the time that each dose was taken • also get control specimens from persons with similar exposure histories that did not become ill obtain clinical specimens at the time of the initial interview during acute illness or as soon as practicable thereafter. even though this is not always possible, take specimens even after recovery because etiologic agents may remain in low populations or concentrations. if a disease has already been diagnosed, collect specimens as listed in table b . if a disease has not yet been diagnosed, choose kinds of speci-mens that are appropriate to the clinical features. laboratory information obtained from the first patients may be useful to physicians in the treatment of cases detected later. apart from the fact that people are more likely to cooperate while they are ill, some pathogens or poisonous substances remain in the intestinal tract for only a day or so after onset of illness. if the patient is reluctant to provide a fecal specimen explain that the specimen will be tested to identify the causative agent and compare it to any agent recovered from the water. if a disease has not yet been diagnosed, choose specimens that are appropriate to the clinical features. laboratory information obtained from the first patients may be useful to physicians in treating cases detected later. some pathogens (e.g., salmonella, parasites) may be recovered for weeks after symptoms have abated. if applicable for the disease under investigation, take specimens even after recovery because some etiologic agents may remain in low numbers, and changes in serologic titers can be detected. before collecting specimens, review table e (guidelines for specimen collection) and, if necessary, get additional instructions from laboratory personnel and seek their advice on how to preserve the stool specimens if you cannot deliver them to the laboratory immediately. many public health agencies have special fecal specimen kits. demonstrate to the patient how to use the materials in the kit, how to complete the form in the kit and how to mail it if you are not going to pick it up. if mailing specimens, make sure that you are aware of the regulatory requirements that may apply to the transport of infectious material. stool specimen containers for intestinal parasite examination are not suitable for bacterial or viral examinations because they ordinarily contain a preservative, such as formalin or polyvinyl alcohol. if an inappropriate transport medium is used, a specimen can be rendered unsuitable for laboratory examination. feces. if the patient has diarrhea or is suspected of having had an enteric disease, obtain a stool specimen (preferred specimen) or a rectal swab. instruct patients to provide you with their own specimens by one of the following means. . if practicable, give the patient a stool specimen container with a wooden or plastic spoon or a tongue depressor. a clean container available in the home (e.g., a jar, or disposable container that can be sealed) and a clean plastic spoon or similar utensil can be used if laboratory containers are not available. . label the specimen container with the patient's name age/date of birth and date of collection. . collect the stool specimen by one of the following methods: (a) put sheets of plastic wrap or aluminum foil under the toilet seat and push them down slightly in the center, but not so far as to touch the water in the bowl. sheets of paper can be tacked on the rise of a latrine and pushed down to form a depression in which to catch feces. take care to ensure that toilet cleaning chemicals and other microorganisms in the toilet bowl do not contaminate the fecal specimen. after defecating, use a clean spoon or other utensil to transfer about g of feces into a specimen container or other clean container. (b) defecate directly into a large clean dry container or bedpan. use a clean spoon or other utensil to transfer about g or the size of a walnut of feces into a specimen container or other clean container. (c) scrape feces off a diaper with a clean spoon or other utensil to transfer about g of feces into a specimen container or other clean container. . collect fecal swabs by twisting the cotton-wrapped end of the swab into the stool obtained in one of the ways described above. follow instructions given in table e . if necessary, use fecal-soiled toilet paper or cloth diaper and twist a swab into the top of feces. take care to ensure that there is no carryover of toilet paper as they are impregnated with barium salts which are inhibitory to some fecal pathogens. dispose of excess fecal material into the toilet and carefully wrap all soiled articles (e.g., by placing them inside two plastic bags) and dispose of in domestic waste. check that the specimen container is tightly sealed and properly labeled and place into a clean outer plastic bag (special zip lock bags for clinical specimens, if available). store the specimen in a cool place, preferably at °c to await pick-up or despatch. do not freeze. feces from rectal swabs. collect rectal swabs by carefully inserting the swab approximately . cm ( in) beyond the anal sphincter. gently rotate the swab. fecal matter should be evident on the swab. vomitus. if the person is vomiting or subsequently does so, arrange to collect vomitus. tell the patient to vomit directly into a sterile specimen container or a plastic bag. otherwise, transfer some vomitus from a clean receptacle into the container with a clean spoon. refrigerate, but do not freeze, this specimen until it can be picked up or delivered to the laboratory. blood. take blood if a patient has a febrile infection or when infectious agents are suspected (see tables b, c, and d) . blood specimens are collected for: • bacterial culture • detection of antibodies to specific agents • detection of certain toxins before collecting specimens, get additional instructions from laboratory personnel and seek their advice. blood should be obtained by an appropriately trained and accredited person (check appropriate laws). collect blood during the acute phase of illness, as soon as the febrile patient is seen (within a week after onset of illness) and, if comparing of serologic titers, again within weeks (usually - weeks later) during the convalescent phase. draw ml of blood (from an adult) or ml (from a child) or - ml (from an infant). if possible, collect the blood from the same patients from which stool specimens were obtained if both specimens are to be examined. label tubes and vials at every step of serum transfer. do not freeze whole blood because the resultant hemolysis interferes with serologic reactions. collect into a sterile syringe or evacuated sterile tube that does not contain anticoagulants. if practicable, centrifuge the blood at , rpm for min; pour off the serum into small screw-cap vials and store at approximately − °c. if the serum cannot be separated immediately, rim the clot with a sterile applicator stick and refrigerate approximately °c to get maximum clot retraction if the specimen is to be stored unfrozen overnight. if centrifugation cannot be done, store the blood specimens in a refrigerator until a clot has formed, then remove the serum and transfer it with a pasteur pipette into an empty sterile tube. send only the serum for analysis urine. instruct patients to collect urine in the following manner. clean the area immediately around the urethral orifice with a paper pad that has been pre-moistened with % tincture of iodine or other appropriate antiseptic. then begin to urinate into a toilet and collect ml (about oz) of midstream urine into a sterile bottle. use either a second antiseptic-moistened pad or an alcohol-moistened cotton ball or tissue to clean any drops from the top or side of the bottle. other instructions. follow applicable instructions given in table e . before or immediately after collecting clinical specimens, use waterproof permanent markers to label each container with the patients name, complaint number, case identification number, specimen number, date and time of collection, tests requested, and other appropriate information. tightly seal all containers. clinical specimen collection report for each specimen. complete form e (clinical specimen collection report). the complaint number, case identification (id) number, and specimen number must be entered on each report so that laboratory results can later be correlated with other data. on form c record the type of specimen collected, and submit both the specimen and a copy of form e to the laboratory. send a copy of the laboratory report to the patient's physician or call if urgent. if the patient/case or other household member collected any water, ice, or beverage as instructed during initial contact, label containers with the complaint/outbreak and sample numbers. proceed as instructed in table f (general instructions for collecting water samples for microbiological analysis) and complete form f (water/ice collection report) and/or forms g -g as applicable. record conditions of collection as called for on the forms. if a hypothesis associates the illness with water, caution these persons not to use the water source unless the water is first boiled and to discard all previously prepared ice and water-containing beverages until notified otherwise. develop a working case definition to classify exposed persons as either cases or non-cases. start with the most specific symptoms (such as diarrhea and vomiting) rather than broader symptoms such as nausea or malaise. for example in an outbreak of gastroenteritis, a case might be defined as a person from whose stool a specific pathogen was isolated. it may be a person who was at risk and developed diarrhea within a specified period of time. diarrhea will have to be defined, perhaps as three or more loose, watery stools during a -hour period. in some cases, a particular pathogen responsible for the outbreak might have been identified from clinical specimens. a case definition, which is developed later in the investigation, might include either a person having specific signs and/or symptoms within a period of time or a person from whom a specific pathogen was isolated. the ultimate case definition has a tremendous impact on the investigator's ability to make illness and exposure associations and to calculate probability of these associations. sometimes the first symptom or sign provides a clue to developing a case definition. information in tables b, c, d, g, and h can be useful in making case definitions. compare newly identified cases with the definition to see whether each is part of the outbreak. classify cases into categories: • a confirmed case is a person with signs and symptoms that are clinically compatible with the disease under consideration and for which there is either (a) isolation of an etiologic agent from (or otherwise identified in) an appropriate specimen from the patient, or (b) serologic evidence of a fourfold or greater rise in convalescent antibody titer. a confirmed case must also have possible exposure to the etiologic agent within the incubation period of disease. see table e . criteria for confirmation of etiologic agent responsible for outbreaks of waterborne illnesses for definitions of confirmed cases for specific waterborne diseases: • a presumptive case is a person with signs and symptoms that are clinically compatible with the disease under consideration, and for which there is laboratory evidence of infection (e.g., an elevated antibody titer but less than a fourfold increase), but the etiologic agent was not found in specimens from patients or no specimens were collected. a presumptive case must also have possible exposure to the etiologic agent within the incubation period of disease. • a suspected case is a person with signs and symptoms that are clinically compatible with the disease under consideration and history of possible exposure, but laboratory evidence is absent, inconclusive or incomplete. • a secondary case is a person who became infected from contact with a primary (outbreak-associated) case or from a vehicle contaminated by a primary case. onset of illness for secondary cases typically is one or more incubation periods after the outbreak-associated cases. it is not essential, however, to classify cases into these categories. do so only if it aids in developing a final case definition or in making comparative analyses of data. consider doing analyses using case definitions of both confirmed and combined confirmed, presumptive, and highly suspect cases, and compare the results. make a preliminary evaluation of the data collected as soon as possible. if you decide that there is an outbreak, use the information you have to develop a hypothesis about the causal factors. an outbreak is an incident in which two or more persons have the same disease, have similar clinical features, or have the same pathogen (thus meeting the case definition), and there is a time, place, or person association among these persons. a waterborne outbreak is traceable to ingestion of contaminated water or ice or to contact with contaminated water. a single case of either chemical poisoning or a disease that can be definitely related to ingestion of drinking water or contact with water can be considered an incident of waterborne illness and warrants further investigation. waterborne methemoglobinemia in an infant who resides in a rural area having a high concentration of nitrates in well water is an example of a single case of waterborne illness due to ingestion. a rare diagnosis such as primary amebic meningoencephalitis in a person who swam in a body of freshwater and inadvertently ingested the ameba, naegleria fowleri, through the nose is an example of a single incident related to water contact. sometimes it will be obvious from an initial report that an outbreak of waterborne disease has occurred simply because of the number of persons displaying certain signs and symptoms at or near the same time. many complaints, however, involve illness in only one or a few persons. it is often difficult to decide whether ingestion or contact with a particular water source and onset of illness was associated or coincidental. certain diseases that are highly communicable (e.g., shigellosis and epidemic viral gastroenteritis) may result in secondary infections from person-to-person spread or from subsequently contaminated food or water. however, if complaints are received from several persons who are associated with ingesting water or contact with water at the same place, water is likely to be involved. routine review of the log pertaining to potential waterborne illnesses for similar complaints can often be useful in detecting time, place or person associations. an investigation may also proceed based upon the suspicion of an intentional contamination of a water source. a time association exists if the time of onset of similar illnesses is within a few hours or days of each other. place associations exist when persons have ingested water from a particular single source, have swum in, worked in or otherwise been exposed to the same water, have attended the same event, or reside in an area common to all. person associations indicate a shared personal characteristic, such as being of the same age group, sex, ethnic group, occupation, social group, or religion. waterborne illnesses transmitted by a community water supply usually afflict persons of both sexes and all ages throughout the community. non-community water sources, such as bottled water, ice, water from individual wells, or water from areas of recreation should also be considered when making associations. keep in mind that water can contaminate foods during washing or freshening, and it can contaminate utensils and vessels that are used to handle or store foods. water may therefore be a source of contamination of another vehicle. also, water can be ingested as aerosols generated by shower heads, whirlpools, hot tubs, fountains, cooling towers, and irrigation devices. once some of these associations become obvious, question other persons who could be at risk because of their time, place, or person associations with the ill persons. from time, place, or person associations that have been established or suggested by the investigation, formulate hypotheses to explain (a) the most likely type of illness, (b) the most likely vehicle involved, (c) where and the manner by which the vehicle became contaminated, and (d) other possible causal relationships. the section "collection and analysis of data" describes calculations that can aid in the formation of these hypotheses. test hypotheses by obtaining additional information to support or reject them. if the hypothesis includes food contamination, the instructions given in the manual, procedures to investigate foodborne illness, might be useful. guidelines for confirmation of waterborne outbreaks are presented in table g and guidelines for confirmation that water is responsible for illness are presented in table h . if there is strong evidence to support a hypothesis that the outbreak is waterborne, take precautionary actions. the choice of action is dictated by the (a) suspected causal agent, (b) size of the water source, (c) availability of alternate water sources, and (d) expected use of the water. on the basis of available information, estimate the population at risk and engage any public relations staff with your organization to help inform all persons potentially impacted. when dealing with a microbiological contaminant or agent, consider issuing a boil-water advisory with water treatment guidelines (e.g., heating water in a covered container to a rolling boil for at least min and keeping it covered until use). other options that can be explored include chlorinators that can be installed in individual and non-community systems. for community and non-community supplies in which chlorine is already used, increasing the chlorine dosage and opening hydrants and taps to draw the super-chlorinated water through the whole system might be an option. increasing chlorine is sometimes not effective because the chlorine contact time is too short or super-chlorinated water does not reach some parts of the system. furthermore, chlorination is ineffective against cryptosporidium oocysts and requires a long contact time to kill other human pathogens like hepatitis a virus and giardia. for suspected chemical contamination contact a specialist for further assessment and remedial strategies, such as activated charcoal filters. as a last resort, shut off the contaminated system until the source of contamination is found and controlled. be cautious when you take this drastic measure, because it may do greater harm than good by causing lack of water for hospitals, nursing homes, or for firefighters to extinguish fires. if the water is shut off or the treatment facility or distribution system disrupted (as in the case of floods or other disasters), consider means to distribute water from an alternative source to healthcare facilities and homes. if an illness could have resulted from water contact, close the offending water source, post warning signs around it, and patrol the area. where there is a swimming pool, hot tub, spa, fountain, or whirlpool, evaluate the recirculation system and its operation. it may be that increasing disinfectant concentration by super-disinfection could resolve the problem. where there may be chronic operational problems, evaluate ph, disinfectant concentration, and bacteriological laboratory records. choose your course of action, including consultation with appropriate professional experts, depending on the contributing factors existing at the time of investigation. verify the effectiveness of these actions (e.g., boil-water advisory, superchlorination, provision of alternate water source) to protect public health by monitoring illness levels in the population to determine if the outbreak terminates. if the outbreak continues unabated, consider the possibility of other transmission routes. also, verify the effectiveness of repairs to the water system, super-disinfection, and other actions by closely monitoring the quality of the water supply or recreational water to determine if laboratory reports indicate that the water is now safe for consumption or contact. if there is a public health threat, work with any available public relations staff to announce the outbreak in the mass media so that the public who consumed or was otherwise exposed to the implicated water can be alerted to take appropriate action including seeking medical consultation or treatment. provide only objective factual information about the outbreak. coordinate among the investigating agencies to assure that a consistent and accurate message is delivered. it is easy for agencies to miscommunicate before and during a water crisis (see box , false alarm; box , the walkerton outbreak; box , the flint water crisis). it is often preferable to have one spokesperson for all agencies. do not release preliminary information that has not been confirmed. the person giving information about an outbreak should be well informed about the etiologic agent being investigated and prepared to deal with questions. if the health hazard warrants a public warning at the hypothesis stage, tell the public why emergency measures are being invoked and that subsequent information may be cause to modify the action. as the investigation proceeds and the etiologic agent is confirmed and contributory factors are identified, consider terminating emergency measures, and give advice on specific control and preventive measures. attempt to reach all segments of the population at risk; this may require communication in multiple languages. route all news releases or statements to all persons involved in the investigation. in situations involving large outbreaks or highly virulent or toxigenic etiologic agents, set up an emergency hotline for the public to call to ask questions. this is likely to occur if there is an intentional contamination event where there is high publicity and public concern. train staff to handle these calls in a consistent manner so that the advice is the same who gives it. faulty information derived from poorly tested hypotheses can lead to severe political, legal or economic consequences. an example of this occurred in sydney, australia, in when an apparent water contamination event was publicized for the public to take precautionary actions. the false alarm was costly because of rebates to water customers, additional water testing, and for hiring extra staff, as well as a loss of confidence in the facility (see box , false alarm). they may then be disseminated by the mass media with inappropriate interpretations of the public health significance. furthermore, this information may be used as an unrealistic base for water programs or water regulations because of either misinterpretations or pressure from misinformed consumer-advocate groups. all involved parties should follow a written protocol for cross-agency communication and release of information to the public. unreasonable delays are unacceptable. test hypotheses by obtaining additional information to either confirm or refute their validity. do this by case-control or cohort studies, additional laboratory investigations, and on-site investigations (e.g., laboratory reports of water testing). sydney water (a new south wales state-owned corporation) supplies million liters of water each day to . million properties in sydney and its outlying areas. the city has a large and complex catchment with nine major dams and several storage reservoirs. about , km of water main, almost pumping stations and many tunnels deliver water from four main river systems. the water is filtered through eleven treatment plants. seven are owned by sydney water and four are privately owned. these plants provide % of sydney's drinking water and one plant, prospect, provides up to %. in , the quality of sydney's drinking water came under acute review when giardia and cryptosporidium were found in the city's main water supply at the warragamba dam. initially, low levels of these parasites were first detected in the water supply on july, but these were within the acceptable health limits. in days following, much higher levels were recorded, and on july the first "boil water" alert (in which residents were instructed to boil their tap water before use) was declared for the eastern central business district of sydney. however, by late on july high readings were found in samples at the prospect filtration plant, in a reservoir and at a location further down the system, and a "boil water" alert was issued for the south of sydney harbour, and on july a sydney-wide "boil water" alert was issued affecting most of sydney's residents. on august the warning was discontinued. however, high levels were again found on august (the second event), although it was believed that most organisms would likely be dead. more positive readings were found on august , although at lower levels. further contamination was identified on august and an extended boil water alert was again declared. this was progressively lifted suburb by suburb until further contamination was reported on september (the third event). a -week alert was then instituted, which was finally lifted on september . it was determined that the parasitic contamination was caused by low-quality surface water entering the dam. this contaminated source was attributed to moderate rainfall in july, followed by heavy rainfall in august and september which caused intermittent supplies of the raw water to enter the dam. despite high levels of cryptosporidium (up to > , oocysts) and giardia (up to > cysts) being recorded in july and august, , no increase in human cryptosporidiosis or giardiasis was detected in the exposed population. the incident was highly publicized and caused major a public alarm because the number of people affected, the on and off boil water alerts, and the fact that the filtration plant had been advertised as one of the best in the world. the economic and political repercussions were extensive. the cost of the crisis to sydney water was estimated at a$ million which included $ million paid in rebates to customers, $ million in lost revenue, water testing and staff costs and at least $ . million for damages claims. these costs exclude those relating to improvements to the system and infrastructure. the lack of cases of cryptosporidiosis, giardiasis or other water-related health problems led to suggestions that the parasites were either not an infectious type, or not as extensively distributed. an inquiry after the event revealed the publicity as an exaggeration of fact, with australian water technologies, part of sydney water, severely overestimating levels of cryptosporidium and giardia present in the water, with the recorded levels exposed to consumers as not harmful to human health. the handling of the crisis by state-owned sydney water was heavily criticized, causing the resignation of both the chairman and the managing director, and bringing up issues of private vs. public ownership and scientific uncertainty. the eventual consequence of the state inquiry was the establishment of the sydney catchment authority in to assume control of sydney's catchments and dams, while sydney water maintained responsibility for water treatment and distribution and for sewage collection, treatment and disposal. if an outbreak investigation requires resources beyond your agency's capacity, request assistance from other health professionals. it is desirable to have a team including, if feasible, an epidemiologist, an engineer, a microbiologist, a sanitarian/ environmental health office/public health inspector, a chemist, a physician and others, to undertake a detailed waterborne illness investigation. such personnel can usually be provided by local, state/provincial, or national agencies concerned with health, environment, or agriculture, depending on the expertise needed. for events suspected to arise from intentionally contaminated food, contact emergency response or law enforcement agencies. continue to search for and interview both ill persons who have had time, place, or person associations with the identified cases (see the section on "make time, place, and/or person associations"). review recently received complaints in the water-related complaint log (form b). contact other nearby health agencies, hospital emergency rooms, elderly care centers, and local physicians to discover other epidemiologically related cases. call previously contacted persons to see whether they know anyone else who has become ill or had a common association suggested by data in the log. the illness you are investigating may be part of a larger multijurisdictional outbreak, and therefore communicate with adjoining local and state agencies to learn if they are seeing similar illnesses. state or provincial public health agencies can check reportable disease records and state/provincial public health laboratories can start looking for clusters in isolates that they are characterizing. for outbreaks where intentional contamination of water is suspected or confirmed, public health and law enforcement agency officials may conduct the investigation jointly. if it becomes apparent that an outbreak is associated with a specific water supply (source) or recreational water or event, use form d for recording information. at this stage of the investigation, interviews can be expedited by reviewing the event itself to stimulate each person's recall. ask about specific symptoms and signs that are known to be common to the syndrome, as well as, time of ingestion or contact with water and onset of illness. mention each source of water to which the person may have been exposed, and ask each person (whether a case and well persons at risk) which of the water sources had been ingested or contacted. the number of persons to be interviewed depends on the number exposed and the proportion of them who are probably affected; if fewer than persons were at risk, try to interview all of them; if several hundred are involved, interview a representative sample. be sure to obtain clinical specimens from these cases and well persons at risk (controls). it is more difficult to obtain positive results if symptoms from persons have ceased. there may be situations where self-administrated questionnaires are sent to cases and persons at risk. use either form c or form d or modified versions for this purpose. after questionnaires have been completed, summarize the data on form d. also, identify and interview secondary cases if they become apparent. because no two waterborne disease outbreaks are identical, the order of the expanded investigation may not always follow the outlined sequence of procedures. some investigative steps can usually be done simultaneously by different investigators. additional procedures may also be required. the principles and techniques described will suffice for most investigations. modify forms, if necessary, to accommodate the type and amount of information to be collected. make on-site observations. prove or refute hypotheses developed during the epidemiological portion of the investigation. focus on sources and modes of contamination and ways contaminants could survive and pass through water treatment. as applicable, conduct an on-site investigation of source (lakes, streams, areas around groundwater, etc.), treatment facilities, distribution lines, cross connections, water reservoirs, places of recreational water contact and/or sites at which aerosols were generated. such an epidemiologically focused investigation is quite different from sanitary surveys done during routine evaluations of water source sites, treatment plants or recreational water facilities. not all drinking water (even municipal and bottled water) is disinfected; so, it is important to identify whether the water source is treated and if so, how. some treatments (filtration, reverse osmosis, membrane treatments, riverbank filtration, and others) may not be complemented with a disinfection step. sanitary survey information can provide information about potential sources of contamination in the area of a usually pristine water source. microbiological records of a water supplier, particularly if any total coliform positive samples were found by the system in the last months, may help identify a contamination pathway. if significant matters relating to water quality are observed or otherwise identified during the investigation, note them and communicate them to those responsible for the water system and to the proper authorities. do not lose the focus and objectivity of the investigation by confusing matters of quality and aesthetics with factors related to contamination by, and survival of, infectious and toxic agents. use the haccp-system, also known as systems analysis, way of thinking in your investigation. contact the person with the highest responsibility for the operation and maintenance for the implicated water source, water treatment facility, and/or distribution lines. identify the types of records that ought to be reviewed during the investigation and their likely source. do not forget that the responsible authorities also can have records (about water quality, if there has been a change of municipal water supply, industrial water pollution, wastewater pollution). they can be good sources of information about recent pipe breaks and other water system issues that could be related. in many cases they will be aware of the potential for contamination upstream of source water intakes. if applicable, obtain water distribution maps and recent water quality reports from appropriate departments. if you are not familiar with the community in which the investigation is to be done, obtain maps of the area to locate streams, lakes, water treatment facilities, and other community features that might have a bearing on the investigation. check if there are water protection areas and their rules. get plans and specifications on design of treatment facilities from consulting engineers or state agencies that approve these facilities. contact weather bureaus, airports, radio/television stations, or newspapers for information on heavy rainfall, flooding, extremely low temperatures, droughts, or other unusual weather conditions that preceded the outbreak, if this information is unknown to investigators. contact police or fire departments about traffic accidents, which can be the source of the outbreak. review all background data pertaining to the suspect water. as information is gathered, record it on applicable parts of form g. discuss with laboratory personnel that a field investigation will be made, and get their suggestions regarding samples and specimens that should be collected (see tables e and f) . confer with them about special analyses, media, and sampling procedures; make arrangements for rapid transport of samples to the laboratory. the samples must maybe be transported at the right temperature. pick up appropriate forms and sample collection equipment (preferably preassembled in a kit-see table a ). the laboratory can probably help assemble this kit. during the investigation, identify factors that contributed to contamination and survival of the etiologic agents and perhaps also to their growth or amplification or another cause of the outbreak. identified factors and situations that have contributed to waterborne disease outbreaks include those listed in table . focus the investigation on the potential situations listed in table , as applicable. remember that other possibilities can occur. describe circumstances that contributed to contamination and that permitted the etiologic agent to survive so that it reached drinking, agricultural, industrial, or recreational water. also describe circumstances that allowed pathogenic bacteria or algae to multiply in the water. write your findings down on the back of form g (illustration of contamination flow) or on a separate sheet. continually update the listing in table with newly available data. introduce yourself (who you are, where you come from, who ordered you there) to the owner, resident, or persons in charge and state your purpose, when you arrive at the place of the suspected contaminated water source. emphasize that your visit is to confirm or eliminate suspicion that this water was a source of illness. tell him or her that a complete epidemiologic study is in progress and that other possible sources (such as food) will be investigated as well as operations of this site. explain that your investigation is not to fix blame but to identify the cause of the outbreak. emphasize that the findings can yield benefits related to the ability to identify needed improvements, to educate staff and to provide public support. try to create an atmosphere of cooperation. maintain an open mind and try to answer all questions. if you can not answer a question, tell the person that you will come back with an answer. come back to the person within or weeks even if you do not have any new information. give the person your phone number and e-mail address and tell the person to contact you if the person has more information later. privately interview key persons responsible for operating or repairing water facilities. do not forget to interview persons from other work shifts. identify persons who were working there at the likely time of contamination and have since left and interview them as well. ask questions to determine the flow of water and operations from intake through distribution through plumbing systems. ask about any changes in operation, unusual events in the watershed or repairs to the water facilities. ask if you can check records, both in paper form and on the computer (monitoring system), analyses of results, and/or incident reports. plant personnel may not describe water treatment or installations exactly as they existed at the time that a mishap occurred. they may fear criticism or punitive action as a result of their possible role in the causation of the outbreak. their descriptions should be plausible and should account for possible sources and modes of contamination and indicate possibilities for survival of pathogens. if a description does not contain all the information desired, reword questions and continue the inquiry. confirm accounts by private interviews with others knowledgeable of water treatment or operation of the facility. be alert for inconsistencies among the accounts told by different persons. seek resolution of discrepancies in accounts by watching actual procedures as they are being carried out, by taking appropriate samples, or by conducting experiments. a communicative working relationship between the plant management and the investigator influences plant workers' attitudes toward the investigative team. consider the position, feelings, and concerns of the manager and staff; defensive reactions are normal on their part. diagram each phase of the water system or situation under study on form g (illustration of contamination flow). insert special symbols and notes for all sites that might be involved in introducing contamination to the water or where contaminants might have survived treatment. record other information gathered on the appropriate parts of forms g - . review and collate appropriate information on quality control and operational records from the water utility and from responsible agencies. as applicable, obtain information on quality of untreated surface or groundwater from a local, state/ provincial, or national pollution control or geological survey agency. also, seek water distribution maps, well logs, descriptions of geological conditions and indices of groundwater quality from them. for surface water supplies, obtain information on upstream discharges and unusual events that may have affected raw water quality. get data on finished water quality in the distribution system from a local, state/ provincial water surveillance or regulatory agency. water suppliers also frequently have records of raw and finished water quality. review data on quality control tests (e.g., ph, chlorine residual, chlorine demand, bacteriological and chemical tests, turbidity, jar test data, incident reports) that are available. obtain data on cross connection control programs and sewer repairs from the water supplier or other local agencies (e.g., building inspectors, sewage departments). review files for data concerning potential sources of contamination for individual or semipublic water supplies (e.g., diagrams of septic tank systems, sewer line locations, well logs, small individual wastewater plants, accidental industrial pollution of water, traffic accidents involving chemicals, salting of roads or sawmills). check if they have any haccp-systems or water safety plan and, if so, how they monitor their ccps (critical control points) and if they are implementing control measures. ask them about haccp, to see if they understand the system and if results are documented. check if the haccp-system is validated (should be documented) and that they are conducting internal audits. get information on all aspects of normal operations as well as unusual events or conditions to determine whether such events were coincidental with the time of suspected contamination as determined from the epidemic curve. also, consider the time it takes for a contaminant in the raw water or treatment plant to reach households in the affected community. ask responsible persons for this information. compare data on heterotrophic plate and total coliform counts of raw and finished water leaving the treatment facility and of water in distribution lines. also, compare data on chlorine residuals within the plant with that in distribution and check, if they have, that the uv-light is functioning. review other test data (e.g., turbidity and chemical analyses) that may indicate a problem situation. identify locations and dates of sample collection. take photos, if it is allowed, of things you suspect are not right. go back more than one incubation period of the disease under investigation. record this information on form g (record review of on-site investigations, and test results prior to and during outbreak). photocopy appropriate records for confirmation and subsequent review and attach them to the record review form. be alert for evidence of falsification of records. while reviewing records, watch for evidence of the following: • potential contamination of groundwater systems because of proximity to septic tank systems, latrines, animals manure or landfills, industrial contamination of the water supply, small sewage plants, especially old and nearly forgotten ones, and recent heavy rain • high heterotrophic plate or coliform counts, or counts that exceeded the average (median) or typical count • sudden changes in water quality or operating practices that suggest the possibility of contamination or treatment failure • high turbidity, unusual odor, color, or taste, or high coliform counts in raw water, which can indicate potential overloading of the normal treatment process • high levels of ammonium, nitrate and nitrite, which can indicate organic and inorganic contaminants • low chlorine residuals in treated water or higher-than-normal amounts of chlorine used, which can indicate either a high chlorine demand or a sudden high level of contamination • a sudden drop in amount of disinfectant used, possibly indicating failure or interruption of a disinfection process. no functioning alarm system • a sudden change in the amount of a chemical (e.g., alum or ferric sulfate) used, suggesting equipment disfunction or inadequate coagulation or flocculation and thus poor filtration • lack of treatment chemicals if a more corrosive water supply is used (see box , the flint water crisis) • pump failures, draining of distribution lines or reservoirs, or massive pumping to fight fires, which can produce low pressures that can cause contamination through cross connections or back siphonage • repairs to water mains, wells or pumps where contamination could have been introduced record this information on form g or other appropriate form in the g series. as applicable, investigate the water source, treatment facility, distribution and plumbing systems, sites where water was contacted, and sites at which microorganisms amplified and aerosols disseminated. use forms in the g series as guides while observing facilities, gathering data, making measurements and collecting samples. google or bing maps or other similar resources' views of the watershed can be very helpful in identifying potential sources of contamination that you will need to investigate further. these maps can also facilitate your own map and diagram making on form g . the water source may be surface or ground or in some cases a combination of the two. verify this by observations at the site and by talking to the property owner or persons responsible for operation or maintenance of water supply or recreational facilities, as applicable. examination of "weather events" such as heavy rainfall may indicate a potential for surface water contamination (see box , the walkerton outbreak). when a surface water is either suspected or implicated as the source of a contaminated supply, get information about the watershed concerning possible sites of contamination of the suspected etiologic agent. this includes, but is not limited to (a) land use, (b) sewage effluent from treatment plants and septic tanks, (c) industrial plants that may be discharging toxic waste, (d) mining wastes, (e) landfill leachates, (f) slaughterhouse discharge wastes, (g) animal feed lots, (h) both domestic and wild animals that use the source water for drinking, (i) sludge disposal from sewage treatment plants or septic tanks (e.g., land spreading or lagoons), (j) storm water discharge. if this information is not available from records or persons familiar with the site, visit the site and observe possible sources of contamination and pollution (e.g., while traveling by foot, vehicle, boat, or helicopter, as applicable). record this information on form g (source and mode of contamination of surface water). diagram the surface water and sites of contamination on form g . note type and location of sources of contamination and their distances from the water. visit groundwater sources. using form g (source and mode of contamination of groundwaters) as a guide, question owner or operator and inspect groundwater installations to ascertain character of the land and surface and subsurface soil and water. when a well or improved spring is under consideration as the source of the contaminated supply, observe its location relative to possible sites of contamination and to whether its construction allows contaminants to reach the water. determine locations of all sewage outflows or disposal sites (e.g., septic tanks and absorption lines, cesspools, privies, and other sewage disposal facilities), gradients, and distances from the well or spring. determine the type of soil at the site. if the soil is limestone or fissured rock or if there is a high ground or perched water table, pollution may travel many miles. in this case, the search for sources of contamination may have to be expanded for a considerable distance from the well or spring. ascertain whether there were heavy rains, heavy snow melts, or sudden discharge in may, , many people in walkerton, a small ontario, canada, community of about , people, began to simultaneously experience bloody diarrhea and other gastrointestinal infections. on may - , torrential rain had unknowingly contaminated the town's water system, but operators failed to check residual levels for a period of several days, allowing unchlorinated water to enter the distribution system. however, the privatelyowned walkerton public utilities commission insisted there was no problem with the water despite other laboratory tests showing evidence of e. coli contamination. illnesses began about may , with the first death occurring on may and the seventh and last on may . by may , however, many more cases had been diagnosed, the infectious agent determined to be e. coli o :h , and contaminated well water was confirmed as the source of the e. coli; all this allowed the region's medical officer of health to issue a boil water advisory, warning residents not to drink the tap water. two days later, laboratory results identified the presence of campylobacter and e. coli o :h and dna testing showed that the contaminating source was a cattle farm a short distance from a well used for the water supply. by the time the outbreak was over, > were ill and had died. the people who died directly from drinking the e. coli-contaminated water might have been saved if the walkerton public utilities commission had admitted to contaminated water sooner. those in charge of the water utilities at the commission had no formal training in their positions, retaining their jobs through three decades of on-the-job experience. they were later found to fail to use adequate doses of chlorine, fail to monitor chlorine residuals daily, make false entries about residuals in daily operating records, and misstate the locations at which microbiological samples were taken. regulations state that water suppliers are required to treat groundwater with chlorine to sufficiently neutralize contaminants and sustain a chlorine residual of . mg/l of water after min of contact. had utility operators adhered to the protocol, the disaster most likely would have been averted. the operators knew that these practices were unacceptable and contrary to ministry of environment guidelines and directives; they eventually admitted falsifying reports and were sentenced to short jail terms. the ontario government was also blamed for not regulating water quality and not enforcing the guidelines that had been in place. the water testing had been privatized in october . an enquiry found that the water supply, drawn from groundwater, became contaminated with the e. coli o :h strain from manure from cattle on a farm washing into a shallow water supply well after heavy rainfall. the risk of contamination from farm runoff into the adjacent water well had been known since . key recommendations from the enquiry included source water protection as part of a comprehensive multibarrier approach, the training and certification of operators, a quality management system for water suppliers, and more competent enforcement, which were incorporated into ontario new legislation. the bottom line of the enquiry was that officials and municipal water facilities operators and managers across north america need to recognize public waters are a most valued but vulnerable public resource. investment in keeping them safe and secure needs to be a community top priority. from dams that could have resulted in flooding within the duration of the incubation period of the disease under investigation. obtain information on the depth of the well in reference to the ground water table from the owner or by referring to any available well logs on public file or from local drillers. observe well construction and get information about depth of casing, depth and method of grouting, and whether there is an underground discharge. observe whether there is an impervious well platform and whether the pump or casing seal was subjected to flooding. illustrate the situation by showing location of the well in this event is considered a disaster, still unfolding, initiated from a political decision to save money, and ending up with acute and chronic illnesses and deaths to residents of a michigan city, as well as high system remediation and health-related costs to the taxpayer. on april , , flint, genesee county, michigan, switched its water supply from detroit's system to the flint river as a cost-saving measure for the struggling, majority-black city on the recommendation of the state-appointed emergency manager. flint agreed to separate from the detroit water and sewerage department and go with the karegnondi water authority, including the decision to pump flint river water. this was to be an interim measure until a new pipeline from lake huron was constructed in to serve the region. soon after the switch, residents begin to complain about the water's color, taste and odor, and to report rashes and concerns about bacteria. in august and september city officials issued boil-water advisories after coliform bacteria were detected in tap water. in october , the michigan department of environmental quality (mdeq) blamed the cold weather, aging pipes and a population decline. in the same month a general motors plant in flint (continued) reference to possible sites of contamination on form g . note distances between the well and contamination sites and elevations. determine whether any pumps were out of order or had been repaired during the interval of concern. if priming of the pump was done, find out the source of the water used. record this information on form g . test hypotheses of modes of contamination by conducting a dye test and/or sampling the water. (see appropriate sections of this manual for directions.) collect samples of water from these sites and submit them for analysis of the suspected etiologic agent or for any physical, bacterial, or chemical tests that will provide evidence of contamination or movement of the contaminants. (see procedures for collecting water samples) record these results on forms g or g and i (laboratory results summary). when appropriate, confirm hypotheses by a dye or other tracer test. (see section on this subject). determine the means by which the etiologic agent survived treatment or was otherwise not eliminated or inactivated. consider the treatment process as a series of barriers placed between contaminants and consumption of the treated water. the operation of each barrier should be optimized. review available data for each step in the treatment process. records of well-maintained and properly calibrated continuous monitoring equipment will be especially valuable. look for failures in the barriers, which could include (a) lack of disinfection, (b) inadequate concentration of disinfectant or contact time, (c) interruption of disinfection, (d) inadequate filtration, (e) lack of corrosion inhibitors, which may follow inadequate pre-filtration treatments. in in flint, michigan excessive levels of lead were found in drinking water from corrosion of water distribution pipes (see box , the flint water crisis). corrosion inhibitor had not been added. also, look for possible introduction of contaminants within the treatment process, such as in treatment chemicals. stopped using municipal water, saying it was rusting car parts. on january , , the city announced that flint's water contained a high level of trihalomethanes, a byproduct from increased disinfection by the city. though this is in violation of the safe drinking water act, officials told residents with normal immune systems that they have nothing to worry about. in january , detroit's water system offered to reconnect to flint, waiving a $ million connection fee but the offer was declined by the emergency manager. by february, state officials continued to play down any water problems saying that the water was not an imminent "threat to public health." on february , parts per billion (ppb) of lead were detected in drinking water at a flint home and the federal environmental protection agency (epa) was notified. the epa does not require action until levels reach parts per billion, but science indicates that there is no safe level for lead in potable water. officials from epa and mdeq discussed the lead level in the sample, and epa found that the state was testing the water in a way that could profoundly understate the lead levels. on march , , a second testing detected ppb of lead in flint drinking water. a consultant group hired by flint, reported that the city's water met state and federal standards, and it did not specifically report on any lead levels. in may, tests revealed high lead levels in two more homes in flint. in july, an epa administrator told flint's mayor that "it would be premature to draw any conclusions" based on a leaked internal epa memo regarding lead. however, in september, flint was asked to stop using the flint water supply or consider corrosion control for it, because it was causing lead to leach from the water pipes and children had high levels in their blood. state regulators insisted the water was safe. nevertheless, on october , the governor of michigan ordered the distribution of filters, the testing of water in schools, and the expansion of water and blood testing after a briefing on the lead problems with the mdeq and federal officials. at the same time, flint city officials urged residents to stop drinking water. on october , flint reconnected to detroit's water system, and residents were advised not to use unfiltered tap water for drinking, cooking or bathing. on october , the director of mdeq reported that his staff had used inappropriate federal protocol for corrosion control, and soon after, the governor announced that an independent advisory task force would review water use and testing in flint. on december flint added additional corrosion controls, and soon after an emergency was declared. at the end of december, the task force found that the mdeq was accountable for its lack of appropriate action, and the director resigned. on january , , the governor asked the national guard to distribute bottled water and filters in flint, and president obama declared a state of emergency in the city and surrounding county, allowing the federal emergency management agency to provide up to $ million in aid. three days earlier the crisis expanded to include legionnaires' disease, because of a spike in cases, including ten deaths, after the city started using river water. on january , the michigan department of health and human services stated it did not have enough information to conclude that the increase in cases was related to the ongoing flint water crisis, although the. head of michigan's communicable disease division had said three months earlier that the number of legionella cases at that time "likely represents the tip of the iceberg." as of february , the number of reported cases was close to . a flint hospital official was surprised that michigan and local health agencies did not inform the public about the legionnaires' outbreak in genesee county in - until january ; the hospital earlier had spent more than $ , on a water treatment system and bought bottled water for patients. the source of legionella is not known but it was likely in the flint river, and possibly extensive flushing of flint's colored water, which had undesirable odors and tastes, by residents may have caused chlorine residual in the pipes to be washed away, leaving the pipes susceptible to growth of the legionella; in addition, aerosols from the extensive flushing from turned-on faucets might have led to close contact between the residents and the pathogen. the investigation of the cause of the illnesses continues with criminal charges laid against michigan departmental employees. observe treatment processes from the water inlet to the finished water discharge. diagram on form g the treatment process; insert notations of hazardous situations that were observed. collect samples of water at the inlet, after each phase of treatment that may have functioned suboptimally or failed, and at the outlet. test the samples for pathogens that cause a syndrome characteristic of that being investigated, for indicator organisms and for physical and chemical characteristics of the water, as appropriate to the situation. evaluate effectiveness of the disinfection process and resulting residuals. determine the type of disinfectant (e.g., gaseous chlorine, hypochlorite, chlorine dioxide, chloramine, ozone, ultraviolet irradiation) used and whether the disinfection treatment was adequate for the volume of water treated. determine, by talking to water treatment plant employees and reviewing records of the plant or regulatory agency, whether there were any interruptions of disinfection during the two weeks prior to the first onset date. determine contact time between the point of addition of the disinfectant and the first point of use. measure the chlorine residual, ph and temperature of the water just before it leaves the plant. observe the condition, operation, and maintenance of disinfectant dispensing equipment. review plant records to identify any sudden changes in disinfectant demand that causes temporary depletion of disinfectant residuals and allows survival of pathogens. review maintenance records for disinfectant dispensing equipment and quality assurance records for online analyzers. record this information on form g a (disinfection failures that allowed survival of pathogens or toxic substances). calculate disinfectant rate applied and usage (see formulae in form a). for example, to calculate disinfectant rate, if the flow rate is , , gal/day and the dosage is lb/day: the destruction of pathogens is dependent on (a) type and condition of microorganisms present, (b) type of disinfectant used, (c) concentration of available chlorine or other disinfectant, (d) contact time, (e) water temperature, (f) ph, (g) degree of mixing, (h) presence of interfering substances (which may be related to turbidity). utilize treatment records that provide small scale time resolution, such as online monitoring data, to determine whether the process was stable during the time period in question. daily averages may provide evidence of massive failures, but will not provide information about whether consistent treatment was being provided. in general, the relative effectiveness of microorganisms' resistance to free chlorine, from high resistance to low resistance, is as follows: • protozoan oocysts (i.e., cryptosporidium) • protozoan cysts (i.e., giardia, entamoeba histolytica) • viruses (hepatitis a virus, poliovirus) • vegetative bacterial cells (shigella, escherichia coli) protozoan oocysts are highly resistant to chemical disinfectants, but not to physical means such as uv light or ozone (gas). microorganisms within each group and strains among the same species differ somewhat in resistance. the state of injury induced by environmental impacts and selection of resistant strains influence survival. aggregation of microorganisms and/or close association with debris shield them to various degrees from lethal effects of disinfectants and attachment to surfaces such as pipe walls to form biofilms that protect organisms from inactivation by disinfectants. a measurement of microbiological inactivation by disinfectants is the ct value (ct calc ), which is the product of the free residual disinfectant concentration (c) in mg/l that is determined before or at the first user (customer) and the corresponding disinfectant contact time (t) in minutes (i.e., c × t). refer to table i (ct . values for inactivation of giardia cysts at different concentrations of disinfectants, temperatures, and ph values) and table i (ct values for inactivation of viruses at ph - , at different temperatures with different disinfectants for comparing disinfectant efficiencies). make residual measurements during peak hourly flow. for comparisons of ct values between the indicated ph, temperature, and concentration values, use linear interpolation. (for example, for free chlorine, °c, concentration mg/l, ph . = [ − = ; / = ; + ] = ). if no interpolation is done, use the ct . value at the higher temperature, at the higher ph and higher concentration. a simple ct calculation, for example, using a disinfectant concentration (c) at the basin effluent of . mg/l and a detention time (t) of min, is as follows: use this calculation for comparing to values in table i or j. the calculated ct value should be higher than the value stated in the table for specific conditions of disinfection, temperature, ph, and concentration (residual). in this situation, if the temperature of the water was °c, the ph and the concentration of chlorine mg/l, a ct value of would be needed for a . reduction of giardia cysts. the ct value of . would have been inadequate to meet the criteria and could explain the survival of the pathogen under investigation. microbial inactivation efficiencies vary considerably among different disinfectants and are influenced by the characteristics of the water and water temperature. tables i and j show that, in general, ozone is more effective than chlorine dioxide, which is more effective than free chlorine, which is more effective than chloramines. also, in general, longer contact time increases the degree of inactivation, and higher water temperatures as well as lower ph values increase rates of inactivation. rapid mixing of the disinfectant with water increases disinfection efficiencies, whereas dissolved organic matter reacts with and consumes the disinfectant and forms products that have weak or no disinfection activity. certain inorganic compounds and particulate matter also react with disinfectants. the ct value must be determined sequentially whenever a disinfectant is added to water. contact time (t) is the duration in minutes for water to move from the point of application of the disinfectant or the previous point of residual disinfectant measurement to the point where residual disinfectant concentration (c) is measured. it is measured from the first point of disinfectant application and from all subsequent applications until or before the water reaches the first user. determine contact time in pipelines by dividing internal volume of the pipe by the maximal hourly flow rate through the pipe. determine the flow rate from (a) plant records, (b) continuous monitoring device readings, (c) measurements at hourly intervals, or (d) if this sort of information is unavailable, measurements at expected high flow periods. use tracer studies to determine contact time within mixing basins and storage reservoirs. these values represent only % effectiveness because of short circuiting. chlorine, fluoride, and rhodamine wt (but not b) are commonly used tracer chemicals. contact time is usually measured by a step-dose method, but a slug-dose method is used where chemical feed equipment is not available at the designated point of addition or where such equipment does not have the capacity to provide the necessary concentration. (see appropriate epa literature for procedures, and consider getting engineering expertise if these matters are too complex.) estimate whether pathogens had been inactivated. to do this, divide the ct calc value by a value (ct x% ) resulting in a certain percentage inactivation (e.g., . % [ -log] or ct . for giardia cysts and . % [ -log] or ct . for viruses). this gives an inactivation ratio. see table i for ct . values for giardia and table j for ct . values for viruses. following is a sample calculation for data in table i when water temperature is °c, ph in a clearwell (reservoir for storing filtered water) is . , time (t) (either calculated or measured by dye test) is min, and the disinfectant used is chlorine: the desired ct value for . % inactivation of giardia for ph at °c is between and depending on concentration of disinfectant. in this case, the disinfectant measured at the clearwell outlet is . mg/l. therefore, the result, , is larger than the value, , required in the table; hence, these disinfection concentration (c) and time (t) conditions should result in a . % or greater inactivation of giardia cysts. for free chlorine, a -log inactivation of giardia cysts provides greater than a -log inactivation of viruses. the following example, using the data in table i , demonstrates a means to calculate the increased disinfectant dosage needed for a plant during the transition from summer to winter, when the water temperature fell from to °c, chlorine dioxide was the disinfectant used and the t value (calculated or measured) is min. using table i in this situation, the plant should have increased the chlorine dioxide concentration from . mg/l to . mg/l to maintain the same efficiency of disinfection. if this had not been done, it may explain the survival of pathogens in the water supply. the sum of these ratios gives the total inactivation ratio, which should equal or more to provide effective disinfection. make calculations and record information on form g a. the following example shows the way this is done. chlorine is added to three basins. chlorine concentration, contact time, temperature and ph are measured at these locations and recorded as shown in table . data from table i is combined to do the calculation. the resulting sum exceeds . . this ensures that the plant met the recommended or required ct. regulations may require that community and non-community public water systems that use surface water or water under direct influence of surface water meet a criterion (e.g., minimum of . % [ -log] removal and/or inactivation of giardia cysts and a minimum of . % [ -log] removal and/or inactivation of viruses of fecal origin that are infectious to humans). removal and/or inactivation of microorganisms may be accomplished by either filtration plus disinfection or disinfection alone, depending on the water source. water systems using chlorine with ct values that attain minimal level or inactivation of giardia cysts will result in inactivation of . % ( -log) of viruses. evaluate the prefiltration processes (e.g., coagulation, flocculation and sedimentation). coagulation is a process that uses coagulant chemicals and mixing, by which colloidal and suspended materials are destabilized and aggregated into flocs. flocculation is the process that enhances agglutination of smaller floc particles into larger ones by stirring. sedimentation is the process by which solids are removed by gravity separation before filtration. observe whether these processes reduce turbidity. calculate detention (transit) time within the settling tank and seek information about frequency and method of cleaning the tank. for example, if an -ft-deep sedimentation basin has a volume of million gal, and the plant flow rate is million gal/day, detention time in the basin is: (in your country you may want to calculate rates based on metric measurements) several different types of filtration may be used in water treatment facilities. these are conventional, direct (both conventional and direct are referred to as "rapid" filtration), slow sand filtration, and diatomaceous earth filtration. conventional filtration consists of a series of processes including coagulation, flocculation, sedimentation, and filtration. direct filtration consists of a series of processes including coagulation and filtration but excluding sedimentation. slow sand filtration is a process involving passage of raw water through a bed of sand at low velocity (usually less than . m/h), utilizing both physical and biological means to remove particles and microorganisms. in diatomaceous earth filtration, water is passed through a precoat cake of diatomaceous earth filter medium while additional filter medium is continuously added to the feed water to maintain the permeability of the filter cake. if done properly, each filtration method results in substantial particulate removal. when rapid sand filters have a head loss of about - ft, they require back washing. filters are backwashed by reversing the flow of the filtered water back through the filter at a rate between and gal/min/ft of sand-bed area. sometimes water jets at the surface aid in loosening and removing deposited material on the sand. observe an actual backwash and look for indications of short-circuiting or areas of the filter material that seem agglomerated or resist being cleaned by the flowing water. if backwash water is not discharged to waste, evaluate where it is released. slow sand filters eventually become clogged. when this occurs, a scraper or flat shovel is used to remove the top layer of clogged sand, and new sand (equivalent to the depth removed by scraping) replaces the old. test the effectiveness of filtration for each filter unit by observing capacity and filtering area relative to volume and turbidity of the filtered water. also, review turbidity, headloss, and filter rate record. look for anomalies, especially in the few hours after a filter is returned to service, and before the filter is backwashed. review criteria that cause a backwash to be initiated, and establish if these criteria were followed during the time preceeding the outbreak. determine the source of backwash water and the frequency of back washing of filters from records and head gauge readings. check whether the water used to back wash or clean filters came from an untreated source and determine the fate of the backwash water. in the case of illness due to chemical substances, evaluate types of chemicals used and condition, operation and maintenance of chemical feeding equipment. consider sampling backwash water for pathogens under investigation. review plant records for results of monitoring and be alert for changes that suggest treatment failure. record this information on form g b (source of contamination and treatment failures that allowed survival of pathogens or toxic substances.) data in table k (estimated removal of giardia and viruses by various methods of filtration), give a summary of expected minimal removal of giardia and viruses in well operated filtration systems. values can be subtracted from ct values required for disinfection. although contamination is likely to be associated with raw incoming surface water, look for bypass connections where raw or partly treated water can get into treated water. also look for common walls that separate treated and untreated water. consider the possibility that a contaminant was introduced in any of the treatment chemicals themselves, or as an act of sabotage. determine whether any flooding has occurred during the interval of concern. check absentee records for possible enteric illness of the water treatment plant staff. such illness may reflect either sources of contamination or victims. record this information on form g b. at domestic locations, evaluate treatment devices (such as chlorinators, filtration units, softening equipment) as described above, but modified to fit the situation under investigation. record observations and measurements on forms g a and g b, as applicable. the water distribution system can be complex, with multiple entrance points for treated water and different pressure zones in which water can enter but not leave. water flows in the direction in which it is being "requested," so can flow in different directions in the same pipes from one hour to the next. contaminated water can enter a potable water supply from a non-potable water supply when the two are directly connected. such interconnections are referred to as cross connections. to evaluate such situations, trace lines of the treated supply from the point of treatment or entrance into a building to points of use and associated plumbing. look for any interconnections of other water supplies, such as wells, waste lines or holding tanks for water intended for fire control. if cross connections are found, look to see whether backflow prevention devices are inserted between the lines and, if so, whether they are functioning properly. also, look to see whether there is an air gap between the water inlet and vessel or tank. evaluate the arrangement and operation of check valves on connections between the two water systems. review inspection report for backflow prevention devices. contaminated water can also enter a treated supply by siphonage from a contaminated vessel or sewerage to the potable water line having negative pressure. this is referred to as back siphonage. examine all water vessels to see whether they contain submerged inlets or hoses connected to water faucets, and if so, whether properly functioning vacuum breakers are in place. without proper air gaps or properly functioning vacuum breakers, there is a possibility of siphonage of water from plumbing fixtures in upper stories to lower stories when line pressure is negative. this may occur when faucets on lower floors are opened after the water supply valve has been turned off for repairs or when the supply line has had a sudden loss of pressure, as can happen with nearby heavy use of water (e.g., to fight fires or irrigate) or when pressure lines are broken. measure water pressure on upper stories of buildings to determine whether negative pressure occurs. (pressure losses may be transient and of very short duration.) interview building managers and residents about whether there were (a) any repairs of water service during the past month, (b) fires that occurred nearby, or (c) other situations that could have caused negative pressure in the water line. also, if appropriate, review fire and utility department records for information about these situations. get dates of line repairs to see whether they correlate with the time of incubation periods of early cases. measure chlorine residual (of chlorinated water systems) and take samples for microbiological tests at several strategic locations in the distribution systems. perform calculation on comparison of disinfectant residual. if a toxic chemical poisoning is under investigation, talk to home owner, building manager or maintenance staff about whether pesticides or other toxic compounds were sprayed with equipment connected to a hose or a sprinkler system. furthermore, interview building managers and residents about whether there are persons residing there who either are or recently were ill with diarrhea. they may represent sources of the etiologic agent or may identify victims. interview those identified about the onset of their illness and symptoms and examine their plumbing systems. record information obtained during the investigation of distribution and plumbing systems, and record related calculations on form g (source and mode of contamination during distribution and at point-of-use). evaluate implicated waters used for swimming, water skiing, bathing, clothes washing by hand, or agricultural activities, in a manner similar to that described under the section on investigation of surface water source. if the potential site of contact was natural surface water, determine whether the water is likely to be infested by parasites and look for the presence of snails (swimmer's itch). for swimming pools, measure the water's ph, chlorine residual, water temperature, and turbidity, if applicable. also, review pool records for previous information on these characteristics. high turbidity in pools, hot tubs, and spas is a sign of either poor filtration or inadequate disinfection. evaluate whether the resulting water would adequately protect those who swam or waded in it or had any physical contact with it. evaluate filter and chlorination equipment as described for water treatment. backwash filters and collect a sample to get an indication of microorganisms present on the filter (thus obtaining historical information). this approach has been useful for identification of pseudomonas aeruginosa. look for the presence of slime on tub, whirlpool, slide and pool surfaces, and collect some of this material for analysis for p. aeruginosa. if the answer is not obvious, ask ill persons whether they had puncture injuries or wounds or scrapes while immersed in water. record this information on appropriate parts of form g (contamination source and survival of pathogens or toxic substances for recreational waters). collect samples of the water (see section on "collect water samples"), and test them for pathogens and/or indicator organisms, as applicable. the agents listed in table d can multiply in water and if such water is aerosolized, they can be transmitted to human beings via the respiratory route. highly susceptible persons (e.g., the elderly, smokers, immunosuppressed individuals) are the usual victims. look for possible sites where water may have been or is being disseminated as aerosols. consider (a) air conditioning cooling towers and evaporative towers, (b) hot water systems (heaters and tanks), (c) shower heads, (d) faucets with aerators, (e) mist machines used to freshen fruits and vegetables in markets, (f) humidifiers, (g) nebulizers/respiratory therapy equipment, (h) whirlpools and spas, (i) dental drills and cleaners, (j) cooling water apparatus for grinders, (k) splash from hoses, (l) water pressure line breaks, (m) decorative water features, (n) outside misters, (o) other aerosol-producing devices. sample water from all suspect sites for legionella or other waterborne agents that may cause illness when inhaled. it is not possible to recognize by visual inspection the potential for water to be contaminated with legionellae. warm temperatures, especially those between °c ( °f) and °c ( °f), are conducive to growth of legionellae. additionally, stagnant water allows time for legionellae to multiply, especially in dead-end lines, reservoirs and hot water tanks, and in water trapped in shower heads and faucet aerators. if it is deemed appropriate or necessary to sample for detection of legionella in the environment, collect water samples from suspect sources. it is important to use a lab with proven expertise in isolating and characterizing legionella, such as those labs in the u.s. certified under the environmental legionella isolation techniques evaluation (elite) program. the centers for disease control (cdc) have a convenient form for recording case histories (http:// www.cdc.gov/legionella/downloads/case-report-form.pdf). it is not appropriate to sample air for detection of legionella hazards. it may, however, be appropriate to use micromanometers or smoke to trace direction of air flow to determine route of dissemination. micromanometers measure pressure differences, and flow can be assumed to travel from high to low pressure areas. smoke moves from areas of higher pressure to areas of lower pressure and is extremely sensitive to air currents. observe direction and spread of smoke movement. record this information on form g (contamination source and sites of amplification and aerosolization of pathogens). prior to the collection of samples, investigators should consult with the testing laboratory that will be used, to receive specific laboratory sampling instructions and sampling kits. sampling protocols for potable and non-potable sources are dependent on the specific etiological agent and the related analytical procedures performed by the testing laboratory. collect samples promptly to test for possible etiologic agents and for microorganisms indicative of fecal contamination. contaminants in water are in a dynamic state; their presence and quantity differ with time and place. see table f (general instructions for collecting drinking water samples) for guidance on collecting and shipping samples for viral, bacterial, and parasitic analyses. samples for bacteriological tests can be collected in one of three ways: (a) by letting a stream of water flow into a container or by submersing a container into a volume of water, (b) by passing a large volume of water through a filter, (c) by putting moore swabs (see table a for description) or similar absorbent materials in surface water or drains for a few days (see table f ). use bottles that have been cleaned, rinsed, and sterilized, or use sterile plastic bags to collect and store samples for bacteriological examination. for a chlorinated water supply, or when in doubt about the presence of residual chlorine, use bottles containing mg/l sodium thiosulfate to combine with any free chlorine in the sample and prevent lethal effects of chlorine on microorganisms in the sample. this compound will not interfere if used for non-chlorinated water. when collecting water samples, first try to get "historical" samples that might give an indication of the condition of the water at the time it was ingested by those who became ill. obtain historical samples from water in bottles in refrigerators, toilet tanks, hot water tanks (for chemical analyses only), fire truck reservoirs, storage tanks, and taps at seldom-used and dead-end locations, and from ice in refrigerators and commercial ice plants. direct the laboratory to test historical samples for pathogenic organisms or toxic chemicals, as well as indicator organisms, because these samples have a chance of still containing the etiologic agent, whereas samples collected during the investigation several days or weeks after the event may be of water that has been flushed free of contamination or has been significantly diluted. take samples from to points throughout the distribution system. sample dead-end locations if they are found. do not neglect to obtain raw water samples even though treatment is provided. this is important, as it suggests possible sources of contamination and reflects the effectiveness of treatment. compare these test results with records of results on previous samples of raw or treated water. before drawing a sample from a water tap, make sure the tap is connected to the supply to be tested. do not collect samples (other than for legionella) from hose connections, sprays, or swivel faucets; uncouple these connections or choose different outlets. it is unnecessary to flame outlets, as this does not improve the quality of the sample. first, ensure your hands have been thoroughly washed then take a line sample by allowing the water to run to waste for - min. adjust the flow of water so that the thiosulfate will not wash out of the bottle or bag (do not overfill-most laboratory bottles indicate a maximum fill line). keep sample containers closed until the moment they are to be filled. hold the bottle near the base, fill to the "fill line" or within an inch of the top without rinsing, and immediately replace the stopper or cap and secure the hood, if attached. if a whirl-pak™-type plastic bag is used instead of a bottle, hold the base, rip off the perforated top, open the bag by pulling the side tabs apart, grasp the end wires, and place the bag under the flowing water. remove the bag before it is completely filled and squeeze most of the air out; fold over the top of the bag several times and secure by twisting the end wires. take a source or a distribution line sample by opening the tap fully and letting the water run to waste for sufficient time to empty the service line (or if in doubt, for min) and proceed as above. collect samples from open shallow wells and step wells by dropping a clean wide mouth container on a string or rope into the well. allow the container to sink below the water surface and then pull it out of the well. pour contents into a sample jar or bag. collect samples from rivers, streams, lakes, reservoirs, springs, toilet tanks, and non-pressurized storage tanks by holding a ml sample bottle near the bottom and plunging it neck down to a depth of cm ( in) below the surface; turn it right side up, and allow it to fill. don a plastic disposable glove when small vessels used for drinking are sampled in this manner. when collecting these samples, move the bottle in a sweeping, continuous, arc-shaped motion, counter to stream flow or in a direction away from the hand. collect samples at locations approximately one-quarter, one-half, and three-quarters the width of the stream or water course. special apparatus can be used for sampling at various depths. samples can then be taken by positioning large bottles on a rod or pole at the desired depth and location before pulling their stoppers with a wire, string or thin rod. samples of bottom sediments are sometimes useful for the detection of certain pathogens. collect surface scum or regions containing dense particulate colored material when seeking cyanobacteria (blue-green algae). collect slime, if present, when seeking pseudomonas. if large amounts of water are needed, seek assistance and obtain specialized sampling equipment from agencies responsible for water quality. if possible, avoid wading when sampling bodies of water because wading often stirs up bottom sediments. if this is the only way to get a sample, however, wade against any current (e.g., upstream in creek or river) and keep moving forward until sample taking is completed. piers or similar structures, or the front end of a drifting or slow moving boat, make good sampling stations. concentration of bacteria by the use of swabs, filters, or by absorption, is particularly important when waterborne pathogens are sought. to concentrate bacterial pathogens from flowing water (e.g., streams, lakes, sewer lines, or drains), suspend moore swabs (or non-medicated sanitary napkins or non-medicated tampons if moore swabs are unavailable) for - days. these can be held in place by wire just below the surface or at other depths. if rodents are about, put moore swabs in wire baskets. after the sampling period, either put swabs or pads into a plastic bag and pack in ice, or put the swabs or pads directly into an enrichment broth for the pathogen sought. take or send these to the laboratory promptly. concentration of microorganisms can be increased by filtration with a variety of filters (e.g., membrane filters, cartridge filters, or other filter media). when membrane filters are used for pathogenic bacteria recovery, pass at least l of water (relatively free of turbidity) through a sterile . μm membrane filter. for viral analysis, use virus-absorbing electropositive cartridge filter to concentrate l or more water (see table f ). keep filters cool (but not frozen) and ship to a reference laboratory for further processing. for giardia cysts and cryptosporidium detection, collect samples by passing at least l water through a cartridge filter (see table f ). for inorganic chemical analyses, use l polyethylene containers. these should be new, or acid-washed if previously used. collect the water without flushing the lines, preferably in the early morning before water is used. for trace metal analyses, preserve one sample with ml of high-grade nitric acid to a ph of or less. this is particularly important whenever it is suspected that metals may have leached from water pipes or vessels. for organic chemical analysis, use l glass containers with teflon-lined caps. clean and rinse the containers with a good quality laboratory solvent and heat at °c for min. rinse the cap thoroughly with distilled water. fill the container so that there is a minimum of air space. for physical analyses, collect at least l, or other amounts requested by the laboratory. collect ice aseptically in sterile plastic bags or jars. use sterile tongs to collect cubes; sterile spoons for collecting chipped or crushed ice; and sterile chisel, hammer, or pick to chip block ice. put block ice or large chips into plastic bags. if legionella is sought, sample water at sites of any source that may have been aerosolized and send to a lab with proven expertise in legionella isolation and characterization, such those in the cdc elite program. this includes cooling towers, evaporative condensers, water heaters and holding tanks, humidifiers, nebulizers, decorative fountains and whirlpool baths (see section on investigating sites where aerosols are disseminated for a more complete listing). turn off fans of condensers before sampling; if this is not possible, wear a respirator. use ml to l polyethylene bottles that have had sodium thiosulfate added if the water to be tested has been chlorinated. for each sample, don disposable plastic gloves and collect the sample by inverting the bottle and moving it in a continuous arc away from the hand. measure and record water temperature. handle samples as described in table f . rub swab over faucet aerators and shower heads if these are considered as sources of aerosols. break stick and allow tip to fall in a tube containing - ml sterile water (not saline). investigators are often requested to test air to demonstrate the presence of legionella in aerosols. although legionellosis is an airborne disease, legionellae are susceptible to low humidity and become non-viable on drying. therefore, air sampling is an ineffective and inefficient way of determining whether a legionella hazard exists, and it can thus be misleading. label each container with sample number, date, time of collection, and your name or initials. complete the water/ice sample collection report, form f, for the first sample. list additional samples with sample numbers and other pertinent information on the back of the form. in those situations where the laboratory needs additional information, attach the appropriate g series forms. send the original form f and list with samples to the laboratory; retain a copy for your files. inform the laboratory of the type and number of samples and specimens; also, consult with the laboratory on methods to preserve and transport samples, if necessary, and on time of their arrival. if legal proceedings are anticipated, deliver sample personally to the analyst, or seal the sample container in such a way that it cannot be opened without breaking the seal. note on form f the method by which the bottle was sealed. maintain a chain-of-custody log to document the handling of the sample, and have the log signed and dated each time it changes hands. consult with state/provincial regulatory agency on complying with legal requirements for chain-of-custody procedures. recipient should record on the form whether the sample was sealed when the laboratory received it. if analysis cannot be done on the day of collection, chill water samples rapidly and hold them at temperatures at or below °c ( °f), but do not freeze, because populations of bacteria such as escherichia coli and of parasites decrease during frozen storage. hold ice samples frozen; if this is not possible, keep the temperature below °c h ( °f). investigators should consult with the testing laboratory that will be used to receive specific laboratory sample packaging, labeling, and transportation instructions as protocols are dependent on specific transportation regulations (iafta, tdgr) within each jurisdiction. ensure each sample is uniquely identified and labeled (as per the receiving laboratories requirements). many laboratories include barcode labels along with the sample containers within the sample collection kits. ensure that the correct label is affixed onto the correct sample container and that this information is transferred to the shipping manifest accurately (chain of custody form). specimens should be packed and the packages labeled according to applicable regulations governing transport of hazardous materials. generally, the transport of samples of water and ice intended for laboratory analyses are packed and shipped in a manner to ensure the sample does not change from the time of sampling to the time received by the testing laboratory and shipped using the most expeditious means (e.g., personal delivery or overnight mail). typically samples of water or ice are packed with refrigerant (ice packs, dry ice, etc.) in insulated and sealed containers (see table f ). several measurements are routinely called for during on-site investigations. brief instructions are given for those that are commonly done; nevertheless, follow manufacturer's instructions if these are available. color comparison kits are available for testing for free, combined and total residual chlorine. the diethyl-p-phenylenediamine (dpd) test is an example (see table a ). check instrument calibration regularly. use dry reagents, because the liquid forms are unstable. chlorine comparators can be used to test for bromine by multiplying the result by the factor . and to test for iodine by multiplying the result by the factor . . measure temperature. measure water temperature by immersing the sensing end of either thermocouples, transistors, or thermometers into the water. sometimes measurements need to be made at various depths; use thermocouples with wire leads of sufficient length for this purpose. calibrate temperature measuring devices periodically. measure ph. calibrate the ph meter as recommended by the manufacturer with at least two standard buffers (e.g., ph . or . ) and compensate for temperature, if the meter does not do it automatically, before each series of tests. remove a sample of water to be tested and immerse the ph electrode into the sample; record the reading. ph can also be measured by color comparators that employ color indicator solutions or discs. (ranges of ph color indicator solutions are bromophenol blue, . - . ; bromocresol green, . - . ; methyl red, . - . ; bromocresol purple, . - . ; bromothymol blue, . - . ; phenol red, . - . ; cresol red, . - . ; thymol blue, . - . ; and phenolphthalein, . - . .) in this case, water containing more than mg/l chlorine in any form must be dechlorinated with sodium thiosulfate before the ph indicator solution is added to prevent decolorization of the indicator. always report temperature at which the ph is measured. measure turbidity. nephelometric turbidity unit (ntu) is the usual standard unit, but other turbidity measurements (such as particle counts) are used. the ntu requires a nephelometer, which measures the amount of light scattered predominantly at right angles and absorbed by suspended particles (e.g., clay, silt, finely divided organic matter, inorganic matter, soluble colored organic compounds, and microscopic organisms) in the water sample. calibrate turbidimeters with a standard reference suspension. make turbidity measures on the day samples are taken. vigorously shake samples, wait until all air bubbles have disappeared, and then pour sample into turbidimeter tube. read directly from scale on instrument or from an appropriate calibration scale. pump chemical smoke into the air at the exit of the device suspected of releasing aerosols. observe the direction and spread of the smoke. otherwise, measure pressure differentials with a micromanometer. measure other attributes of water. follow instructions given by manufacturers or in standard reference books (see further reading). use fluorescein dye, lithium or other tracers in appropriate soils to determine the means by which contamination from sewage, industrial wastes, or other sites of pollution reached the water supply. fluorescein dye is particularly helpful in evaluating flow of contamination through fissured rock, limestone, gravel, and certain other soils. this dye is not readily absorbed or discolored by passage through these soils or sand, as are many other dyes, but it is discolored by peaty formations or highly acid (ph < . ) soils. make a concentrated fluorescein dye solution by mixing g of fluorescein powder into a liter of water. usually, / to l of this solution are sufficient for the test for up to , l of water. fluorescein dye is also available in liquid and tablet form. one tablet will dye approximately l (~ us gal). pour the calculated amount of fluorescein solution or put a sufficient number of fluorescein tablets into a receptacle at a point of potential pollution. usually this point will be located within yards and at a higher elevation than the water source under study. cesspools, latrines, distribution boxes, sink holes, borings, septic tanks, drains, manholes, toilets and plumbing fixtures are typical places to introduce the dye. if dye is poured into a plumbing fixture or dry hole or boring, add water to wash it down. the amount of dye to use varies with the distance the dye must travel, the expected time of the journey, the size of the aquifer or water channel, and the nature of the soil. take samples of the water when the dye is introduced into the test hole or fixture and then hourly for up to h to detect arrival and departure of fluorescein. if no dye is observed, repeat the test with twice the amount of dye. whenever possible, use a fluorescent light or fluorometer to analyze water samples for evidence of fluorescein. a fluorometer can be set up and calibrated, and a continuous recording can be made. this meter can detect fluorescein in concentrations of μg/l (ppb). fluorescein dye will temporarily color water, which discourages use of the water until the dye is sufficiently degraded or diluted. alternate tracers can be used if specific ion meters are available. the dye stains all it touches. methanol is a good solvent for the dye, and hypochlorite solutions decolorize it; both can aid in removing stains. abrasive soaps are useful for cleaning stained skin; fluorescein-stained clothing should be washed separately. appearance of dye in a water supply is conclusive evidence of seepage from the site where the dye was introduced. failure to detect dye, however, is not conclusive evidence that seepage did not or would not occur if more dye had been added or if weather conditions or subsurface flow had been different at the time of the test than during the outbreak event. illustrate source and direction of contaminated water flow as indicated by the dye test on form g . take photographs of sources of contamination and evidence of staining of the ground at the site or dye-stained color of the water. in situations where a single source of contamination is obvious or where multiple sources are readily apparent, dye studies serve little purpose. drinking water, however, is not the only source of water that may contribute to outbreaks. other sources of water that can contribute to outbreaks include water not intended for drinking, recreational water and water used in agriculture during harvesting and packaging. legionnaires disease is the pneumonia caused by the inhalation of contaminated water aerosol containing the bacteria legionella, with legionella pneumophila being responsible for % of all infections. it is also a common cause of healthcare associated pneumonia. legionella can replicate within free-living amebae in water, allowing it to resist low levels of chlorine used in water distribution systems. risk of infection is more common in warm and humid weather, when water droplets are able to drift further due to higher absolute humidity. fifty percent of all legionella outbreaks have been traced to cooling towers with l. pneumophila serogroup responsible for all cooling tower outbreaks. all aerosol generating devices, however, can be potential sources of legionella. some other sources of aerosolization that may have contributed to or be associated with outbreaks include: whirlpool displays, building's air conditioning systems, water spray fountains, public bath houses, vegetable misting systems in grocery stores, evaporative condensers, showerheads, humidifiers, air scrubbers, car washes, ornamental and decorative fountains, potting soil, respiratory therapy equipment, dental units, road asphalt paving machines, car windshield washer fluid and car air-conditioning systems. in the investigation of a legionella outbreak, (see box , the flint water crisis, which describes a likely legionella outbreak from a commercial water source) due to the varied sources, there is a need to use a broad investigative questionnaire and the collection of environmental data. environmental factors such as dry bulb temperature, relative humidity and wind rose data can provide information regarding drift evaporation, deposition (settling) and the size of the affected zone. although aerosol drift can carry legionella up to mi ( km), the risk of infection is usually highest within ft ( m) of the source. there are also air dispersion models that can be used to determine drift zone and the use of human activity mapping in the identification of potential sources. in general, e. coli and norovirus are the most common pathogens responsible for recreational waterborne outbreaks associated with non-treated water such as beaches and lakes. cryptosporidium, which is resistant to chlorination, is the most common pathogen resulting in outbreaks in treated water venues such as swimming pools and water spray parks. it should be noted that e. coli, the indicator of choice of recreational water samples, is not indicative for the presence of norovirus and giardia, cryptosporidium. e. coli can also be "naturalized" and have been found to survive and multiply in beach sand. beach water sampling results therefore may provide false positive or false negative results and may not be the best indicator for the presence or absence of pathogens. recreational waterborne outbreaks are not just traced to the ingestion of contaminated water (table c . illnesses acquired by contact with water: a condensed classification by, symptoms, incubation period, and types of agents). hot tub rash, or pseudomonas dermatitis/folliculitis commonly occurs in public hot tubs or spas such as those found in hotels. the rash is often a result of skin infection from the bacteria pseudomonas aeruginosa colonizing in the hair follicles after exposure to contaminated water. pseudomonas aeruginosa is an opportunistic pathogen that can survive within the biofilm on the tub surface or within the piping system. outbreaks can occur when there is a heavy bather load resulting in an increase in chlorine demand, which in turn reduces the effectiveness of the disinfectant to control the population of pseudomonas. blue-green algae or cyanobacteria bloom can occur in warm, slow-moving or still water. when conditions are favorable, mostly during hot summer weather, cyanobacteria populations may increase dramatically, resulting in a "bloom" as they rise to the surfaces of lakes and ponds. they resemble thick pea soup and are often blue-green in color. although blooms can occur naturally, water bodies which have been enriched with plant nutrients from municipal, industrial, and agricultural sources are particularly susceptible. some cyanobacterial species may contain various toxins, some are known to attack the liver (hepatotoxins) or the nervous system (neurotoxins); others simply irritate the skin. health effects from cyanotoxin exposure may include dermatologic, gastrointestinal, respiratory and neurologic signs and symptoms (table b . illness acquired by ingestion of contaminated water: a condensed classification by symptoms, incubation periods, and types of agents). water can also be an indirect cause of foodborne outbreaks by providing a media for the survival, transportation and the introduction of pathogens into food products. water used during production, including irrigation, pesticides and fertilizers application and washing, frost protection, harvesting, has long been recognized by food safety scientists as one of plausible and probable sources of the contamination of fresh fruits and vegetables. there have been many outbreaks from produce traced to pathogens being introduced by contaminated irrigation water. although harvested products are sometimes washed with chlorine solution, pathogens may still survive the process through internalization. e. coli o :h may migrate to internal locations in plant tissue and be protected from the action of sanitizing agents by virtue of its inaccessibility. experiments have also demonstrated that e. coli o :h can enter the lettuce plant through the root system and migrate throughout the edible portion of the plant. however, this claim has been refuted by others. salmonella and e. coli can also adhere to the surface of plants, and enter through stomata, stem and bud scars and breaks in the plant surface caused by harvesting and processing. water containing bacteria can be drawn into the produce if it is immersed in or sprayed with water that is colder than the produce itself. e. coli o :h may also use its flagella to penetrate the plant cell walls and attached to the inside of the plant. once attached, it may be able to grow and colonize the surface of the plant. the concerns are not just with bacteria. the present of norovirus in the hydroponic water can result in internalization via roots and dissemination to the shoots and leaves of the hydroponically grown lettuce. irrigation water may be contaminated from runoff from nearby domesticated animals and their lagoons, feedlots, ranches into rivers; from feral/domestic animals with direct access to creeks, ditches, rivers, ponds; from sewage flows into waterways and contaminated wells. in some parts of the world sewage contaminated water is preferred for irrigation despite a potential risk of transporting enteric pathogens, since it carries nutrients (n and p) for the plants. there is sufficient information to conclude that the application method of irrigation water to fresh produce can have an effect on the microbiological risks associated with the crop. in general, keeping water away from the edible parts of ready-to-eat crops that are consumed without cooking can result in a lowered risk of a foodborne outbreak. the least to more risky methods for irrigations for microbial contamination are: subsurface irrigation (buried soak hoses) < drip irrigation < indoor flood irrigation (hydroponics) < outdoors flood irrigation (water-filled furrows) < overhead irrigation (sprinklers). an outbreak of illness arising from exposure to water demands immediate epidemiological investigation to assess the situation, gather, evaluate, and analyze all relevant information, with the goal of ( ) halting further spread of this illness, and ( ) predicting, preventing, and/or attenuating future outbreaks. this twofold mandate of epidemiology is usually described as "surveillance and containment." at the commencement of an investigation, the unknowns usually outnumber the known facts. there is no substitute for prompt, thorough, and careful collection of interview data from ill and well persons who ingested or contacted the suspect water, attended a common event, or who were part of a group of persons where illness occurred. careful analysis of these data, particularly with reference to common patterns of "time," "place," and the characteristics of the persons involved, can often eliminate many vehicles, agents, and pathways quite early in the investigation, and focus on the remaining possible vehicles, routes, and agents. later, laboratory results may confirm the agent, the specific pathology, the route taken by the infection or toxic agent, and indicate what is needed to stop the spread, but early epidemiology can often be invaluable in predicting the outcome and taking preventive steps to contain the problem before the lab results are available. lessons can be learned from most outbreak investigations and are invaluable for increasing our understanding of these pathologies, and preventing their future occurrence. an outbreak is defined as either an unusually large occurrence of an expected illness at that time of year in that place, or the occurrence of a type of illness that does not usually appear at that season and location. the "time" factor should be studied immediately by plotting the onset time of each case on a time-based grid, to create the epidemic curve. although any number of cases can be involved, the minimum number for an "outbreak" to be declared is two associated cases, with special exceptions such as naegleria fowleri where, because of the severity and the possibility that cases may have been missed, a single case constitutes an "outbreak." although the epidemic curve is usually measured in hours or days, protracted exposure to agents in water may mean apparent sporadic cases linked to a common source over months or years. if an "outbreak" is suspected by a sudden increase of cases, determining who is to be categorized as a "case" is not necessarily a simple process. many people notoriously fail to report enteric illness for many reasons: embarrassment, lack of time, no clear idea which agency should be notified, mild self-treatable symptoms, or simply because they prefer not to make a fuss. they may therefore be incorrectly classified at least initially as "nonill." consider also that - % of the general population will have experienced some form of "upset stomach" in the last hours, regardless of exposure to the suspect item, and they may be incorrectly classified, at least initially, as "ill." to reduce the "false negatives" and "false positives" that are expected with self-reporting, the investigator needs to establish a working case-definition. a careful case definition categorizes people as "case" or "control" with the best accuracy possible within the time constraints and resources available. a case definition could be considered "too sensitive" if it classifies as a "case" a person who experiences: "… at least one episode of stomach cramps, nausea, vomiting, or diarrhea in the last hours." this would confuse subsequent analysis, and produce more false positives. similarly, a case definition could be considered "too specific" if it classifies as "not-ill" a person who had experienced only three episodes of diarrhea or vomiting, because they failed to satisfy a case definition requiring "…at least four episodes of vomiting or diarrhea in the last hours." should this last individual, having been declared as not fitting the case definition, be taken into the "notill" group, the error and subsequent analysis is confounded even further. a reasonable case definition therefore attempts to reduce both types of errors, and will depend upon the early indications of what the etiology may be. in the instance of a suspected salmonellosis, a case could be defined as "a person who was in good health before attending the event on monday may rd, and who experienced two or more of the following symptoms anytime up to midnight, sunday may th.: nausea, vomiting, stomach-cramps, diarrhea, headache, or fever." note that a case definition should include a place of exposure if known, a timeframe during which symptoms may have been experienced (salmonellosis has a range from to h. usually - h), and the additional footnote that the individual was not already symptomatic before the suspected "exposure." calculate the percentage of ill persons who manifest each symptom by dividing the number of persons reporting the given symptom by the number of cases ( for the example, table ) and multiplying the quotient by . the distribution of symptoms can be used to identify the most likely pathogen, and aids in requests to the laboratory for microbiological assays of samples and specimens. other symptoms (e.g., prostration, lethargy, weakness) may be included if deemed appropriate or helpful, but the six symptoms in table should always be included. headache, for instance, is associated with many viral infections (e.g., norovirus, rotavirus), but much less so with bacterial infections. fever is usually associated with an invasive bacterial infection (such as salmonellosis or campylobacteriosis), and is not usually seen in outbreaks of simple enteritis (such as with cholera). this information helps to determine whether the outbreak was caused by an agent that produced intoxication, an enteric infection, or generalized illness. in the example given, a predominantly diarrheal syndrome without much fever or headache tends to eliminate some of the viral infections (norovirus or rotavirus) or the host-adaptive/invasive serotypes of salmonella (e.g., s. dublin or s. choleraesuis). median onset time calculations may further reduce possible candidate etiologies. in historical investigations, or where no laboratory confirmation is possible, the symptom profile and onset times can sometimes predict the etiology of the outbreak within reasonable certainty. an epidemic curve (also called an onset curve or onset distribution) is a graphic illustration called a histogram that shows the distribution of the time of onset of first symptoms for all cases that are associated with the disease outbreak. paper printed with square "grid" lines will allow the investigator while on site to represent each case as a single "block." the horizontal axis is the sequence of intervals of time and date. the unit of time that defines the width of each interval depends on the characteristics of the illness under investigation. for example, intervals of days or weeks are appropriate for diseases with long incubation periods, such as cryptosporidiosis or hepatitis a. intervals of a day or half-day are appropriate for outbreaks of enterohemorrhagic e. coli strains or shigellosis, while single-hour, -h, or -h intervals will be more suitable for illnesses with shorter incubation periods, such as chemical poisonings. the vertical axis is always the actual count, or "frequency" of cases (blocks) stacked at each interval. it is often necessary to redraw the onset curve as more accurate information becomes available. if the illness is known, a rule of thumb is that the time interval used for each "block" on the x-axis should be no more than ¼ the incubation period of the disease under investigation. if the illness is not known, select an interval where the data produces a bell-shaped curve; not too flat and not too tall. construct this graph using time-of-onset data from forms c or d, employing an appropriate time scale. once all the onset times for the cases have been plotted on the histogram, determine the range as the interval between the shortest and longest incubation periods. in fig. a , the range is the day period from the th to th march. the median onset time is preferred to the mean because the latter is vulnerable to a few or even a single very small or very large value. the median on the other hand, is the midvalue of a list of all individual onset times, including duplicate entries, that are ordered in a series, from shortest to longest. if the series comprises an even number of values, the median is the mean of the two middle values. most standard reference texts on communicable diseases give onset times as median values. the mode is simply the interval having the largest number of observations. a distribution with a single "peak" is called a uni-modal distribution, while an outbreak with two peaks is called "bi-modal." subsequent modal peaks following the first may indicate either a "secondary wave" of cases or the exposure of other people at a later time. the shape of the epidemic curve helps to determine whether the initial cases originated from a single point-source exposure (such as water or food available for only part of a day), or from repeated exposures for a longer time, or even more gradual person-to-person spread. a point-source epidemic curve is characterized by a sharp rise to a peak, followed by a fall that is almost as steep ( fig. a ). an "explosive" outbreak of this type is common where a municipal water supply is the vehicle, affecting large numbers of people in a very short period of time, but without secondary cases occurring, or any evidence of onward spread within the community. propagated outbreaks are those in which the initial victims ("primary cases") manage to spread the agent to other people ("secondary cases") such as family members, patients, clients, or other contacts in crowded places through aerosols, personal contact, or contaminated water/food/utensils/surfaces, etc. propagation following a point-source exposure is demonstrated by a second increase in reported cases following the decline of the first cluster. sometimes this takes the form of a second "modal peak" separated by approximately one incubation period, but this distinction is soon lost. figure a shows no evidence of propagation; fig. b suggests that propagation may have taken place, although care must be taken to consider other explanations. in addition to ( ) true propagation, where the secondary wave can be expected to appear one incubation period after the first, secondary waves may be also explained by ( ) exposure to the same point source (e.g., food or water supply) at different, but specific times by other people; this might be a repeated offering of contaminated food or water at two or more mealtimes; ( ) a second pathogen (perhaps from the same unhygienic food or water source) which may have a different symptom profile and a different (incubation) time. slow propagation from the beginning of an outbreak with neither an obvious point-source, nor any distinctive "waves" separated by an incubation period as in fig. c , usually indicates one-at-a-time person-to-person spread through closecontact, poor personal hygiene, aerosol (e.g., influenza, or sars), or sexual transmission (e.g., hiv/aids). it can also be explained by (non-propagated) continuing exposure, for example drinking of contaminated surface water following a conflict, natural disaster or other breakdown of infrastructure. as such it is commonly associated with waterborne cholera, shigellosis, typhoid fever, or e. coli infection, and characterized by scattered cases which continue until the chain of infection is cut. slow, constant and/or intermittent exposure to persons over time to pathogenic microorganisms can also result from sewage run-off after a series of heavy rainfalls. in addition to revealing whether the outbreak was due to a single point-source, or had been spread steadily through the community by propagation in some way, another important objective in constructing the epidemic curve is to estimate the incubation period of the illness if it is not already known. with waterborne illness especially, the time of exposure may be further obscured because people usually drink water several times a day. hence, the incubation period cannot always be determined for each case, but the actual time of onset is usually available. the incubation period is the interval between exposure to food or water that is contaminated (with enough pathogens or with a sufficient concentration of toxic substances to cause illness), and the appearance of the first sign or symptom of the illness. each etiology is characterized by a typical incubation period (tables b, c, d, and g). individual onset times will vary due to immune factors, co-morbidities, the dose ingested, and other ingested materials, but the investigator can often make a rough estimate of the average incubation time by examining the aggregation of all onset times as an epidemic curve. the modal peak of a single "cluster" or distribution is the time interval in which most cases commence symptoms. in fig. a this occurs on march , and in fig. d that occurs at the double interval feb - th. where two separate modal peaks (a "bi-modal distribution") suggests secondary cases ("propagation"), then the distance between the first two modes is a good estimate of the incubation period. figure b shows about days between primary and secondary modal peaks, suggesting that the initial exposure is likely to have been days before the first mode. in fig. b this would be sometime on or near the th of the month. if the exposure point is known but the agent is not, then that estimation of the median incubation period will allow many etiologic agents to be excluded due to date of onset of symptoms: between march and april , count per day onset histogram for cases of shigellosis, march to april , illustrating slow spread through a community through either propagation (person-to-person spread via poor hygiene), or exposure by many people to a small well at different times (a non-propagated route) incubation periods that are clearly outside the range of times observed. the list of possible candidates can be further reduced by examining the symptom profile and other characteristics of the illness and suspect food or water vehicle. as time passes, the onset curve also provides an ongoing measure of the potential for propagation, and the incidence rate. all this information can be useful in deciding whether the illness in question is an infection or intoxication and thereby determining which laboratory tests should be requested (tables b, c, d, and g). note that not all water or foodborne illnesses listed in a standard reference such as the "control of communicable diseases manual" (apha ), are directly communicable person-to-person; many require a suitable substrate (food or drink) and adequate time/temperature combinations to attain sufficient numbers or the production of enough toxin to induce a pathological condition. an exposure time can sometimes be estimated from a clear, point-source, singleexposure onset distribution (fig. d ). it has no solid basis in statistics, but has sometimes been found to be useful in practice. the typical incubation periods for most foodborne and waterborne illnesses are readily available for comparison (e.g., control of communicable diseases manual, apha/cdc, ), and in this manual in tables b, c, d, and g. an incidence rate is the number of new cases of a specified disease reported during a given time period in relation to the size of the population being studied, multiplied by a constant, usually , to give percentages. thus new cases of e. coli o :h infection among the residents of a children's summer camp in july is an incidence rate of ( / ) × or ( . ) × = . % for that month. if several people have left and their state of wellness is not known, their impact should be expressed in the form of the possible range of values around the known incidence rate within the two extremes whereby they may all be well or they may all be ill. thus, where six children who were at the camp had departed around the time of the outbreak and their health is unknown, the range could be from a possible ( / ) × (or . %) if all of the six had been well, up to ( / ) × (or . %) if all had been ill. note that the "missing" six are added to the denominator only when we speculate that none were ill, whereas they are added to the numerator and denominator if we speculate that they might all have been ill. in this example, the overall incidence rate would be reported as " %, with a possible range from . to . %." depending upon the situation, it is often necessary to identify exposures which may be related to the illness, and to calculate an incidence rate for each such exposure. for example, in the summer camp illustration (above) it might be useful to enquire if gender, age, location, or some other attribute or activity increased the risk of becoming ill. this should not be interpreted automatically as implying that a given exposure would be associated with the outcome in any situation. by hypothesizing that gender was linked to the risk of illness, for example, does not imply that males are more vulnerable to the illness (the outcome) than females, but it can indicate that gender may have been related to the exposure, which in turn increased the risk. as an illustration, suppose that boys at the camp had been swimming, while the girls had gone on a nature walk. the boys may subsequently show increased incidence rate for e. coli o infection, not because they are more susceptible, but because of their activity. every proportion or percentage statement should be made with clear reference to the appropriate denominator used. incidence rates of waterborne illnesses are usually similar for both sexes at any given age group in the population, but differences in activities or dietary habits or susceptibility due to age or underlying health status can change the risk. the very young, the elderly and the immunocompromised can be at more susceptible, while in some instances, previously exposed populations may have developed a measure of immunity to an infection that may still cause more serious illness among visitors. a further complication arises where the "at-risk" population (perhaps residents at an institution, summer camp, or on a cruise ship) have generally consumed all the food and water for the extended period. careful interviewing of affected persons often uncovers one or more persons who entered the subject community shortly before becoming ill or who visited the community for a short time and became ill after leaving it. example: the south-west part of the county is served by three semi-private water systems. thirty cases of waterborne illness are being investigated in the area. when the numbers of cases are displayed for each water system, no clear grouping or clustering is evident, although the delta supply appears associated with about % more cases than the other two (table a) . however, when the analysis introduces the total population of persons who depend upon each water system (as denominators), a different scenario emerges. the incidence rates (expressed here as percentages) now allow a meaningful comparison (table b ). we can see that persons using the bravo system have roughly five times the risk of illness compared to people who are served by the other two systems. the use of the denominator is vital for most calculations. caution: numerous other factors may also explain the outbreak and these should be carefully examined. for example, the households using the bravo supply may be closer to an unhygienic corner store, drink from a cross-connected public water fountain, or their children may swim in a more polluted pond than the other communities. potential sources such as these should be eliminated before the water supply is announced as the source of the illness. sometimes a spot map may be useful in showing the location of the residence of each case, while on a larger scale, the rates of illness can be shown using city blocks, census tracts, townships, or other subdivisions. different colors or symbols to indicate cases with different time of onset periods (such as weeks) may help to support a hypothesis as to where contamination was introduced, inasmuch as the earliest cases tend to cluster around the point where contamination first occurred. the weakness of this procedure is that if the exposure had been at a restaurant, workplace, or school, plotting the relationship to the location of the home would not be useful. the investigation of waterborne or foodborne disease outbreaks invariably commences after both exposure and illness have happened. this is the classical "casecontrol" study, where a group of ill people ("cases") and a group of non-ill people ("controls") are compared in terms of their exposures. to measure the association between exposure and illness, the data are typically displayed in a × contingency table. table compares cases and controls in terms of their exposure to a suspected factor "x." the table is ready for analysis using odds ratio, as well as the chi-square or the fisher's exact tests where appropriate. one × table will be used for each possible exposure (e.g., each beverage, food, or other material). as many cases as can be identified and contacted, and as many non-ill people (controls) as can be found, should be interviewed as quickly as possible about their exposures to each suspect item. fading memories, the chance of obtaining stillavailable samples of implicated food or water, and the opportunity to obtain fecal specimens before the patient is started on the ubiquitous broad-spectrum antibiotics are all reasons for rapid response. case and control numbers do not have to be the same; the calculations compare ratios so equal numbers in each group are not needed. generally a : to : ratio of cases to controls is perfectly adequate. as interview data from cases and controls are accumulated, leading to formation of hypotheses about the source of the illness, human resources should be deployed in two additional essential tasks: ( ) tracking down and confirming the hypothesized source of the illness, and ( ) promptly issuing warnings to all affected groups about the possible risks from any source that is still accessible, with assurances that further bulletins will be issued as soon as confirmation is received. this precautionary principle is a vital component of risk management in modern public health. waiting for absolute confirmation before releasing warnings and advisories should not be an option in the twentyfirst century. the principle holds that while false alarms can be quickly forgiven, further illness should be avoided at the highest priority. failure to heed this step has contributed to needless suffering and severe damage to reputation, trust and credibility. a case-control approach is necessary because unlike the data in table b , we rarely have full information about all the attendees, and therefore the true incidence/attack rate is not available. very rarely, when all cases and controls are available for interview, we would have the true incidence rates for ill and for not-ill persons and this would allow a "retrospective cohort study" to be carried out. under such circumstances, and using table as an illustration, we could state that of persons exposed to item x, persons ( . %) had become ill compared to ill of not exposed ( . %). where incubation times are longer than a day, there is increasing likelihood that only a small proportion of the non-ill people will be available for interview, and on many occasions, not even all the ill persons can be contacted. the point here is that the investigator is usually working with sub-sets of the true cases and controls. the controls in table and possibly even the cases may have been drawn from larger groups, and therefore we cannot state the incidence rate, for example, as: "… of exposed were ill," because the " " had been artificially assembled, and may not resemble the true incidence at all. we can, however, use exposure rates, for example: "…of ill persons, ( . %) had been exposed to x," and, "…of who were not-ill, only ( . %) had been exposed to x." the overwhelming majority of waterborne or foodborne illness investigations are run as "case-control" studies (or to be more accurate, "case-comparison" studies, as very little true "controlling" is accomplished during the selection of the comparison group). a broadcasted invitation to all who might have been exposed to come forward, typically results in few non-ill persons volunteering information, because nonaffected individuals believe they have little if anything to contribute. this reduces validity even further, and more active recruitment is often necessary to convince them that their information is just as essential for the investigation as are the contributions from the less-fortunate attendees. let us examine a waterborne illness suspected as being due to the consumption of water bottled from a certain spring. you have found people who meet the case definition of illness, and another non-ill people in same neighborhood who report no symptoms at all, and who will be your controls. in table we display the data and ask the question: "is drinking this water related to the risk of illness?" whenever a × table appears, the first step is to calculate the odds ratio (or). an odds ratio tells us if there is a relationship (where or ≠ ), and the strength of the relationship (the or value itself). it also clearly indicates the direction of the relationship: was drinking or not-drinking the dangerous activity? this is easily determined by finding the dominant pair from (a × d) or (b × c). in the example above, (a × d) is greater, so cell "a" links the row "drank" with the column "ill," while cell "d" links "not drink" with "not-ill." this assumption is not as obvious as it may seem; the cause of the illness may have been whatever "other" thing was drunk by those who avoided spring water! it is important to clarify that the odds ratio yields the strength of the association, not the statistical significance. most or values (where many exposures are being assessed) will be close to . (= "no association"), while an or clearly exceeding . signifies a positive association between this exposure and illness, such that this exposure increased the risk of illness. an or < . is protective, meaning that exposure to this factor reduced the risk of illness compared to the other group. for example, an or of . means that the exposed group had only one-quarter the risk of illness compared to the non-exposed group. the non-exposed group therefore has a greater risk (by a factor of ). while this protective effect can be due to true therapeutic protection (e.g., exposure to antibiotics when you have an infection), it is frequently explained as "statistical" protection. as an example, consider an outbreak where everyone consumed only one of two possible types of bottled water. one source, a, contains a pathogen, and b does not. if the ill people were found to be five times more likely to have consumed type a (odds ratio = . ) then the not-ill would have five times the rate of consuming water b, and only one-fifth of the rate of choosing water a (or = . or %). this can also be read as the risk of illness for the non-exposed group, or as the risk of staying well by the exposed group. an easier way to interpret an or less than is to place over the or to reveal a value greater than , but clearly labeled "protective." the or is a ratio between numbers, and therefore not sensitive to the actual numbers of people in individual cells, an important consideration when the numbers of subjects are relatively small. this is illustrated by the common question: "how large does an odds ratio have to be before it is considered evidence of an association?" a popular response is "at least . ," but this must be considered with extreme caution. for instance, with very large studies, an or of . (barely more than . ) can be shown to be very highly significant statistically (p = . ), whereas in a small-n study, an or of even . may not achieve statistical significance. the odds ratio is certainly a useful measurement, and should always be used when a × table is encountered. it will quickly advise you ( ) that there is an association, ( ) the strength of that association, and ( ) the direction of the association, none of which are specifically measured by a test of statistical significance. unfortunately, it is not reliable with small cell sizes, and is unable to answer the question: "how likely is it that these numbers could happen just due to chance?" for this, we need to test the statistical significance. the best advice is to use the or (or relative risk where appropriate) together with a test of statistical significance. most online statistic calculators or laptop versions of sas, spss, epiinfo, etc. will give a selection of useful statistics (odds ratio, relative risk, several versions of chi-square, and fisher's exact test, both onetailed and two-tailed.) in keeping with all scientific enquiry, we begin by advancing the notion (the "null hypothesis") that there is no association between the exposure and the illness, and attempt to support that notion. if insufficient evidence is found to support the null hypothesis, we reject it and cautiously consider that an association may exist between the two variables. this can be described as a statistically significant association. two methods of testing are presented: the chi-square test (written χ and pronounced "ky"-square) for most × (or larger) tables, and the fisher's exact test (only for × tables) when chi-square is not valid due to the numbers in the cells being too small (the following sections give advice about this decision). the original data value in each cell we call the observed, or "o" value, and these are compared with the numbers that you would expect ("e" values) if there were no relationship at all; that is, if the variables were not related, and the data were arranged purely by chance (as stated by the null hypothesis). the chi-square test measures the difference between the o and e values. if they are close, we have to accept that there may be no real relationship; if far apart, we can reject the null hypothesis and cautiously declare that exposure and illness were probably related. numerous online statistical calculators can be used to yield ors, rrs, and chisquare values. if you prefer to do the calculation by hand, construct a × table as shown, with "observed" data, marginal totals, and the grand total. the expected "e" values are found from: to make sure the chi-square analysis is appropriate for your table, you must be sure that all "e" numbers are more than . the quickest way is to first calculate for the cell with the smallest e value; (this will be the cell with the smaller column total and the smaller row total.) in table , the smallest e value will be cell "d," and this is calculated as ( × )/ = . . as this is > , all other e values will be greater than this, so chi-sq. is valid. (note that the smallest e value did not coincide with the smallest o value). . for all four cells the sum (χ ) is: . + . + . + . = . an online statistical calculator will give you this same chi-square (χ ) value. to verify by hand whether the o vs. e difference is statistically significant, compare your chi-sq. value (for a × table only) with . . if your calculated value exceeds . , then this is unlikely to be due to chance, and thus you can begin to believe that this exposure did influence the risk of illness, and you can reject the null hypothesis. statistical results usually include a probability (p) statement. this is the probability that the null hypothesis ("no association") is correct. the . value is the minimum needed for statistical significance, where the p is less than % (p < . ). recall that the p is the probability that no real association exists between exposure and illness. by convention, if p > . (more than %) then the relationship is declared not statistically significant. where p = . or < . , then the relationship is statistically significant. the smaller the p value, (p < . , p < . , etc.) the more confidence you have that a relationship really exists. other critical values exist for assessing calculated chi-sq. values, from larger tables than × , and at more extreme levels of significance. a further chi-square calculation is shown as an appendix. where a table greater than × is found to have more than % of the cells with an e value less than , chi-square is not valid. the solution is to collapse either columns or rows to allow the e values to increase. for example in table a , two cells out of six ( %) have e values less than , but if "high dose" is merged with "medium dose" the resulting increase in observed (o) cell sizes is also reflected in greater e values, while the table becomes × (table b) . some outcome information has been lost, but the chi-square analysis can proceed. if, after trying to collapse cells and/or rows, a × table is reached still with an e value < , the fisher's test is indicated. this procedure is reserved only for × tables where one or more expected (e) values is less than , making the chi-square test not valid. our example is taken from an investigation into an outbreak of shigellosis presumed to be due to water from a well ( table ). the odds ratio has been calculated as ( × )/( × ) = . , meaning ill persons were six times as likely to have drunk well water compared to non-ill the "!" denotes a factorial, meaning that number multiplied by the next smallest number, and so on down to . (e.g.: ! = ) p persons. an attempt to use chi-square is prevented by at least one e value less than of . (cells c and d both show e values as ( × )/ = . ). the starting null hypothesis is "that no relationship exists." this is not quite the end of the calculation however. the goal is to calculate the probability of the original data occurring plus all more extreme probabilities. the original data have to be adjusted by increasing the "dominant" pair of cells by + and the others by − , while leaving the margin totals the same (table ) . because no zero has yet appeared in the matrix of cells, we continue to increase the "dominant" pair by + and obtain a zero. the next calculation is the last. (by convention, ! and ! = ) (table ) . no reference table is required. the total calculated probability ( . ) is exactly the probability that the null hypothesis ("that there was no relationship"), is correct: . %. by convention, for a result to be significant statistically, that probability (p) must be less than % (< . ), so in this instance we are not able to reject the null hypothesis and must conclude that the relationship could have occurred by chance alone more than % of the time. the odds ratio of . is explained as the number of times more likely it was for a shigellosis victim to have drunk well water than for a non-ill person. this increased risk would normally be impressive, but because of the small number of persons in the study, it has been found not to pass the test of statistical significance. a basic write up of the results might read: "a relationship exists between drinking well water and developing shigellosis. a shigellosis patient is six times more likely to have drunk water from the well compared to a non-ill person. this relationship is not statistically significant, however, and could have occurred by chance alone more than % of the time. the null hypothesis of no-relationship cannot be rejected." [ df, p > . , or: . , not statistically significant.] with the odds ratio (or) calculated for all the suspected exposures, and the chisquare test or fisher's exact test calculated for the strongest of these, all the results can be displayed in a composite table. earlier protocols for the investigation of waterborne and foodborne diseases encouraged the use of the "factor-specific attack rate table" (for example the "foodspecific attack rate table"), but where only a "convenience sample" of controls and cases are available, we are unable to derive valid incidence/attack rates. investigators are discouraged from using it as it may produce misleading results. the exposurerate table for cases and controls is preferred in all case-control studies, and compares the rates of exposure to each factor between both the ill and non-ill people. table displays six exposure factors from a hypothetical outbreak involving water contamination. exposure rates are calculated from both cases and controls. the "spring-water" data that we used for the odds ratio calculation example in table appears as the first exposure in table . the column headed "differences in exposure rates" subtracts the exposure rate among the non-ill from the exposure rate among the ill. [use: exp. rate (cases) minus exp. rate (controls), keeping the signs correct]. you are looking for a large positive difference to indicate the most likely culprit. the spring water shows the largest positive difference at + %. the odds ratio of . supports this, again the largest value, indicating that ill persons were times more likely to have drunk the spring water compared to non-ill persons in this group. hence both the large positive difference in exposure rates and the large odds ratio point to the spring water being the likely source of the illness, and it is certainly the strongest association between illness and any of the exposures shown. the chi-square value has also been added ( . ), as well as the associated p value. taken together, the evidence clearly points to this factor as the culprit. in those less-common circumstances in which all the ill and non-ill persons can be contacted for interview, the table can be rearranged to show attack rates (incidence rates) for each of the suspect factors (table ) . here, the column of "differences" shows the attack rate (exposed) minus the attack rate (non-exposed), (i e − i n ), and again a large positive difference will point to the culprit. this measure is called the attributable risk and for the spring water example we obtain + %, the largest value of all the risk factors. also, because of the availability of valid attack rates (incidence rates), the true relative risk (rr) is available, and can be substituted the p (probability) value is a statement of statistical significance. it indicates the probability that there is no relationship between the factor and the illness. so as the number becomes very small (as shown here) we can be increasingly satisfied that a real relationship does exist. see the statistical significance section for the correct way to calculate this ns = not significant the ("true") relative risk (also called the risk ratio) is only used when we have the true incidence data. it is calculated as the attack rate (exposed) over the attack rate (non-exposed), or i the p (probability) value is a statement of statistical significance. it indicates the probability that there is no relationship between the factor and the illness. so as the number becomes very small (as shown here) we can be increasingly satisfied that a real relationship does exist. see the statistical significance section for the correct way to calculate this ns = not significant for the odds ratio. the data in table shows the same data as table rearranged for easy comparison. values in the column of "differences" are not the same as for table and of course the relative risks are not the same as the odds ratios in table , but both of these results still point clearly to the suspect exposure. in both analyses, spring water is clearly the factor most strongly associated with illness. it is important to note that in both tables a high rate (exposure-or attack-) on the left side taken by itself is meaningless until it is compared with the rate from the right side of the table. this again underscores the importance of gathering complete data from the non-ill as well as the ill. an interesting phenomenon is visible in the second factor listed (soft drink). the or is listed as . , which is "protective," meaning that this factor is strongly associated with not being ill. it is the equivalent of or equal to . ( / . ), and the chi-square is seen as quite large, although not enough for statistical significance. this is sometimes seen where two factors are "in competition" with each other; if everyone had drunk one item, and the spring water was the contaminated source, then those drinking the other item would be strongly "protected" because they did not drink the spring water, and this shows clearly. all other factors have or values very close to . . most attack rate tables record some persons who did not ingest the suspect vehicle but who nevertheless became ill. plausible explanations are that (a) some people forget which beverages or foods they ingested; (b) some might have become ill from other causes; or (c) some may have exhibited symptoms with a psychosomatic rather than a physiological origin. it is also not unusual for the table to include some persons who ingested contaminated water or food but did not become ill. plausible explanations are that (a) organisms or toxins are not always evenly distributed in water or food and consequently some persons ingest small doses or perhaps none at all; (b) some persons eat or drink larger quantities than others; (c) some are more resistant to illness than others, and (d) some will not admit that they became ill, or fail to report it. whichever table is used, the combined totals for cases (ill) and controls (well) are fixed and should not change for each exposure unless there are "missing" responses from interviewees. while some procedure manuals include confidence limits around both rr and or, this may be omitted here as the use of the chi-square test or fishers exact test yield the statistical significance for both tables. illness caused by ingestion of waterborne toxicants and some pathogenic organisms can be dose-related in that the risk of developing symptoms, and their severity varies with the quantity ingested. where the suspect water is no longer available (for example, the well may have been quickly super-chlorinated to break the chain of infection before samples were taken), attack rates can be based on the amount of water usually drunk per day by each person. this is easily extended to other non-treated sources of water such as ice cubes, water-reconstituted fruit juices, and flavored crystals. a comparison of attack rates at various water intake levels may provide valuable evidence that water is, or is not, the vehicle responsible for the outbreak. for an example, see table . here, the entire group was people and we have interviewed them all, so we are justified in calculating the attack/incidence rates: in this example, the attack rate increased as the consumption of water increased, which suggests that the illness was directly related to water and the agent it contained. this is a trend established from the group as a whole, and an individual's experience may vary with factors such as (a) preferences of water ingestion, (b) intermittent contamination, (c) unequal distribution of the contaminant, or (d) varying susceptibility of individuals. these data can be compared with rates from persons who ingested no water, but only hot tea, hot coffee, soups, and/or other safe sources of liquids. if unheated water was indeed the vehicle, and the agent was a living biological agent, these persons should have attack rates showing no increase in risk of illness. (outbreaks from a toxic agent may be unaffected by chlorination, boiling, and some types of filtering.) the data can be displayed in a contingency table as follows for analysis using chi-square procedure (table ) . for this example, chi-square equals . and if calculated by computer or online, p will be shown as p = . . reference to form j confirms that for a × table ( df), the calculated chi-square ( . ) exceeds the critical value for statistical significance at the . level ( . ), allowing us to claim statistical significance at p < . . odds ratios are normally associated only with × tables, but here, the or can usefully be calculated on selective cells or groups of cells as long as you clearly explain the selection process. for instance, persons who were ill were . times more likely to have drunk three or more glasses of water per day compared to those who were well. for this calculation we collapse cells into a × table and crossmultiply: (a + c) × (f + h)/(b + d) × (e + g) = ( ) × ( )/( ) × ( ) = / = . . alternatively, because we have all people involved, we can compare the attack rates (ar) for each intake level, and observe the increasing attack rate as the intake increases: for five or more glasses/day, ar: %, for - /day, ar: %, for - / day, ar: %, and for < /day, ar: %. we might summarize as follows: "there was a relationship observed between the quantity of water consumed each day and the risk of illness. the incidence rate increased with the quantity consumed from % for < glasses/day to % for five or more glasses/day. this relationship is statistically significant. the null hypothesis of no association can be rejected." , df, p < . ] water as a vehicle can deliver pathogenic organisms in many ways beyond simply drinking a glass of water, or using a drinking fountain. investigators should be sure to ask about the preparation of ice-cubes, the mixing of fruit flavored crystal drinks, reconstituting concentrated orange juice, brushing and rinsing teeth, and washing hands, utensils, or containers. swimming or playing in muddy pools or even swimming pools have caused waterborne poliomyelitis, and naegleriasis, while swimming in saltwater inlets have allowed inadvertent infections from vibrio parahaemolyticus and v. vulnificus. unwashed plastic jugs containing poster paint residue have caused rapid illness when drink crystals are reconstituted in them, while refillable plastic containers and bottles have a long history of contamination from biological and chemical agents. in the late s, an increase of viral ear, nose, and throat infections among people who were using parkland next to a river was hypothesized to have been due to people waterskiing on the river and creating an aerosol. the river was the receiving body for effluent from a water treatment plant upstream. record all laboratory results on form i, laboratory results summary. compare epidemiological and statistical results with on-site observations, laboratory results and the information summarized on form i. the agent responsible for the outbreak can be determined by (a) isolating and identifying pathogenic microorganisms from patients, (b) identifying the same strain and/or pfge pattern or genetic sequence of pathogen in specimens from several patients, (c) finding toxic substances or substances indicative of pathological responses in specimens, or (d) demonstrating increased antibody titer in sera from patients whose clinical features are consistent with those known to be produced by the agent. when implicating the water as a likely (or presumptive) vehicle of transmission, ideally identification of a pathogen in samples of suspect water will correspond to the one found in clinical specimens from ill persons or that produces an illness that is compatible with the incubation period and clinical features of the ill who were exposed to the water. for organisms that are common in the gastrointestinal tract or that have multiple strains, compare strains isolated from ill persons with strains isolated from the suspected water. additionally, specific microbial markers (e.g., serotype, phage type, immunoblotting, plasmid analysis, antibiotic resistance patterns, restriction endonuclease analysis, nucleotide sequence analysis) or chemical markers identified by chromatography or spectrophotometry can be used for this purpose. for confirmation of water-related transmission, the same pathogen strains should be found in both the ill persons and the epidemiologically implicated water. however, due to the period of time that may have passed after the outbreak was actually reported, and to methodological issues, such as the need for concentrating pathogens in water samples, it is often unlikely that the outbreak-associated pathogen will be found in the water samples. laboratories frequently test water samples for indicator organisms, such as fecal coliforms, escherichia coli, or enterococci, rather than pathogens. the finding of these bacteria in high densities in the water may indicate contamination (from a fecal source) and implicate the water was a possible vehicle. however, the finding of increased indicators in water samples alone is insufficient evidence to confirm the water as the source of an outbreak. the probable source of contamination or the situation that allowed contamination to reach and survive in a water supply (e.g., water supply not disinfected or inadequately disinfected, inadequately filtered, or upstream to sewage or agricultural discharges; cross connection between sewerage and drinking water pipes; well improperly constructed; nearby septic tank system; or livestock in water supply) can often be identified, but the etiologic agent in the water may never be found. success in finding the etiologic agent is most likely where (a) the incubation period of the illness is short, (b) the agent is stable in water and the system is static, or (c) large amounts of the agent are being continually added to the water supply. try to recover and identify the specific agent whenever a water supply is suspected to be the vehicle of transmission, even if finding the etiologic agent is likely to be difficult and not considered practical for routine monitoring of water supplies. if water samples do not reveal a likely causal agent, clinical data as well as time, place, and person associations can cast strong suspicion on a water supply, particularly if indicator organisms are found in the water. tests other than those for pathogens, however, are frequently used to evaluate water supplies on a routine basis. organoleptic tests attempt to evaluate the total effect of all compounds present in water that can be measured by the senses of taste, smell, or sight. results cannot be expressed in terms of specific compounds present, and the measured qualities are usually a result of a mixture of compounds. these tests are often empirical and arbitrary, but changes in the physical qualities of water (such as ph, turbidity, color, odor, or taste) can indicate abnormalities of the water. outbreaks have occurred, however, when turbidity readings have met present standards and when water appeared and tasted good. chemical examination of water is useful for (a) detecting pollution (especially from industrial wastes and pesticides), (b) determining effectiveness of treatment processes, (c) evaluating the previous history of the water, (d) determining hardness, and (e) detecting the presence of specific toxins. results are usually expressed in milligrams per liter (mg/l = ppm, parts per million), or micrograms per liter (μg/l = ppb, parts per billion). historically, acute water-related outbreaks seldom involve chemical substances, so chemical tests are not requested routinely unless either (a) circumstances indicate possible chemical contamination or (b) clinical symptoms suggest chemical poisoning. flowing water in a distribution system can be monitored to determine chlorine residual. free available residual chlorine refers to that portion of the total residual chlorine remaining in chlorinated water at the end of a specific contact period that will react chemically and biologically as hypochlorous acid or hypochlorite ion. the reaction is influenced by ph and temperature. total or combined residual chlorine refers to chlorine that has reacted with ammonia or other substances and is not available for further reactions, as well as the free available chlorine. a chlorine demand exists in a chlorinated water until a free available residual is produced. a free available chlorine residual, e.g., mg/l ( ppm) or higher, maintained throughout the distribution system of a community supply is an indicator of safety from enteric bacteria but not necessarily from pseudomonads, viruses or parasites. outbreaks have occurred when chlorine residue levels have met present standards. analyses for microbial indicator organisms provide information on the microbiological quality of water and guidance as to its safety for consumption or contact. indicator organisms are easier to test for than pathogenic organisms, and some serve as a surrogate measure of fecal contamination in water. the absence of indicator organisms in the water, however, does not guarantee water safety; numerous outbreaks of water-related disease have occurred from water in which no indicator organisms were detected. evaluation of the safety of water should be based upon a combination of results of (a) an on-site study to identify sources and modes of contamination and means by which contaminants survived treatment and (b) appropriate laboratory analyses. microbiological results should be compatible with observed sources of contamination and/or treatment failures found during the investigation. although all natural waters contain bacteria, the number and kind vary greatly in different places and under different climatic and environmental conditions. the number of bacteria isolated and reported, however, often represents only a fraction of the total number present, for several reasons. colonies seen on agar plates develop from either single organisms or clusters or chains of organisms. heterotrophic bacteria represent only those that can use organic matter and grow at the selected temperature ( - °c) within - hours under aerobic/microaerophilic conditions in/on a defined medium when the standard test (spread plate, membrane filter or pour plate) is used. the hpc may also be done using different media under different incubation times/conditions. (higher counts are usually found when the longer incubation periods are used.) also, certain microorganisms are unable to grow aerobically either in or on the medium used. because of these variables, the terms total plate count (tpc), standard plate count (spc) and aerobic plate counts (apc) should not be used. hpcs serve as an index of changing sanitary conditions. in general, counts of good-quality well water are fewer than - colonies per ml. densities in surface water are higher, but quite variable, depending on water temperature, sources of pollution, amount of organic matter present, and soil that washes into the water. the sources of pathogens, toxic substances, or fecal contamination may not increase the hpc of a surface water sample as much as washings from soil. nevertheless, marked changes in the number or kind of microorganisms should be viewed with concern, at least until the reason for the change is discovered. heterotrophic plate counts greater than /ml and some specific antagonistic species may interfere with the growth or recovery of pathogenic or indicator organisms. some heterotrophic species are opportunistic pathogens that may pose a health threat to immunocompromised persons. the coliform group of bacteria comprises those from non-fecal environmental sources, and those from animal and human intestines, including escherichia coli. the environmental species of non-fecal bacteria are found in soil, on fruits, leaves, and grains, and in run-off water, especially after heavy rains. some of these species are capable of surviving in water longer than e. coli. furthermore, some coliform strains and can multiply on decaying vegetation in water, in biofilms in pipelines, or on pump packings, washers, and similar materials. therefore, finding coliforms may not be indicative of fecal contamination, although most water utilities have standards for coliforms in water. fecal coliforms are present in large densities in all human and animal feces, normally much higher than pathogens which are typically only present in infected persons and normally at lower levels. as such, high populations of fecal coliforms can indicate recent sewage pollution of water, but are not always indicative of pathogens present, particularly viruses and parasites. none of the coliform group, however persists as long as most viral or protozoan pathogens in water, and indicator bacteria described below (fecal streptococci and clostridium perfringens). typical chlorination or ozonation of water inactivates coliform bacteria. presence of the coliform group or even a high population of coliform bacteria is not proof that a treated water supply contains pathogens. however, coliforms can provide a warning that either the water treatment was inadequate or contamination occurred after treatment, and that some pathogens may be present. as mentioned above, under some conditions, pathogens may be present where there are few or no coliforms. furthermore, unlike coliforms, many parasites and viruses are resistant to normal levels of disinfectants. coliforms have little or no correlation with the presence of parasitic protozoa or pathogenic viruses. the standard test for the coliform group may be carried out by a membrane filtration technique, a multiple-tube fermentation technique (presumptive test, confirmed test, or completed test), or a presence-absence test. results of the membrane filtration technique are reported as colony forming units (cfu) per ml of water. results of the multiple-tube fermentation technique are reported as the most probable number (mpn) per ml of water. this is a statistical estimation of the total number present, but the actual number can fall within a considerable range. counts derived from these two methods are not necessarily the same, but they have the same sanitary significance. false-negative or false-positive results can also occur with the membrane filtration technique because of interfering background growth of nonfecal microorganisms. results of the presumptive test of the multiple-tube fermentation technique can be misleading, because other microorganisms frequently found in water also produce gas in laboratory media, and may thereby give false-positive results. also, especially in waters containing a large number of microorganisms, some coliforms present may produce gas slowly, leading to false-negative results. the presence of coliform bacteria is corroborated by means of the second phase of the multiple-tube fermentation technique, known as the confirmed test. positive results are usually considered confirmation of the presence of coliforms. a third phase of this test, known as a completed test, further ensures the correct identification of coliform bacteria. a simple modification of the coliform test is to analyze for the presence or absence of coliforms in a -ml drinking water sample. the "presence-absence (p/a) coliform test" allows for simple examination of a larger number of samples. when a positive sample is detected, it is advisable to measure coliform densities in repeat samples by one of the other methods to determine the magnitude of the contamination. thermotolerant coliform (fecal coliform). coliform bacteria will frequently grow at a relatively high temperature, . °c, unlike species or strains normally encountered in the environment, which usually have an optimal temperature near °c. this thermotolerant characteristic has been used in an attempt to separate coliform bacteria into those of so-called fecal and non-fecal origin. this test may provide better indication of fecal contamination than the coliform test, but it is however, unreliable. positive results are not proof that either organisms of fecal origin or pathogens are present. the number of thermotolerant coliforms is considerably lower than the number of total coliforms in contaminated water; therefore, the test is less sensitive for testing treated drinking water. furthermore, escherichia coli o :h , which has been implicated as causing water-related illness, does not grow well at . °c. escherichia coli. e. coli is common in feces of human beings, other mammals, and birds. it can also be found to grow naturally in the environment, specifically in tropical waters. comprised of the larger coliform group, its detection in water is a more definitive indicator of fecal contamination, compared to total or fecal coliforms. however, a positive test result does not identify if the fecal source is human or nonhuman. rather, the finding of e. coli in water serves as an indicator that fecal matter reached the water and provides a warning, but not proof, that pathogenic organisms may also be present. it should be noted that some strains of e. coli are pathogenic (see table b ). simple commercial p/a and quantitative tests have been developed to detect the presence of total coliforms and e. coli in hours by observing color changes and fluorescence of the media under daylight and uv light. such tests may be useful for field evaluation of microbiological water quality. another group of organisms, collectively known as fecal streptococci, is also used as an indicator of fecal contamination. enterococci (enterococcus faecalis, enterococcus faecium) are particularly used for testing recreational waters. like coliforms, enterococci are normal inhabitants of the intestinal tract of human beings and other animals. in human feces, they occur in considerably lower numbers than e. coli. some members of the group, such as e. faecalis, subsp. liquefaciens, however, have been associated with vegetation, insects, and certain types of soils. enterococci generally survive longer than coliforms in fresh water, and therefore the source of contamination may be distant in either time or place from the site where samples were obtained. their resistance is, however, less than that of clostridium perfringens, enteric viruses, and parasites. like e. coli, simple commercial p/a and quantitative tests have been developed to detect the presence of enterococci in hours by observing color changes under uv light, which may be useful for field evaluation of microbiological water quality. clostridium perfringens (sulfite reducing clostridia). c. perfringens is also of fecal origin, but it occurs in feces in much lower densities than e. coli and can also be found in soils. being a spore-former, it can survive for long durations in soil and water, and persist when all other bacteria of fecal origin have disappeared. therefore, it is a useful indicator of remote or intermittent contamination in wells that are not frequently examined by the coliform test; but, it is not, by itself, evidence of recent contamination. chlorine, in the concentration typically used in water treatment, does not inactivate all spores; and thus c. perfringens is not valuable in assessing the efficiency of chlorination for bacterial vegetative cells. its long persistence and its resistance to chlorine make this organism a potential indicator for viral and parasitic organisms that have similar resistance and disinfectant susceptibility. coliphage. coliphages, which are viruses that infect e. coli, are simpler to detect and enumerate, compared to other viruses, and are generally associated with fecal contamination. they have been considered as possible indicators of treatment effectiveness for human enteric viruses. coliphages are categorized into two groups: the somatic phages, which enter e. coli via the cell wall and the male-specific phages, which enter e. coli through the sex pili. the somatic and male-specific phages are common in sewage and the feces of human beings and other animals, but in lower densities than the common fecal indicator bacteria, fecal coliforms, e. coli, and enterococci. some strains appear to be more resistant to chemical disinfection than water-related pathogens or indicator bacteria. be aware of local standards for water distribution systems, private water systems, and recreational water. although drinking water standards, such as the total number of coliforms allowed in a water sample, vary from jurisdiction-to-jurisdiction, it is generally agreed that any fecal contamination (e.g. fecal coliforms, escherichia coli) render the water unacceptable for human consumption and may close down recreational bathing waters. there are numerous pathogens that can be transmitted by water, many of which are also able to cause respiratory symptoms, in addition to the classical gastrointestinal symptoms. for a comprehensive summary of waterborne pathogens see "american waterworks association manual of water supply practices, m waterborne pathogens, nd edition ( ) ." for several reasons, analyses for pathogens are not usually conducted during routine water testing, or are only conducted by specialized laboratories. first, tests for pathogens are pathogen-specific, expensive, and often difficult to perform because they may require specialized trained personnel. secondly, the etiologic agent of the outbreak is often unknown at the time of analysis; hence, many analyses would have to be done blindly. thirdly, pathogens are not always recovered because they are heterogeneously dispersed and diluted in the environment, and their numbers decline in water over time. as a result, they may be absent or present in low densities by the time samples are collected following an outbreak. fourthly, recovery efficiencies are often poor because microorganisms are stressed by disinfectants or the method is sensitive to interferences from the source waters environment, hence not easily recovered by routine methods. additionally, recovery efficiencies for viruses and protozoa may be poor because of the interferences of substances within the sample matrix with method reagents (concentrating l of water down to μl will also concentrate inhibitory chemicals and substances). finally, the time required for isolation and identification is often long, and the number of samples is usually too small to allow the investigator to have much statistical confidence in the results when pathogens are not found. negative results should be reported as "not detected" because they do not ensure that the water sampled was not the source of the pathogen. procedures used for many bacterial pathogens are qualitative because enrichment procedures are used. quantitative procedures (e.g., mpn) require considerable work and are less reliable than those used for coliforms because small populations may be present, and these may be unevenly distributed. despite these difficulties, pathogens that cause a syndrome similar to the one being investigated should be sought. see tables b, c, and d, for descriptions of the disease syndrome associated with the pathogens described in the following material. finding the same pathogen in specimens from patients and in water samples confirms water as a vehicle. summarize investigative data in a narrative report. describe in this report situations that led to contamination of the water and survival of etiologic agents up to the time of consumption. include all events that contributed to the outbreak to guide control and preventive measures. compare your data with the listings in table g (guidelines for confirmation of waterborne outbreaks) and table h (guidelines for confirmation of water responsible for illness), and criteria for confirmation of vehicle responsible for waterborne illness before assigning the etiologic agent and the vehicle. outbreak confirmation is based on (a) time, place, person associations, (b) recovery of etiologic agents from clinical specimens from cases and samples of water, and (c) identification of sources and modes of contamination and means by which pathogens or toxic substances survived treatment. all three of these, however, might not be found in any one investigation. complete form k (waterborne illness summary report). attach the narrative and the epidemic curve. also attach form d (case history summaries: water/laboratory data), all applicable parts of forms g, forms h, form i, and other data that will provide supplemental information to reviewers. send this report through administrative channels to the appropriate agency responsible for waterborne disease surveillance. make the final report as complete as possible, so that the agency can accurately interpret the results and develop a meaningful waterborne disease data bank. in the interest of continuing cooperation, give all participants in the investigation due credit and send each a copy of the report. also, send copies of the report through administrative channels to agencies (a) that have jurisdiction over the implicated water, (b) that initiated the alert, and (c) that participated in the investigation. those concerned with water sources, treatment and recreation, as well as with public health, should make every effort to ensure the complete investigation and reporting of waterborne diseases. without reliable, complete information, trends in waterborne disease incidence and causal factors of the disease are difficult to determine. good surveillance is essential for detecting and evaluating new waterborne disease hazards. the primary purposes of a waterborne disease investigation are to identify the cause, establish control measures, and take actions to prevent future illness. prudence may require some action before all the hypotheses regarding the water supply involved and the source of contamination are confirmed. frequently the local health authority will issue a boil water advisory if a microorganism is suspected to have contaminated the water. refer to "possible precautionary control actions" section for a discussion of these precautionary control measures. if these measures have not already been considered, consider them now. once control measures have been implemented, continue to monitor for disease to evaluate whether the measures were effective. in a waterborne event in sydney, australia (see box ) sydney water severely overestimated levels of cryptosporidium and giardia present in the water raising public alarm. boil water advisories were announced and rescinded several times. however, it is better to announce boil-water advisories than to have thousands ill, as has happened in the past, such as the cryptosporidium outbreak in milwaukee in . deficiencies in treatment must be corrected and defective parts of distribution systems must be repaired, beginning with those that either contributed to or had a high potential for contributing to the outbreak. the effectiveness of these efforts will be directly related to the thoroughness of the investigation. document the source and the manner of contamination and survival of the etiologic agent through the water treatment process. provide clear documentation of contributory factors, so that preventive measures taken will be specific to the problem. if previous sanitary surveys have revealed, or if subsequent ones reveal, that conditions which contributed to the outbreak are widespread, initiate a training and education program. these programs can be developed for water treatment plant or recreational water operators and employees, engineers, homeowners, or other appropriate groups. impress upon them the importance of proper construction and operation of facilities and proper protection, treatment, storage, and distribution of water. follow up with periodic inspections and surveys and verify by sampling, as appropriate, to determine whether faulty conditions have been corrected or allowed to be reintroduced. legal action may be necessary to ensure compliance with official standards and accepted sanitary practices. formulate solutions to problems found during outbreak investigations, and incorporate these into regulations for drinking, agricultural, industrial, domestic, and recreational waters. inform the public, through mass media and other means available to your agency, of hazardous conditions that can affect their water supply, but do so only after hypotheses are confirmed. the public must be told of any potential or actual harm that may result from ingesting or contacting contaminated water and must also be informed of measures that they can take and that official agencies are taking to correct these conditions. the water supply and recreational water facilities must be verified periodically to determine whether critical processes are being monitored and operated within limits of appropriate public health standards (see box , the walkerton outbreak). most waterborne illnesses are preventable, but prevention requires that those in the water treatment industry and in health and water-protection regulatory agencies be constantly vigilant to ensure that the hazards are understood and that questionable water treatment or delivery system construction or practices are avoided. examples investigation guidelines and investigative forms iafp manual, "procedures to investigate waterborne illness, rd ed"; copies of form c; one dozen copies each of forms e and f; two copies of form d and all parts of form g, epi-info software (cdc, atlanta). water sample bottles (bottles for chlorinated water should contain enough sodium thiosulfate to provide a concentration of mg of this compound per l of sample), plastic bags (whirl-pak ® type), ml, -l and -gal sized jars and jugs. sterile and wrapped sampling implements moore swabs (compact pads of gauze made from strips cm [ ft] by cm [ in.] tied in the center with a long, stout twine or wire-for sewer drain, stream or pipeline samples), fiberglass-epoxy bacterial filter cartridge, . μm; tongs, scoop or similar utensils for collecting ice. specimencollecting equipment (for human specimens from cases and controls) sterile containers (with lids) for stool specimens, bottles containing a bacterial preservative and transport medium, mailer tubes or styrofoam box, sterile swabs, rectal swab units, tubes of bacterial transport medium, stool preservative medium for parasites, phlebotomy supplies for blood specimens. kits for testing chemical disinfectants and ph dpd (n,n-diethyl-p-phenylenediamine) chlorine comparator with color disc for chlorine ( . ppm) and chlorine test papers; field-type ph meter or ph comparator with color disc or ph test papers; applicable ph indicator solutions and dpd reagent solution; dissolved oxygen testing unit. dye tracing study equipment fluorescein (yellow-green fluorescent) dye in powder form ( packages containing g each), in tablet form ( tablets), or in liquid form (prepared by mixing g in l of water); fluorometer; filters (primary and secondary) for use with fluorometer; sample holder for continuous sampling or individual sampling; fluorometer recorder. disinfectant and neutralizer . % w/v solution of calcium hypochlorite or . % household liquid bleach; % w/v sodium thiosulfate. virus filtration equipment for viruses and parasites b large plastic container for storing water sample prior to concentration; portable electric or gasoline powered water pump with quick disconnect brass or stainless steel plumbing adapters or hose couplings; two filter holders for -in. water filter cartridges fitted for adapters or couplings; portable water meter fitted for adapters or couplings; four lengths of fiber-reinforced garden hose fitted with adapters or couplings; one length of a strong-walled supply hose fitted with adapters or couplings; -in. prefilter ( μm nominal porosity wound polypropylene yarn filter with hollow perforated stainless steel core) cartridge filter; -in. virus absorbing filter pleated . μm porosity nylon membrane type (positively charged) for waters of ph values up to . , or pleated . μm porosity glass fiber membrane type (positively charged) for waters of ph value of . or lower (e.g., virosorb, -mds, amf/cuno meriden, pleated, . μm, glass filter); ml sterile, ph , % beef extract solution in gal wide-mouth screw capped autoclavable polypropylene container for each sample to be collected; stands to support filter holders during filtration; for parasites -in. polypropylene yarn-wound cartridge filter, . μm porosity (e.g., micro wynd ii™, amf/cuno; meriden, ct. . μm normal porosity). (continued) laptop or tablet, with software; thermocouples of varying lengths with either recording potentiometer, data logger, or digital indicator; devices to take samples below surface and sediment samples; chemical smoke kit and/ or micromanometer; occupational safety and health administration (osha) or equivalent approved respirator; sterile plastic gloves; plastic container liners for ice; waterproof marking pens; waterproof test tube rack; pencils, note pad; roll of adhesive or masking tape; labels; waterproof cardboard tags with eyelets and wire ties; flashlight; matches; test tube rack to fit tubes used; insulated chest or styrofoam container; packing material; camera with flash; spare batteries for all equipment; % ethyl alcohol; propane torch; refrigerant in plastic bags, liquid in cans, rubber or heavy plastic bags that can be filled with water and frozen; heavy-duty bags for ice, "canned ice," or cold-packs (blue ice). a assemble a kit to be kept in the agency responsible for investigating waterborne illness. it should include at least ten water sample bottles; ten -l, or gal jars or jugs; ten specimen collection containers or devices; and one each of the following supporting equipment and sterilizing equipment. date of sterilization should be marked. periodic resterilization or replacement of sterile supplies, media, or transport media is required to maintain the kit in a ready-to-use condition b similar equipment for sampling for either viruses or parasites may be available from national water, environmental, or health agencies how much to collect two rectal swabs or swabs of fresh stool from ten ill persons; samples from ten controls also can be submitted. whole stool is preferred if nonbacterial stool testing considered a fresh stool sample from ten ill persons; samples from ten controls can also be submitted. to enhanced detection, three stool specimens per patient can be collected > h apart as much stool sample as possible from ten ill persons (a minimum of ml of stool sample from each); samples also can be obtained from ten controls. a fresh urine sample ( ml) from ten ill persons; samples from ten controls also can be submitted. label each specimen in a waterproof manner and put the samples in sealed, waterproof containers (i.e., plastic bags). batch the collection and send in overnight mail to arrive at the testing laboratory on a weekday during business hours unless other arrangements have been made in advance with the testing laboratory. contact the testing laboratory before shipping, and give the testing laboratory as much advance notice as possible so that testing can begin as soon as samples arrive. when etiology is unclear and syndrome is nonspecific, all four types of specimens may be appropriate to collect c for more detailed instructions on how to collect specimens for specific parasites, please go to http://www.cdc.gov and search the website of key words d for more detailed instructions on how to collect specimens for viral testing, please go to http://www.cdc.gov and search the website of key words e the containers have been tested for the presence of the chemical of interest prior to use f unused specimen collection containers that have been brought in to the field and subjected to the same field conditions as the used containers. these containers are then tested for trace amounts of the chemical of interest table e (continued) isolation of agent from ill persons and from water and laboratory criteria for confirming etiologic agent as stated in table g . combination of on-site investigation, statistical evidence and laboratory analysis. (see entries below) presumptive vehicle on-site investigation demonstrating source and mode of contamination of water and survival of etiologic agent in water. also, desirable to have laboratory isolations from water of etiologic agent that causes syndrome similar to that observed during the investigation and other supportive epidemiologic data. if so, this might provide sufficient evidence for confirmation. or p-value for water < . when other epidemiologic data supports water hypothesis. also, desirable to have either laboratory isolations from water or on-site investigation that demonstrates source and mode of contamination and survival of treatment that supports the hypothesis. if so, this might provide sufficient evidence for confirmation. or odds ratio or relative risk for water greater than and the lower limit of the % confidence level greater than when other epidemiologic data supports the water vehicle hypothesis. also, desirable to have either laboratory isolations from water or on-site investigation that demonstrates source and mode of contamination and survival of treatment that supports the hypothesis. if so, this might provide sufficient evidence for confirmation. for calculation of data when all persons who may have been exposed to the suspect vehicle (but not all persons will have ingested every beverage/food) have been identified and interviewed (retrospective cohort) form j : chi-sq. analysis can be easily completed using on-line calculators or statistics programs such as epi-info. however, to confirm the result or to do the whole thing yourself, here are the steps: ill well totals step : create a x table as shown with observed data (o values) and marginal totals step : calculate odds ratio: (axd)/(bxc) = ( x )/( x )= . step . . chi-sq. (all four cells) = . + . + . + . = . step : compare your calculated chi-sq. value with the critical value to determine significance (table ) : begin at column p < . … your calculated chi-square value must meet or exceed the critical value to be considered statistically significant at that level. if you fail to meet or exceed the minimal value for p < . , the result is p > . , and the relationship is declared "not significant". step : summarize: exposure was related to illness. ill persons were eight times more likely to have been exposed to this factor than non-ill persons. this relationship is statistically significant. the probability of these data occurring by chance alone is less than . %. reject the null hypothesis of "no association." [odds ratio: . , % cl: . < or < . , chi-sq.: . , df, p < . ] form j : fisher's exact test can be easily completed using on-line calculators or statistics programs such as epi-info. however, to confirm the result or to do the whole thing yourself, here are the steps. ill well totals step step : calculate the probability directly... here, cell 'a' has smallest e value at . p = ! x ! x ! x ! = . ! x ! x ! x ! x ! step : the final probability (p) is the sum of all probabilities (in this case p + p + p ) or approximately . . step : summarize: exposure was related to illness. ill people were almost times more likely to have been exposed to this factor compared to non-ill people. the relationship is statistically significant. the probability of these data occurring by chance alone is less than . % (< . ). reject null hypothesis of "no association". [odds ratio: . , % cl: . < or < . , p = . ]. . when deciding which cells to increase by + , always multiply (a) × (d) and compare with (b) × (c). increase each cell of the pair with the higher product and decrease each cell of the pair with the smaller product, while keeping all marginal totals unchanged. . the final p is an "exact" p (probability) and may be reported as such (p = . ). in this example, it is also < . of course, and can be reported in this way if preferred. . the fisher's test is used when the chi-square test is invalid due to any "e" values < in a × table. in all other circumstances, chi-sq. is an excellent approximation for the fe test. . if original data include a zero in one of the cells, you will calculate only one p value. (the o.r. will be reported as "undefinable" but the direction of the effect will be very clear). . this p is calculated for a one-tailed fe test. it is adequate for this application. two-tailed fe test will require further calculation. . should a relationship not meet the critical value for significance (that is, p > . ), it is described as "not statistically significant". note that a relationship may be observed, but this result is telling you that it could have occurred by chance alone more than % of time if you were to repeat the analysis. that may still require further investigation, but from a statistical standpoint, it cannot be claimed as a statistically significant relationship. water treatment plant design, fifth edition american waterworks association. manual of water supply practices, m waterborne pathogens, second edition the flint water crisis confirms that u.s. drinking water needs improved risk management produce safety-what's going on here? national environmental health association neha-cert ep global health -division of parasitic diseases and malaria page last reviewed surveillance reports for recreational water-associated disease & outbreaks. page last reviewed on internalization and dissemination of human norovirus and animal caliciviruses in hydroponically grown romaine lettuce environmental protection agency technical guidance manual lt eswtr disinfection profiling and benchmarking environmental protection agency guidance manual for the compliance with filtration and disinfection requirements public water systems using surface water sources environmental protection agency. manual of individual and non-public water supply systems. epa no. epa no. water treatment manual: disinfection. environmental protection agency health canada risk analysis of the walkerton drinking water crisis algal bloom-associated disease outbreaks among users of freshwater lakes-us microbial resistance to disinfectants: mechanisms and significance. environ health perspect safe drinking water: lessons from recent outbreaks in affluent nations risk management for assuring safe drinking water ensuring safe drinking water: learning from frontline experience with contamination. american water works association cyanotoxin management and human health risk mitigation in recreational waters a massive outbreak in milwaukee of cryptosporidium infection transmitted through the public water supply private water supplies: technical manual good practice guide to the operation of drinking water supply systems for the management of microbial risk: research project two cases of keratosis follicularis squamosa (dohi) caused by swimsuit friction part two report of the walkerton inquiry: a strategy for safe drinking water public health inspector's guide to the principles and practices of environmental microbiology acute otitis externa: an update collect representative distribution system samples guidance on water supply and sanitation in extreme weather events. who regional office for europe effect of irrigation method on transmission to and persistence of escherichia coli o :h on lettuce transmission of escherichia coli o :h from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization acknowledgments the committee and association thank and cite the following persons for chloramines refer to all forms of chloramine. the ct values may be assumed to achieve greater than . % inactivation of viruses only if chlorine is added and mixed in the water before addition of ammonia. if this condition is not met, the system must demonstrate by on-site studies or other information that it is achieving at least this much inactivation of viruses key: cord- -nil vv h authors: alfano, niccolo; dayaram, anisha; axtner, jan; tsangaras, kyriakos; kampmann, marie-louise; mohamed, azlan; wong, seth t.; gilbert, m. thomas p.; wilting, andreas; greenwood, alex d. title: non-invasive surveys of mammalian viruses using environmental dna date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: nil vv h environmental dna (edna) and its subdiscipline, invertebrate-derived dna (idna) have been used to survey biodiversity non-invasively [ , ]. water is ubiquitous in most ecosystems, and, among invertebrates, terrestrial haematophagous leeches are abundant and can be easily collected in many tropical rainforests [ , ]. such non-invasive nucleic acid sources can mitigate difficulties of obtaining wildlife samples, particularly in remote areas or for rare species. recently, edna/idna sources have been applied to monitoring specific wildlife pathogens [ , ]. however, previous studies have focused on known pathogens, whereas most wildlife pathogens are uncharacterized and unknown. non-invasive approaches to monitoring known and novel pathogens may be of particular benefit in ecosystems prone to viral emergence, many of which occur in areas where invasive sampling is challenging, such as tropical rainforests. here, we show that both edna from natural waterholes, and idna from terrestrial haematophagous leeches, can be used to detect unknown viruses circulating in mammalian hosts (figure ). using a curated set of rna oligonucleotides based on the virochip microarray assay [ ] as baits in a hybridization capture system, multiple mammalian rna and dna viruses were detected from both edna and idna samples. congruence was found between host dna assignment and viruses identified in leeches, and between animals observed visiting the waterholes and the viruses detected. our results demonstrate that edna/idna samples may represent an effective non-invasive resource for studying wildlife viral diversity. several of the detected viruses were novel, highlighting the potential of edna/idna for epidemiological analysis of emerging viruses prior to their emergence. highlights environmental dna (water and blood-sucking leeches) provided a non-invasive method of screening wildlife for viruses a comprehensive viral rna oligonucleotide bait set was developed to capture known and unknown mammalian virus diversity leech blood meal host determination and viruses identified were congruent viruses determined from water correlated with known and observed species visiting the water sources in brief alfano, dayaram, et al. demonstrate that environmental dna from southeast asian leech bloodmeals and waterholes from africa and mongolia can be used as to detect viruses circulating in wildlife. these nucleic acid sources may represent an effective non-invasive resource for studying wildlife viral diversity and emerging viruses pre-emergence. in order to test the sensitivity of the viral capture in recovering vertebrate host viruses, the capture system was first applied to a positive control consisting of medical leeches fed with human blood spiked with two rna viruses and two dna viruses at different concentrations [ ] . all four viruses were detected, even if enrichment efficiency (proportion of on-target viral reads) and target genome recovery varied among viruses (suppl. fig. ) . no viral contigs were identified in the negative controls included to monitor laboratory contaminations for either the leech or water experiments. tiger leeches (haemadipsa picta) and brown leeches (haemadipsa zeylanica) were collected in malaysian borneo and processed as pools (bulk samples) consisting of to individual leeches separated by leech species and sampling location. viruses were identified in of the leech pools analysed ( %) ( fig. ; suppl. tab. ). in of these ( %), two to three viruses were identified. sequence data from six vertebrate-infecting viral families were detected, including the anelloviridae, circoviridae, coronaviridae, parvoviridae, retroviridae and rhabdoviridae. the most common viral group detected was rhabdoviridae which was found in % of samples ( samples out of ), followed by coronaviridae which was identified in % of samples ( samples) . members of the anelloviridae were identified in % of samples ( samples) , retroviridae in three samples ( %), and parvoviridae and circoviridae in two samples ( %) (fig. ; suppl. tab. ). rhabdoviridae contigs were genetically similar to three different viral genera (suppl. tab. ). five contigs were most similar ( - %) to the vesicular stomatitis indiana virus (vsiv) (genus vesiculovirus) as determined by blast searches. the limited similarity of the contigs to known rhabdoviruses suggest they may represent a new genus related to fish rhabodviruses (perhabdovirus and sprivirus) or vesiculovirus (suppl. fig. ). the other contigs clustered phylogenetically, suggesting they represent two new species of a rhabdovirus related to lyssaviruses (suppl. fig. ). although in most cases one contig per sample was observed, in five samples (l , l , l , l , l ) two different viruses were found. several viral regions for rhabdoviridae were represented in the baits. however, most of the oligonucleotides were specific for the l gene which encodes the rna-dependent rna polymerase. all the recovered contigs mapped to the l gene (suppl. fig. a -c). the viral contig sequences were confirmed by pcr and sanger sequencing for l and l (suppl. fig. d ). all coronaviridae contigs matched a bat betacoronavirus as determined by blast searches with identities between - % (suppl. tab. ). the resulting sequence did not cluster in any of the four clades representing the known coronaviridae genera, suggesting it may represent a novel coronavirus genus (suppl. fig. ). each contig overlapped with the coronavirus rna-dependent rna polymerase gene (orf ab), the viral region mainly targeted by the rna oligonucleotide baits (suppl. anelloviridae contigs matched either porcine torque teno virus (pttv) ( - % identity), a giant panda anellovirus (gpav) ( - % identity) or a masked palm civet torque teno virus (pl-ttv) ( - % identity) (suppl. tab. ). the pttv contigs were found in two samples (l and l ), while the gpav and pl-ttv contigs were detected in six samples. gpav was the best match in four samples (l , l , l , l ) and pl-ttv in three (l , l , l ). in sample l both were identified. every anelloviridae contig mapped to the non-coding region of the relative reference genome since all anelloviridae baits targeted the same untranslated region (suppl. fig. a , c, e). the non-coding region sequenced is not phylogenetically informative and therefore, phylogenetic analysis could not be performed. viral contigs were confirmed by pcr and sanger sequencing for samples l , l , l and l (suppl. fig. b , d, f). three circoviridae contigs matching a porcine circovirus (pcv) ( % identity) were identified in l and l ( fig. ; suppl. tab. ). two non-overlapping but adjacent contigs were retrieved from l . a single contig overlapping with one of the two contigs determined from l was recovered from l (suppl. fig. a ). the contigs mapped to the pcv replication protein (rep), targeted by the circoviridae baits (suppl. fig. a ). the two overlapping contigs of l and l were confirmed by pcr and sanger sequencing (suppl. fig. b ). since the identity of the contigs with known viral sequences in genbank was %, no phylogenetic analysis was performed. parvoviridae contigs with the highest similarity to porcine parvovirus (ppv) were found in l ( contig with % identity) and l ( contigs with - % identity) (suppl. tab. ). the contig of l clustered within the tetraparvovirus genus, close to ungulate parvoviruses (porcine, ovine and bovine pv), while the contigs of l within the copiparvovirus genus, close to ppv (suppl. fig. ). two of the three contigs mapped to the replicase gene, while one from l mapped to an intergenic region (suppl. fig. c ). whereas the replicase region of ppv was covered by parvoviridae baits, the intergenic region was not (suppl. fig. c ). this portion of the virus may have been recovered by other non-parvoviridae baits targeting that region non-specifically. retroviridae contigs similar to the simian and feline foamy virus (spumaretrovirinae subfamily, - % identity) were detected in three samples (l , l , l ) (suppl. tab. ). phylogenetically the contigs clustered together as a sister group to the feline foamy viruses (felispumavirus genus), potentially being a new genus within the spumaretrovirinae (suppl. fig. ). the contigs mapped to the polymerase gene, which the exogenous retrovirus baits were designed to target (suppl. fig. d ). the mammalian hosts of the leeches were determined by metabarcoding [ ] . five waterholes from tanzania and six from mongolia were tested. from each waterhole, one water filtrate and one sediment sample were collected (except for one waterhole where only a sediment sample was collected), for a total of twenty-one samples. five samples (two water and three sediment samples) in total were positive for viral sequences ( . %). four viral families were identified including: retroviridae, herpesviridae, adenoviridae and papillomaviridae. in filtered water and sediment samples collected from the same waterhole, only one virus per sample was generally identified and in one location (wm and sm ) contigs from different viral families were isolated based on sample type. differences between sediment and water are not unexpected as the sediment likely represents a longer-term accumulation of biomaterial and the water represents more acute contamination at the surface and variable mixing throughout. of the water filtrate samples tested, two samples from mongolia (wm and wm ) ( . %) had viral contigs with % identity to the equid herpesvirus and (ehv- and ehv- ). the contig of wm mapped to the membrane glycoprotein b, whereas the two contigs of wm to the dna packaging protein and membrane glycoprotein g, all regions covered by the herpesviridae baits (suppl. fig. a -d). a nested panherpes pcr targeting the dna polymerase gene and the resulting sanger sequences further confirmed ehv presence (suppl. fig. e ). several equine species including domestic horses inhabit the gobi desert [ ] , which is consistent with the presence of these viruses. from the sediment samples tested, two from mongolia and one from tanzania yielded viral sequences ( %) representing three viral families including: retroviridae, adenoviridae and papillomaviridae. mongolian sediment sample sm was positive for four contigs mapping to the protease (pro) gene of the jaagsiekte sheep retrovirus (jsrv) with % identity (suppl. fig. a ). jsrv from this sample was further confirmed by pcr (suppl. fig. a ). mongolia sediment sample sm was positive for equine adenovirus ( % identity) with a contig mapping to a region comprised between the pvi and hexon capsid genes (suppl. fig. b ). given that multiple equine species are found in the gobi desert in mongolia, it is likely that the water sources sampled may have been frequented by these species [ ] . the sediment sample from tanzania st was positive for a zetapapillomavirus related to the equus caballus papillomavirus and equus asinus papillomavirus ( % identity; e -e genes) (suppl. fig. c ; suppl. fig. ), consistent with the detection of plains zebra's (equus quagga) dna from this water source [ ] . given that both captive and wild zebras have been known to contract bovine papillomaviruses [ , ] it is likely that they are susceptible to different equine papillomaviruses. emerging infectious viruses increasingly threaten human, domestic animal and wildlife health [ ] . sixty percent of emerging infectious diseases in humans are of zoonotic origin [ ] . wildlife trade and consumption of bushmeat, especially in africa and asia, have increasingly played a role in pathogen spillovers into human populations [ , ] . wildlife markets have recently facilitated the spillover of sars-cov- to humans [ ] resulting in a pandemic [ ] . the - sars-cov outbreak [ ] , the ebola outbreak in west africa [ ] and the global emergence of hiv [ ] have all been linked to wildlife trade and bushmeat consumption. early detection of novel infectious agents in wildlife represent a key factor to prevent their emergence. however, identification, surveillance and monitoring of emerging viruses using currently broadly applied approaches based on direct sampling of wildlife requires enormous investment in sampling, particularly for viruses that have low prevalence [ ] . for example, , wild birds were sampled in germany to detect avian influenza prevalence below % [ ] . similarly, sampling of over , animals in poland was required to determine an . % prevalence of african swine fever virus (asf) [ ] . sampling under remote field conditions or in developing countries present additional challenges. we provide evidence that environmental and invertebrate-derived dna samples including waterhole water, sediment and wild haematophagous terrestrial leeches can be used to survey known and unknown viruses. dna and rna viruses could be detected in % and . % of the idna (leech) and edna (waterhole) samples, respectively. the congruence of host dna assignment for leeches and viral families identified suggests that bloodmeals are a useful resource for determining viral diversity. similarly, the detection of primarily equine viruses from african and mongolian waterholes, where intense wild equid visitation rates were directly observed, suggests edna viruses from this resource reflect host utilization of the water and do not derive from other environmental sources such as fomites distributed over long distances. pcr based approaches, as used in earlier studies to detect pathogens from flies [ , ] or, under laboratory conditions, in medicinal leeches [ ] , require prior knowledge about the expected pathogens in the samples. the unknown viral diversity in the wild, and the potential degradation of viral nucleic acids in bloodmeals or in the environment, may affect detection by pcr resulting in high false negative rates. rna oligonucleotide based hybridization capture overcomes such limitations because the short baits can capture divergent and degraded dna. the comprehensive viral group representation in the rna bait set also allows for the determination of both viral presence and viral diversity with a relatively simple workflow. the ability of oligonucleotides with substantial divergence from the target sequence to capture more distantly related sequences is particularly useful in virology since most viruses are uncharacterized in wildlife and many evolve rapidly. using short rna baits to capture highly conserved sequences from every known vertebrate viral genome is a useful and relatively inexpensive approach for providing an initial viral identification. however, to fully characterize each virus, the rna oligonucleotide bait set would need to be customized to retrieve full length viral genomes. initial screening with full length genomes for all viruses is costly and may result in detection of host dna in cases of spurious homology between viruses and host dna sequence. several novel viruses were identified with our short rna bait approach, which is not unexpected as little is known about the virology of wildlife in southeast asia, where the leeches were collected. several viral contigs were phylogenetically distinct from known viruses and may represent new genera. for example, the novel coronavirus identified in leech bloodmeals did not cluster with any of the known coronaviridae clades. this finding highlights the ability of this method to detect unknown viruses. we could also associate the novel corona-and rhabdoviruses with mammal bloodmeals with limited evidence of a cervid association for both. cervids are regularly sold as bushmeat in wildlife markets [ ] and both recent coronavirus epidemics (sars-cov [ ] and sars-cov- [ ] ) spilled over from wildlife. this suggests that edna/idna-based pathogen surveillance approaches may complement efforts to proactively identify viruses that could potentially spillover to humans or livestock. the collection of wild haematophagous invertebrates such as leeches or water and sediments has both advantages and disadvantages compared to invasively collected wildlife samples. large amounts of dna can be extracted from bloodmeals, in particular when leeches are processed in bulk. we pooled up to leeches and many of our leech bulk samples contained a diverse mix of mammalian dna. a disadvantage of leeches is that they cannot be found in all environments: for example haematophagous terrestrial species are restricted to tropical rainforests of asia, madagascar and australia [ ] . in addition, leech feeding biases could influence diversity surveys [ , ] . however, this disadvantage could be overcome in the future by employing additional invertebrates such mosquitoes [ ] or carrion flies [ ] . waterholes are commonly found in almost all environments. in environments with seasonal water shortages, dna from animals can become highly concentrated due to many animals utilizing rare water sources. the disadvantages are that the dilution factor of water, depending on water body size, can obscure rare dna sequences and mixed host species sequences are generally the rule rather than the exception. further experiments with field filtration and sample concentration such as methods used with pathogen detection in waste water may improve detection rates [ ] . environmental dna and in particular its subdiscipline invertebrate-derived dna viral hybridization capture may be a useful and economical tool for identifying and characterizing major viral pathogens particularly in difficult to access sampling environments prior to viral emergence. sampling in environments where direct access to animals is difficult or highly restricted, edna and idna may be the only option to detect viral pathogens in the wild. the current study suggests this approach will be successful in either complementing or replacing invasive approaches. leeches two types of leeches, tiger leeches (haemadipsa picta) and brown leeches (haemadipsa zeylanica) were collected from february to may in the deramakot forest reserve in sabah, malaysian borneo as described in abrams et al. [ ] and axtner et al. [ ] . all leeches of the same type (tiger or brown) from the same site and occasion were pooled and processed as one sample. number of leeches ranged from to per pool (median= ). samples were stored in rna fixating saturated ammonium sulfate solution and exported under the permit 'jkm/mbs. - / jld. ( )' issued by the sabah biodiversity council. a total of pools (l -l ) were selected for viral capture to maximize representation of host wildlife species identified from bloodmeals [ ] . at each waterhole, ml of water was passed through a . µm sterivex filter unit (millipore) using a disposable -ml syringe to remove debris from water. in addition, g of the top - cm of sediment was collected at each waterhole. the samples were stored on ice packs during the respective field trip, and frozen at - °c. in total water filtrate and sediment samples were sampled at waterholes, six respectively from mongolia and tanzania. for each sample, ml of water filtrate was ultra-centrifuged at , rpm for hours to pellet dna and viral particles. the supernatant was then removed, the pellet re-suspended in ml of cold phosphate-buffered saline (pbs) (ph . ) (sigma-aldrich) and left at °c overnight. leeches were cut into small pieces with a new scalpel blade and lysed overnight (≥ hours) at °c in proteinase k and atl buffer at a ratio of : ; . water and sediment µl of the centrifuged filtrate was used to extract viral nucleic acids using the rtp ® dna/rna virus mini kit (stratec biomedical). the following modifications were made to the original protocol: µl of lysis buffer, µl of binding buffer and µl of proteinase k and carrier rna were used per sample. samples were eluted in µl. the nucleospin soil kit (macherey-nagel) was used to extract dna/rna from sediment. mg of soil was extracted according to the manufactures protocol using an elution volume of µl. as a positive control medical leeches (hirudo spp.) were fed human blood spiked with four viruses [ ] . two rna viruses, influenza a and measles morbillivirus, and two dna viruses, bovine herpesvirus and human adenovirus were used (see kampmann et al. [ ] for details). the rna was reverse transcribed using superscript iii and iv (thermo fisher scientific) with random hexamers prior to second-strand synthesis with klenow fragment (new england biolabs). the resulting double-stranded cdna/dna mix was sheared to an average fragment size of bp using a m focused ultrasonicator (covaris). sheared product was purified using the zr- dna clean & concentrator- kit (zymo). dual-indexed illumina sequencing libraries were constructed as described by meyer and kircher [ ] with the modifications described in alfano et al. [ ] . each library was amplified in three replicate reactions to minimize amplification bias in individual pcrs. the three replicate pcr products for each sample were pooled and purified using the minelute pcr purification kit (qiagen). negative control libraries were also prepared from different stages of the experimental process (extraction, reverse transcription, library preparation and index pcr) and indexed separately to monitor any contamination introduced during the experiment. amplified libraries were quantified using the tapestation (agilent technologies) on d screentapes. the targeted sequence capture panel was designed based on the oligonucleotide probes represented on the virochip microarray [ ] . the virochip is a pan-viral dna microarray comprising the most highly conserved mer sequences from every fully sequenced reference viral genome present in genbank, which was developed for the rapid identification and characterization of novel viruses and emerging infectious disease. we retrieved the viral oligonucleotides from the th generation virochip (viro ) [ ] , which are publicly available at ncbi's gene expression omnibus (geo) repository [ ] , accession number gpl https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=gpl ( ). this platform includes ~ , oligonucleotides ( mer nucleotides) derived from ~ , viral species. we excluded sequences from bacteriophage, plant viruses, viral families infecting only invertebrates and endogenous retroviruses. we included viruses that could have both vertebrate and invertebrate hosts, such as vertebrate viruses with insect vectors. exogenous retroviruses were represented but murine leukemia viruses (mlvs) were removed. mlvs sequences may interfere with the capture of other viruses, since mlvs can cross enrich endogenous retroviruses which can represent large portions of several vertebrate genomes and mask rarer viral sequences. control oligonucleotides included in the virochip, such as those from human genes, yeast intergenic sequences, and human papilloma virus sequences present in hela cells were also removed. ninety-two -mer oligonucleotides covering (spaced end-to-end) the entire pol and gb genes of equine herpesvirus (ehv- ) were included as pcr screening of the water samples indicated they were positive for this virus (data not shown). the resulting , oligonucleotides were examined for repetitive elements, short repeats, and low complexity regions, which are problematic for probe design and capture, using repeatmasker. repetitive motifs were identified in oligonucleotides, which were removed. the final targeted sequence capture panel consisted of , unique sequences which were synthesized (as a panel of biotinylated rnas) at mycroarray (ann arbor, usa). in-solution target enrichment via hybridization-based capture was performed according to the manufacturer's protocol (mybaits® custom targeted enrichment, mycroarray, ann arbor, usa), with the following modifications for likely partially degraded samples with an expected low target viral content: ul dynabeads® m- streptavidin beads (invitrogen) instead of ul dynabeads® myone™ streptavidin c (invitrogen); hybridization, bead-bait binding, and wash steps temperature set to °c; hours hybridization time; ng baits per reaction; μl indexed library inputs. for capture, libraries generated from pooled leeches consisting of more than individuals were captured individually, while libraries generated from pools of fewer individuals were combined to have a comparable number ( ) ( ) ( ) ( ) ( ) ( ) of leeches per capture. this was done in order to ensure that each individual leech represented in each library was allocated enough bait for capture. for capture libraries generated from water and sediment samples. samples were pooled in groups of two. sediment and water cdna and dna were pooled separately. per pooled sample, µl of baits were used to ensure enough bait for each sample. the enriched libraries were reamplified using herculase ii fusion dna polymerase (agilent technologies) with p and p illumina library outer primers with the same cycling conditions described in alfano et al. . the re-amplified enriched libraries were purified using the minelute pcr purification kit (qiagen), quantified using the tapestation (agilent technologies) on d screentapes and finally pooled in equimolar amounts for single-read sequencing on two lanes of an illumina nextseq with the tg nextseq® / high output kit v ( cycles). a total of , , sequence reads bp long were generated (average: , , single reads per sample; standard deviation [sd]: , , ) (suppl. tab. ) and sorted by their dual index sequences. cutadapt v . and trimmomatic v . were used to remove adapter sequences and low-quality reads using a quality cutoff of and a minimal read length of nt. after trimming, % of the sequences were retained. three different approaches (a, b, c) were used to analyse the viral capture data: a) leech reads were removed from the dataset by alignment to the helobdella robusta genome v . (assembly gca_ . ), which is the only complete genome of hirudinea available in genbank, and all leech sequences from genbank ( , sequences resulting from "hirudinea" search) using bowtie v . . . [ ] . this left % of the original reads (suppl. tab. ). then, the filtered reads were searched by blast against a database generated from the capture bait sequences. the reads which matched with baits were then extracted and screened against the entire ncbi nucleotide database (nt) using blastn to find the best viral match. the filtered reads were mapped both to the corresponding bait sequence and the genome sequence of the best hit obtained by blast against the complete nt database, in order to generate a consensus sequence. this consensus sequence was again searched against the ncbi nt database using blastn to obtain a viral assignment. b) leech reads were removed as in method a. in addition, rrna reads were removed using sortmerna [ ] , leaving % of the original reads (suppl. tab. ). the filtered reads were de novo assembled using both spades v . . [ ] and trinity v . . [ ] assemblers. the obtained contigs from spades and trinity were pooled and clustered to remove duplicated or highly similar sequences using usearch v . . [ ] with a % threshold identity value. the centroids were then subjected to sequential blast searches against the ncbi nucleotide database and ncbi refseq viral protein database using blastn and blastx, respectively. c) the adaptor and quality trimmed data were uploaded to genome detective [ ] , a web base software that assembles viral genomes from ngs data. the software first groups reads into different buckets based on the proteins similarity to different viral hits. genome detective then de novo assembles the reads of each bucket creating a longer consensus sequence that is then searched against the ncbi refseq viral database using blastx and blastn algorithms. the results of amino acid and nucleotide search are combined and viral hit is assigned based on the best combined score. bacteriophages, invertebrate viruses and retroviruses were excluded from subsequent steps, which only focused on eukaryotic, specifically vertebrate viruses. the results of the three methods were compared and the viral contigs obtained were manually inspected. if more than one method generated a contig with the same viral hit, the contigs from each method were compared. if they had the same sequence or were overlapping, the longest contig was selected. the filtered reads were mapped to the viral contigs to calculate the number of viral reads for each virus. finally, the viral contigs were mapped to the reference genome of the virus corresponding to the best blast hit using geneious v . . (biomatters, inc.) [ ] . the baits were mapped to the same references to determine the genomic positions targeted by our bait panel for each virus. viral contigs were assigned to viral families according to the best blast results. comprehensive sets of representative sequences from these viral families were retrieved from genbank and aligned with the contigs using mafft v . [ ] . phylogenetic analysis was performed using the maximum-likelihood method based on the general time reversible substitution model with amongsite rate heterogeneity modelled by the Γ distribution and estimation of proportion of invariable sites available in raxml v [ ] , including bootstrap replicates to determine node support. phylogenetic analyses were performed only on viral contigs i) showing divergence from known viruses, i.e. with both blast identity and coverage to the best reference below %, to place them into a phylogenetic context, and ii) mapping to phylogenetically relevant genomic regions. therefore, circoviridae and anelloviridae contigs were excluded as were those identified from water. host identification of leeches followed an edna/idna workflow recently published [ ] . in summary, leech samples were digested and short fragments of the mitochondrial markers s, s and cytochrome b were amplified in four pcr replicates each resulting in pcr replicates per sample. we used a twin-tagging -step pcr protocol and pcr products were sequenced using an illumina miseq (for details please see axtner et al. [ ] ). after demultiplexing and read processing, each haplotype was taxonomically assigned to a curated reference database using protax [ ] . taxonomic assignments followed the criteria of axtner et al. . the primers listed in suppl. tab. were designed to confirm by pcr the viral contig sequences generated by the three approaches (a, b, c see above) from the leech samples. for pcrs targeting rna viruses, ul of extract were digested with rdnase i (ambion) following the manufacturer's protocol. the dnase-digested extracts were then purified using the rneasy minelute cleanup kit (qiagen). rna was reverse transcribed into cdna using iscript™ reverse transcription supermix (bio rad). sediment and water samples that tested positive for ehv and jsrv were screened using a previously described pan-herpes pcr [ ] and for jsrv [ ] , respectively. the resulting amplicons were sanger sequenced. environmental dna for wildlife biology and biodiversity monitoring environmental dna-an emerging tool in conservation for monitoring past and present biodiversity terrestrial mammal surveillance using hybridization capture of environmental dna from african waterholes idna from terrestrial haematophagous leeches as a wildlife surveying and monitoring tool-prospects, pitfalls and avenues to be developed tropical rainforest flies carrying pathogens form stable associations with social nonhuman primates design-and model-based recommendations for detecting and quantifying an amphibian pathogen in environmental samples using a pan-viral microarray assay (virochip) to screen clinical samples for viral pathogens leeches as a source of mammalian viral dna and rna-a study in medicinal leeches equus hemionus. the iucn red list of threatened species sarcoids in captive zebras (equus burchellii): association with bovine papillomavirus type infection detection of bovine papillomavirus dna in sarcoid-affected and healthy free-roaming zebra (equus zebra) populations in south africa public preferences for one health approaches to emerging infectious diseases: a discrete choice experiment global trends in emerging infectious diseases toward a quantification of risks at the nexus of conservation and health: the case of bushmeat markets in lao pdr wildlife trade and the emergence of infectious diseases a pneumonia outbreak associated with a new coronavirus of probable bat origin the sars, mers and novel coronavirus (covid- ) epidemics, the newest and biggest global health threats: what lessons have we learned? identification of a novel coronavirus in patients with severe acute respiratory syndrome emergence of zaire ebola virus disease in guinea origins of hiv and the aids pandemic. cold spring harb surveillance of wild birds for avian influenza virus chances and limitations of wild bird monitoring for the avian influenza virus h n -detection of pathogens highly mobile in time and space african swine fever epidemic tracking zoonotic pathogens using blood-sucking flies as' flying syringes empty forests, empty stomachs? bushmeat and livelihoods in the congo and amazon basins debugging diversity-a pan-continental exploration of the potential of terrestrial blood-feeding leeches as a vertebrate monitoring tool shifting up a gear with idna: from mammal detection events to standardised surveys broad surveys of dna viral diversity obtained through viral metagenomics of mosquitoes assessing the feasibility of fly based surveillance of wildlife infectious diseases two-step concentration of complex water samples for the detection of viruses an efficient and robust laboratory workflow and tetrapod database for larger scale environmental dna studies illumina sequencing library preparation for highly multiplexed target capture and sequencing variation in koala microbiomes within and between individuals: effect of body region and captivity status microarray-based detection and genotyping of viral pathogens virus identification in unknown tropical febrile illness cases using deep sequencing gene expression omnibus: ncbi gene expression and hybridization array data repository fast gapped-read alignment with bowtie sortmerna: fast and accurate filtering of ribosomal rnas in metatranscriptomic data spades: a new genome assembly algorithm and its applications to single-cell sequencing trinity: reconstructing a full-length transcriptome without a genome from rna-seq data search and clustering orders of magnitude faster than blast genome detective: an automated system for virus identification from high-throughput sequencing data geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data mafft multiple sequence alignment software version : improvements in performance and usability raxml version : a tool for phylogenetic analysis and post-analysis of large phylogenies unbiased probabilistic taxonomic classification for dna barcoding long term stability and infectivity of herpesviruses in water the long terminal repeat of jaagsiekte sheep retrovirus is preferentially active in differentiated epithelial cells of the lungs we would like to thank joseph derisi for guidance in using his oligonucleotide data. this project received financial support from the german federal ministry of education and research (bmbf fkz: ln a) to na, ja, am and aw and was supported by funds from the leibniz gemeinschaft, saw- -izw- to ad and adg. we thank peter seeber and sanatana-erini soilemetzidou for collection of water and sediment samples in africa and mongolia respectively. we thank the sabah forestry department, especially johnny kissing, peter lagan, and datuk sam mannan, for supporting the fieldwork and the sabah biodiversity council for providing research, collection, and export permits (jkm/mbs. - / jld. ) for the leech work. adg and aw designed the study; am and stw collected the leeches in the field; ja performed the leech nucleic acids extractions and the pcrs on leeches; na and ad performed the capture experiments; na and kt performed the bioinformatics and phylogenetic analyses; na, ad, adg and aw wrote the manuscript; na, ad, ja, kt, mlk, tpg, aw and adg reviewed the manuscript; all authors read and approved the final manuscript. the authors declare no competing interests. in the upper panel the left photo shows a leech feeding on a frog in a rainforest of vietnam (courtesy andrew tilker; leibniz-izw) and the right photo shows an african waterhole in tanzania (courtesy peter seeber; leibniz-izw). the middle panel depicts the hybridization capture protocol. briefly, illumina libraries were produced from reverse transcribed rna and dna from leech bloodmeals or from waterhole surface water and sediments. biotinylated viral rna baits were hybridized to the libraries and non-target dna was washed away. the remaining dna was sequenced, reads assembled into contigs and mapped to reference viral genomes. these contigs were further analyzed to determine viral identity. viral identity was paired with host identity determined either by mammalian metabarcoding of the leech samples, or by observation of waterhole usage. relative abundance of viruses from each family, shown as the percentage of the total number of viral reads in each leech and waterhole sample. in the sample names, s stands for sediment, w for water, t for tanzania, m for mongolia and l for leeches. the leech host assignment for each leech sample is shown on the right (see suppl. tab. for further details). key: cord- -hruq hdw authors: waffo tchounga, c.a.; sacre, p.y.; ciza, p.; ngono, r.; ziemons, e.; hubert, ph.; marini, r.d. title: composition analysis of falsified chloroquine phosphate samples seized during the covid- pandemic date: - - journal: j pharm biomed anal doi: . /j.jpba. . sha: doc_id: cord_uid: hruq hdw the proliferation of falsified medicines can cause serious public health issues, particularly in the context of a global pandemic such as the actual covid- pandemic. our study involved eight chloroquine phosphate medicines seized in cameroon, democratic republic of congo and niger during march and may . these suspect samples were first analyzed in a screening phase using field tools such as handheld raman spectroscopy (truscan) and then in a confirmation phase using laboratory tools such as hyperspectral raman imaging and high performance liquid chromatography (hplc). the results confirmed the falsified nature of the samples, highlighting the presence of metronidazole at low dose in four samples ( . , . , . and . mg/tab), too low levels of chloroquine in two samples ( . and . mg/tab), and substitution of chloroquine phosphate by paracetamol in one sample ( . mg/tab). the results also confirmed that four samples had been adulterated with paracetamol in trace amounts and two of them presented traces of chloramphenicol. on december , a new coronavirus disease emerged from china and gradually spread, becoming a global pandemic. thus, on january , the world health organization (who) announced a state of public health emergency of international scope. today, more than million people have already been infected by this disease [ ] [ ] [ ] [ ] . the world is currently facing many challenges arising from this pandemic. beyond the non-negligible economic consequences affecting governments and international trade in general, there are problems related to access and supply of high quality essential medicines and health products. taking advantage of this opportunity, unscrupulous people are selling substandard and falsified medicines. according to some experts, this phenomenon constitutes a risk of a parallel pandemic to the covid- pandemic [ ] [ ] [ ] [ ] [ ] . substandard and falsified medicines have a negative impact not only in terms of public health, but also in socio-economic terms [ , , ] . aware of these risks, international organizations such as the who and the european medicines agency (ema) have drawn the attention of consumers, professionals and health authorities to the emergence of substandard or falsified vaccines, medicines, as well as falsified screening tests and laboratory reagents claiming to have covid- disease prevention, detection and treatment properties [ , ] . falsifiers who are indeed criminal gangs are always on the lookout for such situations that could provide them large profit margins no matter the impact on human lives. they take advantage of the scarcity of medicines and health products to infiltrate both the formal and informal markets [ ] . thus, a few time after the very controversial announcement of the treatment of covid- disease with chloroquine phosphate and hydroxychloroquine sulfate [ , ] , who has brought to the public's attention cases of falsified chloroquine in burkina faso, cameroon, niger, democratic republic of congo (drc) and france, thanks to the effectiveness of its global surveillance and monitoring system (gsms). a previous publication, at the origin of the who alert, showed about five falsified tableted chloroquine samples collected in cameroon and drc using thin layer chromatography (gphf minilab) as a screening method [ ] [ ] [ ] . after the alert launched by the who, the pharmaceutical analytical chemistry laboratory of the university of liege was contacted in the frame of its who prequalification to carry out tests on seized samples of chloroquine medicines suspected of being falsified. in the s, resistance to this long-used molecule against malaria was observed in most areas of high malaria endemicity, leading to a change in treatment [ , ] . among the phenomena increasing this resistance, falsification plays a non-negligible role. indeed, it has been observed in cameroon a phenomenon of resistance that was supposedly linked to falsified chloroquine tablets [ ] . several studies have highlighted the presence of poor-quality chloroquine on the african pharmaceutical market [ ] [ ] [ ] [ ] . in recent years, special attention has been paid to vibrational spectroscopy methods (mainly raman and near infrared spectroscopies) for the detection of falsified medicines. they have many advantages compared to conventional methods used to control the quality of medicines because of their non-destructive nature (no sample preparation is required), absence of consumable (no use of solvents) and speed [ , ] . time is indeed an important factor in the detection of falsified medicines to reduce its impact on the population [ ] . however, once confirmed falsified, further laboratory analyses must be undertaken to measure the risk to which the population is exposed. our study is focused on eight suspect samples: four from yaoundé and douala in cameroon, one from kinshasa in drc, and three from niamey in niger. these suspect chloroquine samples were seized by local authorities as being part of the who alert list. for the analysis of these suspect samples, we have adopted an analysis strategy based on two types of methods: screening phase using handheld raman spectrophotometer and a confirmatory analysis to obtain information on the qualitative composition of both organic and inorganic chemicals of the seized samples [ , ] . hplc-uv was used to obtain quantitative information of the identified active pharmaceutical ingredients and a screening lc-ms method was used to confirm the hyperspectral imaging results. the suspect chloroquine samples were all in tablets dosage form. they are described in table i . some samples were contained in blisters, some in boxes and some were unpackaged. the samples, once seized, were sent by courier service to the author's laboratory. once received, they were stored at room temperature before analysis. handheld raman analyses were performed using a truscan rm spectrophotometer (thermo scientific, usa). the acquisition parameters were automatically selected by the device. when available, ten tablets were analyzed per sample. otherwise, all tablets were analyzed. the tablets were scanned directly either through the blister (when samples were inside a blister, samples f-g) or out of the blister (when samples were without blisters, samples a-e). spectra were preprocessed by the savitzky-golay first derivative (window size: , polynomial order ) before comparison with the database over the spectral range - cm - using the pearson's correlation coefficient. the database used throughout the study for raman spectra comparison (truscan or raman imaging spectra) is a homemade database containing api and excipients. before being analyzed by raman imaging, tablets were glued on a microscope slide and their surface was milled using a leica em rapid milling system equipped with a tungsten carbide miller (leica microsystems gmbh). raman hyperspectral imaging experiments were performed on a labram hr evolution (horiba scientific) equipped with an emccd detector ( × -pixel sensor) (andor technology ltd.), a leica x fluotar lwd objective and a nm laser with a power of mw at sample (xtra ii single frequency diode laser, toptica photonics ag). a mapping of x pixels per sample was performed and the spectra were then analyzed by multivariate curve resolution-alternating least square (mcr-als) in order to find the pure spectra of the constituents as well as their spatial distribution. the raman data were corrected by an asymmetric least square baseline correction (p: × − , λ: × ). the pre-processed data were first compared to the laboratory database. following the first comparison, the data were analyzed by mcr-als. three mcr-als models were successively computed with , and components respectively. non-negativity on concentration j o u r n a l p r e -p r o o f and spectra were used as constraints. the different resolved spectra were then preprocessed by the savitzky-golay first derivative (window size: , polynomial order ) before comparison with the database over the spectral range - cm - using the pearson's correlation coefficient. a correlation coefficient of one corresponds to a perfect correlation. the identification of chloroquine phosphate was confirmed by a second comparison in the - cm- spectral range. indeed, chloroquine phosphate presented very good discrimination towards chloroquine sulfate (correlation coefficient of . ) and hydroxychloroquine sulfate (correlation coefficient of . ) in this spectral range (see figure s ).the total analysis time was about h per sample. raman imaging was used to detect as much compounds as possible (both organic and inorganic) in order to obtain a composition of the sample as complete as possible. assays were performed on a hplc system comprising a waters separation module coupled to a waters photodiode array detector from waters corporation (eschborn, germany). the hplc methods are detailed in tables s , s and s in supplementary data. the double information of retention times and uv spectra of detected peaks allowed a formal identification of the active ingredients of the different samples compared to reference substances. a screening confirmatory lc-ms analysis of the samples was performed. the lc-ms equipment was composed of a dionex ultimate (thermo scientific) with an ms detector amazon speed etd (bruker). the details of the lc-ms method are given in table s . samples a, b, c and d from cameroon and samples f, g and h from niger correspond to the who / alert samples from these two countries. the samples came from both the informal sector (b, c, d, f, g, h) and the formal sector (a and e). see photos in supplementary data s -s . samples a, c and d have the same reported manufacturing laboratories and lot numbers as samples i, ii, iv and v collected by gnegel et al [ ] . sample d in particular has the same information (manufacturer's laboratory, lot number, date of manufacture and expiry date) as sample v from gnegel et al [ ] , but with differences in the declared active ingredient content ( and mg respectively). not all samples from niger included information on the manufacturing laboratories. for sample h, the secondary packaging did not have a lot number but the inscription "see on blister". on the blisters, this information was not readable, thus highlighting the product traceability problem in medicines falsification. as in the study by gnegel et al [ ] , these suspect samples came from both the formal and informal sectors. considering the results in table , in samples a, b, c and d from cameroon, metronidazole and starch were identified as the major constituents with correlation coefficient > . (see table s for detailed truscan results). the spectra were overlaid with reference raman spectra of chemicals from the database. as one can see, handheld raman spectra have a rather low signal to noise (snr) ratio due to the high fluorescence background (see fig a) . this low snr implies that the detection of the pharmaceutical ingredient is sometimes difficult or may be hindered by a stronger signal by a main constituent. this finding may be seen in table s when looking at the results obtained for different tablets of a sample. nevertheless, this first screening step enabled the confirmation of falsification of samples a -e because of the presence of a wrong api. samples f -h were considered as suspicious since the signal of chloroquine phosphate was very small or undetectable. raman imaging confirmed the presence of metronidazole in samples a -d together with excipients such as starch, magnesium stearate, calcium carbonate, talc, and calcium phosphate. in addition, sample b showed the presence of paracetamol and chloramphenicol and sample d contained paracetamol (see table ). paracetamol and chloramphenicol were present at trace amount since they are present in a very few number of pixels of the chemical images (see figure a ) in sample e, handheld raman spectroscopy identified paracetamol. hyperspectral imaging confirmed this result with a correlation coefficient of . . it also revealed the presence of excipients such as sodium benzoate and microcrystalline cellulose. hplc assay confirmed these results with a paracetamol content of . mg per tablet. lc-ms also identified the presence of chloroquine (in trace amounts), chloramphenicol and aspartame. these compounds were not detected by raman imaging possibly because of the high fluorescence background and high raman scattering character of paracetamol possibly masking the signal of trace level api's [ ] . however, the probable heterogeneity among tablets may also explain the discrepancies between the lc-uv, lc-ms and raman experiments. for samples f and g, the truscan identified only starch. in sample h, the presence of calcium carbonate was detected. however, the presence of chloroquine may be confirmed when looking at the specific spectral range - cm - with an average correlation coefficient of . (see fig b and table s ). raman imaging results showed the presence of chloroquine phosphate in samples f, g and h with correlation coefficients of . , . and . respectively. the spectra were tested against chloroquine sulfate and hydroxychloroquine sulfate in the spectral range - cm - to ensure the identification of chloroquine phosphate salt. the fact that the handheld raman spectrometer detected the presence of chloroquine only in sample h can be explained by the fact that in sample f and g, chloroquine phosphate was present only in small quantities with a content. indeed, hplc-uv estimated the contents at . mg and . mg per tablet respectively. the presence of paracetamol in sample g was demonstrated by hyperspectral raman imaging (see fig b) , which was not the case by lc-uv or lc-ms. on the other hand, lc-uv results revealed the presence of paracetamol in sample h, which was not detected by handheld raman spectroscopy, hyperspectral raman imaging or lc-ms demonstrating the heterogeneity of falsified samples. trace amounts of adulterating apis in samples b, d, e g and h indicate contamination problems certainly resulting from the production of previous batches. this situation highlights the noncompliance with good manufacturing practices. metronidazole and paracetamol have certainly been used as a substitute for chloroquine as stated by gnegel et al to mimic its bitter taste [ ] . concerning sample e from drc where chloroquine was substituted with paracetamol, the falsifiers certainly targeted its analgesic and antipyretic properties that could calm the pain and fever that are part of the symptoms of covid- disease and malaria. metronidazole levels are very low with amounts below mg per tablet in samples a, b, c and d. also in samples f and g, chloroquine levels are very low ( . mg and . mg per tablet respectively). these very low levels of active ingredients are prone to contribute to the emergence of antimalarial resistance problems [ ] . raman spectroscopy has been preferred to near-infrared spectroscopy because of the highly resolved spectra enabling a fast comparison with a pure compound spectral database [ ] [ ] [ ] . in addition, raman hyperspectral imaging has the advantage to give a deep insight in organic and inorganic compound composition of the tablets, making it possible to highlight the presence of substances likely to be toxic without a priori knowledge. however, this technique is long making it complicated to analyze several tablets per sample diminishing the representativity of the analysis. in addition, it suffers from the limitations of raman spectroscopy such as fluorescence and low signal intensity possibly making difficult the analysis of some colored or degraded formulations. falsified samples of chloroquine phosphate seized by cameroon, drc and niger during the covid- pandemic were analyzed. in six samples, we noted the lack of chloroquine and its substitution by metronidazole (at sub-therapeutic levels) or paracetamol. in two samples, chloroquine phosphate was detected but at very low levels. in one sample, the chloroquine phosphate content was in conformity with the declared content. in addition to the major apis present in the tablets, trace levels of paracetamol and chloramphenicol were detected in four and two samples respectively. non-compliance with good manufacturing practices and low levels of active ingredients reflect the desire from the falsifiers to make a profit without worrying about the unfortunate consequences that may happen to those who have consumed their product. this also demonstrates the speed with which falsifiers adapt their production to actual demand such as the chloroquine phosphate in the covid- context. it appears that rather than producing new products with the correct api, falsifiers prefer to print new labels for their old bad quality products increasing their profits. in the frame of the covid- pandemic, the need for specific products (allegedly treating the disease) may increase rapidly at a global scale. this constitute an opportunity for criminal groups and unscrupulous sellers to sell substandard and falsified medicines. in this context, the application of rapid, easy-to-use techniques such as raman spectroscopy should make it possible to combat efficiently the resurgence of poor-quality medicines. however, handheld devices are not sensitive enough to detect trace level contaminants or low-dosed active ingredients. therefore, hyperspectral imaging may help in this context providing also a "signature" of the product composition possibly allowing the grouping of falsification cases. novel coronavirus: an emerging global threat the novel coronavirus -a snapshot of current knowledge covid- : a fast evolving pandemic who covid- dashboard covid- and risks to the supply and quality of tests, drugs, and vaccines the consequence of covid- on the global supply of medical products: why indian generics matter for the world? why the 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médicaments sur le marché pharmaceutique africain counterfeit anti-infective drugs a review of near infrared spectroscopy and chemometrics in pharmaceutical technologies application of nir spectroscopy and chemometrics for revealing of the 'high quality fakes' among the medicines, forensic chem understanding and fighting the medicine counterfeit market investigation of the composition of anabolic tablets using near infrared spectroscopy and raman chemical imaging recent developments of raman spectroscopy for the qualitative analysis of falsified and substandard medicines vibrational spectroscopy in analysis of pharmaceuticals: critical review of innovative portable and handheld nir and raman spectrophotometers a link between poor quality antimalarials and malaria drug resistance? assessment of the performance of a handheld raman device for potential use as a screening tool in evaluating medicines quality comparison of hyperspectral imaging techniques for the elucidation of falsified medicines composition comparing the qualitative performances of handheld nir and raman spectrophotometers for the detection of falsified pharmaceutical products the authors would like to thank the lanspex ( key: cord- - ecsp y authors: griesemer, sara b; van slyke, greta; st. george, kirsten title: assessment of sample pooling for clinical sars-cov- testing date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: ecsp y accommodating large increases in sample workloads has presented one of the biggest challenges to clinical laboratories during the covid- pandemic. despite the implementation of new automated detection systems, and previous efficiencies such as barcoding, electronic data transfer and extensive robotics, throughput capacities have struggled to meet the demand. sample pooling has been suggested as an additional strategy to further address this need. the greatest concern with this approach in a clinical setting is the potential for reduced sensitivity, particularly the risk of false negative results when weak positive samples are pooled. to investigate this possibility, detection rates in pooled samples were evaluated, with extensive assessment of pools containing weak positive specimens. additionally, the frequency of occurrence of weak positive samples across ten weeks of the pandemic were reviewed. weak positive specimens were detected in all five-sample pools but failed to be detected in four of the nine-sample pools tested. weak positive samples comprised an average . % of the positive specimens tested during the pandemic thus far, slightly increasing in frequency during later weeks. other aspects of the testing process should be considered, however, such as accessioning and reporting, which are not streamlined and may be complicated by pooling procedures. therefore, the impact on the entire laboratory process needs to be carefully assessed prior to implementing such a strategy. the covid- pandemic has presented numerous challenges to the health care industry in general and laboratory testing specifically. not least among the latter has been a dramatic increase in sample testing load ( ) . efforts to meet the demand have included increased use of automated instrumentation, multiplexing of molecular detection assays, streamlined testing protocols, as well as increasingly varied acceptable sample types, collection devices and transport media ( ) ( ) ( ) . accommodating the testing workload and reagent shortage during the symptomatic pandemic wave was a significant undertaking ( ) . however, the more recent task, to test returning healthcare workers as well as patients returning for services such as nonessential surgery and other clinical procedures, has generated an even greater challenge. one proposed solution has been sample pooling ( ) ( ) ( ) ( ) , a method previously used for numerous other situations where large scale testing was needed ( ) ( ) ( ) ( ) . when used as an epidemiological surveillance tool, acceptable parameters and limits may be quite different to those when the same system is applied in a clinical testing setting ( , ( ) ( ) ( ) ( ) . in the latter, the issue of detection sensitivity for every individual specimen becomes critical. methods for pooling vary widely and testing large numbers of pooled samples is not possible without an inherent loss of sensitivity. however, there is the potential for more limited pooling, without loss of sensitivity. we sought to investigate to what extent this was possible, while maintaining the detection of weak positive samples. the cdc ncov real-time rt-pcr diagnostic panel ( ) ( ) ( ) was used throughout, with extraction on the biomerieux emag ® (biomerieux inc, durham, nc). for individual specimens, µl of viral transport media (vtm, regeneron, rensselaer) or molecular transport media (mtm, primestore, longhorn, san antonio, tx) from upper respiratory swab, was added to ml nuclisens lysis buffer (biomerieux) and extracted into µl of eluate. the biomerieux emag extraction system will accommodate a maximum volume of ml. therefore, a maximum of nine samples ( µl per sample) could be added to a ml lysis buffer tube without exceeding the maximum volume, while maintaining the same input volume per specimen. if the same extraction efficiency is maintained when nine samples are loaded in the tube as when one is loaded, and the eluate is still µl, theoretically the same total nucleic acid from each sample should be extracted. provided there is no increase in the pcr inhibition of the pooled eluate, the detection sensitivity should therefore also still be the same. pool sizes of five and nine samples were tested, with each pool containing a single positive specimen. for specimen pooling, µl each of one positive, and either four (five-sample pool) or eight (nine-sample pool) negative specimens were added, at random, to ml of lysis buffer, in triplicate, extracted and eluted into µl of elution buffer. in an initial experiment, strong and moderate positive samples in vtm were tested in each size pool. in a second experiment, four weak positive specimens in vtm and four weak positive specimens in mtm, were tested in both five and nine sample pools, in triplicate. additionally, the percentage of positive samples at different viral loads, as assessed by ct value, was reviewed across nine weeks of the pandemic, to determine if these weak positive specimens comprise a significant component of total tested specimens and whether the proportion has changed over the course of the pandemic. pooling of samples with lower ct values did not cause a loss of detection of any of the individual positive samples in either the five-or nine-sample pools (table ) , although in one of the ninesample pools, the detection became inconclusive rather than positive (specimen d). the ct values of positive samples were increased by . to . when pooled with four negative samples. in contrast, pooling with negative samples caused ct increases ranging from . to . . when weak positive samples were pooled with four or eight negative samples (table ) , the positive samples were still all detected in five-sample pools, whether they were in vtm or mtm transport media. when combined in nine-sample pools, detection was more adversely affected for samples in vtm than those in mtm. for samples in mtm, one of three replicates for one sample, failed to be detected in a pool of nine samples. in contrast, for weak positive specimens in vtm transport media, nine-sample pools caused multiple replicates to return negative results. we then sought to assess what component of the total specimens tested are comprised of these weak positive specimens, to evaluate how much of an impact pooling might have overall on testing sensitivity across positive patient detection in the pandemic. further, to assess the positivity rate during the months since the onset of the pandemic in new york, since pooling strategies are not efficient unless sample positivity is low. despite the large range in number of specimens tested per day from late february through mid-may (figure ), the percentages of specimens with viral loads ranging from very strong (ct < ), strong (ct - ), moderate (ct - ), weak (ct - ) and very weak (ct - ) remained remarkably constant, with the exception of those in the very weak range which increased slightly during the last five weeks. overall, this weak positive sample type constituted an average . % of positive specimens, a substantial proportion of the positive specimens received for testing. positivity rates among samples received at this facility rose to almost % during march and has continually dropped since early april, remaining below % since th may and below . % since may . as the pandemic evolves, despite case counts, hospitalization rates and fatalities decreasing in some areas, laboratories continue to face new challenges. workloads have increased with, for example, requirements to perform repeated surveillance screening of asymptomatic health care workers and testing of patients undergoing elective procedures where there is a risk of aerosol production. these policies have pushed test numbers beyond those encountered even at the height of the pandemic wave. suggestions to help manage the load have included pooling of samples to enhance throughput capacity. when being considered for application in a clinical testing environment, of greatest concerns is the potential for this strategy to increase the false negative rate. with that, the greatest risk of false negative results is with weak positive samples. to investigate the potential limits of pooling in this situation, this study focused on pooling weak positive samples in relatively small size sample pools. had these been successful, larger pools would have been attempted. there are multiple methods for pooling samples, some of which carry an inherent risk of reducing test sensitivity and some of which do not. the method described in this paper, where the same volume of each sample is added to lysis buffer as would normally be added if the sample were being tested individually, does not theoretically adversely affect sensitivity, as long as extraction efficiency is maintained and the level of pcr inhibition is not increased by the additional load through the extraction device. when the data was analyzed, despite some minor shifts in ct values, no loss of detection was observed in any of the five-pool experiments, for samples that had been collected in either vtm or mtm. however, when the same weak positive samples were pooled with negative samples rather than , to create pools of , detection failures were observed. in three of the -sample vtm pools and one of the -sample mtm pools, this larger pool size resulted in a complete loss of detection. whether the less frequent occurrence in mtm samples compared to vtm samples is significant is difficult to say based on this limited data. the use of pooling as a throughput enhancement strategy is only efficient if the positivity rate in the samples being tested is low enough that a minimal number of pools will test positive, otherwise, multiple pools will have to be deconvoluted for retesting of individual samples. the optimal or maximum positivity level at which pooling starts to become efficient, depends on the pool size being used. for pool sizes as small as samples, this maximum positivity level is considerably higher than that for very large pool sizes that are sometimes suggested for large scale epidemiological screening studies. for example, at a positivity rate of % and a pool size of , on average, only in every pools will be positive and need to be deconvoluted for retesting. therefore, for every samples, testing could be achieved with a total of tests ( pools and one deconvoluted pool). we noted that the positivity rate in our own lab is now approaching % and therefore such a strategy may be efficient for extraction and detection. moreover, as the pandemic has progressed, there has been an increasing proportion of samples in the weak positive range, and therefore major consideration must be given to the issue of detection sensitivity for these weak samples. it must be noted however, that extraction and detection are not the only components of the laboratory operation and a pooling strategy does not enhance other aspects and may in fact create complications. processes for specimen receiving and accessioning, as well as those for result data management and reporting, are not reduced by pooling strategies. these procedures may be complicated by sample pooling, especially for electronic data transfer programs and laboratory information systems. a large increase in test load facilitated by a pooling strategy, may create serious workload bottlenecks for these and other areas of the operation. therefore, the global implications of a pooling strategy need to be carefully assessed, especially in the clinical testing environment, before implementation. coi sbg and gvs declare no conflict of interest. ksg receives research support from thermofisher for the evaluation of new assays for the diagnosis and characterization of viruses. she also has a royalty generating collaborative agreement with zeptometrix. report from the american society for microbiology covid- international summit overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for covid- detection all in': a pragmatic framework for covid- testing and action on a global scale laboratory testing of sars-cov, mers-cov, and sars-cov- ( -ncov): current status, challenges, and countermeasures assessment of specimen pooling to conserve sars cov- testing resources sample pooling as a strategy to detect community transmission of sars-cov- pooling of nasopharyngeal swab specimens for sars-cov- detection by rt-pcr evaluation of covid- rt-qpcr test in multi-sample pools hepatitis b, hepatitis c and hiv transfusion-transmitted infections in the st century pooling nasopharyngeal/throat swab specimens to increase testing capacity for influenza viruses by pcr large scale screening of human sera for hcv rna and gbv-c rna malaria diagnosis from pooled blood samples: comparative analysis of real-time pcr, nested pcr and immunoassay as a platform for the molecular and serological diagnosis of malaria on a large-scale estimating community prevalence of ocular chlamydia trachomatis infection using pooled polymerase chain reaction testing optimization of group size in pool testing strategy for sars-cov- : a simple mathematical model pooling urine samples for ligase chain reaction screening for genital chlamydia trachomatis infection in asymptomatic women pooling as a strategy for the timely diagnosis of soil-transmitted helminths in stool: value and reproducibility centers for disease control and prevention rvb, division of viral diseases. . research use only -novel coronavirus ( -ncov) real-time rt-pcr primers and probes the authors thank kamran zamani for assistance with data management and the provision of figure . they also thank jennifer laplante, rene hull and steven zink for specimen selection and retrieval. we thank the wadsworth covid response team for months of dedicated, skilled testing and careful specimen archiving that provided the well-characterized samples accessed for this work. key: cord- -n le ix authors: saxena, abhishek; tiwari, archana; kaushik, rinku; iqbal, hafiz m.n.; parra-saldívar, roberto title: diatoms recovery from wastewater: overview from an ecological and economic perspective date: - - journal: nan doi: . /j.jwpe. . sha: doc_id: cord_uid: n le ix alarming water pollution is toxic to the aquatic ecosystem leading to a sharp decline in species diversity. diatoms have great potency to survive in contaminated water bodies, hence they can be compelling bioindicators to monitor the change in the environmental matrices effectively. around the globe, researchers are intended to evaluate the impact of pollution on the diatoms recovery and techniques used for the assessment. the diatoms are precious for futuristic need viz. value-added products, energy generation, pharmaceuticals, and aquaculture feedstocks. all these applications led to a significant rise in diatoms research among the scientific community. this review presents different isolation practices, cultivation, and other challenges associated with the diatoms. a precise focus is given to diatoms isolation techniques from highly polluted water bodies with the main thrust towards obtaining an axenic culture to elucidate the significance of pure diatom cultures. recovery of “jewels of the sea” from polluted water signifies the prospective ecological and economic aspects. diatoms are the most successful contemporary group of photosynthetic eukaryotic microbes that inhabit almost every kind of aquatic ecosystem. they can occur as endosymbionts in dinoflagellates [ , ] . diatoms have arisen because of an endosymbiotic event between red algae and heterotrophic flagellate approximately million years ago [ ] . although there are several types of microalgae present in water, diatoms are the most important and unique as they are the only species with the presence of silica in their cell [ , ] . diatoms are surrounded by a silica cell wall that allowing them to have varied shapes and sizes. their silica cell wall is popularly known as frustules that offer protection from photoinhibition; supply appropriate nutrients uptake; manage sinking rate and playing a significant role as a mechanical barrier against grazers [ ] . their silica content varies from one species to another depending upon their size, growth pattern, and environmental variables such as light, temperature, salinity, nutrients, and growth phase [ ] . there are more than , extant species of diatoms distributed to a different ecosystem [ ] . they are widely present in all aquatic habitat constituting lentic, lotic habitat and sand. it is estimated that % of species occur in freshwater, and % are benthic [ ] . aquatic ecosystems such as rivers, oceans, lakes, etc. support the lives of a large variety of organisms but excessive urbanization and industrialization transform them into the heavily polluted zone [ , ] . the rising pollution level deteriorates the water quality index leading to a huge decline in species diversity due to untreated discharge of metals, pesticides, aromatic polycyclic hydrocarbons, etc., which slows down the survival of an aquatic species, thus posing a big threat [ ] . several organisms have been used as bioindicators such as protozoa, bacteria, fishes, zooplankton, algae, etc. however, diatom-based monitoring for the assessment of ecosystem remedy is in great demand [ ] . because diatoms produce organic matter to a large extent that permits natural inbuilt capacity to withstand toxicity levels in water bodies, extended survival rate, short regeneration time than microalgae, fishes, and other micro invertebrates thus making them one of the best candidate for water quality monitoring, and excellent bioindicators of aquatic biological integrity [ , ] . water quality analysis is essential as it allowed to detect the habitat linked information of the related organisms [ ] . as diatoms also produce eicosapentaenoic acid (epa), docosahexaenoic acid (dha), and other nutritional compounds, they can be used as a food source to every aquatic organism such as zooplankton and fish. they constitute % of oxygen in water bodies, fix excessive chemical compounds and inorganic nutrients in the water, such as carbon, phosphorous, silica etc., [ ] . as fossil fuels are diminishing quickly, therefore, the demand for biofuel production has increased many folds since the last decades. the crop and food-producing plants will never be the top priority for biofuel production. in the future, diatoms will remain as the best candidate for biofuel production. some strain of diatoms produces lipids as % to % of the dry weight that can be used in the production of biofuel thereby focusing on the role of diatoms as an ideal substitute for fossil fuels [ ] . despite having all these important characteristics, they are the least explored aquatic species because isolation and maintenance of axenic culture is a tedious job [ ] . in this review, an effort has been put forward on the details related to the isolation of diatoms from polluted water bodies, the significance of diatoms, and their future potential. rapid industrialization and urbanization releasing a toxic substance into the aquatic environment. thousands of chemicals without any proper risk assessment and legislation are being introduced, which are endangered to aquatic species [ ] . algae, bacteria, protozoa, fishes, micro invertebrates, and zooplankton have been extensively used in biomonitoring purposes, but recently, diatoms have gained much more attention because they are primary producers and plays a crucial role in biogeochemical cycles of the aquatic food web [ , , ] . diatoms survive well in a variety of habitat, such as oceans, lakes, estuaries, wetlands, etc. the use of diatoms in ecotoxicological studies is advantageous to examine any possible effects of toxic elements. this makes diatom a potential tool in bioassay than any other aquatic species because of its easy sampling and frustules identification [ , ] . diatoms are highly sensitive to metal pollution than macroinvertebrates, which could very well explain gradation in metal composition, thus allows early identification of the presence of any heavy metal pollution in an aquatic ecosystem [ ] . diatom responds quickly to any organic matter or nutrient contamination, for example, metolachlor, atrazine, simazine, phenols, nitrate, phosphate, heavy metals, and pahs [ , ] . even though diatoms are ecologically diverse, their potential in biomonitoring is underutilized in evaluating any risk assessment because of traditionally available metrices such as cell density, biovolume, and relative abundance. new and advanced metrices are more advantageous in recognizing the dynamics of diatoms communities by demonstrating the sub-lethal effect that is not evident in diatom counts [ , ] . nuclear abnormalities, cell membrane, cytoplasmic content, and photosynthetic apparatus alteration change in lipid content and diatoms classification using ecological status are the main parameters that were infrequently reported [ ] [ ] [ ] . this behavior demonstrates sensitivity and efficiency in quantifying the toxic elements, stress detection after ultraviolet (uv) exposure, which remains unnoticed by the conventional method [ ] . as reported, calculating live diatom species in the biofilms seems to be a lucrative bioassessment tool. similarly, for assessing weak cytoplasmic content of freshwater species, the ecological association of diatoms is examined for evaluating the environmental health of water bodies because these newer metrices are easy to acquire, rapid detection, low labor-intensive, good reproducibility, standardized and worldwide acceptance [ , ] . diatoms as bioindicators check and observe the integrity of the precious environment by enumerating the change in water quality over large geological areas, provide variability, and any possible backdrop precondition [ ] . investigator worldwide have examined various water bodies using pollution tolerant diatom species to measure the pollution level by employing algal indices such as palmer's algal index; chlorophycean index; nygaard's phytoplankton indices to better realize the water quality. for example, some investigator utilized nygaard's algal index to evaluate the trophic status of hatia dam revealing the oligotrophic condition of water bodies [ ] . mishra and co-workers studied the bhoj wetland, employing nygaard's algal index to illustrate the wetland's eutrophic nature [ ] . palmer index was utilized in determining the seasonal variation in phytoplankton diversity of pichhola lake of udaipur, rajasthan [ ] . sharan and rekha used nygaard's algal index in their evaluation of the trophic status of hatia dam, finding the waterbody to exhibit an oligotrophic status [ ] . but still, diatoms potential is hardly exploited as bioindicators in determining the economic conditions as compared to other groups of microalgae [ ] . the environmental conditions of the target species must be taken into account as understanding their habitat and mimicking them in the laboratory play a crucial role in obtaining pure strains. each aquatic ecosystem has different attributes. river ecosystems may have less alkalinity than a marine ecosystem [ ] . these attributes directly or indirectly affect the occurrence of diatoms in a diverse aquatic ecosystem. thus, several factors, such as seasonal variation, ph, temperature, salinity, etc. should be considered as important factors [ ] . the representatives of the genus gomphonemoid can be examined from both marine and freshwater. gomphonemoid is widely present often in freshwater epiphytic and epilithic communities [ ] . gomphonema, one of the most common diatom strains, its valve and girdle view, the heteropolar cuneate shape can be recognized easily. gomphonema is attached to the substratum by a mucilage stalk, which assists in recognize strain from the rest of the other diatoms found in the freshwater ecosystem. the valves are wide at the head than foot pole with both uniseriate and biseriate nearby amid individual areolae cover-up externally by crescent-shaped volae [ ] [ ] [ ] . the raphe is straight or sinuous with terminal fissures expanding both apices to the valve margin, and inner central endings turn aside to one or the same side, respectively. the raphe is always shorter from the central nodule to the head pole, thus extending towards the foot pole. consequently, a perforated distinct pole field of different structures is also present at the foot pole. in many species, isolated punctatum near the central nodule occurs. the cingulum is arranged into many open bands; every second band is shorter than the next one to form lingual at the foot pole. each band is punctured by more than one row of pores. many other diatom species with the same cuneate shape, growing in the marine ecosystem can be compared to gomphonema by various workers [ ] . benthic diatoms are the significant controller in freshwater owing to their excellent adsorption, absorption, oxidation, decomposition, precipitation capability to store nutrients. they are very well utilized in biomonitoring purposes since the last decade because these diatoms provide crucial linkage in the aquatic ecosystem stipulating the flow of energy, cycling of materials, and reveal the water environment [ ] . diatom based indices were first produced by kolkwitz & marsson [ ] . since more than indices have been created from to such as humic degree, trophic level indicators, eutrophication, biological diatom index to name a few. however, the impact of indices has become less focal owing to the differences between floral geographical area and environment fluctuations guiding species to adapt according to the water quality criteria of the particular region in a particular instance of time [ ] . isolation and identification of benthic diatoms are problematic in comparison with planktonic species due to difficulties in sample treatment, sampling, and microscopic observation though benthic diatoms play the main role as bioindicators in the aquatic ecosystem because they attached to the substratum with secreted mucilage from their cell wall [ , ] . the morphological features help in the classification of particular species. morphological analysis by light microscope (lm) and electron microscope, for instance, transmission electron microscope (tem), scanning electron microscopy (sem) provides the better-detailed result of diatom biodiversity [ ] . the detection of diatom communities on various substrata can be observed as the slimy layer of biofilm appears brown or yellow-brown. although there are several types of substrata for isolating diatoms, the most preferred is from cobbles (epilithon). epiphytes also have diatoms attached to them [ ] . the taxonomy of the epiphyte should be recorded as specific strains attach to specific macrophytes. a typical substratum can be introduced to the water manually if the preferred substratum is absent. the isolated biofilm can be preserved or can be used for the preparation of axenic culture. a well-concentrated sample collected from a highly polluted habitat may have a more significant number of microalgae rather than diatoms. sometimes an association between diatoms and other microalgae can be seen. it may also contain empty or broken frustules. highly polluted water may have a high concentration of biogenic compounds, which may provide less nutrition or over nutrition. this may profit in the growth of those strain, which requires fewer nutrients or high nutrients, respectively. salinity, ph, light, etc. factors also play a role in the growth of such diatoms [ ] . the productivity of diatoms is higher than the other class of microalgae. they have undergone - cell divisions per day. they can survive in adverse climatic conditions since their silica cell wall is a nanoporous structure which allows them to act as a potential buffer system by improving carbonic anhydrase activity and helps in the transformation of bicarbonate to carbon-di-oxide (co ). they actively participate in wastewater remediation and quenching of heavy metal, leading to a degradation of environmental pollution [ ] . though bacteria, fungus, yeast, and algal biomass are commonly used in the treatment of heavy metal, diatoms are most preferred due to their large surface area, mucilage, nutritional requirement, and faster growth rate. bioremediation of heavy metal involves biosorption to metal-binding ligands on the cell surface, followed by bioaccumulation consists of inorganic molecules and related enzymes. in biosorption, the uptake of heavy metals is either carried out by the physiochemical pathway devoid of atp or during metabolism engaging atp energy. this mode of heavy metal remediation is a cost-effective approach through filtration. it is an extracellular passive route of adsorption of heavy metal, which is an energy-independent process occurring at the cell surface in equilibrium [ ] . biosorption is dependent upon temperature, ionic strength, ph, contact time, biomass and metal concentration, the composition of the cell wall, and metallic ions complexation. the principle behind biosorption is that diatoms are equipped with polymers, their outer wall has a negative surface charge due to functional groups like carboxyl, phosphoryl, and amine. the heavy metal with a positive surface charge attracts a negative charge through electrostatic interaction, which finally supports biosorption. bioaccumulation is an active intracellular process driven by energy. the heavy metals grow along with nutrients that assist in bioremediation. when heavy metals are accumulated, the microalgae produce reactive oxygen species (ros) to control cellular metabolism. there is an underlying passive uptake followed by an active process that is brought about by the detoxification of heavy metal at the cell surface by dehalogenation and denitrification process. this triggers the exclusion of heavy metals to maintain equilibrium conditions, thus eliminate toxic effects. despite this, a lot of work needs to be done in the treatment of wastewater by testing more species and study their capability of heavy metals removal. the biological degradation of heavy metal is quite challenging but not impossible [ , ] . previously, diatoms have been differentiating according to shape, size, pattern, and ultrastructure of their exoskeleton, which consists of silica, known as frustules. microscopic identification is based on the shape of their frustules, but still, they are challenging to identify, and improper examination reflects misleading observation upon identification beyond the genus level [ ] [ ] [ ] . to combat such a drawback, deoxyribonucleic acid (dna) sequence analysis has become a more potent approach in diatom research, thus opens a new leaf into their systematic and evolution. the main aim of the taxonomic examination is to make a hierarchical classification accurately reflecting evolutionary relationship, which then converted into a hierarchy of names related to different levels in a hierarchy of taxonomic ranks. recently, diatoms phylogenetic analysis at the generic and infrageneric levels require internal transcribed spacer (its) region of the s- . s- s nuclear ribosomal dna and among all, s ribosomal ribonucleic acid (rrna) sequencing resulting in the largest reference sequence dataset among the different markers used for diatoms [ ] [ ] [ ] . microarray technology enables parallel analysis of many genes in a single reaction. here, ordered matrices of dna sequence marked on the glass slide used for hybridization experiment with fluorescently labeled target genes and have been extensively used in both basic and applied aspects of the biological sciences [ ] . though several papers related to taxonomy and most of them provide line drawings or light microscopic images that are unable to give enough ultrastructure details of the diatom cell [ ] . a complete taxonomic survey can give the entire morphology of lesser-known microalgae or diatoms. as there can be a minor difference in the morphological structure of two or more organisms, such organisms are difficult to identify even under the lm. organisms like cyclotella brebisson may comprise more than complex species, which includes small specimens with a diameter of ≤ μm [ ] . based on frustules morphology, observation of these small specimens under a sem provides notable interspecific similarities that help in taxonomic studies. as some species may have similar morphological characteristics which may be ecologically distinct, their identification should be fundamentally accurate as it can provide quite an information about biodiversity which can be represented as an essential tool for different kind of studies such as ecological and applied studies. proschkinia is a rare diatom of the family proschkiniaceae within the naviculales. based on light microscopy, it was classified as a relative of nitzschia. the phylogenetic position of proschkinia was resolved with sequencing the complete mitochondrial genome of proschkinia complanatoides and compared the data set to other published diatom mitochondrial genes [ ] . to differentiate between diatoms strains that have similar morphology, it is vital to know the taxonomy of the target species. the nutritional requirements of various species also vary. some species require less nutrition composition, light temperature variables, while some require more. the taxonomy also helps in understanding the basic requirements of the target species [ ] . once the culture has been collected and identified, they will be preserved in the world federation of culture collection centers, which then made available to researchers and industry for intending applications and further experiments. table shows the list of available algae, microalgae, and diatom culture collection centers worldwide. the field procedures play a crucial role in the isolation of diatoms, microalgae, or other species. in a riverine environment, small boulders (rocks) and cobbles are the most favored substratum for diatoms screening. almost all diatom indexed to the eplithion community found here where epilithion`s ecology is recognized better than any other group [ ] . bricks, concrete, bridge supports, canal walls, etc. may be used as an alternative substratum, whereas simulated substrate can be set up into the stream if pebbles, cobbles, boulders, or macrophytes do not exist from the site though sampling should be done if they have been plunged for several weeks [ ] . sampling is an important step towards the collection of diatoms as the whole isolation process depends on it. the collected water sample may contain dead cells or damaged cells, which is not considered good, so it becomes very crucial to ideally collect the sample and isolate the most viable cells at the earliest. keeping the water sample for longer times is not recommended because environmental conditions change very frequently, which leads to cell death. some species multiply quickly and suddenly die, in that case, isolation should be done rapidly. sometimes, a planktonic net can be used to concentrate the sample as it removes other unwanted algal species, microorganisms, or debris but should be avoided as more concentrated sample leads to more damaged cells [ , ] . the collected sample should be kept according to the target species` environmental conditions. for example, several marine species are susceptible to sample concentration. for a sampling of these strains of species, special water bottles can be used. as several factors like water depth, pressure, temperature, or light influence the sample conditions, so instead of using a concentrated sample, a normal water sample taken in sterile containers can provide a better result. the collected sample should always be kept in sterile containers at a stable temperature [ ] . often sample collected from different water bodies contains zooplankton or other species which feed on algae or specifically on diatoms. so, it is essential to remove the unwanted organisms gently by filtering the sample at the sample site. however, sometimes tiny organisms may pass through the filter, which may pose threats to target species, in that case, dilution methods can be used to isolate the target species. the health of species in nature also plays a crucial role as an isolated cell will have successful growth only if they were in a good state at the sample collection time. it is advised to collect the sample carefully. the use of sterile equipment is mandatory, as dirty equipment may lead to contamination. all the isolation techniques should be performed under sterile conditions [ ] . a general idea about field procedure, laboratory procedure, culture preparation, and finally aim towards obtaining axenic culture is presented in fig. . the identification of a suitable substrate is a pivotal step to recognize the diatom communities in the natural environment. diatom communities form a slimy layer or thin golden-brown layer on the substratum. it can be more noticeable by feel or touch and can be identified at a specific time depending on species. the substratum can be differentiated into a preferred substrate and an alternative substrate. diatom attaches to different substrata or facilitates locomotion by releasing mucilage from various structures of the cell wall [ ] . diatom community composition is mainly influenced by chemicals present in water, water turbulence, temperature and light present in water, being eaten by large microorganisms, etc. the most preferred substratum is the solid substratum. it mainly includes cobbles/pebbles and small rocks (epilithon). fig. depicts various substrates intended for diatoms sampling. they are widely available in almost every aquatic habitat and throughout the year. in the absence of a solid substrate, submerged or emerging macrophytic plants (epiphyte) should be sampled as it also provides a suitable habitat for diatom communities. it is ideal to note the microhabitat of diatoms species [ ] . the most effective method to eliminate contaminants is achieved by various techniques such as filtration, sedimentation, and centrifugation as gravity separation. the most successful technique is filtration as it removes all unwanted microorganisms or other zooplankton, which feed on diatoms from the sample. in this, a planktonic net of a specific size is used, which does not allow any other organism to pass through except the target species. in gravity separation, diatoms settle down as sediments while leaving other microalgae in the suspension, which can be discarded further [ ] . field operators should always wear thigh waders for the protection of feet. they should wear life jackets while sampling. they should never go in-depth for sampling. they should wear globes when sampling heavily polluted water bodies. they should be very careful in habitats dominated by dangerous animals posing a threat to people and blood- fig. . flow-chart depicting isolation of diatoms from sample collection procedure to obtaining axenic culture of target species. sucking worms like leeches [ ] . anthropogenic pressure from the urbanized area causes changes in the water quality of a river and leads to the eutrophication of reservoirs and the proliferation of algae. the informal settlements without sanitary infrastructure aggravate the deterioration of water quality in urban water sources [ ] . the water quality index, conducted by using the planktonic diatom index (pdi) for monitoring the lake erie's nearshore pelagic zone, provides a robust assessment of water quality [ ] . the collected water sample should be adequately analyzed as it gives full information about the diatom habitat and its environment. several factors should be measured. hydrological characteristics of the stream, which mainly examined with stream velocity and channel depth and channel breadth. physical variables of water mainly involve water temperature and turbidity. physico-chemical variables are examined with ph, conductivity/total dissolved solids. water contains nutrients such as orthophosphate-phosphorus (po [ ] . for monitoring the pollution of water bodies, several indices have been developed, such as the index of saprobity-eutrophication (idse/ ), which utilizes the diatom population in terms of organic pollution [ ] . water can be divided into two types such as groundwater and surface water and both are at the risk of pollutants ranges from heavy metals, pesticides, fertilizers, hazardous chemicals, etc. an accepted water quality criterion according to american public health association (apha), the world health organization (who), indian standard institution (isi), indian council of medical research (icmr), and central pollution control board (cpcb) consisting of various parameters are presented in table [ ]. examination of the collected sample from different substrata should be done as quickly as possible to make sure that diatom assemblage contains live cells and not dead ones. sample with more dead cells should be discarded, and sampling should be performed again. the environmental conditions of samples collected from various aquatic habitats should be mimicked in a laboratory. it should be noted that all equipment like glass-slide, coverslip, pipette, forceps, inoculating loop, centrifuge tube, glass test tube, watch glass, etc. must be sterilized before use. uncleaned equipment may lead to contaminations that further result in the death of cells. after examination of diatoms in the collected sample, standard isolation methods should be performed [ ] . as the collected sample contains various microorganisms and other microalgae, these standard isolation methods play a crucial role in the isolation of target species of diatom. the isolator should focus on finding the target species in the collected sample. viable cells should be cultured as soon as possible while avoiding contaminants. all the following methods should be performed carefully [ ] . the simplest method of single-cell isolation is performed with the help of micropipette, first attempted by zumstein ( ) later improved by pringshiem ( ) [ , , ] . a capillary is attached to the pasteur pipette to make it ideal for single-cell isolation. this method requires a lot of practice. as diatoms have a minimal size, one single cell can also be picked separately with the help of a micropipette. single-cell isolation with micropipette is to pick a single cell of target species without getting it damage and avoiding contaminants and deposit it into a sterile tube or watch glass and then transfer it to a culture tube. the whole process is done with the help of a microscope, especially an inverted microscope, as it allows the isolator to work conveniently [ , ] . this method is very useful while isolating cells from very contaminated samples with a tiny microorganism, which is harder to separate from the sample. the aim of this method to separate one single cell of target species and deposit it in a culture test tube. usually, dilution is repeated serially up to : [ ]. it is probably the oldest and standard method available to isolate microorganisms. most of the diatom species usually grow well in agar. isolation using agar can be used with both "streaking" and "spreading" methods. later single cells or colonies can be picked from the culture plate and observed under the microscope. to study the colony/diatom community characteristics, agar plate method is advantageous. the agar concentration should be between . % to . % and . %. while observing glass slide under a microscope, if it contains a single diatom cell, it can be replicated on the agar plate for culture. agar pour plate method can also be used to isolate cells that do not grow on the surface of the agar plate [ ] . this is a physical technique for the pre-separation of single cells of diatoms from polluted water. the centrifugal technique applies gravity settling to isolate more prominent species from the microalgae. low dense cells, for example, microscopic organisms and others present in the supernatant, are emptied while diatoms species stay at the base as a pellet. the speed and time of centrifugation vary depending upon the target microalgal species. even though reasonably successful, however, it might harm delicate cells through sheer pressure [ ] . this method involves the use of enrichment media. enrichment media is rich in one constitute which provides nutrition to only one/two types of cells (species) while inhibiting the growth of other cells (microorganisms). for example, it has been noted that the presence of silica in a culture media boost the growth of diatoms but inhibit the growth of other microalgae. sometimes even a trace element makes an enormous difference in the growth of species. so, it is necessary to know the nutritional requirement of the target species, and it should be reached as it will improve the growth of the target species [ , ] . this is a pre-isolation step towards the separation of diatoms. samples can break into two portions depend upon particle size difference. sometimes enrichment media can also be utilized for better outcomes while separating fungal cells from diatoms. bigger species can be held at the membrane however, diatom cells pass through the filter quickly. this strategy is helpful, advantageous, and very adaptable [ ] . another technique where alginate beads were utilized to isolate the algal species from the mixed algal culture [ ] . in this method, the contaminated water is placed in nutrient media and incubate for one week or more to affirm the development of different species. a known concentration of sodium alginate solution containing a mixed culture of diatoms is added drop by drop to a calcium chloride solution to form alginate beads and kept for - h to make beads stronger, stiff, and firm. the beads transferred to a well enzyme-linked immunosorbent assay (elisa) microplate (one bead per well), smashed partially containing standard culture media and incubated under controlled conditions in the presence of light for one week to confirm the growth of isolated diatom species. the trapped diatom species in the beads viewed microscopically for the confirmation of isolated species. this methodology is simple, easy, cost-effective compared with existing methods and can be easily applied for the mass cultivation of specific species [ ] . there are other different strategies for the isolation of diatom species like anti-microbials, uv radiation. anti-microbial treatment hinders growth & development by eliminating microorganisms, thus help with getting the confined unadulterated culture of diatoms. uv radiation act as a disinfectant that prevents bacteria and other contaminants since diatoms are better resistant to uv radiation over bacterial cells [ ] . photoinhibition is an immediate strategy that harms biomolecules by engrossing uv light, which prompts the loss of natural capacity of microscopic organisms [ ] . diatoms are valuable for the aquatic food chain and give significant bioactive compounds to human prosperity. segregating unadulterated species or getting axenic culture from a polluted water source is a complicated and tedious procedure with the current strategies. hence, it becomes necessary to search for other advanced isolation techniques. micromanipulation is one such method permitting refined, confining single species in a more cleansed manner. prior fine capillary tubes were used focusing on target cells under microscopic observation, which is a laborious task and threat to contamination [ ] . with the arrival of a modern, sophisticated micromanipulator exploiting micromanipulator and stereomicroscope, a high level of precision can be achieved for screening and isolation of species of interest [ ] . a cell of interest can be captured utilizing a focused laser and transferred to a sterile media of interest. this system is still in its early stages because of the high state of expertise and time required [ ] . the requirement for cutting edge new methods push researcher to redesign the flow cytometer coupling facs (fluorescence-activated cell sorting). the premise of this procedure is light scattering and fluorescence [ ] . cells absorb the laser beam transmit fluorescence and give information on cell size, pigments, and reliability of species, which is identified with the morphology and other characteristics of the species accordingly, thus allowing characterization of up to , cells in a fraction of second [ ] . this technique can be employed to build up an axenic culture and get rid of any bacterial contamination. in some cases, mixed culture or aggregation of cells may make issues in the precise identification of cells, which can be overcome by cell disruption through appropriate sonication under controlled conditions. this technique is generally excellent for quick screening of organisms overproduces metabolites of interest in conjunction with fluorescent dyes. for example, bodipy or nile red facilitates the selection of desired mutants from a mixed population [ ] . an overview of the main techniques used to establish axenic diatom cultures is presented in table . diatoms, the most productive phytoplankton found all over the planet from antarctic glaciers to brick walls, have drawn a tremendous awareness in the research field, i.e., for the quantitative reconstructions of ocean surface conditions to establish palaeoceanographic records [ ] . they are one of the most promising candidates for various applications such as pharmaceuticals, bioenergy, industrial chemicals, nutraceuticals, and aquaculture [ , ] . to make most out of it, an axenic culture of diatom must be a prerequisite bearing in mind that a pure culture of diatoms is undoubtedly required in genome sequencing [ ] , to identify the producer of any novel bioactive compound for large scale manufacturing of nutraceutical [ ] , building a consortium for bioremediation [ ] and to elucidate the relationship between other microalgae using omics tool [ ] . maintaining an axenic culture for a longer duration is very difficult because bacteria are vulnerable, which frequently attacks the diatom. the primary focus is to isolate pure species of diatoms and their maintenance [ ] . nevertheless, an axenic strategy is dependent upon contamination and desirable organisms disclosing the possible relationship between them leading to the understanding, selecting, and developing an axenic culture, which is a critical step, thus alleviate the cultivation method. mimicking the natural environment for optimum growth under laboratory conditions facilitates the development of an axenic culture, but this step requires the correct isolation plan and approach [ ] . the fundamental question is, are axenic culture genuine.? what is the acceptability of their purity level.? with the direct symbiotic association between diatoms and other aquatic organisms, the development of a new advanced technique to assess the purity level cannot be ruled out. therefore, emphasis should be given towards improvement and innovation in isolation methods, to standardize and establish the correct isolation practices to solve future energy crises, nutritional requirement, nutraceuticals, pharmaceuticals by choosing the right technique for right diatom species for the benefit to the mankind, society and last but not least the ecological balance [ ] . therefore, constant effort to develop a new scientific method will surely pave the way towards the isolation of diatoms from mixed culture and maintaining their purity level to a greater extent. diatoms-virus interactions are rather difficult in obtaining pure culture. the virus kills diatoms, thus benefitting other algae. the limitation in silicon levels in the oceans facilitates infection by the virus. accelerating diatoms mortality due to viral infection is a major concern affecting the carbon cycle leading to global warming and ultimately changes climatic conditions drastically. also, ocean study is a difficult task. the main emphasis is given towards the marine environment rather than terrestrial causing disparity about diatoms-virus interactions. as a result, more isolations techniques and characterization is necessary for controlling the action of the virus in the regulation of host populations [ ] . several species of diatoms are found in a highly acidic environment, and they continue to grow consistently near or in the acidic conditions both in marine and freshwater ecosystems. they exhibited a significant response to the alterations in the growth conditions. they are capable of integrating numerous physiological as well as morphological adaptations, which favors their persistence. diatoms growth inhibited majorly in silica limitations than any other nutrients because cell division cannot sustain for a more extended period under silica deprived conditions [ ] . highly acidic conditions resulted in decreased si, indicating that close to the end of this century ocean acidification might persuade the c and si cycle and alter the composition of diatoms. [ ] . considerable human interference is turning aquatic bodies towards the acidic zone, and many countries have seen ocean acidification even in extreme low-temperature conditions such as north america, canada, and italy. due to the high concentration of hydrogen ions, diatoms flora like nitzschia, pinnularia, eunotia, and frustularia are exceptionally rich in habitats within the ph range of . - [ ] . in an acidic environment, a higher cell volume, chlorophyll, and productivity were observed due to a change in water chemistry because in acidic conditions number of grazing macroinvertebrates and microheterotrophs were less in number [ ] . however, it is unattainable to understand the natural influx of pre-acidification, natural fluctuations in conditions, and the level of nutrients that tend to vary from habitat to habitat. abundance and fall in diatom species can be explained based on physiological ph, availability of the essential nutrients, and biological interactions, which could be the factor for the productivity of diatoms in extreme conditions. very little is known about the influence of acidification on diatoms. hence the effect of acidic environment on diatoms changes from the open sea, near to the sea and deep-sea, and indeed not a decrease in diatoms productivity and growth [ ] . since diatoms nurture associated aquatic species to a greater extent within the marine environment so achieving utmost purity and break communication with unwanted organisms is a significant challenge. therefore, emphasis should be given to improve and channelize knowledge towards proper isolation techniques that will separate diatoms from contamination and any possible intervention to promote appropriate growth and development of diatoms [ , ] . an outline of isolation of pure diatom species getting affected by the surrounding contaminants is challenging since they get heavily occupied with different interfering organisms, which pose a significant threat in obtaining axenic culture, as presented in fig. . although there are several culture media are available, but the most recommended culture media for isolation and growth of diatoms are f/ -si, pm, and wc media. the composition of each culture media is prescribed according to the selection of diatoms for culturing for the desired application. f/ -si culture media is widely used as the most effective media, whereas pm media is utilized for culturing diatoms in an acidic environment. wc media is generally used for culturing diatoms existing in an alkaline environment [ ] . all these culture media differ in their composition of major and minor nutrients and provide proper nourishment to diatom culture. f/ -si media is required for marine species while pm and wc media supports the cultivation of freshwater species. for marine, it is advised to prepare both media with seawater and for freshwater prepare media with distilled water [ ] . the chemical composition of different culture media for diatoms cultivation are presented in table . elimination of contaminants is important to maintain the pure culture of isolated species. as stated, the above contamination can be eliminated with gravity separation, dilution techniques, etc. in the case of bacterial contamination, antibiotics should be used in a specific amount in culture. every equipment, glassware used in isolation or culture should be sterilized before performing experiments. culture should be maintained in aseptic and optimum conditions [ ] . after preparing the axenic culture, it should be subcultured after over a specific period. subculturing should be done before the decline phase of the primary culture. each subculture should be prepared in aseptic conditions. after each subculture, it should be observed for contaminations, and in case of occurrence of contamination that specific subculture should be discarded, and the process is repeated [ ] . an axenic culture contains only one target species and is free of all contaminations. after isolation of target species of diatoms, it is necessary to maintain the culture free from contaminants so that a pure culture can be established. pure isolated cultures can be obtained from a combination of techniques such as flow sorting, pasteur pipette, and agar plate methods. the polluted water collected from various habitat will be immediately examined by monitoring ph and temperature at the site and will be carried to the laboratory in - liters plastic bottles. the raw wastewater was filtered twice with a . μm pore size whatman filter paper to remove the large suspended solids particles and debris. the wastewater will be autoclaved for sterilization and further used for the cultivation of the microalgae. the physico-chemical parameters of wastewater will be characterized as per the standard procedure of apha guidelines [ , ] . initially, the water sample will be serially diluted in a microwell plate and test tube to get a pure culture and observed under the microscope to confirm the presence of microalgae species. isolation will also be carried out by spreading or streaking wastewater in a solid agar medium supplemented with silica. once confirmed, the microalgae species were transferred slowly and gradually in different volumes of erlenmeyer flasks containing artificial seawater enriched with f/ -si media at ph . and maintained in the culture room at h dark/ light diurnal cycle with a desired luminous intensity at - • c. finally, strains will be identified, and their taxonomic classification will be established. fig. shows the overview of obtaining pure culture diatoms from polluted water bodies. diatoms are very young as compared to other phytoplanktons but have evolved rapidly over some time. a decline return on investment in research and development (r&d) and slow-growing companies accelerating the demand for diatoms cultivation serving as a superfood. the term nutraceutical refers to nutrition that provides physiological benefits coupled with the protection and prevention of disease. these functional foods promote health by adding novel ingredients that are similar to conventional foods but with rich nutrition or bioactive compounds that may target the physiological mechanism of our body as characterized by the us department of agriculture, agricultural research service [ ] . on account of large content of polyunsaturated fatty acids (e.g., pufas n and n ), essential amino acids (e.g., leucine, isoleucine, and valine) and pigments (e.g., lutein and β-carotene) and vitamins (e.g., b ), the diatoms biomass have gained much popularity across the globe [ ] . nutraceutical industries manufacturing products on a large scale in collaboration with the food and pharmaceutical industry, thus benefitting consumers. diatoms are emerging as leading nutraceutical ingredients from a biotechnological point of view since they are equipped with polyunsaturated fatty acids, essential amino acids, pigments, and vitamins. according to the world health organization who, the most severe challenge of the st century is a lifestyle disease, i.e., noncommunicable disease. the current focus is to develop functional food and their products, which combat the disease development [ ] . diatoms are underutilized among the microalgae, and only a few species have been characterized thoroughly. diatom based products are an fig. . shows the main interfering agents or treats in obtaining axenic culture of diatoms. . × - . × - . × - excellent source for multifaceted use covering the health and nutraceutical sector. therefore, the cultivation of diatoms under different cultural conditions helps to understand their biochemistry to a large extent [ ] . diatoms cells, after degradation, settled down in the form of silica, which is known as diatomaceous earth. these death remains have tremendous applications for industrial and agriculture purposes. ongoing covid- impacted the world economy. the diatomite industry has also suffered significantly but successful in maintaining an optimistic growth for four years. the average annual growth rate of the diatomite industry will reach millions usd in . probably by , the market potential will see a significant expansion [ ] . diatomaceous earth is one of the major causes of their successful existence on the globe and also act as carbon dioxide sequester on the ocean floor. however, more information is needed about nutrient upwellings and the causes of their bloom degradation in their natural habitats. further, a more in-depth study on their self-defense mechanisms will reveal the unfolded truth about the interaction of diatoms and their feeders in the food web. to overcome the constraints of fossil fuels, the generation of bioenergy makes renewable energy particularly interesting. nowadays, renewable resources are contributing % of the total energy of the world. diatoms synthesize oil for human consumption, which is rich in nutrition showing a promising alternative to meet the demand of future growing populations. we need to emphasize the latest, more productive technologies to overcome the cost of their cultivation and harvesting for optimum utilization of diatoms as biofuels, valuable products, wastewater remediation, and aquaculture [ ] . the generation of bioenergy from novel sources like diatoms is incredibly significant as they contribute towards carbon dioxide mitigation generation of renewable energy concomitant with a plethora of value-added products. due to their unique evolutionary history and their adaptations to varied environmental conditions, diatoms have spread all around the world. undoubtedly, diatoms are playing a significant role in the reduction of global warming gases such as carbon dioxide and provide possibilities to change the present climatic conditions. but still more researches are needed on their biogeographic distribution and to learn more about factors which are responsible for their more successful existence. diatoms cells, after degradation, settled down fig. . protocol for adopting isolation strategies employed for diatom axenic culture. a. saxena et al. in the form of silica, which is known as diatomaceous earth. these death remains have tremendous applications for industrial and agriculture purposes. diatomaceous earth is one of the major causes of their successful existence on the globe and also act as carbon dioxide sequester on the ocean floor [ ] . however, more information is needed about nutrient upwellings and the causes of their bloom degradation in their natural habitats. further, a more in-depth study on their self-defense mechanisms will reveal the unfolded truth about the interaction of diatoms and their feeders in the food web. to overcome the constraints of fossil fuels, the generation of bioenergy makes renewable energy particularly interesting. nowadays, renewable resources are contributing % of the total energy of the world. diatoms synthesize oil for human consumption, which is rich in nutrition showing a promising alternative to meet the demand of future growing populations. we need to emphasize the latest more productive technologies to overcome the cost of their cultivation and harvesting for optimum utilization of diatoms as biofuels, valuable products, wastewater remediation, and aquaculture. they are the primary food of most of the herbivores in freshwater, as well as in marine water. furthermore, being the rich source of poly unsaturated fatty acid (pufa), they have a great scope in aquaculture. therefore, diatoms are the cost-effective approach which sequesters co , excess amount of heat, reduce the extra amount of nutrients, and remove metal contaminations from their habitats. they could be a possible solution to energy savers and to reduce the threat of global warming. conversely, they may be a viable source of food and feeder for living beings. diatoms and wastewater are an advanced integrated system that, on the one hand, utilize the potential of diatoms in purifying the wastewater thus, it helps in restoring the original quality of habitat. on the other hand, their biomass can be utilized in the production of antiobesity, antibacterial, antioxidant, biofuels, anticancerous, antiviral compounds. diatoms have received great attention because they are the significant producer of lipids and biomass for various commercial applications such as biofuels comprising of biodiesel and aviation fuel. they participate in cleaning the environment by fixing the atmospheric co and contributes to the reduction of greenhouse gas (ghg) [ ] . diatoms based biofuels are projected to be economically beneficial in the future, but still, some ambiguity exists whether they equate with fossil fuels or not is remain a question. obtaining a biofuel require several steps like harvesting, extraction, conversion of biomass to target biofuel is economically not feasible. one of the key bottlenecks is cell density, which is similar to the water and negative surface charge that put off settling owing to gravity, thus makes harvesting a most crucial and challenging step. there is an urgent need for an efficient harvesting technique to improve the economics and efficiency of the whole downstream process [ , ] . at present, the technologies of the water purification industry have been used for harvest and recovery of microalgae, but still, some technical glitch needs to address that are sole to microalgae harvest. firstly, harvesting techniques must be species-specific. secondly, a blend of different harvesting techniques must result in synergistic outcomes. thirdly, a complete cell separation from a diluted suspension to lower the cost of the downstream process. fourthly, steps such as lipid extraction and biofuel conversion should be minimized. finally, to come up with the idea of more advanced harvesting techniques in the future [ ] . the ultimate goal of economic feasibility is the conversion method by utilizing inexpensive chemicals and the release of the least toxic waste. microalgae biomass is the best option for fossil fuels for transportation, but the commercialization of microalgae-based biofuel is a significant task. new strategies should encourage innovative elements to the existing downstream process that can appreciably reduce the cost towards the realization of biofuel commercially and dispose of harmful co production. to sum up, harvest, extraction, and conversion steps must be environment friendly, while making biofuels as future transportation fuels [ , ] . all the above-mentioned factors enhancing the popularity of diatoms globally strengthen their market potential and growth in the coming years. their medicinal characteristics are likely to provide more interest in diatoms cultivation with a rising population. considering health benefits, the global diatoms market is expected to witness as one of the most emerging economies across the world. a general question raised by everyone about diatom`s attractive potential and advantages but still exploring their true value is a long journey because high rich diversity limits their studies making it obscure and inaccurate. it is high time to investigate new class and species of diatoms and understand their mechanism, research models, and the potential role for future perspectives. the past few decades have seen a tremendous rise in diatoms research, as reflected in the publication. an undercurrent excitement is moving around, but challenges are roaring high. practically, a good isolation practice is the only solution that will fill this void. smart isolation practices are prerequisites that must help quick isolations and speedy recovery of diatoms from polluted water. diatoms`axenic culture is the first step towards a complete study of an organism and maintaining their pure culture can be further used in different applications such as bioindicators, nutraceuticals, cosmetics, phycoremediation, aquaculture, etc. thus, growing 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diatoms a morphological classification capturing functional variation in phytoplankton protocols for collection, preservation and enumeration of diatoms from aquatic habitats for water quality monitoring in india protocol for seaweed decontamination to isolate unialgal cultures challenges regarding water quality of eutrophic reservoirs in urban landscapes: a mapping literature review validation of a diatom-based index of water quality confirms its utility in monitoring of the lake erie's near shore area a new species of pleurosigma from western ghats, south india biomonitoring of selected freshwater bodies using diatoms as ecological indicators a review of permissible limits of drinking water protocol to establish axenic cultures for diatoms of fresh water zur morphologie und physiologie der euglena gracilis klebs jahrb pure culture of algae a note on the isolation of small marine algae and flagellates for pure cultures traditional microalgae isolation techniques microalgae isolation and basic culturing techniques. handbook of marine microalgae aseptic laboratory techniques: plating methods coagulant derived from waste biogenic material for sustainable algae biomass harvesting bioprospecting for hyper lipid producing microalgal strains for sustainable biofuel production characterization of soluble algal products (saps) after electrocoagulation of a mixed algal culture a new method to isolate algal species from mix algal culture survey and isolation of marine cyanobacteria from eastern coast of india as a biodiesel feedstock potential use of flow cytometry in microalgae-based biodiesel project development facilitating harmful algae removal in fresh water via joint effects of multi-species algicidal bacteria flow cytometry for the development of biotechnological processes with microalgae diatoms in arctic regions: potential tools to decipher environmental changes, polar sci a novel growth method for diatom algae in aquaculture wastewater for natural food development and nutrient removal applications of diatoms and silica nanotechnology in biosensing, drug delivery, and formation of complex metal nanostructures comparative genome and transcriptome analysis of diatom, skeletonema costatum, reveals evolution of genes for harmful algal bloom indigenous marine diatoms as novel sources of bioactive peptides with antihypertensive and antioxidant properties bioremediation of chromium contaminated water by diatoms with concomitant lipid accumulation for biofuel production omics approaches for microalgal applications: prospects and challenges an efficient screening method for the isolation of heterotrophic bacteria influencing growth of diatoms under photoautotrophic conditions the biogeography and ecology of common diatom species in the northern north atlantic, and their implications for paleoceanographic reconstructions first viruses infecting the marine diatom guinardia delicatula silicon metabolism in diatoms: implications for growth a review of diatoms found in highly acidic environments functional plasticity of benthic macroinvertebrates: implications for trophic dynamics in acid streams effects of acidification on diatom communities as revealed by analyses of lake sediment the phaeodactylum genome reveals the evolutionary history of diatom genomes interactions between diatoms and bacteria culture media for mass production of microalgae algal-based removal strategies for hazardous contaminants from the environment-a review -microbiology laboratory techniques rheological property analysis of pyrolytic liquid fuel (plf) using astm and apha standards microalgal derivatives as potential nutraceutical and food supplements for human health: a focus on cancer prevention and interception bioactive compounds from microalgae: current development and prospects diatomite (diatomaceous earth) market covid- impact analysis global industry analysis by trends, size, share, company overview, growth and forecast by a review of the potentials, challenges and current status of microalgae biomass applications in industrial wastewater treatment preparation, characterization and catalytic performance of mesoporous silicates derived from natural diatomite: comparative studies ocean acidification interacts with variable light to decrease growth but increase particulate organic nitrogen production in a diatom methods of downstream processing for the production of biodiesel from microalgae innovative microalgae biomass harvesting methods: technical feasibility and life cycle analysis dr. archana tiwari is thankful to the department of biotechnology (dbt), new delhi, india for providing financial support under project grant no: bt/pr/ /aaq/ / / . all listed authors are grateful to their representative universities for providing the literature access. key: cord- -e phs jd authors: ford, richard b.; mazzaferro, elisa m. title: section diagnostic and therapeutic procedures date: - - journal: kirk & bistner's handbook of veterinary procedures and emergency treatment doi: . /b - - - - . - sha: doc_id: cord_uid: e phs jd nan for anorexic dogs or when pills must be given without food, give medications quickly and decisively so that the process of administering the medication is accomplished before the dog realizes what has happened. with cooperative dogs, insert the thumb of one hand through the interdental space, and gently touch the hard palate. this will induce the cooperative dog to open its mouth (figure - ) . using the opposite hand (the one holding the medication), gently press down on the mandible to open the mouth further (figure - ). note: oral medication frequently is dispensed to owners without regard for the client's knowledge of how to administer a pill or tablet or without asking whether the client is even physically able to administer medications. clear instructions that include having the client perform the technique in the hospital significantly improve compliance. quickly place the tablet or capsule onto the caudal aspect of the tongue. quickly withdraw the hand and close the dog's mouth. when the dog licks its nose, the medication likely has been swallowed. dogs that offer more resistance can be induced to open their mouths by compressing their upper lips against their teeth. as they open the mouth, roll their lips medially so that if they attempt to close the mouth, they will pinch their own lips. alternatively, dripping water onto the nostrils or blowing into the patient's nose sometimes encourages the patient to accept and swallow oral medications (tablets or capsules). pilling syringes are also available and in some dogs seem to work well. critical to the oral administration of medication is the ability of the owner to effectively administer the medication at home. animals that aggressively resist oral medication should be treated by alternative methods-for example, parenteral administration of medication. it is inappropriate, and unsafe, to delegate treatment responsibilities to the owner of a dog (or cat) that might injure the individual who is attempting to treat the patient. none required. caution: only experienced individuals should attempt this technique of administering tablets or capsules to cats. even cooperative cats that become intolerant will bite. therefore, this is not a technique recommended for most owners to try at home, even if specific instructions have been given. two methods of pill administration are used in cats. in both methods the cat's head is elevated slightly with the nose pointed upward. success in administering pills and tablets to a cat entails a delicate balance between what works well and what works safely. in cooperative cats, it may be possible to use one hand to hold and position the head (figure - ) while using the opposite hand (the one holding the medication) to open the mouth gently by depressing the proximal aspect of the mandible (figure - ) . press the skin adjacent to the maxillary teeth gently between the teeth as the mouth opens, thereby discouraging the alternatively, some cats will tolerate a specially designed "pilling syringe" in an attempt to administer a tablet or capsule. the pilling syringe works well as long as it is inserted cautiously and atraumatically into the cat's mouth. however, if resistance ensues, the rigid pilling syringe may injure the hard palate during the ensuing struggle. subsequent attempts to use the syringe may be met with increasing resistance and increasing risk of injury. success with a pilling syringe depends largely on the cat. pill pockets treats are also available for use in cats and are manufactured in chicken and fish flavors. in addition, as is the case in dogs, some cats will respond to the application of water drops on the nostrils or blowing into the nostrils to encourage swallowing. when dispensing oral medications for home administration to cats, do not expect clients to force a tablet or capsule into a cat's mouth. although some clients are remarkably capable and confident with their ability to administer oral medications to cats, the risk of injury to the client can be significant. whenever feasible, liquid medications or pulverized tablets should be mixed with the diet or an oral treat readily accepted and consumed (see the following discussion). none required. technique is appropriate for owners to perform at home. small amounts of liquid medicine can be given successfully to dogs and cats by pulling the commissure of the lip out to form a pocket . deposit the liquid medication into the "cheek pouch, " where it subsequently flows between the teeth as the head is held slightly upward. patience and gentleness, along with a reasonably flavored medication, contribute to the success. spoons are ineffective, as fluids are easily spilled. a disposable syringe can be used to measure and administer liquids orally. depending on the liquid administered, disposable syringes can be reused several times, assuming they are rinsed after each administration. in addition, disposable syringes can be dispensed legally to clients for home administration of liquid medication. mixing of medications in the same syringe is not recommended. however, dispensing of a separate, clearly marked syringe for each type of liquid medication prescribed for home administration is recommended. compounding pharmacies are also available and can mix many medications into palatable flavors to help facilitate the oral administration of medications. dogs with swallowing disorders should not be treated at home with liquid medications because this could cause complications associated with aspiration. none required. administration of medications, contrast material, and rehydrating fluids can be accomplished with the use of a well lubricated feeding tube passed through the nostrils into the stomach or distal esophagus. when a feeding tube is placed for long-term use (multiple days) and repeated use (described under gastrointestinal procedures later), it is generally recommended to avoid passing the tip of the tube beyond the distal esophagus. the reason for recommending nasoesophageal intubation over nasogastric intubation is based on the fact that reflex peristalsis of the esophagus against a tube passing through the cardia can result in significant mucosal ulceration within hours. this is not a factor in patients receiving a single dose of medication or contrast material. note: this procedure is reserved for in-hospital use only. the technique should be performed only by individuals trained to perform this procedure. the narrow lumen of tubes passed through the nostril of small dogs and cats limits the viscosity of solutions that can be administered through a tube directly into the gastrointestinal tract. nasoesophageal intubation can be done with a variety of tube types and sizes (table - ). newer polyurethane tubes, when coated with a lidocaine lubricating jelly, are nonirritating and may be left in place with the tip at the level of the distal esophagus. when placing the nasogastric tube, instill to drops of . % proparacaine in the nostril of the cat or small dog; . to . ml of % lidocaine instilled into the nostril of a larger-breed dog may be required to achieve the level of topical anesthesia needed to pass a tube through the nostril. with the head elevated, direct the tube dorsomedially toward the alar fold (figure - ) . pushing dorsally on the nasal philtrum and pushing the nostril from lateral to medially will help facilitate passage of the tube into the ventromedial nasal meatus. caution: the tip of the feeding tube can be inadvertently introduced through the glottis and into the trachea. topical anesthetic instilled into the nose can anesthetize the arytenoid cartilages, thereby blocking a cough or gag reflex. after inserting the tip to cm into the nostril, continue to advance the tube until it reaches the desired length. if the turbinates obstruct the passage of the tube, withdraw the tube by a few centimeters. then readvance the tube, taking care to direct the tube ventrally through the nasal cavity. occasionally it will be necessary to withdraw the tube completely t a b l e - the french catheter scale equivalents * millimeters inches from the nostril and repeat the procedure. in particularly small patients or patients with obstructive lesions (e.g., tumor) in the nasal cavity, it may not be possible to pass a tube. do not force the tube against significant resistance through the nostril. gavage, or gastric lavage and feeding, in puppies and kittens can be accomplished by passing a soft rubber catheter or feeding tube into the mouth, tilting the puppy's or kitten's head, and watching it swallow the tube. most puppies or kittens will struggle and vocalize. they usually will not vocalize if the tube has been placed into the trachea. a f catheter is of an adequate diameter to pass freely, but it is too large for dogs and cats less than to weeks of age. mark the tube with tape or a pen at a point equal to the distance from the mouth to the last rib. merely push the tube into the pharynx and down the esophagus to the caudal thoracic level (into the stomach). verify the placement of the tube using the same dry syringe aspiration technique to ensure that the tube is positioned in the esophagus or stomach rather than the trachea. attach a syringe to the flared end, and slowly inject medication or food. depending on the feeding tube type, the end of the tube may or may not accommodate a syringe. for example, soft, rubber urinary catheters are excellent tubes for single administration use. however, the flared end may not accommodate a syringe. to affix a syringe to the outside end of a tapered feeding tube or catheter, insert a plastic adapter (figure - ) into the open end of the tube. none required. intranasal administration of liquids in dogs and cats is usually limited to a single dose of a vaccine specifically labeled for intranasal administration. there is little indication for routine instillation of liquids into the nostrils of dogs and cats. rarely, administration of isotonic solutions directly into the nostrils is indicated. in contrast to singledose vaccines, lavage solutions applied intranasally are usually multiple-dose containers. therefore the tip of the administration device should not be allowed to directly contact the patient's skin or nose. doing so may result in contamination of the entire bottle. oily drops are not advised because they may damage the nasal mucosa or may be inhaled. the technique for intranasal administration of vaccine is straightforward and usually works quite well…the first time. some animals, dogs more than cats, will aggressively resist intranasal administration of vaccine. attempts to overcome this resistance include covering the eyes with a towel or otherwise distracting the patient with noise or other visual cues. concerns expressed over the loss of vaccine immediately after intranasal administration are generally unfounded. manufacturers of intranasal vaccines typically include a greater antigen (virus or bacteria) titer per dose than is necessary to induce a protective immune response. if the patient resists aggressively and the vaccine is indicated, parenteral preparations are available for all intranasal vaccines and should be considered. several objectives should be considered when treating dermatologic disorders with topical medication: ( ) eradication of causative agents; ( ) alleviation of symptoms, such as reduction of inflammation; ( ) cleansing and debridement; ( ) protection; ( ) restoration of hydration; and ( ) reduction of scaling and callus. many different forms of skin medications are available, but the vehicle in which they are applied is a critical factor (box - ). technique in all cases, apply topical medications to a clean skin surface in a very thin film, because only the medication in contact with the skin is effective. in most cases, clipping hair from an affected area enhances the effect of medication. when dispensing medications to owners for home administration, the owner should be instructed to wear disposable examination gloves if using fingers and hands to apply the medication. with the widespread availability of compounding pharmacies, prescribing compounded medications for topical and oral administration recently has become a popular dispensing technique for dogs and cats requiring long-term, daily medication. caution is warranted. some compounding pharmacies that serve the veterinary profession are using inappropriate or ineffective vehicles in which the drug has been compounded, or the drug itself, purchased in bulk, is of a lower grade and possibly an ineffective product once compounded. studies on the quality and efficacy of compounded drugs for use in veterinary patients are limited. however, of those studies that have been performed, serious questions are being raised over the bioavailability of the drug administered. it would be admirable to prepare the skin surgically before making needle punctures to administer medications. because such preparation is not practical, carefully part the hair and apply a high-quality skin antiseptic such as isopropyl alcohol. place the needle directly on the prepared area, and thrust the needle through the skin. although the use of antiseptics on the vial and skin is not highly effective, the procedure removes gross contamination and projects an image of professionalism. before aspirating medications from multiple-dose vials, carefully wipe the rubber diaphragm stopper with the same antiseptic used on the skin. observe this basic rule with all medication vials, even with modified live virus vaccines. technique dogs and cats have abundant loose alveolar tissue and easily can accommodate large volumes of material in this subcutaneous space. the dorsal neck is seldom used for subcutaneous injections because the skin is somewhat more sensitive, causing some patients to move abruptly during administration. a wide surface area of skin and subcutaneous tissue over the dorsum from the shoulders to the lumbar region makes an ideal site for subcutaneous injections. administration of drugs, vaccines, and fluids by the subcutaneous route represents the most commonly used route of parenteral administration in dogs and cats. for small volumes (< ml total), such as vaccines, a -to -gauge needle generally is used. the site most often used is the wide area of skin over the shoulders. the large subcutaneous space and the relative lack of sensitivity of skin at this location make it an ideal injection site. cleaning of the skin with alcohol or other disinfectant generally is performed before injection. several injection techniques are used. a common technique entails grasping a fold of skin with two fingers and the thumb of one hand. gently lift the skin upward. using the opposite hand, place the needle, with syringe attached, through the skin at a point below the opposite thumb. aspiration before injection is not typically necessary when using this route of administration. after administration and on removal of the needle from the skin, gently pinch the injection site and hold it for a few seconds to prevent backflow of medication or vaccine onto the skin. when larger volumes are to be administered-fluids in dehydrated dogs and cats-the skin directly over the shoulders is the injection site most commonly selected. generally, only isotonic fluids are administered by the subcutaneous route. depending on the patient's size, needles ranging from to gauge can be used. because of the larger volumes of fluid involved, warming of the fluids before administration is recommended. doing so can enhance significantly the patient's tolerance for the displacement of skin during the period of administration and, in small patients, prevent hypothermia. depending on the rate of administration and breed of dog, relatively large volumes of fluid generally can be given in one location. cats typically tolerate to ml/kg body mass in a single location. large dogs can tolerate volumes greater than ml of fluid in a single location. when administering large volumes, it is usually not necessary to use multiple injection sites for purposes of distributing the total fluid volume. doing so actually may increase the risk of introducing cutaneous bacteria under the skin. because the administration time required to deliver larger volumes is longer, and the injection needle will be placed in the skin for extended periods, it is appropriate to cleanse and rinse the skin carefully before actually inserting the needle. isotonic, warmed fluids may be administered by large syringe or through an administration tube attached to a bag. monitor skin tension and the patient's comfort tolerance throughout the procedure. although fluid absorption begins almost immediately on subcutaneous administration of fluids, significant pressure caused by the bolus of fluid delivered can develop within the fluid pocket. on removal of the needle, firmly grasp the injection site with the thumb and forefinger for several seconds. the procedure is not complete until one has verified that back-leakage of fluid from the subcutaneous space onto the skin is not occurring. depending on the patient's hydration status and physical condition, fluid absorption may take from to hours. the rate of absorption of fluid administered by the subcutaneous route largely depends on the patient's hydration state and vascular and cardiac integrity. for that reason, the subcutaneous route is not recommended to manage patients in hypovolemic shock. exceptions to this do exist-for example, when in a life-or-death situation access to a vein is simply not possible. subcutaneous or intraosseous (see the following discussion) fluid administration may be the only option available. recently, implantable subcutaneous ports* have been introduced for use in patients requiring regular administration of subcutaneous fluids at home. a -inch silicon tube is preplaced under the skin and is sutured in place by a veterinarian. objectively, this offers easy access to the subcutaneous space without the need for needle penetration. owners simply attach a syringe or extension tube tip to the port and administer the appropriate volume of fluids at an appropriate rate and frequency. because of the usual requirement for long-term placement of an implantable fluid administration tube, there is some risk of infection under the skin and around the incision site. some cats do not tolerate the device. *gif-tube single implant kit for subcutaneous fluid administration, various models available. phoenix, arizona, www.practivet.com (owner instruction guide is also available). note: not all parenteral medications can be administered safely by the subcutaneous route. when administering any compound by the subcutaneous route, verify that the product to be administered is approved for subcutaneous administration. serious reactions, including abscess formation and tissue necrosis, can occur. none required. because the tightly packed muscular tissue cannot expand and accommodate large volumes of injectables without trauma, medications given by the intramuscular route should be small in volume. these medications are often depot materials that are poorly soluble, and some may be mildly irritating. unless the animal is extremely thin, give injections into the lumbodorsal muscles on either side of the dorsal processes of the vertebral column. after proper preparation of the skin, insert the needle through the skin at a slight angle (if the animal is thin) or perpendicularly (if the animal is obese). when injecting any medication by a route other than the intravenous one, it is imperative to retract the plunger of the syringe before injecting to be certain that a vein was not entered by mistake. this is especially crucial with oil suspension, microcrystalline suspension, or potent-dose medications. never give intramuscular injections in the neck because of the fibrous sheaths there and the complications that may occur. also, intramuscular injections into muscles of the rear legs can cause severe pain, lameness, and occasionally peroneal nerve paralysis because of local nerve involvement. intracutaneous (or intradermal) injections are used for diagnostic testing purposes. prepare the skin by carefully clipping the hair with a no. clipper blade. if the skin surface is dirty, gently clean it with a moist towel. scrubbing and disinfection are contraindicated because they may produce iatrogenic trauma and inflammation, which interfere with the test. stretch the skin by lifting a fold, and use a -to -gauge intradermal needle attached to a -ml tuberculin syringe. insert the point of the needle, bevel up, in a forward lifting motion as if to pick up the skin with the needle tip. advance the needle while pushing the syringe (levered) downward until the bevel is completely within the skin. inject a bleb of . to . ml of fluid. if the procedure is done correctly, the small bleb will appear translucent. intradermal injections generally are used in patients subjected to intradermal skin testing for allergenic antigens. administration of compounds by the intradermal technique is not necessarily simple. inadvertent administration of medications into the subcutaneous tissues is easy when attempting intradermal injection. for that reason, specific training and experience are recommended before attempting intradermal skin testing of allergic patients. none required. intradermal administration of vaccine and drugs in veterinary and human medicine largely has been limited to the complexities of accurately delivering the desired dose into, and not under, the skin. in a transdermal administration system* was introduced for cats (recombinant feline leukemia virus [felv] vaccine) that was designed after a similar device *vet-jet transdermal administration system, merial, duluth, georgia. used in human (pediatric) medicine. recently the transdermal administration system used for administration of the recombinant felv vaccine has been re-designed. this same administration system is now used for the transdermal administration of the oral melanoma vaccine. the transdermal administration system consistently delivers a precise volume of vaccine into the skin, subcutaneous tissues, and muscle. use of the transdermal administration system should only be used to administer those vaccines approved for this method of delivery. administration of vaccine using the transdermal administration system requires training to understand proper procedure for loading and administering vaccine. at this writing, sale of the transdermal administration system for delivery of the canine oral melanoma vaccine is limited to select specialists in veterinary medicine. see section . none required. generally, two techniques are used. oscillometric blood pressure (bp) measurement entails use of an automated recording system. a cuff is applied to the base of the tail or a distal limb for access to an artery. this technique generally is regarded as being most accurate in dogs. when oscillometric bp measurements are performed in dogs, the patient should be in lateral recumbency. this places the cuff at approximately the same level as the heart. in cats the patient generally remains in sternal recumbency (and minimally restrained). most patients experience a brief acclimation period to the cuff placement. for this reason, at least three to five separate readings are obtained at -to -minute intervals. this technique can be used on awake or anesthetized patients (figure - ) . the doppler-ultrasonic flow detection system is most accurate in cats for measuring systolic bp. again, the ventral tail base or a dorsal pedal artery (hindlimb) or the superficial palmar arterial arch (forelimb) can be used. apply and inflate an occluding cuff. the readings are obtained by a transducer as the pressure on the cuff is reduced. caution is recommended in interpreting results from dogs that are reported as hypertensive but have no overt clinical disease. the higher reported occurrence of falsely elevated bp in normotensive dogs measured by this method justifies additional scrutiny when interpreting doppler bp results in dogs. clinically, the most common use of indirect bp measurement is in assessing cats for the presence (or absence) of systemic hypertension caused by renal insufficiency or hyperthyroidism (thyrotoxicosis). a common finding among untreated hypertensive cats is retinal detachment and blindness. early detection and therapeutic intervention (e.g., enalapril and or amlodipine) is critical. in dogs, bp measurement is indicated in patients with chronic renal insufficiency and/or protein-losing nephropathy, hyperadrenocorticism, and diabetes mellitus. in veterinary medicine, interpretation of bp centers on the systolic bp reading, not the diastolic reading (table - ) . stepien rl: blood pressure assessment. results dictate that even routine bacterial cultures and identification are best reserved for the commercial laboratory equipped to carry out these increasingly complex procedures and experienced in doing so. what follows are fundamental methods and techniques used to properly collect diagnostic specimens and the appropriate methods for transporting samples to a laboratory in order for the best possible diagnostic result to be obtained. before actually collecting and submitting a sample to a laboratory for bacterial culture, it is appropriate (whenever feasible to do so) to prepare, stain, and examine, under direct microscopy, exudates or fluid from the suspect material or tissue. staining the air-dried sample with a rapid romanowsky-type stain (e.g., diff-quik stain) or a gram stain may reveal evidence of neutrophilic inflammation (neutrophilia, especially with a left shift) and occasionally degenerative neutrophils with intracellular bacteria visible. these findings greatly facilitate patient management by documenting the immediate need for interventive empiric antimicrobial therapy until definitive culture and antimicrobial susceptibility results are obtained. the absence of cytologic evidence of bacterial infection does not rule out the possibility that the patient is infected or bacteremic (table - ) . collecting diagnostic samples for bacterial culture should be attempted as early in the disease process as possible. it is also critical to accomplish the sample collection under aseptic conditions. it is appropriate, therefore, to perform a surgical scrub of the skin or tissue from which the sample is to be collected in advance. this is especially true for tissue biopsies and fluid samples collected by needle aspiration through intact skin. failing to adequately prepare the collection site can result in significant contamination and complicate diagnostic interpretation of results. in addition, it is recommended to collect the diagnostic sample before the administration of antibiotics in order to minimize the risk of false-negative culture results. in the event antimicrobials have been administered to a patient with a suspected infection, and that is not responding to treatment, discontinuing treatment for hours before attempting sample collection is generally recommended. collection of an adequate amount, or volume (fluid), is equally important in obtaining meaningful result. for example, a single sterile cotton-tipped swab of contaminated tissue should be considered inadequate sampling and inappropriate for any patient. multiple specimens are always recommended when feasible. also, biopsy material, surgically removed tissue, and several milliliters of fluid (e.g., urine) should be collected and placed in a sterile container that can be appropriately sealed (leak-proof container) before transport. a "clean catch" of urine in a "clean cup" is not appropriate. inexpensive commercial containers specifically designed for the transport of infectious material are readily available today and should be used. many containers designed to hold bacterial samples contain buffered, nonnutritive transport media to sustain the growth of pathogenic bacteria yet minimize overgrowth of bacterial contaminants during the time required to transport the sample. most commercial laboratories provide appropriate containment devices for the transport of bacterial samples. because most diagnostic specimens collected for bacterial culture are submitted to commercial laboratories for bacterial isolation, identification, and antimicrobial susceptibility testing, it is important to prepare the sample properly for shipping. special transport media are generally not required for routine aerobic culture specimens as long as the sample can remain moist and relatively cool and the sample can t a b l e - common bacterial culture results-cont'd be inoculated onto culture medium within to hours only. for samples that must be shipped overnight to a laboratory, it is imperative that the specimen be kept cool (not frozen) and moist. elevated temperatures during shipping contribute to bacterial overgrowth of nonpathogenic bacteria, making isolation and identification of diseaseproducing organisms difficult. special transport media may be required. contact the individual laboratory regarding information pertaining to shipping of specimens for bacterial culture. specimens submitted for anaerobic culture need to be inoculated onto culture media within minutes after collection. although special anaerobic transport media are available, they may not be well suited for extended shipping times (> hours). among the most frequently tested fluids for bacteria, urine supports the growth of several types of bacteria. therefore it is necessary that the genitalia be cleaned before collection of urine (free-catch specimen) or cystocentesis (preferred). use of a urinary catheter to collect urine is likely to introduce urethral bacteria and may result in false-positive culture results. bacteria will survive for only a limited time in urine. samples collected must be sealed, and unless processed within hours the sample must be refrigerated. samples held for longer than hours may not contain viable bacteria. if extended transportation times are required to reach a laboratory, a urine reservation tube (vacutainer brand urine transportation system, bd, franklin lakes, new jersey) will allow storage for up to hours at room temperature (table - ) . collection from fluid filled compartments (e.g., abscesses, seromas) requires collection with a needle and syringe. the maximum quantity possible should be collected and submitted. the skin or tissue overlying the area from which the sample is to be collected should be surgically prepared. if it becomes necessary to flush an open lesion (or perform tracheatranstracheal aspiration or bronchoalveolar lavage [bal]), it is recommended that a buffered solution of sterile ringer's lactate be used. use of fluids that contain preservative may actually inhibit the growth of bacteria. if it is necessary to submit fecal material for specific bacterial isolation, at least to g of feces should be submitted. a single cotton-tipped swab inserted rectally is unlikely to yield meaningful results. multiple (up to three) samples are recommended when attempting to isolate specific pathogens (e.g., salmonella). samples should be submitted in a sealed, leakproof container (always appreciated by the lab). the containerized sample should refrigerated if there is a significant delay (several hours) involved in submission to the laboratory. confirmation of the presence of bacteria in the blood (bacteremia) can be difficult and requires some patient preparation before collection of a series of samples. in addition, samples should be collected only in vials clearly marked for the collection of blood. furthermore, there are several reasons for an infected patient to have a negative blood culture result-for example, prior or concurrent antimicrobial therapy, chronic (low-grade) infection, and intermittent shedding of bacteria into blood. sample volume, numbers of samples submitted, skin preparation, and timing of collections are variables that can directly affect results. clip and surgically prepare the skin over the cephalic, recurrent tarsal, and/or jugular veins. do not draw blood for culture through an indwelling intravenous or intraarterial catheter. collection vials are available for aerobic and anaerobic bacterial culture. it is generally recommended that three blood samples be collected from separate veins over a -hour period. there is no advantage to collecting arterial blood. it has been suggested that samples collected during times when the patient is febrile may improve the likelihood of isolating bacteria. the volume of blood collected is determined by the size of the patient, the collection vials (adult, pediatric, infant) used, as well as the laboratory equipment used to propagate the culture. in addition to adult human blood culture collection vials ( ml), pediatric blood collection vials ( to ml) and infant collection vials ( . to . ml) are available. it is appropriate that sterile technique be adhered to during collection of all samples. this includes the use of sterile gloves by the individual collecting the sample. once blood has been collected, air should not be allowed to enter the collection vial. the vial should be gently inverted (never shaken) two to four times. vials may be maintained at room temperature (the laboratory maintains samples at ° c). the opportunity to submit complementary cultures (e.g., from urine) from patients in which blood cultures are being collected can help to confirm the infecting bacteria and may lead to identification of a likely source (boxes - and - ). samples collected from patients suspected of having fungal infections of the nasal cavity (e.g., aspergillosis) or systemic (also called "deep") mycotic infections (e.g., histoplasmosis, cryptococcosis) are usually assessed by cytopathology or serology (see section ) or with tissue biopsy and histopathology involving special stains. direct cytologic assessment of samples from patients suspected of having fungal infections is always indicated. you certainly get credit for trying! however, experience in recognizing diagnostic elements of individual fungi and spores is essential, as is the availability of special stains for wet mount ( % potassium hydroxide) cytopathology. scrapings of skin and plucked hair shafts are commonly selected for fungal culture. the area of skin and hair to be sampled should be cleaned with % alcohol. iodine-based soaps and solutions should not be used. hair shafts, particularly those immediately adjacent to the lesion, are removed from skin with a sterile hemostat. skin scrapings can be collected with a sterile surgical blade or the edge of a clean (unused) microscope slide. scrapings from healthy, normal-appearing skin as well as abnormal skin should be collected. skin biopsy may be required if results of attempts to culture hair and skin scrapings are negative. sterile cotton-tipped swabs should not be used to collect samples for fungal culture. hair and skin scrapings can be placed directly into a sterile, dry container without need for any type of media as long as the sample can be processed within hours. refrigeration is generally not required. if transport times are extended, it is reasonable to place samples in a vial containing bacterial transport medium and refrigerate for up to hours. samples should never be frozen. skin and hair samples from patients suspected of having superficial fungal infections can be inoculated directly on a commercially available substrate called dermatophyte test medium (dtm). because samples can remain at room temperature and do not require special handling, the use of dtm is ideal for in-hospital use. the medium contains phenol red as a ph indicator. if a dermatophyte is present, characteristic colony morphology will be observed and the medium underlying the colonies will turn red. vials are unreliable after weeks; color change noted weeks or more after inoculation of the dtm should be disregarded. ultraviolet light filtered through nickel oxide produces a beam called wood light. if an animal is taken into a dark room and its hair and skin are exposed to a wood lamp, fluorescence may occur for several reasons. hair shafts affected by some species of microsporum fluoresce a bright yellow-green (like the color of a fluorescing watch face). however, iodide medications, petroleum, soap, dyes, bacteria, and even keratin may produce purple-, blue-, or yellow-colored fluorescence. the positive fungal fluorescence is a valuable aid in selecting affected hairs for culture inoculation. however, a negative fluorescence does not preclude a possible diagnosis of fungal infection. false-negative and false-positive interpretations are common. various laboratory techniques are currently available for the identification of viral infections in dogs and cats. excellent qualitative testing platforms are commercially available for in-practice use. molecular diagnostic tests, viral culture, histopathology, and serology, all of which are routinely available to veterinarians, require that samples be submitted to commercial laboratories for assessment. among the in-hospital test systems used to identify virus, the enzyme-linked immunosorbent assay (elisa) is the most common testing platform used. elisa testing can be performed quickly (minutes) with little or no patient preparation and with relatively high sensitivity and specificity. virus (antigen) detection tests are available as point-of-care tests for felv antigen in blood or serum and canine parvovirus (cpv) antigen in feces. in addition, these point-of-care tests for viral infections are capable of identifying patients that have not been exposed, enabling the clinician to rule out infection and viral shedding. test sensitivity refers to the likelihood that a patient with known infection will have a positive test result (a test with high sensitivity is expected to have few false-positive results). test specificity refers to the likelihood that a patient that is free of the infection will have a negative test result (a test with high specificity is expected to have few false-negative results). in addition, many commercial and point-of-care nonquantitative serologic assays are available that detect antibody to many of the viruses that affect dogs and cats. however, the positive predictive value of antibody tests is typically lower than that of antigen tests. for example, a positive antibody test result to a particular viral pathogen typically does not constitute a diagnosis of infection, especially in the absence of clinical signs. it may merely reflect recent vaccination (e.g., feline immunodeficiency virus). on the other hand, a negative antibody test result generally does indicate that the patient has had no prior exposure to the virus (or vaccine). serology refers to the use of serum to detect the concentration of antibody and is widely used in veterinary medicine. the value of antibody titers in diagnosing a viral infection is dependent on a number of factors, including the infecting virus, vaccination history, and time since exposure. use of acute and convalescent antibody titers in a patient suspected of having an acute viral infection can be a reliable diagnostic tool if a fourfold or greater increase in titer can be demonstrated over to weeks. acute and convalescent viral titers in individual patients are rarely performed in veterinary medicine. virus isolation, however, is a valuable diagnostic tool that is underused in veterinary medicine, perhaps because of the limited number of commercial and university laboratories that provide viral isolation services and the increased availability of molecular diagnostic testing services. diagnosis of viral upper respiratory infection in cats (herpesvirus and/or calicivirus) is perhaps among the situations for which virus isolation can be most useful, especially in cluster households where many shedding carrier cats exist and kittens may be at risk. to obtain a sample for viral isolation from the oral cavity of a cat, quickly insert a sterile cotton swab into the oral cavity to the level of the tonsil or oropharynx. by rolling the swab across the epithelium, it is possible to harvest cells and virus from infected cats. immediately place the swab into a virus transport medium (usually provided by the laboratory). antibiotics added to the solution prevent bacterial overgrowth of the sample. for short-term transit ( days or less), hold specimens for viral isolation at ° c rather than frozen. on reaching the laboratory, the specimen will be inoculated into a suitable tissue culture. within a few days it is usually possible to establish, based on the cytopathic effect on the tissue culture, whether a virus infection is present. fluorescent antibody testing can be done subsequently to confirm the isolate. although availability is limited, direct assessment of specimens (e.g., feces for cpv or canine or feline coronavirus) can be accomplished by electron microscopy. these methods can be useful for infections in which the virus concentration in the specimen reaches to organisms per milliliter. specimens such as feces, vesicle fluid, brain tissue, urine, or serum can be submitted for electron microscopy. tissue specimens and exfoliative cytologic preparations can be submitted for viral identification by histopathology, immunohistochemistry, and direct fluorescent antibody testing. such testing has limited application in patients with active disease because of the limited availability of these types of services and the time required for samples to be processed and reviewed by a pathologist. these tests can be particularly useful in postmortem diagnostics when multiple animals are potentially at risk. molecular diagnostics refers to the use of nucleic acid-based tests for the detection of viral dna or rna. polymerase chain reaction (pcr) is a laboratory technology that offers exceptional test sensitivity. through its ability to amplify trace amounts of dna or rna from pathogenic organisms millions of times, pcr facilitates identification of the "target" sequence of nucleic acid and therefore the infecting organism. this technology is also available commercially for the detection of dna from selected bacteria and rickettsiae. pcr technology is particularly useful in the very early stages of a viral infection, when the level of antibody has not yet reached levels that are detectable with conventional antibody tests. in addition, pcr testing may detect healthy virus carrier animals that pose a risk to a larger population of susceptible animals yet cannot be identified by conventional virus isolation or identification technologies. it should be noted, however, that pcr technology is still subject to false-positive and false-negative test results. therefore such testing is not necessarily indicated as a primary or exclusive test method for an individual patient. serology serum, plasma, or other fluids (e.g., cerebrospinal fluid [csf]) can be tested for the presence of antibodies to selected pathogenic viruses. whole blood samples should be allowed to thoroughly clot and retract (or the sample should be centrifuged) before serum is collected. samples are submitted in a leak-proof vial. refrigeration is appropriate for samples that must be held for several hours before testing. samples are limited to tissue obtained during surgical biopsy. as with conventional histopathology, samples (no more than . mm thick) should be placed in % buffered formalin and submitted in a leak-proof vial. it is recommended that the volume of formalin used be at least times greater than the tissue sample submitted. testing can be performed on tissues collected during surgical biopsy or from tissue impressions (exfoliative) made from tissue imprints on a clean microscope slide. it is recommended that tissue impressions on slides be fixed in alcohol or acetone before submission. fresh tissue is submitted on wet (not dry) ice and is not subjected to formalin fixation. small amounts of tissue suitable for electron microscopy should be no larger than × mm thick. fixation in % to % glutaraldehyde for hours at ° c is required. feces and body fluids collected for electron microscopy should be submitted fresh, not frozen or fixed in preservative. if shipping is required, feces and body fluids may be refrigerated or shipped on wet ice. samples should be viable for to hours. sterile swabs may be used to collect samples for viral culture and isolation. samples should be inoculated into a sealed vial containing viral transport medium (usually provided by the laboratory). samples should not be frozen or fixed in preservative. laboratories offering pcr testing typically accept serum or anticoagulated (ethylenediaminetetraacetic acid [edta]) whole blood in leak-proof vials. samples should be refrigerated and shipped on wet ice. samples should not be frozen. in most instances, a -to -ml sample of anticoagulated whole blood is adequate for routine hematology; some laboratories will accept as little as ml. for routine biochemical analyses, the volume of serum requested can vary from to ml, depending on the number and type of tests requested. plan ahead which samples are required to prevent the need for further venipuncture at a later time. in small dogs and cats, using the jugular veins facilitates collection of an adequate volume of blood. if smaller samples are required, the cephalic, lateral saphenous, or medial saphenous vein can be used for sample collection. do not use the jugular vein if a coagulopathy is suspected, as hemorrhage may be difficult to control after venipuncture. for successful venipuncture, proper restraint of the animal is important. details for the proper restraint for various venipuncture locations are discussed with each specific topic throughout this text. the patient must remain comfortable yet relatively motionless to avoid iatrogenic vessel laceration. stretch the skin tightly over the selected vessel without causing vascular occlusion to help anchor the vessel in place during penetration by the needle. the specific venipuncture will vary somewhat depending on the specific vein selected. the following sections describe venipuncture technique for each of four commonly accessed veins: the cephalic vein, jugular vein, lateral saphenous vein, and medial saphenous vein. to restrain a dog or cat for venipuncture of the cephalic vein, place the dog or cat on the table, sitting or in sternal recumbency. if the right vein is to be tapped or catheterized, the assistant should stand on the left side of the animal and place the left arm or hand under the animal's chin to immobilize the head and neck. the assistant should reach across the animal and grasp the leg just behind and distal to the right elbow joint. the assistant should use the thumb to occlude and rotate the cephalic vein laterally while the palm of the hand holds the elbow in an immobilized and extended position. make sure that the animal stays on the table if struggling occurs. the person performing the venipuncture then grasps the leg at the metacarpal region and begins the venipuncture on the medial aspect of the leg, just adjacent to the cephalic vein proximal to the carpus. for a jugular venipuncture in the dog, place the patient in sternal recumbency, with the hands of the assistant placed around the patient's muzzle to extend the neck and nose dorsally toward the ceiling. in short-coated dogs, the jugular vein usually can be seen coursing from the ramus of the mandible to the thoracic inlet in the jugular furrow. the vessel may be more difficult to visualize in dogs with long hair coats or if excessive subcutaneous fat or skin is present. the person performing the venipuncture should place the thumb of the nondominant hand across the jugular vein in the thoracic inlet or proximal to the thoracic inlet to occlude venous drainage from the vessel and allow it to fill. with the dominant hand, the person performing the venipuncture should insert the needle and syringe or vacutainer (bd, franklin lakes, new jersey) into the vessel at a -to -degree angle to perform the venipuncture. for smaller and very large animals, the jugular vein also can be tapped by placing the patient in lateral recumbency. the assistant should pull the animal's front legs caudally and extend the head and neck so that the jugular vein can be visualized. the venipuncture then can be performed as previously described. a jugular venipuncture is contraindicated in patients with thrombocytopenia or vitamin k-antagonist rodenticide intoxication. place cats in sternal recumbency. the assistant should stand behind the patient so that the patient cannot back away from the needle during the venipuncture. the assistant should extend the cat's head and neck dorsally while restraining the cat's front legs with the other hand. the cat's fur can be clipped or moistened with isopropyl alcohol to aid in visualization of the jugular vein as it stands up in the jugular furrow. the person performing the venipuncture should occlude the vessel at the thoracic inlet and insert the needle or vacutainer apparatus into the vessel as previously described to withdraw the blood sample. alternately, place the cat in lateral recumbency as described in the previous paragraph. to perform a lateral saphenous venipuncture, place the patient in lateral recumbency. the lateral saphenous vein can be visualized on the lateral portion of the stifle, just proximal to the tarsus. the assistant should extend the hindlimb and occlude the lateral saphenous vein just proximal and caudal to the tarsus. the person performing the venipuncture should grasp the distal portion of the patient's limb with the nondominant hand and insert the needle or vacutainer apparatus with the dominant hand to withdraw the blood sample. to perform a medial saphenous venipuncture, place the patient in lateral recumbency. move the top hindlimb cranially or caudally to allow visualization of the medial saphenous vein on the medial aspect of the tibia and fibula. the assistant should scruff the patient, if the patient is small, or should place the forearm over the patient's neck to prevent the patient from getting up during the procedure. with the other hand, the assistant should occlude the medial saphenous vein in the inguinal region. the person performing the medial saphenous venipuncture should grasp the paw or hock of the limb and pull the skin taut to prevent the vessel from rolling away from the needle. the fur may be clipped or moistened with isopropyl alcohol to aid in visualization of the vessel. the needle or vacutainer apparatus can be inserted into the vessel at a -to -degree angle to withdraw the blood sample. incorrect proportions of blood to anticoagulant may result in water shifts between plasma and red blood cells (rbcs). such shifts may alter the packed cell volume (pcv), especially when small amounts of blood are added to tubes prepared with volumes of anticoagulant sufficient for much larger volumes of blood. erroneous laboratory results also may be obtained when small volumes of blood are placed in a relatively large container. evaporation of plasma water and adherence of the cells to the surface of the container can produce artifactual changes in hematologic results. refrigerate liquid blood mixed with anticoagulant after collection if the sample is to be held before being transported to a laboratory. white blood cell (wbc) and rbc counts, pcv, and hemoglobin level can be measured within hours of sample collection. platelet counts, however, should be done within hour of collection. dried, unfixed blood smears can be stained with most conventional stains to hours after being made. if a considerable delay is anticipated between the time that the blood smear is made and the staining process, the blood smear should be fixed by immersion in absolute methanol for at least minutes. blood smears fixed by this method are stable indefinitely. never place unfixed blood smears in a refrigerator because condensation forming after the smear is removed from the refrigerator will ruin the blood smear and make it unusable for cytologic evaluation. take care to leave unfixed blood smears face down on a countertop or in a closed box. special stains, such as peroxidase, may require fresh blood films. the anticoagulant of choice for hematologic testing is edta. heparin is especially to be avoided if blood films are to be made from blood mixed with anticoagulant because contact with whole blood will distort the morphology of cells significantly. heparin is acceptable for most procedures requiring blood plasma. the anticoagulant effect of heparin is transitory. specimens still may clot after to days. make blood films immediately after collection because cell morphology rapidly deteriorates after sample collection. although blood films can be made after introducing blood to edta, a better practice is to make blood smears (films) immediately from the collection needle before the blood comes in contact with any anticoagulant. never use blood exposed to heparin to make blood smears. incorrect proportions of blood to anticoagulant may result in water shifts between plasma and rbcs. such shifts may alter the pcv, especially when small amounts of blood are added to tubes prepared with volumes of anticoagulant sufficient for much larger volumes of blood. erroneous laboratory results also may be obtained when small volumes of blood are placed in a relatively large container. evaporation of plasma water and adherence of the cells to the surface of the container can produce artifactual changes in hematologic results. refrigerate liquid blood mixed with anticoagulant after collection if there is a delay in making the laboratory determinations. wbc and rbc counts, pcv, and hemoglobin level can be measured within hours of sample collection. platelet counts, however, should be done within hour of collection. dried, unfixed blood smears can be stained with most conventional stains to hours after being made. if a considerable delay is anticipated between the time that the blood smear is made and the staining process, the blood smear should be fixed by immersion in absolute methanol for at least minutes. blood smears fixed by this method are stable indefinitely. never place unfixed blood smears in a refrigerator because condensation forming after the smear is removed from the refrigerator will ruin the blood smear and make it unusable for cytologic evaluation. take care to leave unfixed blood smears face down on a countertop or in a closed box. special stains, such as peroxidase, may require fresh blood films. prepare the selected vein as described earlier. most clinical chemistry procedures are performed on serum. the serum is obtained by collecting blood without any anticoagulant and allowing the blood to clot in a clean, dry tube. separate serum from cells within minutes of sample collection (venipuncture). special vacuum vials are available that produce a gel barrier between the clot and the serum (serum separator tubes) which avoid the need to draw off the serum into a separate vial. clotting of the blood and retraction of the clot occur best and maximum yields of serum are obtained at room or body temperature. refrigeration of the sample delays clot retraction. some samples clot and retract faster than others. if a serum separator tube is not used, it is recommended to free the clot from the walls of the container by rimming with an applicator stick. after the clot is freed, allow clot retraction to occur, and then centrifuge and draw off the clear supernatant serum using a pipette or suction bulb. allow whole blood samples to completely clot before attempting to remove serum. failing to do so may result in a mixture of plasma and serum in the submitted sample. serum yield is usually one third of the whole blood volume. patients that are hypovolemic or dehydrated can have a significantly lower serum yield. many clinical chemistry procedures can be performed on plasma and on serum. the advantage of using plasma is that separation of cells can be accomplished immediately after centrifugation or sedimentation, without the need to wait for clot formation and retraction. the disadvantage of plasma is that the presence of the anticoagulant interferes with many of the chemistry assay procedures. plasma is less clear than serum, which may be an additional disadvantage for colorimetric assays. plasma and serum are virtually identical in chemical composition except that plasma has fibrinogen and the anticoagulant. for many procedures in which plasma or whole blood is to be used, heparin is the anticoagulant of choice. heparinized blood is the only acceptable specimen for blood ph and blood gas analyses. although blood containing edta is acceptable for certain chemical procedures, it cannot be used for determination of plasma electrolytes because it contributes to and sequesters them from the specimen. in addition, edta can interfere with alkaline phosphatase levels, decrease total carbon dioxide, and elevate blood nonprotein nitrogen. refer to the tube selection guide in section to assure use the proper collection tube is used for the appropriate test requested. separate serum or plasma and remove it from the cells as soon as possible after blood is collected, because many of the constituents of plasma exist in higher concentrations in rbcs. with time, these substances leak into the plasma and cause falsely elevated values (positive interference) and falsely lower values (negative interference) ( table - ) . under no circumstances should whole blood be sent via the mail; serum derived from such specimens usually is hemolyzed, and results are often inaccurate. separate serum and transfer it to a clean, dry tube for shipment. bone marrow aspiration collection of bone marrow may prove valuable in diseases of the blood in which examination of the peripheral blood reveals abnormal cells or cell counts. conditions such as leukopenia, thrombocytopenia, nonregenerative anemia, agranulocytosis, pancytopenia, leukemias, other bone marrow cancers, and infectious diseases (e.g., histoplasmosis, ehrlichiosis) may be confirmed only by assessment of bone marrow cytology. bone marrow in the young animal is cellular and exists in the flat bones (sternum, ribs, pelvic bones, and vertebrae) and in the long bones (humerus and femur). as the animal ages, the cellular content of the marrow decreases, especially in the long bones. in older animals, bone marrow cells still exist in the flat bones; however, in conditions of stress in which new blood cells must be produced in large numbers, primitive cells in the bone marrow of the long bones again become active. interpretation of the bone marrow smear may be limited by ( ) technique used to obtain a bone marrow specimen or ( ) the specialized knowledge necessary to interpret bone marrow cells. bone marrow aspiration is much underused in clinical practice. the procedure does require some degree of skill if high-quality samples are to be obtained, but the procedure is of low risk to the patient and can be highly valuable in establishing a diagnosis or prognosis. a short-acting anesthetic occasionally may be needed, but tranquilization together with infusion of local anesthetic is usually sufficient. the site selected for aspiration or biopsy must be shaved and surgically prepared. bone marrow aspiration or biopsy is a percutaneous procedure conducted using sterile technique. the techniques involved include marrow aspiration and bone marrow core biopsy alone or in combination. when aspiration biopsy fails to produce adequate cytology (as in advanced myelofibrosis, neoplasia, or marrow aplasia), a core biopsy of bone marrow is indicated. the bone marrow aspiration needle may be a -gauge rosenthal needle or illinois needle for a medium-sized dog; an -gauge rosenthal needle for a small dog or a cat; or a jamshidi (pronounced yam-she-dee) bone marrow biopsy needle, gauge for most adult dogs and gauge for small dogs and cats. the selection of needles for aspiration biopsy of bone marrow is based on the biopsy site, the depth of the biopsy site, and the density of cortical bone. for bone marrow aspiration, the modified disposable illinois sternal-iliac bone marrow aspiration needle works well (figure - ) . for a core biopsy of bone marrow, the jamshidi bone marrow biopsyaspiration needle (pediatric, . inches, gauge) can be used (figure - ). the iliac crest is a commonly used site for marrow aspiration in dogs. place the animal in lateral recumbency, and prepare the aspiration site. to aspirate marrow, have the needle enter the widest part of the iliac crest and stop the needle just after penetration of the bone. remove the stylet, place a -ml syringe on the needle, and aspirate . ml of marrow. alternatively, the head of the humerus offers easy access to abundant bone marrow. sedation may be required. with the patient in lateral recumbency and the humerus flexed (the humerus is positioned parallel to the patient's thorax), instill local anesthetic into the skin and subcutaneous tissues to the level of the head of the humerus. the site of needle insertion is on the most proximal facet of the humoral head ( figure - ). direct the needle into the bone toward the elbow and parallel to the humeral shaft. if the needle is positioned too far medially over the humeral head, it is easy to penetrate the joint capsule. although this is a common occurrence, it does not pose a risk of injury to the patient (assuming the skin was surgically prepared). if joint fluid contaminates the bone marrow aspirate, the sample will be rendered useless. contamination of the bone marrow with peripheral blood results if ( ) the marrow is not aspirated immediately after the needle enters the marrow cavity or ( ) if aspiration time is sustained and a large volume of blood enters the syringe subsequent to the rupture of small blood vessels in the bone marrow. perhaps the least desired technique is to obtain marrow from the proximal end of the femur by insertion of the bone marrow needle into the trochanteric fossa. make a small skin incision over the trochanteric fossa just medial to the summit of the trochanter major. insert the bone marrow aspiration needle medial to the trochanter major, and place the long axis of the needle parallel to the long axis of the femur. once the site has been selected, grasp the needle firmly. apply steady, slight pressure while alternately rotating the needle tip against the bone (fast, -degree clockwise and then counterclockwise movements). begin with gentle pressure until the needle begins to seat into the bone. gradually increase the pressure as the needle penetrates into the bone. insert the bone marrow needle ⁄ inch into the femoral canal. remove the stylet from the needle, and aspirate using a -or -ml syringe that contains a small volume (approximately . ml) of % edta. use significant negative pressure, for example, by withdrawing the plunger of a -ml syringe to the -or -ml mark. collection of more than ml of bone marrow is unnecessary. collection of larger volumes may cause greater amounts of peripheral blood to enter the syringe, leading to hemodilution of the sample. once collection is complete, immediately transfer the aspirate to a watch glass containing approximately . ml of % edta. immediately mix the sample well using the end of the syringe. this is also a good time to remove the bone marrow needle from the patient. prepare slides in a manner similar to that used for peripheral blood smears. preparation of five to eight high-quality slides for submission is customary. smears are air-dried. slides may be stained using the same stains used for peripheral blood smears. bone marrow biopsy samples, usually obtained as a core, should be placed directly into % buffered formalin. it is generally recommended not to roll the core across a microscope slide (exfoliative cytology), as this may significantly disrupt the architecture of the sample and influence histopathologic interpretation. when submitting a bone marrow aspirate or bone biopsy, a complete blood count (cbc) should also be collected from that patient on the same day. the bone marrow sample and the cbc should be submitted together in order to obtain maximum diagnostic information. a thorough patient history should accompany the submitted samples. depending on the volume of bone marrow aspirate obtained, any additional aspirated bone marrow remaining after slides have been made can be mixed with edta in the same type of tube used to collect whole blood for a cbc. tubes may be refrigerated for short periods but never frozen. prompt shipping and processing of liquid samples of bone marrow is encouraged, as these cells tend to rapidly undergo degeneration. bone marrow biopsy core samples, after fixation in % buffered formalin, require decalcification before processing and interpretation. when feasible, a short-acting anesthetic administered to a cat before bone marrow aspiration or biopsy is recommended owing to the difficulty of adequately restraining a cat, even if sedated. the site selected for aspiration or biopsy must be shaved and surgically prepared. infusion of local anesthetic at the aspiration or biopsy site is appropriate. supplemental oxygen may be indicated. accessible sites for bone marrow sampling and biopsy in the cat are the iliac crest, the head of the humerus, and the proximal end of the femur via the trochanteric fossa. the techniques described for the dog can be used. smears of bone marrow are made immediately after aspiration. extrinsic thromboplastin present in bone marrow tissue will cause the marrow to clot within seconds. unstained slides should be submitted. a core of bone marrow can be fixed in % buffered formalin before submission for decalcification and histologic preparation. another method is to aspirate the sample of bone marrow into a syringe containing . ml of % edta solution. expel the aspirate, up to . ml, into a sterile petri dish, from which the marrow particles can be isolated easily by aspirating an aliquot with a glass pipette, placing an appropriate volume onto several glass slides, making the appropriate number of smears. slides prepared from bone marrow aspirates should be allowed to air-dry and then labeled appropriately. slides should never be refrigerated, as moisture from condensation can alter or destroy the appearance of individual cells. cytology collection techniques (see also section for additional information on slide preparation of samples to be submitted for cytopathologic examination.) cytopathology involves a simple, direct, and inexpensive technique that can yield significant diagnostic information within a short time at minimal direct cost. cytologic examination can be made of material obtained from pustules, vesicles, or the raw, ulcerated, or cut surfaces of a lesion. to make the smear, press a clean microscope slide firmly against a raw or ulcerated lesion to transfer cellular material to the slide. exudates may be collected by sterile swab or may be aspirated into a sterile syringe. roll the swab gently across the slide, or place a drop of fluid from the syringe onto the slide and carefully spread the fluid in a uniform film. transfer material from a block of tissue to the slide by gently pressing the tissue onto the slide in several locations. use various stains for different conditions. rapid stains such as new methylene blue or a quick romanowsky-type stain (e.g., diff-quik) are useful and convenient for office procedures. even wright and gram stains for evaluation of bacteria in tissues and fluids are easy to use. the presence of many bacteria, especially mixed types, may mean only surface contamination, whereas single types of bacteria, abundant polymorphonuclear wbcs, and especially phagocytosis support the diagnosis of infection and the host response to it. a few acantholytic cells (loose epidermal cells) in the smear may be compatible with infectious processes, but large numbers, or "rafts, " of acantholytic cells are highly suggestive of pemphigus and imply the need for more complex tests for positive diagnosis. large numbers of eosinophils sometimes are found in stained smears. contrary to popular opinion, they usually do not mean allergy. these cells are seen most commonly with furunculosis and may be associated with the eosinophilic granulomas, eosinophilic plaques, sterile eosinophilic pustulosis, pemphigus complex, and ectoparasites. yeasts (usually malassezia, rarely candida) commonly are found as budding cells in masses of wax and debris from ear smears. tumor cells may be recognized in some impression or aspiration samples where giemsa is a preferred stain. although special expertise is needed, cases of mastocytoma, histiocytoma, and lymphoma are recognized most easily. always prepare formalin-fixed tissues for histologic diagnosis in tumor evaluations (box - ). fine-needle aspiration, the use of needle and syringe to remove cells from normal and abnormal tissue, apply them to a glass slide, stain the smear, and review the results immediately is among the most useful, cost-effective procedures available in clinical practice. in most cases there will be no specific requirements for patient preparation. shaving hair over the aspiration site is generally not required. surgical preparation of the site is optional. lymph node aspiration is a procedure that can, and should, be performed routinely in clinical practice. follow proper technique to maximize the diagnostic utility of this procedure. lymph node aspiration typically is indicated ( ) in patients with generalized lymphadenomegaly, ( ) to evaluate abnormally enlarged solitary lymph nodes, and ( ) in suspected instances of tumor metastases to lymph nodes. surgically prepare the skin over the node from which a biopsy specimen is to be taken. with one hand, localize and immobilize the lymph node; with the other hand, guide the aspiration biopsy needle into the affected node. affix a -ml syringe onto a -to -gauge needle (a -gauge needle can be used when the site to be aspirated is particularly small), and advance the needle into the lymph node. withdrawal of the syringe to approximately . ml before inserting it into the tissue is recommended. doing so helps to prevent expelling material when removing the sample from the tissue. when the needle is in position in the approximate center of the node, gradually draw negative pressure on the syringe to a level of to ml. hold the negative pressure in place for a few seconds. release, and then repeat two or three times. before removing the needle from the tissue, release the negative pressure in the syringe (this is why it is recommended to have . ml of air prepositioned inside). do not remove the syringe from the tissue while maintaining negative pressure, because this can enlargement of nucleus or nuclei larger than nm decreased nuclear/cytoplasmic ratio multinucleation because of abnormal mitosis abnormal or frequent mitosis variations in size and shape of nuclei increase in size and number of nucleoli increased basophilia of cellular cytoplasm; increased rna content anisokaryosis or pleomorphism multinucleated giant cells box - cytologic features of malignancy result in the aspiration of significant amounts of blood from the skin, thereby significantly diluting the sample with peripheral blood. eject cellular material within the needle onto clean glass slides. handle all aspirates gently. to make slides, place two slides together and pull the slides apart to avoid shearing the cells. do not compress or force slides together. in addition, a biopsy of the lymph node can (and usually should) be performed as a means of confirming or supporting diagnostic decisions made on aspirates. lymph node biopsy samples can be obtained easily and safely by punch (core) techniques (e.g., -mm skin biopsy punch) or tru-cut biopsy needle. the most significant limiting factors are ( ) the technical ability to prepare high-quality slides and ( ) the ability to interpret the cytologic findings. some experience is needed to obtain the skills needed to aspirate cells and make diagnostic preparations. significant training is required to interpret the slides adequately. however, access to cytopathologists affiliated with diagnostic laboratories today makes fine-needle aspiration a highly useful diagnostic tool. the lymph node aspiration technique, described next, illustrates the finer points of the fine-needle aspiration technique. also called "touch impression cytology, " exfoliative cytology entails preparing cytologic slides directly from the cut surface of biopsy samples. requirements for patient preparation depend on the location of the tissue from which the sample is taken. the number and quality of cells collected are best when the procedure involves the freshly cut surface of tissue. attempting to collect samples directly from skin lesions on the patient is much less likely to yield diagnostic cytology. preparation, therefore, depends on the target tissue from which slides are needed. preparation may entail local anesthesia and collection of a tissue biopsy specimen from a lesion or suspect tissue (e.g., lymph node or cutaneous tumor) or general anesthesia and an exploratory abdominal procedure (e.g., liver biopsy). once the tissue has been collected, a scalpel blade is used to make a full-thickness linear cut through the biopsy specimen. a fresh surface of the tissue of interest is exposed. using forceps or a sterile needle, gently lay the tissue on a clean glass slide. do not force the tissue onto the slide, because this can significantly damage cells. several imprints can be made from the same surface. as needed, make new cuts to obtain a fresh surface from which to exfoliate cells. allow the slide to air-dry completely. apply conventional staining, and examine the specimen when it is dry. the remaining tissue, if not significantly damaged, can be submitted for histopathologic examination (recommended). once slides have air-dried and are labeled, they may be stained and reviewed immediately or submitted for review and interpretation by a pathologist. unstained slides should not be refrigerated, as moisture from condensation can alter the cytology of the preparation. several slides should be submitted. any remaining tissue may be placed in % buffered formalin and submitted for histopathology. the number of diagnostic cells obtained when making slides by way of exfoliative cytology depends on the tissue. epithelial cells (e.g., carcinoma), mast cells (cutaneous mast cell tumor), and lymph nodes readily exfoliate abundant numbers of cells. excessively thick slides can make interpretation difficult. on the other hand, biopsy specimens of tissue composed predominantly of mesenchymal cells (e.g., granuloma, fibrosarcoma) do not readily exfoliate cells, and slides made from these types of tissues are typically hypocellular. contamination of the cytology specimen with peripheral blood is a common mistake that can make interpretation of the sample difficult. it may be appropriate to gently blot the cut surface of the tissue sample, thereby removing excessive blood, before making slides. depending on the tissue type and lesion, it may be possible to obtain diagnostic cytologic samples from scrapings (e.g., conjunctival epithelium for virus inclusions), brushes (e.g., material obtained during endoscopy), and swabs (e.g., ear and vaginal swabs). the cells, once harvested, can be applied delicately directly to a clean glass slide by carefully rolling or even by just touching the material to the slide to create a thin layer. allow the sample to airdry thoroughly before staining. cytologic examination of fluids obtained with needle and syringe from body cavities, cysts, and urine typically requires additional preparation to obtain adequate cell concentration to make diagnostic decisions. analyze fluid specimens with respect to protein and nucleated cell count and a morphologic description of the cells. if overall cell counts are low, centrifugation will be required to concentrate cellular material for analysis. after centrifugation, remove the supernatant (and save it). resuspend the cells in two or three drops of the supernatant. apply a single drop of the mixture to a glass slide and allow it to air-dry. i prefer not to smear the liquid onto the slide; instead, i allow the liquid to run, by gravity, from one end of the slide to the other. after the liquid is thoroughly air-dried, it can be stained and reviewed. none required. skin scrapings frequently are obtained to find and identify microscopic parasites or fungal elements in the skin. material required includes mineral oil in a small dropper bottle, a dull scalpel blade, glass slides, coverslips, and a microscope. select undisturbed, untreated skin for a scraping site. the best method is to scrape the periphery of skin lesions and avoid the excoriated or traumatized center areas. in scraping for demodectic mange, pinch a small fold of affected skin firmly and collect the surface material for examination. this procedure forces the mites out of the hair follicles and onto or near the skin surface. for sarcoptic mange, scrape large areas. select sites on the elbows, hocks, and ear margins when searching for sarcoptic mange. many or frequent scrapings may be necessary to demonstrate sarcoptic mange mites or their fecal pellets or eggs. place the accumulated material on a microscope slide and mix it with mineral oil. examine the entire area with a × objective thoroughly and carefully. none required. acetate tape preparation is one of the simplest diagnostic procedures to perform when looking for the presence of ectoparasites, especially the nits of cheyletiella. use clear (not frosted) acetate tape. bend the tape into a loop around the fingers with the sticky side facing out. part the animal's hair coat, and press the tape firmly onto the skin and hair around suspect lesions. the sticky tape picks up loose particles with which it makes contact. cut the loop of tape and place the strip of tape sticky side down on a clean microscope slide. use a low-power microscope to look through the tape at the collected particles. this technique is excellent for trapping and identifying biting and sucking lice, otodectes and cheyletiella mites, flea dirt and larvae, fly larvae, and dandruff scales. acetate tape also is useful for studying hair abnormalities. use a strong hemostat to securely clamp and quickly avulse a group of to hair shafts. press the pointed distal ends onto sticky acetate tape (lined up like pickets in a fence), and cut the hair shafts off in the middle with scissors. likewise, press the butt ends with the hair roots onto another piece of tape. then press the tape holding the hair onto a microscope slide to allow low-power examination of the hairs through the clear tape. the tips of the hairs will be well oriented and controlled; thus, it is easy to evaluate whether the hairs are split, broken, or bitten off and whether the hair roots are in the anagen or telogen growth stage. urine can be removed from the bladder by one of four methods: ( ) voided (the "free catch"), ( ) manual compression of the urinary bladder (expressing the bladder), ( ) catheterization, or ( ) cystocentesis. for routine urinalysis, collection of urine by voiding (micturition) is satisfactory. the major disadvantage is risk of contamination of the sample with cells, bacteria, and other debris located in the genital tract and the perineal hair coat. the first portion of the stream is discarded, as it is most likely to contain debris. voided urine samples are not recommended when bacterial cystitis is suspected. compressing the urinary bladder is occasionally used to collect urine samples from dogs and cats. critical: do not use excessive pressure; if moderate digital pressure does not induce micturition, discontinue the technique. excessive pressure can culminate in forcing contaminated urine (bladder) into the kidneys, or, worse, in patients with a urethral obstruction the urinary bladder can rupture. the technique is most difficult to accomplish in male dogs and male cats. several types of urinary catheters are currently available for use in dogs and cats. the catheter types most often used today are made of rubber, polypropylene, and latex-free silicone. stainless steel catheters are occasionally used but unless placed with care these can cause damage to the urethra and/or urinary bladder. generally, urinary catheters serve one of four purposes: . to relieve urinary retention . to test for residual urine . to obtain urine directly from the bladder for diagnostic purposes . to perform bladder lavage and instillation of medication or contrast material the size of catheters (diameter) usually is calibrated in the french scale; each french unit is equivalent to roughly . mm. the openings adjacent to the catheter tips are called "eyes. " human urethral catheters are used routinely in male and female dogs; f to f catheters are satisfactory for most dogs (table - ) . polypropylene catheters should be individually packaged and sterilized by ethylene oxide gas. equipment needed to catheterize a male dog includes a sterile catheter ( f to f, inches long, with one end adapted to fit a syringe), sterile lubricating jelly, povidone-iodine soap or chlorhexidine, sterile rubber gloves or a sterile hemostat, a -ml sterile syringe, and an appropriate receptacle for the collection of urine. proper catheterization of the male dog requires two persons. place the dog in lateral recumbency on either side. pull the rear leg that is on top forward, and then flex it ( figure - ) . alternatively, long-legged dogs can be catheterized easily in a standing position. before catheter placement, retract the sheath of the penis and cleanse the glans penis with a solution of povidone-iodine % or chlorhexidine. lubricate the distal to cm of the appropriate-size catheter with sterile lubricating jelly. never entirely remove the catheter from its container while it is being passed because the container enables one to hold the catheter without contaminating it. the catheter may be passed with sterile gloved hands or by using a sterile hemostat to grasp the catheter and pass it into the urethra. alternatively, cut a -inch "butterfly" section from the end of the thin plastic catheter container. this section can be used as a cover for the sterile catheter, and the clinician can use the cover to grasp and advance the catheter without using gloves. if the catheter cannot be passed into the bladder, the tip of the catheter may be caught in a mucosal fold of the urethra or there may be a stricture or block in the urethra. in smallbreed dogs, the size of the groove in the os penis may limit the size of the catheter that can be passed. one also may experience difficulty in passing the catheter through the urethra where the urethra curves around the ischial arch. occasionally a catheter of small diameter may kink and bend on being passed into the urethra. when the catheter cannot be passed on the first try, reevaluate the size of the catheter and gently rotate the catheter while passing it a second time. never force the catheter through the urethral orifice. effective catheterization is indicated by the flow of urine at the end of the catheter, and a sterile -ml syringe is used to aspirate the urine from the bladder. walk the dog immediately after catheterization to encourage urination. equipment needed to catheterize a female dog includes flexible urethral catheters identical to those used in the male dog. the following materials also should be on hand: a small nasal speculum, a -ml sterile syringe, lidocaine . %, sterile lubricating jelly, a focal source of light, appropriate receptacles for urine collection, and ml of povidone-iodine or a dilute chlorhexidine solution. use strict asepsis. cleanse the vulva with a solution of povidone-iodine or dilute chlorhexidine. instillation of lidocaine . % into the vaginal vault helps to relieve the discomfort of catheterization. the external urethral orifice is to cm cranial to the ventral commissure of the vulva. in many instances the female dog may be catheterized in the standing position by passing the female catheter into the vaginal vault, despite the fact that the urethral papilla is not visualized directly. in the spayed female dog, in which blind catheterization may be difficult, the use of a sterilized otoscope speculum and light source (figure - ), vaginal speculum, or anal speculum with a light source will help to visualize the urethral tubercle on the floor of the vagina. in difficult catheterizations it may be helpful to place the animal in dorsal recumbency (figure - and - ). insertion of a speculum into the vagina almost always permits visualization of the urethral papilla and facilitates passage of the catheter. take care to avoid attempts to pass the catheter into the fossa of the clitoris because this is a blind, possibly contaminated cul-de-sac. before attempting urinary bladder catheterization of the male cat, administer a short-term anesthetic (e.g., ketamine, mg/kg im), but only after a careful assessment of the cat's physical, acid-base, and electrolyte status (see treatment of hyperkalemia in section ). in some cases, drugs to treat hyperkalemia may be required before anesthetic induction. once the patient's electrolyte status has been evaluated and hyperkalemia, if present, addressed appropriately, anesthesia can be induced with a combination of propofol ( to mg/kg intravenously [iv]) and diazepam ( . mg/kg iv); then the patient is intubated and maintained on gas anesthesia. place the anesthetized patient in dorsal recumbency. gently grasp the ventral aspect of the prepuce and move it caudally in such a manner that the penis is extruded. withdraw the penis from the sheath and gently pull the penis backward. keeping sterile catheters in a freezer will help them become more rigid to facilitate passage into the urethra. pass a sterile, flexible plastic or polyethylene (pe to ) catheter or -to -inch, . f urethral catheter into the urethral orifice and gently into the bladder, keeping the catheter parallel to the vertebral column of the cat. caution: never force the catheter through the urethra. the presence of debris within the urethral lumen may require the injection of to ml of sterile saline to back-flush urinary "sand" or concretions so that the catheter can be passed. in some instances the presence of cystic and urethral calculi will prevent the passage of a catheter into the urethra. for this reason a lateral radiograph of the penis, with the patient's hindlimbs pulled caudally, may help document the presence of a urethral stone. urinary bladder catheterization of the female cat is not a simple procedure. when indicated, and after a preanesthetic examination has been performed, attempt the technique only in the anesthetized cat. urinary bladder catheterization can be accomplished with the use of a rubber or plastic, side-hole (blunt-ended) urinary catheter. the same catheter type used in male cats is effective in female cats. instilling lidocaine . % has been recommended as a means of decreasing sensitivity to catheter insertion in sedated (not recommended) cats. cleanse the vulva with an appropriate antiseptic. catheterization can be accomplished with the cat in dorsal or ventral recumbency. experience and size of the cat dictate which technique works best. after cleansing of the perineum and vaginal vault, place the patient in sternal recumbency, and gently pass the catheter along the ventral floor of the vaginal vault. conversely, if the patient is placed in dorsal recumbency, direct the catheter dorsally along the ventral vaginal floor. if a catheter cannot be placed blindly, a small otoscopic speculum can be placed into the vagina, and the catheter pushed into the urethral papilla once it is visualized directly. for continuous urine drainage in the awake, ambulatory patient, use a closed collection system to help prevent urinary tract infection. a soft urethral or foley catheter can be used, and polyvinyl chloride tubing should be connected to the catheter and to the collection bag outside the cage. the collection bag should be below the level of the animal's urinary bladder. place an elizabethan collar on the animal to discourage chewing on the catheter and associated tubing. the urinary bladder is catheterized as described previously. despite the quality of care of the catheter, urinary tract infection still may develop in any patient fitted with an indwelling urinary catheter. ideally, remove the catheter as soon as it is no longer necessary, or if there are clinical signs of a urinary tract infection or previously undiagnosed fever. a urinary catheter is generally changed after it has been in place for more than hours. observe the patient for development of fever, discomfort, pyuria, or other evidence of urinary tract infection. if infection is suspected, remove the catheter and submit urine for culture and sensitivity or determination of minimum inhibitory concentration (mic). previously, culture of the catheter tip was recommended to diagnose a catheterinduced infection. however, culture of the catheter tip is no longer recommended, as it may not accurately reflect the type of microorganisms in a urinary tract infection. the empiric use of antibiotics to help prevent catheter-induced infection is not recommended, as their use can allow colonization of resistant nosocomial bacteria in the patient's urinary tract. cystocentesis is a common clinical technique used to obtain a sample of urine directly from the urinary bladder of dogs and cats when collecting a voided, or free-catch, aliquot is not preferred. the procedure is indicated when necessary to obtain bladder urine for culture purposes. urine that is collected by free catch has passed through the urethra and may be contaminated with bacteria, thereby making interpretation of the culture results difficult. cystocentesis also is performed as a convenience when it is desirable to obtain a small sample of urine but the patient is not ready or cooperative. cystocentesis involves insertion of a needle, with a -or -ml syringe attached, through the abdominal wall and bladder wall to obtain urine samples for urinalysis or bacterial culture. the technique prevents contamination of urine by urethra, genital tract, or skin and reduces the risk of obtaining a contaminated sample. cystocentesis also may be needed to decompress a severely overdistended bladder temporarily in an animal with urethral obstruction. in these cases, cystocentesis should be performed only if urethral catheterization is impossible. warning: penetration of a distended (obstructed) urinary bladder with a needle could result in rupture of the bladder. to perform cystocentesis, palpate the ventral abdomen just cranial to the junction of the bladder with the urethra, and trap the urinary bladder between the fingers and the palm of the hand. use one hand to hold the bladder steady within the peritoneal cavity while the other guides the needle. next, insert the needle through the ventral abdominal wall into the bladder at a -degree angle (figure - ) . although this procedure is relatively safe, the bladder must have a reasonable volume of urine, and the procedure should not be performed without first identifying and immobilizing the bladder. for the procedure to be performed safely and quickly, the patient must be cooperative. if collection of a urine sample by cystocentesis is absolutely necessary, sedation may be indicated to restrain the patient adequately for the procedure. generally, cystocentesis is a safe procedure, assuming the patient is cooperative and the bladder can be identified and stabilized throughout the procedure. however, injury and adverse reactions can occur. in addition to laceration of the bladder with the inserted needle (patient moves abruptly), the needle can be passed completely through the bladder and into the colon, causing bacterial contamination of the bladder or peritoneal cavity. there is also risk of penetrating a major abdominal blood vessel, resulting in significant hemorrhage. obtaining a skin biopsy from abnormal skin only to receive a nondiagnostic result as reported from a pathologist suggests that improved biopsy technique may result in collecting a specimen with higher diagnostic value. the following guidelines apply when skin biopsies are performed: ❏ consider obtaining multiple samples from multiple sites, which is especially useful when different stages of similar lesions are identifiable. ❏ do not perform a surgical scrub before collecting the sample; shaving the hair away is fine, but surgically prepared skin removes superficial lesions that, had they been left in place, might have been diagnostic. ❏ biopsy of lesions that are depigmenting should be done before they have turned white; the absence of color usually denotes absence of active skin lesions. biopsies from completely depigmented skin are less likely to demonstrate active lesions. ❏ biopsy of lesions associated with alopecia should be done in the center of the most alopecic area. ❏ also, biopsy of lesions associated with alopecia should be done at junctional (between normal-and abnormal-appearing) skin. ❏ consider submitting biopsy samples from completely unaffected, normal-appearing skin. ❏ avoid biopsies of ulcerated skin areas. biopsy samples may be obtained with a scalpel blade (incisional or excisional) or via a dermatologic punch biopsy. punch biopsy instruments are circular blades available in -mm, -mm, and -mm diameter sizes (figure - ) . hold the punch perpendicular to the skin site of interest. a back-and-forth motion that rotates the circular blade cuts through the skin. when the skin no longer moves as the punch is rotated, the biopsy is complete and the skin sample may be removed (from the skin or from the biopsy instrument). avoid grasping the dermis or epidermis of the sample with any instrument to prevent crushing of the sample and causing artifact. if the sample must be lifted, use the attached subcutaneous fat only. if the lesion of interest is deep, the punch biopsy technique may not be effective. in this situation, an incisional or excisional biopsy using a sterile no. or no. surgical blade is indicated. biopsies of ulcerated skin and solitary nodules are best done by removing a wedge of skin (incisional biopsy). in some cases it is possible surgically to remove all visible, palpable parts of the lesion (excisional biopsy). place each sample of skin in buffered formalin, using a volume that is at least times that of the sample size. if particularly large areas of skin are harvested during biopsy, cut these into -cm thick pieces before placing into formalin. (note: placing larger tissue samples into formalin may result in inadequate or incomplete fixation of the tissue and the inability to properly prepare the tissue for examination). alternatively, it is possible, and in many cases important, to evaluate a biopsy specimen of skin or subcutaneous tissue at the time of collection. when the lesion of interest is suspected to be neoplastic, quickly differentiating between inflammatory cells and neoplasia may be possible by simply performing an exfoliative cytologic examination (described in this section) on one of the biopsy samples in addition to fixing a separate sample in formalin and submitting that for histopathologic examination. small biopsy samples that have been subjected to the additional handling required to make impressions on a glass slide are not good candidates for subsequent fixation and histopathologic examination. it is generally recommended to perform exfoliative cytologic and histopathologic examinations on separate samples. among the most common diagnostic procedures carried out on the skin of dogs and cats is a routine skin scraping. yet despite the frequency with which this test is used, doing a skin scraping in such a manner that the sample recovered maximizes the opportunity to establish a diagnosis can be anything but routine. a skin scraping, properly done, does require using consistent techniques appropriate to the suspected diagnoses, and as such, superficial or deep scrapings, or both, may be indicated. skin scraping is indicated whenever ectoparasite infestation is suspected. superficial scrapings are appropriate for detecting mites that live on the skin surface, such as cheyletiella species and otodectes cynotis, as well as mites that burrow within the outermost layers of skin (stratum corneum), such as sarcoptes species and notoedres cati. because the area to be scraped is relatively large (≥ cm ), shave dogs and cats with long hair coats before attempting the procedure, unless cheyletiella infestation is suspected. make the scraping over healthy-appearing skin. do not cleanse the skin of superficial scale or crusts. the technique for superficial skin scraping entails the use of mineral oil or pyrethrin ear drops applied to a clean scalpel blade and directly onto the area of skin to be scraped. scraping begins as a gentle motion made in the direction of the hair coat. gradually increase the pressure of the blade against the skin with repetitive scrapings over the same area. take care not to lacerate the skin, although minor capillary bleeding at the site is common. transpose material collected on the edge of the blade to a clean glass slide, cover it with a coverslip, and thoroughly examine the material under low magnification for evidence of ectoparasites. note that for mites such as cheyletiella or scabies, finding just one mite or one egg is diagnostic and justifies implementing treatment. same as described for superficial skin scraping (earlier). a slightly different technique is indicated in dogs and cats suspected of having an infestation that includes demodex canis mites. the mites are known to live predominantly in sebaceous glands and hair follicles. they can survive in the skin of animals without manifesting lesions. hair loss and skin lesions develop where overgrowth of the mite population occurs. demodex infestations can be localized or generalized; infestations can occur in either dogs or cats but the most severe, generalized infestations are much more likely to occur in young dogs. although both superficial and deep skin scrapings may reveal the presence of mites on the skin, deep scrapings may reveal demodex mites in some patients when superficial scrapings are negative. the technique for deep skin scraping targets a small area of skin (< cm ). it may be helpful to apply gentle pressure to the skin or actually to squeeze the area of interest between the thumb and a finger in an attempt to force mites from the deeper to the more superficial skin. in some breeds (e.g., old english sheepdogs and shar-peis) recovering mites on a skin scraping can be particularly difficult. in such cases, when demodex infestation is highly suspected but the results of repeated skin scrapings are negative, a skin biopsy is appropriate. alternatively, a procedure called a trichogram that involves pulling (plucking) a few hairs from the hair follicles using a hemostat may be diagnostic. once the hairs have been plucked from the skin, place them on a glass slide that has been preprepared with a drop of mineral oil, add a coverslip, and examine the hair shaft under low magnification. half of all dogs with demodex infestation will have a positive trichogram. not all dogs and cats with otitis externa require comprehensive ear flushing and debridement before or as part of otic therapy. in many cases, home treatment is sufficient to resolve the problems effectively, assuming the underlying diagnosis has been established. however, in patients with chronic or particularly severe infections, topical treatment administered by the owners at home may not be sufficient. in such cases, the external ear canal requires a careful and comprehensive cleaning before administration of topical medications. properly performed, flushing and cleaning of the external ear canals is not a quick procedure. anesthetize the patient. attempting to perform a thorough ear cleansing under sedation usually will not be successful. once the animal is anesthetized, perform a careful otoscopic (or video otoscopic) examination to establish the integrity of the ear canal, evaluating, for example, for the presence or absence of tumors or parasites. in severe cases the tympanic membrane (ear drum) may not even be visible. with the patient in lateral recumbency, flush the ear canal (figure - ) or lavage it with warm saline initially, and then aspirate the material from the canal. if this procedure is not successful in removing the debris attached to the epithelium of the ear canal, use ceruminolytic ear solutions to facilitate breakdown and removal of this material. a -minute instillation and soak is recommended, followed by thorough flushing to remove debris and the ceruminolytic material. remove hair growing inside the ear canal with forceps. a suction apparatus is recommended for removal of debris and liquid remaining. reintroduce an otoscope to examine the integrity of the skin in the ear canal and to look for any evidence of stenosis, foreign body, or tumor. the flushing process is not complete until it is possible to visualize the tympanic membrane. carefully remove any remaining debris with an otologic loop (figure - ) , not a cotton-tipped swab. repeat the procedure on the opposite ear as indicated. at the conclusion of the examination, apply appropriate topical medication into the ear canal before allowing the patient to recover from anesthesia. systemic therapy or surgical intervention may be required in some patients for complete resolution of the problem. however, a thorough examination and cleaning is critical before actually making decisions regarding medical versus surgical intervention. use of a cotton-tipped swab to remove debris from the external ear canal, although commonly done, is not generally recommended. repeated attempts may ultimately force debris deeper into the external ear canal. general anesthesia and otic lavage or flushing may be required to effectively correct the problem created by the use of cotton-tipped swabs. in selecting an appropriate endotracheal tube, consider the size of the animal and select a tube that has the largest diameter that can be inserted without force (table - ). the length of tube selected must not extend beyond the bifurcation of the trachea (carina). always check the cuff of the endotracheal tube to ensure there are no leaks and that the cuff is working properly before intubation. lubricate the selected endotracheal tube with sterile lubricating jelly before inducing anesthesia. after induction, place the patient in sternal recumbency and elevate the head. the individual inserting the tube should grasp and extend the tongue with a piece of gauze. the tongue is extended to facilitate visualization of the larynx. avoid excessive downward pressure on the tongue in order to prevent inadvertent laceration or injury from the lower incisors. if a laryngoscope is used, place the tip of scope at the base of the tongue. gently press the tip of the laryngoscope ventrally to move the epiglottis and expose the glottis. contact during an attempt to intubate, one or two drops of % lidocaine can be applied to the arytenoid cartilages. once the tube has been inserted into the trachea, never advance it farther than the carina. doing so may result in intubation of either the right or the left main bronchus (endobronchial intubation). once the tube is in place, secure it in place with a loop of ⁄ -inch white tape or muzzle gauze. overinflation of the endotracheal tube cuff can cause tracheal ulceration, tracheitis, hemorrhage, tracheomalacia, fibrosis, stenosis, and subcutaneous emphysema. abdominal paracentesis refers to the aspiration of fluid from the abdominal cavity for both diagnostic and therapeutic purposes. always weigh the animal before and after removing abdominal fluid. any subsequent gain in weight indicates a reaccumulation of abdominal fluid. place the animal in left lateral recumbency and restrain it in this position. clip and surgically prepare a -to -inch square between the bladder and the umbilicus just lateral to the midline. if the bladder is distended, empty it before performing paracentesis. infiltrate the paracentesis site with lidocaine . % using a -to -gauge needle. in most cases, local anesthesia is not necessary. abdominal puncture can be made with an -to -gauge needle (figure - ) . gently insert the needle through the skin and external abdominal oblique muscles while simultaneously pushing and twisting, to push viscera away from the tip of the needle. blind abdominocentesis without the use of ultrasound to guide needle placement into a fluid pocket can have negative results if there is less than ml of fluid per kilogram within the peritoneal cavity. when the abdominal puncture has been made, allow the animal to rest quietly to facilitate drainage of the fluid. some clinicians recommend tapping while the patient is in a standing position in the hope of obtaining more complete drainage. changing the patient's position after the tapping may result in needle-tip laceration of intraabdominal organs. aspiration may be easier if a specially adapted needle with multiple holes drilled in the shaft is used because it is less likely to become plugged with omentum. ideally, tap four quadrants of the abdomen. if four-quadrant abdominocentesis has been performed and no fluid has been obtained, and if there is suspicion for peritonitis, diagnostic peritoneal lavage can be performed. the abdomen is clipped and aseptically scrubbed as previously described. next, insert an over-the-needle catheter or hypodermic needle just caudal to the umbilicus, and instill ml of warmed . % sterile saline or lactated ringer's per kilogram. after instillation of the fluid, walk the patient around or gently roll the patient from side to side to distribute the fluid throughout the abdominal cavity. next, perform four-quadrant abdominocentesis as previously described. only a small amount of the fluid infused will be obtained. the fluid will be diluted by the saline or lactated ringer's infused, so biochemical analysis will not be accurate. however, the fluid can be examined microscopically for the presence of plant material, bacteria, wbcs, or bile pigment to help diagnose various forms of peritonitis. hackett numerous biopsy techniques are available, and the selection of the appropriate technique is based on the tissue to be examined, the condition of the patient, and the skill of the examiner. inserted cranial and to the right/left of the umbilicus, and caudal and to the right/left of the umbilicus. when the needle is inserted, it should be twisted while penetrating into the abdominal cavity, to prevent iatrogenic puncture of hollow organs. in some cases it may be necessary to place more than one needle before fluid will flow from the needle hub. appropriate locations are marked on the skin with an "x." excisional biopsy refers to the surgical removal of the entire lesion or organ with subsequent histologic examination. excisional biopsy is used most frequently for skin lesions and cases in which an entire organ may have to be removed (such as an eye or an internal organ that has developed a tumor). incisional biopsy refers to the surgical removal of a portion of a lesion with subsequent histologic examination. choose a representative area of the lesion for biopsy. include lesion margins, if possible. needle aspiration refers to the use of needle and syringe to remove representative cells from the tissue or organ of interest. specialized needles are available that allow removal of very small biopsy specimens that can be submitted for histopathologic examination. (see also fine needle aspiration.) needle biopsy techniques: general considerations needle biopsy or aspiration techniques refer to a variety of techniques used to obtain diagnostic tissue or cells from internal organs, including the lung, liver, spleen, pancreas, abdominal lymph nodes, and mass lesions within the abdomen and thorax. in contrast, fine-needle aspiration is a technique generally used to recover cytopathologic samples (cells only) from skin or subcutaneous tissues (e.g., superficial lymph nodes). the advantage of needle biopsy is related directly to how well the abnormal tissue has been characterized and how easily it can be identified during the procedure. in addition, depending on patient cooperation, most procedures can be performed safely with the patient sedated only. shortterm intravenous anesthesia and general anesthesia eliminate undesired patient movement during the biopsy procedure. potential lesions or abnormal tissues from which aspirate or biopsy samples are to be taken are located using palpation, radiographs, or ultrasound-guided imaging techniques. shave the skin over the site of needle penetration and surgically prepare it. the type of sedation or anesthesia depends on the temperament of the animal and the site on which the biopsy will be performed. attach a -gauge needle without stylet to a -ml syringe prefilled with . to . ml of air. optionally, affix a flexible extension set to the needle and connect it proximally to the syringe. needle length may vary from to ⁄ inches depending on the required depth of penetration and size of the patient. guide the needle into the tissue or organ of interest. stabilize the tip of the needle to avoid random movements through organs, especially highly vascular tissue such as liver and spleen. once the needle has been inserted, the aspiration technique entails withdrawing the plunger of the syringe to the -or -ml level. hold that position for to seconds, and then release. repeat the procedure. depending on the nature of the lesion, it may not be indicated to thrust the needle into the tissue at multiple and different angles. neutralize the pressure in the syringe, and withdraw the needle rapidly. expel any material within the needle onto glass slides using the air in the syringe. this same procedure can be repeated with a new needle to obtain an additional three to five samples from alternative sites. this technique allows samples to be obtained without applying negative pressure to the syringe, which may damage cells. ultrasound guidance for needle aspirations from abdominal tissues greatly enhance the safety of this technique, especially when obtaining samples from smaller animals. automatictrigger needles such as cook or temno biopsy needles ( to gauge) are available for use in human beings but are seldom used in veterinary medicine. the risks associated with fine-needle aspiration include rupture of an encapsulated inflammatory process, dissemination of an infectious agent, seeding of neoplastic cells in the needle tract, and hemorrhage. larger volumes of fluid and cells can be placed directly into a vial containing edta to prevent clot formation. prepare and examine direct and sedimentation specimens. needle biopsy of internal organs using the tru-cut needle is particularly useful in patients with subcutaneous ( figure - ) or cutaneous masses and for localized abdominal and thoracic mass lesions and diffuse liver, kidney, and splenic disease. serious complications, usually hemorrhage or laceration of the gallbladder (during liver biopsy), can occur when the procedure is performed blindly. therefore ultrasound-guided needle biopsy is strongly recommended whenever a percutaneous biopsy of internal organs is performed. additional safety factors provided by ultrasound guidance include the ability to image, and avoid, large aberrant blood vessels. risk of complications associated with needle aspiration of the lung is considerably higher than for most abdominal procedures. pneumothorax can occur after a single, "clean" aspiration attempt. see respiratory tract procedures for a detailed description of performing fine-needle aspiration of the lung. histologic examination of diseased skin can serve as a means for diagnosis of cutaneous lesions. the causative agent often is found in acute and chronic skin infections. punch biopsy of the skin is a quick and accurate means of removing a small sample of diseased skin for histopathologic examination. select a site that is well developed but not traumatized or excoriated. the sample should include little or no normal tissue. if the lesion (pustule, vesicle) can be identified early in its development and if the biopsy sample is taken only from the lesion, one may obtain a superior specimen. it is best not to take too large a sample that contains much normal skin; by mistake, the technician might take a section that misses the lesion. proper selection of the biopsy site is crucial to accurate diagnosis. carefully clip the hair from the lesion. lightly blot the skin with % alcohol. avoid superficial trauma while cleaning the skin. inject a small subcutaneous bleb of . % lidocaine to deaden the area. special equipment needed for the biopsy includes a -mm, -mm, or -mm biopsy punch and % buffered formalin solution. after the area has been anesthetized with lidocaine, press and rotate the biopsy punch through the skin until the subcutaneous tissue is penetrated. remove the biopsy specimen by "spearing" the subcutaneous fat with a fine needle. do not grasp the specimen with a forceps. blot the specimen gently between two paper towels. spread the tissue out gently (like a pancake), place the specimen epidermal side up on a piece of cardboard or tongue depressor, press the specimen gently to cause adhesion, and drop the specimen into the formalin fixative. the skin defect may be closed with one or two simple interrupted sutures. if deep subcutaneous tissue or large biopsy samples are needed, a punch biopsy is inadequate. use a small (no. ) scalpel blade to obtain an appropriate sample. in all cases in which skin biopsies are made, take multiple samples to increase the odds that at least one will have diagnostic lesions. specimens submitted to laboratories should be accompanied by extensive, detailed clinical information, including a differential diagnosis. skin biopsies routinely are stained with hematoxylin-eosin; however, periodic acid-schiff, gomori methenamine silver, and verhoeff stains are used for special problems. the diagnosis of liver disease is generally confirmed on the basis of the patient's clinical signs coupled with laboratory findings, radiography, and abdominal ultrasound. the development of a more specific diagnosis and prognosis in liver disease may be aided greatly by information obtained in a liver biopsy. percutaneous liver biopsies are of much greater value in generalized liver disease such as cirrhosis, generalized acute hepatic necrosis, or amyloidosis than in focal hepatic disease. the major indications for performing a liver biopsy are (l) to explain an abnormal liver profile, ( ) to define reasons for abnormal liver size, ( ) to identify a possible liver tumor, ( ) to arrive at a prognosis and rational approach to management, and ( ) to identify the cause of ascites. the procedures for obtaining liver tissue are numerous; however, needle biopsy of the liver, when performed properly, can be helpful. careful physical and clinicopathologic examination should precede a liver biopsy. a normal coagulation profile should be documented on every patient undergoing liver biopsy. detect and correct abnormalities in normal hemostatic mechanisms, if feasible, before needle biopsy of the liver. liver biopsy should be performed only in the fasted patient and only after removal of ascitic fluid. percutaneous needle biopsies and fine-needle aspirations of the liver can be performed with local anesthesia in the sedated, and cooperative, patient. general anesthesia is a reasonable alternative whenever feasible. biopsy sites in the liver can be selected best when needle biopsy techniques are used along with laparoscopy or ultrasound techniques. blind percutaneous needle biopsies of the liver can be performed with relative safety if the liver is significantly enlarged and easily palpated. however, blind biopsies do carry the risk that the operator will be unable to determine the impact of penetrating the liver if only an abdominal radiograph and impression of abdominal palpation are available. in cases in which the liver is not palpable, blind biopsy carries significantly higher risk and should be performed only when no alternative exits. a modified percutaneous liver biopsy can be performed by the following method. place the animal in dorsal recumbency, and place a local block in the midline of the skin and abdomen at the caudoventral aspect of the left hepatic lobe. the incision into the peritoneal cavity should be large enough to accommodate the gloved index finger. make a separate skin puncture site in the abdominal wall to accommodate the biopsy needle. use the index finger manually to fix the left hepatic lobe (or other desired hepatic lobe) against the diaphragm or other adjacent structures, and insert the outer cannula and stylet through the abdominal wall in the isolated hepatic lobe. remove the stylet, and rapidly insert the cutting prongs. if properly placed, the cutting prongs should not go through the entire hepatic lobe. advance the outer cannula over the blades of the cutting prongs, thus entrapping the hepatic tissue material within the cutting prongs. remove the biopsy needle. using a wooden applicator stick, carefully place the biopsy specimen into fixative. biopsy samples can be used to prepare slides for cytologic examination, and the biopsy needle may be cultured. close the abdominal incision in the routine manner. another liver biopsy technique entails use of a tru-cut biopsy needle. place the dog in dorsal recumbency. clip a -cm area over the triangle formed by the xiphoid cartilage and left costal arch, and prepare the area as for aseptic surgery. make a small paramedian incision large enough to accommodate a sterile otoscope head mm in diameter. use a sterilized halogen-illuminated otoscope speculum to visualize the liver. pass a tru-cut biopsy needle through the otoscope cone to directly obtain a biopsy specimen of the liver. the technique for performing diagnostic nasal biopsies is sufficiently complex (and bloody) that it is generally recommended to refer patients in need of this procedure to a specialty or referral hospital that has rhinoscopic and/or computed tomography capabilities. blind biopsies of dogs and cats with chronic nasal disease, especially if associated with bleeding, can be associated with significant risk, including penetration of the cranium. no one likes nasal biopsy results that indicate "normal brain. " renal biopsies can be valuable in confirming or eliminating a diagnosis of renal disease that is based on history, physical examination, and radiographic and laboratory data (box - ). in addition, biopsy may be a way of arriving at a prognosis in generalized renal disease and a better means of evaluating the type of treatment to be instituted. ultrasonographic guidance can prove valuable during renal biopsy for placing the needle into the tissue desired and avoiding complications. the liver biopsy, although a critical diagnostic tool in patients with laboratory evidence of liver disease, can be a fatal event, even in the hands of the experienced clinician. abdominal ultrasound imaging of the liver before and during biopsy is strongly recommended. before renal biopsy, the animal should have a baseline coagulation profile that includes, at the very least, an activated coagulation time and platelet count. a buccal mucosal bleeding time may be indicated if there is any history of spontaneous bleeding in a patient with a normal platelet count. obtain biopsies from the renal cortex. administer fluids to patients before and after biopsy. many patients with generalized renal disease are critically ill and debilitated, and general anesthesia is contraindicated. in these cases, a neuroleptanalgesic agent may be used for sedation. if the animal is a good anesthetic risk and renal function will permit it, use inhalation anesthesia. when bilateral renal disease is documented, select the left kidney for biopsy because it is more accessible than the right kidney. with the anesthetized patient in right lateral recumbency, surgically prepare the skin behind and below the junction of the costal arch at the level of the second and third lumbar vertebrae. make a -inch paralumbar incision parallel to, but just behind, the costal arch. dissect muscle and fascia until the peritoneum is visible. carefully open the peritoneal cavity. digitally feel for and examine the caudal pole of the left kidney. guide the needle toward the posterior pole of the kidney with the index finger. immobilize the kidney against the body wall and insert the tru-cut biopsy needle, with the biopsy notch exposed into the parenchyma of the kidney. capture the biopsy specimen by sliding the outer sleeve of the needle over the (now embedded in the kidney) biopsy notch. remove the needle and gently lift the biopsy sample from the needle and place it into formalin. evaluate the site for hemorrhage. once bleeding is controlled, a second biopsy specimen may be collected. once bleeding from the biopsy site has stopped, the incision can be closed. in dogs, renal biopsy can be performed under ultrasound guidance using probes with channels for biopsy needle insertion. evaluation of bone marrow is indicated in patients with evidence of persistently diminished cell counts of any or all cell lines (wbcs, rbcs, platelets) or evidence of morphologically abnormal cells in peripheral blood. bone marrow aspiration and bone biopsy are extremely helpful but underused diagnostic procedures. the availability of inexpensive, high-quality biopsy needles makes these procedures safe and easy to perform (once experience is gained). conventional practice today is to obtain a bone marrow aspirate (cytopathologic examination) and a bone biopsy specimen from the same patient during the same procedure when changes in the peripheral blood justify this level of diagnostic testing. bone marrow aspiration technique is described earlier in this section. two types of bone biopsy needles are available. the most commonly described procedure involves use of the jamshidi biopsy needle, an -to -gauge needle that ranges in length from to cm (see figure - ). the needle contains a stylet that extends beyond the needle tip by to mm. because of the size of the jamshidi needle, its use is limited to medium and large dogs. for bone biopsies in cats and small dogs, the illinois bone marrow aspiration needle is preferred (see figure - ), which is a -to -gauge needle available in lengths ranging from . to . cm. the patient usually is sedated or anesthetized for the procedure. although some patients will tolerate this procedure when performed under local anesthesia only, the additional manipulation required to obtain a high-quality sample justifies sedation. in some cases the patient is sufficiently obtunded that sedation is neither indicated nor required. the technique for bone biopsy is the same regardless of the needle used. once the site has been selected (usually the same sites selected for bone marrow aspiration: head of the humerus, wing of the ileum, ischial tuberosity, proximal femur), clip the hair and surgically prepare the skin. make a small stab incision in the skin over the site selected. pass the needle, with stylet in place, through the incision and subcutaneous tissues until the needle tip makes firm contact with bone. advance the needle using steady, increasing pressure and stable rotation. rotation, in this case, means rotating the needle back and forth to the left degrees and then to the right degrees. once the needle is situated in the bone (about . cm penetration only), stop. carefully remove the stylet. continue the penetration by gradually applying additional pressure and simultaneously rotating the needle. the usual depth of penetration varies from inch to as much as inches. on reaching the desired depth, remove the needle by continuing to rotate as described but gradually withdrawing the needle from the bone. an obturator is provided to push the sample out of the bone. place the core of bone directly into buffered formalin and submit it for histopathologic examination (decalcification will be required, which takes a little longer). some authors recommend carefully rolling the bone core across a glass slide (for cytopathologic examination) before placing the bone in formalin. most pathologists do not recommend this because additional handling of the biopsy sample can sufficiently disrupt the architecture of the tissue and compromise the quality of the biopsy (besides upsetting the pathologist). note also that the needle can, with a little gentle manipulation, be reinserted into the hole from which the biopsy sample was obtained. because the illinois needle and the jamshidi needle accommodate a syringe, it is possible to obtain (if done quickly, to prevent clotting) a bone marrow aspirate from the same site. place that sample directly onto glass slides or (recommended) into % edta and mix it before making slides. there are no specific requirements for postbiopsy care of the patient. clean the blood from the skin using hydrogen peroxide; sutures generally are not required. the femoral and dorsal pedal arteries can be punctured to obtain an arterial blood sample for blood gas and electrolyte analyses (see section ) for information on indications and interpretation of results). to obtain a sample of arterial blood gas, place the patient in lateral recumbency and restrain the patient in a manner similar to that for a medial saphenous venipuncture. a -gauge needle affixed to a tuberculin syringe is preferred for arterial puncture. prepare the tuberculin syringe by coating it with heparin and forcing all the heparin out except for that left in the hub of the needle. pull back on the plunger of the syringe slightly to facilitate visualizing the point at which the artery is entered. arterial blood initially will enter the syringe without the plunger being drawn back. for collection of blood from the femoral artery, and once the patient is sufficiently restrained, the individual collecting the arterial blood sample should palpate the medial aspect of the limb over the proximal medial femur until the femoral pulse is palpated. direct the needle at a -to -degree angle, inserting the needle slowly, watching for a flash of blood in the hub of the needle (figure - ) . gradually withdraw the plunger to facilitate blood entering the syringe. collect . to . ml and immediately submit the blood for analysis, or place it on ice until the analysis can be performed. to obtain blood from a dorsal pedal artery, place the patient in lateral recumbency and extend the rear limb as for a medial saphenous blood sample collection. the person obtaining the blood sample should pull the paw of the down leg in the nondominant hand toward his or her body, rotating the limb slightly in a medial direction to palpate the arterial pulse. palpate the pulse in the dorsal pedal artery on the dorsomedial aspect of the tarsus. gently insert the needle at a -degree angle into the artery, watching carefully for a flash of blood into the syringe. when the necessary amount of blood has filled the syringe, remove the needle and place pressure over the site of arterial puncture for a minimum of minutes. evacuate excess air from the syringe and needle, and cap the needle with a red rubber stopper to prevent air from entering the needle and syringe. place the sample on ice until analysis, if arterial blood gas analyses cannot be performed immediately. in the event that percutaneous access to a peripheral artery is not possible, the femoral artery can be isolated and prepared for surgical cutdown. after appropriate aseptic skin preparation, make a -to -cm incision in the skin over the femoral artery. find the caudal edge of the sartorius muscle by blunt dissection and then reflect it anteriorly to expose the underlying femoral artery, vein, and nerve. taking care to avoid tearing any vessel branches, gently isolate up to cm of the femoral artery from the surrounding fascia. visually direct the needle into the artery at this point. alternatively, catheterize the artery in the event repeated arterial samples are required. elevate the femoral artery by preplacing two stay sutures beneath the artery and then elevating the vessel to the level of the skin. insert a long catheter-overthe-needle system into the lumen of the artery without penetrating the deep wall. gently insert the catheter into the vessel, remove the needle, and cap and flush the catheter. close the incision and affix the catheter to the skin via a tape tag sutured to the skin. the collection of csf is an important diagnostic procedure indicated for patients suspected of having significant intracranial or certain spinal diseases. however, it is our opinion that the technique to safely perform this procedure requires hands-on training and, preferably, prior experience before attempting the procedure in a clinical patient. attempting to perform csf collection from a written description in a text is not recommended. although this procedure is generally safe when performed correctly, significant injury and even death are possible, despite the experience of the individual performing the procedure. the electrocardiogram provides a fast, efficient way to obtain considerable data about a patient's cardiovascular status. electrocardiography is a clinical test and must be correlated with clinical findings (box - ). keep in mind that an electrocardiogram measures only electrical activity of the heart as seen on the body surface at any one instant. electrical disorders of the myocardium can be transient or intermittent and, as such, can be missed on a single electrocardiogram. if the answer to any of these questions is no, proceed to identify the abnormality. next, determine the rate, rhythm, and wave character-that is, evaluate measurements of the p wave, pr interval, and qrs complex. evaluate the st segment, t wave, and qt interval. use all leads to determine the axis and any miscellaneous criteria. depending on the type of electrocardiographic equipment used, there are several methods for determining heart rate from the electrocardiographic tracing. many electrocardiographs compute the heart rate and print that on the tracing. however, in patients with a significant dysrhythmia, these calculations can be flawed and should be verified manually when a question exists. small linear lines or demarcations at the top of the electrocardiogram paper can be used to determine the heart rate. at a paper speed of mm/second, the time between adjacent marks is . seconds. counting the number of qrs complexes (or r waves) between just two of these divisions and multiplying by equals the heart rate in beats per minute (figure - ) . for those inclined to higher mathematics, the heart rate also may be determined by counting the number of small squares between r waves (at a paper speed of mm/sec) and then dividing into (box - ) . the normal heart rhythm is sinus in origin. for every qrs complex there is a p wave (figure - ). the p waves are related to qrs complexes (p-p interval is constant). sinus arrhythmia, sinus arrest, and wandering pacemaker are normal rhythm variations. in sinus arrhythmia, the p-p interval is irregular. the pauses are never longer than twice the usual p-p interval (figure - ) . a wandering pacemaker means that the p waves vary in height and may even be negative temporarily (figure - ) . sinus arrest is defined as a prolongation of the p-r interval longer than twice the usual p-p interval. the normal p wave is . second × . mv (two boxes wide × four boxes tall) for the dog and . second × . mv for the cat. in p mitrale (left atrial enlargement), the p wave is wider than . second. in p pulmonale (right atrial enlargement), the p wave is taller than . mv for the dog and . mv for the cat. the pr interval is measured from the beginning of the p wave to the beginning of the qrs complex. the normal interval is . to . second ( to . boxes wide) for the dog and . to . second for the cat. in first-degree atrioventricular heart block, the pr interval is prolonged. the pr interval is sometimes useful in monitoring the effects of digitalis therapy. the qrs complex duration is measured from the beginning of the q wave to the end of the s wave. normal duration is up to . second in cats, . second in small dogs, and . second in large dogs. a qrs complex that is too wide indicates left ventricular enlargement (figure - ). an r wave that is too tall indicates left ventricular enlargement. the amplitude is measured from the baseline to the top of the r wave (figure - ) . the normal r wave can be up to . mv tall in cats, . mv in small dogs, and . mv in large dogs. figure - : a, the wandering pacemaker in this recording is suggested by the slightly negative p waves in some of the complexes. negative p waves of this nature result from vagal depression of the sinoatrial node and the development of a junctional atrioventricular nodal rhythm. b, marked sinus arrhythmia and a wandering pacemaker result in a decreased heart rate (increased r-r interval) and negative p waves in the fifth complex. as the pacemaker returns to the sinoatrial node, the rate increases, and positive p waves of varying amplitude result in the sixth and seventh complexes. the st segment is between the end of the s wave and the beginning of the t wave. the qt interval is measured from the beginning of the q wave to the end of the t wave. the normal interval is . to . second ( to boxes wide) in dogs and up to . second in cats. a lengthened qt interval may be seen with hypokalemia or hypocalcemia. the qt interval varies with heart rate and tends to be prolonged when bradycardia occurs. a decreased qt interval may be seen with hypercalcemia. the mean electrical cardiac axis measures the direction (vector) of the cardiac ventricular impulse during depolarization. therefore the qrs complex is examined in leads i, ii, iii, av r , av l , and av f . these six leads determine the axis. they are arranged in a manner known as bailey's hexaxial lead system (figure - ) . the procedure is as follows: . find an isoelectric lead-that is, a lead for which the total number of positive (upward) and negative (downward) deflections of the qrs complex is equal to zero (figure - ) . when there is no perfectly isoelectric lead, use the one that comes closest. . find the lead that is perpendicular to the isoelectric lead: lead i is perpendicular to av f ; lead ii is perpendicular to av l ; and lead iii is perpendicular to av r . . determine whether the perpendicular lead is positive or negative on the patient's electrocardiogram. if the perpendicular lead is negative, the axis is at the negative end of that lead (each lead has a plus and a minus pole marked). if the perpendicular lead is positive, the mean electrical axis is at the positive end of the perpendicular lead. for example, if avl is isoelectric (normally it is), lead ii is its perpendicular. if lead ii is positive on the electrocardiogram, the axis is + degrees. if lead ii is negative on the electrocardiogram, the axis is − degrees. the mean electrical axis in the normal dog is + to + degrees; for the cat it is more variable, at to ± degrees. right axis deviation (axis more than + ) indicates right ventricular enlargement in the dog (figure - ) . left axis deviation (axis to + degrees) indicates left ventricular enlargement in the dog. when there is biventricular enlargement, the axis usually remains normal. axis determinations are of less value in the cat because the normal range is so wide (boxes - to - ). inappropriate use of endoscopic equipment not only can damage expensive equipment but also can cause serious injury to the patient. endoscopy of the upper respiratory tract is among the most important advanced diagnostic and therapeutic tools used in the evaluation of patients that have stertor (snorting), reverse sneeze, stridor (wheezing), and chronic cough. laryngoscopy is of value in the diagnosis of upper airway obstructions such as eversion of the lateral ventricles, collapsed arytenoid cartilages, hyperplasia of the vocal cords, nodules on the vocal cords, elongated soft palate, collapsed proximal trachea, and traumatic injuries to the neck. note also, however, that a careful visual examination of the larynx in the anesthetized patient (only) can be highly valuable even without the use of endoscopic equipment-for example, for assessment of laryngeal movement in patients with laryngeal paralysis. suspected lesions inside the larynx may be difficult to visualize with or without endoscopic equipment. examination of the trachea and main stem bronchi requires endoscopic evaluation to assess the integrity of the airway for conditions such as collapsed trachea, mediastinal tumors, hilar lymph node enlargement, parasitic nodules (filaroides osleri), and foreign body aspiration. in addition, tracheobronchoscopy is a valuable technique that permits culturing and cytologic examination of material from bronchi involved in chronic respiratory disease. upper airway obstruction that is not responsive to conservative therapy is an indication for more extensive diagnostic procedures, such as bronchoscopy. note: the discussion that follows centers around indications and capabilities of endoscopy in clinical practice. the discussion is not intended to be used as a "how-to" instruction guide on performing endoscopic procedures in dogs and cats. today, numerous types of endoscopes and accessory materials are available for use in clinical practice. specific hands-on training and complete familiarity with the equipment package available is essential before attempting to perform any of the procedures outlined. endoscopes of varying sizes are appropriate for use in examining the larynx and trachea. however, in cats and small dogs, examination of the trachea using equipment as small as a (human) bronchoscope may limit the examination because the endoscope nearly occludes the tracheal diameter. additional training and/or experience is recommended for performing tracheoscopy in small patients. one of the most important endoscopic techniques performed in dogs and cats involves examination of the nasopharynx, the upper respiratory compartment above the soft palate. sometimes called pharyngoscopy, examination entails retroflexion of a small-diameter endoscope (e.g., bronchoscope) to degrees to allow visualization of the space between the posterior nares (choanae) and the larynx (figure - ) . this is a common location for foreign body entrapment and occasional tumor development in cats and dogs (figure - ) . pharyngoscopy is the only effective means of examining this portion of the upper respiratory tract in patients that have a history of stertor (snorting) and so-called "reverse sneeze." lower respiratory tract: bronchoscopy endoscopic examination of the bronchi and lower airways is a highly diagnostic, occasionally therapeutic procedure indicated in patients presented with persistent cough. as in all endoscopic procedures, the patient is anesthetized for the examination. however, examination of the lower respiratory tract requires considerable attention to patient oxygenation and respiratory status during the examination. the requirement for oxygen to be administered throughout the procedure may be a significant limiting factor unless special accessories are used. in the ideal situation, the patient is a medium-to large-sized dog and the endoscope can be passed through the endoscope using a t adaptor while oxygen and anesthetic are administered simultaneously. however, in cats and small dogs it is usually not possible to pass an endoscope through the endotracheal tube. the procedure must be done by passing the endoscope directly into the trachea to the level of the right and left main bronchi and probably not much farther. supplemental intravenous anesthetic is likely to be required because of the time required to complete the examination. training and/or experience is essential before performing bronchoscopy, particularly in cats and small dogs. the greatest advantage in performing bronchoscopy is to visualize the integrity of the trachea and, to a limited extent, the lower airways. airway collapse, not visible on conventional radiography, can be strikingly apparent. foreign body entrapment, tumors, respiratory parasites, and airway trauma also can be identified with bronchoscopy. in addition, the bronchoscopic examination allows for collection of cytologic samples from discrete areas (airways) within the lower respiratory tract. the ability to perform bal in patients with reactive airway disease, subclinical or clinical infections, and certain types of tumors can be highly diagnostic. flexible fiberoptic endoscopy is a noninvasive, atraumatic means of visualizing the mucosal surfaces of the esophagus, stomach, and colon. flexible endoscopes are available from several companies at a wide range of prices. to minimize the risk of injury to the animal and to reduce the possibility of damage to the endoscope, place animals undergoing endoscopic examination under general anesthesia after routine preanesthetic preparation. a fast of to hours is recommended for most patients undergoing upper gastrointestinal endoscopy. however, for patients with indications of delayed gastric emptying, a longer fast ( to hours) may be needed to empty the stomach completely. in preparation for colonoscopy, a -to -hour fast is recommended. give a high warm-water enema the evening before and again to hours before the procedure. give such enemas until the return is clear. the clinical signs indicating esophageal disease and a potential benefit of esophagoscopy include repeated regurgitation, excessive drooling, ballooning of the esophagus, anorexia or dysphagia, and recurrent pneumonia. esophagoscopy allows visualization of the mucosal lining of the esophagus and makes it possible to detect inflammation, ulcerations, dilatations, diverticula, strictures, foreign bodies, tumors, and parasite infestations. endoscopic examination of the mucosal aspect of the stomach is indicated when the clinical signs or physical findings suggest the presence of gastric disease or when there is a need for confirmation or clarification of radiographic findings. in most cases, persistent vomiting is the chief complaint. other clinical signs suggestive of serious gastric disease include hematemesis, melena, weight loss, anemia, and abdominal pain. gastroscopy allows visualization of the mucosal lining of the stomach and enables detection of inflammation, ulceration, foreign bodies, and tumors. in most dogs and cats the endoscope can be passed into the proximal duodenum. depending on the patient size and length of the scope, it may be possible to evaluate as much as inches or more of the proximal duodenum. colonoscopy is endoscopic examination of colon, rectum, and anus. the technique is helpful in the definitive diagnosis of lower bowel lesions, such as granulomatous colitis, foreign bodies, tumors, lacerations, and other mucosal abnormalities. the primary indication for colonoscopy is the presence of signs of large bowel disease, which typically include tenesmus and the passage of small, frequent stools containing fresh blood or excess mucus. endoscopic examination of the colon allows direct visualization of the effects of mucosal inflammation, ulceration, mucosal polyps, malignant neoplasia, and strictures. histologic examination of mucosal biopsy material will confirm the diagnosis of colonic disease. the large bowel must be empty for the colonic mucosa to be visualized. the bowel can be emptied by withholding food for hours and performing a colonic irrigation the evening before and again hours before the examination. the material used for the enema must be nonirritating and nonoily. mildly hypertonic saline solutions such as fleet enemas work well if given hours before examination so that gas and fluid can be passed completely. however, do not use fleet enemas in cats or small dogs. if the general physical condition of the animal is poor and withholding food is not possible, feeding a low-residue diet for to hours before colonoscopy can be helpful. this diet could consist of cooked eggs, small amounts of cooked beef or chicken, and small amounts of carbohydrates, such as a slice of toast or ⁄ to ⁄ cup of moist kibble. maintain good hydration. if all food is contraindicated, oral electrolyte solutions such as gatorade (pepsico, purchase, new york) can be used to maintain hydration without moving solids through the intestinal tract. give the animal a short-acting anesthetic and place the animal on a tilted table in lateral recumbency with the hindquarters elevated. perform a digital examination of the rectum and pelvic cavity to ensure that there are no strictures, polyps, or other obstructions. lubricate the proctoscope thoroughly with water-soluble jelly and pass it gently through the anal sphincter. press the proctoscope forward slowly and carefully with a spiral motion. if any resistance is encountered, stop the motion, remove the obturator, and inspect the bowel to determine the cause of the resistance. if possible, replace the obturator and continue forward motion until the instrument is passed its full length. withdraw the obturator, and observe the mucosa. the major portion of the examination is conducted as the instrument is withdrawn. to view the colonic and rectal walls completely, one must move the anterior end of the proctoscope around the circumference of a small circle while withdrawing the proctoscope. occasional insufflation with the inflating bulb is helpful in smoothing out folds of tissue. repeated instrumentation may produce petechiae and minor hemorrhages that are not pathologic. for examination of the terminal rectum and anus, the hirschman anoscope provides adequate, convenient visualization. newer techniques for visualizing the upper and lower gastrointestinal tract are being used in dogs. the flexible fiberoptic endoscope enables one to visualize and photograph the esophagus, colon, and stomach. one is able not only to visualize lesions of the gastrointestinal tract directly but also to assess motility, take biopsies of lesions, and remove foreign bodies. the ability to visualize directly the vestibule, the vagina to the level of the cervix, and the urethral orifice in female dogs is of particular value in evaluating patients with known or suspected congenital urinary tract disorders, such as incontinence or ectopic ureters and vaginal strictures (congenital or traumatic). numerous vaginal malformations and chronic infections cause visual changes that are identified easily during endoscopic examination. frequently the procedure can be conducted in the standing awake patient. sedation or general anesthesia is indicated when extensive manipulation, catheterization of the bladder, or a vaginal biopsy are indicated. position the sedated or anesthetized patient in dorsal or ventral recumbency to facilitate orientation during the procedure. if catheterization of the urinary bladder is required during the procedure, dorsal recumbency seems to facilitate visualization of the urethral papilla and insertion of the catheter. vaginoscopy entails use of a relatively small, flexible endoscope to mm in diameter or a -to -mm rigid scope. the flexible scope offers the advantage of a larger biopsy channel and the ability to view the lateral vaginal wall easily. vaginoscopy is considered an invasive procedure and should be conducted under sterile conditions. before insertion of the sterilized endoscope, the vulva should be free of obvious debris, should be clipped if necessary, and should be cleaned gently with a surgical soap and rinsed. insert the scope such that initial position of the tip of the scope is directed toward the anus. as insertion proceeds, the tip of the endoscope reaches the horizontal portion of the vestibule and vagina. when feasible, pass the scope to the level of the cervix. slight insufflation of the vagina may be useful in dilating the vagina, greatly facilitating the examination. conducting the examination from the level of the cervix caudally is recommended. this maximizes the ability to visualize critical anatomic features. the relatively recent introduction of very small ( -mm diameter) flexible and rigid endoscopes into veterinary medicine allows visual examination of the urethra, trigone, urinary bladder, and right and left ureterovesicular junctions of female dogs and even cats. such examinations are most useful when obstructive lesions (tumor or calculi) of the urethra or trigone are suspected. visual examination of the interior surface of the bladder and the capability of collecting biopsy samples make this a particularly useful diagnostic tool in the hands of the experienced clinician. numerous techniques are described for administering calories and nutrients to patients that are unable or unwilling to take in, chew, or swallow food. one method, intravenous hyperalimentation, is reserved for patients that are not able to tolerate any food being introduced via the gastrointestinal tract and represents a radical, and ideally transient, departure from normal. however, enteral feeding, which is always preferable to intravenous hyperalimentation, allows the clinician several options for administering food directly into the gastrointestinal tract. consideration of several variables is critical when one is initiating enteral feeding programs, such as the patient's diagnosis and attitude, the status of the gastrointestinal tract, and the ability of the patient to digest and absorb food once introduced. in addition, consideration of the type and constituency of the diet provided is important. although the options available for enteral nutrition are much greater that those for intravenous hyperalimentation, the clinician must consider dietary requirements carefully when planning enteral nutritional support. when evaluating enteral feeding for the individual patient, the clinician has four basic options: nasoesophageal tube, pharyngostomy tube (least recommended), esophagostomy tube, and percutaneous gastroscopy tube (which can be introduced using an endoscope or with the so-called "blind" technique). all techniques involve use of a polyurethane or silicone feeding tube. the nasoesophageal tube placement technique does not require general anesthesia, and the tube may be inserted using a topical anesthetic only. each of the other techniques described requires that the patient be anesthetized to ensure proper and safe placement. for temporary, short-term feeding, nasoesophageal intubation is a simple technique that works well in cats, puppies, and adult dogs. patients that are comatose; have severe, persistent vomiting; have esophageal disease or dysfunction; or are unable to swallow are not candidates for this procedure. the objective of the procedure is to place a small-diameter tube ( f to f for dogs weighing more than kg and f to f for small dogs and cats) through the nasal cavity into the distal esophagus. the tube does not have to enter the stomach. when measuring the tube length, measure from the tip of the nose to the eighth or ninth rib (figure - ) . administer to drops of a topical ophthalmic solution ( . % proparacaine) directly into one nostril. hold the head gently upward for a few seconds to allow the solution to reach the back of the nasal cavity. in most patients, it is desirable to wait to minutes and then to repeat the instillation in the same nostril. for larger dogs, % lidocaine solution ( . to . ml) gradually instilled into the nostril is an alternative technique to achieve topical anesthesia. lubricate the tube with a thin coat of a water-soluble lubricant, such a % lidocaine lubricating gel. pass the tube into the nasal cavity while directing the tube tip medially and ventrally into the ventral meatus. the anatomic shape of a dog's nostril usually requires directing the tip medially but almost perpendicular to the plane of the nasal cavity to facilitate insertion. initial resistance (pressure, not pain) usually is perceived, and the patient's head as expected quickly retracts, leaving the operator holding the tube tip some inches away from the patient's nose. be persistent. repeat the procedure, as necessary, by quickly inserting the first inch or more of the tube into the nostril. with the other hand, push the nasal philtrum up, and with a finger, push the lateral portion of the nostril medially. this will help facilitate movement of the tube into the ventromedial nasal meatus. once started, the remainder of the technique is relatively straightforward. as the tube reaches the caudal aspect of the nasopharynx, it should pass directly into the esophagus with little or no resistance. affix the tube remaining outside the patient to the head or face using a "butterfly" tape, gauze, suture (figure - ) , or skin glue (skin glue [superglue] generally is not recommended because this can result in loss of hair and skin pigment when the glue becomes dislodged). caution: the tip of the tube can be introduced inadvertently through the glottis and into the trachea. topical anesthetic instilled into the nose can anesthetize the arytenoid cartilages, thereby blocking a cough or gag reflex. i prefer to check the tube placement with a dry, empty syringe. attach the test syringe to the end of the feeding tube. rather than inject air or water in an attempt to auscultate borborygmus over the abdomen, simply attempt to aspirate air from the feeding tube (figure - ) . if there is no resistance during aspiration and air fills the syringe, it is likely that the tube has been placed in the trachea. completely remove the tube and repeat the procedure. however, if repeated attempts to aspirate are met with immediate resistance and no air enters the syringe, the tube tip is positioned properly within the esophagus. if there is any question regarding placement, a lateral survey radiograph is indicated. less invasive and not requiring endoscopy equipment, esophagostomy tube placement in dogs and cats is an alternative technique to use in patients that have long-term feeding needs. use a f to f rubber, polyurethane, or silicone feeding tube placed at the level of the middle of the cervical esophagus to the level of the eighth rib. the technique does require general anesthesia or, in the hands of an experienced individual, shortterm intravenous anesthesia. the technique has been described in detail in textbooks (see marks sl; additional reading). to place an esophagostomy tube, first assemble the necessary supplies: large curved rochester-carmalt forceps, clipper and clean blades, antimicrobial scrub, gauze squares, red rubber tube, permanent marker, scalpel blade and handle, needle holder, suture scissors, and nonabsorbable suture ( nonabsorbable, cutting needle. after placing the patient under general anesthesia and intubating the patient, place the patient in right lateral recumbency and clip the lateral left side of the neck from the ramus of the mandible caudally to the thoracic inlet, and dorsally and ventrally to midline. note that the left side of the neck is preferred because of the normal anatomic location of the esophagus. however, if there is injury, infection, or mass that prevents placement of the esophagostomy tube in the left lateral cervical region, the right lateral side of the neck can alternately be used. next, aseptically scrub the clipped area, and push the rochester-carmalt forceps through the mouth into the esophagus. direct the curved tips of the instrument laterally, so the tips can be visualized under the skin. use care to note where the external jugular vein lies, to avoid laceration of the jugular vein. measure the tube from the proposed site of tube entrance to the mid thorax, then label the tube with a permanent marker. next, open the curved tips of the instrument, and make a stab incision through the skin, through the open tips of the instrument, into the esophagus. push the tips of the instrument through the skin incision. grasp the distal end of the tube with the instrument, and clamp the instrument. pull the tube through the skin incision and rostrally out of the front of the mouth. if the tube does not come easily, usually the hinges of the forceps are caught on tissue within the oropharynx or pieces of the endotracheal tube. once the distal end of the tube is through the front of the mouth, push the distal end of the tube caudally into the esophagus with a finger or the instrument. as the distal end of the tube is pushed into the esophagus, pull the proximal end of the tube, to add tension to the tube. the proximal end of the tube will flip toward the patient's nose when the tube is situated in the esophagus. the tube can be taped in place while radiographs are taken to confirm placement. after radiographs confirm placement in the esophagus, suture the tube in place with two sutures, one purse-string and finger-trap around the tube entrance site, and another deep suture near the atlas, with a second finger-trap. finally, place antimicrobial ointment over the tube entrance site, and a loose bandage around the neck. unlike gastrostomy tubes, esophagostomy tubes can be used immediately, and removed immediately, if the patient chooses to start eating voluntarily after tube placement. it is important that one first observe the technique being performed by someone with experience before attempting to place an esophagostomy tube for the first time. although postplacement complications generally are limited to local irritation or minor infection at the site of the stoma in the midcervical region, tube placement into the mediastinum or subcutaneously can occur. percutaneous gastrostomy tube placement percutaneous gastrostomy tubes are used routinely to administer nutrients and medications orally over days or weeks to cats and dogs that cannot have nutrients administered by mouth or that will not eat (e.g., because of feline hepatic lipidosis, oropharyngeal neoplasms, maxillary or mandibular fractures, oral reconstructive surgery, esophageal masses or foreign bodies, or severe pharyngitis). the percutaneous gastrostomy tube is placed so that it extends through the skin and left cranial abdominal wall of the abdomen into the body of the stomach. catheter preparation for percutaneous gastrostomy tube is as follows: . use the french-pezzar mushroom-tipped catheter. stomach tube preparation for percutaneous gastrostomy tube is as follows: . use a smooth-ended vinyl stomach tube. . measure the length of the tube needed to reach the stomach by laying the tube along the animal's side with the rounded end to cm caudal to the last rib. . mark the tube with an indelible marker or adhesive tape at the tip of the muzzle and cut off the excess tube. . put the tube in the freezer for minutes to stiffen the tube before beginning the procedure. placement of a percutaneous gastrostomy tube is as follows: . clip and surgically prepare the skin over the left abdominal wall. . place the mouth speculum between the right canine teeth. . place the stomach tube in the esophagus to the level of the cardia. . rotate the tube counterclockwise while carefully advancing it through the cardia. . turn the tube back clockwise and advance the tube until it can be visualized through the abdominal wall to cm caudal to the last rib (figure - ). . rotate the tube so that the tip lies against the stomach and abdominal wall one third of the distance between the epaxial muscles and the ventral midline. . make a -to -mm skin incision directly over the lumen of the stomach tube. . use a sovereign catheter (over the needle) and puncture the abdominal and stomach walls, placing the catheter inside the lumen of the stomach tube. remove the needle (figure - ) . . thread a long, rigid suture through the catheter and advance it through the stomach tube until the end is observed at the mouth end of the tube (figure - ) . . carefully remove the plastic catheter from the stomach tube opening and place a hemostat clamp at the end of the suture material. . remove the stomach tube over the oral end of the stiff introduction suture line. . attach the open, beveled end of the french-pezzar catheter stomach tube to a plastic sovereign catheter using a mattress suture (figure - ) . . force the tip of the rubber stomach tube into the large end of the sovereign catheter. . advance the catheter tube through the mouth and esophagus into the stomach by placing traction on the abdominal end of the introduction line. . the catheter will emerge through the skin incision, followed by the rubber tube. grasp the tube with forceps and pull it through the incision opening (figure - , a) . . remove the catheter by cutting it off cm below the beveled tip. pull the rubber tube through the abdominal wall until slight resistance is felt (figure - , b) . . slide the outer flange over the end of the tube down to the skin level (figure - ) . . apply antimicrobial ointment and a sterile gauze sponge over the skin incision. . bandage the gastrostomy tube in place (figure - ) . rights were not granted to include this figure in electronic media. please refer to the printed publication. rights were not granted to include this figure in electronic media. please refer to the printed publication. tear production comes predominantly from the tarsal and conjunctival glands and from the accessory tarsal glands. the reflex tear secretors are the main lacrimal gland and the accessory lacrimal glands. the production of normal lacrimal secretions can be tested by using the schirmer tear test, a standardized filter paper (figure - ) that effectively measures the rate of tear production in millimeters per minute. schirmer tear strips now are impregnated with a blue dye to facilitate visualization of the distance (in millimeters) that the tear migrates during the -minute test. none required. technique each eye can be tested independently, or both eyes can be tested simultaneously in the cooperative patient. carefully fold the notched end of the test strip before removing it from the plastic package. insert the folded end into the lower conjunctival cul-de-sac (figure - ) and begin the timing. maintain the schirmer test strip in position by gently holding the eyelids closed but not touching the paper. at the end of minute, note the degree (distance) of wetting that occurred and record it in the medical record. the normal dog and cat should produce wetting over to mm in minute for each eye. amounts less than that are consistent with keratoconjunctivitis sicca. amounts greater than mm may be normal or may be consistent with excessive tear production, or epiphora. the cornea is composed of various layers of specialized avascular epithelium and stroma. the outer layer, the corneal epithelium, is a highly sensitive, thin layer overlying the corneal stroma, the thickest layer. descemet's membrane is a distinct, thin layer of tissue beneath the stroma. the innermost layer of the cornea is the endothelium. damage to the corneal epithelium occurs frequently in dogs and cats. clinical presentation typically is characterized by blepharospasm of the affected eye with or without a visible ocular discharge or conjunctivitis. whenever superficial corneal injury is suspected, assessment of the integrity of the corneal epithelium is indicated. fluorescein dye-impregnated test strips can be used to determine whether the epithelial barrier overlying the corneal stroma has been disrupted and thus can establish the presence or absence of a corneal ulcer (figure - ) . none required. the test is simple to accomplish. moisten the dye-impregnated tip of the test strip with a drop of balanced saline solution (or commercial ocular irrigation solution). gently allow the tip of the test paper to touch the cornea, or sclera, of the affected eye. (in patients with particularly painful, sensitive eyes, use a topical anesthetic to moisten the test strip or apply the anesthetic directly to the cornea before testing.) immediately rinse the eye with a sterile irrigation solution to remove the excess dye (the test strip has a lot of dye; be prepared to catch the excess fluid with -× -inch gauze). promptly examine the eye with a direct, focal light source. evidence of green dye uptake in the stroma indicates that an ulcer is present. the absence of staining generally indicates that the corneal integrity is intact. one exception exists. the descemet membrane will not take up fluorescein dye. a patient with a deep corneal ulcer that penetrates through the corneal stroma and allows herniation of the descemet membrane (descemetocele) will not demonstrate a positive stain. careful visualization of the cornea, however, is likely to reveal the presence of a such a serious, deep ulcer. none required. fluorescein dye can also be used to assess patency of the nasolacrimal duct. to perform this examination, place a drop of fluorescein dye from a sterile fluorescein strip into the eye and add or drops of a sterile eye wash. after to minutes, examine the external nares with the aid of a cobalt blue filter or wood light for the presence or absence of fluorescence. a clean, -× -inch white gauze square touched against the nasal planum also will pick up the greencolored dye if the duct is patent. if dye is present, the lacrimal excretory system is patent and functioning. if epiphora exists but the primary dye test indicates that the lacrimal excretory system is patent, hypersecretion of tear fluid may be implicated as the cause of the epiphora. irrigation of the nasolacrimal system is indicated if the primary dye test result is negative. in the dog the nasolacrimal puncta are located to mm from the medial canthus on the mucocutaneous border of the upper and lower lids. in the dog, use a -to -gauge (in the cat, a -gauge) nasolacrimal cannula (figure - ) . topical anesthesia often is required. fill a -ml syringe with saline, and attach the lacrimal cannula and pass it into the lacrimal puncta of the upper lid. several points should be made about evaluating the nasolacrimal system. brachycephalicbreed dogs and cats occasionally may have a negative primary dye test result, although no blockage in the nasolacrimal system exists. in flushing the nasolacrimal system of some animals, fluid may not appear at the nose; however, the animal may gag and exhibit swallowing movements, indicating that the fluid has entered the mouth and the system is patent. none required. this procedure is preferably performed without administration of topical anesthesia. topical anesthetics not only are bacteriostatic but also may distort the cells and compromise the cytologic examination. in performing conjunctival scrapings, use a platinum spatula (kimura spatula), the tip of which has been sterilized. gently scrape the inferior conjunctival cul-de-sac (figure - ) . place the material on two glass slides. fix one slide in acetone-free % methanol for to minutes, then stain the slide with giemsa stain. heat-fix the other slide, and apply gram stain. to culture the conjunctiva, use sterile cotton-tipped applicators, fluid thioglycolate medium, and blood agar medium. evert the palpebral conjunctiva of the lower lid, and pass one side of a sterile cotton applicator, previously moistened with sterile broth or thioglycolate medium, over the palpebral conjunctival surface. streak the swab onto a sterile blood agar plate, then place the plate in a tube of thioglycolate broth. no topical anesthesia is used before culturing because preservatives present in anesthetics can inhibit the growth of bacteria. glaucoma is an increase in intraocular pressure incompatible with normal ocular and visual functions. one method used to measure intraocular pressure is tonometry, in which the tension of the outer coat of the eye is assessed by measuring the impressibility, or applanability, of the cornea. because the measurements based on tonometry involve calculations that have a wide base of variations, tonometry readings are always approximations. the schiøtz tonometer consists of a corneal footplate, plunger, holding bracket, recording scale, and . -, . -, . -, and . -g weights. the principle of the schiøtz tonometer is that the amount that the plunger protrudes from the footplate is related to the indentability of the cornea, which in turn is related to the intraocular pressure. however, use of applanation tonometry today has virtually replaced use of the schiøtz tonometer. in applanation tonometry, a very small area of the cornea is flattened by a known force, usually a calibrated burst of air. the advantage of this technique over the indentation (schiøtz) method is that the errors resulting from ocular rigidity and corneal curvature are greatly reduced. special equipment is required to perform applanation tonometry (figure - ) . the presence or absence of glaucoma, an increase in intraocular pressure, can be determined using applanation tonometry. on the other hand, gonioscopy permits one to visualize and examine the iridocorneal angle and potentially establish the cause of glaucoma. however, specific training and equipment are required not only to perform gonioscopy but also to interpret the result. this procedure is most appropriately performed by an ophthalmologist. barnett kc, crispin sm: feline ophthalmology, philadelphia, , wb saunders. barnett kc, sansom j, heinrich c: canine ophthalmology, philadelphia, , wb saunders. gastrointestinal studies when considering a contrast study of the gastrointestinal tract, it is not unreasonable to question the value of doing the procedure. at issue is the fact that abdominal ultrasound and/or gastrointestinal endoscopy has largely replaced contrast radiography of the gastrointestinal tract and for good reason. diagnostic modalities such as ultrasound (in the hands of an experienced individual) and endoscopy have a much greater diagnostic yield than the less sensitive contrast study. so why even try? endoscopes are not available in every practice, and limited access to ultrasound equipment, much less someone who is qualified to use it, puts routine use of advanced diagnostic modalities out of reach for many practices. however, it must be appreciated that with regard to diagnostic value, a radiographic contrast study of the gastrointestinal tract is a far less sensitive diagnostic modality than abdominal ultrasound or endoscopy. the procedure for the gastrointestinal radiographic contrast study is outlined next. contrast agents available for gastrointestinal studies include barium suspension preparations or micropaque (guerbet, villepinte, france), and water-soluble agents (gastrografin [bracco diagnostics, princeton, new jersey], which is % meglumine and % sodium diatrizoate). water-soluble agents are used if bowel perforation is suspected. undiluted water-soluble agents are hypertonic and should be diluted at a ratio of one part gastrografin to two parts water. no single procedure is appropriate for all gastrointestinal cases. the clinician must select procedures based on the clinical history and physical findings, apparent location of the lesion within the gastrointestinal tract, endoscopic findings, and results from other imaging studies, such as abdominal ultrasound. the contrast esophagram also is called barium swallow. the decision to perform a contrast esophagram is based on physical evidence of dysphagia (difficulty or pain while attempting to swallow) and/or persistent regurgitation (reflux of swallowed food without effort). the procedure necessitates that the animal fast for hours before radiography. remove all leashes from around the animal's neck, and obtain survey radiographs of the thorax. in esophageal contrast studies, administer barium suspension contrast medium, to ml/kg body mass. administration of barium as a contrast material is contraindicated if a perforation of the esophagus is suspected. when the esophagus has been coated with radiopaque material, take lateral, ventrodorsal, and right ventrodorsal oblique thoracic radiographs to visualize the esophagus. properly prepared, the barium should be relatively thick and of a pastelike consistency. position the patient and cassette, and have the radiographic technique set. give a tablespoonful of barium orally. make the exposure when the animal takes its second swallow after the barium has been given. for esophageal studies and barium swallows, sedation with acepromazine and buprenorphine (iv, im, sq) will produce no adverse alteration in gastrointestinal motility. for cats, ketamine mg iv and midazolam . mg/kg (combined) can be administered intramuscularly (im) with no significant effect in esophageal motility. caution: patients with significant swallowing disorders have a risk of aspiration if contrast material is regurgitated. sedation can increase that risk. in some cases of incomplete esophageal stricture, barium liquid will pass through the esophagus unobstructed, whereas food will not. veterinarians should mix kibbled food with the barium in this case and allow the patient to eat the mixture just before the radiograph is taken. ideally, contrast esophagrams are performed using fluoroscopy rather than conventional radiographs. in this manner it is possible not only to identify strictures and dilatations, if present, but also to obtain a dynamic study of the esophagus that provides valuable information pertaining to swallowing and esophageal motility and function and an opportunity to evaluate sphincter activity at the level of the cardia. contrast studies of the upper gastrointestinal tract are used to facilitate diagnosis of persistent vomiting, hematemesis, unexplained and chronic diarrhea, suspected enteric foreign bodies, and suspected neoplasms and obstructions and for confirmation of displaced intestinal organs, as may be seen in diaphragmatic hernias. that said, abdominal ultrasound has become sufficiently available to largely replaced the upper gastrointestinal series. with an experienced ultrasonographer, the diagnostic value of abdominal ultrasound far exceeds that derived from evaluating sequential radiographs of a patient after oral administration of a contrast medium such as barium. in the event that ultrasound capability is not available, a contrast study of the upper gastrointestinal tract still can be performed. however, the clinician must appreciate that a barium contrast study of the stomach, duodenum, jejunum, and ileum has a low sensitivity as a diagnostic test. that is, negative findings are not expected to correlate well with the absence of clinical disease. a negative study does not rule out disease. likewise, a contrast study of the upper gastrointestinal tract is not recognized for its ability to confirm a diagnosis of gastrointestinal tract disease, even when disease is present. perhaps the greatest value in performing the upper gastrointestinal series in a dog or cat today centers on the need to identify a displacement of the stomach and/or small intestine because of an extraluminal mass lesion or congenital defect in the patient. in addition, the use of a microfine barium suspension may facilitate identification of intestinal ulcers, irregularities (e.g., intraluminal neoplasia), and radiolucent foreign bodies. however, variable-diameter, solid-phase radiopaque markers called barium-impregnated polyethylene spheres (bips) can be used to assess gastric emptying time, gastrointestinal transit times, and, to some extent, obstructive disorders. if an upper gastrointestinal study is indicated, follow the technique described: . ensure that the hair of the animal is free from dirt, paint, and foreign material. bathe the animal if necessary. . withhold food for to hours. . if the colon is filled with feces, administer a cleansing enema the evening before performing the procedure. in dogs, give a second enema to hours before the start of the gastrointestinal series. . at the start of an upper gastrointestinal series, obtain survey radiographs of the abdomen. administer a barium sulfate (micropulverized) preparation by stomach tube, or induce the animal to swallow the fluids. flavored, prepared barium suspensions are available, but they taste bad (personal experience). dosage levels vary, but for barium suspensions, give approximately ml/kg. as an alternative to barium, use an organic iodide liquid preparation. administer . ml/kg by stomach tube. obtain lateral and dorsoventral radiographs of the abdomen immediately after administration of the contrast material and at -minute, -hour, and -hour intervals. watersoluble contrast material passes through the gastrointestinal tract in to minutes. barium suspensions take to minutes to traverse the intestine. the colon usually is filled with barium hours after oral administration and may contain barium for to days after administration. barium contrast radiography is contraindicated if perforation of the stomach or upper gastrointestinal tract is suspected. in these cases, use water-soluble contrast media such as the oral diatrizoates because leakage into the abdomen will produce no foreign body granuloma. in addition, do not administer barium sulfate when an obstruction of the lower bowel may be present. in these cases, barium may only contribute to the obstipation. the following radiographic views are recommended after administration of radiographic contrast material: . immediately after administration of contrast material, obtain ventrodorsal, right lateral, and left lateral views. the right lateral view shows the pylorus of the stomach filled with barium, and the left lateral view shows the cardia and fundic portion filled with barium. the objective is to evaluate the distended stomach and initial gastric emptying. . twenty to minutes after administration of contrast material, obtain ventrodorsal and right lateral views to assess the stomach, pyloric emptying, and the proximal duodenum. . sixty minutes after administration of contrast material, repeat the ventrodorsal and right lateral recumbency views to assess the small intestine. . two hours after administration of contrast material, repeat the ventrodorsal and right lateral views to evaluate passage of contrast material into the colon and complete emptying of the stomach; contrast material should be in the terminal portion of the small intestine. the passage of contrast material through the normal gastrointestinal tract is variable; however, the following guidelines have been suggested: . contrast material is in the duodenum within minutes in most patients. excitement can delay gastric emptying time to to minutes. . contrast material reaches the jejunum within minutes and is within the jejunum and ileum at minutes. . contrast material reaches the ileocecal junction in to minutes. . at to hours after administration, contrast material has cleared the upper gastrointestinal tract and is within the ileum and the large intestine. in evaluation of gastrointestinal contrast studies, consider the following criteria: ( ) the size of the intestinal mass, ( ) the contour of the mucosal surface, ( ) the thickness of the bowel wall, ( ) the flexibility and motility of the bowel wall, ( ) the position of the small intestine, ( ) the continuity of the opaque column, and ( ) the transit time. clinical disorders for which the barium enema is indicated in dogs include ileocolic intussusception and cecal inversion (intussusception), mechanical and functional large bowel obstruction, invasive lesions of the large bowel, a mass outside the large bowel compressing the bowel, and inflammation of the lower intestinal tract. barium sulfate enemas are contraindicated in suspected obstruction of the colon and rupture or perforation of the colon. however, these same disorders also can be identified by ultrasonic examination or colonoscopy, either of which is the preferred diagnostic modality over a barium enema. twenty-four hours before radiographs, administer a liquid diet only, preferably water. during the to hours before the radiographs, administer a mild high colonic enema or give a saline laxative orally. do not give any irritating enemas within hours of the scheduled radiographic examination; however, administer isotonic saline solution or plain water enemas before the examination to ensure that the bowel is clear. obtain survey radiographs of the abdomen, and examine the colon to ensure that this portion of the bowel is clear. sedation or anesthesia may be indicated. barium may be infused through a catheter into the colon or allowed to flow in by gravity through an enema bag. do not force barium into the colon under pressure. do not elevate the enema bag more than inches above the animal. cuffed rectal catheters (bardex cuffed rectal catheters, f to f, and the bardex cuffed pediatric rectal catheter, f [c.r. bard, murray hill, new jersey]) can be used in dogs (figure - ) . for very small dogs and cats, use smaller catheters. a plastic catheter adapter and a three-way stopcock are needed. various barium sulfate preparations can be used; however, the final concentration should be % to % w/v. a commercially available barium enema kit is helpful. place the cuffed rectal catheter so that the inflated bulb is cranial to the anal sphincter. place the animal in right lateral recumbency and fill the colon with contrast material at a dose of to ml/kg. take the radiographs after infusion of a two-thirds dose of barium. if the colon is not filled, infuse more contrast agent. obtain radiographs in the ventrodorsal and lateral positions, and determine whether the colon is distended adequately. remove as much of the contrast material as possible from the colon, and repeat the radiographs. insertion of room air at ml/kg into the colon facilitates the evaluation of the colonic surface. deflate the cuff on the catheter, and remove the catheter from the rectum. throughout the procedure of filling the colon with contrast material or air, take care not to overdistend the colon, which may lead to rupture. when reviewing individual radiographs, look for the following radiographic lesions: ( ) irregularity of the barium-mucosal interface; ( ) spasm, stricture, or occlusion of the bowel . the arterial phase demonstrates renal blood flow; the nephrogram demonstrates the accumulation of contrast agent in the renal tubules and is used to evaluate renal parenchyma; the pyelogram phase evaluates the urinary collecting system, including the ureters; and the cystogram reveals the collection of contrast agent in the urinary bladder. excretory urography does not result in any quantitative information about renal function and is not a substitute for renal function tests. the degree of visualization of contrast material within the renal excretory system depends on the concentration of iodine in the contrast medium, the technique of excretory urography performed, the state of hydration of the patient, renal blood flow, and the functional capacity of the kidneys. an intravenous catheter is prepositioned. the contrast medium most commonly used is a diatrizoate or iothalamate compound. administer mg/kg of an iodine compound iv by syringe. continuous iv "push" is indicated. obtain a ventrodorsal radiograph at seconds after injection, and repeat ventrodorsal and lateral radiographs , , , , , and minutes after injection. this method is the current standard technique. if the patient's blood urea nitrogen level is greater than or equal to mg/dl or the creatinine level is greater than mg/dl, double the dose of contrast material. lesions that can be detected by using intravenous urography are renal mass lesions; neoplasia; renal cysts; renal and ureteral traumatic lesions; pyelonephritis; hydroureter; hydronephrosis; renal agenesis; hypoplasia; pelvic and ureteral obstructions (calculi, blood clots); renal parasites; ectopic ureter; and duplication of the collecting system. retrograde urethrography is a diagnostic tool used to localize diseases of the lower urinary tract of dogs and cats. this method can reveal conditions such as urethral neoplasms, strictures, trauma, calculi, or other anomalies. the procedure involves the injection of an aqueous iodine contrast medium into the urethra through a ureteral or balloon-tipped catheter. the radiopaque contrast material is mixed to a threefold to fivefold dilution with sterile lubricating jelly to increase the viscosity. a dilution of : contrast medium with sterile distilled water or saline also can be used. before retrograde contrast urethrography is performed, give the animal a cleansing enema. sedation or anesthesia may be necessary. inject to ml of contrast medium. near the end of the injection, while the urethra is still under pressure, obtain a lateral radiograph. if the urinary bladder is to be distended with contrast material or air, remove urine from the bladder. in the male dog, position the catheter so that the tip of the catheter is distal to the os penis. inject lidocaine to ml into the urethral lumen to anesthetize the urethra adjacent to the balloon-tipped catheter. in male cats, retrograde contrast urethrography can aid in defining the extent of urethral damage (stricture) or the presence of urethral calculi. in male cats, use a f balloon catheter or a . f tomcat open-ended urethral catheter. insert the catheter . cm into the penile urethra. if the urethra is patent, to ml of contrast material will enable visualization of the urethra, but increased amounts of contrast material ( to ml/lb) injected into the bladder are needed for maximum distension of the preprostatic urethra. a voiding positive contrast urethrogram is necessary to visualize the distal (penile) urethra. apply external pressure to the bladder (using a wooden spoon or other external compression device), and radiograph the distal urethra. take extreme care with the amount of fluid placed in the bladder if the urethra is occluded by a balloon catheter. overdistension of the bladder results in hematuria, pyuria, urinary bladder rupture, and mild to severe bladder inflammation. palpate the bladder carefully during distension, and note the backpressure on the syringe used in filling the bladder. cystography refers to contrast radiographic procedures that facilitate visualization of the lumen and/or contents of the urinary bladder and trigone (box - ). three procedures can be used to image the urinary bladder: positive contrast cystography, negative contrast cystography (also called pneumocystography), and double-contrast cystography (combination of positive and negative cystography performed in the same patient). note: many of the indications for performing contrast cystography are also indications for ultrasound examination. contrast cystography is most useful for characterizing congenital and acquired alterations in the normal anatomy and function of the ureters and lower urinary tract, such as ectopic ureter. abdominal ultrasound, when available, remains the preferred method for imaging abnormalities within the bladder lumen (e.g., calculi and tumors) and changes within the bladder wall. pneumocystography, also called negative-contrast cystography, involves the insufflation of a soluble gas into the lumen of the urinary bladder to facilitate imaging of any material or tissue within the bladder lumen that otherwise would be obscured by the presence of urine or positive contrast material. prepare the patient as described previously. once a urinary catheter has been placed and the urethra is occluded, use a syringe and a three-way stopcock to inject to ml of carbon dioxide or nitrous oxide per kilogram. palpate the bladder while filling it with gas to avoid overdistension or rupture. inject air until there is pressure on the syringe barrel or leakage of air around the catheter. replace any air that escapes during the procedure. take lateral and ventrodorsal views of the abdomen. caution: room air is the most accessible contrast material for pneumocystography and generally can be found in most practices. however, an increased risk of air emboli is associated with the placement of room air into the bladder under positive pressure, particularly in patients with hematuria. pneumocystography is not an innocuous procedure; fatal venous air emboli have occurred in dogs and cats. this complication is seen most commonly in cases of severe hematuria. ultrasound or positive contrast cystography is preferred over pneumocystography in such cases if a soluble gas is not available. if possible, use a gas that is readily soluble in blood (such as carbon dioxide or nitrous oxide) for bladder insufflation. the injection of radiographic contrast material into the urinary bladder is referred to as contrast cystography or positive contrast cystography. when ultrasound examination is not available or not feasible, the clinical and radiographic findings noted in box - justify the use of contrast radiography to image the bladder. the same principles of preparation apply as for obtaining a pneumocystogram. use a urethral catheter with a three-way valve or a small foley catheter with an inflatable cuff. organic iodides are the contrast material of choice and should be used in % to % concentrations. double-contrast cystography also can be performed in patients for which a positive contrast study is not diagnostic, yet there is reasonable indication for an intraluminal lesion. in this case, the same urinary catheter as used for the contrast study, remove all remaining urine and contrast material. if necessary, inject to ml of an aqueous organic iodine contrast material into the bladder. gently roll the patient over in an attempt to coat the bladder with contrast material. then distend the bladder with air in the same manner as described for pneumocystography. some of the lesions routinely diagnosed with the aid of cystography are calculi ( vaginal examination is indicated for collection of material from the mucosal wall for culture and exfoliative cytologic examination and for vaginoscopic examination of vaginal and cervical mucosa (box - ). examination of the vagina for culture and cytologic or vaginoscopic examination occasionally can be performed in the cooperative patient without the use of sedation or anesthesia. an assistant is used to restrain the patient on an examination table. bitches that can be restrained for other minor examinations (ears, teeth, toenails, anal sacs, and blood samples) often will tolerate vaginal examinations. those that need further restraint may require sedation or administration of a short-acting barbiturate anesthetic. trim long perivulvar hair and cleanse the perineum with a germicidal or surgical scrub such as povidone-iodine. water and germicidal soap usually will not control surface contamination by pseudomonas and proteus species, which frequently contaminate culture swabs. in dogs with long tail hair, it is appropriate to wrap the tail with gauze before the procedure to prevent bacterial contamination. if vaginal culture is indicated, this procedure should be conducted first to avoid contamination induced by the general examination. pass a sterile, warm vaginal speculum with only a thin coating of lubricating gel into the posterior vagina while an assistant spreads the vulva. guide the speculum into the vagina by placing the speculum into the vulva just at the dorsal commissure of the vulva and applying pressure up and out against the commissure. direct the speculum dorsally toward the rectum until meeting resistance, and then direct it horizontally into the cranial vagina. this procedure bypasses the clitoral fossa and enables visualization of the urethral opening and pelvic arch. take a guarded culture swab (swab covered by a protective plastic pipette) from its individual sterile bag and pass it inside the vaginal speculum to the anterior vagina or cervical area. then expose the swab from the protective plastic tubing and rotate it against the mucosa. retract the swab into the protective plastic tubing and carefully remove it from the vagina. the protected swab then may be placed back in its original sterile bag until it is processed for culture ( minutes) or placed in amies transport medium with charcoal. amies transport medium with refrigerator packs and a styrofoam-insulated mailing box will retain fastidious organisms for to hours. process bacterial, mycoplasma, and ureaplasma cultures for potential infectious agents. viral transport medium can be used for a separate sterile swab if viral agents such as the genital form of canine herpesvirus are suspected. immediately after the swabbing for culture, while the vaginal speculum is still in place, advance a clean or sterile swab moistened with sterile physiologic saline solution carefully into the anterior vagina to make a smear for cytologic examination. gently scrape vaginal epithelial cells from the ceiling of the vagina at or cranial to the region of the external urethral orifice. collect samples from the region of the clitoral fossa, which is lined by stratified squamous epithelium at all stages of the estrous cycle. gently rub the swab on the vaginal mucosa. remove the swab and roll it smoothly onto two or three clean glass slides. sterile vaginal speculum (e.g., adjustable spreading, stainless steel, or disposable plastic; cylindric; glass, plastic, stainless steel, or nylon) sterile otoscope heads of variable size for small dogs sterile protected culture swabs (teigland type or other) sterile culture swabs (culturettes) amies transport medium with charcoal viral transport media glass slides and coverslips sterile proctoscope (welch allyn, human pediatric type) or other endoscope, flexible or rigid sterile offset biopsy punch the smears may be fixed immediately in % alcohol, sprayed with a commercial fixative or hair spray, or left to dry in air. a drop of new methylene blue stain placed on a coverslip and inverted on the smear can be used to examine a wet mount preparation immediately. this stain is not permanent and precipitates when it dries, and new methylene blue-stained smears cannot be used for comparison with other smears made later in the cycle. the diff-quik or leukostat stain is a permanent stain that can be submitted for review by a pathologist. examine the smear for stage of estrous cycle and evidence of active inflammation. compare these findings with culture results and vaginoscopic findings to interpret evidence for an active genital tract infection, a carrier state of a potential infectious agent, or a possible contaminant at culture. a diagnostic laboratory with the ability to isolate specific infectious agents should indicate the number of organisms (few, moderate, many, or heavy) and report whether the isolates are pure or mixed and their significance. the vagina of the bitch is long in comparison with that of other domestic animals, hence digital examination of the cervix, and in many cases the urethral orifice, is not feasible. the mucosa forms longitudinal folds. the clitoris is in a well-developed fossa in the floor of the vestibule. the vagina can be visualized completely with a small, sterile proctoscope or flexible endoscope. lubricate the warmed, sterile instrument, and pass it to the region of the cervix. examine first without insufflation for true color and vaginal fluids or discharge. when insufflation is performed while the vulva is compressed around the sterile proctoscope, the vagina expands and its entire wall can be viewed completely as the instrument is withdrawn. the normal canine vagina has a uniform light pink color and longitudinal folds. during proestrus and estrus, the folds become more prominent and cross-striations give the surface a cobblestone appearance. this cobblestone appearance remains smooth when estrogen levels are high but quickly becomes angular (cobblestone appearance) when estrogen levels drop during the luteinizing hormone peak (ovulation), and progesterone levels increase. this change can be used to indicate ovulation and the ideal time for breeding. the hyperemia causes the vagina to appear reddish and congested. the pressure of air insufflation balances the mucosa. the canine vulva has a large cranial dorsal median fold that may obscure the cervix. in fact, ridges near the dorsal fold may give a false impression that this fold is the cervix. during estrogen stimulation, the cervix may be open and uterine blood may be escaping. in the management of dystocia, the vaginoscope can be used to detect puppies in the birth canal and to diagnose malpositions and aid in the correction of these conditions. during the endoscopic examination, small tumors or polyps can be removed or large masses can be sampled with the biopsy punch. ulcers or erosions can be cauterized, and foreign bodies can be removed. a complete vaginal examination must include careful palpation of the vaginal wall and pelvic canal. this palpation is accomplished by digital examination through the vulva (using a sterile glove) and is assisted by palpation through the posterior abdominal wall. incomplete hymen rings, vaginal fibrous stenotic rings, or pelvic malformation can be diagnosed. a digital rectal examination may be needed for vaginal masses or pelvic deformities. the canine reproductive cycle begins at the age of to months and repeats at intervals of to months. in the average bitch, ovulation occurs spontaneously to days after the onset of estrus; in normal bitches ovulation may occur days before to days after the onset of estrus. sperm live in the uterus of the estrous bitch up to days, and the ovum lives up to days after ovulation. the fertilized ovum takes to days to reach the uterus, and implantation takes place to days after ovulation. the gestation period from the first breeding is to days and from the luteinizing hormone peak is to days. anestrus is characterized by dryness of the mucosa and a thin vaginal wall with stratified squamous epithelial cells a few cells to several layers thick but without cornification. noncornified epithelial cells and wbcs are present in a ratio of : in the vaginal smear. the wbcs are polymorphonuclear. the noncornified epithelial cells are to nm in diameter and have round free edges, granular cytoplasm, and large nuclei with distinct chromatin granules. the period of anestrus is to months or longer in some breeds. in proestrus the vaginal wall is thicker than in anestrus, and the mucosa shows prominent cornified squamous epithelium ( to cells thick) with rete pegs. the longitudinal and transverse vaginal folds are thick, smooth, and round. the vaginal wall becomes impervious to wbcs, but there is extravasation of rbcs to the surface epithelium. the rbcs are discharged. vaginal smears show predominantly rbcs and noncornified epithelial cells, which become cornified as proestrus progresses. wbcs are present, but their numbers decrease as estrus approaches. debris and bacteria are abundant for to days. the vagina is thick with longitudinal and transverse folds that become angular as estrogen levels decrease and progesterone levels increase. fluid is abundant, often tinged with blood. noncornified epithelial cells and wbcs are absent. cornified epithelial cells, which are polyhedral and contain pyknotic nuclei or no nuclei, are predominant; their presence seems to be related to the appearance of flirting by the bitch and acceptance of the stud. wbcs reappear about to hours after ovulation. bacteria and debris are absent during estrus, but they are seen again in the smears after ovulation when wbcs reappear to days later. the number of wbcs increases rapidly, the number of cornified epithelial cells decreases, and the number of noncornified epithelial cells increases. after to days, the number of wbcs may decrease to to per field. after parturition, much cellular debris, wbcs, rbcs, and a few epithelial cells are present for several days, until placental sloughing is complete. the presence of masses of degenerate wbcs (and bacteria) indicates metritis or endometritis. the continued presence of blood-tinged fluids containing abundant rbcs, a few noncornified epithelial cells, and occasional wbcs (nontoxic) plus necrotic cells for months postpartum is evidence of subinvolution of placental sites. most of the characteristics just discussed that apply to bitches also pertain to queens. however, the small size of the feline vagina precludes palpation and early vaginoscopy. a sterile, warm, small-animal otoscope speculum enables fairly good visualization of the vaginal mucosa and can be used with a small, -mm-diameter sterile swab to obtain smears for culture procedures. use of the speculum is easiest after parturition or during estrus. vaginal cells for cytologic examination can be obtained with a moistened -mm cotton swab (calgiswab) inserted cm into the vagina. in some cases, flushing the vagina with sterile saline injected and aspirated with a clean glass eyedropper is more successful. use of an eyedropper may trigger ovulation, as it simulates coitus. unlike the bitch, the queen shows no diapedesis of rbcs during proestrus or throughout the estrous cycle. cytologic examination of feline vaginal smears reveals the following by stage of the estrous cycle. cytologic examination reveals scarce debris and numerous small, round epithelial cells with a high nuclear/cytoplasmic ratio, frequently in groups (seasonal: from september to january in the northern hemisphere). cytologic examination reveals increased debris and fewer but larger nucleated epithelial cells with a low nuclear/cytoplasmic ratio ( to days). cytologic examination reveals markedly less debris and numerous large polyhedral cornified cells with curled edges and small dark pyknotic nuclei or loss of nuclei ( to days) after coitus or induced ovulation. cytologic examination reveals hazy, ragged-edged cornified cells and zero to numerous wbcs with numerous bacteria and increased debris. cytologic examination reveals increasing numbers of small basophilic cells with wbcs still present (total period of metestrus, to days). if ovulation does not occur, the smear will return to an anestrous stage with few to no wbcs. the feline estrous cycle is continuous every to days if to hours of light are present daily. ovulation is induced to hours after coitus. sperm require to hours for capacitation in the uterus. implantation is expected to days after coitus. the procedure for artificial insemination in dogs includes the following steps: . determine the correct time to inseminate by test-teasing with a stud, by cytologic examination of vaginal smears, or by vaginoscopic examination to determine the day when vaginal folds change from round to angular. breed the day after the bitch first stands staunchly to accept service and "flags" her tail or during cytologic indications of estrus (complete cornification of vaginal epithelial cells) but before wbcs reappear in the smears. breed at -hour intervals until the female dog goes out of heat or for three or four inseminations. . if the vulva is soiled, clean it thoroughly with alcohol swabs (box - ). . gently aspirate semen through the inseminating pipette into the warm syringe. applying the artificial vagina to the shaft of the penis, apply pressure with the thumb and forefinger proximal to the exposed glandis. this usually can be done with one motion as the stud is thrusting. if the stud is shy and not interested, massage the penis slightly in the prepuce or in the artificial vagina to cause erection. when erection of the bulbus is felt, reflect the prepuce posteriorly to free the bulbus. apply pressure with the thumb and forefinger behind the bulbus, circling the shaft of the penis. after completion of the most rapid pelvic thrusting and ejaculation of the sperm-rich fractions of semen ( to ml), twist the penis degrees backward in a horizontal plane, between the hind legs, so that the penis remains in the same plane as in the forward position, with the thumb and forefinger still applying pressure around the circumference of the penis proximal to the bulbus. the penis cannot be twisted unless the prepuce is reflected posterior or proximal to the bulbus glandis. twisting the penis in this position simulates a natural "tie" and allows the person collecting the semen to better visualize the collection (artificial vaginas are widely available now and are much preferred because they simulate the natural pressure of the vagina). the first drops of ejaculate may be discarded, especially if any urine is present. collect the sperm-rich fraction separately. a clear ejaculate is prostatic fluid, which may be collected separately for examination. . after semen collection, place the penis in the forward position, straighten out the prepuce to avoid paraphimosis, and remove the bitch from the room. allow the stud to lick the erect penis and lose the erection. check the stud for evidence of paraphimosis before it is released or caged. the ejaculate consists of three fractions: first fraction: urethral secretion (usually clear fluid)- . to ml within seconds, ph . . if evidence of urine is present, discard this fraction and do not add it to the sperm-rich fraction. in most ejaculates collected from dogs, the first and second semen fractions are collected together. second fraction: sperm-containing secretion (milky opaque fluid)- . to ml within to minutes, ph . . third fraction: prostatic secretion (usually clear fluid)- to ml within minutes, ph . . the total specimen is . to ml, ph . . because the first and third fractions are clear, waterlike material and the second fraction is milky-opaque, the clinician can separate them by changing collecting tubes as each fraction is ejaculated. collection of only enough prostatic fluid to rinse the sperm fraction into the test tube is best. too much prostatic fluid may be detrimental to the longevity of sperm in storage. collecting individual fractions may be important in determining the site of an inflammatory reaction, but for artificial insemination only the sperm-rich, low-volume ejaculate is needed for insemination, dilution, or freezing. . return the stud to his cage. retain the bitch until the semen is examined, if insemination is to be performed. immediately after semen collection, slowly invert the tube several times to mix the semen gently. determine the motility of sperm by placing one drop of semen on a warmed microscope slide. cover the slide with a coverslip, and observe the specimen under low power for progressive motility. there will be no "waves, " but general vigorous forward motion should be evident. if the sample is too concentrated for individual sperm to be found, mix one drop of semen with one drop of saline at body temperature on a warmed microscope slide. using high power, count different groups of sperm, observing the numbers of motile and nonmotile sperm. total motility for a suitable sample should be % or greater. motility less than % is not satisfactory. determine the number of sperm in the total ejaculate. sperm concentration may be determined in a hemocytometer with a : blood cell dilutor kit (unopette), and concentration then is multiplied by volume to determine sperm numbers per ejaculate. remember that more dilute samples will be obtained when prostatic fluid is collected, but total sperm numbers in the ejaculate will be only marginally influenced by dilution with prostatic fluid. total sperm per ejaculate should exceed million in a normal male dog and may approach billion in large dogs. a minimum number of million sperm per insemination is needed on average for conception. determine morphology. make a smear of a drop of semen like a blood smear and allow it to air-dry. then stain the smear with diff-quik stain; dip the slide into the fixative and solutions and for to minutes each. then count sperm at × magnification, noting normal and abnormal sperm. if there is any question about abnormality, examine sperm cells. normal canine sperm are nm long; the heads are nm long. the percentage of abnormal sperm should be less than %. differential abnormality is important, and the following abnormalities should not be exceeded in any sperm count: abnormality of the head, % to %; midpiece abnormalities, % to %; tail abnormalities, % to %; and retained protoplasmic droplets, % to %. figure - shows abnormalities that should be counted and recorded. the presence and location of distal or proximal protoplasmic droplets, which may indicate cell immaturity, are important to note. defects of the cells within the testes are generally more serious than defects that occur in the sperm during epididymal transport or after ejaculation (such as fractured heads, retained protoplasmic droplets, or bent tails). usually a biopsy should not be done on material from testes unless the testes are azoospermic. damage produced after the sperm have left the testes may indicate epididymal disease or may be the result of cold, trauma, or osmotic or urinary contamination. when abnormalities are found, it is wise to obtain two or three semen samples within a few days for baseline evaluation and then repeat the studies in to weeks to determine whether there is a healing or regressing trend. there are usually days from the date of sperm formation to the date of ejaculation: days in the testes and days in transport and maturation in the epididymis. normal male dogs can be used at stud once every other day indefinitely or once every day for to days, after which sperm numbers in the ejaculate will decline but not to less than the numbers needed to achieve conception. although castration is a common first recommendation for any male dog with known or suspected prostatic disease, a number of prostatic disorders are recognized for which cytopathologic and histopathologic examination, rather than castration, is indicated. benign prostatic hyperplasia is recognized as the most common prostatic disorder of male dogs. in half of the dog population, changes consistent with benign prostatic hyperplasia are present by to years of age, especially in older intact dogs. because benign prostatic hyperplasia is androgen dependent, routine castration is the recommended treatment. however, at least three differential diagnoses justify additional diagnostic tests: prostatic neoplasia (usually adenocarcinoma), acute and chronic bacterial prostatitis, and prostatic cysts (septic and nonseptic). in male dogs with prostatomegaly and associated signs (dysuria and/or dyschezia), further evaluation of the prostate is indicated. several techniques have been described. abdominal ultrasonography is the preferred technique for evaluating prostate size, shape, and consistency. distension retrograde contrast urethrocystogram has been described as a means for evaluating the internal integrity of the prostate and is moderately effective in distinguishing normal from abnormal. however, this technique is not known to distinguish among various types of prostate disease. cytologic examination and quantitative bacterial culture of the ejaculate (especially the third fraction) of a male dog is recommended in any patient with prostatomegaly. however, sample collection can be difficult and is frequently not successful. in addition to lumbar radiographs and abdominal ultrasonography, performing a prostatic wash is a simple, noninvasive technique that may yield diagnostic information. using aseptic technique, place a conventional urinary catheter into the bladder and remove all urine. lavage of the urinary bladder with up to ml of sterile saline is recommended. recover the saline and save it (sample no. ). subsequently, retract the catheter tip, but only to the level of the prostate gland (immediately caudal to the trigone). position of the tip usually can be verified by tactile placement and the detection of increased resistance to catheter movement during retraction. position can be confirmed with a lateral radiograph of the pelvis. with the catheter in place, identify the prostate on a digital rectal examination and gently massage for approximately minute to force prostatic fluids into the urethra. infuse ml of sterile saline through the catheter. the objective is to wash prostatic fluids and cells into the urinary bladder and recover the saline from the bladder (sample no. ). examine fluid from both samples cytologically by distributing a drop of fluid across a glass slide, air-drying, and staining; submit a small aliquot ( . ml) for bacterial culture. cytologic examination is used to detect the presence of inflammatory cells versus neoplastic cells. low numbers of neutrophils (< cells per high-power field) are present in ejaculates and prostatic washes from normal dogs. quantitative bacterial culture, with a yield of greater than log , of one or more bacterial species in sample no. confirms bacterial prostatitis. complications from this procedure are unlikely, but conceivably a patient with septic prostatitis and prostatic abscesses could become bacteremic after this procedure, which in some patients could lead to sepsis. ultrasound examination is an important first step, when available, in assessing the size, shape, and internal integrity of the canine prostate gland and for detecting any changes in structures adjacent to the prostate. however, ultrasonography generally will not distinguish among different types of prostatic disease. further diagnostic tests are especially indicated in castrated, middle-aged to older male dogs with evidence of prostatomegaly. percutaneous fine-needle aspiration and/or prostatic biopsy are indicated. fine-needle aspiration of the prostate is performed through a ventral abdominal approach. use aseptic technique, and surgically prepare the skin at the level of needle insertion. because needle movement, once the needle has been inserted, could damage the urethra or adjacent structures, perform the procedure in the sedated or anesthetized patient. use an approach similar to that used for cystocentesis in a male dog with the exception that needle entry is at a point caudal to that used to enter the urinary bladder but is cranial to the pubis. the procedure can be performed with or without ultrasound guidance. in the absence of ultrasound guidance, determine needle position by tactile placement and detection of resistance as the needle enters the prostate. multiple needle penetrations and aspirations are attempted without withdrawing the needle from the skin. relieve negative pressure in the syringe before removing the needle. apply any material collected to a glass slide and allow it to air-dry before staining. any conventional stain used for peripheral blood is appropriate. a transrectal approach to fine-needle aspiration of the prostate has been used in dogs and is performed routinely in men. however, the distance from the anus to the prostate, visualization, and the risk of infection generally are cited as reasons for not performing this technique in dogs. fine-needle aspiration may not be diagnostic, particularly in patients with isolated, discrete lesions (cysts or neoplastic nodules) within the prostatic parenchyma. in such cases, ultrasound-guided needle (tru-cut) biopsy of the prostate is indicated. specific training and experience are indicated for performing this procedure because significant complications can result. complications associated with prostate biopsy and fine-needle aspiration are not insignificant. hematuria and periprostatic hemorrhage are described. postaspiration and postbiopsy abscess also have been described. consider the risk of urethral penetration and subsequent stricture at the site of penetration. kutzler ma, yeager a: prostatic diseases. in ettinger sj, feldman ec, editors: textbook of veterinary internal medicine, ed , st louis, , elsevier. upper respiratory tract for purposes of this discussion, the anatomic limits of the upper respiratory tract of the dog and cat extend caudally from the nasal planum to the first tracheal ring. key anatomic structures that principally can cause clinical signs include the anterior (external) nares, nasal cavity, nasal turbinates, frontal sinuses, maxillary recesses, upper dental arcade (especially the roots of the maxillary canine teeth), choanae (posterior nares), nasopharynx, soft palate, arytenoid cartilages, glottis, larynx, and vocal folds (see table - ). clinical signs related to the upper respiratory tract in dogs and cats are among the most common presenting complaints encountered in small animal practice and, interestingly, are frequent reasons for referral to specialty practices and veterinary teaching hospitals. the oral and nasal cavities are important portals of entry for foreign body entrapment and infectious agents. in addition to the occurrence of nasal neoplasia and trauma, it is not surprising that upper respiratory tract diseases in dogs and cats are common presentations. however, upper respiratory signs can be associated with significantly different underlying causes. localizing the problem amid a variety of clinical signs in an anatomically complex area presents significant diagnostic and therapeutic challenges to even the most astute clinician. the presentation addresses upper respiratory disease in the dog, with specific emphasis on clearly defining the presenting clinical signs, localizing the problem, and establishing the diagnosis. the first and most important step in establishing a diagnosis of canine upper respiratory disease is to define the presenting sign. experience has shown that an owner's ability to describe the patient's clinical signs accurately, particularly when signs are not present at the time of examination, is usually inconsistent and inaccurate, although it can be most entertaining. the four localizing clinical signs characteristically associated with upper respiratory diseases are sneezing and/or nasal discharge, stertor, stridor, and cough. each sign, considered independently, will focus the examination to the appropriate anatomic region of the upper respiratory tract. definition of the clinical signs sneezing and nasal discharge may seem intuitive. this is the most common presenting sign in dogs with upper respiratory disease. owners that present a dog with sneezing are likely to be accurate in their description of the problem. however, the presence or absence of a nasal discharge may be more difficult to establish. volume, character, and frequency of the discharge ultimately determine whether the owner will have even observed this sign. the astute owner will report whether the discharge is unilateral or bilateral. in the patient that has a history of sneezing and nasal discharge, instillation of a topical nasal decongestant into each nostril occasionally will provoke sneezing and elicit the nature of any discharge that is present. sneezing and/or nasal discharge localizes the problem to the nose, nasal cavity, and paranasal sinuses. however, thorough examination of the nose and nasal cavity can be difficult, even with the availability of appropriate endoscopy equipment. in addition to careful examination of facial symmetry, the first part of the examination begins in the oral cavity, with emphasis on the maxilla, the hard palate, and the canine teeth. examine the hard palate for evidence of trauma (penetrating or nonpenetrating) and congenital cleft palate (puppies). carefully probe the medial aspect of the maxillary canine teeth for evidence of oronasal fistulas. despite normal-appearing teeth and gingiva, severe, occult periodontal disease with resulting necrosis of bone does result in a septic communication between the oral and nasal cavities. the owner characteristically describes paroxysms of sneezing associated with a sanguineous nasal discharge or spray. if these findings are negative, radiographs of the skull are indicated. three views, obtained in the anesthetized patient, are indicated: lateral, ventrodorsal, and occlusal (open mouth) view. radiographic interpretation of the nasal cavity and sinuses dictates that the clinician have a thorough understanding of the anatomy of the upper respiratory tract. subsequently, with the patient still anesthetized, attempt a visual examination of the nasal cavity. radiographs are always performed before visual examination of the nasal cavity. manipulation of the tissue may result in intranasal bleeding, which will significantly complicate radiographic interpretation. a simple otoscope speculum placed into each nostril allows an adequate examination of the proximal % to % of the nasal cavity in most dogs. visual examination of the caudal % of the nasal cavity can be attempted only with a small-diameter endoscope. flexible and rigid scopes are available; each has advantages and disadvantages that will be discussed. computed tomography and magnetic resonance the second most common clinical sign associated with upper respiratory disease in dogs, stertor is intermittent, yet persistent or continuous snorting, also called stertorous breathing. paroxysms of stertor, typically called reverse sneezing, are characterized by rapid, consecutive inspiratory bursts through the nose. seldom actually seen during examination, reverse sneezing is likely to result from the patient's attempt to displace matter trapped in the nasopharynx and move it into the oropharynx, where it can be swallowed. visualization of the nasopharynx and choanae is essential in the patient that has chronic or persistent stertor. the examination can be accomplished only in the anesthetized patient. sedation is not sufficient to conduct the examination. a flexible endoscope with the ability to flex approximately to degrees is recommended. examination allows visualization of the nasopharynx and associated mucosa, the choanae (posterior nares), and the top of the soft palate (see figure - ). nasopharyngeal foreign bodies are by far the most common finding. sticks, plant material (grass and juniper twigs), peas, cotton balls, and thread are just a few examples. neoplasia is the second most common finding. in cats, lymphoma (felv related) obstructing the choana most commonly is observed (see figure - ). in dogs, neoplasia is uncommon, but (in my experience) sarcomas in young dogs have been seen most frequently. the least commonly encountered of the upper respiratory signs is stridor, or stridulous breathing. stridor is audible wheezing and is associated with restriction to airflow, usually at the level of the larynx. therefore stridor is the most critical and potentially life-threatening upper respiratory sign. this is especially true when stridor is continuous. the patient that has continuous stridor deserves immediate attention. make every effort to discern the cause once the clinical sign is characterized. in obtaining the history, owners generally describe wheezing accurately; however, some patients actually may have severe dyspnea or orthopnea. careful questioning of the client is indicated to determine whether wheezing is associated with the additional effort to breath. the clinician also should make an effort to discern whether the owner has observed any change in the ability of the dog to vocalize or bark. simply listening to the patient breath in a quiet room is the first step in assessing stridor. a stethoscope is not required to hear wheezing but should always be used to examine the cervical trachea, the larynx, and the lungs. any restriction to airflow in the larynx or cervical trachea can cause stridor. however, in the majority of cases the stridor will be significantly louder at the level of the larynx, indicating a restrictive lesion at that level. if any indication of respiratory distress is reported or manifests during the examination, subject the patient to a visual examination under general anesthesia. sedation is not sufficient to conduct the examination. be prepared. these patients are not routine. emergency resuscitation may be required on induction of anesthesia, including the need to perform a tracheostomy. on induction, carefully place an endotracheal tube. if there are no complications associated with inserting the tube, once anesthesia has been effectively induced and the patient's condition is stable, lateral and dorsoventral radiographs of the larynx and cervical trachea are indicated. metallic objects (e.g., fish hooks) can become buried in the mucosa and may not be observed during a visual examination. remove the endotracheal tube in order to conduct a visual examination. a focal, handsfree light source directed into the oropharynx is strongly recommended. carefully examine the epiglottis, arytenoid cartilages, glottis, and vocal folds using a cotton-tipped applicator. careful observation of the symmetry and function of the arytenoid cartilages is essential. the left and right cartilages normally respond to tactile stimuli when the patient is in a light plane of anesthesia; both sides should move to the medial plane rapidly and at the same time. they may not close, depending on the depth of anesthesia. it should be possible to visualize the cartilage on the inside of the tracheal rings while looking through the glottis. in large breed, middle-aged and older dogs, laryngeal paralysis is the most common cause of stridor. associated signs may include exercise intolerance and collapse during exertion. laryngeal paralysis and stridor also may be observed in young breeds as a congenital disorder (dalmatian, rottweiler, bouvier des flandres, siberian husky, and bull terrier). foreign body penetration of the laryngeal tissues can cause serious and lifethreatening obstruction because of infection and swelling. neoplasia may cause obstructive mass lesions involving the larynx, especially squamous cell carcinoma and lymphoma. granulomatous laryngeal disease and fungal mycetoma have been reported. the presence of a mass lesion, assuming there is no foreign body detected, warrants biopsy of the lesion. additional effort to control postbiopsy bleeding is important. i use a cotton-tipped applicator saturated with a : , dilution of epinephrine held against the biopsy site for to seconds. this is time well spent. postbiopsy administration of systemically effective dexamethasone has been suggested to control laryngeal swelling, but i have not found this to be effective or important. the following diagnostic procedures are elective and are indicated in patients with chronic disorders of the lower respiratory tract that are not considered life-threatening. transtracheal aspiration is a safe and clinically useful method for obtaining material for cytologic and bacteriologic examination from the lower respiratory tract of medium-sized to large dogs without invading the oval cavity. this procedure is not indicated in cats. the technique can be performed on the unanesthetized animal, although some sedation may be indicated. the hair overlying the larynx is clipped and prepared surgically. in small dogs and cats, tracheal aspirates are collected by passing the catheter through sterile tracheal tubes. light levels of anesthesia are used to accommodate coughing and tracheal intubation. place the animal in sternal recumbency or in the sitting position. elevate and extend the head. locate the cricothyroid membrane by moving the finger along the proximal trachea until the large ventral ridge of the cricoid cartilage is felt. use a -gauge, ⁄ -inch intravenous catheter to collect material through the trachea (figure - ) . puncture the cricothyroid membrane with the -gauge needle, and pass the catheter into the trachea until it reaches the distal trachea or main stem bronchus. (alternatively, in large dogs, insert the catheter between the tracheal rings at the junction of the middle third and distal third of the cervical trachea.) withdraw the needle, and leave the catheter in place. attach a -ml syringe containing sterile saline solution to the catheter. expel to ml of saline from the syringe. when the animal coughs, aspirate with the syringe to collect cells and mucus for bacteriologic and cytologic examination. when material has been collected, remove the catheter and bandage the animal's neck. culture material present in the syringe in blood agar and in thioglycolate medium. prepare material from aspiration for cytologic examination. press large plugs of mucus between two clean glass slides, and stain thin smears with wright or giemsa stain. complications of transtracheal aspiration biopsy include catheter trauma to the lower airway or needle trauma to the larynx, resulting in bleeding, subcutaneous emphysema, pneumomediastinum, pneumothorax, or airway obstruction. in cats and small dogs and in dogs for which general anesthesia is not contraindicated, tracheal aspiration (or tracheal wash) is a relatively safe, easy-to-perform procedure that can yield excellent diagnostic cytologic and culture specimens. the procedure has some advantages over transtracheal aspiration in that it allows sample collection from airways beyond note: cats and dogs with acute, severe dyspnea must be regarded as having a life-threatening condition until proved otherwise. immediate therapeutic and diagnostic intervention is indicated. section describes appropriate interventive procedures for the management of these patients. the bifurcation of the trachea (carina) and avoids complications associated with patient discomfort and movement during the procedure. however, cough reflexes are eliminated completely, thereby decreasing potential sample yields from deep in the airway structure. in either case, transtracheal and tracheal aspirations provide the best diagnostic material from large airways, not small airways and alveoli. the anesthetized dog or cat usually is placed in sternal recumbency. lateral recumbency (affected side down) may facilitate recovery of specimens from patients with focal or regional lung disease. use a sterile endotracheal tube to administer the anesthetic and oxygen. introduce a sterile red rubber catheter (long enough to extend beyond the carina) through the endotracheal tube (figure - ). (note: disposable adapters for use with endotracheal tubes are available that allow continuous administration of anesthetic gases while passing the rubber catheter through the tube [ figure - ].) introduce the catheter blindly until resistance is met as the tube attempts to enter smaller airways. use aliquots of warmed, sterile saline in prepared syringes to wash and retrieve samples. aliquots of to ml can be used per collection attempt in small dogs and cats, whereas volumes up to and ml are appropriate for larger dogs. with the catheter positioned as deep as practical in the airway, infuse the entire volume of saline. gentle agitation (intermittent aspiration and injection) may facilitate sample collection. if a -ml quantity is infused, retrieval of only to ml as a final volume per collection attempt is not unusual. the remaining fluid is rapidly (seconds) absorbed into the pulmonary vasculature. important: when performing this procedure, do not withdraw the rubber catheter while maintaining a high negative pressure on the syringe. doing so actually may tear mucosa away from the airway and could lead to pneumothorax or pneumomediastinum. the procedure can be repeated safely in the same patient several times. collection of three to five samples is routine. more samples may be indicated depending on the patient's condition and response to the procedure. monitoring of patients undergoing a tracheal wash procedure for oxygen saturation (pulse oximetry) throughout the procedure is recommended. in some patients with reactive airways, infusion of saline may cause significant bronchoconstriction, detected by a rapid decline in oxygen saturation. process collected samples immediately. submit at least one sample of liquid (not a swab of the liquid) for bacterial culture and sensitivity or mic. quantitative cultures are impractical because specimens will be diluted. if the sample appears to be highly cellular (characterized by turbidity), place aliquots into tubes containing edta. syring rs: tracheal washes. in king lg, editor: textbook of respiratory disease in dogs and cats, st louis, , elsevier. bronchoalveolar lavage bal is an alternative diagnostic procedure to transtracheal aspiration and endotracheal wash. bal has the advantage of retrieving fluid samples from distal airways and alveoli. this is a highly diagnostic procedure indicated in patients with generalized lung and regional (interstitial and/or airway) disease that are not in respiratory distress. patients suspected of having allergic or infectious respiratory disease or neoplasia are candidates for bal. although bal is used as a therapeutic procedure in human beings with chronic lung disease associated with accumulations of surfactant in the alveoli, there is no therapeutic indication for bal in dogs or cats. bal must be performed in the anesthetized patient and consequently may be contraindicated in some patients with severe respiratory disease. technique bal entails instilling sufficiently large volumes of fluid into the distal airways to reach, and recover, reasonable cytologic samples representative of small airways and alveoli. several variations on the technique have been described, but all recommend blind or visual placement of a catheter or bronchoscope into an airway of a lung lobe such that the airway is occluded. sterile, nonbacteriostatic . % saline, warmed to approximately body temperature and drawn into prepared syringes, is the fluid of choice. the volume of fluid varies with the size of the patient. defined doses of saline per kilogram of body mass have not been described. in large dogs, two -ml aliquots ( ml total) can be infused into each lobe sampled. in small dogs and cats, total volumes per lobe generally are restricted to -ml aliquots. recovery may be as low as to ml with each attempt. ensure that the animal's hair is free of dirt and debris try to limit the animal's fluid intake in the hours preceding radiography empty the animal's bladder immediately before taking radiographs understanding the most commonly diagnosed causes of sneezing and nasal discharge is especially helpful in patient management. in no particular order, the most common differential diagnoses for sneezing and/or nasal discharge include the following: . oronasal fistulas: especially common in middle-aged to older dogs, despite a history of recent dental prophylaxis. empiric treatment with an orally administered antibiotic typically results in rapid and complete resolution of clinical signs no breed is predisposed, but the condition is uncommon in brachycephalic breeds. persistent nasal discharge, sneezing, and intermittent epistaxis are common presenting signs. nasal radiographs may demonstrate lytic bone lesions. lysis of the vomer strongly supports neoplasia versus mycotic rhinitis. exposure to tobacco smoke has been associated with . times greater risk in long-nosed dogs. no or minimal response of the discharge to antibiotics occurs. eighty percent of nasal tumors are malignant. adenocarcinoma is most common, followed by squamous cell carcinoma persistent and voluminous mucoid nasal discharge, with or without sneezing, and nasal pain are reported. erosion of the external nares is an important physical finding. discharge is not responsive to antimicrobial treatment. occlusal view radiographs of the nasal cavity may demonstrate evidence of turbinate destruction and/or increased fluid density on the affected side. forty percent of patients are years of age or younger; % are years of age or younger. the diagnosis is uncommon in brachycephalic breeds lymphoplasmacytic rhinitis: poorly described clinical syndrome associated with chronic sneezing and nasal discharge (bilateral or unilateral) nebulization is used (l) in combination with oxygen therapy upper respiratory disease of cats, pneumonia, and postoperative atelectasis and pneumonia; and ( ) in chronic respiratory diseases such as chronic bronchitis, bronchopneumonia, collapsed trachea with secondary tracheobronchitis, emphysema, and bronchiectasis. aerosol therapy, however, is a limited-use therapeutic technique used in dogs and cats to administer antimicrobials, bronchodilators (aminophylline, mg), or corticosteroids. the advantage of doing so is to achieve relatively high levels of drug in the respiratory tract in patients with defined lower respiratory tract disease. in addition, administration of potentially toxic antimicrobials (aminoglycosides) by this route has been shown to be associated with minimal or insignificant uptake into the general circulation drugs that can be applied by jet nebulizer (figures - and - ) include the following: . bronchodilators: always use bronchodilators when administering drugs that may be irritating and constricting systemic administration of most antibiotics produces adequate pulmonary concentration for antibacterial effect. for bordetella species that are located at the tips of bronchial cilia, topical contact via nebulization may be useful. antibiotics that have been used successfully and safely include kanamycin ( mg in ml saline twice daily); gentamicin ( mg in ml saline twice daily) bland solutions: use these in large volume for prolonged mist effect: . % sterile saline ( to ml as needed); glycerin ( % in saline) detergents and mucolytics: these compounds are irritating and currently are not recommended by most authors antifoaming agents: administer ethyl alcohol ( % solution, to ml twice daily) figure - : disposable jet nebulizer used to administer humidified air and/or medication drugs affecting the respiratory system advanced and expensive techniques recently have been described: laparoscopic-assisted cystotomy, use of the ellik evacuator, use of stone "baskets" (through a cystoscope), and lithotripsy are examples. however, for removal of uroliths from dogs and cats with partial or complete urinary obstruction, urohydropulsion is among the more effective yet inexpensive techniques available. urohydropulsion is a therapeutic procedure for removal of foreign material, namely, uroliths, from the bladder and/or urethra of dogs canine lower urinary tract diseases canine and feline nephrology and urology . using a gloved left index finger (not lubricated) as a guide, insert the pipette through the vulva and dorsally into the vagina and forward to the cervix. elevate the bitch's rear quarters to a -degree angle by having an assistant pick up the bitch by holding the hock region so that no pressure is applied to the ventral abdomen and uterus. eject the semen gently and slowly. eject a bubble of air to push all the semen through the pipette. deposit the semen in the anterior vagina. . remove the pipette, and hold the bitch in an elevated position for minutes. during this time, use the finger encased in a sterile glove to "feather" the ceiling of the vagina to stimulate constrictor activity. this may be important to simulate a "tie" and transport semen into the uterus. . lower the bitch to the normal position, and immediately walk her for minutes so that she does not sit down or jump up on a person and allow semen to run back out of the vagina. . for best conception, inseminate undiluted fresh semen immediately. . refrigerated extended semen is best used within to hours if possible. however, refrigerated semen has been kept viable for up to days with proper care. skim milk has been used as an economical and adequate extender. heat milk to ° to ° c for minutes, cool it, and skim it at room temperature. to each milliliter, add units of crystalline penicillin. if pseudomonas species affect the semen, polymyxin b may be added at units/ml of extender. dilute semen with extender at a semen/extender ratio of : to : . extend canine semen for freezing with a diluent containing % lactose, % glycerin, and % egg yolk. refrigerate the : diluted semen; then pipette . -ml portions into depressions in a block of dry ice and hold them for minutes to freeze. store the frozen pellets in liquid nitrogen. frozen semen can be thawed in buffered saline at ° to ° c. good semen may be stored in liquid nitrogen for many years without significant loss of motility. conception is best when large numbers of thawed motile sperm are deposited in the cervix or uterine cavity. conception is poor when thawed semen is placed in the anterior vagina, as done in artificial breeding with raw semen. memon ma, sirinarumitr k: semen evaluation, canine male infertility, and common disorders of the male. in ettinger sj, feldman ec, editors: textbook of veterinary internal medicine, ed , st louis, , elsevier. semen collection: canine semen is collected for examination for breeding soundness, for investigation of infertility or prostatic disease, and for artificial insemination (box - ).the following steps outline the procedure for collecting semen from a male dog: . take the stud and an estrous teaser bitch (if available) to a quiet room where there will be no distractions and where there is good traction (rubber mats or rug) for mounting by the stud. . hold the bitch, and allow the stud to "flirt" (become aroused) for several minutes. if the bitch is in heat, a brief period of foreplay ("foreplay" may not be the appropriate word to describe the mating behavior of dogs, but it illustrates the point) with both dogs unrestricted will help the process. . if necessary, have assistants restrain the muzzled bitch and control the stud by a collar and leash. bring the stud up to the rear end of the bitch, and allow him to mount her or keep his nose in the region of her perineal area. . attach the artificial vagina to the semen collection tube, and apply a scant amount of lubricant to the opening of the artificial vagina. . if mounting occurs, allow the stud to grasp the bitch and start to thrust his pelvis in an attempt to copulate. gently, from the side of the sheath, grasp the penis by the prepuce and move the prepuce back over the engorged bulbus glandis; while for dogs undergoing bal, particularly when reactive (allergic) airway disease is suspected, pretreatment with a bronchodilator is appropriate and is recommended. aminophylline can be administered at mg/kg (cats) or mg/kg (dogs) orally to hours before the procedure. alternatively, terbutaline, . mg/kg, can be administered subcutaneously to cats minutes before the procedure.bronchoscopic bal allows direct visualization of the airway or lobe of interest. in medium to large dogs, place the bronchoscope directly through a sterile endotracheal tube. use of an inexpensive, disposable endotracheal tube adaptor permits simultaneous administration of oxygen and anesthetic throughout the procedure. saline can be infused from a syringe directly through the biopsy channel of the endoscope. the bronchoscope serves as the infusion catheter. using this technique, samples can be collected effectively from multiple lobes. blind placement (nonbronchoscopic) bal using a rubber end-hole catheter is required in cats and small dogs. blind placement is also appropriately used in patients with generalized lung or airway disease when discrete placement of the bronchoscope cannot be accomplished reliably. as with the endotracheal wash procedure described before, gentle agitation with the syringe (intermittent aspiration and injection) may facilitate sample collection. do not withdraw the bronchoscope or catheter while maintaining significant negative pressure because this may lacerate the airway, leading to pneumothorax or pneumomediastinum.bal is an invasive diagnostic procedure that is not without risk of injury or death. after completion of bal, administration of % oxygen for to minutes via endotracheal tube is recommended for all patients. evaluate the patient carefully for breathing effort and oxygen saturation (pulse oximetry) during recovery. although significant quantities of fluid remain in the airways after bal, most of the volume is absorbed rapidly. residual amounts of fluid, however, can be retained for to hours after the procedure. during this time, some patients will manifest cough. crackles may be auscultated. percutaneous aspiration needle biopsy can be helpful in establishing a diagnosis in conditions such as (l) chronic inflammatory disease of the lung-for example, granulomatous lung disease caused by mycotic organisms; ( ) chronic inflammatory disease; ( ) metastases to the lung; and ( ) primary lung tumors. the biopsy may provide enough diagnostic information to preclude performing an exploratory thoracotomy. lung biopsy is contraindicated in animals with hemorrhagic disease or thoracic disease that produces forceful breathing and coughing. clip and surgically prepare the biopsy site. infiltrate the skin, subcutaneous tissue, muscle, and parietal pleura with % to % lidocaine. in patients with diffuse parenchymal lung disease, taking biopsy material from the diaphragmatic lobes is recommended. the dorsal portions of the seventh to ninth intercostal spaces are preferred for percutaneous biopsies. in diffuse lesions, take biopsy material from the right or left thorax. caution: understanding of the risks associated with performing fine-needle aspiration of the lung is important, and these risks must be clearly communicated to owners whose pets undergo this procedure. lung aspirates will yield only cells, fluid, and trace amounts of tissue, yet there is a significant risk of inducing pneumothorax with the procedure, even when performed without difficulty or complications. for the procedure, a -to -gauge disposable needle (such as a -inch spinal needle) with stylet is preferred. leave the stylet within the needle until the lung has been penetrated. then quickly remove the stylet and immediately attach a sterile -to -ml syringe. the amount of air that might enter the lung between the time the stylet is removed and the syringe is attached is negligible. holding the syringe carefully and steadily against the patient's thorax, establish negative pressure in the same manner as when obtaining an aspirate from a lymph node. as much as the patient will permit, attempt three to four aspirations without withdrawing the needle.alternatively, insert a conventional -gauge needle attached to a -ml syringe subcutaneously over the area of interest. then establish significant negative pressure while the tip of the needle is still positioned in the subcutaneous tissues outside the parietal pleura. while maintaining the same amount of negative pressure in the syringe, direct the needle into the lung, leave it in place for to seconds, and withdraw it completely. apply any material collected directly to glass slide. this procedure is best conducted in patients that are awake. attempting the procedure in anesthetized dogs or cats could result in an unsuccessful aspiration, or, if the lungs were under positive pressure (ventilation or bagging), the risk of causing pneumothorax could be increased. other reported complications include hemothorax (always exciting), lung laceration caused by patient movement during the procedure, pulmonary hemorrhage, and hemoptysis. contraindications to fine-needle aspiration include patients with a known bleeding diathesis and coagulopathy, thrombocytopenia, uncontrolled coughing, pulmonary hypertension, pulmonary cysts, and bullous emphysema.ultrasound-guided techniques for fine-needle aspiration or biopsy of the lung recently have been described and generally are associated with fewer procedural complications. however, additional training and experience, in addition to having access to the proper size and type of ultrasound probe, are critical. cole sg: fine needle aspirates. in king lg, editor: textbook of respiratory disease in dogs and cats, st louis, , elsevier. inhalation therapy can be defined as nebulization (humidification of the inspired air) and aerosol therapy (the process whereby drugs are vaporized in a solution and delivered directly into the respiratory tract). in companion animals, inhalation therapy is most useful for humidifying air in the respiratory tract and moistening the mucous membranes (nebulization). sustained inspiration of dry air or gases causes irritation to the respiratory epithelium, which in turn results in swelling, bronchial gland hypertrophy, goblet cell proliferation, and loss of ciliary epithelium over time. respiratory secretions become thick and tenacious, and efficient bronchial drainage is impaired. the objectives of inhalation therapy include the following: . humidification of bronchial mucous membranes . deposition of miniscule amounts of potent drugs in smaller airways to achieve optimal topical therapeutic effects with minimal systemic side effects (e.g., bronchodilators) . deposition of moderate amounts of potent agents or agents that are effective only topically (e.g., antibiotics and mucolytics) . deposition of relatively large quantities of bland substances that promote bronchial drainage with minimal irritation (e.g., saline, propylene glycol, glycerin, and detergents) the objective of voiding urohydropulsion is to induce forceful voiding of urine by manually compressing the bladder to facilitate removal of cystic uroliths in female dogs. caution: do not perform this procedure until it can be confirmed by catheterization or cystoscopy that the urethra is patent.with the bladder filled with urine or saline (via catheterization), lift the patient (preferably sedated or anesthetized, although this procedure can be done in the awake patient) into a position such that the tail and perineum are ventral and the head is upright. the spine should be approximately perpendicular to the working surface. using one or both hands, gradually increase pressure on the bladder to induce and maintain a forceful stream of urine. objectively, small uroliths will be extruded. if the procedure is only partially successful, it can be repeated as necessary. obviously, voiding urohydropulsion has limitations and cannot be used in male dogs or in dogs with urethral obstructions or strictures. this procedure is indicated for male dogs and cats with partial or complete urethral obstruction caused by uroliths or accumulations of "sand. " this procedure should be performed in the anesthetized patient.note: there are discrepancies in the literature regarding whether to empty the urinary bladder of urine before performing this procedure. because patients with urethral obstructions may have a significant volume of urine in the bladder at the time of presentation, some authors recommend performing cystocentesis to relieve the internal pressure before attempting urohydropulsion. however, in patients that have had a profoundly distended bladder for several hours (even days), penetrating the urinary bladder with a needle presents significant risk of rupturing a fragile bladder. the next step, of course, is abdominal surgery. i recommend avoiding cystocentesis whenever possible. the volume of saline required to flush uroliths into the bladder is inconsequential considering the total volume already present.with the patient positioned in lateral recumbency, retract the prepuce and expose the penis as for conventional bladder catheterization technique. use sterile technique to pass an appropriately sized flexible catheter, which is advanced to the point of obstruction. attach a catheter-tipped -ml syringe filled with warmed (my preference) sterile saline and a water-soluble lubricant mixture (approximately two parts saline to one part lubricant) to the urinary catheter. an assistant places a gloved (always preferred) finger into the rectum to identify and occlude the lumen of the pelvic urethra at the level of the pubis. subsequently, infuse saline forcefully into the catheter to dilate the urethra proximal to the obstructing urolith. at that point, release the digital pressure on the proximal urethra while the solution continues to be infused through the catheter. objectively, the pressure within the urethra forces small stones retrograde into the urinary bladder, thereby relieving the obstruction. the objective of this procedure is not to push the calculi into the bladder with the catheter, because this can substantially injure the urethral mucosa, nor is it to force the calculi around the catheter and move it antegrade. key: cord- -nxut ov authors: grädel, c.; terrazos miani, m. a.; baumann, c.; barbani, m. t.; neuenschwander, s.; leib, s. l.; suter-riniker, f.; ramette, a. title: whole genome sequencing of human enteroviruses from clinical samples by nanopore direct rna sequencing date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: nxut ov enteroviruses are small rna viruses that affect millions of people each year by causing an important burden of disease with a broad spectrum of symptoms. in routine diagnostic laboratories, those viruses are identified by pcr based methods, often combined with partial sequencing for genotyping. in this proof-of-principle study, we assessed direct rna sequencing (drs) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. results of the approach were complemented with those obtained by sequencing the corresponding viral cdna via illumina miseq sequencing. drs of total rna extracted from three different enterovirus-positive stool samples produced long rna fragments, covering between % to . % of the best reference genomes. the identification of the enterovirus sequences in the sample was confirmed by the short-read cdna sequencing. sequence identity between drs and illumina miseq enterovirus consensus sequences ranged between - %. here we show that nanopore drs can be used to correctly identify the genotypes of enteroviruses from patient stool samples with high viral load. enteroviruses (evs) present a major burden for human health and healthcare systems worldwide, with large outbreaks consisting of hundreds of thousands of hospitalized cases occurring periodically [ ] . evs are associated with a wide variety of symptoms, ranging from mild respiratory diseases to severe neurological infections, leading potentially to death [ ] . these single-stranded rna viruses that belong to the picornaviridae family possess a relatively small genome size ranging from . to . kb. for routine diagnostics, ev presence is generally determined by real-time, reverse-transcription pcr (qrt-pcr) assays, complemented by partial sequencing of the vp -vp coding regions for genotyping [ , ] . due to the lack of proofreading mechanism in rna replication, ev genomes are highly variable and the likely subject of within-and between-genome recombinations [ , ] . therefore, when diagnostic tests solely rely on pcr using conserved primer sequences, false-negative results cannot be excluded. there is thus a need to generalize the assessment of ev diversity and evolution via a whole-genome approach, rather than from the limited information gained from sequencing short sequences alone. the main limitation towards a general adoption of whole-genome sequencing (wgs) for better ev characterization and better understanding of their evolution and epidemiology, is mostly of technological nature: wgs approaches are more expensive, technically demanding and hence time consuming than standard molecular assays; as such they are generally not routinely used in diagnostic laboratories. in the case of rna viruses, the challenge is even more exacerbated because various molecular steps (rna purification, extraction, cdna synthesis, and optionally amplification) add to the turnaround time and final cost of the assays. clinical wgs applications are mostly based on sanger sequencing and on second-generation sequencing platforms, with illumina miseq/hiseq and ion torrent machines leading the market, as benchtop sequencing technologies enable highthroughput sequencing at an affordable price per sample when samples are multiplexed [ ] . third-generation sequencers, i.e. smrt technology (pacific biosciences) and nanopore sequencing (oxford nanopore technologies, ont), have recently emerged as complement to or replacement of second-generation sequencers, by enabling the sequencing of single, long dna molecules, a feature that is particularly attractive in the context of sequencing full-length viral genomes [ ] . nanopore sequencing technology has been successfully applied to genome sequencing of rna viruses. based on viruses propagated on cell lines, viral transcriptomes have been examined by nanopore sequencing for instance for porcine circovirus [ ] , herpes simplex virus [ ] , hepatitis c virus [ ] , cultured influenza virus a, human cytomegalovirus / (hcmv) [ ] . additionally, nanopore sequencing has been used successfully to sequence whole ev genomes from viral cdna extracted from cell cultures: in their proof-of-concept study [ ] , the authors demonstrated that nanopore sequencing could be applied for rapid routine whole genome sequencing of ev with sufficient accuracy compared to sanger sequencing. metagenomic nanopore sequencing of influenza virus was performed directly from randomly amplified viral cdna obtained from clinical respiratory samples [ ] . furthermore, ont has presented a new, potentially revolutionary nanopore sequencing application, which allows sequencing of rna molecules directly, i.e. without the prerequirement of cdna synthesis or pcr amplification [ ] . this method, direct rna sequencing (drs), yields full-length, strand-specific rna sequences and enables the direct detection of nucleotide modifications in native rna molecules. drs has been used in several studies, including human poly(a) transcriptome [ ] and to analyse the transcriptome of dna viruses, such as hsv herpes simplex virus type (hsv- ) during productive infection of primary cell [ ] . drs was also used for sequencing rna genomes of e.g. pseudorabies virus propagated on immortalized porcine kidney epithelial cell line [ ] , influenza a virus (h n ) from infected chicken eggs [ ] , human coronaviruses viral rnas produced in cell cultures [ ] , and many other examples of complete sequencing of multiple, single-stranded rna (ssrna) viruses obtained with or without poly(a)-tailing of rna viruses obtained from cell cultures [ ] . here, in a proof-of-concept study, we apply nanopore drs to ev-positive stool samples and show that whole rna genomes of enteroviruses can be retrieved with enough genomic information for the characterization of the infectious agents. we further analyse the metatranscriptomic data provided by the drs approach and compared it with that obtained by illumina miseq sequencing of the same samples. sample description. in this study, we used three independent patient stool samples (e , e , e ), which were sent for enterovirus diagnostics to the clinical diagnostic laboratory of the institute for infection diseases, bern. the samples were collected in (e , e ) and (e ) and had a cycle threshold (ct) value of . (e ), . (e ) and . (e ) via real-time pcr using enterovirus-specific primers (see description below). ethics approval was granted by the swiss ethics committee on research involving humans on march to conduct sequencing of enteroviruses in clinical samples stored in the ifik biobank (basec-nr: req- - ). figure . analytical workflow. the workflow used for diagnostic assays is indicated by greyed boxes and that followed for ngs techniques by white boxes. routine diagnostic approach. after homogenization with a sterile pipette, about . g of stool sample was added to ml of transport medium [ ] containing - glass beads (diameter mm; merck ag, zug, switzerland) ( figure ). after s of vortexing, the suspension was centrifuged min at g and the resulting supernatant . -µm filtered with % penicillin/streptomycin (biochrom, berlin, germany). this preparation was further extracted on the easymag platform (biomérieux, geneva, switzerland) for real-time pcr and with trizol ls reagent (thermofisher, reinach, switzerland) for illumina miseq sequencing (see below). rna extraction and purification. a total of µl of trizol ls reagent (thermofisher) was added per µl of stool suspension in pbs. following homogenization by pipetting, the sample was transferred to phasemaker tubes (thermofisher). after incubation for min, µl of chloroform were added, and the tubes shaken vigorously by hand for seconds. after min incubation, the sample was centrifuged for min at , g at °c. the aqueous phase was transferred to a new tube and µg of carrier rnase-free glycogen (thermofisher) were added, followed by µl isopropanol. after incubation for min, the sample was centrifuged min at , g at °c, and the supernatant was discarded. the total rna precipitate was resuspended in ml of % ethanol. the sample was vortexed briefly, and then centrifuged for min at × g at °c. the supernatant was discarded and the rna pellet was air-dried for min, before being resuspended in µl of rna storage solution (thermofisher). after incubation at °c for min, the rna sample was either used directly in downstream applications, or stored at - °c. alternatively, total nucleic acid extraction was performed with nuclisens easymag from µl of the routine diagnostic stool preparation, to which . µl carrier rna (qiagen) was added and eluted in µl. this extract was used for real-time pcr and sanger genotyping. pre-treatment (chloroform/beads/centrifugation). we used the who recommended protocol for pre-treating stool samples for enterovirus rna isolation (enterovirus surveillance guidelines, [ ] ) adapted from nix et al., [ ] as follows: a low amount ( . to g) of stool sample was added to pbs (thermofisher) up to ml volume, to which . g of glass beads ( mm diameter, sigma) and . ml of chloroform (applichem, aesch, switzerland) were added. the mixture was shaken vigorously using a tissuelyser (qiagen ag, hombrechtikon, switzerland) for min at . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint / maximum speed. the suspension was centrifuged at × g for min at °c, and approximately ml of the supernatant was transferred to a new . ml tube and continued with rna extraction. real-time rt pcr. one-step rt-pcr was done with the agpath-id one-step kit (ambion, reinach, switzerland) using published primers and probes [ ] , following the protocol described previously [ ] . primers and probes were synthesized at microsynth ag, balgach, switzerland. enterovirus genotyping. genotyping of the samples was performed by vp amplicon sanger sequencing as described previously [ , ] and carried out at microsynth. nanopore sequencing. for nanopore sequencing, we followed the manufacturer's instructions of the protocol for kit sqk-rna (version drs_ _v _revn_ dec ), but substituted superscript iii with superscript iv (thermofisher). the input rna and amounts loaded on flow cells for each experiment is specified in supplementary table . quantification was done using a qubit rna hs assay kit (rna) or dna hs assay (cdna) kit on a qubit fluorometer . (thermofisher). the reverse-transcribed and adapted rna was loaded onto r . . flowcells and sequenced on minion sequencer. illumina miseq. primer design for coxsackievirus a cdna synthesis. the following three specific primers for coxsackievirus a were designed for this project to hybridize to coxsackievirus a sequence kj : r_a : ′-cccgtttctgccgctt- ′ adapted from primer by oberste et al. [ ] , r_a : ′-atatctctgaatttctcatt- ′ adapted from primer hev. c.d by bessaud et al. [ ] , ev- utr _a _rc: ′-catattcacgaccagattcctggtg- ′ (this study). all primers were synthesized at microsynth. cdna synthesis for illumina miseq. first strand synthesis was performed with superscript iv first-strand synthesis system as follows: for reverse transcription, a reaction mixture was prepared containing . µl of the specific primers ev- utr _a _rc, r_a , r_a ( µm), or µl of random hexamers ( µm) or µl of oligo(dt) ( µm) together with µl of mm dntp mix and filled with rna extract and depc-treated water to a total volume of µl. after heating at °c for min and snap cooling, a mixture of µl ssiv buffer, µl mm dtt, µl ribonuclease inhibitor and µl superscript iv reverse transcriptase ( u/µl) were added. the samples were incubated for min at °c and min at °c (preceded by min at °c when containing random hexamer primers). subsequently, µl of e. coli rnase h ( u/µl) was added and incubated for °c for min. to this mixture, µl of second strand synthesis reaction buffer nebnext (new england biolabs (neb)), µl nebnext second strand synthesis enzyme mix and µl nuclease free water were added. the reaction mixture was incubated at °c for min. clean up with µl agencourt ampure xp magnetic beads (beckman coulter, nyon, switzerland) was performed according to the manufacturer's instructions, with an elution volume of µl for samples e and e and µl for sample . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint / e . for sample e , an additional end-prep step was performed: to µl of the elution, µl of ultra ii end-prep buffer (neb), µl of ultra ii end-prep enzyme mix (neb), and µl of nuclease free water were added and the mixture was incubated for min at °c and mins at °c. after an additional purification step using µl agencourt ampure xp magnetic beads the cdna was eluted in µl of nuclease-free water. illumina miseq sequencing. libraries were prepared from unamplified cdna using nextera xt dna library prep kit (e ) and nextera dna flex library prep kit (e , e ) (illumina) before sequencing using an illumina miseq benchtop sequencer generating × bp paired-end reads (v ), according to the manufacturer's protocols. sequencing was performed at the next generation sequencing platform of the inselspital, bern. bioinformatic analysis. raw fast files produced by minion sequencing were basecalled under high accuracy mode using the ont basecaller guppy version . . with the parameter: "guppy_basecaller --input_path path --recursive --save_path path --qscore_filtering --min_qscore --flowcell flo-min --kit sqk-rna --cpu_threads_per_caller --num_callers ". statistics for nanopore sequencing output are summarized in supplementary table . basecalled nanopore reads were classified using blastn against ncbi's nucleotide (nt) database (downloaded . . ), using blast version . . . blast results were classified using megan (v. . . , [ ] ). rna sequences were mapped to the best scoring reference genome using minimap (version . -r -dirty, [ ] ). for illumina miseq data, adapter trimming was done with bbduk.sh from the bbmap package (v . , [ ] ), and human and rrna reads were removed by mapping reads against the corresponding databases (grch genome, cdna and ncrna, and silva lsu ssu https://www.arb-silva.de/documentation/release- /) using minimap [ ] . non-human, non-rrna reads were then assembled using spades (parameters -k --rna --only-assembler; version . . ; [ ] ). the resulting contigs were subsequently analysed using blastn and megan as described for nanopore data. sequence identity between illumina miseq and nanopore drs enterovirus genome consensus sequences was calculated using legacy blast . . . coverage plots were made with bbmap and plots created using r statistical computing environment (version . . ). data availability. after removal of any human reads, all illumina miseq sequencing data (fastq), raw and basecalled ont data (fast and fastq, respectively), and sanger sequences have been deposited on the european nucleotide archive (ena) under the project reference prjeb . drs was performed with enterovirus-positive stool samples with similar viral load (ct values between - ) in three independent experiments, consisting of samples from three different patients. the samples were prepared using the who recommended protocol for viral enrichment using chloroform/bead treatment, followed by rna extraction using trizol or easymag . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . table ). we sequenced the total polyadenylated rna using drs on minion nanopore sequencer. the runs were continued until there was only negligible sequencing output or until the maximum recommended duration of the run was reached. for validation purposes, all three samples were also subjected to cdna sequencing using illumina miseq from samples prepared by the routine diagnostic procedure (figure ) . the cdna was produced with either genotype specific primers (e ), or with both oligo-dt primers and random hexamers (e , e ). table . taxonomic classification of reads (oxford nanopore) and contigs (illumina miseq) using megan based on blastn matches against ncbi's nucleotide (nt) database at the domain level. sample e . drs reads from extracted rna were obtained up to hours after the beginning of the sequencing run, and sequencing was stopped after hours. out of a total output of , raw nanopore reads, , reads were successfully basecalled into rna sequences, with an average length of , bases (range - , bases). more than % of basecalled rna reads ( , reads) were taxonomically classified using blastn against ncbi's nucleotide (nt) database. the large majority (> %) of those hits matched eukaryotic sequences, and only . % bacterial ( reads) and . % viral sequences ( reads) ( table ). the majority of eukaryotic reads were assigned to the yeast species saccharomyces cerevisiae ( , reads; %). all other eukaryotic species had < reads assigned. within the reads assigned to bacteria, the only species with notable number of reads were escherichia coli ( reads), faecalibacterium prausnitzii ( reads) or bacteroides vulgatus ( reads), all of which are known to be commensal species in the human gut. as far as viral sequences were concerned, all ( . % of , ) reads matched the enterovirus genus, species ev-a ( table ) . they were on average , bases long (range - , bases). alignment of the rna sequences to the best scoring reference genome (coxsackievirus a kj ; figure ) demonstrated that the long reads covered almost the entire genome of coxsackievirus a , with one single read covering alone . % of the reference genome (with only bases missing at the ' end). the top two longest rna sequences ( , and , bases long) were obtained within the first hour of sequencing, and most of the larger sequences (> kb) were . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . obtained within the first hours of sequencing. after hours of sequencing, sequences matching enterovirus were all < bases long. table . taxonomic classification of viral reads (oxford nanopore) and contigs (illumina miseq) using megan based on blastn matches against ncbi's nucleotide (nt) database at the species and genotype levels. grey cells indicate absence of hits in the dataset. asterisks (*) indicate that > % of the respective genome sequence was covered, while thick boxes indicate ev species whose identity was also confirmed by sanger sequencing of the vp gene. classical amplicon sequencing of the vp region was attempted for sample e , but it did not yield good results. in order to confirm the genotype identification and to obtain a highly accurate whole genome sequence of the enterovirus genome, we subsequently subjected the sample e also to cdna sequencing using illumina miseq. in this case, the cdna was produced using genotype-specific primers given that low number of reads was obtained by drs and miseq . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint / sequencing library preparation was done with the routine diagnostic pre-treatment given that low amount of original material was available (figure ). after removal of reads mapping to human genome and bacterial rrna, metatranscriptomic reads were assembled into contigs and further subjected to blastn similarity analysis against known sequences in the nt database, and the top hits were taxonomically summarized using megan. most contigs of sample e were classified as bacteria ( ) or viruses ( ) ( table ). such differences in species composition when compared to the results of the drs run may be explained by the genotype-specific approach used for cdna synthesis and different pre-treatment of the sample. all viral sequences matched again uniquely to ev sequences, however, megan analyses revealed the presence of two enterovirus species consisting of ev-a ( contigs; contigs lengths ranging from to bases) and ev-b ( contigs; contigs lengths ranging from to , bases) in the cdna sample, of which the majority of reads were assigned to coxsackievirus a and echovirus ( table ) . mapping of the viral sequences to a vp database confirmed the presence of these two genotypes and the identification was verified by using the online rivm bioinformatic platform [ ] . alignment of the reads to the closest reference genome revealed that the coverage of cv-a was % with an average depth of . (± . ), while for echovirus , only % of the reference genome were covered (figure ) . the consensus sequence for cv-a obtained from illumina miseq was compared to the one from drs sequencing, which showed . % identity ( , / , ) , with % gaps ( / , ). sample e . based on the promising results obtained for sample e , the drs transcriptomic approach was repeated on two other clinical samples: for the drs of rna extracted from sample e , the total yield of the sequencing run was , reads ( . mb) after h of sequencing. a total of , reads passed basecalling, with an average length of . bases (range of - , ). only % ( , ) of the basecalled sequences were taxonomically classified using blastn. interestingly, there was a drastic difference in read composition as compared to the first sample (e ), and the majority of reads classified as of bacterial origin ( . %, , ), and only very few reads matched to eukaryotic ( , . %) or archaeal ( , . %) species. for bacteria, drs reads were assigned to a large variety of species. the most prominent phyla were bacteroidetes ( , reads) and proteobacteria ( reads). a total of viral reads, corresponding to . % of classified reads, were found. as seen for the previous sample, only matches to the enterovirus genus were found. although many more viral reads were found in comparison to sample e , they were, with an average of , bases, much shorter, and with a range of - , . no reads spanned the complete genome sequence of any known species or genotype, and altogether could cover % of the reference genome sequence (figure ) . the short fragment length might result from increased fragmentation of the viral rna during extraction or library preparation. due to the directional sequencing used in the drs process of nanopore sequencing, higher coverage at the ' end of the sequence may be obtained, and fragments without poly-a tail cannot be sequenced. therefore fragmented, incomplete rna molecules may hinder the recovery of the 'end of the rna genome. nevertheless, although the covered region did not span the vp sequence, we were able to identify the enterovirus as echovirus with the best scoring reference genome. this . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint / species identification was independently confirmed by standard genotyping approach that uses sanger-based sequencing of the partial vp amplicon. figure . coverage plots for both drs nanopore runs (blue lines) and illumina miseq (grey lines) for all three samples for each ev genotype. shown are the depth of coverage after mapping the corresponding reads to the best reference genomes in sample e (ca : kj . , e : hm . ) and e (mh . ). for e , the coverage plot was produced by using the assembled contig sequence of the illumina sequencing run (oligo-dt primers) as reads did not map well any other reference ev genome. for samples e and e , illumina miseq reads based on cdna produced with oligo-dt and random hexamers are indicated by light and dark grey lines, respectively. the cdna from sample e was also sequenced by illumina miseq, although in this case with non-specific primers for cdna synthesis for a more unbiased approach towards ev detection and . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint / not to miss possible ev co-infections. both oligo-dt and random hexamer primers were used to ensure a better chance of obtaining the full genome, with the idea that oligo-dt may lead to results more comparable to the those of drs due to polyadenylation requirements, and random hexamers may offer better chance of obtaining a well-covered ' end of the enterovirus genome. the distribution of contigs assigned on the domain level did not vary much between random hexamer and oligo-dt produced cdna: most contigs were assigned to bacteria ( . % or . % respectively), with only a small minority mapping to eukaryotes ( . %, . %) and viruses ( . %, . %). within bacteria, most contigs were assigned to the phyla proteobacteria ( , , , ), followed by bacteriodetes ( , ) and actinobacteria ( , ). taking a closer look at all the detected viral genotypes ( table ) , we found that, besides echovirus , there were also contigs mapping to other virus genotypes, such as rhinovirus a, and a variety of other bacterial or plant viruses, albeit only with few short contigs. the echovirus genome, however, was well covered with either approach (figure ) . comparison of the drs nanopore sequencing consensus to the one obtained from illumina miseq showed . % identity ( , / , ), with . % gaps ( / , ). sample e . the drs run of the rna extract from sample e was stopped after the maximal run duration of h. notably, this run had much higher output compared to two previous samples, resulting in a total of . m sequenced reads ( . gb), from which . m reads passed basecalling. the average length was also considerably higher than in previous runs with , bases (range - , bases). although the rna input material used for library preparation was measured to be of a similar amount for all three samples ( ng), cdna measurement ( ng) before loading on the flow cell indicated that the initial rna concentration might not have been the same. overall, % of basecalled reads ( . m reads) were taxonomically classified using blastn. of those, . % ( , , ) mapped to eukaryotic sequences. as seen for sample e , the vast majority of eukaryotic reads mapped to saccharomyces cerevisiae ( . %), while . % ( , ) were classified as of bacterial origin, and only a negligible amount as archaea ( , . %). viral sequences comprised . % ( , ) of the reads, the majority of which were classified as plant viral pathogens ( table ) , mostly belonging to the tomato mosaic virus ( , reads, % of viral reads). other, more rarely detected species, included melon necrotic spot virus ( ) , and cactus virus x ( ) . in total, reads mapped to enteroviruses. although sequences were mapped to several genotypes, mapping the reads to the best scoring reference genome and to a vp database identified genotype echovirus as the most likely and only ev genotype present in the sample. although there were relatively few hits to enteroviruses considering the high total output of the run, there were some very long sequences present that covered up to . % of the reference genome ( figure ). illumina sequencing of the cdna from sample e produced using oligo-dt primers and random hexamers showed similar distributions on the domain level: the majority of contigs belonged to bacterial species ( . % and . %, respectively for oligo-dt and random hexamer approaches), with a minority of reads mapping to eukaryotes ( . %, . %) and viruses ( . %, . %). this major shift from yeast to bacteria as compared to the drs approach, which was also observed for . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint / sample e , could be explained by the different pre-treatment using . -µm filtration. additionally, comparison of number of contigs and nanopore reads are of limited value when not considering the coverage depth. for e , the genome of echovirus genotype, whose presence was also confirmed by sanger sequencing of the vp gene, was well covered with both types of cdna synthesis (figure ) . some contigs also mapped to another genotype, echovirus , but these were only short contigs, which may have been wrongly taxonomically assigned due to the short sizes of the illumina reads. otherwise, we also found a variety of viral sequences, with the most prominent being the tomato mosaic viruses and other plant pathogens, reflecting the results of the drs run. the consensus sequence identity for echovirus sequenced by drs to illumina miseq was calculated as . % ( , / , ) , with % gaps ( / , ). given the high cost associated with drs at the present time, those experiments were designed to assess the potential of the technology on clinical samples, while refining wet laboratory approaches, so that maximum information could be obtained as more samples were analysed. using rna extracted from clinical samples, we were able to repeatedly identify human enteroviruses in stool samples from three independent patients by nanopore-based drs. beyond identifying ev species and genotypes correctly, we showed that the approach may also provide rich metatranscriptomic information on sample composition for all life domains. clear differences in overall species compositions, with either yeast or bacteria dominating the majority of obtained reads, were observed between samples. viral reads constituted between . - . % of total passed reads. by complementing drs data with illumina miseq sequencing data for the same samples, we were able to validate the obtained enterovirus sequences and the further analysis of the metatranscriptomic data. illumina miseq sequencing revealed a higher diversity in viral species in these samples ( table ) , which was not captured by the drs approach. in all samples, % to . % of the ev reference genome sequences were covered. the average identity of the drs consensus was - % compared to the illumina miseq consensus sequence. other studies using drs have achieved similar values with - % identity when sequencing viruses from cell culture [ ] , or up to % consensus identity for influenza a [ ] . for applications with samples with low target concentration such as patient samples, getting enough sequencing depth to obtain accurate consensus sequences constitutes a challenge for the method. the resulting consensus sequences might therefore be of limited use for applications which require high accuracy, such as phylogenomic analysis. however, for the purpose of genotype identification the approach is sufficiently accurate, especially as it can provide long reads, which can facilitate mapping to the correct reference, and expedite downstream bioinformatic analyses. the good overall agreement in ev detection between the two ngs approaches suggests that the variability observed in number of reads and composition between samples may be attributable to the natural biological variability that may exist between the sampled patients. yet, finer differences . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint / in composition between samples may be explained by the sequencing technology and by the different pre-treatments of the samples: on the one hand, smaller cdna molecules produced by the wet laboratory procedure (cdna synthesis, bead cleaning), followed by short-read sequencing, may be easier to detect, than larger, intact rna molecules via drs. on the other hand, unreliable mapping and poor taxonomical identification may be produced by short reads aligning to genomic regions that are conserved among genotypes, but also by long, error-prone nanopore reads [ ] aligning incorrectly to reference sequences. therefore, further large-scale comparisons of the two ngs approaches would be needed to confirm or reject the hypothesis about the effect of read length on organism detectability in metagenomic or metatranscriptomic studies. while enteroviruses were detected in all tested samples using drs, it should be noted that these were all samples with relatively high viral load, i.e. low ct values. with a range of only - total reads mapping to enteroviruses for the three samples, the approach was yet close to the limit of detection. in the case of sample e , this sensitivity issue may explain why co-infection with another enterovirus was not detected, as compared to the short-read sequencing-based approach. low sensitivity of nanopore sequencing in a viral metagenomic approach has been reported previously in patient samples with low viral titers, even when sequencing viral genomes via sispa-based amplification [ ] . an improvement in sensitivity would be necessary before attempting to sequence samples with low viral load in general. a limiting factor of the approach was the amount of polyadenylated rna used for library preparation, as in two of the sequencing runs the full flowcell sequencing capacity was not used and the sequencing was stopped before maximal run duration, because low amounts of rna library were loaded on the flowcell. if sufficient amounts of patient samples were available, increasing the input concentration might have provided better coverage of the target sequences. furthermore, drs sequencing is expensive if only one sample is loaded per flowcell. recent progress with barcoding for drs [ ] might however reduce this problem in the future. additionally, results might be improved with adaptations in viral enrichment steps. we used a chloroform/bead pre-treatment in drs samples for enrichment of viral capsid and although it might be efficient in enriching viral reads, it could also reduce the overall amount of rna extracted. as the maximum sequencing yield of the flowcell was not always reached, perhaps milder enrichment methods might be preferable, especially if the broader diversity of pathogens is to be studied. currently the drs method is restricted to sequencing of polyadenylated rna, however, performing polyadenylation of the total rna fraction could expand its applications. another characteristic of the drs method is the expected sequencing bias towards the ' end of the poly-adenylated molecules [ ] . hence, coverage of the ' region of the ev genomes was generally low, which may be problematic given that the vp region is the main region used for ev genotyping (e.g. located at position , - , in echovirus kt . ). thus, correct genotyping would require at least around , bases long rna reads from the ' towards the ' end of the genome sequence. we encountered this problem in the case of sample e , although in that instance we were able to identify the same genotype as found by the gold standard vp . cc-by-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. 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enables modification analysis. biorxiv rapid sequencing of multiple rna viruses in their native form. front microbiol evaluation of -sp transport medium for detection of chlamydia trachomatis and neisseria gonorrhoeae by two automated amplification systems and culture for chlamydia enterovirus surveillance guidelines: guidelines for enterovirus surveillance in support of the polio eradication initiative. ; world health organization (who) regional office for europe sensitive, seminested pcr amplification of vp sequences for direct identification of all enterovirus serotypes from original clinical specimens rapid routine detection of enterovirus rna in cerebrospinal fluid by a onestep real-time rt-pcr assay rapid and cost-efficient enterovirus genotyping from clinical samples using flongle flow cells improved molecular identification of enteroviruses by rt-pcr and amplicon sequencing characterization of the genome of human enteroviruses: design of generic primers for amplification and sequencing of different regions of the viral genome megan analysis of metagenomic data minimap : pairwise alignment for nucleotide sequences bbmap: a fast, accurate, splice-aware aligner spades: a new genome assembly algorithm and its applications to single-cell sequencing an automated genotyping tool for enteroviruses and noroviruses barcoding and demultiplexing oxford nanopore native rna sequencing reads with deep residual learning. biorxiv pcr-based approach. however, unambiguous identification relies on the capsid region and its presence is crucial in case of recombinations or co-infections. therefore, great care must be applied during extraction and library preparation to avoid shearing or degrading extracted rna. the known high variability of ev genome sequences makes it necessary to have suitable alternatives for genotype identification available if pcr-based approaches fail. in such cases, drs of patient samples could be a valuable alternative, as the sequencing is primer-independent and the sequences of long, native rna molecules are recovered. additionally, as is the case with all nanopore sequencing, the data is available in real-time, which can provide faster identification and a sequencing-on-demand approach to molecular assays.overall, our proof-of-concept study demonstrated to the possibilities offered by the drs technique for genotype identification of human enteroviruses directly from clinical samples. the method requires further optimization to improve the overall sensitivity and to lower costs for future possible applications in routine diagnostics. however, with the rapid advancement of the technologies those issues will likely improve in the near future. key: cord- -u y bznk authors: jaluria, pratik; konstantopoulos, konstantinos; betenbaugh, michael; shiloach, joseph title: a perspective on microarrays: current applications, pitfalls, and potential uses date: - - journal: microb cell fact doi: . / - - - sha: doc_id: cord_uid: u y bznk with advances in robotics, computational capabilities, and the fabrication of high quality glass slides coinciding with increased genomic information being available on public databases, microarray technology is increasingly being used in laboratories around the world. in fact, fields as varied as: toxicology, evolutionary biology, drug development and production, disease characterization, diagnostics development, cellular physiology and stress responses, and forensics have benefiting from its use. however, for many researchers not familiar with microarrays, current articles and reviews often address neither the fundamental principles behind the technology nor the proper designing of experiments. although, microarray technology is relatively simple, conceptually, its practice does require careful planning and detailed understanding of the limitations inherently present. without these considerations, it can be exceedingly difficult to ascertain valuable information from microarray data. therefore, this text aims to outline key features in microarray technology, paying particular attention to current applications as outlined in recent publications, experimental design, statistical methods, and potential uses. furthermore, this review is not meant to be comprehensive, but rather substantive; highlighting important concepts and detailing steps necessary to conduct and interpret microarray experiments. collectively, the information included in this text will highlight the versatility of microarray technology and provide a glimpse of what the future may hold. although, the principles behind microarray technology were conceived almost years ago and developed from southern blotting, they did not gain wide spread attention for nearly a decade when researchers were first able to utilize high quality slides with precision robotics resulting in reproducible results [ ] [ ] [ ] . for instance, a quick pubmed search with the words, 'microarray and ' results in total articles, of which are review articles. similar searches with the words, 'microarray and ' and 'microarray and ' result in total articles ( review articles) and total articles ( review articles), respectively. despite this relative surge in microar-ray-related articles, few recent publications address core issues regarding design, implementation, and subsequent data analysis. in covering these and related issues, the present text aims to illustrate the strengths, weaknesses, and application of microarrays, especially to those unfamiliar with the technology. today's arrays are vastly superior to their predecessors in terms of quality, probe density, and structural layout [ , ] . before dealing with these and other characteristics, it is important to discuss, at some length, what microarrays are as well as the fundamental concepts behind the technology. the term microarray is both descriptive and somewhat ambiguous as it is commonly used to describe a variety of platforms including protein microarrays and tissue microarrays [ , ] . a microarray is typically defined as a collection of microscopic spots arranged in an array or grid-like format and attached to a solid surface or membrane, hence the term [ , ] . these spots typically referred to as probes, are designed such that each probe binds a specific nucleic acid sequence corresponding to a particular gene through a process termed hybridization [ ] . the sequence bound to a probe, often referred to as the target, is labeled with some kind of detectable molecule or dye such as a fluorophore [ ] . the level of binding between a probe and its target is quantified by measuring the fluorescence or signal emitted by the labeling dye when scanned. this signal, in turn, provides a measure of the expression of the specific gene containing the target sequence [ , ] . although, there are several different types of dna microarrays, for the purposes of this text only two will be considered; spotted microarrays and oligonucleotide microarrays [ ] . details regarding these two platforms are highlighted in table . spotted microarrays are often referred to as dual-channel or two-color microarrays because two samples, each labeled with a different fluoro-phore, are hybridized onto a single slide [ , ] . as a result of combining two samples onto a single slide, only relative expression levels can be determined using spotted arrays [ ] . the probes in spotted arrays are oligonucleotides, complementary dna (cdna), or fragments of polymerase chain reaction (pcr) products; each type conferring different properties to the spotted array. despite these differences, all spotted arrays are similar in terms of: array construction, target preparation, and data analysis [ , ] . in contrast, oligonucleotide microarrays also referred to as single-channel microarrays are hybridized with only one sample and therefore generate absolute expression levels. these arrays utilize probes designed to complement mrna sequences and are produced using various methods including in situ synthesis, some type of deposition method, or photolithography [ , ] . as alluded to earlier, two important elements of microarray technology are target preparation and probe construction. depending on the type of microarray being used, different cellular components can be used for target generation including: rna, genomic dna, cdna, complementary rna (crna), and pcr products [ , ] . regardless of which of these are used, ensuring the quality, stability, and reproducibility of the generated targets is paramount for subsequent processing. similarly, probes can consist of any of the following: cdna, oligonucleotides, fragments of pcr products, restriction-enzyme digested fragments, oligomers, or expressed sequence tags (ests) [ , , ] . irrespective of the exact composition of the probes, they all serve the same basic function; binding very specific sequences. although, probes are constructed in a variety of ways, depending on the type of array and the specific application, the same public databases are referenced for sequencing information [ , , ] . typically, arrays are fabricated with duplicates of each probe, enhancing the likelihood of observing hybridization for each gene. a simple schematic of the entire process for a spotted cdna microarray experiment can be seen in figure a [ ] . briefly, total rna, once isolated from a sample, is reverse transcribed to produce cdna, labeled with fluorescent dyes, and then hybridized onto the spotted array [ , ] . hybridization is quantified using the intensities of the fluorescent dyes at particular wavelengths. by comparing fluorescence intensities, genes that are differentially expressed between the two samples can be identified, along with the direction of that difference (i.e. overexpression or under-expression relative to a control) [ , ] . for example, figure b illustrates the significance of each color when the test sample is labeled with cy and the control sample is labeled with cy . in this case, black represents no binding (i.e. no signal), green indicates greater binding of the control sample than of the test sample referred to as down-regulation, yellow indicates equal binding between the two samples, and red indicates greater binding of the test sample than of the control sample referred to as up-regulation. if the dyes are used in reverse (i.e. cy is used to label the control sample and cy is used to label the test sample) the colors would have the opposite representations [ , ] . although a multitude of microarrays are commercially available, each designed for a specific species or general family of organisms; these arrays are limited by the information available in genomics databases [ , ] . though, the genomes of only a few species have been entirely sequenced and made available to the public, microarrays for a large number of species are available [ , ] . for instance, checking the website for affymetrix reveals genome-wide arrays are available for the following microbes: bacillus subtilis, escherichia coli, pseudomonas aeruginosa, members of the genus plasmodium, staphylococcus aureus, and members of the genus saccharomyces. small, custom arrays can be designed for many more species as long as genomic sequences are available for a particular organism or family of organisms [ , , ] . continued genome exploration has resulted in the need for frequent updating and re-organization of spotted arrays. with more information constantly coming online, microarrays are continually refined to enhance reproducibility and detection levels of weak signals by modifying the positioning and sequences of the ests spotted [ , ] . as previously mentioned, ests are essentially unique segments of cdna identical to a portion of a gene, thereby acting as binding domain. in addition, valuable information can still be ascertained by hybridizing samples onto arrays designed for other species [ , ] . so, even without an entire genome being spotted onto commercially available arrays for a given species, microarray experiments can still yield important results. any discussion regarding microarray technology would be incomplete without a detailed examination of the various limitations and complexities inherently present. such a discussion is vital to properly conduct microarray experiments and analyze microarray data; overcoming technological limitations in the process. before conducting microarray experiments, the following questions need to be addressed: what are the goals of the experiment, what biological comparisons are most relevant to these goals, how should the experiments be designed and performed taking into account the various sources of variability, which platform should be used, what controls need to be in place, and how can the results be verified [ , ] . in approaching these and other relevant questions, a great deal of information regarding microarray technology can be ascertained. to answer the first two questions regarding goals and relevant comparisons, a number of resources can be referenced. several organizations such as the microarray gene expression data (mged) society and the european bioinformatics institute (ebi) have established guidelines to aid researchers in the design and implementation of microarray experiments [ , , ] . in general, narrowing the objectives of a microarray study can provide insight into which biological samples should be compared. clear and concise goals also help define the scope of the study, providing a framework within which subsequent experiments can be proposed and implemented. one of the most commonly sited proposals is the minimum information about a microarray experiment (miame) that includes a series of recommendations and standards on collecting and analyzing microarray data [ , ] . this document was designed to allow data generated by microarray experiments to be interpreted and reproduced with certainty. in addition, repositories such as the gene expression omnibus (geo) created by the national center for biotechnology information (ncbi) and arrayexpress created by the ebi have been established to store and share gene expression data [ , ] . array even extending to other experiments as long as the same reference sample is used [ , ] . however, this setup can become costly if the goals of the experiment require multiple comparisons to be made. in contrast, the saturated design involves making every possible comparison exactly once [ ] . this approach is balanced and simple to establish, however, it is not applicable to all conditions and is not appropriate when a series of experiments are planned. ultimately, the design selected must address the goals and requirements of the experiment being conducted. without these and other considerations, errors in analysis including the identification of false positives can result, masking underlying patterns and incorrectly deciphering biological behavior. there are multiple sources of variability such as differences in: arrays, dye labeling, efficiency in reverse transcription, and hybridization [ , ] . some of these issues relate back to the actual production of arrays and how probes are prepared; elements of quality control on the part of the manufacturer. the remaining issues are best overcome by: incorporating replicates to generate statistical significance (i.e. averages and variance), performing dye-swapping experiments, and pooling samples to minimize biological variation [ , , ] . both technical and biological replicates are commonly employed, each with a different purpose in mind. technical replicates aim to quantify procedural variations such as sample preparation and handling [ ] . in contrast, biological replicates aim to identify variation in the biological system being studied [ ] . similarly, dye swapping involves switching the dyes used for labeling in a manner that prevents one type of sample from being labeled by a single dye. this setup helps account for the dye effect; an important systematic error that stems from differences in the properties of the dyes. the pooling of samples also reduces inherent variation in biological samples while at the same time generating sufficient sample quantities for subsequent processing [ ] . as discussed earlier, there are two main platforms to consider when designing microarray experiments; spotted microarrays and oligonucleotide microarrays. the advantages and disadvantages of each are outlined in table along with examples of when a particular platform is most beneficial [ , ] . for example, oligonucleotide microarrays are ideal for time-course experiments because each array is hybridized with only one sample, allowing any array to be compared to any other array. this translates into requiring a smaller number of total samples for the same number of duplicates while at the same time more accurately representing the control for a given condition. similarly, for static conditions in which a basic comparison between treated and untreated cell populations is needed, dualchannel microarrays may be the best fit. each application has its own set of criteria that should be carefully evaluated to determine the best platform to use [ , ] . for instance, if specific genes are to be investigated, it should be verified that the platform includes those particular genes with the desired number of replicates. a simple search online will reveal a multitude of companies that manufacture microarrays and allow customers to construct their own custom arrays using specialized software. in addressing the various sources of error, systematic or otherwise, proper controls need to be implemented. there are two types of controls, as they pertain to microarray technology; internal controls and external controls [ , ] . internal controls check for the quality of the printed microarray whereas external controls account for performance in terms of sensitivity and robustness. the internal controls often used include: hybridization controls, poly-a controls, normalization control sets, and housekeeping genes [ , ] . each type of control is commonly found in commercially available arrays and serves a distinct function relating to one specific aspect of microarray processing. in addition, samples can be spiked with particular agents to isolate or quantify detection limits, non-specific noise, and similar parameters [ ] . similarly, a number of approaches can be taken to minimize external variables such as discrepancies in: growing and preparing biological samples, isolating and purifying rna, cell synchronization, hybridization protocols, and target preparation [ , ] . in general, standardizing procedures can greatly reduce these errors introduced during the course of the experiment. although, the preparation of control samples used in a microarray experiment is typically not critical, the samples must be stable throughout the experiment and be reproducible. to verify the quality of purified rna and/or cdna gel electrophoresis and/or spectrophotometry should be used. with regards to cell synchronization, whole-culture methods such as serum starvation (a method in which cells are deprived of animal serum, a commonly used media supplement, to direct cells towards quiescence) and dna arrest (a general method of using chemical or pharmacological agents to prevent one or more phases of dna replication, suspending cells in a particular stage of the cell cycle) are typically used [ , ] . however, selective methods such as mitotic shake-off, a method that involves shaking a flask or plate to remove cells undergoing mitosis because these cells are loosely attached, have also been used due to questions about the validity of whole-culture methods [ , ] . whatever synchronization method is used should be applied to all of the biological samples to ensure a valid comparison is being made. typically to validate microarray results any one of a number of techniques such as rt-pcr, northern blotting, western blotting, and even the use of multiple microarray platforms can be employed [ , , ] . figure illustrates which of these methods are relevant to which aspects of microarray analysis. as mentioned earlier, verification is critical in order to assign distinct expression patterns to specific genes with certainty (i.e. statistical significance) because of the inherent variability present in microarray data. since a single microarray experiment evaluates the expression levels of tens of thousands of genes simultaneously, it would be extremely impractical to verify each and every gene using any of the methods listed above. instead, what is typically done is that a number of key genes are verified depending on the purpose and scope of the experiment [ , ] . in addition, not every gene can be assayed using each verification method because the necessary components may not be available such as monoclonal antibodies necessary for western blotting or labeled primers for rt-pcr. as a result, multiple methods are often used to verify the results of microarray experiments. as described previously, expression levels for a given gene are determined using intensity values. one distinction between dual-channel microarrays and single-channel common steps employed to ensure quality and validity of microarray results common steps employed to ensure quality and validity of microarray results. from a quality control standpoint, replicates should be performed using rna samples prepared at the same time under the same conditions. various features of the arrays being used should also be known, especially the controls. to verify the results generated from microarray experiments, a combinatorial approach is usually needed; checking the statistical significance associated with the expression levels of specific genes, reviewing the literature, and conducting additional experiments such as rt-pcr or northern blotting. microarray quality microarrays is that the former generates relative expression levels whereas the latter generates absolute expression levels [ , ] . this distinction stems from the fact that dual-channel microarrays are hybridized with two different samples; one considered the test sample and the other considered the control sample. as a result, the expression level determined for a specific spot or gene is dependent upon both samples and is a ratio of the form: therefore, the expression level for a gene in a dual-channel microarray is relative, not absolute. in contrast, singlechannel arrays are hybridized with only one sample and therefore the expression level for a given gene is absolute [ , ] . once a scanned image for a hybridized microarray has been generated, visual inspection of the data can proceed, prior to normalization. this entails using imaging software to exclude specific spots with poor signaling and adjust the size/shape of grids that encompass the spots [ , ] . next, normalization procedures can be applied to the data. essentially, normalization accounts for differences in labeling efficiencies and detection levels for the fluorescent dyes as well as differences in the quantity/ quality of rna samples [ , , ] . as such, normalization can be thought of as the first level of filtering applied to the data. advanced statistical software packages offered by companies such as partek and acuity are commonly used. private research institutes such as the institute for genomic research (tigr) and the sanger institute along with academic facilities around the world also provide free software for microarray analysis [ ] . although a number of normalization techniques can be applied to microarray data, the most commonly used are: total intensity, regression, and ratio statistics [ , , ] . all three of these techniques assume that for some group of genes on the array, the average expression ratio is equal to one [ ] . total intensity normalization assumes both samples (test and control) are comprised of the same amount of rna and the total amount of rna hybridized from each sample is the same. therefore, the total intensity calculated from all the spots on an array should be the same for both fluorescent dyes (channels) [ , ] . conversely, normalization using regression presumes that a significant number of genes are expressed to the same extent in both samples; a reasonable assumption for samples that are fairly similar [ ] . if the labeling and detection efficiencies for the two samples were equivalent, then the slope of the plot shown in figure would be one [ , ] . figure was constructed from unnormalized data obtained from a single, spotted cdna array. two dif-ferent samples were hybridized onto the array, each labeled with a different dye. the graph illustrates inherent differences between the dyes in terms of labeling and detection efficiencies due to the characteristics of each dye such as stability. using regression techniques, the best-fit slope is calculated and modified to be equal to one by adjusting gene intensities. lastly, normalization using ratio statistics assumes that there exists some subset of genes with the same expression levels in both samples [ , ] . these housekeeping genes, as they are often referred to, are used to calculate probability densities which in turn allow the mean expression ratio to be adjusted to one. each of these techniques calculates a normalization factor that is then used to scale the data, accounting for the variations previously mentioned [ , , ] . following normalization the data can be probed using a host of statistical techniques that evaluate and ultimately decipher microarray data. for the purposes of this text, only two types will be touched upon briefly; clustering and hypothesis testing [ ] . in general, both types of statistical methods strive to categorize, shape, and illuminate underlying patterns and therefore can be very useful in analyzing microarray data [ , ] . however, both methods rely on different underlying principles and assumptions that directly influence their employment. clustering algorithms rely on calculating some kind of 'distance metric' to position gene expression levels into a expression level test sample intensity control sample inten = s sity scatter plot of measured intensities for both fluorescent dyes on a log-log scale prior to normalization figure scatter plot of measured intensities for both fluorescent dyes on a log-log scale prior to normalization. the measured intensities are in arbitrary units. each point in the graph represents a single spot on a hybridized microarray. in addition, the red line shown is the best-fit line calculated for the data with a slope that is close to unity. matrix of sorts with a certain level of commonality [ ] . both differentially expressed genes and groups of genes with similar expression patterns can be highlighted using clustering techniques. the most widely used clustering algorithms include: hierarchical, self-organizing maps (soms), k-means, and principle component analysis (pca) [ , , ] . the mathematical formulations behind each of these methods are too complex and lengthy to be dealt with here, and so for the sake of brevity, very basic information will be covered in this text with a strong recommendation to consult specific references [ , , ] . typically, these and other algorithms are used to create a more accurate and meaningful interpretation of the data. figure illustrates how four different algorithms (when applied to the same data set) can generate vastly different groupings; each providing a different perspective on patterns present in the data. the data shown in figure was obtained by hybridizing human cell lines grown under varying conditions onto cdna microarrays. by applying clustering algorithms in sequence, one after another, synergy is possible, lessening the shortcomings in each individual method. for example, it is common to apply pca to data prior to analysis with either k-means clustering or soms in order to generate an estimate for the number of clusters to be formed [ ] . hierarchical clustering, quite possibly the most commonly used clustering algorithm, links every gene in an array to every other gene through a series of expanding brackets that collectively form a dendrogram [ ] . genes deemed to be closely associated, referring back to the concept of a distance metric which in fact can be computed using several different statistical frameworks, are connected by a node [ , ] . each node links to other nodes of various sizes, in a repetitive process until every possible pair of genes are linked, as illustrated in figure b . this type of clustering is popular due to its simplicity and ability to visualize the data. however, the statistical framework has several disadvantages including: not being able to account for multiple ways in which expression patterns can be similar, having difficulty assimilating large quantities of data, and forcing a hierarchical system upon a data set that does not truly exhibit a hierarchical lineage [ , ] . unlike hierarchical clustering, soms require initialization and are much less rigid in terms of structure while at the same time remaining robust and unique. initialization involves defining a particular geometry, typically a grid or ring, with a specified number of groups or nodes [ , ] . these nodes are mapped into a high dimensional space and successive iterations, usually tens of thousands, look to reduce the number of dimensions [ ] . the algorithm also makes use of weighted vectors to select and group similar data entries together, essentially training itself after each phase. the end result of this process is a selforganized network that can be visualized [ , ] . these and other features make soms a powerful tool in exploratory studies with an emphasis on visualization. similarly, k-means clustering aims to partition gene expression data into a specified number of disjoint clusters. again, a distance metric is used in these calculations and can be specified by the user. genes within a cluster are deemed similar to one another, but clusters are deemed dissimilar to one another producing a series of clusters that are not related or connected, opposite of the structure produced in hierarchical clustering [ , ] . essentially, each gene is placed in one of the clusters initially specified and distances between clusters are calculated. next, genes are moved from one cluster to another until a local stability is reached in which the distance between clusters is maximized while at the same time minimizing the distance between members of a given cluster [ , ] . this method is reliable and relatively simple and therefore is useful in analyzing data for which there is some prior knowledge such as classifying serotypes or strains. ensuring that the partitions constructed using k-means clustering have some type of real or actual significance is where the difficulty lies [ ] . pca is an algorithm that relies on visually highlighting similarities in data in a manner that reduces the number of dimensions. it can be applied to any number of data sets from a small group of genes within a single array to groups of experiments each with a number of arrays [ , , ] . as seen in figure a , the plot generated from pca allows patterns in data to be visualized by examining the proximity of clusters. the method implements a series of calculations to best separate the data and project that final analysis onto a or dimensional plot [ , ] . when combined with other clustering methods, pca can be a very useful tool, as described earlier. besides clustering algorithms another statistical approach typically used to analyze microarray data is hypothesis testing which aims to establish statistical significance associated with divergent findings. if a group of genes, perhaps genes that constitute a particular pathway, are differentially expressed between two samples, hypothesis testing can quantify the extent of those differences. hypothesis testing is comprised of the following steps: specify the null hypothesis and the alternative hypothesis, select a significance level, calculate a statistic analogous to the parameter designated in the null hypothesis, calculate the probability value (p-value), compare the p-value with the significance level, and finally accept or reject the null hypothesis [ , ] . at the end of these steps, an observed outcome is associated with a statistical likelihood indicating whether or not the observed outcome is the result of chance and not some real difference or phenomenon [ ] . application of hypothesis testing is most useful when evaluating microarray data with specific genes or groups of genes in mind as opposed to discovery or exploration. a large number of microarray-related studies in the past have aimed to either characterize diseased cells in comparison to healthy cells or highlight the genes involved in a particular biological pathway [ , ] . infrequently, studies were undertaken for other purposes such as gene discovery or examining distinct cellular properties [ , ] . however, in recent years, the number of studies utilizing microarrays in some capacity has increased greatly. more and more studies are relying on microarrays to provide insight into observed physiology, essentially using microarrays to further characterize biological systems [ , ] . in most of these cases, microarray analysis has generated interesting results, but also raised additional questions requiring further investigation, limiting its successful implementation. for instance, the application of bio-informatics tools such as microarrays to characterize microbial populations exposed to toxins and pollutants has been explored [ ] . being able to understand the catabolism of xenobiotics could enhance bioremediation processes with a direct impact on pollution control and environmental organization [ ] . in addition, the exploration of previously uncharacterized microbes using microarrays could identify novel genes with relevant functionality [ ] . in this context, a number of studies have focused on specific issues such as investigating how candida albicans, a human fungal pathogen, is able to protect itself from the toxic effects of nitric oxide produced by the immune system [ , ] . microarray analysis revealed a group of nine genes were over-expressed during exposure to nitric oxide. of these nine genes yhb , which produces a flavohemoglobin that detoxifies nitric oxide, was the most highly expressed [ ] . evolutionary studies using microarrays have also gained prominence with the use of species-specific arrays in parallel. for example, researchers hybridized dna from the progeny of two yeast strains, one with a particular evolved trait (i.e. mating discrimination) and the other without, onto oligonucleotide microarrays [ ] . the arrays used in this study were designed to detect a multitude of polymorphisms between the two strains. adaptive mutations were identified by linking polymorphisms to the evolved parental strain [ ] . investigators then mapped known genes and constructed a computer simulation capable of evaluating various parameters impacting mapping precision [ ] . finally, the researchers applied their method to yeast strains adapting to a changing glucose-galactose feed illustrating mutations in the same gene can lead to parallel adaptation [ ] . similarly, scientists compared community-acquired invasive staphylococcus aureus strains to isolates from healthy people using microarray constructed from previous sequencing projects [ ] . ten dominant lineages were identified; each with a distinct group of genes with potential functions related to virulence and resistence. subsequent analysis suggested a common ancestor could be traced back for all of the strains studied, but evolutionary divergence must have occurred early on [ ] . the development of therapeutics has also benefited from the implementation of microarrays as evidenced by a number of recent publications. for example, scientists examined gene expression profiles from patients with chronic drug abuse, intending to better understand addiction and therefore formulate better treatments [ ] . analysis of the array data revealed very little overlap in the expression patterns for heroin and cocaine users [ ] . these findings were contrary to widely held views regarding the shared effects of heroin and cocaine on dopamine, thus prompting reassessment of previous assumptions [ ] . another study, examined the mechanism behind acquired nisin resistance in bacteria [ ] . researchers found genes involved in the following pathways to be expressed differentially between resistant and non-resistant lactococcus lactis strains: cell wall biosynthesis, energy metabolism, fatty acid and phospholipid metabolism, regulatory functions, and metal/peptide transport and binding [ ] . using this information, the researchers established mutant strains that either had genes knocked down or over-expressed and found these mutants had varying levels of nisin resistance as compared to the parental, wild-type strains [ ] . in terms of disease characterization and detection, microarrays are also finding use. for instance, the pathogenicity of coxasackievirus b (cvb ) was examined; in humans this virus adversely affects the heart muscle [ ] . using cdna microarrays, researchers compared murine hearts infected with the virus against non-infected murine hearts. in addition, oligonucleotide microarrays were used to compare infected hela cells over time [ ] . together, these experiments identified a number of differentially expressed genes, providing clues as to the precise sequence of events following infection. similarly, the use of custom microarrays to characterize unknown samples from water treatment centers as part of a quality control measure was examined [ ] . the microarray was constructed to target s ribosomal rna (rrna) from several groups of nitrifying bacteria and tested against reference samples with some success [ ] . using microarrays in the capacity of diagnostics has also become relatively popular especially in the context of outbreaks for which rapid diagnostic tools are needed to quickly evaluate pathogens and identify specific strains or serotypes [ , , ] . for example, a microarray was constructed specifically to probe single nucleotide polymorphisms (snps) for foot and mouth disease virus (fmdv) [ ] . the results were classified using statistical methods in order to develop a procedure to test for specificity with diagnostic application [ ] . similarly, a study combined the use of microarrays with reverse transcription-pcr to differentiate between two genetically similar enteroviruses; enterovirus (ev ) and coxsackievirus a (ca ) [ ] . this approach had a diagnostic accuracy of at least % for each of the two viruses as compared to reverse transcription-polymerase chain reaction (rt-pcr) and neutralization testing [ ] . currently, studies are being conducted to explore the feasibility and implementation of similar methods for other pathogens [ , ] . with advancements in software and robotics technology, microarrays are becoming inexpensive, robust, and reliable [ , ] . the availability of custom arrays designed to probe a small subset of genes (usually several hundred) or specific pathways have also enhanced the potential utilization of microarray technology [ , , ] . this section was designed to highlight the latest advances in the technology, speculate on novel applications of microarray technology, and outline areas of research that have just begun to use microarrays. together these aspects portray the potential of microarrays in terms of applications as well as from a technical standpoint. breakthroughs in various aspects of the technology from fabrication to commercialization are continually influencing the kinds of microarrays and techniques researchers are using. currently, microarray experiments are conducted in a series of steps with each step being distinct and in a particular order. however, newly developed chips equipped with electronic circuitry are circumventing a number of these steps particularly sample labeling [ , ] . in addition, a number of companies and research facilities now offer specialized arrays for detection, sequencing, and/or diagnostic purposes [ , ] . by commercializing such highly specific arrays, data gathering is being expedited for studies with explicit purposes. an integrated platform like the lab-on-a-chip (a system that combines multiple manipulations including sample mixing, labeling, and separation onto a single chip) is also influencing microarray technology. the miniaturization and automated techniques used to construct the lab-on-a-chip system are being applied to microarrays leading to arrays that can be readily used for high-throughput applications [ ] . one of the most promising areas of research includes classification; particularly in the context of diseases and/or pathogens [ ] [ ] [ ] [ ] . for instance, in researchers at the national cancer institute used microarrays to organize biopsy samples of diffuse large-b-cell lymphoma from more than patients [ ] . they identified subgroups with varying expression of distinct genes; constructing a model capable of predicting survival rates following chemotherapy [ ] . in another study, researchers used microarrays to confirm previous classifications of nonpathogenic, low-pathogenic, and high-pathogenic types for different yersinia enterocolitica strains [ ] . researchers identified clusters of genes as being representative of each type (i.e. being present in one group, but not in another) with functional implications [ ] . another arena in which microarrays may prove beneficial is discovery; primarily in the context of gene functions and the identification of novel organisms. for instance, in a recent study researchers analyzed an escherichia coli strain, a , with a mutation in the rnpa gene making it sensitive to temperature and therefore unable to grow at or above °c [ ] . under varying growth conditions, researchers found a number of genes differentially expressed. careful review of these genes revealed rnase p, the mutated gene product, may have more functions than what had been proposed previously, especially in the context of handling precursor rnas [ ] . researchers in constructed a custom array with highly conserved arrangements from every fully sequenced viral genome available in genbank [ ] . next, they hybridized a viral isolate from a severe acute respiratory syndrome (sars) patient onto the array and found a previously unidentified coronavirus [ ] . subsequent work involving viral sequencing verified these findings and showcased the potential of custom arrays to expedite the identification of pathogens; a virtual necessity in combating future outbreaks [ ] . in terms of biological products, particularly vaccines and therapeutic proteins, microarrays may also find use. as detailed in various governmental regulations, slight variations in a biological process may result in distinct final products; requiring further testing and validation [ ] . microarrays may very well provide a means of avoiding these procedures by establishing criteria (i.e. expression patterns for a small set of genes) that can be used to verify consistency and reproducibility. extensive research would, however, be required to first establish the necessary criteria. in addition, it should be stressed that in this particular application, microarray results would have to be viewed in terms of patterns for a group of genes rather than the expression levels of individual genes [ , ] . this is because the variability associated with a single gene can exceed levels needed to verify or validate biological proc-esses, whereas the variability in groups of genes where an overall pattern is decoded is much less [ ] . another area that has and continues to find microarray technology beneficial is pathway probing; illumination of biological pathways. often, microarray data alone cannot decipher the sequential steps necessary for a particular mechanism to occur, however, it can provide insight into what genes or groups of genes should be investigated further [ , ] . for instance, a paper published in used microarrays together with other experimental techniques to decipher a pathway responsible for regulating the expression of cyclooxygenase- (cox- ), a pro-inflammatory protein associated with arthritis and pain [ ] . a continuation of this work was published in further illuminating the pathway and possible feedback mechanisms with important therapeutic implications [ ] . perhaps the greatest potential lies in combining two fields within the scope of bio-informatics; genomics and proteomics. genomics is the study of genes and their function whereas proteomics is the study of proteins and their functions [ , ] . by utilizing tools from each of these two disciplines, researchers may be able to construct more accurate and comprehensive models depicting specific biological processes. for example, in a recent study both two-dimensional gel electrophoresis and microarrays were used to identify genes involved in the acclimation of changing visible light in cyanobacteria [ ] . focusing on the organism fremyella diplosiphon, researchers found approximately proteins with different levels between cells grown in green light vs. red light as well as genes not previously thought to be regulated by light [ ] . further exploration revealed a number of these genes had homologs in other organisms, though their functionality had not been fully deciphered [ ] . in another study, both microarrays and proteomics were used to evaluate an escherichia coli mutant secreting more α-hemolysin (hlya) than the parent strain [ ] . the researchers found decreased levels of trna-synthetases in the mutant as compared to the parent strain [ ] . based on this information, the researchers designed a modified hlya gene to reduce the rate of translation by incorporating rare codons leading to the same amino acid sequence [ ] . when the parent strain was transformed with this modified hlya gene, it secreted even more hlya than the mutant [ ] . in other words, the study indicated it was possible to engineer cells using an approach that combined genomics and proteomics. microarrays are a powerful genomics tool, designed to illuminate differences in the expression of genes within cells. despite being a relatively new technology, the scientific community has quickly adopted its use in a variety of fields including drug development, evolutionary biology, and disease characterization [ , ] . the strength of the technology rests on the several factors including: ease of use, availability of platforms and lower cost relative to other exploratory methods such as northern blotting or ribonuclease protection assay (rpa), implementation of statistical methods for detailed analysis, and most importantly a global view of a gene expression encompassing an entire genome. as previously eluded to, the technological limitations associated with microarrays manifest themselves in terms of variability typically seen as systematic errors. improvements in robotics, array fabrications, and continued genome sequencing can certainly address these issues, but not entirely remove them. this places limits on what microarray technology can achieve, although a comprehensive understanding of microarrays can help establish meaningful and reproducible data. an effort to: properly design the experiment, establish quality control steps such as checking rna purity, analyze the data, and verify the results can also combat technological challenges [ , , ] . in addition, archiving databases and files is a consideration often overlooked, though quite important in being able to return to data with new leads and directions for subsequent research or simply cross-compare with new data. there are, of course, other limitations, inherently present that restrict the scope of microarray analysis just like any other tool. for example, microarrays only present a snapshot of the transcriptome which is continually changing and responding to cellular needs and signals. as such, microarrays only illuminate a part of what is going on inside a cell or a population of cells [ , ] . in addition, there does not necessarily have to be a tight correlation between the expression of a gene and the amount of translated protein. therefore, differentially expressed genes may not translate into varying protein levels with functional implications [ ] . furthermore, the complexity of microarray analysis makes it exceedingly difficult to ascertain meaningful data with real biological significance without clearly defined goals or targets. an intricate aspect of genomic analysis is the interplay between genes or groups of genes (i.e. mechanisms) and that information is not easily deciphered using microarrays. and finally, the functionality of a gene cannot be determined solely using microarrays [ , ] . indeed, other methods and experimental tools are needed to decipher the proteome, understand the varying interactions between genes and/or proteins, and develop a more complete picture of cellular behavior. ultimately, microarrays will continue to be used in a variety of research areas as more options in the design of custom arrays become available along with an increase in the assortment of species-specific arrays. technological advancements may help bring down the cost as well as enhance reproducibility and reliability promoting the applicaton of microarrays in new and diverse fields. in the end, the questions raised by microarray results are often just as vital as the answers they produce; a key to expanding 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identification of new genes regulated by light color in the cyanobacterium fremyella diplosiphon engineering hlya hypersecretion in escherichia coli based on proteomic and microarray analyses genetic analysis and attribution of microbial forensics evidence quantitative oligonucleotide microarray fingerprinting of salmonella enterica isolates funding was provided by the intramural program at the national institute of diabetes & digestive & kidney diseases, national institutes of health.the authors would also like to thank members of the biotechnology unit for their input and willingness to proofread the manuscript. the authors declare that they have no competing interests. pj formulated the content, performed the literature search, and drafted much of the manuscript. kk contributed to revising the manuscript and adding content. mb contributed to formulating the content and layout. js contributed to formulating the content, revising the manuscript, and drafting portions of the manuscript. key: cord- -w efsshj authors: han, tae-hee; chung, ju-young; hwang, eung-soo title: human bocavirus in children, south korea date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: w efsshj nan to the editor: in , kapoor et al. and arthur et al. published reports on the prevalence of the newly identified parvovirus, human bocavirus (hbov- ), in fecal samples ( , ) . hbov- had been discovered in ( ), and reports indicate its possible role in respiratory diseases such as upper respiratory tract infections, lower respiratory tract infections (lrtis), and in exacerbation of asthma ( ) ; in these diseases, the virus co-infects with other respiratory viruses ( ) . systemic infection with hbov- and possible association of this virus with other diseases such as gastroenteritis, kawasaki disease, and hepatitis have been reported ( ) ( ) ( ) . we looked for hbov- in clinical samples from children with various diseases, including acute lrtis, kawasaki disease, henoch-schönlein purpura, and hepatitis. during september -january , a total of nasopharyngeal aspirates were collected from children (median age months, range - months) hospitalized with acute lrtis at sanggyepaik hospital in seoul, south korea. previously, during january -june , a total of serum samples had been obtained from children (age range month- years) with hepatitis (hepatitis b, samples; hepatitis c, samples; unknown hepatitis, samples), kawasaki disease ( samples), and henoch-schönlein purpura ( samples) and from healthy children (same age range, samples) ( ) . the study was approved by the internal review board of sanggyepaik hospital. dna was extracted from serum samples, and rna and dna were extracted from nasopharyngeal aspirates by using a qiaamp viral rna mini kit (qiagen, hilden, germany) and a qiaamp dna blood mini kit (qiagen gmbh), respectively. all nasopharyngeal aspirates were tested by pcr for common respiratory viruses such as respiratory syncytial virus, influenza viruses a and b, parainfluenza virus, and adenovirus, as described previously ( ) . pcrs to detect hbov- were performed by using primers for the nonstructural (ns) and nucleocapsid protein (np) genes, as described previously ( ) . additional pcrs for rhinovirus, human metapneumovirus, human coronavirus (hcov)-nl , hcov-oc , hcov- e, hcov hku- , wu polyomavirus, and ku polyomavirus were performed, as described, for hbov- -positive samples ( ) . hbov- was detected by performing first-round pcr with primers based on the ns gene, hbov -sf , and hbov -sr . second-round pcr was performed by using primers hbov -sf and hbovsr , as described previously ( ). the pcr products were sequenced by using an abi xl autoanalyzer (applied biosystems, foster city, ca, usa). the nucleotide sequences were aligned by using bioedit . (www.mbio.ncsu.edu/ bioedit/bioedit.html) and presented in a topology tree, prepared by using mega . (www.megasoftware.net). of the samples tested, the following viruses were detected: human respiratory syncytial virus ( hbov- was not detected in the study population. hbov- dna was found in ( . %) of the samples collected; all positive samples had been obtained in october . the age range of the children with hbov- -positive samples was - months (median months), and all were male. the diagnoses were bronchiolitis for children and bronchopneumonia for . the most frequently codetected virus was human respiratory syncytial virus, found in ( %) of samples. one sample that was negative for respiratory syncytial virus and positive for hbov- was negative for all other respiratory viruses. nucleotide sequences were determined for the ns- gene, and phylogenetic analyses, which included hbov- , a new lineage designated by arthur et al. ( ) , showed that the ns- gene was relatively well conserved and that there were major groups of the virus, the uk strain and the pakistan strain. hbov- strains isolated from south korea belonged to the hbov-pk (fj ) cluster (figure) . recent studies have detected hbov- in serum samples of children with kawasaki disease and of an immunocompromised child with hepatitis ( , ) . however, neither hbov- nor hbov- was detected in the serum samples from patients with hepatitis, with kawasaki disease, with henoch-schönlein purpura, and healthy children. the absence of hbov- in the samples examined was unexpected because hbov- was detected in > % of respiratory samples collected from a demographically similar study population during the winter years earlier ( ) . future studies, with larger populations and over longer periods, are needed to delineate seasonal variations between hbov- and hbov- . we demonstrated hbov- dna in the respiratory tract secretions of children with acute lrtis. in most positive samples, the virus was found in addition to other respiratory viruses. a limitation is that the study did not consider health control measures and other clinical disease such as gastroenteritis and was conducted for a short time. the role of hbov- in lrtis remains unclear; further studies are needed to clarify whether this virus is only shed from the respiratory tract or whether it replicates in the gastrointestinal tract. this study was partly supported by a research grant ( ) from inje university. to the editor: mycobacterium haemophilum is an aerobic, slowgrowing microorganism with optimal growth at °c to °c. it has a unique requirement for ferric iron-containing compounds ( ), from which it acquired its name (i.e., haemophilum). infections with m. haemophilum are rare, but cervicofacial lymphadenitis caused by m. haemophilum has been described in children ( ) . besides cervicofacial lymphadenitis, extrapulmonary signs of m. haemophilum disease include subcutaneous noduli, arthritis, and osteomyelitis, which generally affect immunocompromised patients ( ) . recently, cases of cutaneous m. haemophilum infections after alemtuzumab treatment were reported ( ) . a small number of pulmonary m. haemophilum infections associated with aids or solid organ or bone marrow transplantation have been described ( ) . we report pulmonary m. haemophilum infection in a woman who had been immunosuppressed by tumor necrosis factor-α antagonist (tnf-αa) (adalimumab) treatment for rheumatoid arthritis. a -year-old woman with a history of rheumatoid arthritis and obstructive sleep apnea syndrome had signs and symptoms of fatigue, mild fever episodes, and a nonproductive cough months after treatment for rheumatoid arthritis had begun with methotrexate (mtx) and tnf-αa. physical examination was unremarkable except for a body temperature of . °c. laboratory testing showed an increased erythrocyte sedimentation rate (esr) ( mm/h), an increased creactive protein (crp) level ( mg/l), a normal leukocyte count ( , cells/ μl), and relative monocytosis ( %). hiv serologic testing results were negative. chest radiograph showed an infiltrate in the right upper lobe. chest computed tomography confirmed this finding and showed lymphadenopathy in the right hilus and mediastinum. notably, the tuberculin skin test result was negative at screening before she began the tnf-αa treatment, but was now positive ( mm), suggesting mycobacterial infection. auramine and ziehl-neelsen staining of sputum and bronchoalveolar liquids showed no acid-fast bacilli, and m. tuberculosis infection was not confirmed by pcr or culture. eventually, a mediastinal lymph node biopsy was taken by endoscopic ultrasound guidance. granulomatous inflammation and acid-fast bacilli were seen by microscopy. corresponding cultures yielded a strain identified as m. haemophilum at the netherlands national institute for public health and the environment (rivm) by using the inno-lipa mycobacteria v reverse line blot assay (innogenetics, ghent, belgium). strain identity was confirmed by sequencing of the complete s rdna gene, which was identical to that of m. haemophilum available in the genbank sequence database (national center for biotechnology information; www.ncbi.nlm.nih.gov; accession no. x ). the rivm performed drug susceptibility testing by using a modified agar dilution method ( ) . middlebrook h media were enriched with % sheep blood hemolyzed by : dilution with water and subsequent freezing-thawing. historic drug susceptibility data was reviewed (table) . initially, adalimumab was discontinued, and our patient was treated with isoniazid, ethambutol, rifampin, and pyrazinimide because m. tuberculosis infection was suspected. after identification of m. haemophilum, our patient was treated with rifampin and azithromycin. a total treatment duration of months resulted in complete resolution of the a newly identified species in human stool a novel bocavirus associated with acute gastroenteritis in australian children cloning of a human parvovirus by molecular screening of respiratory tract samples human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus frequent detection of bocavirus dna in german children with respiratory tract infections human bocavirus, a respiratory and enteric virus detection of human bocavirus in children with kawasaki disease hepatitis and human bocavirus primary infection in a child with t-cell deficiency small anellovirus infections in korean children wu polyomavirus in children with acute lower respiratory tract infections key: cord- -epxd pt authors: eckermann, m.; frohn, j.; reichardt, m.; osterhoff, m.; sprung, m.; westermeier, f.; tzankov, a.; werlein, c.; kuehnel, m.; jonigk, d.; salditt, t. title: d virtual patho-histology of lung tissue from covid- patients based on phase contrast x-ray tomography date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: epxd pt we present a new approach of three-dimensional ( d) virtual histology and patho-histology based on multi-scale phase contrast x-ray tomography, and use this to investigate the parenchymal-architecture of unstained lung tissue from patients who succumbed to covid- . based on this first proof-of-concept study, we can propose multi-scale phase contrast x-ray tomography as a novel tool to unravel the patho-physiology of covid- , extending conventional histology by a third dimension and allowing for a full quantification of tissue remodeling.by combining parallel and cone beam geometry, autopsy samples with a cross section of mm are scanned and reconstructed at a resolution and image quality which allows for the segmentation of individual cells. using the zoom capability of the cone beam geometry, regions-of-interest are reconstructed with a minimum voxel size of nm. we exemplify the capability of this approach by d visualisation of the diffuse alveolar damage with its prominent hyaline membrane formation, by mapping the d distribution and density of lymphocytes infiltrating the tissue, and by providing histograms of characteristic distances from tissue interior to the closest air compartment. severe progression of the coronavirus disease is frequently accompanied by the clinical acute respiratory distress syndrome (ards) and respiratory failure, an organ manifestation responsible for the majority of covid- fatalities. lung injury associated with ards can be readily detected by radiographic chest imaging and clinical computed tomography (ct), which have assisted the diagnosis and management of covid- patients [ , , ] . here, so-called peripheral lung ground-glass opacities are the main radiological hallmark of ards, and can be linked to the histological observation of diffuse alveolar damage with edema, hemorrhage, and intraalveolar fibrin deposition [ ] . these findings have also been reported for infections by middle east respiratory syndrome coronavirus (mers-cov), sars-cov, and influenza viruses. distinctive features of pulmonary involvement of covid- include severe endothelial injury associated with the presence of intracellular virions and inflammation, disrupted cellular membranes, as well as widespread thrombosis with microangiopathy. as reported in [ ] , alveolar capillary microthrombi were found to be times as prevalent in patients with covid- as in patients with the also very aggressive h n influenza a virus, also referred to as swine flu. importantly, in covid- lungs, a specific variant of new vessel growth -intussusceptive angiogenesis -was significantly more prevalent, i.e. . times as high as in lungs of patients with h n influenza a. histomorphological assessment of formalin-fixed, paraffin-embedded (ffpe) tissue stained with haematoxylin and eosin still represents the gold standard in histological diagnostics of non-neoplastic lung diseases, including diffuse alveolar damage and virus induced pneumonia. in order to unravel the corresponding pathophysiology of the lung, digitalisation, visualisation and quantification of the morphological changes associated with covid- represent a key challenge, and require both high resolution and the capability to screen larger volumes. for this reason, imaging the intricate three-dimensional ( d) tissue architecture of the lung and its pathological alterations on multiple length scales calls for d extensions of well-established histology techniques. in this work, we use propagation-based phase contrast xray tomography as a novel tool for virtual d histology. we present first results obtained from postmortem lung samples of six patients who succumbed from covid- . we exemplify the capability of this approach by d visualization of the diffuse alveolar damage with hyaline membrane formation, by mapping the d distribution and density of angiocentric inflammation (perivascular t-cell infiltration), and by providing histograms of characteristic distances from the tissue interior to the closest air compartment. propagation-based x-ray phase-contrast tomography (pc-ct) has been introduced before for d virtual histology [ , , , ] . in contrast to conventional histology based on thin sections, it offers a full d visualization with isotropic resolution and without destructive slicing of the specimen. the interaction of x-rays with the object is described by the continuous complex-valued index of refraction n(r) = −δ(r)+iβ(r). phase contrast capitalizes on the fact that for hard x-rays, the real-valued decrement δ is several orders of magnitude higher in soft biological tissues than β which accounts for absorption [ ] . contrast is formed by transformation of the phase shifts into measurable intensity variations by self-interference of the exit wave during free-space propagation between sample and detector [ , ] . by careful optimization of photon energy, illumination function, and phase retrieval algorithms, the phase sensitivity is high enough to probe the small electron density variations of unstained tissue, for example tissue embedded in paraffin, ethanol, or even aqueous buffer [ ] . in contrast to other full-field phase contrast techniques, e.g. based on grating interferometry or analyzer crystals, propagation based imaging (pbi) can reach a resolution below optical microscopy [ ] . the d virtual pathohistology approach for covid- presented here was realized by implementing a novel multi-scale phase contrast x-ray tomography concept, with dedicated xray optics and instrumentation to image the tissue structure on multiple length scales, while at the same time covering large reconstruction volumes. to this end, overview and regions-of-interest (roi) scans were recorded on the same paraffin-embedded sample, covering a maximum tissue cross section of mm by stitching different tomograms, and with a minimum voxel size of nm in certain rois. scale-bridging and dynamic roi selection in close spatial and temporal proximity was implemented with dedicated instrumentation the ginix endstation of the beamline p /petra iii (desy, hamburg) [ ] . specifically, we combined two optical geometries, which has only been realized at different synchrotron beamlines before: (i) parallel beam tomography, covering a large field of view, with a pixel size of nm. in this setting, a volumetric throughput on the order of µm s was achieved, while maintaining the ability to segment isolated cells in unstained tissue. (ii) cone beam geometry for recording of highly magnified holograms, based on advanced x-ray waveguide optics, providing mode filtering, i.e. enhanced spatial coherence and smooth wavefronts. based on the geometrical magnification, the effective pixel size can be adjusted in the range - nm. the two imaging schemes are shown schematically in fig. (c) and (d), respectively. further, using this particular optics, together with appropriate choices of photon energy and geometric parameters, we can reach extremely small fresnel numbers f of the deeply holographic regime, well below the typical range exploited at other nano-tomography instruments. this offers the advantage of highest phase sensitivity sufficient to even probe the small electron density variations of unstained tissue, at relatively low dose [ ] . to exploit this sensitivity, we use advanced phase retrieval methods including non-linear generalizations of the ctf-method [ ] based on tikhonov regularisation [ ] . in total we investigated six post mortem lung samples from covid- patients [ ] . a tissue micro-array paraffin block with samples of all six patients and the corresponding he stain is shown in fig. a ). information about age, gender, hospitalization, clinical, radiological and histological characteristics of all patients are shown in tab. . all patients suffered from hypertension and were treated with raas (renin-angiotensin-aldosterone-system) interacting drugs. heterogeneous ground glass and consolidation were observed in all patients clinical ct scans and the cause of death was also related to respiratory failure in each separated for their stain, six tissue samples were dehydrated and embedded in the same multi-sample paraffin block. the size of the post-mortem tissue samples made available for the study varied between the different patients (i-vi), with maximum cross-section of about mm after dehydration. from all six samples, biopsy punches were taken by either a mm or a . mm punch, depending on the individual size. the punches were then transferred onto a holder for the parallel-beam local tomography acquisition, followed by a further reduction in size (after measurement of the entire sample) to a mm biopsy punch, for further tomographic recordings. a sketch of the sample preparation is shown in fig. b) . the control lungs were first mounted in eppendorf tubes, and mm biopsy punches were then transferred to polyimide tubes similar to the paraffin-embedded ones, but scanned in fixative buffer solution. for covid- lung tissue, the scans were recorded at the ginix endstation of the petra iii storage ring (desy,hamburg). the projections were acquired at two different photon energies e ph , kev and . kev, using the first and third harmonic of the m p undulator and a si( ) channel-cut monochromator, respectively. data is shown here only for kev, which gave highest contrast for the unstained lung tissue. two tomography configurations were combined to cover a larger range of length scales: ( -parallel beam) recordings with the unfocused quasiparallel beam illumination and a high resolution microscope detection system, resulting in in a field-of-view (fov) of . mm× . mm sampled at a pixel size of nm. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . ( -cone beam) holographic recordings with the divergent and coherence filtered beam emanating from a compound focusing system composed of a kirkpatrick-baez (kb) mirrors system and an x-ray waveguide, resulting in a fov of . mm× . mm sampled at a pixel size of about nm (depending on exact geometry). both configurations were implemented side-by-side, using the same fully-motorized tomography stage and mounting, as detailed in [ ] . a sketch of both configurations is shown in fig. . first, parallel-beam overview scans were acquired of the entire tissue volume embedded in paraffin. to this end a . mm biopsy punch was taken from the multi-sample tissue block, and then scanned in a stitching-mode yielding a large overview reconstruction, composed of up to individual tomograms (depending on tissue size). to increase image quality and avoid artifacts related to region-of-interest (roi) or local tomography, a selected mm punch was then taken from the already scanned larger tissue cylinder, and re-scanned, first in the parallel-and then the cone-beam geometry. experimental and acquisition parameters used for the data shown are listed in tab. . the configuration for ( -parallel beam) is depicted in fig. c) . the high-resolution microscope detection system (optique peter, france) was based on a µm thick luag:ce scintillator imaged with a × magnifying microscope objective onto a scmos sensor (pco.edge . , pco, germany), resulting in an effective pixel size of . µm. the high photon flux density allowed for image acquisition with continuous motor movement, short acquisition time of ms per frame, and a framerate of fps. single-distance tomogram recordings with about projections, and flat images before and after the scan, took less than min. for these scans, the focussing optics (kb-mirrors and waveguide) as well as the fastshutter (cedrat technologies) were moved out of the beam, and beam size was adjusted by the upstream slit systems. to avoid detector saturation, the beam was attenuated by × µm single crystal silicon wafers. the configuration for ( -cone beam) is depicted in fig. d ): the beam was focused by the kb-mirrors to about nm× nm. to further reduce the secondary source size, to increase coherence, and to achieve a smooth wavefront for holographic illumination, an x-ray waveguide formed by mm long lithographic channels in silicon with a cross-section of about nm× nm was positioned in the focal plane of the kb-mirrors, resulting in an exit flux of - × photon/s (depending on alignment and storage ring), as measured with the single photon counting detector (pilatus, dectris). the sample was positioned at variable (defocus) distances behind the focus (waveguide exit), typically z = mm for the first distance. the geometrically magnified holograms were recorded by a fibre-coupled scmos sensor (zyla hf . detector, andor technologies) with a customised µm thick gadox scintillator, and . µm pixel size. the detector position at about z = mm behind the focus resulted in a magnification of about m = (for the first defocus distance), and . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint hence an effective pixel size of nm. projetions were recorded, with a typical exposure time of s per projection. phase retrieval and reconstruction phase retrieval was performed from dark and empty beam corrected holograms, using both linearised single step ctf-approach [ , ] , and nonlinear generalisations of the ctf-method based on tikhonov regularisation (nl-ctf), using our code package holoto-motoolbox as described and deposited [ ] . importantly, both ctf and nl-ctf implementations can be augmented by imposing support and range constraints, when needed. when available, projections recorded at several defocus distances were first aligned with sub-pixel accuracy and then used for multi-distance phase retrieval. for these purposes, the holotomotoolbox provides auxillary functions, which also help to refine the fresnel number or to correct for drift in the illumination function. after phase reconstruction of all projections, the tomographic reconstruction was carried out with the matlab implemented iradon-function (ram-lak filter) for the parallel geometry and with the fdk-function of the astra toolbox [ , ] for the cone beam geometry. hot pixel and detector sensitivity variations as well as strong phase features in the parallel beam illumination resulting from upstream window materials, which persist after empty beam correction, can all result in ring artifacts in the tomographic reconstruction, in particular as these flaws can increase by phase retrieval. to correct for this, the extra information provided by • scans was used to mask out the corresponding pixels and replace them with values of the opposing projection. stitching of reconstructions from different tomographic scans was performed using [ ] . image segmentation and quantification for each patient the stitched overview scans were analysed with regard to structural characteristics. the d-reconstructions were first binned ( × × ), and the tissue was then segmented from the surrounding paraffin using the segmentation software ilastik [ ] , which was then further refined with matlab. in order to exclude single macrophages or detached tissue, only voxels connected to the tissue block were considered for the distance map. further, the areas of the paraffin which represent air compartments were linked to the outside of the tissue block. individual self-contained areas of paraffin (not connected to air) were excluded from the mask. based on this segmentation, the distance to the nearest voxel containing oxygen was calculated for each tissue voxel. the tissue volume v is given by the sum of all voxels containing tissue. the surface area sa is defined by all tissue voxels with a distance of pixel to the air. from this information we calculated the specific surface with vx edge length of a voxel was determined for each sample. additionally, the mean distance (do ) from all tissue voxels to air and its standard deviation was calculated. hyaline membranes and capillary networks were extracted using semi-manual segmentation functions in avizo (thermo fisher scientific, usa). beside hyaline membranes and the capillary network, different cell types can be readily identified based on the d reconstructions of the electron density. in particular, inflammatory cell subpopulations -i. e. macrophages and lymphocytes -can be distinguished. to this end, an automatic and parameter-controlled algorithm denoted as blobfinder was used (arivis). based on its segmentation output, the amount and position of the lymphocytes in the d-reconstructions from parallel beam scans was calculated. the algorithm is able to identify roundish structures with a given size. for the segmentation of the lymphocytes, we chose a characteristic size of . µm. the structures identified in this step also include macrophages and parts of the capillary system. for the unbinned datasets, a distinction between lymphocytes and the nuclei of the macrophages was made based on the difference in electron density. in the tomographic reconstructions, the lymphocytes appear denser compared to the nuclei of the macrophages. further, nuclei from endothelial cells and parts of the capillaries filled with blood residues were excluded based on their elongated shape. only structures with a sphericity higher than . were included. based on the segmentation of lymphocytes, the total number of lymphocytes n l was obtained and the mean concentration of lymphocytes within the lung tissue c l = n l /v was estimated. before presenting the results for the six covid- patients for each of the imaging levels (scales), we give an overview on the typical datasets for one exemplary sample (i), see fig. . the typical field-of-views, image quality, and appearance of the lung structure as well as the amount of data can be inferred, and inspected in the reconstructions provided online (https://doi.org/ . /zenodo. ). the datasets are denoted by patient i-vi, respectively. gray values of the tomographic reconstruction represent phase shift per voxel with edge length vx, the local electron density difference to the average paraffin can be computed by with wavelength λ and r classical electron radius. next, we present representative slices through the reconstruction volumes of all samples for all acquisition scales. conventional he-stained histology images of all samples are shown in the supplementary information (fig. ). fig. presents the stitched reconstruction volumes, recorded under conditions of local tomography, see tab. . since these volumes are computed from stitching up to individual tomograms, the question arises to which extend the image quality is limited by potential artifacts of local tomography, i.e. errors due to the fact that part of the sample is outside the reconstruction volume. for this reason, mm punches were taken after the stitched overview and rescanned in the parallel beam configuration, without local tomography conditions, since they fitted within the fov. the results are presented in fig. , and validate the previous stitching results. the mm punches then also provided an appropriate size for the cone-beam recordings, which are shown in fig. . importantly, in each scan the previous level guided the choice for the next fov . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint and informed about the larger environment. in the following, we briefly discuss the samples one-by-one, with regard to all acquisition scales. a comparison of morphological features between conventional and virtual histology is shown in the supplementary (fig. ). sample i: by conventional histopathological assessment, the peribronchial alveolar parenchyma of sample i showed dad with focal formation of hyaline membranes adjacent to the epithelial lining, moderate lymphocytic interstitial pneumonia and singular thrombi in small pulmonary veins. there is a moderate hypertrophy of the muscular media in smaller pre-and post-capillary blood vessels with desquamation of the endothelial cell layer as well as mild centrilobular emphysema (original magnification x). in pc-ct, enlarged alveolar septa with pronounced lymphocytic inflammation are displayed. the reconstruction volume contains a large artery filled with erythrocytes (fig. , lower left) , which bifurcates into two vessels. this area was then selected for the mm biopsy punch extraction. the cone-beam zoom tomogram was then centered around the perimeter of the blood vessel. this volume is particularly well suited to investigate the connective tissue including elastic fibers and collagen, as well as smooth muscle. sample ii: histomorphological analysis shows peribronchial alveolar parenchyma with hyperemia of capillary and postcapillary blood vessels, as well as a moderate centrilobular emphysema (original magnification x). on the level of blood vessels, both blood-filled and empty vessels are discernible. it should be noted that septa with signs of parallel capillaries are visible. in the reconstruction volume of the zoom tomogram, a single vessel can be easily tracked over large distances. sample iii: the sample consists of peribronchial alveolar parenchyma showing prominent multifocal neutrophilic capillaritis as well as a moderate centrilobular emphysema (original magnification x). in pc-ct, septa with again similar physiological size and distribution emerge, with moderate emphysema. the bottom part of the sample contains a fibrous area near a larger blood vessel. the zoom tomogram shows a single septum, a blood vessel and a fibrous region. sample iv: histomorphological analysis shows peribronchial alveolar parenchyma with marked lymphocytic interstitial pneumonia, multifocal venous thrombi and focal intraalveolar fibrin deposition in terms of dad. furthermore, there is a mild centrilobular emphysema (original magnification x). in pc-ct, a network of thin septa, thrombi and emphysema, as well as a large empty blood vessel appears. electron-rich diffuse black granules are also visible. the biopsy punch was selected to contain the empty blood vessel, some small thrombi. it also includes thin septa and tissue embedded dirt-particles. the zoom tomogram covered tissue with black granules as well as a band of inflammatory cells next to a cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . sample v: by conventional histological assessment, sample v consists of peribronchial alveolar parenchyma showing massive lymphocytic interstitial pneumonia with ubiquitous hyaline membranes superimposed on the alveolar walls, neutrophilic capillaritis and multifocal post-capillary thrombi in terms of severe dad. furthermore, bronchialized alveolar epithelial cells show cytopathic changes and multifocal desquamation, as does alveolar macrophages. focally, accumulation of intraalveolar neutrophilic granulocytes in the sense of a florid bronchopneumonia can be observed (original magnification x). pc-ct data give rise to alterations of the overall morphology due to covid- , including substantial inflammation, pronounced hyaline membranes, and high load of lymphocytes. the biopsy punch was chosen to include areas with increased presenced ofhyaline membrane and lymphocytes. a blood vessel splitting into several smaller blood vessels is easily recognized when browsing through the reconstructed volume. noteworthy, different cell types as macrophages, t-cells or erythrocytes can be distinguished in the zoom tomogram. sample vi: histomorphological analysis shows peribronchial alveolar parenchyma with lymphocytic interstitial pneumonitis and a singular thrombus in a small vein. the interstitium of the alveolar septae are widened by myogenic metaplasia. adjacent, centrilobular emphysema and anthracosis are observed. the bronchial mucosa shows varying degrees of lymphocytic inflammation in the sense of chronic bronchitis / bronchiolitis (original magnification x). from pc-ct reconstructions, the sample consists of thin alveoli (fig. vi, upper left) evolving into compact, fibrotic tissue (fig. , lower right). the amount of lymphocytes is rather low in this sample. black granules and some thrombi are embedded within the bulky tissue parts. the biopsy punch covers the region of transition from alveoli to fibrotic tissue, containing also a thrombus and capillaries as identified from the zoom tomogram. the overview scans of all paraffin embedded covid- positive samples and one biopsy of a hydrated control lung were analyzed in terms of structural characteristics. the top row of fig. shows the workflow of the analysis on the example of the d reconstruction of sample v. based on the tissue mask the distances for each tissue voxel to the next voxel containing oxygen were determined. for the analysis of the tissue it is mandatory to consider the three-dimensionality of the samples. this can also be seen in fig. d , which shows a zoom of the distance analysis around a small blood vessel which is marked with a yellow box. while the wall thickness appears quite homogeneous in the d slice, the distance analysis reveals that . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint the vessel is thicker on the top right. the distribution of the distances obtained for a given slice is shown in fig. e . the swelling of the alveolar walls as well as the inflamed blood vessels can be identified by comparing the reconstructed electron density and the d-distance map (see fig. f and g) . fig. h shows the distribution of the tissue-air distances (histogram) for all samples, following the workflow illustrated in fig. e . the binning of distances was set to one voxel length. the figure underlines the high diversity of the tissue structure which could already be seen in the d histology. further, it directly informs about the specific surface sv , which is given by the first point of the graph. table : results of the analysis of tissue characteristics: specific surface area sv , characteristic length lc and mean distance do and standard deviation from all tissue voxels to air as well as the mean concentration of lymphocytes c l for all six covid- positive samples as well as for one control sample. patient no. the corresponding parameters and metrics are tabulated in tab. for all samples. additionally, the mean concentration of lymphocytes c l within the lung tissue is listed for all samples. the values quantify the general structure of the tissue which is qualitatively discernibel by eye. samples with a high amount of swollen, inflamed blood vessels and thick hyaline membranes exhibit a larger characteristic length. note, that the control lung was prepared in a hydrated environment and shrinking due to further preparation of the sample does not occur. hence, the results cannot be directly compared to the paraffin embedded samples. further, the analysis of the lymphocyte concentration was be performed since no lymphocytes were found in the reconstructed volume. the low values of lc and do for sample ii correlate with the lack of ground-glass opacification in clinical ct. based on the extracted structural parameters, the degree of inflammation and swelling of lung tissue can be evaluated. e.g. patient ii has the highest surface area volume-ratios while sample i and vi have a relatively low specific surface. larger characteristic lengths may also be indicative of inflammation and the formation of hyaline membranes, which will be evaluated in the following based on roi and high-resolution reconstructions. fig. illustrates the aggregation of hyaline membrane in the vicinity of a single alveole. volumetric renderings in (a) and (b) demonstrate particular attachment of fibrin to the alveolar walls. in cases of severe hyaline membrane formation as for this patient, this pathological alteration can be tracked throughout the volume (c-e). in (f), hyaline membranes of neighboring alveoles are indicated. in the d-context, their locations with respect to blood vessels can be inspected, see (g), which exemplifies a direct connection of hyaline membranes to the vasculature. the severeness of hyaline membrane formation is casespecific, as the yellow rendering in fig. (a, b & d) demonstrates reduced amounts of deposits for patient i in a subvolume of parallel-beam reconstructions. further, lymphocytes (red) were identified based on the automated cell segmentation (see methods). for clearer visualisation, each cell is rendered as a sphere with a size corresponding to the mean cell volume in (a, b & d) . based on convolution of the cell positions with a sphere of µm in radius, the local cell density was calculated [ ] and presented as d-maps of cells/mm in (c & e). this concept was then translated to a d-stitched volume of an entire tissue block as shown in (f & g). next, the segmentation of blood vessels is demonstrated for the example of a splitting blood vessel in the zoom tomogram of sample v. the segmentation was performed manually. to give an impression of the separation of a single capillary, a series of virtual slices in the xy-plane (magnified views) is shown in fig. a . the separation starts with the creation of a branch from the blood vessel (arrow in slice ). in slice , . µm above slice , this branch evolves into an empty and separated capillary. another µm above, the capillary is entirely filled with cells. further . µm, the capillary is empty again and has a diameter of about . µm. the segmentation of the blood vessel with all its separated branches is indicated for slice in fig. b by the red lines. in this slice, three capillaries have already separated from the main vessel, while the fourth starts to emerge, indicated by the red arrow. the d shape of the blood vessel is illustrated by the d rendering of the segmentation, shown in fig. c . this segmentation of the blood vessel shows the potential of the datasets, which may be fully exploited in future with more advanced segmentations. in summary, we have demonstrated that multi-scale x-ray phase contrast tomography can firstly augment pathohistology of the lung to full three dimensions, and secondly provide a link between microscopic and macroscopic scales. for the first time, characteristic morphological changes associated with diffuse alveolar damage such as hyaline membranes and pronounced inflammation have been imaged in three dimensions, in particular in covid- , the most severe pandemic which mankind has faced for decades. moreover, we conducted our study in ffpe material, a fixation method established for well over a century and ubiquitously available. on the side of imaging technology and exploitation, there is still ample room for improvement: higher resolution could be achieved by further geometric zooms, waveguide optics with higher numerical aperture, in combination with pixel detector technology, and further improvements in holographic reconstruction. equally important to extend the length scales covered to small scales is the further upscaling of the field of view (fov) based on stitching different sub-tomograms. in this way, one could bridge scales to cover the entire lobe of a lung. to this end, optimized recording and data flow is a larger bottleneck than photons and optics, in particular since th generation synchrotron radiation sources are about to deliver unprecedented brilliance. finally, in view of exploitation and information gain, specific labels for different cell types and d immunostaining coupled to radiocontrast agents should be developed. here, lung offers an advantage over other tissue in view of volume accessibility and label diffusivity. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint most importantly, efforts have to be directed to exploit the full information contained in the reconstruction volumes based on advanced segmentation. tracing of capillaries are an obvious important next step, in view of unraveling the role of intussusceptive angiogenesis in covid- [ ] . the current data, which is made fully available at https://doi.org/ . /zenodo. , is likely to already provide such clues once that more advanced segmentation approaches based on machine learning are applied. more generally, it may advance our understanding of diffuse alveolar damage in the particular case of covid- , as an intrinsically volumetric phenomenon. beyond the current data, further studies could shed further light on the differences between moderate and severe progression. possibly, phase contrast tomography of lung biopsies could in future also help diagnosis and treatment. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted june , . the imaging workflow may also be applied to hydrated and/or healthy lung tissue for systematic pathological analysis, as indicated in fig. . (a) shows the rendering of truncated hydrated control tissue volume. (b) and (c) show virtual slices through the volume, demonstrating the parenchymal architecture in unaffected lung autopsies. before taking tomograms of the samples at the synchrotron setup, tissue slices of . µm thickness were cut from the top, afterwards he (hematoxylin and eosin) stained and imaged with a microscope. figure shows the histological slice of each sample. a comparison of morphological features between conventional he histology and virtual histology is presented in fig. . artery lumen, artery wall, erythrocytes, thrombus, alveolar septum, marcophage, hyaline membrane and black granules (anthracosis) are shown for both imaging methods. contrast of the hyaline membrane is homogenous in both histologies, making it simple to recognize and segment. erythrocytes are easy recognizable by eye in the conventional histology, due to the he staining, while they are less eye catching in the virtual histology. this results in a difficult differentiation between thrombus and blood stasis as well as a difficult identification of blood capillaries in the alveolar septum in virtual histology. for each of the six unstained and ffpe tissue samples, there is also a ua-stained block sample of similar size. stitched overview scans in ( -parallel beam) configuration have been recorded, analogue to fig. . from the ua-labelled tissue blocks of patients i, iii, iv and v, mm biopsy punches have been inspected in the same configuration (cf. fig. ). using the ( -cone beam) setup, these samples from i, iii and iv were imaged at . kev x-rays, while v has been examined at . kev, as shown for fig. . further, scans of the unstained tissue block from patient ii have been performed at different propagation distances (z = , and kev) and different x-ray energies ( . , . , . and . kev). in cone-beam configuration, the unstained biopsy punch from patient i was scanned using . kev x-rays. from all healthy, hydrated tissue blocks, overview scans covering the entire samples have been recorded. hydrated mm biopsy punches (two for ctrli, one for ctrlii & ctrliii, where ctrlii & ctrliii have been extracted from the same patient) have also been recorded in ( -parallel beam) configuration. those from ctrlii & ctrliii were also examined in ( -cone beam) mode. prior to the synchrotron experiment, some of the samples have been examined with a laboratory phase-contrast µctsetup in large mm -sized fov-configuration (kα = . kev, px eff = µm, z = . m, projections of s exposure time with a flat panel cmos detector with µm gadoxscintillator, perkinelmer, usa) [ ] . ua-staining of the tissue proved advantageous in feature contrasting when working with laboratory x-ray sources. aiming at overview data with no claim for refined resolution, structures can be well-correlated with histological sections in fig. . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted june , . . https://doi.org/ . / . . . doi: medrxiv preprint covid- pneumonia: what has ct taught us? radiological findings from patients with covid- pneumonia in wuhan, china: a descriptive study ct imaging features of novel coronavirus ( -ncov) pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid- synchrotron inline phase contrast µct enables detailed virtual histology of embedded softtissue samples with and without staining three-dimensional virtual histology of human cerebellum by x-ray phase-contrast tomography extending two-dimensional histology into the third dimension through conventional micro computed tomography x-ray based virtual histology allows guided sectioning of heavy ion stained murine lungs for histological analysis coherent methods in the x-ray sciences noninterferometric phase imaging with partially coherent light holotomography: quantitative phase tomography with micrometer resolution using hard synchrotron radiation x rays contrast enhancement for visualizing neuronal cytoarchitecture by propagation-based x-ray phasecontrast tomography hard x-ray nanoholotomography: large-scale, label-free, d neuroimaging beyond optical limit compound focusing mirror and x-ray waveguide optics for coherent imaging and nano-diffraction coherenceresolution relationship in holographic and coherent diffractive imaging a phase-retrieval toolbox for x-ray holography and tomography post-mortem examination of covid patients reveals diffuse alveolar damage with severe capillary congestion and variegated findings of lungs and other organs suggesting vascular dysfunction frauke alves, and tim salditt. d virtual histology of human pancreatic tissue by multi-scale phase-contrast x-ray tomography x-ray phase imaging: demonstration of extended conditions with homogeneous objects the astra toolbox: a platform for advanced algorithm development in electron tomography fast and flexible x-ray tomography using the astra toolbox nrstitcher: non-rigid stitching of terapixel-scale volumetric images ilastik: interactive machine learning for (bio)image analysis phase contrast tomography of the mouse cochlea at microfocus x-ray sources we thank maximilian ackermann and florian länger for their helpful suggestions, patrick zardo for providing control specimen, emily brouwer for help in sample preparation, bastian hartmann and jan goemann for technical help with instrumentation and it, and jakob koch for help in segmentation. it is also our pleasure to acknowledge desy photon science management for the covid- beamtime call and beamtime. the authors declare no competing interests. the study was approved by and conducted according to requirements of the ethics committees at the hannover medical school (vote nr. bo k ). key: cord- - wmp ti authors: lewandowski, kuiama; xu, yifei; pullan, steven t.; lumley, sheila f.; foster, dona; sanderson, nicholas; vaughan, alison; morgan, marcus; bright, nicole; kavanagh, james; vipond, richard; carroll, miles; marriott, anthony c.; gooch, karen e.; andersson, monique; jeffery, katie; peto, timothy e. a.; crook, derrick w.; walker, a. sarah; matthews, philippa c. title: metagenomic nanopore sequencing of influenza virus direct from clinical respiratory samples date: - - journal: j clin microbiol doi: . /jcm. - sha: doc_id: cord_uid: wmp ti influenza is a major global public health threat as a result of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. metagenomic viral sequencing offers the potential for a diagnostic test for influenza virus which also provides insights on transmission, evolution, and drug resistance and simultaneously detects other viruses. we therefore set out to apply the oxford nanopore technologies sequencing method to metagenomic sequencing of respiratory samples. we generated influenza virus reads down to a limit of detection of ( ) to ( ) genome copies/ml in pooled samples, observing a strong relationship between the viral titer and the proportion of influenza virus reads (p = . × (− )). applying our methods to clinical throat swabs, we generated influenza virus reads for / samples with mid-to-high viral titers (cycle threshold [c(t)] values, < ) and / samples with low viral titers (c(t) values, to ). no false-positive reads were generated from influenza virus-negative samples. thus, nanopore sequencing operated with % sensitivity ( % confidence interval [ci], to %) and % specificity ( % ci, to %) compared to the current diagnostic standard. coverage of full-length virus was dependent on sample composition, being negatively influenced by increased host and bacterial reads. however, at high influenza virus titers, we were able to reconstruct > % complete sequences for all eight gene segments. we also detected a human coronavirus coinfection in one clinical sample. while further optimization is required to improve sensitivity, this approach shows promise for the nanopore platform to be used in the diagnosis and genetic analysis of influenza virus and other respiratory viruses. adults, infants, young children, pregnant women, those with underlying lung disease, and the immunocompromised ( ) . the burden of disease disproportionately affects low-/middle-income settings ( ) . influenza virus diagnostics and surveillance are fundamental to identify the emergence of novel strains, to improve the prediction of potential epidemics and pandemics ( , ) , and to inform vaccine strategy ( ) . diagnostic data facilitate real-time surveillance, can underpin infection control interventions ( , ) , and can inform the prescription of neuraminidase inhibitors (nai) ( ) . currently, most clinical diagnostic tests for influenza virus depend on detecting viral antigen or on pcr amplification of viral nucleic acid derived from respiratory samples ( ) . these two approaches offer trade-offs in benefits, as follows: antigen tests (including point-of-care tests [poct] ) are typically rapid but have low sensitivity ( ) ( ) ( ) , while pcr is more time-consuming but more sensitive ( ) . irrespective of the test used, most clinical diagnostic facilities report a nonquantitative (binary) diagnostic result, and the data routinely generated for influenza diagnosis have limited capacity to inform insights into epidemiological linkage, vaccine efficacy, or antiviral susceptibility. on these grounds, there is an aspiration to generate new diagnostic tests that combine speed (incorporating the potential for poct [ , ] ), sensitivity, detection of coinfection ( , ) , and generation of quantitative or semiquantitative data that can be used to identify drug resistance and reconstruct phylogeny to inform surveillance, public health strategy, and vaccine design. the application of oxford nanopore technologies (ont) sequencing to generate full-length influenza virus sequences from clinical respiratory samples can address these challenges. ont offers a "third-generation," portable, real-time approach to generating long-read single-molecule sequence data, with demonstrated success across a range of viruses ( , ( ) ( ) ( ) . to date, nanopore sequencing of influenza virus has been reported using high-titer virus from an in vitro culture system, producing full-length genome sequences through direct rna sequencing ( ) , or using targeted enrichment by either hybridization of cdna ( ) or influenza virus-specific pcr amplification ( ) . we therefore aimed to optimize a metagenomic protocol for detecting influenza viruses directly from clinical samples using nanopore sequencing. we determine its sensitivity compared to that of existing diagnostic methods and its accuracy compared to short-read (illumina) sequencing, using clinical samples from hospital patients during an influenza season and samples from a controlled laboratory infection in ferrets. further optimization is required before the nanopore method can be rolled out as a diagnostic test, but we highlight the potential impact of this technology in advancing molecular diagnostics for respiratory pathogens. study cohort and sample collection. we collected respiratory samples from the clinical microbiology laboratory at oxford university hospitals nhs foundation trust, a large tertiary referral teaching hospital in southeast england. we worked with anonymized residual material from throat and nose swabs generated as a result of routine clinical investigations between january and may . samples were collected using a sterile polyester swab inoculated into to ml of sterile viral transport medium (vtm), using a standard approach described on the cdc website ( ) . during the study, respiratory samples submitted to the clinical diagnostic laboratory were routinely tested by a pcr-based test using the genexpert assay (cepheid) to detect influenza a and b viruses and respiratory syncytial virus (rsv). the workflow is shown in fig. . samples from patients in designated high-risk locations (hematology, oncology, and critical care) were tested using the biofire filmarray (biomérieux) to detect an expanded panel of bacterial and viral pathogens. quantitative data (cycle threshold [c t ]) were generated by the genexpert assay, and we used the influenza virus c t value to estimate the viral titers in clinical samples. using the genexpert assay, up to pcr cycles are performed before a sample is called negative (i.e., positives have a c t value of Ͻ ). quantification was not available for the biofire results. for methodological assessment, we focused on four categories of samples, as follows: positive pool, negative pools, individual positive samples, and individual negative samples. for the positive pool, we pooled throat swab samples that had tested positive for influenza a virus in the clinical diagnostic laboratory to provide a large enough sample to assess reproducibility (fig. b) . for the negative pools, we generated three pools of throat swab samples that had tested negative for influenza virus (consisting of , , and individual samples) (fig. b) . for the individual positive samples, we included individual samples ( throat swabs and nasal swabs) that had tested positive for influenza a or b virus, selected to represent the widest range of genexpert assay c t values ( . to . ; valid test result range, to ). for the individual negative samples, we selected individual throat swab samples that were influenza virus negative. quantification of viral rna in samples. we quantified viral titers in hazara virus stocks and pooled influenza a virus-positive throat swabs by quantitative reverse transcription-pcr (qrt-pcr), using previously described assays and standards ( , ) . optimization of methods. prior to establishing the protocol detailed in full below, we assessed the impact of two possible optimization steps, centrifugation versus filtration and reduced time for cdna synthesis. for centrifugation versus filtration, we investigated two approaches to deplete human/ bacterial nucleic acid from our samples, i.e., filtration of the raw sample via a . -m filter (sartorius) before further processing versus using a hard spin ( , ϫ g for min). cdna libraries for this comparison were produced as described previously ( ) . for the reduced time for cdna synthesis, to assess the possibility of time saving in the cdna synthesis steps, we compared performance of the previously described protocol ( ) to that of a modified version with two alterations, first using superscript iv (thermo fisher) in place of superscript iii (thermo fisher) for reverse transcription, with the incubation time reduced from min to min at °c, and second, reducing the cdna amplification pcr extension cycling time from min to min. positive control. prior to nucleic acid extraction, each sample was spiked with hazara virus virions to a final concentration of genome copies per ml as a positive internal control. this is an enveloped negative-stranded rna virus (genus orthonairovirus, order bunyavirales) with a trisegmented genome of , , , , and , nucleotides in length (genbank accession numbers kp to kp ). it is nonpathogenic in humans and would therefore not be anticipated to arise in any of our clinical samples. cultured virions from an sw cell line were provided by the national collection of pathogenic viruses (ncpv; catalog no. v). nucleic acid extraction. samples were centrifuged at , ϫ g for min. the supernatant was eluted without disturbing the pelleted material and was used in nucleic acid extraction. total nucleic acid was extracted from l of supernatant using the qiaamp viral rna kit (qiagen) eluting in l of h o, followed by a dnase treatment with turbo dnase (thermo fisher scientific) at °c for min. rna was purified and concentrated to l using the rna clean & concentrator- kit (zymo research), following the manufacturer's instructions. randomly amplified cdna was prepared for each sample using a sequence-independent single-primer amplification (sispa) approach, adapted from our previously described workflow ( ) , based on the round a/b methodology ( ) . for reverse transcription, l of rna and l of primer a ( =-gtttcccactggaggata-n - =, pmol/l) ( ) were mixed and incubated for min at °c and then cooled to room temperature. first-strand synthesis was performed by the addition of l superscript iv first-strand buffer, l of . mm dinucleoside triphosphates (dntps), . l of . m dithiothreitol (dtt), l h o, and . l superscript iv (thermo fisher) before incubation for min at °c. second-strand synthesis was performed by the addition of l sequenase buffer, clinical sample collection (orange), clinical diagnostic testing (yellow), sample processing and sequencing using oxford nanopore technologies (blue), and processing of sequence data (purple). (b) outline of pooled influenza virus-positive samples into an influenza virus-negative background to generate various titers of influenza virus (from to genome copies/ml), undertaken in triplicate, and spiked with a standard titer of hazara virus control at genome copies/ml. flua, influenza a virus. . l h o, and . l sequenase (affymetrix) prior to incubation for min at °c, followed by the addition of . l sequenase dilution buffer and . l sequenase and a further incubation at °c for min. amplification of cdna was performed in triplicate using l of the reaction mixture as input to a -l accutaq la (sigma) reaction mixture, according to the manufacturer's instructions, using l primer b ( =-gtttcccactggaggata- =) ( ) , with pcr cycling conditions of °c for s, cycles of °c for s, °c for s, and °c for min, followed by °c for min. amplified cdna was pooled from the triplicate reaction mixtures, purified using a : ratio of ampure xp beads (beckman coulter, brea, ca), and quantified using a qubit high-sensitivity double-stranded dna (dsdna) kit (thermo fisher), both according to the manufacturers' instructions. nanopore library preparation and sequencing. multiplex sequencing libraries were prepared using ng of cdna from up to samples as input to the sqk-lsk or sqk-lsk kit and barcoded individually using the exp-nbd native barcodes (oxford nanopore technologies) and a modified one-pot protocol (https://www.protocols.io/view/one-pot-ligation-protocol-for-oxford-nanopore-libr -k acz e). libraries were sequenced on flo-min flow cells on the minion mk b or gridion device (oxford nanopore technologies), with sequencing proceeding for h. samples were batched according to the genexpert c t value (see file s in the supplemental material). illumina methods. nextera xt v kit (illumina) sequencing libraries were prepared using . ng of amplified cdna, as per the manufacturer's instructions, and sequenced on a ϫ -bp paired-end illumina miseq run by the genomics services development unit of public health england. bioinformatic analysis. nanopore reads were base called using guppy (oxford nanopore technologies, oxford, uk). output base called fastq files were demultiplexed using porechop v . . (https:// github.com/rrwick/porechop). the reads were first taxonomically classified against the refseq database using centrifuge v . . ( ) . the reads were then mapped against the reference sequence selected from the centrifuge report using minimap v . ( , ) . a draft consensus sequence was generated by using a majority voting approach to determine the nucleotide at each position. the resulting draft consensus sequences were subjected to a blast search against an influenza virus sequence database that included Ͼ , h n and h n seasonal influenza virus sequences between and and were downloaded from the influenza research database ( ) . the reads were again mapped against the reference sequence using minimap v . , and the number of mapped reads was calculated using samtools v . ( ) and pysam (https://github.com/pysam-developers/pysam). the subtype of the influenza a virus derived from each clinical sample was determined by the subtypes of the ha and na reference sequences. a consensus sequence was built using nanopolish v . . ( , ) and the margin_cons.py script ( ) (https://github.com/zibraproject/zika-pipeline). for the illumina data, reads were quality trimmed to a minimum score of q across the read with trimmomatic ( ) . bwa-mem v . . ( ) was used to align the reads to reference genomes using mem defaults. samtools v . was used to compute the percent reads mapped and coverage depth ( ) . mapping consensuses for illumina sequencing were generated using quasibam ( ) . maximum likelihood phylogeny was generated for the ha gene segment using raxml v . . ( ), in which a general time-reversible model of nucleotide substitution and a gamma-distributed rate variation among sites were applied. sequence alignments were performed by using muscle v . ( ) . ferret study. we applied our sequencing approach to residual samples collected in a previous time course experiment undertaken in a controlled laboratory environment ( ) . we tested ferret nasal saline wash samples from three independent animals over an -day time course, from days prior to first exposure with influenza h n pdm virus and at days , , , and postinfection. sampling and plaque assays of the viral titer were described previously ( ) . ethics approval. the study of anonymized discarded clinical samples was approved by the london-queen square research ethics committee ( /lo/ ). ferret samples were residual samples from an existing study ( ) for which the project license was reviewed by the local animal welfare and ethics review board of public health england (porton) and subsequently granted by the home office. data availability. following the removal of human reads, our sequence data have been uploaded to the european bioinformatics institute (https://www.ebi.ac.uk/) under bioproject number prjeb . method optimization to increase the proportion of viral reads derived from throat swabs. our method protocol is shown in fig. a . we first sequenced five influenza a virus-positive and five influenza virus-negative throat swabs, each spiked with hazara virus control at genome copies/ml. using a sequence-independent single-primer amplification (sispa) approach ( ), followed by nanopore sequencing, we produced metagenomic data dominated by reads that were bacterial in origin, with extremely few viral reads detected. passing the sample through a . -m filter prior to nucleic acid extraction increased the detection of viral reads by several orders of magnitude (fig. s ). filtration is relatively expensive, so we also assessed the alternative approach of adding a rapid-centrifugation step to pellet bacterial and human cells, followed by nucleic acid extraction from the supernatant. we used a pooled set of influenza a virus-positive samples (concentration, genome copies/ml) to provide a large enough sample to assess reproducibility, with the hazara virus control spiked in at genome copies/ml. enrichment for influenza virus and hazara virus was similar for filtration versus centrifugation, based on read mapping to the viral genome (fig. s ). as centrifugation is simpler and less expensive, we selected this approach for all further testing. method optimization to reduce time for cdna synthesis. synthesis of tagged randomly primed cdna and its subsequent amplification via sispa ( ) required lengthy reverse transcription and pcr steps ( h and h min), respectively. optimizing these stages upgraded the reverse transcriptase from superscript iii to superscript iv (thermo fisher), reduced the incubation time to min (processing time reduction, min), and reduced the pcr extension time within each cycle from min to min ( h min processing time reduction). comparing this final method with our original protocol, using triplicate extractions from the pooled set of influenza a virus-positive samples demonstrated no significant loss in performance in the more rapid protocol (fig. s ) , and we adopted this approach as our routine protocol, giving a wet-lab processing time of ϳ h. consistent retrieval of hazara virus by nanopore sequencing. starting with an influenza a virus-positive sample pool ( genome copies/ml), we made three volumetric dilution series using three independent influenza virus-negative pools (fig. b) . the total quantity of cdna after preparation for sequencing was consistently higher in all samples using negative pool as the diluent ( fig. a) , indicating the presence of a higher concentration of nonviral rna within pool . this is likely due to host cell lysis or higher bacterial presence and demonstrates the variable nature of throat swab samples. we consistently retrieved hazara virus reads from all three dilution series by nanopore sequencing, independently of influenza virus titer in the sample (fig. b) . sequencing from dilution series and gave a consistent proportion of total reads mapping to the hazara virus genome, across dilutions and between the first two pools, with mean Ϯ standard deviation values per pool of . ϫ Ϯ reads per million (rpm) of total reads and . ϫ Ϯ rpm, respectively. the pool dilution series generated Ϯ rpm hazara virus reads across samples and showed a decreasing trend associated with increased dilution factor as increasingly more nonviral rna was introduced from this high-background pool. limit of influenza virus detection by nanopore sequencing from pooled samples. nanopore sequencing of the triplicate sispa preparations of the influenza a virus-positive pool produced mean Ϯ standard deviation of . ϫ Ϯ . ϫ rpm mapping to the influenza a virus genome (fig. b) . across the dilution series, the proportion of influenza virus reads was strongly associated with influenza virus titer (p value ϭ . ϫ Ϫ ) but was also influenced by which negative pool was used for dilution, consistent with the pattern observed for the hazara virus control. sequencing the negative controls (pools with no influenza virus spike) generated no reads mapping to influenza virus. at influenza virus titers of Ͻ copies/ml, influenza virus reads were inconsistently detected across the samples (fig. b) , suggesting that the limit of detection is between and influenza virus copies/ml. retrieval and reconstruction of complete influenza virus genomes from pooled/spiked samples. for the hazara virus control ( genome copies/ml spike), genome coverage was . to . % (at ϫ depth) for pools and . coverage in the high-background pool was more varied ( . to . %; fig. a ). influenza a virus genome coverage at copies/ml was Ն . % for each segment in all samples (fig. a) . at genome copies/ml of influenza virus, a mean ϫ coverage per segment was . % for pools and but was substantially reduced in the high-background pool to . % (fig. a) . at influenza virus titers of Ͻ copies/ml, coverage was highly varied across genome segments. however, when present at copies/ml, / pools had sufficient data for correct subtyping as h n (table ) . having demonstrated our ability to retrieve influenza virus sequences from pooled influenza virus-positive material diluted with negative samples, we next applied our methods to individual anonymized clinical samples, with samples testing influenza virus positive and samples testing influenza virus negative in the clinical diagnostic laboratory. data yield varied between flow cells (range, . ϫ to . ϫ reads from up to multiplexed samples). within flow cells, barcode performance was inconsistent when using a stringent, dual-barcode, demultiplexing method ( ) . from each clinical sample, the range of total reads generated was . ϫ to . ϫ (median, . ϫ reads) (table s ) . reads mapping to either the influenza a or b virus genome were present in all samples with a c t of Ͻ (range, to , reads). at a c t of Ͼ , / samples generated influenza virus reads (range, to , reads) (difference between sensitivity at a c t threshold of , p Ͻ . ; fig. ). the highest c t value at which any influenza virus reads were detected was . (sample ; reads of influenza a virus). no reads classified as influenza virus were obtained from sequencing the genexpert assay-negative samples (table s ). based on this small data set, sensitivity is % and specificity is % ( % ci, to % and to %, respectively). there was a strong correlation between c t value and both the reads per sample classified as influenza virus (r ϭ . ) and the number of influenza virus reads per million reads (r ϭ . ) (fig. ) . the consensus genome sequences generated (at ϫ minimum depth) covered over % of the influenza virus genome for samples, with another two generating over % coverage. the highest c t value of a sample from which Ͼ % of an influenza virus genome sequence was generated was . (fig. s ) . (table s ). four ( %) of samples generated no detectable hazara virus reads, two with high numbers of influenza virus reads (for sample , c t of . and . ϫ influenza b virus reads, and for sample , c t of . and . ϫ influenza a virus reads) acting to dilute the control signal. the other two samples contained no detectable influenza virus reads (for sample , c t of . , and for sample , influenza virus negative). the lack of control detection therefore indicates a loss of assay sensitivity due to high levels of background nucleic acid present in some samples. comparison of nanopore and illumina sequencing. we selected a subset of samples from across the viral titer range and resequenced on an illumina miseq platform. the proportions of reads generated that mapped to the influenza virus genome were similar between the two sequencing technologies (fig. s ) . from of the samples, nearly complete genomes were obtained. a comparison of consensus sequences derived from nanopore and illumina sequencing showed % concordance, except one sample that showed nucleotide differences (identity, . %) (table s ) . influenza virus phylogeny. we reconstructed the phylogeny using consensus sequences for the ha gene (fig. ) . this demonstrates closely related sequences, as expected within one geographic setting in a single influenza season. detection of other rna viruses in clinical samples. within the clinical samples sequenced, we found limited evidence for the presence of other rna viruses. sample produced reads mapping to human coronavirus in addition to Ͼ . ϫ influenza a virus reads, suggesting coinfection. we also derived Ͼ . ϫ reads from human metapneumovirus from an influenza virus-negative sample, providing a nearly complete genome ( . % coverage) from one sample (fig. s , sample i), further detailed previously ( ) . animal time course study. finally, we used samples collected from a previous animal experiment ( ) to test the reproducibility of our methods across a time course model of influenza a virus infection (three ferrets swabbed preinfection [day Ϫ ] and then sampled at days , , , and following laboratory infection with influenza a virus). the proportion of viral reads present at each time point was highly congruent with viral titer (titer is shown in fig. a and sequencing reads in fig. b ). we generated consensus genome sequences from nanopore data at days , , and postinfection; these were % concordant with illumina-derived consensus sequences from the same cdna (table s ) . to our knowledge, this is the first report of successfully applying metagenomic nanopore sequencing directly to respiratory samples to detect influenza virus and generate influenza virus sequences. the approach demonstrates excellent specificity. sensitivity varies by viral titer but is comparable to that of existing laboratory diagnostic tests for c t values of Ͻ . our optimized protocol depletes human and bacterial nucleic acids and reduces the time from sample to sequence. this method has the potential to be further optimized and validated to improve sensitivity for influenza virus, identify other rna viruses, detect drug resistance mutations, and provide insights into quasispecies diversity ( , ) . at a population level, these sequence-based diagnostic data can, in addition, provide phyloepidemiological reconstruction, insights into transmission events, the potential to estimate vaccine efficacy ( ) , and approaches for public health intervention ( ) . whole-genome viral sequencing, coupled with phylogenetic analysis and appropriate clinical metadata, can contribute to the accurate tracking of outbreaks across time and space ( ) . the metagenomic method employed here produced Ͼ % complete genomes for / samples with a c t value of Յ (fig. s ) , demonstrating the ability of metagenomics to produce sufficient data for influenza virus diagnostics and genome characterization, while also detecting and sequencing other common rna viruses. despite time reductions in wet-laboratory processing, this method requires further modification to simplify and accelerate the protocol if it is to become viable as a near-to-patient test. high error rates are a recognized concern in nanopore sequence data, and cross-barcode contamination can create challenges when low-and high-titer samples are batched ( ) . to avoid these problems, we batched samples according to c t value and applied stringent barcode demultiplexing criteria; however, this reduces the total data available for analysis, typically by ϳ % but with variation between sequencing runs ( ) . for future primary diagnostic use, it would be preferable to sequence samples individually using a lower-throughput flow cell, e.g., ont flongle (each paired with a negative-extraction-control sample, for which a prior spike with hazara virus, using the same methods described here, would remain appropriate). careful optimization of laboratory and bioinformatic methods is required to resolve individual sequence polymorphisms, particularly for drug resistance alleles. infectious diseases society of america (idsa) guidelines ( ) recommend nasal/ nasopharyngeal specimens for influenza diagnosis, but throat swabs are easier to collect in clinical practice and therefore account for the majority of diagnostic samples processed by our clinical microbiology laboratory. further work is needed to investigate the sensitivity and specificity of our protocol for a wider array of respiratory sample types (also including bronchoalveolar lavage fluid, sputum, and saliva), which may contain different degrees of contaminating bacterial and/or human reads. loss of assay sensitivity due to the presence of high-level background dna from either the host or bacterial origin is a fundamental issue for metagenomic approaches, even in cell-free sample types such as cerebrospinal fluid ( ) . this challenge is exacerbated in throat swabs, as seen in our data. our use of hazara virus as an internal positive control allows us to identify those samples in which sensitivity has dropped to Ͻ viral genome copies per ml. in our test set, % of samples showed insufficient sensitivity for hazara virus; however, half of these contained a high titer of influenza virus, so only % were true sensitivity failures. this figure is in line with the reported % failure rate due to high background for rna virus detection from a clinically validated metagenomic sequencing assay for pathogen detection in cerebrospinal fluid ( ) . at the higher c t values in our clinical samples (c t , to ), the sensitivity of nanopore sequencing was reduced compared to that of the current pcr-based test (genexpert assay; cepheid). further optimization will be required to maximize the diagnostic yield from this group of samples without sacrificing specificity. the correlation between c t value and nanopore reads confirms semiquantitative output. using samples from the ferret influenza virus model, collected under standardized laboratory conditions, we demonstrated excellent reproducibility of viral read proportions at a given viral titer across biological replicates. however, we observed heterogeneity in output between clinical samples as well as between nanopore flow cells, suggesting that the current platform is not yet sufficiently reliable for reproducibly generating quantitative data. in addition, the detection of positive controls can be impaired in high-background samples. future application of this method will involve real-time laboratory testing of respiratory samples, running the platform head to head with existing clinical diagnostics to further assess sensitivity and specificity, and using influenza virus sequence data to investigate transmission events. identifying instances of nosocomial transmission may shed light on health care-acquired infection, thus helping to improve infection control practice. assessment of diversity within deep-sequence data sets provides an opportunity to investigate the relationship between within-host polymorphisms and clinical outcomes. long-read sequences confer the potential advantage of identifying viral haplotypes and ascertaining the extent to which significant polymorphisms are transmitted together or independently ( ) . we have shown that the method is robust for the identification of commonly circulating influenza virus strains in human populations, but further investigation is required to ascertain the extent to which it performs reliably in other (avian and animal) strains. comparison with existing/alternative approaches. the current standard assay for influenza diagnosis employed within the large tertiary referral teaching hospital in which this study was performed is the genexpert assay (cepheid), which detects influenza a and b viruses and respiratory syncytial virus (rsv). wider testing is performed on a subset of samples using the biofire filmarray respiratory panel (biomérieux), targeted at common respiratory pathogens ( viruses and bacteria). these assays have the advantages over a metagenomic approach of higher sensitivity, shorter handling times, simpler laboratory workflow, and very rapid time to result ( and min, respectively). compared to a metagenomic approach, their limitations are that no sequence data informative for molecular epidemiology and drug resistance typing are generated for the target pathogens, and that the assay will only detect the small number of pathogens targeted. periodic refinement of such assays is required in the event of newly emergent pathogens or diverse strains of established pathogens leading to assay escape. this is not an issue affecting metagenomic sequencing, which has the ability to detect all rna viruses in a sequence-independent manner. at the time of undertaking this laboratory work, the materials costs of the metagenomic sequencing were ϳ£ per sample when multiplexing six samples per flow cell and purchasing flow cells together. the cost of the current influenza a/b and rsv genexpert test is ϳ£ , and the biofire rp panel costs ϳ£ per sample. the existing tests have the significant advantage of short handling times and simple processing, whereas the metagenomic sequencing requires ϳ h and skilled laboratory staff. alternative approaches to generate sequence data include amplicon-based sequencing of the influenza virus genome ( , ) . however, this approach detects only the target pathogen, requiring multiple assays or more complex multiplex primer schemes to add targets and capture diverse strains of the original target. the use of short-read illumina sequencing instead of nanopore sequencing for metagenomic sequencing of influenza virus ( ) provides the current gold standard of sequence quality and some potential cost savings per base of sequence generated. however, our data show that at a relatively modest minimum coverage depth of ϫ, nanoporegenerated consensus viral genome sequences are . to % identical to illumina sequences. of the samples compared, samples were % concordant. the few bases that differed between the technologies in a single sample appear to be clear in each case and a genuine disagreement between the long-and short-read approaches, rather than simply being due to the higher per-base error rate of nanopore per se. larger and more diverse data sets will be required to set more rigorous thresholds for base calling. currently, it is wise to bear such potential issues in mind when comparing genome sequences generated by different technological platforms. the per-read differences in base accuracy are also compensated for by the increased read length, providing further confidence in the case of individual read taxonomic assignment. consideration of relative costs must also take into account other costsaving attributes. these include the substantially lower infrastructure and startup costs of nanopore sequencing, the unique ability to interrogate sequence data as they are generated in real time, and the potential for the portable minion device to be utilized near to patient, potentially decreasing turnaround time, particularly for high-virus-load samples which may be identified within minutes. the throughput of the nanopore flow cells allows for a small number of samples to be run immediately rather than requiring samples to be batched to reach a number sufficient to cost efficiently run a greaterthroughput short-read sequencing device. in the future, the use of an even lowerthroughput flow cell with the ont flongle adaptor may allow individual samples to be run per cell, offering quicker turnaround per sample and minimizing cross-sample contamination. a summary table comparing the different approaches is included in supplemental material (table s ) . limitations of the method. the current limitations of metagenomic methods are their sensitivity in the context of low-pathogen-titer samples. pcr-based methods measure the absolute count of viral genome copies present within a sample. metagenomic sequencing measures the proportion of total rna that is viral. metagenomic sequencing is therefore affected by the level of nontarget rna within a given sample, whereas pcr is not. as demonstrated here ( fig. b and table ), detection of as little as genome copies per ml is possible from throat swab samples (a level comparable with pcr-based methods), but variation in the level of background nucleic acids between individual samples makes detection at this level inconsistent. further development of methods to deplete host and bacterial rna within the samples is required to improve the performance of the assay at c t values of Ͼ . enrichment of pathogen sequences within libraries through either target capture or amplification is also an effective method to reduce the limit of target detection ( , ) but requires the same a priori knowledge of both which pathogens are to be targeted and the full range of circulating viral diversity as other targeted methods discussed above, albeit with increased tolerance for diversity over pcr-based methods. a further limitation compared to alternative sequencing technologies is the lack of confidence in determining the presence of minority variants due to the limited per-read accuracy, although we expect this to be addressed in future iterations of the ont sequencing. in summary, while substantial further work is needed, our methods show promise for generating influenza virus sequences directly from respiratory samples. the "pathogen-agnostic" metagenomic sequencing approach offers an opportunity for simultaneous testing for a wide range of potential pathogens, providing a faster route to optimum treatment and contributing to antimicrobial stewardship. longer term, this approach has promise as a routine laboratory test, providing data to inform treatment, vaccine design and deployment, infection control policies, and surveillance. supplemental material is available online only. supplemental file , pdf file, . mb. the study was funded by the nihr oxford biomedical research centre. computation used the oxford biomedical research computing (bmrc) facility, a joint development between the wellcome centre for human genetics and the big data institute supported by health data research uk and the nihr oxford biomedical research centre. the views expressed in this publication are those of the authors and not necessarily those of the nhs, the national institute for health research, the department of health, or public health england. p.c.m. is funded by the wellcome trust (grant ). d.w.c., t.e.a.p., and a.s.w. are nihr senior investigators. the ecology and adaptive evolution of influenza a interspecies transmission the influenza of : evolutionary perspectives in a historical context origins and evolutionary genomics of the swine-origin h n influenza a epidemic the pandemic threat of emerging h and h avian influenza viruses evolutionary dynamics of avian influenza a h n virus across five waves 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outbreak the ability of single genes vs full genomes to resolve time and space in outbreak analysis laboratory validation of a clinical metagenomic sequencing assay for pathogen detection in cerebrospinal fluid whole genome sequencing of influenza a and b viruses with the minion sequencer in the clinical setting: a pilot study minion nanopore sequencing of an influenza genome a metagenomic analysis of pandemic influenza a ( h n ) infection in patients from north america enhanced virome sequencing using targeted sequence capture capturing sequence diversity in metagenomes with comprehensive and scalable probe design key: cord- -wszo s d authors: zhu, hanliang; zhang, haoqing; ni, sheng; korabečná, marie; yobas, levent; neuzil, pavel title: the vision of point-of-care pcr tests for the covid- pandemic and beyond date: - - journal: trends analyt chem doi: . /j.trac. . sha: doc_id: cord_uid: wszo s d infectious diseases, such as the most recent case of covid- , have brought the prospect of point-of-care (poc) diagnostic tests into the spotlight. a rapid, accurate, low-cost, and easy-to-use test in the field could stop epidemics before they develop into full-blown pandemics. unfortunately, despite all the advances, it still does not exist. here, we critically review the limited number of prototypes demonstrated to date that is based on a polymerase chain reaction (pcr) and has come close to fulfilling this vision. we summarize the requirements for the poc-pcr tests and then go on to discuss the pcr product-detection methods, the integration of their functional components, the potential applications, and other practical issues related to the implementation of lab-on-a-chip technologies. we conclude our review with a discussion of the latest findings on nucleic acid-based diagnosis. st century has witnessed exponential growth in science and technology; however, sadly and ironically, it has succumbed to devastating coronavirus infections: severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and now coronavirus disease . [ , ] as of july , , a total of , , covid- cases had been reported across the world, together with , deaths. [ ] the sars and mers coronaviruses were transmitted indirectly from bats to humans through civets and camels, respectively, as intermediate hosts, [ ] but the one responsible for covid - , has yet to be identified. [ ] one of the main reasons that outbreaks such as these occur is the ability of viruses to evolve rapidly and change their animal hosts; this was seen with spanish influenza, the single-largest pandemic of the last century, which was probably caused by a virus that was transmitted from ducks to domestic pigs and then to humans, in the densely populated region of southern china. [ ] the virus was eventually transmitted to the usa and europe after world war i. recombination among viruses as they change their hosts is a natural process. there is, therefore, always a strong probability of new epidemics emerging from novel types of viruses, especially in densely inhabited regions ( figure ). the emergence of a coronavirus epidemic in the southern provinces of china has been predicted for some time, given the presence of multiple bat species that are infected with many different coronaviruses, in these highly populated areas. [ ] coronaviruses and influenza viruses tend to be diagnosed through the detection of their specific genomes, in particular their ribonucleic acid (rna) sequences. rapid and precise screening of the public is essential to identify and quarantine infected people, thereby limiting or potentially eliminating the spread of the virus. the viral load of coronaviruses is high, making it easily detectable in symptomatic patients. unfortunately, there is also a high percentage of asymptomatic individuals, making an accurate disease diagnosis of the population a challenging job unless the entire population is tested. [ ] a low level of limit-of-detection (lod) is crucial to shift the diagnostic window of opportunity toward the start of the infection process, to detect newly infected individuals. the test can be based on immunoassays, using antibodies to detect a specific antigen produced by the body's immune system or polymerase chain reactions (pcr) to detect a viral genome sequence. the pcr technically, once a disease emerges, researchers identify the infectious agent by sequencing its genome, after which the newly obtained sequence is compared against sequences in the databases to evaluate its similarity to other known viruses. determination of a sequence that is truly unique to the newly characterized virus represents a crucial step in the development of an effective diagnostic procedure. furthermore, such a sequence should be selected from a region of the viral genome that is not variable between its different isolates and is not expected to evolve and mutate quickly. when a suitable sequence is determined, the design and synthesis of the relevant pcr primers can be performed routinely. in the case of rna viruses, the reverse transcription that converts viral rna to cdna represents a crucial step, allowing the subsequent application of all methodologies based on pcr ( figure ). the raw sample and the reagents used in the sample preparation often contain a significant level of pcrinhibiting factors; thus, proper sample preparation-whether manual or automated-is an essential step for successful detection. [ ] the success of virus detection in real-world biological samples is highly dependent on the volume of the analyzed sample. during the initial phases of infection, the viral loads in patients may be near or below the test lod. [ ] the analysis of larger volumes of biological fluids with a low viral load is likely to reduce the risk of false-negative results, but microfluidic point-of-care (poc) devices are typically unable to handle ml-scale volumes in a short time due to the requirement of slow flow rates. multiple approaches have been developed to address this problem. flow-through capture membranes [ ] have been used to capture nucleic acids in a short period of time, allowing the direct amplification of the captured material without any elution step, detecting as few as ≈ copies of dna with flow rates as high as ≈ ml·min - . magnetic beads [ , ] were used to isolate the rna of avian influenza h n and sars from a ≈ µl sample of blood; this is an insufficient volume on which to perform reliable coronavirus diagnostics. carbon nanotubes [ ] or aptamers [ ] have been used to capture rna from influenza a and h n from avian flu viruses, respectively, and to promote their enrichment in samples. here, we look back and identify those technologies that appeared since the emergence of sars in late . it is our aim to put landmark developments under the spotlight, critically evaluate them, and raise questions to identify the gaps that are currently hindering a commercial poc-pcr test. there have also been other recent outbreaks of infectious diseases that have threatened global human health, such as ebola virus disease in the democratic republic of the congo in , measles in burundi, and yellow fever. additionally, the lower respiratory infections, tuberculosis, and human immunodeficiency virus (hiv) disease were listed among the top ten global causes of death in . [ ] the pcr test on samples prepared from blood and urine specimens is capable of detecting the virus in the early stages of the disease, resulting in early diagnosis and subsequent isolation of infected patients to block transmission. for a comprehensive snapshot of the field, we refer the reader to in-depth reviews on the topic that have appeared in recent years. [ ] [ ] [ ] figure . a conceptual demonstration of the detection of viruses in the field, based on real-time rt-pcr at poc. the process starts with a nasal swap to obtain a sample that might contain the virus. the sample is purified using paramagnetic beads and viruses are captured, followed by virus lysis to release the rna, and purification then, the rna is reverse transcribed to cdna; the number of cdna molecules is multiplied by the pcr, and the results are displayed and possibly transferred to a centralized laboratory via a mobile device. [ ] . pcr and its technology a. the principles and brief history of pcr single-step rt-pcr is a technique derived from the original pcr [ ] to perform a reverse transcription followed by the pcr temperature cycling. it requires a mixture of reverse transcriptase, dna polymerase, short oligonucleotides complementary to the cdna target, called primers, free nucleotides, and bivalent salts, such as mgcl . the rt-pcr starts with reverse transcription, typically conducted at ≈ °c, followed by a hot start performed at ≈ ˚c, destroying the reverse transcriptase and activating the polymerase. typically, there are - cycles consisting of a double stranded dna (dsdna) denaturation at °c, primer annealing at a temperature ranging between °c and °c, and extension at °c to finish the dsdna. the three temperature protocols can be simplified by a two-step temperature protocol, whereby the annealing and elongation are conducted at the same temperature. [ ] the classical endpoint detection system, which required an external pcr product detection by hybridization or electrophoresis, was modified by adding a fluorescent agent, [ , ] either intercalating dye or a fluorescent probe that converts the endpoint pcr into a real-time pcr, also known as a qpcr. pcr-based systems are, in principle, capable of detecting a single copy of rna or dna, with the assumption that the single dna/rna copy is presented in the sample and not lost during the sample preparation. the pcr/rt-pcr is a far superior technique in comparison with antibody-based assays due to the much smaller diagnostic window, i.e., it stops providing false-negative results earlier than the antibody-based assays. an alternative to pcr is a ligase chain reaction with short templates, whereby the products are only one base pair longer than the combined length of both the primers. [ ] b. other techniques based on the amplification of nucleic acids a group of new techniques, called isothermal amplification of nucleic acids, has been introduced with loopmediated isothermal amplification (lamp) [ ] and nucleic acid sequence-based amplification (nasba) [ ] as its main representative, with an important aim of bypassing pcr-related patents. papers that have described these techniques start with the claim that lamp is far superior to pcr because complicated temperature cycling is not required for isothermal amplification. however, temperature cycling is a very simple engineering method that helps to improve the pcr specificity. proportional-integral-derivative controllers that include temperature sensors and solid-state relays are cheap and readily available. in addition to that, lamp still needs a closed-feedback loop system heater to maintain the system's temperature in a range of °c to °c. both real-time pcr and real-time lamp require a fluorescence detection system, and due to the rather weak fluorescent output signal the fluorescent signal related electronic processing circuits is far more demanding than those for temperature control. although there is no requirement for temperature cycling, lamp is a complicated process. given that nothing comes for free, a simple lamp requires six primers, but a pcr needs only two; thus, it does not seem to be correct to assume the simplicity of lamp. nevertheless, the lamp system has its merits, which include the fact that thermal cycling is not required, resulting in better thermal isolation and, therefore, less power consumption compared with pcr. also, lamp can be designed to simplify the readout by colorimetry or nephelometry, instead of conventional fluorescence detection. [ ] there are other isothermal nucleic acid amplification techniques using different principles and temperatures, such as strand displacement amplification, [ ] rolling circle amplification, [ ] nicking enzyme amplification reaction, [ ] recombinase polymerase amplification, [ ] and catalytic hairpin amplification. [ ] c. microfluidics and pcr miniaturization following in the footsteps of microelectromechanical systems (mems), the field of microfluidics-or lab-on-achip-emerged in the early s, with the grand vision of producing a micro-total-analysis system (μtas). [ ] two analytical techniques pioneered the field: capillary electrophoresis (ce) [ ] and pcr. [ ] the first demonstrations of both techniques independently on the microchip format led to the pcr-ce integration [ ] later on by implementation of a real-time pcr to a portable unit for a poc-pcr. [ ] since then, important innovations have taken place in terms of materials, [ ] as well as the method of implementation, [ ] extending the boundaries of this field. however, nearly three decades on, these efforts have yet to bear fruit in daily life outside the laboratory. one cannot help but wonder: why has the development and deployment of practical poc-pcr testing taken so much time? this is a pressing question, especially at a dire time like the present, when the world is battling another once-in-a-century pandemic, covid- . [ ] this is the raison d'être of μtas. this background should not be taken as a criticism of the slow development because the task of making portable systems for viral diagnostics with minimal false-positive and -negative results is complex, and great progress has indeed been made in recent years. pcr runs on a platform which cycles the temperature of the reaction between two or three set points for over to cycles. the reaction time, is often limited by heating and cooling rate within each cycle determined by power dissipation (p) and system thermal time constant (τ). it is reduced with a reduced thermal mass (inclusive of the sample and the chamber) and with decreased isolation of this thermal mass from surrounding. both features can be fulfilled by miniaturization, through mems technology and microfluidics, which has lowered the reaction time from > min to several min in the past decade [ ] or even less [ ] as: where Δt is the time taken by the system to change the temperature from one pcr step to another (Δt), and c is its thermal capacitance. the total reaction time depends on a series of parameters, such as the chip size, the pcr master mix volume determining the value of c, the thermal conductivity of the substrate (g), and the temperature cycling rate. here, the miniaturization of the pcr results in a smaller value of c, which proportionally lowers the ∆t. recently, an extremely fast pcr with a . s·cycle - has been demonstrated and resulted in an incredible total reaction time of less than s. [ ] this fast pcr could be combined with a fast melting curve analysis to achieve a total detection time of less than min. [ ] the pcr can also utilize passive cooling, with a cooling rate determined by its τ value: = . ( ) increasing the value of g leads, ultimately, to a faster system, unfortunately, the cooling-rate increase comes with a cost, making the system power demanding as: and power demanding systems are not suitable for battery-operated poc devices. nevertheless, the system heating/cooling rate can still be increased without compromising the power increase by minimizing the sample volume. isothermal amplification systems benefit from the absence of a cooling requirement, making the τ value irrelevant. then, there is an option to thermally isolate the heated part with a g value as low as possible to consume very little energy-a valuable feature of portable, battery-operated systems. poc-pcr requires fast thermal cycling and precise temperature control. there are demanding requirements for the design of temperature modules for miniaturized pcr devices. heating methods determine the temperature ramping rate and are currently divided into contact and noncontact heating. the contact heating method utilizes embedded/external heat sources, such as deposited thin films or external peltier elements; [ ] this method increases thermal mass, inevitably hindering fast thermal transitions during reactions. the non-contact heating method was developed using hot air [ ] or infrared radiation as a heat source and directly heats the sample in small volumes. [ ] these contact and non-contact heating methods employed for microchips depend on the material of the substrate, the reaction volume, and the structure of the chip. thermal isolation must also be considered in a miniaturized pcr system because thermal cross-talk deteriorates the chip's performance, especially in multifunctional reaction chambers on a single chip. [ ] furthermore, poor thermal isolation results in heat loss from the temperature zones to the surroundings, while the suspended pcr chamber enables good thermal isolation. a hollow reaction chamber has been made out of micromachined silicon (si) that is integrated with both the heater and the sensor and is connected by a narrow beam to the substrate to achieve thermal isolation. [ ] in addition, isolated layers with high thermal resistance have been applied to provide excellent thermal isolation; [ ] however, they result in more complex fabrication and additional stress in different layers. materials with low thermal conductivity, such as glass or plastics, could provide excellent thermal isolation for pcr chips, thereby avoiding the temperature cycling of the entire device. [ ] . materials the implementation of microfluidics has led to the development of miniaturized pcr systems that offer portability and save time, thereby facilitating applications in the realm of poc diagnostics. researchers have developed a family of miniaturized devices based on different types of pcr. materials such as si, [ ] glass, [ ] polydimethylsiloxane (pdms), [ ] polycarbonate, [ ] and polymethylmethacrylate (pmma), [ ] used for fabricating microfluidic chips, are also available for the substrate of pcr chips. each material has a number of merits and demerits, based on its individual properties. glass is used to make pcr chips due to its transparency and low auto-fluorescence. a multifunctional platform suitable for the on-chip detection of biomolecules consists of different thin films integrated on a single glass substrate that can also be optically and thermally coupled with another glass. [ ] however, the low thermal conductivity of glass leads to an uneven temperature distribution and slow heating/cooling rates. pdms is currently one of the most widely used polymers for microfluidic devices due to its biocompatibility, low cost, and simple processing. [ ] a disposable pdms-glass bonding chamber has been utilized to perform pcr with an intercalating reusable electrode part for thermal cycling. [ ] however, the evaporation of the pcr solution, located in the pdms chamber, at a denaturation step may lead to cross-talk because pdms is porous. pmma is another popular candidate. [ ] inexpensive and versatile pmma pcr chips can be fabricated by laser ablation technology, followed by bonding methods such as low-temperature bonding that uses optically clear adhesive film and liquid optically clear adhesive. [ ] additionally, the nonspecific adsorption between pmma and dna is minimal. the surface properties of microfluidic channels/chambers must be considered because the interaction between biomolecules in the pcr solution and the channels/chambers affects pcr efficiency. a high surfaceto-volume ratio of microchannels/chambers leads to the non-specific adsorption of the enzymes, limiting or even prohibiting the reaction. [ ] two methods have been employed to solve this problem. one involves the use of silanization to modify the surface permanently. silanization is accomplished by filling the channel with a silanizing agent and heating the filled chip for a period of time, then removing the agent and washing the chip. alternatively, solutions such as bovine serum albumin (bsa), [ ] polyvinylpyrrolidone, [ ] glycerol, [ ] polyethylene glycol , [ ] and tween [ ] are commonly added into the pcr solution to reduce the nonspecific adsorption, thereby improving the pcr efficiency. chips made of si and glass can be cleaned using solutions such as piranha (h so /h o ), since the solution performs the mineralization of organic materials, and no rna/dna can be left behind. nevertheless, general practitioners prefer disposable systems because they are safer in terms of cross-contamination. in the future, pcr chips could well be able to be cleaned and reused, once the cleaning process has shown its consistency. the first conventional thermal cycler, called mr. cycle, was a time-domain system, as are most current pcr devices. samples are placed at fixed locations on a heater whose temperature is modulated according to the required pcr protocol. the heaters can be based on dissipating joule heat with active heating and passive cooling, or by utilizing thermoelectric coolers with active heating, as well as cooling. the pcr master mix is dispensed into a stationary location, such as droplets or microwells, and microheaters are then used to conduct the thermal cycling beneath the droplets or wells. there are several techniques that can be employed to split samples to form droplets. a few µl of pcr master mix is pipetted onto a hydrophobic/oleophobic glass coverslip and covered by mineral oil, forming a virtual reaction chamber (vrc). [ ] alternatively, the droplets, including cells separated by mineral oil, form an emulsion and are loaded into the microfluidic channel and are then transferred to different positions within the chip, after which they are subjected to a series of procedures such as cell lysis, dna extraction, and purification. [ ] then, the thermal cycling is conducted by heating this emulsion or vrc, and cameras capture the subsequent fluorescence. a variety of chip structures are fabricated by micromachining with different wells or microfluidic channels in materials such as si, glass, and polymers, typically pdms or pmma. samples are loaded into either wells or channels for subsequent dna amplification by pcr. [ ] ii. space domain the pcr master mix is pumped into a channel or chamber in the chip, typically with two/three zones maintained at constant temperatures. [ ] the reaction is performed when the solution is passing through these temperature areas by denaturation, annealing, and extension. this is an old technique that was used in the early days of pcr systems, with three baths with different temperatures and a laboratory technician or robot to move a basket with samples from bath to bath. currently, this technique is still utilized in miniaturized systems, such as flow-through and rotational types. the flow-through method is a typical space-domain pcr method. the solution is pumped into a microfluidic channel made of polymers with different temperature regions, and a number of microheaters are applied to provide different temperature zones for denaturation, annealing, and elongation when performing the pcr. these zones maintain constant temperatures, and only the temperature of the samples is changed while it is moved through the zones with different temperatures. [ ] iii. integration with sample preparation the nucleic acids to be detected are first isolated from the bacteria or viruses using technologies such as lysis, pre-concentration, and purification. these procedures can be fulfilled on-chip or off-chip using bench-top instruments. a number of fully integrated systems have so far been developed, starting with the non-portable genexpert from cepheid, which was first utilized for anthrax detection by the united states postal service, [ ] and then with different primers to perform hiv and tuberculosis diagnostics in south africa. [ ] despite being nonportable, it is a true sample-to-answer system that uses injection-molded plastic preloaded cartridges with lyophilized compounds, including a pcr master mix, as well as infectious agent-specific primers. their prohibitive capital cost has unfortunately restricted the spread of these systems, given that those employed in south africa were heavily subsidized. we demonstrated the first monolithic si-based chip integrating a micromixer for diluting blood sample, a microfilter for isolating and chemical lysing of leukocytes, a binder for capturing and purifying nucleic acids, and microvalves for fluidic control. [ , ] the captured dna was amplified in a pcr chip followed by the product detection using si nanowires. [ ] a large sample volume to be processed needed to be stored in a plastic cartridge next to the si chip, and the si chip experienced processing problems due to its small channel cross section. the first step is to filter out red blood cells, while effectively capturing white blood cells for subsequent lysis. the dna-capturing system was based on a sio surface inside the chip, was orders of magnitude smaller than the one based on microfiber membranes or silica beads. finally, the use of si nanowires as dna sensors offers somewhat questionable repeatability. the replacement of a conventional pcr with a qpcr would make life easier because there would be no sample manipulation after pcr, and qpcr is well established technique. nevertheless, the system had limited portability. standard pcr (end-point) typically utilizes electrophoresis of either a gel or capillary type to detect pcr products after thermal cycling. a pcr master mix is pumped into a microchannel fabricated in a microfluidic chip, and pcr is performed using microheaters. after the reaction, the solution is taken out, and gel electrophoresis (ge) is conducted to check the presence and specificity of the amplicons. [ ] alternatively, the solution is transferred to a ce column on a chip with electrodes on both sides, and the products are detected by ce. [ ] detection by ge/ce made the chip or system design complicated, hindering the system miniaturization for poc diagnostics. inspired by commercial real-time pcr instruments, optical detection modules, such as optical fibers [ ] and lock-in amplifiers, [ ] were then integrated into miniaturized qpcr systems while intercalating dyes such as sybr green or probe were added into the pcr master mix, and the reaction was monitored in real time. furthermore, additional types of qpcr systems combined with other technologies have been developed to improve performance. a pico-liter droplet array generated by double-inkjet printing was demonstrated to be capable of performing parallel qpcr with an enhanced throughput. [ ] a complementary metal oxide semiconductor was integrated with the pcr chip used for multiplexing detection, which enhanced detection sensitivity. [ ] electrowetting on dielectric technology is well adapted for single-cell isolation, mrna purification, and subsequent multiplex qpcr at the single-cell level. [ ] . the vision and the reality the miniaturized pcr was first envisioned in the early s, and since then numerous pcr microchips have emerged. most of these pcr microchips have required off-chip sample preparation and even off-chip detection. these undesired features hinder complete freedom from laboratory settings and give rise to the tongue-in-cheek expression for these devices: chip-in-a-lab. [ ] the sample preparation, which involves the extraction and purification of nucleic acids from bodily fluids, plays a determining role in the accuracy of the test, which heavily depends on the effective removal of pcr inhibitors such as hemoglobin. however, the diversity and complexity of bodily fluid samples, together with the sample volume requirement, make it difficult to combine sample preparation with pcr on a microchip or in a cartridge. few studies have undertaken the challenge of demonstrating a fully integrated pcr system, where sample preparation, amplification, and detection occur in one platform. in this section, we describe these integrated solutions and the issues preventing their poc use. the first of these was arguably a unit reported ≈ years ago based on the esensor microarray chip ( figure a ). [ ] the esensor was a printed circuit board featuring a thin-film metal electrode array ( × array), with dna capture probes to hybridize the pcr products for multiplexed electrochemical detection. the overall unit was a fully integrated system made up of pumps, valves, and fluidic micromixers, all made free from any fabricated moving parts and based on thermopneumatic expansion and electrolysis for pumping, cavitation microstreaming for mixing, and phase transition for fluidic valving. eliminating such parts simplified not only the design but also the fabrication, thus lowering the overall cost. the cost was further reduced by patterning the electrodes through the standard printed circuit board (pcb) process and forming the polycarbonate channels and chambers through computerized numerical control-machining. cavitation mixing was induced by a pair of piezoelectric disks mounted on the sample chamber and the microarray detection chamber, while electrolytic pumping was activated by platinum wires inserted into the pumping chambers. a magnet was mounted on the pcr chamber to trap and enrich the immunomagnetic capture beads that carried target pathogens or cells from the sample chamber for subsequent thermal lysis and asymmetric pcr. the unit was shown to work for pathogenic bacteria detection from whole rabbit blood by spiking it with escherichia coli, after which a single nucleotide polymorphism analysis was performed. despite the device's simple fabrication process, it entailed post-fabrication steps, such as introducing phase-changing material wax into the valves and conditioning the channel's surface. despite the claim that it is completely self-contained in terms of reagents and waste storage, the reagents were loaded at the time of sample introduction, which added to the in-use handling. other technical issues included the low efficiency of the pcr due to the immunomagnetic beads (≈ %) and their low cell-capture efficiency of ≈ %, as well as the low sensitivity of electrochemical detection. furthermore, the overall assay time was relatively long as sample preparation, amplification, and detection took ≈ h each. a biochip featuring an integrated pcr with solid-phase extraction (spe) and ce detection has been proposed and tested. [ ] bacillus anthraces (anthrax) and bordetella pertussis have been detected within ≈ min,. while this is impressive, the biochip was not a self-contained unit with reagents and waste storage. reagents had to be externally supplied during each test and the sample had to be lysed off-chip. both these steps have to be integrated when providing a biochip or a cartridge for a portable diagnostic system. the possible inhibition of pcr by reagent traces of spe was avoided through the integration of membrane valves that decoupled the two process sites. the valves, however, involved multiple structural layers, adding further complexity to the fabrication. moreover, the valve activation required compressed air from an external line, which increased the hardware requirement, in addition to the infrared mediated thermal cycling, high-voltage product separation, and laser-induced fluorescence detection. the spe site had to be packed with silica beads, which added to the post-fabrication handling. despite all these technical issues, however, the study demonstrates the feasibility of a highly rapid sample-in answer-out capability. a portable and affordable real-time pcr system has been developed. the core of the system is a micromachined si chip integrated with a thin-film metal heater and a resistive temperature detector type of sensor. [ ] the basic philosophy the researchers had in mind was to develop a cheap system with disposable parts in contact with the sample to avoid sample-to-sample cross-contamination, and, therefore, the mems chip was separated from the sample by a disposable microscope coverslip. the sample was placed together with a volume of a few μl, covered with mineral oil to prevent evaporation, forming a vrc. the first device was rather slow with sample and mineral oil volumes of µl and µl, respectively. the temperature was controlled externally via a personal computer, and the fluorescence was captured by an external microscope. separate to the pcr system, the same group also developed a miniaturized fluorescent system with the vision of integrating it later on with the pcr system. [ ] then, the mems system was redesigned, and the sample volume was lowered to increase the speed to perform pcr cycles in less than min, [ ] after which everything was integrated into a single system, demonstrating real-time rt-pcr detection in a sample containing the rna of the h n avian flu virus in a single vrc-which is clearly insufficient for actual testing. [ ] the system was evolved into a world-smallest real-time pcr capable of detecting four samples at a time and demonstrated the ability to perform real-time pcr of cdna from the h n avian influenza virus, [ ] rt-pcr from the ebola virus rna, [ ] and finally, it was integrated with a bluetooth communication system that detected cdna from the dengue fever virus. [ ] the system was even combined with the sample preparation step to detect rna from the h n avian flu [ ] or sars, [ ] although they were far from perfect and required further improvement. furthermore, the vrc looked like an open system, but the system should be converted into a closed system to increase user comfort and avoid the possibility of accidental contamination. a sample-to-answer system that runs an automated sample preparation and real-time rt-pcr in an all-in-one cartridge has been developed and tested for influenza (h n ), achieving an lod of copies·ml - . [ ] the cartridge was a three-dimensional acrylic block with chambers that extended between the top and bottom surfaces and featured millimeter-sized interconnecting channels ( figure b) . one chamber contained a silica membrane for nucleic acid adsorption, while another was designated for waste storage. all the remaining chambers were preloaded with liquid reagents, and the cartridge was completely sealed to reduce the risk of viral exposure or sample-to-sample cross-contamination. a nasopharyngeal swab sample, diluted in viral transport media, was injected, together with a lysis buffer, into the silica membrane chamber from a syringe equipped with a needle by punching the seal. the sample and reagent liquids were pneumatically manipulated within the cartridge under pressure, and a vacuum was applied from syringe pumps through the seal-piercing connectors. further fluidic control was provided by built-in surface tension valves. the amplification took place in three separate vials, each containing a frozen pcr master mix, which were inserted into the cartridge prior to the test. all the hardware was built in a desktop system that completed a fully automated test within . h. the test duration could be further shortened through miniaturization, which could also minimize the large dead volume introduced by the commercial silica membrane, as well as avoid diluting the viral rna due to a large eluent volume for elution consistency. moreover, the absence of mixing strategies implemented within the pcr vials led to a slightly lower pcr efficiency. otherwise, the cartridge appears to be simple and amenable to injection molding for mass dissemination. the fully automated sample-to-answer detection of influenza type h n was based on a centrifugal microfluidic disk [ ] named the labdisk ( figure c ). the disk was fully enclosed with preloaded reagents comprising liquid stick packs for nucleic acid extraction, air-dried specific pcr primers with fluorescence probes, and a lyophilized rt-pcr master mix. after loading the sample, the disk started to rotate creating a centrifugal force, triggering a series of events. first, the content of each stick pack was released into a designated chamber at a well-defined number of revolutions per min, breaking the fragile seals of the stick packs. then, the silica-coated magnetic beads captured and transported the nucleic acids from the lysis chamber into subsequent chambers for washing and release, with the aid of an external stationary magnet. the subsequent eluent was aliquoted into eight reaction cavities, dissolving the lyophilized rt-pcr master mix before thermal cycling through convective heating. the entire test took less than ≈ . h, with an lod of ≈ . × of viral rna copies per ml of sample. all the hardware was packed into a lightweight shoebox-sized unit suitable for poc use, with the only manual step being the sample loading. the centrifugal pumping, together with the surface tension valving, simplified the overall design, allowing the disk to be fabricated by microthermoforming a polymer foil via hot embossing. the thermoformed foil was then sealed by a pressuresensitive adhesive tape. although the lod shown is fourfold lower than the clinically relevant level, the disk needs to be further developed to process viscous clinical specimens, such as sputum, through sample liquefaction and homogenization. a further reduction in analysis time and a demonstration of the ability to conduct multiplexing for sub-typing viral strains would be likely to bring the disk closer to practical poc use. the detection of the hepatitis c virus (hcv) in serum was demonstrated using a system based on a thermoformed plastic cartridge [ ] where the reagents were confined into discrete wells isolated from each other by a layer of oil. the first well contained an acidic binding buffer that induced a positive surface charge on the ph-responsive magnetic particles mixed into a serum sample before being introduced. accordingly, the nucleic acids were captured by the charged particles and transported from well to well as the cartridge went through a series of translations and rotations between a pair of opposing stationary magnets ( figure d ). the surface charge was neutralized by an alkaline pcr solution, and the nucleic acid release was triggered. following the removal of the particles from the well, rapid thermal cycling took place, administered by the surrounding heat block through the thermoformed thin layer of plastic. a fluorescent signal was captured via a confocal detector placed above the well. more than a dozen clinical serum samples were tested, each analyzed within an hour and generating lods as low as iu per µl. while these results are encouraging, the unit gave rise to some of the same concerns as the others. first, the rt-pcr enzymes required refrigeration; this issue might be difficult to address by keeping the enzymes lyophilized because the pcr solution itself served as the rna eluate necessary for rehydration. second, the unit analyzed blood serum, which must be extracted beforehand, while the unit must be evaluated for other bodily samples relevant to respiratory infections such as sputum or a nasopharyngeal swab. third, the dynamic range should match the entire range of viral loads encountered clinically. fourth, the unit faced the issue of equally allocating particles among the wells, each of which has a distinct set of primers specific to the target serotype. fifth, the greater spread of the data was an issue relevant to the retrieval of viral rna but could be addressed by increasing the eluate volume by an order of magnitude, at the expense of test sensitivity. the processes of dna purification, pcr, post-pcr clean up, and inline injection, as well as ce separation, were all combined into a single microchip for forensic human identification ( figure e ). [ ] the microchip included a miniaturized pdms pump and valves, a fluidized bed of immunomagnetic beads in bifurcating channels, a nl pcr chamber, and a capillary electrophoresis channel with a double-t junction. the dna purification was realized through a sequence-specific capture of dna by a fluidized bed of streptavidin-coated magnetic beads. this, however, required off-chip handling, fragmenting the genomic dna, and then modifying the fragments with biotin-labeled capture probes. moreover, the dna capture efficiency was rather low ( . %). an external magnet was applied to arrest the beads in the bifurcating channels and then manually displaced to transport the beads into the pcr chamber. the pcr master mix, including biotin-labeled primers, was introduced, and the pcr products were then concentrated into a narrow band within a streptavidin-modified gel plug inside the double-t junction. the thermal release of the band led the ce separation and detection. the microchip was accompanied by a small-footprint, battery-powered instrument capable of a full nineplex short tandem repeat (str) typing from . ng input dna, as well as forensic str analysis from oral swabs within h. nevertheless, the high level of integration creates complex fabrication and post-fabrication handling, including the alignment and bonding of multiple layers of glass and a pdms membrane. all reagents had to be externally supplied and the sample lysed outside the microchip. table lists the prototypes discussed above and compares their important features. the labdisk stands out as the sole cartridge that is fully enclosed with reagents and a waste storage. it can be stored at room temperature owing to the lyophilized pcr master mix. reagent evaporation and cross-mixing are addressed by the individually sealed stick packs that keep the reagents isolated. this isolation, along with the plastic housing, renders the cartridge robust against handling and transportation. the use of centrifugal microfluidics and silica-coated magnetic beads facilitates on-chip pumping, valving, and fluidic mixing that are realized in a simple design that can be produced through thermoforming. moreover, the cartridge runs on an instrument very similar to the cd or dvd players of the past. however, several challenges remain. the cartridge must be further developed to handle clinical specimens. the overall test duration should be reduced to below h. finally, all these prototypes, albeit underdeveloped and very limited in number, are encouraging. however, it is imperative that many more studies are geared towards a poc-pcr test, instead of stand-alone pcr microchips, so that the next outbreak can be contained promptly. the time is running out for the vision before it fades away into a dream and a legacy for future generations. table a comparison between the different systems currently available or under development. "cfu" stands for "colony forming unit," "c" for "copy," "hcv" for "hepatitis c virus," and "str" for "short tandem repeat," "na" for "not applicable," "ns" for "not stated," "lod" for "limit of detection", "ma" for "microarray", "ce" for "capillary electrophoresis," and "iu" for "infection unit" waste one of the most important practical challenges to be resolved for any poc molecular diagnostic device is sample preparation. ideally, it should be a sample-to-answer system with no human interaction; this is especially important for poc diagnostics, such as for sars in the past and currently for covid- . during the sars outbreak in singapore, which was one of the worst-hit countries, the tan tock seng hospital was almost the only clinic capable of carrying out routine sars diagnostics. prospective patients' only option for testing was to get there, leading to a high chance of cross-infection. it was seen to be greatly beneficial to have a system for poc to ensure that prospective patients did not have to go anywhere, but rather stay at home and call health care providers to come to their location to conduct infectious disease diagnostics, and not simply collect a sample. this would demand a small system that is easily portable, with samples sealed to avoid sample-to-sample cross-contamination, thus suppressing the number of false-negative results. sample preparation is definitely a problem, and many attempts have been made to simplify these crucial steps. there are too many pcr inhibitors in human body fluid samples, such as blood and saliva. over the years, there has been continuous research into the development of pcr reagents, and especially into methods aimed at simplifying the sample preparation steps and fully integrating the sample preparation with pcr. single-step direct pcr has also been developed, greatly simplifying the sample preparation process; only hemoglobin has to be removed before performing pcr to diagnose malaria infection. [ ] from the standpoint of practicality, the extraction and isolation of the dna/rna of pathogens from saliva are most desirable for poc applications. several studies have explored this area, including standard labor-intensive techniques. [ ] figure the systems currently available for covid- poc testing (poct). (a) a system performing reactions at a time using magnetic beads for sample preparation, followed by a lamp. [ ] (b) cobas® liat® system by roche originally designed as a universal molecular biology platform for diagnoses based on a real-time pcr. [ ] (c) id now, made by abbott laboratories, originally sought to diagnose seasonal flu; it has currently been converted into a covid- system, based on an isothermal dna amplification originally developed for the seasonal flu test. [ ] (d) bosch's new microfluidic system is capable of virus diagnostics; the sample preparation is integrated with a multiplexed end-point pcr. [ ] pcr systems that offer multiplexing capability are preferred because they can reduce the test time for multiple diseases. this can be particularly beneficial during the flu season because different virus strains often present during one season. the differential diagnostics of community-acquired pneumonias can also benefit from this methodological approach, allowing not only the detection of multiple viruses and bacteria in one pcr reaction but also their relative quantification in the examined sample. such a quantification may lead to the correct determination of the disease-causing agent by selecting the most suitable therapy, [ ] thus tackling the spread of a pandemic disease. the application of the broad-range pcr in multiplex arrangements can be used to detect a suspect group of viruses, giving rise to more detailed analyses in the initial phases of outbreaks. [ , ] the pcr for poc and other applications should fulfill the essential requirements of portability, operability by untrained personnel, fast and adequate accuracy for screening reliability, and giving the minimum information for the publication of quantitative real-time pcr experimental guidelines. [ ] miniaturization enjoys a big advantage in poc applications due to its portability and excellent accuracy and specificity; however, the complications arising from system operation outside the specialized laboratory environment as well as by untrained people pose the biggest challenge for the development of such devices. developments in the past have led to the commercialization of sample-to-answer systems that are more or less portable. one of the early birds in this regard was genexpert, [ ] a cartridge-based system that performed a fully automated nucleic acid amplification technique. it was designed for the detection of healthcare-associated infections, critical infectious diseases, sexual health, viruses, and cancers. its development was accelerated by concerns over anthrax in the usa [ ] and via the call for a proposal by the bill and melinda gates foundation to tackle hiv and tuberculosis in south africa. [ ] the system simplifies the testing procedure as it integrates sample preparation with qpcr/rt-pcr. the system is designed to be employed in areas lacking sophisticated facilities to conduct conventional testing. it is not easily portable but it does offer the option of multicolor multiplexing. there was another push to release pcr systems for poc applications and this has contributed to disease diagnosis during pandemics such as the ebola outbreak. starting in early , an unprecedented ebola outbreak occurred in west africa. on august , , the world health organization (who) declared it a public health emergency of international concern[ ] and initiated an emergency use assessment and listing (eual) mechanism to encourage research and development (r&d) of early access to unregistered products of in-vitro diagnostics [ ] . cepheid received a $ . million grant, co-financed by the paul g. allen family foundation and the bill & melinda gates foundation, to develop the genexpert ebola assay based on the genexpert system. it took only ≈ months from development for it to get listed in eual by who. the genexpert ebola assay is fully automated and only requires the placing of the patient sample into the cartridge and inserting the cartridge into a compact desktop-level instrument. it takes ≈ h for an entire assay, from sample to answer, and features high detection accuracy. [ ] filmarray biothreat-e is another product that was given emergency use authorization from both the food and drug administration (fda) and who. the assay is also a cartridge-based automatic rt-pcr system with a turnaround time of only h. [ ] the covid- global pandemic has greatly boosted the determination and motivation of researchers worldwide to speed up the development of poct systems, as well as the number of companies prepared to fund such work. some have adapted existing devices, while others have made new ones. the systems that were available in march mostly contained immunoassays [ ] ; one was pcr-based, involving clusters of regularly interspaced short palindromic repeats, known as clustered regularly interspersed short palindromic repeats (crispr), with a crispr-associated protein, known as cas, performing the detection of the isothermal amplification products. other companies that have developed systems to diagnose seasonal flu have made use of different primers to convert these systems into poct for covid- . the eiken chemical co. in japan has developed a system for multiple testing that combines sample preparation with lamp, and the optical detection of the results with rna purification is based on magnetic force ( figure a ). [ ] it employed a microfluidic cartridge with wells, allowing for multiplexed detection. roche created a poct system called cobas® liat®, a fast and fully automated sample-to-answer system based on a pcr capable of testing samples in min or less ( figure b ). it utilized a sealed-tube design that eliminated the operators' contact with the reagents. its closed system with multiple controls also reduces the potential for human error. [ ] id now, from abbott laboratories, is a fully integrated sample-to-answer system that was originally developed to diagnose a seasonal flu ( figure c ), [ ] and is currently available with modified primers to diagnose the covid- virus. [ ] it uses the isothermal amplification of nucleic acid, and it is capable of processing two samples at a time, with a claimed min wait required for positive and min for negative results. recently, bosch began r&d of a poct system; the result of their efforts is the vivalytic system ( figure d ), a molecular diagnostic device based on a microwell array that is capable of detecting the coronavirus in less than . h, using a fully sealed cartridge. the vivalytic system performs sample preparation, after which it performs a multiplex pcr, followed by microarray-detection, to allow the identification of sars-cov- . [ ] currently, the large-scale practical deployment of the above-mentioned devices to combat the pandemic spread of covid- will allow their advantages and disadvantages to be quickly determined, after which further development can be encouraged. more systems will emerge in the near future in the context of the covid- pandemic, after which companies are likely to switch their interest to systems that are more viable from an economic perspective. as of july , , there were approved test kits and systems approved by the u.s. fda for emergency use authorization, with the vast majority being based on a real-time rt-pcr technique with or without sample preparation. [ ] given the development of communication technology, more and more devices are envisioned to be connected to form a network called the internet of things (iot), a burgeoning industry that is projected to change human society tremendously by bringing about convenience, ubiquitous information, and intelligence to improve our lives in a fundamental way; the health care industry can greatly benefit from this technological advancement. there have also been developments in personal healthcare monitoring devices linked with wearable healthcare monitoring, such as electrocardiogram and blood o measurement devices. the data collected can be used for remote and/or real-time disease monitoring and predictive model building. furthermore, it is easy to predict that a diagnostic device such as pcr, with high connectivity and data transmission capabilities, can become highly impactful in pandemic management. we have demonstrated such a pcr in a pseudo dengue fever test; [ ] however, in order to 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notebook thermal cycler for rapid multiplex real-time pcr analysis effect of materials for micro-electro-mechanical systems on pcr yield microfluidic platforms for lab responding to covid- -a once-in-a-century pandemic? ultra fast miniaturized real-time pcr: cycles in less than six minutes extreme pcr: efficient and specific dna amplification in - seconds integrated extreme real-time pcr and high-speed melting analysis in to seconds multiplex polymerase chain reaction (pcr) on a su- chip automated polymerase chain reaction in capillary tubes with hot air a point-of-need infrared mediated pcr platform with compatible lateral flow strip for hpv detection a novel miniaturized pcr multi-reactor array fabricated using flip-chip bonding techniques disposable real-time micropcr device: lab-on-a-chip at a low cost precise temperature control and rapid thermal cycling in a micromachined dna polymerase chain reaction chip all-plastic, lowpower, disposable, continuous-flow pcr chip with 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polymerase chain reaction based on double-inkjet printing a fully integrated cmos fluorescence biochip for multiplex polymerase chain-reaction (pcr) processes an ewod-based microfluidic chip for single-cell isolation, mrna purification and subsequent multiplex qpcr from chip-in-a-lab to lab-on-a-chip: towards a single handheld electronic system for multiple application-specific lab-on-a-chip (asloc) self-contained, fully integrated biochip for sample preparation, polymerase chain reaction amplification, and dna microarray detection a fully integrated microfluidic genetic analysis system with sample-in-answer-out capability an integrated fluorescence detection system for lab-on-a-chip applications rapid detection of viral rna by a pocket-size real-time pcr system handheld real-time pcr device esensor® a microarray technology based on electrochemical detection of nucleic acids and its application to cystic fibrosis carrier screening a self-contained all-in-one cartridge for sample preparation and real-time pcr in rapid influenza diagnosis labdisk with complete reagent prestorage for sample-to-answer nucleic acid based detection of respiratory pathogens verified with influenza a h n virus sample-to-answer droplet magnetofluidic platform for point-of-care hepatitis c viral load quantitation integrated dna purification, pcr, sample cleanup, and capillary electrophoresis microchip for forensic human identification human malaria diagnosis using a single-step direct-pcr based on the plasmodium cytochrome oxidase iii gene consistent detection of novel coronavirus in saliva fully automated molecular diagnostic system "simprova" for simultaneous testing of multiple items lab-in-a-tube: real-time molecular point-ofcare diagnostics for influenza a and b using the cobas® liat® system comparison of the id now influenza a & b , cobas influenza a/b, and xpert xpress flu point-of-care nucleic acid amplification tests for influenza a/b virus detection in children a sample-in-digital-answerout system for rapid detection and quantitation of infectious pathogens in bodily fluids comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia identification of a novel coronavirus in patients with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome minimum information for publication of quantitative real-time pcr experiments use of the genexpert tuberculosis system for hiv viral load testing in india ebola virus disease norms and standards: assessing new medical products in health emergencies: the eual procedures performance of the genexpert ebola assay for diagnosis of ebola virus disease in sierra leone: a field evaluation study clinical evaluation of the biofire filmarray((r)) biothreat-e test for the diagnosis of ebola virus disease in guinea fast, portable tests come online to curb coronavirus pandemic laboratory diagnosis of emerging human coronavirus infections-the state of the art emergency use authorizations. , pp. approved kits and tests for sars recently there is a boom in development of portable microfluidic based system for sars-cov- molecular diagnostics.most of those techniques approved by us fda is based on a reverse transcription polymerase chain reaction (rt-pcr) drawback of the rt-pcr methods such as sample preparation to remove pcr inhibitors and perform sample preconcentration ☒ the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☐the authors declare the following financial interests/personal relationships which may be considered as potential competing interests: key: cord- -xflhxb authors: manso, carmen f.; bibby, david f.; mbisa, jean l. title: efficient and unbiased metagenomic recovery of rna virus genomes from human plasma samples date: - - journal: sci rep doi: . /s - - - sha: doc_id: cord_uid: xflhxb rna viruses cause significant human pathology and are responsible for the majority of emerging zoonoses. mainstream diagnostic assays are challenged by their intrinsic diversity, leading to false negatives and incomplete characterisation. new sequencing techniques are expanding our ability to agnostically interrogate nucleic acids within diverse sample types, but in the clinical setting are limited by overwhelming host material and ultra-low target frequency. through selective host rna depletion and compensatory protocol adjustments for ultra-low rna inputs, we are able to detect three major blood-borne rna viruses – hiv, hcv and hev. we recovered complete genomes and up to % of the genome from samples with viral loads of ( ) and ( ) iu/ml respectively. additionally, we demonstrated the utility of this method in detecting and characterising members of diverse rna virus families within a human plasma background, some present at very low levels. by applying this method to a patient sample series, we have simultaneously determined the full genome of both a novel subtype of hcv genotype , and a co-infecting human pegivirus. this method builds upon earlier rna metagenomic techniques and can play an important role in the surveillance and diagnostics of blood-borne viruses. a complex host-enriched sample was prepared by diluting in negative human plasma (nhp, negative for each hiv, hcv, and hev) stored plasmas from four samples previously characterized by routine diagnostic testing to contain hcv (x ), hiv and hev (see table for details). nhp was obtained by centrifuging negative human blood for minutes at × g to remove cell debris. the final concentration of each virus in the primary panel sample was iu/ml (copies/ml for hiv -implied by iu henceforth for convenience), and three serial tenfold dilutions in nhp were prepared from this stock. virus multiplex reference (vmr) panel. a reagent comprising a suspension in pbs of rna viruses with different genomic and structural characteristics was provided by the national institute for biological standards and controls (nibsc, potters bar, uk). each viral component and its approximate relative concentrations is given in mee et al. . prior to extraction, the panel was mixed : with nhp. duplicate μl extractions were performed , as was genotyping of the hcv and hev clinical samples of indeterminate hcv genotype. four plasma samples collected from a patient between and were submitted to public health england (phe) for metagenomic analysis as previous genotyping results had been inconsistent. the most recent such test employed ns b sequencing , and reported the presence of a virus belonging to genotype but was unable to resolve the subtype with any further precision. rna extraction and quantification. before extraction, all samples were centrifuged for min at , × g to remove cell debris. triplicate, duplicate and single extractions were performed on the diluted vmr panel samples (referred to as 'vmr panel a/b/pbs'), the bbv panel samples (' - -a/b'), and the patient sample series, respectively. a negative control comprising μl of the same plasma used to dilute the panels was also extracted. the split rna extraction kit (lexogen) was used to extract μl of each sample input, according to the manufacturer's instructions. acidic phenol was used to preferentially recover the large rna fraction, which was eluted in μl of nuclease-free water. rna eluates were quantified using qubit rna hs assay kit (thermo fisher scientific), which is accurate for concentrations between pg/μl and ng/μl. depletion of ribosomal rna and dna digestion. ribosomal rna depletion and dna digestion was achieved using the riboerase kit (kapa biosystems). as all sample extracts were below the detection limit of the qubit quantification system, the total rna input was less than the recommended ng. the manufacturer's specifications were followed with the exception of using the entire μl of the extract, and after the dna digestion reaction clean up, eluting the residual rna was in μl of nuclease-free water. in the case of the bbv panel, two of the three extracts of each dilution were treated with the riboerase kit before rna library preparation. the third set of extracts remained untreated and was used to monitor the effect of the rrna depletion and dna digestion upon the subsequent library preparation and sequence analysis. in the case of the vmr panel (the two duplicates) and the negative control, rrna and dna depletion was performed on all extracts. in the case of the uncharacterized hcv strain, extracts from all four samples were treated with riboerase. an additional, untreated, extract of sample was included, again to monitor the process. rna library preparation with ultra-low rna input. libraries were constructed from μl of extracted rna or μl of rrna-depleted dnase-digested rna, using the nebnext ultra directional rna library prep kit (new england biolabs). as the protocol is designed to use a minimum rna input of ng, several modifications were made to adapt it to an ultralow rna input. these are listed in table . libraries were analysed for size distribution using the high sensitivity dna kit (agilent) on a bioanalyser instrument, and were quantified using the kapa sybr fast universal qpcr kit for illumina libraries (kapa biosystems) on a real-time pcr system (applied biosystems). to determine the relative abundances of viral inserts, libraries constructed from the bbv panel were analysed by qpcrs with primers and probes targeting each of the three viral components (refs - and supplementary table s ). reactions were performed using the quantitect virus kit (qiagen) according to the manufacturer's instructions. libraries labelled with different indexes were diluted to nm and pooled. sequencing was performed on an illumina miseq instrument using the miseq reagent kit v ( cycles) (illumina) according to the manufacturer's guidelines, with the following minor modifications. the library pools were denatured with . n sodium hydroxide for minutes rather than , diluted in kit reagent ht to produce a pm solution and then these were further diluted to pm. of this library pool dilution, μl were loaded onto the miseq cartridge. data analysis. all paired end fastq files were processed with trimmomatic v . , removing the illumina adaptor sequences, then trimming leading and trailing bases with phred scores below . reads were discarded where the length of either trimmed end was below bases. for the determination of genome sequences of blood-borne viruses, trimmed fastq sets were normalised using the normalise-by-median.py script in the khmer package (k = , c = ) and submitted to the spades de novo assembler without error-correction, applying the default kmer sizes of , , and . output contigs that matched each virus were identified with the nhmmer function of the hmmer v . b package using hidden markov models (hmms) built from alignments of each virus (detailed in supplementary table s ) . where necessary, the ends of contigs were trimmed to the whole genome alignment. bwa mem (v . . a, default parameters) was used to map the original trimmed fastqs to the genome sequence, and the sam files were converted to bam files using samtools v . . while discarding reads with either × and/or × flags set (i.e. retaining only fully-mapped paired-end reads). base frequencies at each nucleotide position within each component virus sequence were obtained from bam files using quasibam v . , an in-house c++ program that tabulates base frequencies at each nucleotide position within a reference and generates consensus sequences based upon user-defined depth and variant percentages . mapping of trimmed paired-end fastq to one or more virus reference genomes was also performed using bwa mem . . a. in each case, two independent mappings were performed, using as a reference the viral sequences, supplemented firstly by the march 'grch ' release of the human genome, and secondly by a set of human rrna sequences (nr_ . , nr_ . , v . , nr_ . , gij : - , and gij : - , as per malboeuf et al. ). the second file was used solely to derive counts for reads mapping rrna which would otherwise be subsumed into the human genome mapping results. from the filtered sam files, the numbers of reads mapping to each reference sequence were counted. counts for each of the constituent sequences of the human genome and rrna were pooled into a "human" count and an "rrna" count. quasibam was used to derive nucleotide frequencies from which depth and coverage data were calculated. a minimum depth of was required for inclusion in a derived consensus sequence for the bbv panel ( for the vmr panel). bbv and vmr panel sequences. the members of the multi-fasta reference file for the bbv panel were obtained by submitting fastq sets from the rrna-depleted sample with the highest virus concentrations to the spades-hmmer-mapping approach described in the previous paragraph. vmr panel references were derived from sequences obtained from genbank using accession numbers from mee et al. . additionally, the complete genome sequence of a human pegivirus (hpgv) present in the plasma diluent was discovered in the spades contigs file. a hmm profile was constructed from an alignment of genbank sequences (supplementary table s ). sample with uncharacterised hcv. to obtain full-length hcv genomes, each fastq set was submitted to the spades-hmmer-mapping process. where a complete genome was not obtained, hcv-matching contigs were aligned to the full-length genomes using mega . in addition, contigs with length > kb that did not align to the hmm profile were submitted to blast for identification. following this analysis, an additional pegivirus genome was derived in similar fashion to the hcv genomes, using the same hmm profile as above for the nhp pegivirus. when calculating the read percentages and coverage plots, both sample-derived full-length genome sequences (hcv and hpgv) were used as the reference sequence when mapping that sample's corresponding trimmed paired-end fastqs, as well where only incomplete hcv genomes were obtained. virus (bbv) panel was prepared, comprising two strains of hcv (genotypes a and b), and one each of hiv and hev diluted in nhp to iu/ml. three tenfold serial dilutions in plasma were made from this original panel. step manufacturer's recommendations protocol modification riboerase rna input . . ribosomal rna depletion was performed on two of each set of triplicate extractions prior to all three being subjected to the modified library preparation protocol. data from the most concentrated rrna-depleted samples were used to generate individual virus genome sequences for use in reference mapping. during this data analysis, an unexpected human pegivirus (hpgv) was found and traced to the nhp diluent. the full genome sequence of this hpgv was determined from the -b data and included in the mapping references. table gives the read counts, genome coverages and median depths for each virus-dilution combination, across each of the three samples per dilution ( - -untreated/a/b). each test sample yielded over , reads with the exception of -a, which gave just over , reads. with the exception of the two samples, in which only a very small volume of nhp was added to the clinical samples, the percentage of reads mapping to the hpgv remained relatively constant at - %. the exception is -b, in which the overall viral read percentage was lower than expected, with a corresponding elevation in reads mapping to the human genome suggesting possible incomplete dnase digestion during the rrna depletion step (supplementary table s ) . with increasing dilution, the total viral read percentages (excluding hpgv) decline from over % to . %. complete and near-complete genome coverages with depths greater than were achieved at and iu/ml for all four viruses. a few short regions in hiv had low coverages (< ) with -b, reflecting the reduced overall viral reads in this dataset, but at a minimum depth of , . % coverage was achieved with this sample, with only a -base sequence in pol having no coverage. at iu/ml, hcv a and hev continued to give . - . % coverage with median depths over . hcv b and hiv gave . - . % coverage ( - median depth) and . - . % coverage ( - median depth) respectively, and in -b, despite only . % of all reads mapping to the four viruses collectively, genome coverages of . - . % were achieved, with median depths up to . figure illustrates coverages and depths across target genome at each dilution, showing even distributions of reads across all four target genomes and hpgv. pooling duplicates consistently improved coverages (final column, table ). this is most clearly seen at the lower viral loads, where at iu/ml, three of the four viruses achieve combined coverages of > . % each, and . % in hiv. at iu/ml, the combined coverages for the four viruses are effectively what would be expected were the individual coverages independent, i.e. cov aub depletion of rrna substantially enhances the recovery of blood-borne virus sequences. the percentage of reads mapping to rna virus genomes in the rrna-depleted bbv panel samples was between and -fold higher than in corresponding untreated controls. individual target virus ratios decreased as they became more dilute, from over -fold for hcv in -a to . -fold for hiv at the lowest dilution. concomitantly, the ratio for hpgv rose markedly, from . -fold in -a to in -b, reflecting an effectively constant viral load against decreasing quantities of panel viruses (table and fig. ). genome coverage and median depth values were also much higher in the treated samples than untreated comparators. at the two highest virus concentrations, median depths were between -and -fold higher in the treated versus the untreated samples. only short fragments of hev were recovered from the untreated dilution, and almost no hiv or hcv sequences. by contrast, near complete genomes from all four target viruses were recovered from the treated comparators, with median depths of between and (as noted above, hiv in -a was an exception at . % coverage and a median depth of ). recovery of partial and complete genomes of diverse virus types from human plasma. the ability of our method to recover genome sequences from a range of rna viruses in the context of human plasma was evaluated using a virus multiplex reference (vmr) panel, putatively containing genomically and physicochemically diverse viruses. two plasma-diluted panels and one pbs-diluted panel were tested (table ). no reads from either of the three samples mapped to either of the two norovirus genomes, coronavirus e or influenza b virus. by the panel distributor's qpcr , the threshold cycle (c t ) of the coronavirus was > and the other three were not detected, hence these four targets were excluded from further analysis. notwithstanding influenza virus a h n and parainfluenza virus type also not being detected by the qpcr, we recovered reads from both, with genome coverages ranging from . % to . %. almost no reads belonging to the panel's dna viruses were found. sixty-nine percent of all reads obtained from the pbs-diluted panel mapped to vmr panel genomes, dropping to - % for the plasma-diluted samples, although the distribution of reads between targets was very uneven. parechovirus and rotavirus accounted for . - . % and . - . % of all viral reads respectively, with the other viruses collectively accounting for . - . %. depths and genome coverages showed some inverse correlation with the given c q values (fig. ) . as with the bbv panel data, coverage plots of the samples diluted in plasma were largely unbiased, giving pooled genome coverages close to those expected by independent distributions of reads between replicates ( table , final column). rotavirus and coxsackievirus were exceptions, where despite large numbers of mapped reads, almost identical patterns of read coverages and gaps were observed between their replicates, with minimal additive effect. the pbs-diluted sample gave larger read numbers, but their distribution was less even throughout the genomes, resulting in relatively lower coverages. characterisation of a new subtype belonging to hcv genotype and discovery of a second virus in a patient sample series. four plasma samples from a patient with hcv were used as starting material. all extracts were subjected to riboerase treatment; a second extract of sample remained untreated for comparison. de novo assembly analysis of fastq sets from samples , and each gave a full-length hcv genome sequence as a single contig. for sample , partially-overlapping contigs were obtained, covering % of scientific reports | : | doi: . /s - - - the hcv sequence. additionally, in all four samples, a single contig was obtained that was determined by blast and subsequent hmmer analysis to comprise an hpgv genome. the hcv and hpgv full genome sequences were combined in a single file to carry out reference mapping and nucleotide frequency determination on the four sample fastq sets (table ). samples , and had hcv read percentages ranging from . to . %, and gave complete genomes with median depths greater than . sample had the lowest viral load ( , iu/ml), had . % of reads mapping to hcv giving a genome coverage of % at a minimum depth of ( . % at depth ≥ ) and a median depth of . full coverage of the hpgv genome was obtained from all samples, with median depths over , , and read percentages ranging from . to . %. the depth plots in fig. again show unbiased and even coverages across both genomes, and the percentages of reads mapping to viral targets was again much higher in the rrna-depleted sample than in the untreated comparator ( -fold and -fold for hcv and hpgv respectively). table . detailed sequencing data from the bbv panel. for each of the three samples (untreated, a and b) at each dilution ( - ), the number and percentage of reads mapping to each virus are given, together with the genome coverages (depth ≥ ) and median depths. the final column gives these last two metrics from the combined data sets of both the a and b samples. included in the analysis are data for the hpgv discovered in the sample diluent. analysis of the hcv sequence showed it to belonging to a new subtype within genotype of which the details are presented in a separate manuscript (in preparation). the hpgv clustered with genotype strains, and is distinct from the nhp strain. including the nhp negative control were subjected to virus-specific qpcr for the detection and quantification of hcv, hiv and hev. all were detectable in the sample libraries, but were undetectable in the riboerase-treated negative control library (supplementary table s ). all samples were mapped against reference sequences that included human genome and human rrna sequences to evaluate the efficiency of riboerase treatment. the average ratio of the percentages of reads mapping to rrna in the untreated versus the treated samples was -fold with an approximate halving of the number of reads mapping to the human genome, across all panels (fig. ) . with the exception of the expected human pegivirus, mapping of the negative control fastq set against the reference sequences of the four bbv panel viruses, the two pegiviruses, the vmr panel and the patient hcv gave table . detailed sequencing data from the patient sample series. for each of the four samples - , the number and percentage of reads mapping to both the hcv and hpgv genomes are given, genome coverages (depth ≥ ) and median depths. the analysis of sample extracted without host rrna depletion is in the untreated column. very low numbers of reads mapped to viral genomes and no consensus sequences could be derived. further data for this section are found in supplementary tables s and s . in light of the large and ever-increasing number of human rna virus pathogens, it is perhaps unsurprising that standard serological assays and nucleic acid tests suffer from a lack of sensitivity to diverse variants of target viruses, overlook the presence of new or unexpected viruses, and provide only limited information about those targets they do successfully detect. hence the three main aims of metagenomic virology are to detect & identify known agents irrespective of their diversity, to discover novel agents of disease, and to obtain complete sequence information of detected viruses. most existing protocols achieve a maximum of two of these aims, but difficulties in selectively isolating viral rna species and short read sequences from those of the super-abundant host nucleic acid have limited the utility of metagenomic approaches in diagnostic virology. this study has addressed these limitations by establishing a novel methodology suitable for the agnostic detection and characterization of blood-borne rna viruses in plasma samples. by depleting host-derived nucleic acids and making modifications to an existing library preparation protocol to account for ultra-low rna input quantities, we have been able to reconstruct effectively full-length genomes of hcv, hev and hiv from plasma samples with viral loads of iu/ml (copies/ml for hiv) and substantial fractions of complete genomes at iu/ml. when applied to a series of clinical samples, we could elucidate simultaneously the full genome sequences of both a novel subtype belonging to hcv genotype and a hitherto-undetected human pegivirus. additionally, our system was able to recover viral sequences from a panel of diverse rna viruses diluted in human plasma, with a broad correlation between the genomic coverage and depth metrics and approximate concentration. although full genomes were not assembled in many cases, the independence of read distribution gave sufficient genome coverage for identification. the vast majority of rna molecules in a human plasma sample are host-derived, of which up to % comprises the six species of human rrna. their presence in our libraries was minimised by two key protocol steps in our modified protocol. firstly, we selected an extraction method that combined a phenol/chloroform step with a column format (lexogen split rna) which increased the amount of extracted viral rna by up to one log when compared to other extraction methods (data not shown). perhaps more importantly, by controlling the final precipitation step, small rna molecules below nt such as s rrna and trna are excluded from the eluates, as are the majority of molecules of human genomic dna. secondly, we employed dna probes complementary to human rrna such that hybridisation and subsequent digestion by rnase h dramatically reduced their frequency in the finished libraries. whilst this methodology has been successfully used in the detection and characterisation of two haemorrhagic fever viruses, the frequency of viral reads was often below % and an additional hybrid-capture step was employed to elevate read numbers . methods that do not deplete rrna generally give poor recovery of viral reads, yielding viral genome fragments that necessitate further work , , , , low read numbers even at viral loads over iu/ml [ ] [ ] [ ] , or at best, requiring dilution of both host and virus in pbs in order to recover full hiv genomes at low copy numbers . the resultant rrna-depleted sample extracts typically contain quantities of nucleic acid in the low picogram range. library preparation through hexamer-mediated reverse transcription followed by multiple displacement amplification constitutes an easy and effective means of amplifying very low amounts of dna , , , but in several studies (and in the authors' laboratory), significant amplification biases have been observed, leading to gaps in target genome coverage , [ ] [ ] [ ] . consequently, we adopted an approach using a standard rna library preparation kit, but with substantial modification to compensate for their minimum rna input requirements of at least ng and optimally ng- µg. we made key changes to the rna fragmentation and adaptor-ligation steps of the nebnext ultra directional rna library prep kit protocol. while prior rna fragmentation with heat and divalent cations improves sequence coverage, over-fragmentation of target genomes leads to the loss of material during the library preparation process . lower amounts of rna thus require shorter optimum fragmentation times and we found that minute at °c was optimal in terms of breadth of genome coverage. under standard kit conditions, our ultra-low rna inputs dramatically skewed the ratio of cdna to adaptor. the resulting adaptor excess led to the preferential amplification of adaptor dimers during the pcr step, and despite increasing cycle number to amplify low rna inputs, we were generally unable to generate sufficient quantities of target-specific material. accurate quantification and consequent equimolar pooling of libraries was compromised, as was the miseq clustering efficiency. we found that a reduced final adaptor concentration of . nm was crucial in reducing the amount of adaptor dimers in libraries from rrna-depleted samples whilst simultaneously extending the pcr cycle number. in the present study, serial dilutions of the blood borne virus panel were prepared in negative human plasma, reducing both the absolute quantity and relative frequency of the viral rna targets while maintaining the complexity of the sample in terms of host nucleic acid, thus mimicking that of a clinical sample. with rrna depletion, the number and diversity of viral reads was consistently high, with over % of all reads mapping to constituent virus genomes. throughout the three sample series, we obtained relatively high genome coverages of low-frequency viral targets. co-infections with multiple blood-borne viruses are common , so whilst we speculate that the depths and coverages of target viruses would be greater yet in these samples had it not been for the confounding effect of the unexpected human pegiviruses in both the plasma diluent and the patient sample series, it was reassuring to see the method performed well under such conditions. in our experiments using negative human plasma as sample diluent, we were able to recover levels of viral genomes comparable to previous work using pbs, both for bbv panel viruses and for the vmr panel , and we were able to recover from a patient sample a large percentage of the genome of a previously uncharacterised subtype of hcv genotype when present at × iu/ml, a diagnosis not possible using existing genotyping assays. the presence of an undiagnosed pegivirus in this sample further demonstrated the utility of the method in metagenomic analysis of blood-borne virus co-infections where the relative abundances of each virus can be highly variable . furthermore, in three of the four samples, depths greater than , were routinely obtained, which are likely to be sufficient to call minority variants for clinical resistance . a full description of the patient series and the new hcv strain are provided in a separate manuscript (in preparation). our approach can therefore not only accurately characterise rare or novel variants of existing viruses, but also generates the same level of information regarding unexpected viruses present in the sample. by comparison, vidisca , , and other random amplification-ngs techniques , have detected novel viruses in diverse clinical samples, but all have required further techniques to achieve full genome sequences. together with the vmr panel results, we were able to recover identifying sequence from both enveloped viruses (hcv, hiv, hev, influenza, and several paramyxoviruses), and non-enveloped viruses (several enteroviruses, astrovirus, rotavirus, and sapovirus). for the majority of viruses in the vmr panel, whilst dilution in plasma reduced the total percentage of reads recovered when compared to the panel diluted in pbs, a greater breadth of genome coverage was achieved. in the absence of any host nucleic acid background, it is possible that the pbs extracts had such ultra-low quantities of rna that despite the adjustments made to the library preparation protocol, the rna was over-fragmented, leading to a smaller number of genome fragments that were individually amplified to a greater extent than the larger array of fragments surviving the plasma extraction. in developing a similar approach, kohl et al. were only able to recover a percentage of reads exceeding % at a viral load over copies/ml. at an influenza a virus concentration of over copies/ml, this dropped to just . %, and at a reovirus concentration of - copies/ml, no viral reads were detected . with our method, whole genomes were obtained for those with the highest viral loads, and for minority viral targets, there was a correlation between ostensible quantity and coverage, including for two viruses undetectable by the panel distributor , a result superior to that recently obtained from influenza in clinical respiratory samples . again, the presence of high quantities of one or more target is likely to have inhibited the representation of the minority species such that if tested individually, superior depths and coverages would seem likely. with further reduction to the fragmentation time, or even its abolition, it may be possible to use this method to reconstruct genomes from old, partially degraded samples such as those recently used to re-evaluate the early hiv epidemic in the americas . our negative control data suggest that the level of contamination is low, with most viral reads therein belonging to the most abundant vmr panel member. nelson et al. identified a second source of contamination consisting of incorrect reads from other libraries that were sequenced during the same sequencing run due to truseq index misassignment (~ . % of reads, . % here). although cross-contamination between samples during the library preparation can be another source of contamination, the qpcr results suggest no bbv panel genomes were present after library preparation in the negative control sample. to conclude, by applying the three adaptations of selective large rna extraction, rrna depletion-dnase treatment, and the extensively modified library preparation in combination, ngs data sets can be produced from plasma samples that are rich in rna virus sequence data. complex bioinformatic processing has been employed to identify viruses within a metagenomic dataset , , , , , , but here, only simple bioinformatic processing is needed for detection and identification of known viruses, and by applying only moderately more advanced tools, an agnostic approach to virus detection can be taken, together with characterisation of the full genome even at low viral loads. the role of mutational robustness in rna virus evolution rna virus quasispecies: significance for viral disease and epidemiology ecological origins of novel human pathogens isolation of a 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analysis, optimization and verification of illumina-generated s rrna gene amplicon surveys a novel virus in swine is closely related to the human hepatitis e virus a modified rna-seq approach for whole genome sequencing of rna viruses from faecal and blood samples the authors would like to acknowledge the contribution of nibsc for kindly providing the viral multiplex reference panel and the blood borne virus unit of virus reference department, phe, for providing the hev sample. the genomic services unit at phe are acknowledged for running the miseqs. the authors are grateful to dr. brendan healy, dr. owen seddon and dr. nicola price from university hospital of wales for providing additional information regarding the patient sample series. this work was supported by funding from public health england. supplementary information accompanies this paper at doi: . /s - - - competing interests: the authors declare that they have no competing interests.publisher's note: springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons license, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this license, visit http://creativecommons.org/licenses/by/ . /. key: cord- -a t u authors: nan title: alphabetic listing of diseases and conditions date: - - journal: handbook of autopsy practice doi: . / - - - - _ sha: doc_id: cord_uid: a t u part ii begins with a list of special histologic stains, their for use and their corresponding references. at the end of this list is a procedure for removal of formalin precipitate from tissue sections. diseases. there may also be a list of possible associated conditions. these entities are generally linked pathogenetically to the main disease entry. any asterisk after a related disease indicates that that disorder is also listed as a disease entry. many disease entries will be followed by a three-column table that provides the reader with a listing of the pathologic findings to be expected with the disease as well as the prosection and dissection procedures necessary to demonstrate those findings. it is expected that routine hematoxylin-eosin stains will be done on all sections submitted for histologic examination. special stains will be recommended in the procedures column of the tables, when indicated. any table immediately following the two columns of disease entries always refers to the disease in the right column. prepare smears of undiluted blood. obtain blood for molecular studies for preservation of small intestinal mucosa and for preparation for study under dissecting microscope, see part i, chapter . submit sample for histologic study. submit stool for chemical analysis. record weight and submit sample for histologic study. freeze liver for molecular studies record appearance of spine (see also chest roentgenogram). for removal and specimen preparation, see chapter . request luxol fast blue stain. for removal and specimen preparation, see chapter . below-normal weight in infants. kyphoscoliosis. very low concentrations of cholesterol and decreased triglycerides; serum~-lipoprotein or absent; a.-lipoproteins present. acanthocytosis (spiny red cells). gene mutations ( ) . abnormal shape of villi; vacuolation of epithelial cells. fatty stools fatty changes. gene mutations ( ) systemic manifestations of malabsorption syndrome* and of vitamin a deficiency. * kyphoscoliosis. axonal degeneration of the spinocerebellar tracts; demyelination of the fasciculus cuneatus and gracilis ( ) . possible involvement of posterior columns, pyramidal tracts, and peripheral nerves. atypical retinitis pigmentosa ( ) with involvement of macula. angioid streaks ( ) . synonym: cerebral abscess. note: for microbiologic study of tissues and abscesses, see part i, chapter . include samples for anaerobic culture. it is best to study the brain after fixation but if specimen is examined fresh, aspirate and prepare smears of abscess content. photograph surface and coronal slices of brain. request giemsa stain, gram stain, pas stain, and grocott's methenamine silver stain for fungi. external examination if there is evidence of trauma, see also under "injury, head." prepare roentgenograms of chest and skull. submit for microbiologic study. for removal and specimen preparation, see chapter . for microbiologic study, photography, and special stains, see under "note." for exposure of venous sinuses, see chapter . sample walls of sinuses for histologic study. for exposure of paranasal sinuses, mastoid cells, and middle ears, see chapter . for removal and specimen preparation, see chapter . procedures depend on suspected lesions as listed in right-hand column. skin infections in upper half of face. edema of forehead, eyelids, and base of nose, proptosis, and chemosis indicate cerebral venous sinus thrombosis. * trauma; craniotomy wounds. skull fracture and other traumatic lesions. for possible intrathoracic lesions, see below under "other organs." the national transportation safety board (ntsb)* has authority over aircraft wreckage and the legal authority to investigate and to determine the cause of air crashes. ( ) the dead are the responsibility of the medical examiner or coroner. local police will seal off the area of the crash. other than for the purpose of determining that death has occurred, no one should be allowed to approach the bodies or any objects until the identification teams and the medical examiner or coroner have taken charge. the sudden influx of bodies after a commercial air carrier accident and the request for speedy identification of the victims would overburden almost any institution. managing such a disaster is eased by writing a contingency plan beforehand. temporary morgue facilities may have to be established near the scene of the crash. refrigerated trucks may serve as storage space. a practical approach is to deal first with those bodies that seem to be the easiest to identify, in order to narrow the field for the more difficult cases. if bodies are scattered, their locations can be referenced to stakes in the ground or spray paint on pavement; only then should these bodies (or parts) and personal effects be collected. for large-scale crashes a locations can be referenced to a string-line grid benchmarked to gps coordinates. records and diagrams of the relative positions of victims are prepared during this phase. if bodies are still within the airplane, their positions are recorded, and photographed. the personnel of the medical examiner or coroner can augmented by d-mort team staffed by forensic pathologists, anthropologists, dentists, morgue technicians, and investigators supplied by the national disaster medical system. ** the airline will provide a list of the passengers and the federal bureau of investigation (fbi) disaster team will make itself available to take and identify fingerprints and aid in the acquisition of other identifying data such as age, race, weight, height, and hair color and style. if dental records can be obtained, this provides one of the most certain methods of identification. a medical history indicating amputations, internal prostheses, or other characteristic surgical interventions or the presence of nephrolithiasis, gallstones, and the like will be helpful. fingerprints (and footprints of babies) should be taken in all instances. wallets with identification cards,jewelry, name tags in clothing, or other personal belongings may provide the fastest tentative identification. the medical examiner may elect to autopsy only the flight crew but not the passengers of an aircraft crash. however, the grossly identifiable fatal injuries should be described, photographed, and x-rayed. this may reveal identifying body changes. if comparison of somatic radiographs, dental records, fingerprints, or photographs do not identify the victim, dna comparison must be considered. burned or fragmented bodies of passengers and the bodies ofcrew members, and particularly the pilots, must have a complete autopsy, including roentgenographic and toxicologic examinations, which must always include alcohol and carbon monoxide determinations. internal examination might reveal a coronary occlusion, or roentgenograms may disclose a bullet as evidence that violence preceded the crash. in some airplane crashes, particularly in light airplane accidents, suicide must be considered. in such cases police investigation is required to determine if the pilot exhibited suicidal ideation in the recent past.. when resources permit, autopsies should be performed on all deceased occupants of aircraft crashes, including passengers, in order to distinguish among blunt impact trauma, smoke inhalation, and flash fires as causes ofdeath, and to answer future questions concerning pain and suffering, intoxication, and sequence of survivorship. after a crash victim has been identified, the coroner or medical examiner will issue a death certificate. if remains of a decedent cannot be found, a judge can, upon petition, declare a passenger dead and sign a death certificate prepared by a medical examiner. *phone # ofntsb command center: - - **phone # of dmort: - - . entry should be followed. usually, the circumstances that led to drowning are not apparent from the autopsy findings but can be reconstructed from reports of witnesses and the police. because the reflex drive to seek air is triggered by hypercarbia, not hypoxia, loss of consciousness and drowning can ensue after hyperventilation and breath-holding by experienced swimmers who then drown without a struggle. there are no specific autopsy findings. a search for trauma, including a posterior neck dissection, should be made in all instances. head and cervical injuries may be responsible for loss of consciousness and drowning, usually in individuals diving into shallow water. toxicologic examination as described below for scuba diving accidents is always indicated. with scuba diving fatalities, investigation of the equipment and circumstances is usually more important than the autopsy. scuba fatalities should be studied by or with the aid of diving experts-for instance, members of a diving club or shop (not the one providing the gear used by the decedent) or the u.s. navy. ( ) careful investigation of the scene and study of reports of witnesses and the police are essential. the investigation should ascertain the site of diving (currents and other underwater hazards), the estimated depth, the water temperature (exposure to cold), and a description of water clarity. electrocution should be considered if the site has electric underwater cables (see "injury, electric"). cerebral concussion should be considered if explosives were used in the vicinity. knowledge of the method of recovery of the body and the type of resuscitation efforts can aid in the interpretation of apparent wounds. the medical history of the diving victim should be sought, as it may lead to a diagnosis for which the autopsy is typically silent, such as seizure disorder, or may reveal asthma, emphysema, or chronic bronchitis, all of which increase the risk of air trapping and arterial air embolism. although drowning may be the terminal event in some scuba deaths, the investigation should be focused on the adverse environmental and equipment factors that place a capable swimmer at risk of drowning (see "embolism, air" and "sickness, decompression"). because scuba divers risk arterial air embolism if they ascend with a closed glottis, on can attempt to document gas bubbles at autopsy, but their interpretation is problematic: bodies recovered immediately are subjected to resuscitation efforts, which can by themselves produce extra-alveolar air artifacts, and bodies not recovered immediately tend to be found in a putrefied condition, full of postmortem gas. in the remaining cases, the pathologist must consider the potential of introducing artifactual gas bubbles by the forcible retraction of the chest plate and by sawing the calvarium. the following procedures apply primarily to scuba diving accidents. interrogation of witnesses is important; the behavior and complaints of the decedent, if any, might help distinguish between a natural death by heart disease and an unnatural death by air embolism. external examination eyes and ears head (skull and brain) chest blood (from heart and peripheral vessels) heart tracheobronchial tree and lungs a procedures photograph victim as recovered and after removal of wet suit and other diving gear. record condition of clothing and gear. impound all diving equipment for study by experts, particularly scuba tank, breathing hoses, and regulators. residual air in tank should be analyzed. record color of skin (including face, back, soles, palms, and scalp). palpate skin and record presence or absence of crepitation. record extent and character of wounds. prepare histologic specimens. record appearance of face (including oral and nasal cavities) and of ears. prepare roentgenograms. if air embolism must be expected, as in the presence of pneumomediastinum, follow procedures described under "embolism, air." for evaluation of findings, see also above under "note." if decompression sickness (caisson disease) is suspected, also prepare roentgenograms of the elbows, hips, and knees. otoscopic examination. funduscopic examination. save vitreous for possible toxicologic and other studies. for removal of brain, see chapter . record contents of arteries of the circle of willis and its major branches and basilar artery. strip dura from base of skull and from calvarium. for removal and specimen preparation, see chapter . for demonstration of pneumothorax, see under "pneumothorax". if gas is visible in coronary arteries, photograph. photograph and aspirate gas in heart chambers. submit samples of heart blood and peripheral blood for toxicologic study and drug screen. examine lungs in situ. save bronchial washings for analysis of debris. fresh dissection is recommended. if decompression sickness is suspected, prepare sudan stains from fresh-frozen lung sections. complete toxicologic sampling should be carried out (see chapter ). record nature of gastric contents. remove neck organs toward end of autopsy. for posterior neck dissection, see chapter . incise tongue. for removal, see chapter . for removal, see chapter . for removal, prosthetic repair, and specimen preparation, see chapter . consult roentgenograms. in decompression sickness, fatty change of liver, and ischemic infarctions of many organs. interstitial emphysema. aspiration (see above). trauma to cervical spine. mottled pallor of tongue after air embolism. contusion of tongue after convulsive chewing. nitrogen bubbles in spinal cord arteries may occur after rapid ascent. air embolism;' cerebral edema in decompression sickness. aseptic necroses (infarcts, "dysbaric osteonecrosis"), most often in head of femur, distal femur, and proximal tibia. infarcts indicate repeated hyperbaric exposures. nitrogen bubbles in and about joints and in periosteal vessels ("bends") occur during rapid ascent. related terms: automobile accident; motorcycle accident. note: a visit to the scene can make the interpretation of the autopsy findings easier. the vehicle can also be inspected in a more leisurely fashion at the impound lot. this is particularly useful for correlating patterned injuries with objects in the vehicle. most vehicular crashes occur as intersection crashes or because a vehicle with excessive speed left a curved road. the medical examiner or coroner should gain a basic understanding of the crash mechanism so that informed descriptions can be rendered, e.g., "impact to the b pillar of the decedent's automobile by the front of a pickup truck which failed to stop for a stop sign at an intersection, resulting in a -feet intrusion into the cabin; restraint belts not employed; air bag deployed; extrication required which took minutes." police are responsible for determining mechanical and environmental risk factors for the crash and for determining some human risk factors such as suicidal or homicidal intent. the pathologist determines other risk factors for crashes such as heart disease, a history of epilepsy, and intoxication by carbon monoxide, drugs, and alcohol. suicide as a manner of death should be considered when a single-occupant vehicle strikes a bridge abutment or a large tree head-on, with no evidence of evasive action or braking. in such a situation, the standard police traffic investigation should be supplemented of interviews of the victim's family and friends. the ambulance run sheet is an invaluable source of observations that often are not available from the police. this document should be acquired in all instances, even if the paramedics determined that death occurred and did not transport. the basic autopsy procedures are listed below. most traffic victims who die at the scene or who are dead on arrival at the hospital died from neurogenic shock caused by wounds of the head or vertebral column, or from exsanguination from a tom vessel or heart. as such, they have little lividity, and little blood is found in the vehicles. presence ofintense lividity may indicate suffocation or heart disease as a cause of death. if postural asphyxia is suspected, the first responders to the scene should be interviewed to determine the position of the decedent in the vehicle, and the vital signs, ifany, ofthe decedent from the time of the crash to the time of extrication. posterior neck dissection is indicated in these instances. if manifestations of heart disease, intense lividity, and absence oflethal wounds suggest that a crash occurred because the driver was dead, other drivers on the road may have observed that the victim was slumped at the wheel before the crash. the determination of heart attack at the wheel is usually simple, because most such victims realize that something is wrong, and bring the vehicle to a stop at the side of the road, or coast gently into a fixed object. in such instances, damage to the vehicle is minor, and wounds to the decedent are usually trivial. while pattemed wounds can often be matched to objects (see below), patternless wounds usually cannot be visually matched to specific objects, although an opinion can sometimes be given as to what object was struck, based on the direction of motion and position ofthe body with respect to the vehicle. impacts with the a-pillar produce narrow vertical zones of facial laceration and fractures extending from forehead to jaw. tempered glass shatters into small cubes on impact, and leaves so-called "dicing" wounds, which are abraded cuts arranged in a somewhat rectilinear pattern. windshield glass leaves shallow, abraded, vertically oriented cuts on the face or scalp. with pedestrians, the lower extremities are of particular forensic interest, to determine the height and direction of impact from vehicles that left the scene. scalp hair and blood should be collected from such "hit and run" victims and from occupants of a suspect car if police have a question as to which occupant was the driver; these exemplars can be compared to fibers and tissue recovered from the vehicle in question. likewise, foreign material in wounds can sometimes be matched to suspect vehicles, and should be sought and retained as evidence. for pedestrians, the distance between the impact point on the lower extremities and the soles of the feet should be recorded. the legs should be opened to inspect tibial fractures; cortical fractures initiate propagation opposite to the side of impact, where they usually have a pulled-apart appearance, and then splinter the cortex at the side of impact. abrasions are better impact markers than contusions, because subcutaneous blood extravasation can be caused not only by impact to the skin, but also from blood extravasating from underlying fractures. if no cutaneous abrasions or fractures of the leg bones are found, the skin of the legs should be incised to expose contusions. fracture descriptions should include location in the bone (e.g., proximal metaphysis or shaft), whether the fracture is complete or incomplete, and whether the fracture is displaced or distracted. lacerations of intervertebral disks, facet joint capsules, and ligamenta flava should not be loosely termed "fractures." the presence or absence of blood extravasation in soft tissue adjacent to the fractures should be recorded, and its volume estimated if it appears severe enough. venous air embolism from tom dural sinuses cannot be diagnosed without a pre-autopsy chest radiograph or an in situ bubble test. if an x-ray machine is readily available, an anterior-posterior chest radiograph should be obtained in every traffic victim who dies at the scene or after a failed resuscitation attempt. if a hemothorax is suspected, the rib cuts should be placed further lateral and the chest plate reflected so that the internal mammary vessels can be inspected before the chest plate is removed. after measuring and removing the bloody effusion, the underlying serosal surfaces should be inspected for defects. lacerations of the heart and aorta will be obvious. tamponaded lacerations of the aorta, around which the adventitia still holds, must be noted as such. if no lacerations are found at the usual sites, lacerations of the azygous veins must be considered, especially in association with fracture dislocations of the thoracic vertebral column; other sites are the internal mammary arteries, especially with fractures of ribs i and or of the sternum, and intercostal arteries with displaced rib fractures. only after the serosal defect is identified should the organs be removed, because that procedure creates many more holes in the serosa. for that reason, as much information as possible should be gained by in situ observation. the only evidence of concussion of the heart may be a cardiac contusion or a sternal fracture. the usual clinical history suggests cardiovascular instability that is not associated with craniocerebral trauma and which does not respond to the infusion of intravenous volume agents. the autopsy assistant may saw but should not retract the skull cap and remove the brain. the pathologist should observe in situ whether shallow lacerations of the pontomedullary junction with stretching of the midbrain are present. these lesions cannot be distinguished from artifact by examining the brain later. thus, only after appropriate in situ inspection should the pathologist remove the brain. a posterior neck dissection is required if no lethal craniocerebral or cardiovascular trauma is found, or if suffocation is suspected; neck trauma must be ruled out to diagnose suffocation in a traffic fatality. sudden death in a patient with seemingly trivial wounds may be caused by undiagnosed trauma of the craniocervical articulation. a posterior neck dissection is required in these instances. the diagnosis of diffuse axonal injury of the brain in victims with no appreciable survival interval requires that suffocation be ruled out and that no resuscitation from a cardiac arrest has been attempted. clinicians are quick to apply the label "closed head injury" when a victim of a traffic crash has cerebral edema on a computerized axial tomogram of the head, even if no cerebral contusions, scalp contusions, or skull fractures are evident. this may be a misinterpretation, because cerebral edema can be caused by hypoxic encephalopathy made evident after resuscitation from a cardiac arrest, or from hypoxia caused by suffocation. procedures possible or expected findings record presence of lividity. photograph all external wounds; measure all lacerations and any abrasions or contusions with a pattern. collect scalp hair and blood (see below) from victims of hit and run accidents. collect foreign material in wounds. intense lividity and absence of lethal wounds may indicate that the crash occurred because the driver was dead from heart disease or suffocation. wound documentation. patterned injuries often sometimes be matched to objects in or about the vehicle (the most common patterned wound is that from tempered glass; see above under "note"). impact patterns in pedestrians may help to reconstruct the accident. hair and blood of the victim may be matched to transfer evidence on a vehicle suspected of having left the scene. part ii / diseases and conditions internal examination of body cavities heart and great vessels abdomen skull and brain; neck soft tissue compartments at any location prepare roentgenograms of chest is cases with head impact and skull fractures. collect samples for toxicologic study from all victims, including passengers. create pleural window to detect pneumothorax. if blood is seen, examine internal mammary vessels (see under "note"). measure volume of blood in cavity bleeds, and note whether chambers of heart and great vessels are collapsed or filled. record evidence of cardiac contusion, sprain of intracardiac inferior vena cava, laceration of pericardial sac, and fracture of sternum. laceration of heart or great vessels (measure volume of blood). follow routine procedures for dissection of heart and great vessels (see chapter ) . in situ bubble test to confirm venous air embolism. record evidence of trauma and volume of blood in peritoneal cavity; estimated volume of blood in retroperitoneal soft tissues. autopsy assistant may saw the skull but pathologist should inspect brain in situ and remove it personally. for removal and specimen preparation of brain, see chapter . record brain weight. posterior neck dissection is indicated if there is no craniocerebral or cardio-vascular trauma, or if suffocation is suspected. record evidence of trauma and estimate volume of blood. venous air embolism.' evidence of alcohol or drug intoxication. pneumothorax, hemothorax, e.g., after laceration of internal mammary vessels. evidence of significant hemorrhage. indirect evidence of cardiac concussion. evidence of exsanguinating wounds. evidence of cardiovascular disease that may have felled the driver before the crash. in european countries, the concentration is expressed in promille (grams per liter). in the united states, it has become customary to refer to concentration by percentage (grams per deciliter), and values in these units have been written into legislation and included in the uniform vehicle codes. unless qualified, the use of promille or percentage does not indicate whether the result of the analysis is weight/weight, weight/ volume, orvolume/volume. another common way ofexpressing concentration, milligrams per deciliter, has also been used to indicate alcohol concentrations. the method ofexpressing concentration must be clearly specified whenever the alcohol level is mentioned. the desired expression canbe derived from the toxicologic report by using the following equation: i, ~g/ml = mg/dl = . g/dl = . mmolll = . promille = . % what is the legal interpretation of alcohol (ethanol) intoxication? objective impairment of driving ability is observed at threshold blood alcohol concentrations of . -. g/dl. as of august all states and the district of columbia have adopted laws that make it criminal offense for a driver to operate a motor vehicle with a blood alcohol concentration of . g/ dl or greater. many states have an enhanced penalty for high concentrations such as . g/dl or above. several states have zero tolerance laws, under which drivers who are minors are legally operating only if their blood alcohol concentration is . g/dl or less, and in some states, not detectable at all. blood alcohol concentrations obtained at autopsy are valid until putrefaction begins. specimen tubes with sodium fluoride should be used, and the specimen should be stored in the refrigerator. if the air space above the blood samples in the container is large, alcohol can evaporate and a falsely low blood alcohol level can result. putrefactive changes before autopsy or during storage may cause a falsely high blood alcohol concentration. ethanol can be produced in the specimen container; this is more likely in the absence of a preservative. because fluoride inhibits bacteria far more than fungi, higher fluoride concentrations are required for the inhibition of fungal growth ( ) . although there is no major difference in the alcohol concentrations ofblood samples from the intact heart chambers and the femoral vessels ( ), autopsy samples from pooled blood in the pericardial sac or pleural cavity are unsatisfactory. we therefore recommend that blood be withdrawn from peripheral vessels. is there normal "endogenous" blood alcohol (ethanol) in a living person? blood alcohol concentrations are generally believed to be negligible in the absence of ingested alcohol. "endogenous" ethanol in human blood exists at a concentration of about . g/dl, which is below the limit of detection for most methods ( ) . first in such a list would be postural asphyxia, for example, in drunks who fall asleep face down. also, depressant drugs in the tricyclic, analgesic, barbiturate, and benzodiazepine classes all potentiate the effect of alcohol ( ) . also included in such a list would be infancy and childhood; ischemic heart disease;' chronic bronchitis and emphysema;' other chronic debilitating diseases; poisoning with carbon tetrachloride' or carbon monoxide;' and other causes of hypoxia.' how can one estimate blood alcohol (ethanol) concentrations from vitreous, urine, or tissue alcohol levels and from alcohol in stomach contents? the ratio of serum, plasma, urine, vitreous, and various tissues has been compiled by garriot ( ) . the values may vary considerably. for vitreous, the ratios varied from . - . . these variations may depend on whether blood alcohol concentrations were increasing or decreasing at the time of death. most other body fluids and tissues showed ranges closer to . most urine values were above the blood alcohol concentrations. in another study ( ) , the blood/vitreous (bn) ratio in the early absorption phase was . (range, . - . ; sd . ) and in the late absorption and elimination phase, the bn ratio was . (range, . - . ; sd . ). blood ethanol concentrations probably can be estimated using b = . v for early absorption and b = . v for later phases. a urinelblood ethanol ratio of . or less indicates that the deceased was in the early absorption phase. how can one use alcohol (ethanol) concentrations in postmortem specimens to estimate the blood alcohol concentration at various times before death? with certain limitations, one can base calculations of this kind on the assumption that the blood alcohol level decreases from its peak at a fairly constant rate of . -q. g/dl/h until death ( ) . if blood is not available, conversion factors (see above) must be used. alcoholics have been reported to metabolize at a rate of up to . g/dl/h ( ) . example: the driver of an automobile drinks at a party until midnight. he leaves his host at about : a.m. and is involved in a head-on collision at : a.m. he dies in the emergency room at : a.m. there are multiple injuries and the patient exsanguinates. the autopsy is done at : p.m. although this appears quite unlikely, let us assume that no satisfactory blood sample was obtained before death and that no blood or plasma expanders were given. if under such circumstances the alcohol concentration in the vitreous was found to be . g/dl, what was the alcohol concentration in the blood at the time of the accident? vitreous and blood alcohol concentrations may be assumed to have remained unchanged after death. therefore, the blood alcohol level at the time of death must have been approx . (vitreous humor alcohol) x . (conversion factor, see above) = . g/dl. the time interval between the accident ( : a.m.) and death ( : a.m.) is hand min or / h. if we assume that the decedent was not an alcoholic and that the blood alcohol concentration was decreasing from its peak at a constant rate of . g/dl/h, then the concentration at the time ofthe accident is estimated to have been . (concentration at time of death) + ( / x . ) = . + . = . g/dl or . %. the blood alcohol concentration at the time of the accident could have been lower if the victim stopped drinking later than h or / h before the accident. in the latter case, the peak alcohol level would have occurred after the accident, reflecting the time to absorb the latest drink. the blood alcohol concentration at the time of the accident could have been lower or higher if the time when the patient stopped drinking, the time of the accident, or the time of the death is uncertain. the blood alcohol concentration at the time of the accident could have been higher if the victim was a chronic alcoholic. the elimination rate in such persons may be as high as . mg/dl, which would change the figures in our example above to . + ( / x . ) = . + . = . g/d or . %. only rough estimates are possible. first, the peak blood alcohol level must be determined or calculated, as described in the previous paragraphs. tables (see below) are available that relate blood alcohol level to the minimal amounts of whiskey, wine, or beer that must have been consumed ( ) . however, tables of this type are often based on the minimum amount of alcohol circulating in the body after specific numbers of drinks; such tables do not yield reliable results if used conversely. furthermore, inasmuch as drinking and elimination of alcohol may take place concomitantly, over a longer period the total amount of alcohol consumed may have been much greater than the tables would indicate. it cannot be lower. according to these tables, pints of ordinary beer or fl oz of whiskey would be the minimal amounts needed to produce a blood alcohol level of about mg/dl in a person weighing - pounds. the total body alcohol can be calculated from the blood alcohol level by using widmark's formula: average concentration of alcohol in entire body = . concentration of alcohol in the blood in a person weighing kg, the blood alcohol concentration would be increased mg/dl ( . %) by the absorption of oz of ethanol ( z of -proof whiskey). strength of alcohol is measured in "proof'; absolute alcohol is proof. therefore, in the united states, alcohol content as volume percent is half the proof (for example, -proof whiskey contains % alcohol by volume). the alcohol content of various beverages is shown in the following table. approximate alcohol content in various beverages t toata from glaister, rentoul e. medical jurisprudence and toxicology, th ed. e & s livingstone, edinburgh, with permission. twithin h after consumption of diluted alcohol (approx %) on an empty stomach, assuming body weight of - pounds ( . - . kg) reproduced from ( ) with permission. *one ounce (about ml) of whiskey or z (about ml) of beer. what is the toxicity of alcohol other than ethanol? in general, the toxicity increases as the number of carbon atoms in the alcohol increases. thus, butyl alcohol is two times as toxic as ethyl alcohol: but isopropyl alcohol is only twothirds as toxic as isobutyl alcohol and one-half as toxic as amyl alcohol. primary alcohols are more toxic than the corresponding secondary isomers ( ) . anemia, hemolytic synonyms and related terms: acquired hemolytic anemia; extracorpuscular hemolytic anemia; hereditary hemolytic anemia (hereditary elliptocytosis, pyropoikilocytosis, stomatocytosis. spherocytosis); immunohemolytic anemia; intracor-puscular hemolytic anemia; microangiopathic hemolytic anemia; spur cell anemia. possible associated conditions: disseminated intravascular coagulation;* eclampsia;* glucose- -phosphatase deficiency (g pd); hemolytic uremic syndrome;* malignant hypertension; lymphoma* and other malignancies; paroxysmal nocturnal hemo-globinuria; sickle cell disease;*thalassemia;* thrombotic thrombocytopenic purpura.* (see also below under "note.") note: hemolysis also may be caused by conditions such as poisoning with chemicals or drugs, heat injury, snake bite,* or infections or may develop as a transfusion reaction* or be secondary to adenocarcinoma, heart valve prostheses (see below), liver disease (see below), renal disease, or congenital erythropoietic porphyria. * procedures prepare skeletal roentgenograms. jaundice; skin ulcers over malleoli. in young patients: thickening of frontal and parietal bones with loss of outer table ("hairon-end" appearance); paravertebral masses caused by extramedullary hematopoiesis; deformities of metacarpals, metatarsals, and phalanges. osteonecrosis* of femoral heads. remove and place in fixative as early as possible in order to minimize autolysis (alternatively, formalin can be injected in situ; see below). samples should include oxyntic corpus and fundus mucosa. record weights. submit tissue samples for histologic study. record weight of thyroid gland. for removal and specimen preparation, see chapter . request luxol fast blue stain. for removal and specimen preparation, see chapter . if there is a clinical diagnosis of anemia-related amblyopia, follow procedures described under "amblyopia, nutritional." jaundice. manifestations of malnutrition. * stomatitis with cheilosis and perianal ulcerations due to folic acid deficiency. chronic exfoliative skin disorders. vitiligo. macrocytosis; poikilocytosis; macroovalocytes; hypersegmentation of leukocytes; abnormal platelets. atrophic glossitis with ulcers. pharyngoesophagitis (folic acid deficiency). previous total or subtotal gastrectomy. carcinoma of stomach. autoimmune gastritis (diffuse corporal atrophic gastritis) with intestinal metaplasia. crohn's disease;* sprue;* other chronic inflammatory disorders; jejunal diverticula; intestinal malignancies; fish tapeworm infestation; previous intestinal resection or blind intestinal loop; enteric fistulas. hepatosplenomegaly. alcoholic liver disease. * giant epithelial cells. hyperthyroid goiter; thyroiditis. demyelination of cerebral white matter (in advanced cases). demyelination in posterior and lateral columns of spinal cord, most frequently in thoracic and cervical segments. demyelination of peripheral nerves. retinal hemorrhages; demyelination of optic nerves. hypercellular; megaloblastic. myeloproliferative disorder. brain other organs if mycotic aneurysms are expected and microbiologic studies are intended, follow procedures described below under "aneurysm, mycotic aortic." request verhoeff-van gieson, gram, and grocott's methenamine silver stains. for cerebral arteriography, see chapter . if arteriography cannot be carried out, rinse fresh blood gently from base of brain until aneurysm can be identified. record site of rupture and estimated amount of extravascular blood. for paraffin embedding of aneurysms, careful positioning is required. expected findings depend on type of aneurysm. mycotic aneurysms are often multiple and deep in brain substance. berry aneurysms are the most frequent types and often are multiple. most frequent sites are the bifurcations and trifurcations of the circle of willis. saccular atherosclerotic aneurysms are more common than dissecting aneurysms, which are very rare. with congenital cerebral artery aneurysm: coarctation of aorta;* manifestations of hypertension;* and polycystic renal disease. with mycotic aneurysm: infective endocarditis;* pulmonary suppurative processes; and pyemia. aneurysm, dissecting aortic (see "dissection, aortic.") aneurysm, membranous septum of heart note: for general dissection techniques, see chapter . most aneurysms ofthe membranous septum probably repre-sent spontaneous closure of a membranous ventricular septal defect by the septalleafiet of the tricuspid valve. aneurysm, mycotic aortic note: (i) collect all tissues that appear to be infected. ( ) request aerobic, anaerobic, and fungal cultures. ( ) request gram and grocott methenamine silver stains. ( ) no special precautions are indicated. ( ) no serologic studies are available. ( ) this is not a reportable disease. chest and abdominal organs aorta other organs submit blood samples for bacterial culture. en masse removal of adjacent organs is recommended. photograph all grossly identifiable lesions. aspirate material from aneurysm or para-aortic abscess and submit for culture. prepare sections and smears of wall of aneurysm and of aorta distant from aneurysm. request verhoeffvan gieson and gram stains. septicemia and infective endocarditis. * streptococcus, staphylococcus, spirochetes, and salmonella can be found in mycotic aneurysm. para-aortic abscess. septic emboli with infarction or abscess formation. aneurysm, syphilitic aortic part ii / diseases and conditions heart and aorta other organs en masse removal of organs is recommended. for coronary arteriography, see chapter . request verhoeff-van gieson stain from sections at different levels of aorta, adjacent great vessels, and coronary arteries. see also under "syphilis." aneurysm usually in ascending aorta. may erode adjacent bone (sternum). syphilitic aortitis may cause intimal wrinkling, narrowing of coronary ostia, and shortening of aortic cusps. disruption of medial elastic fibrils. aortic valvulitis and insufficiency;* syphilitic coronary arteritis; syphilitic myocarditis. external examination aorta prepare chest and abdominal roentgenograms. open aorta along line of blood flow, or bisect into anterior and posterior halves. photograph tear(s). measure bloody effusions in body cavities. measure or estimate amount of blood in mediastinum. request verhoeff-van gieson stain. cutaneous impact trauma. mediastinum widened by hemorrhage in case of tarnponaded dissection. a bleed into a body cavity of less-thanexsanguinating volume should point to an alternate mechanism of death such as neurogenic shock or lethal concussion; a posterior neck dissection may be required in such instances. microscopy may show transmural rupture, false aneurysm, or localized dissection. angiitis (see "arteritis, all types or type unspecified.") angina pectoris note: see under "disease, ischemic heart" and chapter . angiokeratoma corporis dittusum (see "disease, fabry's.") angiomatosis, encephalotrigeminal (see "disease, sturge-weber-dimitri.") angiopathy, congophilic cerebral synonyms and related terms: beta amyloid angiopathy due to~-amyloid peptide deposition (~a ) (associated with alzheimer's disease; hereditary cerebral hemorrhage with amyloid angiopathy of dutch type; or sporadic beta amyloid angiopathy); hereditary cerebral amyloid angiopathy, due to deposition of other amyloidogenic proteins such as cystatin c (icelandic type) and others (e.g., transthyretin, gelsolin) ( ). procedures possible or expected findings request stains for amyloid, particularly congo red, and thioflavine s (examine with polarized and ultraviolet light, respectively). request immunostain for~a . some tissue should be kept frozen for biochemical studies. multiple recent cerebral cortical infarctions or small cortical hemorrhages, or both, or massive hemispheric hemorrhages, both recent and old. amyloid deposition in leptomeninges and cortical blood vessels. senile plaques are usually present. in some cases, angiopathy is part of alzheimer's disease. * other organs a prepare material for electron microscopy. electron microscopic study permits definite confirmation of diagnosis. organs and tissues may be minimally affected by amyloidosis. anomaly, coronary artery possible associated conditions: with double outlet right ventricle; persistent truncal artery; tetralogy of fallot;* and transposition of the great arteries.* note: coronary artery between aorta and pulmonary artery, often with flap-valve angulated coronary ostium. coronary artery may communicate with cardiac chamber, coronary sinus, or other cardiac veins, or with mediastinal vessel through pericardial vessel. saccular aneurysm of coronary artery with abnor-mal flow, infective endarteritis of arteriovenous fistula, and myocardial infarction may be present. ifone or both coronary arteries originate from pulmonary trunk, myocardial infarction may be present. heart perform coronary angiography. if infective endarteritis is suspected, submit blood sample for microbiologic study. ectopic origin of coronary arteries or single coronary artery. sudden death. for a detailed description of possible additional findings, see above under "note." anomaly, ebstein's (see "malformation, ebstein's") anorexia nervosa note: sudden death from tachyarrhythmias may occur in advanced cases and thus, autopsy findings may not reveal the immediate cause of death. external examination all organs record height and weight, and prepare photographs to show cachectic features. record abnormalities as listed in righthand column. follow procedures described under "starvation." record weight of endocrine organs and submit samples for histologic study. cachexia, often with preserved breast tissue; hirsutism; dry, scaly, and yellow skin (carotenemia). mild edema may be present. parotid glands may be enlarged. manifestations of starvation.* ovaries tend to be atrophic; other endocrine organs should not show abnormalities. synonyms: cutaneous anthrax; gastrointestinal anthrax; pulmonary (inhalational) anthrax. note: ( ) collect all tissues that appear to be infected. this is a reportable disease. bioterrorism must be considered in current cases. external examination and skin blood photograph cutaneous papules, vesicles, and pustules. prepare smears and histologic sections. submit samples for bacteriologic study. submit sample for serologic study. disseminated anthrax infection may occur without skin lesions. edema of neck and anterior chest in nasopharyngeal anthrax. anthrax septicemia. see above under "note." part ii i diseases and conditions lungs gastrointestinal tracts and mesentery neck organs record character and volume of effusions. after sampling for bacteriologic study (see above under "note") perfuse one or both lungs with formalin. extensive sampling for histologic study is indicated. extensive sampling for histologic study is indicated. photograph meningeal hemorrhage in situ. pleural effusions;* hemorrhagic mediastinitis; anthrax pneumonia (inhalational anthrax; woolsorter's disease). histologic sections reveal hemorrhagic necrosis, often with minimal inflammation and gram-positive, spore-forming, encapsulated bacilli. gastrointestinal anthrax with mucosal edema and ulcerations. hemorrhagic mesenteric lymphadenitis. tongue, nasopharynx, and tonsils may be involved. hemorrhagic meningitis (hemorrhage tends to predominate). external examination distal colon and rectum photograph perineum. measure depth of anal pit, if any. dissect distal colon, rectum, and perirectal pelvic organs in situ (as much as possible). search for opening of fistulous tracts from lumen. use roentgenologic study or dissection, or both, to determine course of tract. absence of normally located anus; anal dimple. abnormal termination of the bowel into the trigone of the urinary bladder, the urethra distal to the verumontanum, the posterior wall of the vagina, the vulva, or the perineum. aortitis note: see also under "arteritis" and "aneurysm, ascending aortic." heart and aorta other organs and tissues remove heart with whole length of aorta and adjacent major arteries. record width and circumference of aorta at different levels. describe and photograph appearance of intima and of orifices of coronary arteries and other aortic branches. submit multiple samples for histologic study and request verhoeff-van gieson stain. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. secondary aortic atherosclerosis or intimal fibroplasia. widening of aorta; syphilitic aneurysm. * giant cell aortitis; rheumatoid aortitis; syphilitic aortitis; takayasu's arteritis.* manifestations of rheumatoid arthritis, * syphilis,* systemic sclerosis,* hodgkin's lymphoma, and many other diseases associated with vasculitis. external examination brain spine and spinal cord other organs prepare roentgenogram of spine. for removal and specimen preparation, see chapter . for removal of spinal cord and specimen preparation, see chapter . expose nerve roots. record appearance and photograph spinal cord in situ. submit samples of spinal cord and inflamed tissue for histologic study. request gram, gomori's iron, and grocott's methenamine silver stains. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. signs of previous spinal surgery or lumbar puncture (myelography). evidence of previous trauma or previous myelography. cerebral arachnoiditis. fibrous arachnoidal adhesions and loculated cysts. tuberculosis;* syphilis;* fungal or parasitic infection. systemic infection (see above). ascending urinary infection or other manifestations of paraplegia. arch, aortic, interrupted synonym: severe coarctation. note: the basic anomaly is a discrete imperforate region in the aortic arch, with a patent ductal artery joining the descending thoracic aorta. type a interruption is between the left subclavian and ductal arteries; type b between the left subclavian and left common carotid arteries; and type c (rare) between the left common carotid and brachiocephalic (innominate) arteries. for general dissection techniques, see part i, chapter . possible associated conditions: bicuspid aortic valve (with type a); di george syndrome* with thymic and parathyroid aplasia (with type b); hypoplasia of ascending aorta (with all types); persistent truncal artery (truncus arteriosus); ventricular septal defect. arrhythmia, cardiac note: see also under "death, sudden cardiac." toxicologic studies may be indicated, for instance, if digitalis toxicity (see "poisoning, digitalis") is suspected. if a cardiac pacemaker had been implanted, the instrument should be tested for malfunction. arteriosclerosis (see "atherosclerosis.") arteritis, all types or type unspecified synonyms and related terms: allergic angiitis and granulomatosis (churg-strauss);* allergic vasculitis; anaphylactoid purpura* and its synonyms; angiitis; buerger's disease;* cranial arteritis; giant cell arteritis;* granulomatous arteritis (angiitis); hypersensitivity angiitis; infectious angiitis; necrotizing arteritis; polyarteritis nodosa;* rheumatic arteritis; rheumatoid arteritis, syphilitic arteritis; takayasu's arteritis;* temporal arteritis; thromboangiitis obliterans; and others (see also below under "note"). note: autopsy procedures depend on ( ) the expected type of arteritis, such as giant cell arteritis,* polyarteritis nodosa,* or thromboangiitis obliterans (buerger's disease*); and ( ) the nature of suspected associated or underlying disease, such as aortic arch syndrome,* beh~et's syndrome,* cogan's syndrome, degos' disease,* dermatomyositis,* erythema nodosum and multiforme,* goodpasture's syndrome,* polymyositis, rheumatic fever, * rheumatoid arthritis,* syphilis,* and other nonspecific infectious diseases, systemic lupus erythematosus,* systemic sclerosis (scleroderma),* or takayasu's disease. for histologic study of blood vessels, verhoeff-van gieson stain or a similar stain is recommended. temporal and ophthalmic arteritis. arteritis of ciliary and retinal vessels. clinically, polymyalgia. anemia. arteritis, takayasu's synonyms: aortic arch syndrome; pulseless disease. external examination heart, aorta, and adjacent great vessels kidney eyes and optic nerve brain for in situ aortography, clamp distal descending thoracic aorta and neck vessels as distal as possible from takeoff at aortic arch. remove heart together with aorta and long sleeves of neck vessels. for coronary arteriography, see chapter (method designed to show coronary ostia). test competence of aortic valve. open aortic arch anteriorly and measure (with calipers) lumen at origin of great neck vessels. photograph aorta and neck vessels and submit samples for histologic study. request verhoeffvan gieson stain. submit tissue for histologic examination. for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . facial muscular atrophy and pigmentation. narrowing at origin of brachiocephalic arteries. dilated ascending aorta. narrowing of coronary arteries at origins. myocardial infarction. aortic insufficiency. * aortic atherosclerosis. thromboses of brachiocephalic arteries. giant cell arteritis. * diffuse mesangial proliferative glomeulonephritis ( ) . atrophy of optic nerve, retina, and iris; cataracts; retinal pigmentation. ischemic lesions. artery, patent ductal synonym: patent ductus arteriosus. note: the basic anomaly is persistent postnatal patency of the ductal artery, usually as an isolated finding (in % of cases in infants, and in % in adults). it is more common in premature than full-term infants and at high altitudes than at sea level. possible complications in unoperated cases include congestive heart failure, * plexogenic pulmonary hypertension,* ductal artery aneurysm or rupture, fatal pulmonary embolism,* or sudden death. in some conditions, such as aortic atresia* or transposition with an intact ventricular septum,* ductal patency may be necessary for survival. possible associated conditions: atrial or ventricular septal defect;* coarctation ofthe aorta;* conotruncal anomalies; necrotizing enterocolitis in premature infants; postrubella syndrome; and valvular or vascular obstructions. artery, persistent truncal synonym and related terms: type i, pulmonary arteries arise from single pulmonary trunk (in %); type , pulmonary arteries arise separately but close-by (in %); type , pulmonary arteries arise separately but distal from one another (in %). note: the basic anomaly is a common truncal artery, with truncal valve, giving rise to aorta, pulmonary arteries, and coronary arteries, usually with a ventricular septal defect. interventions include complete rastelli-type repair, with closure of ventricular septal defect, and insertion of valved extracardiac conduit between right ventricle and detached pulmonary arteries. possible associated conditions: absent pulmonary artery (in %); atrial septal defect (in %); absent ductal artery (in %); coronary ostial anomalies (in %); di george syndrome;* double aortic arch; extracardiac anomalies (in %); interrupted aortic arch* (in %); right aortic arch (in %); truncal valve insufficiency (uncommon) or stenosis (rare); trun-cal valve with three (in %), four (in %), or two (in %) cusps. heart and great vessels if infective endocarditis is suspected, follow culture procedures for endocardial vegetation described in chapter . request verhoeff-van gieson stain. infective endocarditis,* usually of truncal valve. late postoperative conduit obstruction. postoperative late progressive truncal artery dilation with truncal valve insufficiency. hypertensive pulmonary vascular disease. cerebral abscess,* if right-to-ieft-shunt was present. arthritis, all types or type unspecified note: for extra-articular changes, see under the name of the suspected underlying conditions. infectious diseases that may be associated with arthritis include bacillary dysentery, * brucellosis, * gonorrhea, rubella,* syphilis, * tuberculosis, * typhoid fever, * and varicella. * noninfectious diseases in this category include acromegaly,* beh<;et's syndrome,* felty's syndrome,* gout,* rheumatoid arthritis,* and many others, too numerous to mention. remove synovial fluid and prepare smears. submit synovial fluid for microbiologic and chemical study. for removal of joints, prosthetic repair, and specimen preparation, see chapter . for removal and specimen preparation, see chapter . in the polyarticular variant, facial asymmetry may be noted. rheumatoid factor positive in some cases. pericarditis.* interstitial pneumonitis; pleuritis. (see also under "arthritis, rheumatoid.") lymphadenopathy. splenomegaly. monarthritis or severe, erosive polyarthritis; see also under "arthritis, rheumatoid" and above under "externalexamination and skin." ankylosing spondylitis* may be present. chronic iridocyclitis. see "arthritis, rheumatoid." arthritis, rheumatoid synonyms and related terms: ankylosing spondylitis;* felty's syndrome;* juvenile rheumatoid arthritis* (still's disease); rheumatoid disease; and others. possible associated conditions: amyloidosis;* polymyositis (dermatomyositis*); psoriasis;* sjogren's syndrome;* systemic lupus erythematosus;* systemic vasculitis, and others. subcutaneous rheumatoid nodules on elbows, back, areas overlying ischial and femoral tuberosities, heads of phalangeal and metacarpal bones, and occiput. deformities and subluxation of peripheral joints (see also below under "joints"). subaxial dislocation of cervical spine may be cause of sudden death. pneumothorax;* pleural empyema.* t-cell abnormalities ( ) . bacteremia. positive rheumatoid factor. rheumatoid granulomas in myocardium (septum), pericardium, and at base of aortic and mitral valves; constrictive pericarditis;* aortic stenosis;* coronary arteritis. systemic vasculitis (arteritis*). rheumatoid granulomas in pleura and lung (with pneumoconiosis*); bronchopleural fistula; rheumatoid pneumonia with interstitial pulmonary fibrosis and honeycombing; bronchiectasis;* bronchiolitis with cystic changes; pulmonary arteritis. pneumoconiosis* in caplan arthrogryposis ( ) may be a primary muscle disease, or it may involve abnormalities of the brain, spinal cord, and/or peripheral nerves. etiologies are numerous, as are the modes of inheritance. critical to making the appropriate diagnosis is the collection of muscles from various sites for routine histology, muscle histochemistry, and electron microscopy. portions of peripheral motor nerves must also be prepared for histology and electron microscopy. abdominal cavity intra-abdominal lymphatic system puncture abdominal cavity and submit fluid for microbiologic study. record volume of exudate or transudate and submit sample for determination of fat and cholesterol content. prior to routine dissection, lymphangiography (see below) may be indicated. possible associated conditions: with pulmonary aspergillosis-bronchiectasis; * bronchocentric granulomatosis;* sarcoidosis;* tuberculosis. * with systemic aspergillosisleukemia;* lymphoma;* and other conditions complicated by immunosuppression (l, ) . other organs a carefully make multiple parasagittal sections through the unperfused lungs. culture areas of consolidation. if diagnosis was confirmed, perfuse lungs with formalin. prepare histologic sections from walls of cavities, cavity contents, and pneumonic infiltrates. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. assault note: all procedures described under "homicide" must be followed. asthma note: spray death* may occur in asthma sufferers from pressurized aerosol bronchodilators. record thickness and position. perfuse one lung with formalin. because mucous plugs may block bronchial tree, attach perfusion apparatus to pulmonary artery or to bronchus and pulmonary artery. monitor perfusion to ensure proper inflation. prepare photograph of fixed cut section. submit samples of pulmonary parenchyma and bronchi for histologic study. request azure-eosin and verhoeff-van gieson stains. record weight and thickness of walls. leave attached to stomach. photograph and submit samples for histologic study. eczema. conjunctival hemorrhages and subcutaneous emphysema may be present after fatal attack. pneumothorax;* mediastinal emphysema. low diaphragm (see below). increased igeconcentrations in fatal asthma; postmortem tryptase determination is of doubtful value in this regard ( ) . hypertrophy. low position of diaphragm. hyperinflated lungs. thick-walled bronchi with prominent viscid mucous plugs. typical microscopic inflammatory changes ( ) . asthmatic bronchitis with eosinophilic infiltrates. bronchocentric granulomatosis.* pulmonary atherosclerosis with breakup of elastic fibers. paucity of ecosinophils in mucous ( ) . cor pulmonale. refl ux esophagitis ( ) . peptic ulcer. * pneumatosis of small intestine; emphysema of colon. centrilobular congestion and necrosis. petechial hemorrhages in hypothalamus; necrosis of cerebellar folia; anoxic changes in cortex, globus pallidus, thalamus, sommer's sector of hippocampus, and purkinje cells of cerebellum. suspected changes in anterior hom cells of spinal cord in patients with asthma-associated poliomyelitis-like illness (hopkins syndrome) ( ). allergic polyps and other allergic inflammatory changes ( ) . increased erythropoiesis. atresia, aortic valvular synonym: aortic atresia; aortic atresia with intact ventricular septum; hypoplastic left heart syndrome. note: the basic anomaly is an imperforate aortic valve, with secondary hypoplasia ofleft-sided chambers and ascending aorta. for possible surgical interventions, see two-stage norwood and modified fontan procedures in chapter . possible associated conditions: atrial septal defect* (or patent foramen ovale, usually restrictive); dilatation of myocardial sinusoids thatcommunicate with coronary vessels; dilatation of right atrium, right ventricle, and pulmonary trunk; fibroelastosis ofleft atrial and left ventricular endocardium; hypertrophy of ventricular and atrial walls; hypoplastic left atrium, mitral valve, left ventricle, and ascending aorta; mitral atresia* with minute left ventricle; patent ductal artery (ductus arteriosus); small left ventricle with hypertrophic wall; tubular hypoplasia of aortic arch, with or without discrete coarctation. synonyms and related terms: congenital biliary atresia; extrahepatic biliary atresia; infantile obstructive cholangio-pathy; syndromic (alagille's syndrome) or nonsyndromic paucity of intrahepatic bile ducts ("intrahepatic" biliary atresia). possible associated conditions: alpha]-antitrypsin deficiency;* choledochal cyst;* congenital rubella syndrome;* polysplenia syndrome* ( ); small bowel atresia; trisomy - ; trisomy ; turner's syndrome;* viral infections (cytomegalovirus infection;* rubella*). dissect extrahepatic bile ducts in situ or leave hepatoduodenalligament intact for later fixation and sectioning (see below). record appearance and contents of gallbladder and course of cystic duct. in postoperative cases, submit sample of anastomosed hepatic hilar tissue for demonstration of microscopic bile ducts. remove liver with hepatoduodenalligament. prepare horizontal sections through ligament and submit for histologic identification of ducts or duct remnants. prepare frontal slices of liver and sample for histologic study. request pas stain with diastase digestion. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. jaundice. congenital rubella and other viral infections. alpha]-antitrypsin deficiency;* defects in bile acid synthesis. chromosomal abnormalities. in atresia of the hepatic duct, the gallbladder will be empty. in isolated atresia of the common bile duct, the gallbladder contains bile but it cannot be squeezed into the duodenum. atresia or hypoplasia of bile duct(s); choledochal cyst(s). biliary drainage created by kasai operation. obliterative cholangiopathy ( ) . intrahepatic cholelithiasis; postoperative ascending cholangitis; secondary biliary cirrhosis; giant cell transformation; paucity of intrahepatic bile ducts. pas-positive inclusions in alphal-antitrypsin deficiency.* polysplenia syndrome* ( ) with malrotation, situs inversus, preduodenal portal vein, absent inferior vena cava, anomalous hepatic artery supply, and cardiac defects. for other abnormalities outside the biliary tree, see under "possible associated conditions"). nephromegaly ( ) . atresia, cardiac valves (see "atresia, aortic valvular," "atresia, mitral valvular," "atresia pulmonary valvular, with intact ventricular septum," "atresia, pulmonary valvular, with ventricular septal defect," and "atresia, tricuspid valvular.") atresia, duodenal possible associated conditions: with membranous obstruction of the duodenum-annular pancreas; atresia of esophagus* with tracheoesophageal fistula; congenital heart disease; cystic fibrosis;* down's syndrome;* hirschsprung's disease; imperforate anus* or other congenital obstructions of the intestinal tract ( ); intestinal malrotation; lumbosacral, rib-, and digitllimb anomalies; single umbilical artery; spinal defects; undescended testis ( ). see also under "atresia, small intestinal." the basic anomaly is an imperforate pulmonary valve, with a hypoplastic right ventricle. in unoperated cases, ductal closure is the most common cause of death. for possible surgical interventions, see modified blalock-taussig shunt, mod-ified fontan procedure, and pulmonary valvulotomy in chapter . for general dissection techniques, see chapter . possible associated conditions: dilated myocardial sinusoids that may communicate with epicardial coronary arteries or veins; patent ductal artery (ductus arteriosus); patent oval foramen (foramen orale); tricuspid atresia with minute right ven-tricle; tricuspid stenosis with hypoplastic right ventricle (in %); tricuspid insufficiency with dilated right ventricle (in %). synonym: tetralogy of fallot with pulmonary atresia. note: the basic anomaly is atresia of the pulmonary valve and ofvariable length ofpulmonary artery, and ventricular septal defect (membranous or outlet type), with overriding aorta, and with pulmonary blood supply from ductal or systemic collateral arteries. for possible surgical interventions, see rastelli-type repair and unifocalization of multiple collateral arteries in chapter . possible associated conditions: right ventricular outflow tract a short blind-ended pouch ( %) or absent ( %); atresia of pulmonary artery bifurcation, with nonconfluent pulmonary arteries; right aortic arch ( %); atrial septal defect ( %); persistent left superior vena cava; anomalous pulmonary venous connection; tricuspid stenosis or atresia; complete atrioventricular septal defect; transposed great arteries; double inlet left ventricle; asplenia, polysplenia, or velocardiofacial syndromes; dilated ascending aorta, with aortic insufficiency. related term: jejuno-ileal atresia. possible associated findings: esophageal atresia* with tracheoesophageal fistula; lumbosacral, rib-, or digit/limb anom -alies; undescended testes (l) . note: see also under "atresia, duodena ." fascia lata, blood, or liver these specimens should be collected using aseptic technique for tissue culture for chromosome analysis (see chapter ) . intestinal tract for mesenteric angiography, see chapter . leave mesentery attached to small bowel, particularly to the atretic portion. trisomy . multiple atresias; proximal dilatation; volvulus; malrotation; meconium impaction; other evidence of cystic fibrosis. anorectal malformation (l) . annular pancreas ( ). atresia, tricuspid valvular note: the basic anomaly is an absent right atrioventricular connection ( %) or imperforate tricuspid valve ( %), with a hypoplastic right ventricle ( %), muscular ventricular septal defect ( %) that is restrictive ( %), and a patent oval atresia, urethral foramen ( %) or secundum atrial septal defect ( %). for possible surgical interventions, see modified fontan or glenn procedures in chapter . for general dissection techniques, see chapter . possible associated conditions: juxtaposed atrial appendages; large left ventricular valvular orifice; large left ventricular chamber; persistent left superior vena cava; pulmonary atresia; transposition of the great arteries ( %), with aortic co-arctation ( % of those); anomalies of musculoskeletal or digestive systems ( %); down's,* asplenia, or other syndromes. heart aorta and cervical arteries brain if infective endocarditis* is suspected, culture using the method described in chapter . for dissection of carotid and vertebral arteries, see chapter . for removal and specimen preparation, and cerebral anteriography, see chapter . if a foreign body is discovered during a medicolegal autopsy or if the discovery of a foreign body may have medicolegal impli-cations (e.g., presence of a surgical instrument in the abdominal cavity), the rules of the chain of custody apply. for the handling of bullets or bullet fragments, see "injury, firearm." if analysis offoreign material is required, commercial laboratories may be helpful. bolus (see "obstruction, acute airway!') burns note: fatal bums should be reported to the medical examiner's or coroner's office. the questions to be answered by the pathologist depend on whether the incident was accidental, sui-cidal, or homicidal, and whether the victim survivied to be treated in the hospital. a pending death certificate should be issued if the fire and police investigators are not sure of the circumstances at the time of the autopsy. for electrical bums, see under "injury, electrical." for victims who were treated at the hospital, autopsy procedures should be directed toward the discovery or confirmation of the mechanism of death, such as sepsis or pulmonary embolism.* death can be caused primarily by heart disease, with other-wise minor bums and smoke inhalation serving as the trigger that leads to lethal ventricular arrhythmia. because carbon monoxide concentrations are halved approx every min with % oxygen therapy, the pathologist must obtain the first clinical laboratory test results for co-hemoglobin. soot can be detected with the naked eye or d after inhalation of smoke. ambulance records should be examined to determine whether a persistent coma might have been caused by hypoxic encephalopathy following resuscitation from cardiac arrest at the scene. admission blood samples should be acquired to test for cohemoglobin and alcohol. this may not have been done in the emergency room. persons suffering from chronic alcoholism succumb to fire deaths more often than persons who do not drink. a very high initial serum alcohol concentration suggests a risk factor for the fire and presence of chronic alcoholism. patients with chronic alcoholism typically are deprived of alcohol when they are in the bum unit and this can cause sudden, presumably cardiac, death,just as it occurs under similarcircum-stances, not complicated by bums. under these circumstances, the heart fails to show major abnormalities. this mode of dying seems to have no relationship to the presence or absence of liver disease. if the body is found dead and charred at the scene, prepare whole body roentgenograms, before and after removal of remanants of clothing. see also under "identification of the body" and "external examination" in chapter ). one or two fingerpads may yield sufficient ridge detail for identification. if this is not possible, ante-and postmortem somatic and dental radiographs must be compared for identification, or dna comparison must be used. external examination, heart and lungs abdominal cavity and liver see below under "cardiomyopathy, dilated." record volume of ascites. record actual and expected weight of liver. request iron stain. see below under "cardiomyopathy, dilated." alcoholic cirrhosis and alcoholic cardiomyopathy rarely coexist. however, in genetic hemochromatosis,* cirrhosis and heart failure are common findings. cardiomyopathy, dilated (idiopathic, familial, and secondary types) note: for general dissection techniques, see chapter . external examination heart other organs and tissues record actual and expected weights. record ventricular thicknesses and valvular circumferences. evaluate relative atrial and ventricular chamber sizes. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. note: huntington's disease maps to the short arm of chromosome . the gene is widely expressed but of unknown function; it contains a cag repeat sequence, which is expanded (range, to ) in patients with huntington's disease. a sensitive diag-nostic test is based on the determination of this cag sequence, which can be done on fresh-frozen tissue or blood ( ) . in the absence of genetic confirmation, sampling of organs and tissues cannot be excessive because a complex differential diagnosis must be resolved. note: disseminated intravascular coagulation (dic) often is a complication of obstetrical mishaps such as abruptio placentae or amniotic fluid embolism,* or it complicates malignancies (such as adenocarcinomas or leukemia*) or bacterial, viral, and other infections. other conditions such as aortic aneurysm* or hemolytic uremic syndrome* are known causes also. ifthe nature of the underlying disease is known, follow the procedures under the appropriate heading also. note: this is a cause of diarrhea. microscopic colitis is associated with older age; collagenous colitis is associated with female sex ( ). the colon is grossly normal but microscopically, increased lymphocytes in the lamina propria and a subepithelial band of collagen is found. if only the lymphocytic infiltrate is found, the term "lymphocytic colitis" or "microscopic colitis" should be applied. a trichrome stain should be ordered in all instances, because the collagen band may be difficult to see without the special stain. i death, anaphylactic synonym: generalized anaphylaxis. note: autopsy should be done as soon as possible after death. neck organs should be removed before embalming. if death is believed to be caused by drug anaphylaxis, inquire about type of drug(s), drug dose, and route of administration (intravenous, intramuscular, and oral or other). this will determine proper sampling procedures-for instance, after penicillin anaphylaxis. allergy to bee stings, wasp stings, fire ants, and certain plants may also be responsible for anaphylaxis. however, envenomation also can be fatal in the absence of anaphylaxis. external examination search for injection sites or sting marks. if such lesions are present, photograph and excise with -cm margin. freeze excised tissue at - °c for possible analysis. prepare chest roentgenogram. foam in front of mouth and nostrils. swelling of involved tissue. antigen-antibody reaction in involved tissues. antibodies against suspected antigen. laryngeal edema may recede soon after death. foamy edema in trachea and bronchi; diffuse or focal pulmonary distention ("acute emphysema") alternating with collapse; pulmonary edema and congestion; accumulation of eosinophilic leukocytes. eosinophilic leukocytes in red pulp. death, anesthesia-associated. note: there are many possible causes of anesthesiaassociated death that are not drug-related, such as acute airway obstruction* by external compression, aspiration, arrhythmia of a heart not previously known to be diseased, tumor, or an inflammatory process. some ofthe complications are characteristically linked to a specific phase of the anesthesia, and many are not revealed by customary morphologic techniques. the task for the pathologist charged with investigating an anesthesia-associated death is to reconstruct the chain of physiologic events culminating in cessation of vital signs. autopsy morphology plays a supporting role; the main investigations center around the record left by the anesthesiologist, testing of anesthesia equipment, and toxicological testing. a consulting anesthesiologist can divine much more information from the anesthesia and recovery room records than can the pathologist, and can suggest avenues of further investigation. therefore, the most important step in these autopsies is to obtain the anesthesiaassociated records and to secure the consulting services of an independent anesthesiologist. the changes in the vital signs during and after anesthesia will help to focus the investigation toward a cardiac mechanism ofdeath or depression ofbrainstem function as a terminal mechanism. when information is gathered about drugs and chemical agents that have been administered or to which the victim may have had access, the pathologist must keep in mind that some non-medical chemicals and many drugs are known to affect anesthesia. drugs and their metabolic products, additives, stabilizers, impurities, and deterioration products (one of which can be carbon monoxide) may be present and can be identified in postmortem tissues. therefore, all appropriate body fluids and solid tissue should be submitted for toxicological examination. if the anesthetic agent was injected into or near the spinal canal, spinal fluid should be withdrawn from above the injected site into a standard toxicologist's collection tube with fluoride preservative. if the anesthetic agent was injected locally, tissue should be excised around the needle puncture marks at a radius of - em. serial postmortem analysis of specimens may permit extrapolation to tissue concentration at the time of death. the time interval between drug administration and death sometimes can be calculated from the distribution and ratio ofadministered drugs and their metabolic products. for a review of anesthetic death investigation, see ref. ( ) . halothane anesthesia and some other anesthetic agents may cause fulminant hepatitis and hepatic failure. the autopsy procedures suggested under "hepatitis, viral" should be followed. note: for special autopsy procedures in postoperative deaths, see chapter . in some instances, procedures described under "death, anesthesia-associated" may be indicated. for a review of investigational procedures and autopsy techniques in operating-room-associated deaths, see ref. ( ) . if the autopsy will involve anatomy or dissection techniques that are unfamiliar, the pathologist should not hesitate to invite the surgeon to the autopsy. in patients who develop a cerebral infarction after open heart surgery, arterial air embolism should be considered as a possible cause. the diagnosis often must be based on excluding other causes because the air has been absorbed prior to death. if a patient dies rapidly, the hospital records may be incomplete or scanty. for example, if a patient bleeds to death despite attempted repair of hepatic lacerations, hospital records may not suffice to reach the correct cause-of-death opinion; personal accounts from the surgeon and anesthesiologist may be needed. autopsy data on patients dying following thoracic surgery may be found in ref ( ) . d death, restaurant (see "obstruction, acute airway.") death, sniffing and spray related terms: glue sniffing; sudden sniffing death syndrome. note: no anatomic abnormalities will be noted at autopsy. sudden death may occur after cardiac dysrhythmia or respiratory arrest. procedures possible or expected findings lungs brain if poison had been inhaled at the time when death occurred, tie main bronchi. submit lungs in glass container for gas analysis. submit samples of small bronchi for histologic study. for removal and specimen preparation, see chapter . submit samples of fresh or frozen brain for toxicologic study. submit samples in glass containers (not plastic) for toxicologic study. trichloroethane, fluorinated refrigerants, and other volatile hydrocarbons are most often involved in the "sudden sniffing death syndrome." spray death may occur in asthma sufferers using pressurized aerosol bronchodilators. freons and related propellants may also be responsible for sudden death. toxic components of glue-such as toluene-accumulate in the brain of glue sniffers. also present in various glues are acetone, aliphatic acetates, cyclohexane, hexane, isopropanol, methylethyl ketone, and methylisobutyl ketone. aerosols may occlude the airway by freezing the larynx. carbon tetrachloride sniffing may cause hepatorenal syndrome (see also under "poisoning, carbon tetrachloride"). death, sudden unexpected, of adult note: medicolegal autopsies are usually indicated, and appropriate procedures should be followed. ifanaphylactic death is suspected, see also under that heading. for all unexpected deaths, the pathologist should learn the circumstances of the death, in order to determine whether the mechanism of death was rapid or slow, and to guide the selection of ancillary tests. whenever paramedics attended a person, the run sheet should be obtained to look for a history of recent drinking or ofchronic alcoholism may be an important clue. the combination of a history ofalcoholism, a negative test for ethanol, and absence ofcardiovascular disease, should suggest alcohol withdrawal as the cause ofa sudden death. the list of"possible or expected findings" below is not complete. for general toxicologic sampling, see chapter . possible associated conditions: atrial septal defect;*bicuspid aortic valve;* coarctation,* hypoplasia, or interruption (type a) of aortic arch; coronary artery from main pulmonary artery; right atrial arch; patent ductal artery;* right pulmonary artery from ascending aorta; subaortic stenosis;* tetralogy of fallot;* ventricular septal defect. * (in approx % of the cases, one or more of these associated conditions are found.) defect, atrial septal note: the basic anomaly is a defect of the atrial septum, usually at the oval fossa (in %). possible complications in unoperated cases include atrial arrhythmias, congestive heart failure; paradoxic embolism; plexogenic pulmonary hypertension « %), and pulmonary artery aneurysm. possible surgical interventions include surgical and transcatheter closure of defect. for deficiency, vitamin c synonyms: hypovitaminosis c; scurvy. external examination and skin other organs bones, joints, and soft tissues record extent and character of skin lesions; prepare sections of skin. describe appearance of gums, and prepare sections. record evidence of bleeding. for removal, prosthetic repair, and specimen preparation of bones and joints, see chapter . hyperkeratotic hair follicles with perifollicular hemorrhages (posterior thighs, anterior forearms, abdomen); petechiae and ecchymoses (inner and posterior thighs); subcutaneous hemorrhages. gingivitis. in rare instances, gastrointestinal or genitourinary hemorrhages. hemorrhages into muscles and joints. subperiosteal hemorrhages occur primarily in distal femora, proximal humeri, tibiae, and costochondral junctions (scorbutic rosary). deficiency, vitamin d synonyms: hypovitaminosis d; rickets. note: features or rickets may be found in familial hypophosphatemia (vitamin d-resistent rickets; fanconi syndrome). vitreous or blood (serum) other organs prepare skeletal roentgenograms. in infants with suspected rickets, record size of anterior fontanelle and shape of head; state of dentition; and shape of costochondral junctions, wrists, long bones, and spine. submit samples for calcium, magnesium, and phosphate determination. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. weigh parathyroid glands and submit samples for histologic study. submit samples of intestine for histologic study. for removal, prosthetic repair, and specimen preparation, see chapter . in infantile rickets, diagnostic sites for histologic sampling are costochondral junctions, distal ends of radius and ulna, and proximal ends of tibia and humerus. for adults, see under "osteomalacia." in infants, rachitic changes at costochondral junctions; in adults, osteoporosis* and osteomalacia*-with or without pseudofractures (milkman's syndrome ( ) . note: the term spinocerebellar degeneration encompasses a variety of lesions whose classification is controversial. a new approach has come from linkage analysis and molecular biology. for instance, friedreich's ataxia, the classic form of hereditary ataxia, is due to an intronic expansion of a gaa tri-nucleotide repeat. other forms are also identified by their specific gene loci. neuropathologic examination still is important and ample sampling is suggested, which should include cerebral cortex, basal ganglia (caudate nucleus, putamen, and globus pallidus), thalamus, subthalamic nucleus, midbrain (red nucleus and substantia nigra), pons (pontine nuclei), spinal cord (at cer-vical, thoracic, and lumbar levels), optic tract, optic nerves with lateral geniculate nucleus, and sensory and motor peripheral nerves. for removal and specimen preparation, see chapter . enlargement of head. poor demarcation between cortex and gelatinous white matter. extensive demyelination and vacuolation of white matter, particularly subcortically. optic atrophy. degeneration, striatonigral (see "atrophy, multiple system.") related term: thirst. note: possible underlying conditions not related to inaccessibility of water include bums, exposure to heat, gastrointestinal diseases, recent paracentesis, renal diseases, and use of diuretic drugs. see also under "disorder, electrolyte(s)." external examination vitreous urine prepare histologic sections of blisters, ulcers, or skin abrasions. submit sample for sodium, chloride, and urea nitrogen determination. skin turgor may be decreased and eyes may be sunken. microscopic changes help to decide whether skin lesions are antemortem or postmortem. sodium concentrations more than meqll, chloride concentrations more than meq/ and urea nitrogen concentrations between and meq/dl indicate dehydration. absence or minimal amount of urine. dementia (see "disease, alzheimer's.") drug abuse, amphetamine(s) note: methamphetamine abuse may be suggested by poor condition of the dentition. methylenedioxymethamphetamine ("ecstasy") abuse is often suggested by friends with whom the decedent was abusing drugs. follow procedures described under "dependence, drug(s)." drug abuse, cocaine note: cocaine is spontaneously hydrolyzed by blood esterases, even after death. however, one of its major metabolite, benzoylecgonine, is routinely identifiable by immunoassay screening tests. when cocaine is abused concurrently with heroin or other depressant drugs, it may be difficult to ascribe deth to a single agent, unless circumstances clearly point to a rapid cardiac mechanism or a slow brainstem depression mechanism. note: if narcotic paraphernalia and samples of the drug itself are found at the scene of the death, they should be submitted for analysis. helpful information about the nature of a drug may be obtained from witnesses. state crime laboratories may provide much assistance. if name of drug is known, see also under "poisoning,..." the slang name of a drug may be insufficient for identification because these names often are used for different compounds at different times of places. opoid narcotics can be injected intravenously, or subcutaneously, or snorted. death may occur with such speed that the bodies may be found with needles and syringes in the veins or clenched in the hands. drug abuse may be associated with a multitude of local (see below) or systemic complications, including malaria* and tetanus. * as stated in chapter , for a growing number of analytes, most notably tricyclic antidepressants, peripheral blood is preferred over central blood. peripheral blood is aspirated by percutaneous puncture before autopsy, from the femoral vein or the subclavian vein. the authors prefer the femoral approach in order to avoid any question of artifact in the diagnosis of venous air embolism. it may be pru-dent to add naf to some of the samples. related term: childhood dermatomyositis (or polymyositis) associated with vasculitis; dermatomyositis (or polymyositis) associated with neoplasia or collagen vascular disease; primary idiopathic dermatomyositis; primary idiopathic polymyositis. possible associated conditions: carcinoma (lung, stomach, intestine, and prostate in males; breast, ovary, and uterus in females; miscellaneous sites in both sexes); lymphoma* (rare) and other malignancies ( ); lupus erythematosus;* mixed connective tissue disease; progressive systemic sclerosis;* rheumatoid arthritis;* sjogren's syndrome;* and others. vasculitis of childhood polymyositis (dermatomyositis). external examination and skin heart lungs esophagus and gastrointestinal tract photograph grossly involved skin. prepare sections of involved (anterior chest, knuckles, knees) and grossly uninvolved skin and subcutaneous tissue. prepare roentgenograms. submit samples from myocardium for histologic study. perfuse one lung with formalin. submit samples from all segments for histologic study. arteritis* and phlebitis* with thrombosis, fibrosis, and infarctions. steatohepatitis and manifestations of diabetes mellitus* may be found ( ) . myositis with muscular atrophy and fibrosis; vasculitis in childhood cases. polyneuropathy (rare) ( ). arthritis. diabetes mellitus synonyms: type i (insulin-dependent or juvenile-onset) diabetes mellitus; type ii (insulin-independent or adult onset) diabetes mellitus; secondary diabetes mellitus (e.g., due to drugs or pancreatic disease). note: in infants of diabetic mothers, macrosomia and congenital malformations must be expected. record size and weight of placenta and total weight and length, crown to rump length, and crown to heel length of infant. compare with expected measurements (see part iii). expected histologic finding in-clude hyperpla-sia with relative increase ofb cells of the islands of langerhans with interstitial and peri-insular eosinophilic infiltrates, decid-ual changes of the endometrium, enhanced follicle growth in the ovaries, and leydig cell hyperplasia. possible associated conditions: acanthosis nigricans; acro-megaly;* amyotrophic lateral sclerosis; * ataxia telangiectasia;* fanconi's anemia;* friedreich's ataxia;* gout;* hemochro-matosis; *hyperlipoproteinemia; * hyperthroidism;* obesity;* turner's syndrome;* and many others, too numerous to mention. note: the term "caroli's syndrome" often is used for cases that also show histologic features of congenital he-patic fibrosis or other manifestations of fibropolycystic liver disease,* whereas the name "caroli's disease" refers to idiopathic dilatation of intrahepatic bile ducts, without associated abnormalities. possible associated conditions: choledochal cyst* and related extrahepatic biliary abnormalities ( ); congenital hepatic fibrosis; * cysts of kidneys (renal tubular ectasia or medullary sponge kidney; autosomal-recessive polycystic kidney disease, and rarely, autosomal-dominant polycystic kidney disease [ ] )* and of pancreas. record volume of effusions. prepare smears of fresh blood or of buffy coat, or make thick-drop preparation. submit sample for xenodiagnosis or animal inoculation and for serologic study. record weight. in chronic chagas' disease, perfuse intact heart with formalin (chapter ) and slice fixed heart in a frontal plane so as to create anterior and posterior halves. prepare photographs. histologic samples should include conduction system. include several sections of atrial (auricular) walls for histologic study of autonomous ganglia. perfuse at least one lung with formalin. leave affected hollow viscera intact and fill with formalin. cut fixed organs in half, photograph, and cut histologic sections on edge. record liver weight and submit samples for histologic study. record weight. prepare photographs of abnormalities. weigh and examine. prepare histologic sections. for removal and specimen preparation, see chapter . autopsy is desirable in suspected cases because the diagnosis can only be firmly established after neuropathologic examination. serologic studies are not available. unfortunately, all tissues (not just the brain and spinal cord) may remain infectious even after prolonged fixation and histologic processing. thus, the autopsy recommendations for most other infectious diseases do not apply here. this is a reportable disease in some states. special precautions are indicated and therefore, the procedures described here should be followed strictly ( ) ( ) ( ) ( ) : all persons in the autopsy room must wear disposable long-sleeved gowns, gloves, and masks. contamination of the autopsy table should be prevented by covering it with a disposable, non-permeable plastic sheet. autopsy generally should be restricted to the brain. if organs in the chest or abdomen need to be examined, this is best done in situ. to prevent aerosolization of potentially infectious bone dust, a hood or other protective device should be used while opening the skull with a stryker saw. after completing the autopsy, instruments and other potentially contaminated objects should be autoclaved in a steam autoclave ( h at °c). porous load is considered more effective than gravity displacement autoclaves. immerse autopsy instruments in distilled water before and during autoclaving, in order to protect them from corrosion. ifno autoclave is available, chemical disinfection (see below) is a satisfactory alternative. disposable items should be put in a container for infectious hospital waste and ultimately incinerated. contaminated objects not suitable for autoclaving (such as the stryker saw) should be soaked with a nnaoh solution for h (alternatively, nnaoh may be used for h). contaminated surfaces should be thoroughly washed with the same solution. aluminum should be treated for h with a fresh % naoci (sodium hypochlorite) solution with at least , ppm free chloride. wash waters should be collected; if no autoclave is available, n naoh or > volumes of % sodium hypochlorite bleach should be added to the water and left for a minimum of h before being discarded. before removing the body from the autopsy room, it should be sponged with % sodium hypochlorite. to deactivate cjd infectivity, tissue blocks, mm or less in thickness, should be fixed in formalin in a formalin-totissue ratio of at least : for at least h and then soaked in concentrated formic acid ( - %) for i h, followed by another h of formalin fixation. the fixation fluid should be collected and decontaminated, as described earlier for wash water. glassware and tissue carriers should also be decontaminated as previously described. after this deactivation, the tissue blocks can be processed in a routine fashion. at any stage of these procedures, special care must be taken to avoid cuts with potentially contaminated glassware, blades, or other objects. parenteral exposure to potentially contaminated material also should be avoided. remains of patients who have died of the disease should not be accepted for anatomy teaching for students. if specimens are prepared for pathology collections, they should be handled with great caution. morticians and mortuary workers should be warned of possible hazards posed by tissues of patients with transmissible spongiforme encephalopathies; they should be advised about proper use of disinfectants. clinical laboratories that receive autopsy tissues or fluids must be warned about the infectious nature of the material. if possible, decontamination should be done at the site where the autopsy was done. for the shipping of potentially infected material, see chapter . increased concentrations of nse ( ). spongiforme changes, astrocytosis, neuronal loss, amyloid plaque formation, prp deposition, and proliferation of activated microglia ( ). cerebrospinal fluid brain submit sample for neuron-specific enolase (nse). for removal and specimen preparation, see chapter and above under "note." submit fresh-frozen material for confirmation of diagnosis by histoblot technique on protease k-digested frozen tissue or western blot preparations on brain homogenates. immunohistochemical localization ofprp and hla-dr protein on paraffin-embedded tissue is possible. disease, demyelinating (see "degeneration, spongy, of white matter," "encephalomyelitis, all types or type unspecified," "leukodystrophy, globoid cell," "leukodystrophy, sudanophilic," "sclerosis, multiple;' and "sclerosis, schilder's cerebral.") disease, diffuse alveolar synonym: diffuse pulmonary disease. note: autopsy procedures are listed under the more specific diagnoses, such as "hemosiderosis, idiopathic pulmonary," "lipoproteinosis, pulmonary alveolar," "microlithiasis, pulmonary alveolar," "pneumonia, lipoid," and "syndrome, goodpasture's." glycosphingolipid storage in cornea; lens opacities; dilated vessels in conjunctiva and lens; thrombi in blood vessels ( ). disease, fibropolycystic, of the liver and biliary tract note: "fibropolycystic disease of the liver and biliary tract" comprises a group of well defined conditions, which may occur together and hence need a collective designation. the conditions include autosomal-recessive (infantile) and auto-somal dominant (adult) polycystic disease of the liver; caroli's disease or syndrome;* choledochal cyst,* congenital hepatic fibro-sis,* multiple biliary microhamartomas, and related disorders. for autopsy procedures, see also under more specific designations. disease, glycogen storage synonyms: andersen's disease or brancher deficiency (glycogenosis, type iv); cori's or forbes' disease (glycogenosis, type ill); cyclic amp dependent kinase (type x); glycogen synthetase deficiency (type ); hers' disease (glycogenosis, type vi); mcardle's disease (glycogenosis type v); phosphorylase b kinase deficiency (types ixa, b, and c); pompe's disease (glycogenosis, type it); tarui disease (glycogenosis type vii); von gierke's disease (glycogenosis, type ia); x-linked glycogenosis (type vill). note: if the diagnosis had not been confirmed prior to death, samples of liver, skeletal muscle, blood, and fascia (for fibroblast culture, see below) should be snap-frozen for enzyme assay, which will determine the specific deficiency. types ia and b, iii, vi, and hepatic phosphorylase b kinase deficiency (types ixa, b and c) are hepatic-hypoglycemic disorders, whereas types v and vii affect muscle energy processes. type ii also affects the musculature, whereas type iv may cause cirrhosis and death in infancy from extreme hypotonia. determination of type of glycogenosis usually can be based on (i) pattern of glycogen storage in liver, ( ) presence or absence of nuclear hyperglycogenation in liver, ( ) cytoplasmic lipid in liver, ( ) presence or absence of liver cirrhosis, and ( ) presence or absence of glycogen and basophilic deposits in skeletal muscles. possible associated conditions: fanconi syndrome* or gout* with type ia glycogenosis; neutropenia, recurrent infections, and crohn's disease with types ib or ie. glycogen primarily in retinal ganglion cells and ciliary muscle. glycogen in sympathetic nerve ganglia and neurons of cranial nerves in type vii. gouty arthritis. disease, graft-versus-host note: this disease occurs most commonly after bone marrow transplantation. the disease has also occurred after transfusion of viable lymphocytes, for example, to patients with cancer or leukemia. * in patients with graft-versus-host disease (gvhd), autopsy also may reveal recurrence of the underlying disease such as leukemia. possible associated conditions: alphal-antitrypsin deficiency;* amyloidosis;* ankylosing spondylitis;* primary sclerosing cholangitis;* sjogren's syndrome. * see also below under "possible or expected findings." note: in many instances, either chronic ulcerative colitis or crohn's disease* had been diagnosed clinically, but sometimes, the distinction is difficult to make, even at autopsy. many features described below occur in chronic ulcerative colitis but some manifestations of crohn's disease or conditions that may occur in all types of inflammatory bowel disease also are listed so that both positive and negative findings can be recorded properly. osteoporosis;* ankylosing spondylitis;* arthritis of peripheral joints; periarthritis; hypertrophic osteoarthropathy;* tendinitis (particularly of ankle and achilles tendons). disease, iron storage (see "hemochromatosis.") related terms: atherosclerotic heart disease. note: the most common anatomic finding at autopsy in subjects older than yr is coronary atherosclerosis. unusual under-lying or associated conditions include chronic aortic stenosis or regurgitation; coronary artery anomalies; coronary artery dissection; coronary embolism; coronary ostial stenosis (due to calcification of aortic sinotubular junction or, rarely, to syphilitic aortitis); coronary vasculitis (for instance, in polyarteritis nodosa* or acute hypersensitivity arteritis); hyperthyroidism,* gastrointestinal hemorrhage; * hypothyroidism, * idiopathic arterial calcification of infancy; intramural coronary amyloidosis; pheochromocytoma, polycythemia vera; * pseudoxanthoma elasticum,* radiationinduced coronary stenosis; severe pulmonary hypertension (with right ventricular ischemia); sickle cell disease;* and others. if bypass surgery had been performed, see "surgery, coronary bypass." macular rash ( ). multifocal fibrinopurulent pneumonia with sparing of the bronchi and bronchioles. exudate is rich in phagocytes, fibrin, and karyorrhectic debris. synonym: lyme arthritis note: this infection is caused by the spirochete, borrelia burgdoiferi, which is transmitted from rodents to human by the hard deer ticks, ixodes dammini, . ricinus, and others. brain and spinal cord for removal and specimen preparation, see chapter . request luxol fast blue stain for myelin. symmetric and zonal demyelination in corpus callosum, anterior commissure, optic chiasm, optic tracts, and white matter of frontal lobes. external examination and skin; oral cavity lungs aorta record distribution of skin lesions and submit tissue samples for histologic study. for preparation of angiograms of the pulmonary arterial and venous vasculature, see chapter . if aneurysm or dissection is present, follow procedures described under those headings. telangiectatic (often papular) lesions most commonly found in cheeks, scalp, nasal orifices, oral cavity, ears, neck, shoulders, fingers, toes, and nail beds. cyanosis and clubbing may be prominent. arteriovenous malformations/fistulas. aneurysm; * aortic dissection. * if cirrhosis is present, prepare angiograms of hepatic arteries and veins (chapter ). photograph and prepare sections of angiomatous lesions. note: parkinson's syndrome is caused by conditions that may simulate parkinson's disease; these include carbon monoxide* and manganese poisoning, corticobasal degeneration, druginduced parkinsonism, huntington's disease, multiple system atrophy,* progressive supranuclear palsy* (steele-richardson-olszewski syndrome), space-occupying lesions (rare), trauma (dementia pugilistica), and causes related to tumors and vascular diseases. brain for removal and specimen preparation, see chapter . histologic sections should include midbrain (substantia nigra), upper pons (locus ceruleus), medulla, nucleus basalis (substantia innominata), and basal ganglia. if parkinsonian syndrome was diagnosed, follow procedures described under the name of the suspected underlying condition (see above under "note"). depigmentation of substantia nigra and locus coeruleus; neuronal loss and reactive gliosis; eosinophilic intracytoplasmic inclusion bodies (lewy bodies) in some of the surviving neurons; no significant changes in basal ganglia. disease, pelizaeus-merzbacher synonyms: sudanophilic (orthochromatic) leukodystrophy. brain and spinal cord for removal and specimen preparation, see chapter . request luxol fast blueipas stain for myelin and bielschowsky's stain for axons. prepare frozen sections for sudan stain. brain generally atrophic. myelin loss in centrum ovale, cerebellum, and part of brain stem, with a tigroid pattern of residual myelin near vessels. axons are preserved. diffuse gliosis with relatively few lipoid-containing macrophages, compared to the myelin loss. lipoid material stains with sudan. brain and spinal cord for removal and specimen preparation, see chapter . request silver stains (bielchowsky or bodian stain). histochemical stains in pick's cells and bodies reveal phosphorylated neurofilaments, ubiquitin, and tubulin. some tissue should be kept frozen for biochemical studies. severe cerebral atrophy, involving primarily frontal and anterior temporal lobes (knifeblade atrophy; walnut brain). microscopically, severe neuronal loss accompanied by astrocytosis. characteristic argyrophilic, intracytoplasmic inclusions (pick's bodies), particularly in hippocampus and swollen, distended "ballooned" neurons (pick's cells). these changes are not always present. external examination, skin, and adipose tissue blood cerebrospinal fluid heart liver and kidneys brain, spinal cord, and peripheral nerves eyes submit sample for determinaion of phytanic acid concentration and for molecular studies. for obtaining a sample, see chapter . sample for histologic study. for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . ichthyosis. phytanic acid accumulation in adipose tissues. phytanic acidemia, mutation of phyh or pex ( ). increased protein concentrations. cardiomyopathy.* phytanic acid accumulation. axonal neuropathy. retinitis pigmentosa. hypoalphalipoproteinemia. lymphadenopathy with diffuse deposition of cholesterol esters. premature atherosclerotic cardiovascular disease ( ). hepatosplenomegaly with foam cells. enlarged tonsils with characteristic orange discoloration. polyneuropathy ( ) . in adults, corneal infiltrates. foam cells. request pas stain. in granulomas, bacilli are not always pas positive ( ) . section all grossly involved tissues for histologic examination. submit section for electron microscopy. emaciation. hyperpigmentation, particularly of exposed skin and in scars. hyperkeratosis. arthritis involving ankles, knees, shoulders, and wrists. ascites; fibrinous peritonitis. * nodules in peritoneum containing sickle-form particlecontaining cells (spc cells submit sample for determination of sodium, potassium, chloride, glucose, urea nitrogen, and creatinine concentrations. calcium and phosphate concentrations can also be tested. if sample is small, indicate priority for testing. if indicated, submit sample for chemical study. submit tissue samples for histologic study. considerably increased or decreased values for sodium (more than meqll or less than meqll) and chloride (more than meqll or less than meqll) indicate that changes were present before death. for further interpretation, see chapter . postmortem electrolyte concentrations are quite unreliable. may be useful for calcium determination. vacuolar nephropathy (vacuolar changes in proximal convoluted tubules) in potassium deficiency (may also occur after infusion of hypertonic solutions). disorder, hemorrhagic (see "coagulation, disseminated intravascular," ''disease, christmas:' ''disease, von willebrand's," "hemophilia," and "purpura,.••") disorder, inherited, of phagocyte function note: several conditions represent phagocyte function disorders. autopsy procedures for one of these disorders can be found under "disease, chronic granulomatous." consult this entry for other phagocyte function disorders. synonyms and related terms: fabry's disease* (angiokeratoma corporis diffusum); gangliosidosis;* gaucher's disease;* glycogenosis,* type ii; leukodystrophies (krabbe's or globoidcell,* metachromatic leukoencephalopathy*); mucopolysaccharidoses* (hunter, hurler, morquio, and sanfilippo disease); mucolipidosis; niemann pick disease* (type a, b, c, or sphingomyelinase deficiency); neuraminidase deficiency; neuronal ceroid lipofuscinosis (batten's disease or kufs' disease). hypopharyngeal pulsion diverticulum (zenker's diverticulum) at lower margin of inferior constrictor muscle of pharynx. traction diverticulum at midesophagus after an inflammatory process-for instance, tuberculous lymphadenitis. epiphrenic diverticulum may also occur. luxtacardiac or juxtapyloric diverticulum. heterotopic tissue in meckel's diverticulum, with or without peptic ulceration. colonic muscular hypertrophy and stenosis, usually in sigmoid colon. diverticulitis with perforation, fistulas, or peritonitis. * diving (see "accident, diving (skin or scuba).") related terms: dry drowning; fresh-water drowning; near-drowning; salt (sea)-water drowning (see the following table). primary drowning ("immediate drowning") deaths occurring within minutes after immersion, before or without resuscitative measures deaths from hypoxia and acidosis caused by glottal spasm on breath holding. there may be no evidence of water entering stomach or lungs and no appreciable morphologic changes at autopsy. note: the diagnosis is one of exclusion. the pathologist should help the police to determine: i) how did the person (or dead body) get in the water, and ) why could that person not get out of the water? it is not enough to ask if a person could swim but investigators should find out how well (what strokes did the victim know?) and how far he or she could swim. the inquiry must include the depth of the water and must address hazards such as undertow or underwater debris, and the behavior deaths occurring from within min to several weeks after resuscitation, because of metabolic acidosis, pulmonary edema, or infective or chemical pneumonitis deaths from hypoxia and acidosis caused by obstruction of airway by water related to: hypervolemia hemolysis hyponatremia hypochloremia hyperkalemia of the victim immediately before submerging. deaths of adults in bathtubs and swimming pools are usually from natural, cardiac causes, or they are suicides, unless the victim was drunk. diatom tests ( ) have not proven useful in the united states but there is enthusiasm for such tests among european pathologists. the distinction between hyponatremic deaths in fresh water and hypernatremic deaths in salt water derives from experimental studies; in practice, one cannot reliably predict the salinity of the immersion medium from autopsy studies. because many bodies of drowning victims are recovered only after the body floats to the surface, decomposition will often obscure even the nondiagnostic findings such as pleural effusions, which are often associated with drowning. external examination and skin (wounds) organ samples for diatom search serosal surfaces and cavities if identity of drowning victim is not known, record identifying features as described in chapter . prepare dental and whole-body roentgenograms. submit tissue samples for histologic study of wounds. inspect inside of hands. collect fingernail scrapings. record appearance and contents of body orifices. record features indicative of drowning. photograph face from front and in profile. take pictures of all injuries, with and without scale and autopsy number. remove vitreous for analysis. if diatom search is intended, clean body thoroughly before dissection to avoid contamination of organs and body fluids with algae and diatoms (see below). submit sample for toxicologic study. sample early during autopsy, before carrying out other dissections. use fresh instruments for removal of specimens to avoid contamination. submit subpleural portion of lung: subcapsular portions of liver, spleen, and kidneys; bone marrow; and brain. store samples in clean glass jars. for technique of diatom detection, see below. record volume of fluid in pleural spaces. photograph petechial hemorrhages. photograph layerwise neck dissection if strangulation* is suspected. open airways posteriorly, and photograph, remove and save mud, algae, and any other material in tracheobronchial tree. record size and weight of lungs. there may be wounds that were inflicted before drowning occurred-for instance, in shipwrecks or vehicular and diving accidents. other wounds may be inflicted after deathfor instance, from ship propellers or marine animals. sometimes, premortem and postmortem wounds can be distinguished histologically. object (hair?) held by hands in cadaveric spasm. cutis anserina and "washerwoman" changes of hands and feet are of no diagnostic help. foreign bodies; semen (see also under "rape"). foam cap over mouth and nose. in the autopsy room, water running from nose and mouth is usually pulmonary edema or water from the stomach. high concentrations of alcohol indicate intoxication (see under "alcoholism and alcohol intoxication"). evidence of alcohol intoxication may be found. diatoms may occur in the liver and in other organs of persons who have died from causes other than drowning. comparison with diatoms in water sample from area of drowning may be helpful. penny-sized or smaller hemorrhages may indicate violent respiratory efforts or merely intense lividity. presence of pleural fluid suggests drowning. for diatom detection (l) , boil - g oftissue for -- min in rnl of concentrated nitric acid and . rnl of concentrated sulfuric acid. then, add sodium nitrate in small quantities until the black color of the charred organic matter has been dispelled. it may be necessary to warm the acid-digested material with weak sodium hydroxide, but the material must soon be washed free from alkali to avoid dissolving the diatoms. the diatoms should be washed, concentrated, and stored in distilled water. for examination, allow a drop of the concentrate to evaporate on a slide, and then mount it in a resin of high refractive index. all equipment must be well-cleaned, and distilled water must be used for all solutions. there are several variations and adaptations of this method. drug abuse, amphetamine(s) note: methamphetamine abuse may be suggested by poor condition of the dentition. methylenedioxymethamphetamine ("ecstasy") abuse is often suggested by friends with whom the decedent was abusing drugs. follow procedures described under "dependence, drug(s)." ductus arteriosus, patent (see "artery, patent ductal.") synonyms and related terms. achondroplastic dwarf; asexual dwarf; ateliotic dwarf; micromelic dwarf; normal dwarf; pituitary dwarf; true dwarf; and many other terms, too numerous to mention. external examination bones and joints record height and weight. prepare skeletal roentgenograms. for removal, prosthetic repair, and specimen preparation, see chapter . growth retardation. abnormal growth of epiphyseal cartilage with enlargement of metaphysis. long bones and pelvis most commonly affected. cavernous hemangiomas (maffucci's syndrome). see above under "external examination." chondrosarcoma. dyscrasia, plasma cell note: these conditions are characterized by abnormally proliferated b-immunocytes that produce a monoclonal immunoglobulin. multiple myeloma, * plasma cell leukemia, plasma-cytoma, and waldenstrom's macroglobulinemia* as well as heavy-chain diseases and monoclonal gammopathies of unknown type belong to this disease family. amyloidosis* is closely related to these conditions. for autopsy procedures, see under "amyloidosis," "macroglobulinemia," or "multiple myeloma" and under name of condition that may have caused the plasma cell dyscrasia. such conditions include carcinoma (colon, breast, or biliary tract), gaucher's disease,* hyperlipoproteinemia, * infectious or noninfectious chronic inflammatory diseases, and previous cardiac surgery. synonym: shigella dysentery. note: (i) collect all tissues that appear to be infected. blood bowel eyes joints submit sample for culture and for serologic study. submit sample of feces or preferably bloodtinged mucus for culture. if bacteriologic diagnosis has already been confirmed, pin colon on corkboard, photograph, and fix in formalin for histologic study. submit sample of vitreous for study of sodium, potassium, chloride, and urea nitrogen concentrations. for removal and specimen preparation of eyes, see chapter . for removal, prosthetic repair, and specimen preparation, see chapter . escherichia coli septicemia. colitis with microabscesses; transverse shallow ulcers and hemorrhages, most often in terminal ileum and colon. dehydration* pattern of electrolytes and urea nitrogen. serous arthritis* of knee joints is a late complication. external examination record extent of pigmentation, facial features, and primary and secondary sex characteristics. prepare skeletal roentgenograms. for removal, prosthetic repair, and specimen preparation, see chapter . record size of apertures of cranial nerves in base of skull. unilateral skin pigmentation and precocious puberty in females (albright's syndrome), less commonly in males. synonyms and related terms: becker's muscular dystrophy; congenital muscular dystrophy; duchenne's progressive muscular dystrophy; dystrophinopathy; em-ery-dreifuss mucular dystrophy; facioscapulohumeral dystrophy; limb girdle dystrophy; myotonic muscular dystrophy. external examination record pattern of scalp hair. record status of skeletal musculature. obtain sections for histologic examination. dystrophin staining of the sarcolemma is absent in duchenne's muscular dystrophy and patchy in becker's dystrophy. frontal baldness (in myotonic muscular dystrophy). atrophy and wasting of muscles (generalized or local: predominantly distal in myotonic muscular dystrophy). pseudohypertrophy of calf muscles in duchenne's muscular dystrophy. dystrophic changes include variations in fiber size, fiber degeneration and regeneration, peri-and endomysial fibrosis, and fatty replacement of muscle. the liver, especially the right lobe, is the most common site of involvement. secondary infection or calcification may be present. the lung is the second most common site of involvement. fluid and air may be visible on the roentgenogram. cysts may be present in the abdominal cavity, muscles, kidneys, spleen, bones, heart, and brain. eosinophilia. edema, angioneurotic synonym: angioedema. note: possible causes and suggested autopsy procedures are described under "death, anaphylactic." related term: silo-filler's disease. n<yfe: this condition is causedby inhalationoftoxic gases, such as oxides of nitrogen (silo-filler's disease) and phosgene (coci ). see also "bronchitis, acute chemical" and "poisoning, gas." ( ) gunshot and shotgun wounds of the head involving dural sinuses; ( ) injury to large veins, particularly cranial sinuses (during neurosurgical procedures) and veins ofthe neck (knife wounds, surgery) or uterus (criminal abortion); ( ) insufflation offallopian tubes (particularly in pregnancy or during menstrual period); ( ) infusion of blood components or crystalloid; ( ) malfunctioning of dialysis machines; ( ) subclavian vein catheterization in the semi-fowler position; and ( ) fracture in the hub of a central venous catheter used for parenteral nutrition. possible causes of arterial air embolism include: ( ) open heart surgery involving the aorta, left atrium, or left ventricle; ( ) positive-pressure ventilation in newborn infants; and ( ) underwater ascent with closed glottis (see accident, diving) ifair embolism is suspected, the autopsy should be performed as soon after death as possible. decomposition gases may be produced within a few hours. roentgenography of the whole body may detect large quantities of air, and the roentgenograms may serve as a guide to the most advantageous way of dissection. the postmortem diagnosis of arterial air embolism is made largely on the basis of medical history and circumstances. the autopsy serves to rule out competing conditions. the diagnosis of venous air embolism is made by chest roentgenography before the autopsy ( ). the air in the right chambers of the heart can be confirmed at autopsy by aspiration into a syringe. the formerly recommended procedure of clamping the internal mammary vessels below the sternoclavicular joints, and then cutting across the sternum distal to these clamped vessels so that the sternoclavicular joint area remains intact, is designed to prevent the production of a vacuum that pulls air into the veins. the procedure is not necessary if the air is welldocumented by roentgenography before autopsy. at autopsy, a large fatal pulmonary air embolism is readily apparent. the right atrium and ventricle are distended with fine, frothy, brightred blood, which also may distend the pulmonary arteries and superior vena cava. microbiologic examination ofblood and pericardial sac contents may help to rule out the presence of gas-forming bacteria that may simulate air embolism (fig. ii-i). however, the presence of putrefactive emphysema in any part of the body points toward a bacterial origin of the gas. differentiation of air and decomposition gases at the autopsy table with the pyrogallol test is described below. a % pyrogallol solution is prepared (it should be waterclear). two lo-ml syringes (syringe a and syringe b) are loaded with ml of the pyrogallol solution in each, without permitting any air to enter the system. immediately before the solution is used, drops of . n naoh is aspirated through the needle of syringe a to adjust the ph to about ( drop per ml of solution); the mixture will turn faint yellow. six ml of gas is then aspirated from the heart or blood vessels. the needle is immediately sealed with a cork or replaced by a cap, and the syringe is vigorously shaken for about min. in the presence of air, the pyrogallol solution will turn brown. if the solution remains clear, decomposition gases were present. in the latter instance, drops of . n naoh and ml of room air should be aspirated into syringe b, which is then also sealed and shaken for min. the mixture should turn brown, thus serving as a fig. · . gas-forming bacteria simulating air embolism. the pericardial sac is opened and filled with water. the heart is kept submerged with a pair of scissors. the coronary arteries have been incised with a scalpel. note gas bubbles and foam on the water surface. no discoloration of % pyrogallol was noted. blood cultures were positive for enterococcus organisms. microscopically, gram-negative rods were found in most tissues. control that the pyrogallol solution had been properly prepared. syringe b may also serve as a reserve. if only one syringe is used, the decomposition gas can be expel ed and room air can be aspirated for the control test. if only small amounts of gas can be aspirated, the volume of the pyrogallol solution should be decreased so that the gas-fluid volume ratio is at least : . if no bacterial gas formation is present, the edges of the pericardial incision are elevated and the pericardial sac is fil ed with water. clamping of the ascending aorta and venae cavae prevents the escape of gas into these vessels. the heart is held under water while the coronary arteries are incised, and the escape of bubbles is recorded. when the right coronary artery is opened, care must be taken that the right atrium is not incised. air in the coronary arteries indicates systemic embolism. the heart chambers are then incised. when there is gas in any of the arteries or heart chambers, gas bubbles rise to the surface of the water in the pericardial sac ("bubble test"). sometimes the vessels have to be somewhat compressed in order to cause the gas to escape. because large amounts of air or other gases cause the heart to float, it must be kept submerged before the vessels and chambers are incised. basically the same procedure is used for demonstrating the presence of gas in the superior or inferior vena cava and the pelvic veins (for example, in cases of criminal abortion). in this situation, the abdominal cavity is filled with water and the inferior vena cava and its tributaries are incised. in support of the diagnosis of systemic arterial air embolism, the skull vault may be removed without puncturing the meninges, so that the cerebral arteries can be inspected for gas bubbles. the demonstration of gas bubbles in the meningeal vessels and in the circle of willis is meaningful only when the neck vessels are still intact and the internal carotid artery and basilar artery have been clamped before the brain is removed. in acute cases, gas bubbles will be visible within the cerebral vessels. they are released under water when the clamps are removed and the vessels are slightly compressed. for the collection of gas from blood vessels or cavities, a system oflittle quantitative reliability is an air-tight, water-filled glass syringe with a needle. the needle is inserted into the vessel or cavity in question and gas is carefully aspirated. a combined qualitative and quantitative method has been described by kulka ( , ) (see fig. - ). he devised an apparatus for gas collection and described it as shown in fig. - and caption. the entire system is filled with mineral oil so that, when the funnel is level with the upright bottle, the oil fills only about half of the funnel. in operation, the funnel is first raised to a position - cm above the level of the upright bottle. all the cocks are opened and the position is held until every trace of gas has been driven from the system through the needle, which is thereby coated on the inside by a film of oil. after all air has been expelled, the cocks are closed and the funnel is lowered to its original position. as a precautionary measure and control, the air-tightness of the whole system should be tested before operation. this is done by inserting the needle into musculature or skin and attempting aspiration in the manner described in the next paragraph. to make the test, the bottle is inverted and the needle is inserted into the cavity in question. when the needle is in position, all cocks are opened. the funnel is lowered about - cm, or until adequate suction is created. this aspirates the contents of the cavity, which may consist of air or other gases, either pure or mixed with blood or other liquid. any gas or liquid entering this system may be observed through the wall of the short bent glass tubing. in a positive test, gas bubbles will collect in the bottle above the level of the oil. if desired, this gas can be saved for further examination by closing all the cocks and returning the bottle to its upright position ( ). the volume ofintravenous air needed to cause death in adults depends of the cross sectional area of the cardiac cavity or great vessel that is the site of the gas lock, which in tum depends on the position of the decedent at the time of the embolism ( ). ml of gas can pass through an intravenous hub in just a few seconds. small amounts of air entering the systemic circulation may cause death within minutes. delayed air embolism with fatal outcome may also occur. other organs if purpura is present, prepare photographs and record extent. submit sample (from right atrium) for microbiologic study. collect blood from right atrium and right ventricle. after the heart has been removed, allow blood in pulmonary vessels to pool in the pericardial sac. centrifuge this blood and submit sample of flocculent layer above buffy coat for microscopic study ( ) . submit a section of lung for bacteriologic study. dissect pulmonary arteries; prepare histologic sections of all lobes; request mucicarmine stain and the aldan blue and phloxinetartrazine stain of lendrum. also request sudan stain on frozen sections. skin purpura. vernix, lanugo hairs, and meconium can be found in pericardial blood pool. meconium-type material in blood vessels. in histologic sections, squamous epithelium, meconium, and fat from vernix caseosa. complete or incomplete lower uterine tear; chorioamnionitis. manifestations ofdisseminated intravascular coagulation* and fibrinolysis. intrauterine pneumonia. large amounts of debris in the blood vessels of all sections of the lungs may be considered to be lethal if there is no other cause of death. small amounts in one or more blocks of pulmonary tissue are more likely incidental ( ). a small uterine tear is more likely followed by a fatal amniotic fluid embolization than is a large tear, which may result in fatal hemorrhage or fibrinogen depletion. chorioamnionitis, intrauterine pneumonia, and positive lung cultures of the mother indicate infection of the amniotic fluid. record weight and sample for histologic study. for removal and specimen preparation, see chapter . photograph horizontal sections through brain, brain stem, and spinal cord. prepare frozen sections and request sudan stain. prepare frozen sections of one-half of gland and paraffin sections of the other half. petechial hemorrhages of skin (chest, neck, and face). wounds; other traumatic lesions. bone fractures. petechiae of conjunctivas and retinas. fat may accumulate on surface ofblood pool. no useful technique is available for estimating the amount of fat globules in the blood. fat emboli in lumen of small vessels and in pulmonary air spaces. severe fatty changes may be the cause offat embolism. fractures are the most common cause of fat embolism. petechial hemorrhages, fat emboli ( ). abdominal cavity submit sample of subphrenic exudate for gram stain and cultures. record location and volume of subphrenic exudate. possible causes of subphrenic empyema include appendicitis, cholecystitis, * diverticulitis, intrahepatic abscess, pancreatitis,* ruptured viscus; penetrating abdominal wound(s), perforated ulcer of stomach or duodenum,* and other conditions. pleural cavities and lungs e procedures record volume of effusion or exudate in pleural space. basal pleuritis and pneumonia, adjacent to empyema. encephalitis, au types or type unspecified synonyms and related terms: acute disseminated encephalomyelitis;* acute hemorrhagic encephalitis; acute infective encephalitis or encephalomyelitis; acute poliovirus encephalitis or encephalomyelitis; amoebic encephalitis; arbovirus encephalitis (japanese encephalitis; eastern encephalitis, western encephalitis, venezuelan equine encephalitis, st. louis encep-halitis); bulbar encephalitis;* brain stem encephalitis;* herpes encephalitis (cytomegalovirus encephalits, epstein-barr virus encephalitis, varicella-zoster encephalitis); herpes simplex ence-phalitis; hiv encephalitis; measles encephalitis; measles inclusionbody encephalitis; postinfectiousencephalitis; postvac-cinal encephalitis; progressive multifocal leukoencephalitis or leukoencephalopathy; rabies* encephalitis; subacute encephalitis; subacute sclerosing panencephalitis; viral encephalitis, west nile encephalitis and other terms ( ), too numerous to mention. see also under "note" and under "possible or expected findings." note: if the condition that caused the encephalitis is known, see also under that heading. if the cause of the encephalitis is unknown, submit samples of tissue for microbiologic and toxicologic study, particularly if there is a suspicion of lead poisoning.* see also under "encephalitis, brain stem," "encephalomyelitis,...," "encephalopathy" and "myelopathy, myelitis." encephalitis, brain stem synonyms and related terms: brain stem abscess; infectious brain stem encephalitis; listeria monocytogenes brain stem encephalitis; viral brain stem encephalitis. note: see also under "encephalitis, limbic." brain for removal and specimen preparation, see chapter . necrotizing encephalitis, with or without abscess formation ( enterocolitis, other types or type undetermined note: a multitude of infectious and noninfectious agents may cause inflammation of the small bowel, large bowel, or both. if the condition is not listed under "colitis," "enteritis," or "enterocolitis" or under another specific heading such as "dysentery, bacillary," obtain sufficient material for microbio-logic and histologic study to identify organisms such as clos-tridium, chlamydia, shigella, salmonella, yersinia, helicobacter, verotoxic e. coli, and others. if lymphogranuloma venereum,* or tuberculosis* are suspected, see also under these headings. see also under "disease, inflammatory bowel" and "disease, crohn's." synonyms and related terms: c. difficile colitis; darmbrand; hemorrhagic necrosis (gangrene; infarction) of intestine; ischemic enteritis or enterocolitis;* neutropenic enterocolitis;* pseudomembranous colitis. note: the name "pseudomembranous enterocolitis" is descriptive; the condition may be infectious, ischemic, or both. if the intestinal changes are clearly ischemic, see above under "enterocolitis, ischemic." if the cause is in doubt and if pseudomembranes can be identified, follow the procedures described here. (l) collect all tissues that appear to be infected. for other infectious intestinal diseases, see under specific names, such as "enterocolitis, neutropenic" or "enterocolitis, staphylococcal." intestinal tract and mesentery other organs collect material from pseudomembranes for culture and for c. difficile toxin assay. sample intestinal wall with pseudomembranes for histologic study. if the condition is suspected to be caused by ischemia, follow procedures described above under "enterocolitis, ischemic." note: myoclonic seizures also have been described in a number of progressive encephalopathies with complex neurological symptoms, such as gmi and gm gangliosidosis,* and niemann-pick* and krabbe's disease but also acquired disorders, including alzheimer's disease,* creutzfeldt-jakob epilepsy, myoclonus (continued) disease,* posthypoxic encephalopathy, and subacute sclerosing panencephalitis. mitochondrial encephalomyopathy also can present with myoclonus epilepsy and a mitochondrial myopathy with ragged red fibers (merrf syndrome) in skeletal muscles. lafora-body-type material in the heart, liver, retinas, peripheral nerves, skeletal muscles, and sweat gland ducts (especially axillary). ragged red fibers in mitochondrial myopathies. epilepsy, symptomatic note: possible causes or underlying conditions include cerebrovascular diseases, congenital malformations ofthe brain, degenerative and demyelinating diseases of the brain, head injury,* intracranial and cerebral infections, toxic or metabolic disorders (alcoholism,* barbiturate,* carbon monoxide,* and lead poisoning,* hemodilution, hypocalcemia, or hypoglycemia),* and tumors of the brain. * f failure, congestive heart note: coronary atherosclerosis and manifestations of ischemic heart disease, * valvular heart disease, congenital cardio-vascular diseases, and manifestations of systemic or pulmonary hypertension are the most frequent findings in patients dying of or with congestive cardiac failure. other causes include cardiomyopathies* and secondary myocardial disease (such as amyloid or pericardial constriction). if the cause of the congestive cardiac failure is unknown or not immediately evident after dissection of the heart and of the great vessels, myocardium and other appropriate tissues may be submitted for microbiologic study-including viral cultures-and for electron microscopy. specimens can also be snap-frozen for possible immunofluorescent, biochemical, or histochemical studies, particularly of the myocardium. possible causes of congestive cardiac failure are too numerous to mention. dilatation of heart, with or without mural thrombosis. myocardium may be soft, normal, or firm. pulmonary congestion, with or without hemosiderosis; congestion of viscera with organomegaly. other organ manifestations include bowel edema or hemorrhagic enteropathy (without mechanical vascular occlusion) and zonal hepatic steatosis, fibrosis, or necrosis, with or withoutevidence of liver failure. acute renal tubular necrosis may be present also. synonyms and related terms: acute kidney failure; chronic kidney failure; renal failure; uremia. note: if acute kidney failure had been diagnosed, the autopsy procedures will depend on the expected causes, such as poisoning with ethylene glycol,* lead,* mercury,* or methyl alcohol;* disseminated intravascular coagulation* and its various underlying conditions; glomerulonephritis* and its various underlying conditions; diabetes mellitus;* or multiple myeloma. * the procedures described below deal primarily with chronic renal failure. if the patient had had dialysis, see also under "dialysis (for chronic renal failure )." if transplantation had been carried out, see also under "transplantation, kidney." lassa fever is a highly communicable disease and autopsy studies are not recommended in the usually available surround-ings. if lassa fever is suspected, contact the state health department and the centers for disease control and prevention, atlanta, ga, for disposition and further studies ( ).ifan autopsy is done, disinfection can be accomplished by washing instruments with . % phenol in detergent (i.e., lysol), . % hypochlorite solution, formalin, or paracetic acid. for shipping procedures, see chapter . this is not a reportable disease. synonyms: borreliosis; louseborne (epidemic) relapsing fever; tickborne (endemic) relapsing fever. note: (i) collect all tissues that appear to be infected. ( ) rat/mouse inoculation with infected blood is the most sensitive method for the detection of the organism. consult the state health department. ( ) before the autopsy, consultation with the microbiology laboratory is advised. ( ) request direct dark-field examination and giemsa or wright stain. ( ) no special precautions are indicated. ( ) serologic studies are of questionable value due to the antigenic variability of the organism. ( ) fever, scarlet note: usually, this disease is a nonfatal group a streptococcal tonsillitis and pharyngitis with skin rash. potentially fatal complications include suppurative otitis media,* mastoiditis, and pharyngeal abscess ( ), with or without septicemia. ( ) collect all tissues that appear to be infected. ( ) atresia, but may also be seen with viral myocarditis, type ii glycogen storage disease (pompe disease) and carnitine deficiency. thus, a metabolic disorder must be considered. it is ideal to perform the autopsy as soon after death as possible ( ) . urine plasma for removal, prosthetic repair, and specimen preparation, see chapter . malnutrition ( ) muscle necrosis (clostridial myonecrosis) and accumulation of gas; little leukocytic infiltration. other organs g procedures depend on expected findings or grossly identified abnonnalities as listed in right-hand column. see also above under "note" and under "skeletal muscles." synonyms and related terms: acute postinfectious glomerulonephritis (nonstreptococcal postinfectious glomerulonephritis;*minimalchangedisease;mesangialproliferativeglomerulonephritis;* focal and segmental glomerulosclerosis with hyalinosis (focal sclerosis); poststreptococcal glomerulonephritis; idiopathic nephrotic syndrome; iga nephropathy (berger's disease); membranous glomerulonephritis; membranoproliferative glomerulonephritis; mesangial proliferative glomerulonephritis; rapidly progressive glomerulonephritis (associated with systemic infectious or immunologic multisystem diseases; drug idiosyncrasy; or as primary crescentic glomerulonephritis or superimposed on another primary glomerular disease). possible associated conditions: acquired immunodeficiency syndrome (aids);* alport's syndrome;* amyloidosis; anaphylactoid purpura; bee stings; chronic allograft rejection; dennatomyositis;* dennatitis herpetiformis; diabetes mellitus;* drug dependence;*fabry's disease;* goodpasture's syndrome;* guillain-barre syndrome;* henoch-schonlein purpura;* hemolytic uremic syndrome;* infective endocarditis;* leprosy;* malig-nancies;mixedconnectivetissuedisease;myxedema;polyarteritis nodosa;* rheumatoid arthritis;* preeclamptic toxemia; renovascular hypertension; sarcoidosis;* serum sickness;* sjogren's syndrome;*syphilis;*systemiclupuserythematosus;*systemic sclerosis;* thrombotic thrombocytopenic purpura;* thyroiditis;* vasculitis; viral hepatitis;* wegener's granulomatosis;* and many other conditions. urate deposits in scleras and corneas. granulocytopenia note: midline granulomas may belong to the angiocentric immunoproliferative lesions, which are related to lymphomatoid granulomatosis* and malignant lymphoma. the name "idiopathic midline granuloma" should be reserved for the few cases without evidence of malignant lymphoma or wegener's granulomatosis* ( ). the diagnosis of idiopathic midline granuloma also can be ruled out if studies reveal fungal organisms or features of leishmaniasis;* leprosy,* rhinoscleroma, pseudotumor of the orbit or tuberculosis. * complications of nasal cocaine abuse also may mimic midline granuloma ( ). eosinophilic pneumonitis (degenerating eosinophils with charcot-leyden crystals) and granulomas. angiitis (mostly arteritis), typically with giant cells in tunica media ( ) . findings resemble those in polyarteritis nodosa. * heart ( ), grastrointestinal tract ( ), skin, muscles, and joints are commonly involved. however, renal disease is often (but not always) mild or absent. necrotizing vasculitis of small arteries and veins is present, with extravascular granulomas and eosinophilic infiltration of vessels and perivascular tissues. optic neuritis may be found ( ). may be affected by the vasculitis ( ). pcr studies on paraffin sections via rna in situ hybridization may confirm presence of epstein-barr virus-positive b-cell proliferations combined with dense t-cell accumulations ( ) ( ) ( ) . the condition closely resembles angiocentric t-/nk cell lymphoma ( ). infiltration of lymphocytoid cells, plasma cells, and macrophages with necroses and granulomatous features, which are found primarily in the vicinity of blood vessels. special stains may reveal evidence of infection. lymphoreticular and granulomatous infiltrates (see "lung"). lymphoreticular and granulomatous infiltrates (see "lung"). characteristic infiltrates may be present in all organs and tissues. involvement of spleen, lymph nodes, and bone marrow is uncommon. in rare instances, the disease is confined to the abdomen. in most instances, characteristic infiltrates are present ( ). septicemia; circulating immunoglobulin complexes, and antiendothelial antibodies ( ). angiocentric granulomatosis ( ) hemorrhage, intracranial (see under "hematoma, subdural" and under name of suspected underlying condition, such as "aneurysm, cerebral artery," "infarction, cerebral," "injury, head," and "tumor of the brain." hemorrhage, subarachnoid (see under name of suspected underlying condition, such as "aneurysm, cere-bral artery," "infarction, cerebral," "injury, head," and "tumor of the brain." hemosiderosis, idiopathic pulmonary note: this diagnosis is made by exclusion; involvement of organs other than the lungs (see below under "kidneys") suggests another disease. hepatitis, chronic synonyms and related terms: autoimmune hepatitis; chronic viral hepatitis b (with or without d) and c. note: the term "chronic hepatitis" is not a complete etiologic diagnosis and related names such as chronic active (aggressive) hepatitis, chronic active liver disease, and chronic per-sistent hepatitis are obsolete. most cases in these categories represent autoimmune hepatitis or chronic viral hepatitis b or c. many other liver diseases, including drug-induced hepatitis, inborn errors ofmetabolism (e.g., alphal-antitrypsin deficiency* or wilson's disease*), developmental disorders, and chronic biliary diseases such as primary biliary cirrhosis or primary sclerosing cholangitis also may present as chronic hepatitis. ifchronic hepatitis was the cause ofdeath, submassive hepatic necrosis or cirrhosis* is usually present, often with manifestations ofportal hypertension,*hepatic encephalopathy, hepatorenal syndrome,* and with ascites or spontaneous bacterial peritonitis. possible associated conditions: see also below under "possible or expected findings." conditions that may be associated with hepatitis c include ( ): autoimmune hepatitis; behcet's disease; diabetes mellitus (type );* glomerulonephritis;* guillain-barre syndrome;* idiopathic pulmonary fibrosis; idiopathic thrombocytopenic purpura; iga deficiency; lichen planus; mixed essential cryoglobulinemia; mooren's corneal ulcers; polyarthritis; porphyria cutanea tarda;* thyroiditis. * note: coinfection with other hepatitis viruses (e.g., c and g) and/or systemic viruses such as the immunodeficiency virus are common, particularly in drug addicts ( , ) . in many cases of fulminant hepatitis, tests for known hepatitis viruses are negative ( , ) . homicide note: not all of the following procedures can be carried out or will be required in all cases, nor will this checklist be sufficient in all instances. consult also the entries indicating the cause of death, such as "injury, firearm" or "injury, stabbing." if the victim is an infant, see all under "infanticide." before the body arrives or before autopsy is begun: . investigate the scene where the body was found and where the crime may have been committed (these may be two separate locations). if this is not possible, study the report and photographs submitted by the investigator or the police. study all available information. request previous medical records and roentgenograms of the victim. . emphasize to first responders and autopsy personnel that the body of the victim should not be undressed, washed, or otherwise disturbed until it has been inspected by the pathologist. embalming is not permitted before completion of the autopsy. advise first responders or transporters to put paper bags over the hands of the victim. this will protect possible evidence, such as hair from the assailant. . prepare record sheets to document the time of arrival and release of the body; chain of custody for the body and specimens; the name, age, sex, and other information about the victim; the names ofthe technician, pathologist and guests; and the specimens taken. . prepare roentgenographic and photographic equipment. after body arrives but before autopsy is begun: . if the body is left unguarded-for instance, overnight-a lock should be placed on the refrigerator or cool room where the deceased is kept, and the key should be retained by the technician. . if the pathologist is not accustomed to a busy autopsy room, he or she should feel free to ask all persons other than the pathologist(s), technicians, and law enforcement personnel who are not immediately involved in the actual performance of the autopsy to leave the morgue. . the body temperature of the victim can be recorded at the autopsy facility, but this is rarely useful, particularly if the victim seems to have been deadfor longer than a day ortwo or ifthe body had previously been stored in arefrigeratoror cool room. insert thermometer deep into the anus (about - cm). record temperature and manner and time of procedure. . aspirate vitreous from one or both eyes and store the specimen in a refrigerator. . prepare roentgenograms. in each case, a decision must be made whether roentgenograms should be made of the entire body or only of parts of it-or not at all-and whether they should be made before the victim is undressed, after the victim is undressed, or at both times. . take overall survey photographs that depict the anterior surfaces of the body as it is on arrival to the facility, before any undressing, manipulation for roentgenographs, or cleaning. . take close-up photographs of any trace evidence such as fibers or flakes of gunpowder before undressing or cleaning. . photograph the face of the victim for identification purposes after it has been cleaned of any blood and foreign matter. to avoid perspective distortion from wide angle lenses, the lens should be about ft from the face. . photograph all injuries and other forensically significant findings after blood and foreign matter have been cleaned off the body. a moist towel is useful; brushes are too abrasive. two photographs should be taken of each finding. one should include the autopsy number and a scale. a second photograph should be taken without any extraneous objects. collection and documentation of evidence: . fingerprinting can be done before or after the autopsy but should be done in all homicide cases. in cases involving close contact between assailant and victim, fingernail scrapings or clippings (use a new clipper) and hair with roots should be collected, and its source should be identified (hair pulled from scalp, axillae, and pubis is identified as that of the victim; hair in hands or under fingernails or on clothing of victim may be from the assailant). hair exemplars can be collected in cases of homicide by firearm but are rarely of use. if the body is decomposed or mutilated or for any other reason has not been identified, prepare roentgenograms ofthe head, neck, and torso before the autopsy (the radiographic appearance is altered by the autopsy). these can be used for comparison purposes if antemortem films are located. if films of the extremities are needed they can be prepared after the autopsy. dental roentgeno-.grams are taken, usually after the autopsy, when investigators have located antemortem dental records. detailed descriptions and sizes of clothing, with photographs of clothing, can be useful then there is no putative identity. . in cases in which sexual assault may have been involved, collect pieces of clothing that may contain seminal fluid stains. follow procedures described under "rape." . preserve clothing or part of clothing as evidence. wet clothing should be dried before it is stored in labeled and sealed bags. the autopsy: . the measured length and weight, extent of rigor, and color and distribution of livor should be recorded, along with the state of preservation, nutrition, and hydration. do not omit examination of the hands, particularly of the volar surfaces. . if air embolism is suspected, see procedure described under part ii "embolism, air". if pneumothorax is suspected, see procedure described under part ii "pneumothorax". if there is evidence of strangulation or other neck injury, perform a layerwise dissection of the neck that includes the hyoid bone and tongue. . prepare diagrams of wounds and identify their location by anatomic region. for wounds of the torso, state the distance from the soles of a foot and the distance laterally from to the right or left of the midline. . submit samples of wounds for histologic study when there is any question of the age of the wounds. . the records should show when body fluids and tissues were collected for laboratory analysis, the volume and appearance of such specimens, and what was done with them. if most of the toxicology specimens are collected immediately after the internal examination has commenced, the time of the internal examination serves as the collection time. in all instances, the following items should be collected: blood ofvictim for dna comparison and toxicologic study (determination of alcohol, carbon monoxide, and drug concentrations will be requested most frequently); vitreous; urine; gastric contents; at least one solid organ specimen for toxicologic study (liver and brain have the most comparison data if an extracerebral congenital malformation is suspected, follow procedures described under "malformation, arnold-chiari." if cerebrospinal fluid is aspirated with a syringe, record volume. record weight of brain; record size of brain in relation to inner dimensions of skull. describe size of ventricles. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. if a surgical shunt is present, its location should be recorded and both ends of the implanted specimen that was used for shunting may be submitted for microbiologic study. if the shunt is not patent, record site and nature of obstruction. related terms: antibody-mediated hydrops fetalis; erythroblastosis fetalis;* nonimmune hydrops fetalis. note: the classic example of antibody mediated (rh incompatibility) hydrops fetalis is hemolytic disease of the newborn (erythroblastosis fetalis*). however, nonimmune hydrops fetalis also may be caused by hematologic disorders, e.g., alpha-thalassemia* ( ) or it may have no known cause. infec-tions, e.g., with human parvovirus bl ( ), cytomegalovirus, or syphilis;* heart and vascular diseases ( ) (cardiac tumors, cardi-omyopathy,* myocarditis,* arterial calcification, and others); storage disease ( ); tumors (including neonatal leukemia*); and many other fetal (e.g., congenital chylothorax* or lymphatic dysplasia; pulmonary sequestration and cystic adenomatoid malformation) or maternal conditions, e.g., maternal thyrotoxicosis, also may cause nonimmune hydrops fetalis. if coronary insufficiency or myocardial infarction is suspected, follow procedures described under "disease, ischemic heart." for coronary arteriography, see chapter to. record distribution of atherosclerotic lesions in aorta. samples for histologic study should include aorta, coronary arteries, and peripheral arteries. see also above under "heart." submit samples of head, corpus, and tail for histologic study. if applicable, see also under "diabetes mellitus." for special procedures and stains, see above under "heart." other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for removal and specimen preparation, see chapter . obesity* is a common finding. eruptive xanthomas (palms, elbows, knees) in some but not all types of hyperlipoproteinemia. most prominent in apoprotein cii deficiency. xanthomas of tendons (knees, elbows, dorsum of hands), xanthelasmas, and arcus comeae in familial hypercholesterolemia. gangrene of lower extremities (see below under "arteries"). severe coronary atherosclerosis and myocardial infarcts in type hyperlipoproteinemia and multiple lipoprotein-type hyperlipidemia. atherosclerosis of abdominal aorta and its branches and of carotid arteries. coronary atherosclerosis (see above). pancreatitis* in familial apoprotein cii deficiency. foam cells with triglycerides in liver, spleen, and bone marrow in familial lipoprotein lipase deficiency. manifestations of diabetes mellitus* and hypothyroidism* in some cases of type hyperlipoproteinemia. cerebral infarct (stroke*) in type hyperlipoproteinemia. hypernatremia (see "disorder, electrolyte(s).") hyperoxaluria synonyms and related terms: oxalosis; primary hyperoxaluria type i (a anineglyoxylate aminotransferase deficiency); primary hyperoxaluria type ii (d-glyceric acid dehydrogenase deficiency); secondary hyperoxaluria (see under "note"). note: in ethylene glycol poisoning,*oxalatecrystals in media ofsmall arteries, with associated ischemic lesions. similardeposits may occur after long-term hemodialysis ( ). these conditions must be distinguished from the genetic disease. also, oxa-iate nephropathy may be a complication of short bowel syndrome. if patient with congenital hyperoxaluria underwent liver or combined liverlkidney transplantation ( ) , see also under these headings. remove vitreous for biochemical evalvation. submit tissue (i.e., fascia lata) for karyotype analysis. polycystic ovaries, testicular adrenal rests ( note: there are no diagnostic autopsy findings for hypoxia. possible causes of acute hypoxia include anesthesia-associated death,' compression of the torso by a vehicle, diving accident,' exposure to toxic gases-forinstance, carbon monoxide poisoning;' mechanical failure ofan oxygen supply system, as in an airplane; and sudden mechanical airway obstruction,' as in aspiration ofa foreign body orstrangulation. causes ofchronic asphyxia include prolonged exposure to high altitude and chronic pulmonary disease. profound medial hypertrophy of small pulmonary arteries and of pulmonary veins from chronic exposure to hypoxia. acute pulmonary edema may also occur in chronic hypoxia. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. malnutrition. empty and collapsed colon; small amounts of gray and dry meconium in terminal ileum; meconium masses in dilated and hypertrophied mid-ileum; liquid contents in proximal intestine ( ); appendicitis; bowel perforation. glandular inspissation and atrophy of intestinal mucosa. volvulus, infarction, necrosis, perforation, peritonitis, and acquired intestinal atresia may be present. absence of neural plexuses in congential megacolon* (hirschsprung's disease). manifestations of cystic fibrosis, with or without cirrhosis;* biliary atresia* ( ). immunodeficiency (see "syndrome, acquired immunodeficiency" and "syndrome, primary immunodeficiency.") impression, basilar note: basilar impression constitutes an upward bulging of the margins of the foramen magnum. when occipital condyles are displaced above the plane of the foramen magnum, basilar invagination is present. possible associated conditions: klippel-feil syndrome;* osteogenesis imperfecta;* osteomalacia;* paget's disease of bone;* rheumatoid arthritis; rickets; syringobulbia; syringomylia. * compression and secondary ischemic injury to lower brain stem and spinal cord. see also above under "skull and spine" and under "possible associated conditions." incompetence,••• (see "insufficiency,..•") related terms: battered-child syndrome; child-abuse or child-neglect death. note: vitreous should not be aspirated until the skull has been opened and trauma ruled out, because there is a risk of artifactual damage to the retina. in the presence of craniocerebral trauma the pathologist can opt to examine the eyes by removal, fixation, and histologic study; or retinal photography. follow general procedures described under "homicide" and, if necessary,. the procedures described under "rape." external examination see above under "note." prepare roentgenograms of entire body. see above under "note." in other situations, or after study of the fundus, submit sample of vitreous for chemical and possible toxicologic study. umbilical cord attachment prepare histologic sections of umbilicus. and end of umbilical cord blood submit sample for toxicologic study. consider retaining dried blood on filter paper for possible dna testing for paternity investigation. if there is a possibility that the cadaver represents a stillbirth, see part ii, "stillbirth". if infant is thought to have died shortly after birth, determine the location of air in the intestinal tract; roentgenograms may be helpful. save stomach contents and record amount. other organs see also under "homicide." in the abused child, bruises, hematomas, burn marks, or other patterned injuries. in any child, diaper rash, mongolian spots, self-inflicted fingernail scratches, skin infections, and emaciation. in the previously battered child, fractures in various states of healing. in hypertonic dehydration,' sodium concentrations more than meq/l. this may be caused by organic disease, improper medical treatment, or physical neglect. if infant was born alive, inflammatory changes may be found, depending on survival interval. for the hydrostatic lung test, see "stillbirth." the test is unreliable. extraneous material in air passages indicates that child was alive. air reaches the stomach after min, the small intestine after - h, the colon after - h, and the rectum after l h. there is no difference in the speed of gas propulsion between full-term and premature infants. bacterial gas production and previous resuscitation attempts are potential sources of errors. in questionable stillbirth, presence of milk proves that infant was alive and had been nursed. traumatic lesions and signs of neglect may be present. unilateral ulcers in visceral zoster. in rare instances, diffuse meningoencephalitis may occur. limited necrotic and inflammatory lesions in the cord or brain stem at the level of the affected ganglion are common. posterior hom myelitis ( ). ganglion cell necrosis; lymphocytic infiltration, hemorrhage, and, later, fibrosis. as a rule, only one ganglion is severely involved, but less severe lesions may occur in the ganglia that are immediately adjacent. diffuse lymphocytic infiltration; demyelination; axonal destruction; fibrosis. conjunctivitis; keratitis; iridocyclitis; retrobulbar neuritis; neuroretinitis; occlusion of retinal vessels. evidence of hematologic malignancies ( ) or other conditions, as listed above under "note." infection, middle ear (see "otitis media.") infection, pneumocystis carinii synonym: pneumocystosis. note: ( ) collect all tissues that appear to be infected. ( ) this organism cannot be cultured at present but is frequently associated with viral, bacterial, or fungal infections that can be diagnosed by culture. ( ) for rapid staining of aspirates, use gram-weigert stain, which will stain cysts of pneumocystis carinii and also fungi and bacteria. pneumocystis carinii is best demonstrated with grocott's methenamine silver stain. ( ) no special precautions are indicated. ( ) usually, no serologic studies are available. ( ) this is not a reportable disease. injury, fire (see "burns.") injury, firearm note: protect all drains of autopsy table, which will prevent accidental loss of bullets or bullet fragments. recovery of bullet fragments and pellets can also be improved by passing tissue fragments, blood, and other appropriate materials through a fine nonmetallic sieve. caution must be exercised because sharp edges andjagged projections ofbullets and bullet fragments may cause injury ( ). this applies particularly to the black talon bullet. bullets and bullet fragments should not be touched with forceps or other metal instruments that may produce artifactual markings; they must be placed in properly labeled evidence containers, and the chain of custody must be preserved. from a birdshot wound, at least pellets should be recovered. the firearms examiner will divide the total pellet weight by and derive median pellet weight. from a buckshot wound, all pellets should be recovered. in many of these cases, procedures described under "homicide" must also be followed. do not excise wounds before completion of autopsy (see below). as an academic exercise, excised firearm wounds may be used later for analysis of metal traces by neutron activation or for tests for carboxyhemoglobin. in practice, however, a high quality photograph is adequate to establish the presence or absence of gunpowder stippling or soot deposition toward the end of establishing the range of fire. the dimensions of the stippled areas and soot deposits should be recorded. after completion of external examination, dry the wet clothing and keep as evidence. clothing may also be used for possible test firing or for stain, powder, or particle analysis. external examination if identity of victim is unknown, follow procedures described in chapter . prepare initial roentgenograms of the body regions that have been shot, before disrobing of body, and then lateral films of body regions with bullets. follow up with films of other body regions as needed in the course of autopsy. if the body is decomposed to the extent that the external examination cannot be relied upon to determine cutaneous gunshot perforations, prepare roentgenograms of the entire body before autopsy. record location and number of bullet holes in all layers of garments, indicating whether the involved area is bloodstained or shows gunpowder or soot. if there are several cutaneous perforations, number or letter each consecutively and refer to these numbers in all records. location of bullet holes should be described by recording distance from soles of feet or top of head, and from midline of body. prepare diagrams and photograph holes, with and without labels. photographs should show surrounding garments and extent of blood stains. in hairy areas, shave around bullet wounds for better photographic documentation. for evaluation of distance from where a shot had been fired, see fig. additional roentgenograms during the autopsy may be needed to find bullets embolized to the femoral veins or down the spinal canal. systemic roentgenograms may reveal bullets from obscure entrance wounds, particularly in decomposed bodies, old bullets, bullets in the skin flaps reflected at autopsy, or bullets external to the body in clothing. bullet(s) may have been arrested in hollow viscus or blood vessel and may have been transported to distant sites by peristalsis or bloodstream ("bullet embolus"). bullets may be deflected in unexpected directions from bony surfaces. soot ("smudging") and gunpowder stippling may occur around entrance wound, depending on the muzzle-to-skin distance. there may also be an impression from a recoiling handgun or from a power piston. wounds may be stellate, round, jagged, or slit-like, with or without wide margins of abrasion. primer residues on hand of suicide victim after use of a handgun. fatal secondary injuries, particularly hemorrhages. alcohol intoxication is a frequent finding. fig. · . bullet wounds. differences in the appearance of cutaneous wounds of entrance and of exit and differences in wounds of entrance according to the distance between muzzle and skin at the moment of fire. entrance wounds often differ from exit wounds in that the former are usually surrounded by a narrow zone of abrasion. if the muzzle of a gun is in close contact with the skin at the moment of fire, the combustion products will be blown into the wound and will not be visible on the surface. for close-range wounds, the stippling of the skin around the wound, produced by particles of burned and unburned powder, becomes progressively more dispersed as the range of fire increases and is ordinarily not perceptible if the range has been greater than inches ( synonyms and related terms: acute radiation syndrome; chronic (delayed) radiation injury; radiation enterocolitis; radiation nephritis; radiation pneumonitis. note: procedures and expected findings depend on type of radiation damage, whether acute or chronic, localized or whole-body irradiation. if suspected radiation injury was associated with the administration of radionuclides such as p, , or au, follow procedures suggested in chapter . in fatal whole-body irradiation, findings are most likely related to bone marrow injury, and the suggested procedures and expected findings are those described under "pancytopenia." record extent of oropharyngeal and intestinal ulcerations and hemorrhages. late complications include malignant tumors (carcinoma, leukemia,* lymphoma*), manifestations of hypothyroidism,* and cataracts. organ changes in localized radiation injury depend on site of irradiation (lung, brain, kidney, intestine). the skin is involved in both acute (erythema) and chronic (atrophy, epilation) radiation injury. related terms: cutting injury; knife injury. note: in many of these cases, procedures described under "homicide" must also be followed. insuffiency, adrenal synonyms and related terms: adrenocortical insufficiency; polyglandular autoimmune syndrome (type i, usually in childhood, with parathyroid insufficiency and chronic mucocutaneous moniliasis; type ii, usually in adults, with two or more autoimmune endocrine disorders such as thyroiditis and diabetes mellitus*); primary adrenal insufficiency (addison's disease); secondary adrenal insufficiency (in hypothalamic-pituitary disease or steroid induced); x-linked adrenal hypoplasia. is caused by anatomic changes (see below under "adrenal glands"), drugs (enzyme inhibitors or cytotoxic drugs), or acth-blocking antibodies. possible associated conditions: aids with systemic infections ( ); antiphospholipidantibody syndrome ( ); autoimmune hepatitis; chronic lymphocytic thyroiditis; type i diabetes mellitus;* hypoparathyroidism;* hypothyroidism;* megaloblastic anemia;* surgical procedures, such as heart surgery or orthopedic procedures. large cell lymphoma ( ). should be removed as soon as possible (before embalming), either from cervical midline incision or from chest (neck of cadaver must be well-extended). photograph obstruction before larynx is opened, and inside of larynx and epiglottis after larynx is is opened in posterior midline. make gram-stained touch preparations. make histologic sections of larynx and epiglottis. hemophilus injluenzae (cannot always be isolated from larynx synonyms and related terms: acute or chronic lymphocytic leukemia, acute or chronic myelogenous leukemia, and hairy cell leukemia. multiple subtypes have been identified; they are characterized by cell surface markers, chromosomal abnormalities, staining reactions, and morphologic features. note: at the time of autopsy, most leukemias have been properly classified and often treated. in these cases, the goal of the autopsy is to document the extent of the disease and the presence of complications. if the leukemia had not been classified or if the features might have changed from the time of the last work-up (e.g., in suspected cases of superimposed diffuse large cell lymphoma [richter's syndrome]), material should be snap-frozen and studied in more detail. if the patient was treated by bone marrow transplantation,* see also under that heading. possible associated conditions: agnogenic myeloid metaplasia with myelofibrosis; bloom's syndrome;* cutaneous mastocytosis; down's syndrome;* immunodeficiency syndromes* (bruton's disease; louis-bar syndrome; wiskott-aldrich syndrome); infantile genetic agranulocytosis; klinefelter's syndrome;* lymphoma;* multiple myeloma;* polycythemia;* and many others. for sampling and specimen preparation, see chapter . increased protein concentration. material in sediment stains red with toluidine blue. metachromatic material. arylsulfatase-a deficiency. excessive loss of myelin with large amounts of metachromatic material (see above under "urine") in white matter and also in some neurons. demyelination with metachromatic material (see above under "urine"). leukoencephalopathy, progressive multifocal possible associated conditions: aids* and other immunosuppressed states; carcinoma; malignant myeloproliferative or lymphoproliferative disorder; sarcoidosis;* tuberculosis. * procedures submit portion of fresh brain for viral culture. fix remainder of brain and spinal cord in formalin and submit samples for histologic study. in situ hybridization for ic virus is available on paraffin-embedded tissue. small patches of demyelination with tendency to form confluent areas in the cerebral white matter. white matter necrosis may be present. eosinophilic intranuclear inclusions occur in affected oligodendroglia cells. subsequently, large, bizarre astrocytes develop. no inflammatory (lymphocyte) cell reaction is present. lightning (see "injury, lightning.") lipoproteinemia (see "hyperlipoproteinemia.") lipoproteinosis, pulmonary alveolar synonym: idiopathic alveolar lipoproteinosis. note: the condition has been described in allografts ( ); in such cases, see also under "transplantation, lung." elephantiasis of penis, scrotum, or vulva (in chronic cases); skin rash (l) and conjunctivitis (in acute cases); genital ulcers. suppurative or fibrosing inguinal, iliac, or pelvic lymphadenitis, with or without sinus tracts. rectal strictures and fistulas in chronic cases ( ) . systemic involvement in the acute stage with pericarditis,* and arthritis,* and meningitis,* proctitis ( ) lymphoma synonyms and related terms: aids-related lymphoma; adult t-cellieukemiallymphoma; angioimmunoblastic lymphadenopathy; b-celllymphoma; burkitt's lymphoma; cutaneous t-cell lymphoma (includes mycosis fungoides and sezary syndrome); hodgkin's disease; non-hodgkin's lymphoma (low grade, intermediate grade, high grade, all with multiple subtypes too numerous to list, which vary in natural history); natural killer cell neoplasms; post-transplant lymphoproliferative disorders (ptld); t-cell lymphoma. note: at the time of autopsy, most lymphomas have been properly classified and often treated. in these cases, the goal of the autopsy is to document the extent ofthe disease and the presence of complications. if the lymphoma had not been classified or if the features might have changed from the time of the last work-up, material should be snap-frozen and studied in more detail. if the patient was treated by bone marrow transplantation, * see also under that heading. for current terminologies of hodgkin's disease and non-hodgkin lymphomas as well as for staging criteria, appropriate hematologic textbooks should be consulted. possible associated conditions: acquired immunodeficiency diseases (e.g., after organ transplantation; human immunodeficiency virus-l infection); autoimmune disease (e.g., celiac sprue,*rheumatoid arthritis,* systemic lupus erythematosus,* or sjogren's syndrome*); chemotherapy with or without radiation treatment; epstein-barr virus infection; human t-cell leukemia virus infection; inherited diseases with immunodeficiency (e.g., klinefelter syndrome; * common variable immunodeficiency disease, wiskott-aldrich syndrome); radiation. the lesions are found most commonly in the urinary bladder and other parts of the urinary tract, but may also occur at many other sites, including skin ( ) and upper respiratory tract ( ). note: (i) collect all tissues that appear to be infected. ( ) usually, cultures are not indicated. ( ) request giernsa or wright stain. tissue blocks should be rinsed of blood and be as thin as possible before fixation in refrigerated buffered and neutral % formalin solution. this is to avoid precipitationofformalin pigment that may be confused with malaria pigment. the formalin solution should be used in a ratio of parts formalin to one part tissue and should be agitated every few hours. if one is dealing with tissues that contain formalin pigment, this pigment may be removed with a bleaching solution. ( ) usually, no special precautions are indicated. ( ) possible associated conditions: histoplasmosis* and other chronic fungal infections; immune connective tissue diseases such as rheumatoid arthritis* (l) ; malignancies (l); sarcoidosis;* silicosis;* tuberculosis* (l) . note record location and extent of skin changes or skin defects on back. prepare skeletal roentgenograms. if meningitis is suspected, submit sample of cerebrospinal fluid for culture. dse the posterior approach to remove the spinal cord. atrophic skin over meningocele, lacking rete pegs and skin appendages; skin defect in complete rachischisis. bony defects of spine. in meningocele and spina bifida occulta, arachnoid and dura herniate through the vertebral defect. spinal cord and roots are generally not involved. lumbosacral mass in meningomyelocele, with a highly vascular mass (area medullovasculosa) in spina bifida aperta. neural defects in complete rachischisis. diastematomyelia, hydrocephalus ( ) . pyelonephritis;* enlarged urinary bladder ("neurogenic bladder"). external examination heart lungs procedures prepare chest roentgenogram. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. record weights. prepare photographs and roentgenograms of fresh and fixed lung specimens. perfuse one lung with formalin. for preparation of paper-mounted sections, see chapter . submit one lobe for microbiologic and chemical study. decalcify tissue for histologic study. request van gieson's, hale's colloidal iron, pas, and sudan stains. miliary mottling in lower lung fields. mitral stenosis* may be a cause of microlithiasis (mostly intra-alveolar ossification). * cor pulmonale and heart failure ( ) (l) , see also under that heading. ( ) collect all tissues that appear to be infected. ( ) viral isolation, especially with ebv, is not diagnostically useful, due to long incubation periods. ( ) note: these diseases are characterized by a deficiency of a variety of hydrolases, resulting in accumulation of gly-cosaminoglycans (mucopolysaccharides) and glycolipids within lysosomes of fibroblasts, macrophages, white cells, and parenchymal cells of many organs ( , ) . mucopolysaccharides are also excreted in the urine. the general approach is similar in all types. formalin may dissolve all of the stored material and leave empty vacuoles in the involved cells. therefore, frozen sections should be utilized or tissues should be fixed in absolute alcohol. the accumulated material will show intense metachromasia if stained with toluidine blue. it will also stain with pas, alcian blue, and colloidal iron. oil red a will also stain the material from frozen sections. for specimen preparation for elec-tron microscopy, see chapter is. characteristic external features include dwarfism; thickened long bones; coarse facial features; coarse hair; macrocephaly; prognathism; hypertelorism; malformed teeth, and scaphocephalic skull with hyperostosis of sagittal suture; short neck; chest deformity; umbilical and inguinal hernias; lower thoracic and lumbar gibbus; genu valgum and coxa valga; pes planus and other joint deformities; wide hands (clawhands) and feet. coarse thickened skin, covered with lanugo-like hair. hyperlordosis; ovoid deformities of vertebrae; odontoid hypoplasia; large, shoe-shaped sella turcica; kyphosis; hypoplasia of femoral heads; osteoporosis.* if patient underwent bone marrow transplantation ( ), see also under that heading. chronic upper respiratory infections. large cytoplasmic granules in neutrophils. storage of mucopolysaccharides in osteocytes, chondrocytes, and fibroblasts of periosteum, tendons, fasciae, and other connective tissues; dysostosis multiplex. other tissues histologic sampling cannot be excessive. ( ) collect all tissues that appear to be infected. ( ), see also under that heading. blood; other organs and tissues record extent of skin lesions and prepare photographs; prepare sections of affected and unaffected skin. request gram stain of sections and smears. submit samples of blood and grossly affected organs or tissues for microbiologic study. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. extensive epidermal necrosis. shedding of granular and horny layers of epidermis. septicemia; staphylococcal infection of nose, throat, ears, eyes, heart valves, urogenital tract, and other sites. bronchial epithelial detachment and bacterial pneumonia ( ). synonyms and related terms: acute kidney failure; *acute tubular necrosis; lower nephron nephrosis. note: the morphologic diagnosis of this condition may be difficult to discern from autolysis. the features that suggest tubular necrosis are: tubular dilation with epithelial flattening, intertubular edema and necrotic epithelial cells in the collecting ducts. the autopsy should be performed as soon as possible. needle specimens of the kidneys obtained in the immediate postmortem period may yield acceptable material. ifnephrotoxic drugs or chemicals are thought to be responsible for tubular necrosis, submit samples for toxicologic study (see also under synonyms and related terms: multiple endocrine neoplasia (men), type (parathyroid hyperplasia or adenoma; pancreatic islet cell hyperplasia, adenoma, or carcinoma; pituitary hyperplasia or adenoma); or type a (medullary thyroid carcinoma, parathyroid hyperplasia or adenoma; and pheochromocytoma); or type b (medullary thyroid carcinoma, pheochromocytoma, mucosal and gastrointestinal neuromas, and marfanoid features). note: in men, type , foregut carcinoids and subcutaneous and visceral lipomas also may be found. in type a, cutaneous lichen amyloidosis may be observed. mixed syndromes include (i) familial pheochromocytoma and islet cell tumor, ( ) von hippel-lindau syndrome, pheochromocytoma and islet cell tumor, ( ) neurofibromatosis* with features of men or , and myxomas, spotty skin pigmentation, and generalized endocrine overactivity (carney complex). in all instances, the autopsy should be done as soon as possible so that tissues for biochemical study can be frozen without delay. nephritis note: see under specific designation, such as "glomerulonephritis" and "pyelonephritis," or under name of suspected underlying condition, such as "gout" or "lupus erythematosus, systemic." nephroblastoma (see "tumor of the kidney(s).") synonyms and related terms: renal stones; urolithiasis. possible associated conditions: carcinomatosis; cushing's syndrome;* cystinuria;* fanconi syndrome;* hyperoxaluria;* hypervitaminosis d;* gout;* multiple myeloma;* osteoporosis;* polycystic renal disease;* primary hyperparathyroidism;* rheumatoid arthritis* ( ); sarcoidosis* ( ). kidneys ureters, urinary bladder, and urethra other organs remove kidneys, ureters, and urinary bladder en bloc. excise and bivalve kidneys to reveal renal pelves. open ureters. record size, number, and appearance of stones, and save for chemical analysis ( ). record heart weight. dissect and record weights of all parathyroid glands. other procedures depend on suspected underlying conditions, as listed above under "possible associated conditions." nephropathy note: see under name of suspected underlying condition, such as "diabetes mellitus," "disorder, electrolyte(s)" (hypercalcemia, potassium depletion), "gout," "hypertension (arterial), all types or type unspecified," or "poisoning,.. ." (heavy metal). if kidney failure was present, procedures under that heading should also be followed. renal tissue may need decalcification or fixation in water-free solution. ( ) note: in this condition, lesions in the brain and cranial nerves, spinal cord, and spinal roots may be schwannomas (including bilateral vestibular schwannomas); multiple meningiomas; gliomas (generally of spinal cord), mostly ependymomas ( %) and pilocytic astrocytomas; or schwannosis of spinal dorsal root entry zones. intracortical meningioangiomatosis; glial hamartia (intracortical, basal ganglia, thalamus, cerebellum, and dorsal horns of spinal cord) and cerebral calcifications also may be found. cutaneous abscess (j). acute necrotizing nocardial pneumonia with abscesses or sinus formation into surrounding tissues; empyema. pulmonary and mediastinallymph nodes other organs submit samples for culture and histologic study. submit samples from all organs and tissues with suspicious gross lesions for culture and histologic study. poorly encapsulated abscesses of any organ; endocarditis ( ) . related term: total parenteral nutrition. note: air embolism* or blood loss may have occurred if line became detached from the catheter hub. metabolic complications such as fluid overload or disturbances of acid-base and electrolyte balance often cannot be diagnosed reliably at autopsy. the disease(s) that may have necessitated parenteral nutrition therapy are not considered here; they include the acquired immunodeficiency syndrome (aids);* cancer cachexia; inflammatory bowel disease; liver or kidney failure;* severe pancreatitis;* short bowel syndrome, and others. obstruction, acute airway synonyms and related terms: aspiration; bolus death; "cafe coronary"; croup; restaurant death. note: ifpermission has been obtained, remove neck organs through straight incision from chest to chin to avoid dislodging a foreign body. ifdissection has to be accomplished from chest, neck of unembalmed cadaver should remain well extended during procedure. inspect larynx and trachea from above and below, respectively, before opening them carefully along the posterior midline. for removal, prosthetic repair, and specimen preparation, see chapter . submit samples of osteocartilaginous and synovial tissues for histologic study; sagittal or frontal saw section through spine provides for best routine evaluation. heberden's nodes at interphalangeal joints of fingers. degenerative changes, primarily of spine, hip joints, and knee joints. histologic and macroscopic degeneration of cartilage; exposure of subchrondral bone; formation of marginal osteophytes; bone cysts; synovial fibrosis; hypertrophic synovitis. o procedures if artificial joints (e.g., hip or knee) had been implanted, record state of implant site. loose joint prostheses. synonyms: hypertrophic pulmonary osteoarthropathy; pac-hydermoperiostosis (idiopathic hypertrophic osteoarthropathy [ j). note: in all instances, an underlying disease must be identified; they include cardiac disease (cyanotic congenital heart dis-ease with right-to-left shunt; infective endocarditis*); gastroeso-phageal reflux ( ); neoplasms of lungs, esophagus, intestine, and liver; chronic liver disease; inflammatory bowel disease;* pulmonary disease (e.g., abscess;* bronchiectasis;* cystic fibrosis;* emp-yema;* emphysema;* lipoid pneumonia* ( ); pneumocystis pneumonia; sarcoidosis;* tuberculosis*); hyperthyroidism. * external examination elastic arteries other organs prepare roentgenograms of extremities. for removal, prosthetic repair, and specimen preparation, see chapter . record presence of aneurysms (thoracic aorta, subclavian) or arteriovenous fistula of brachial vessels. if abdominal aortic aneurysm had been surgically repaired, search for evidence of graft infection ( ) . procedures depend on expected findings or grossly identified abnormalities as listed above under "note." clubbing of fingers or toes (see below under "elastic arteries"); swelling of extremities. periosteal new bone formation in distal shafts of bones of forearms and legs. all bones of extremities may be involved. see above under "external examination." unilateral clubbing. clubbing of toes but not of fingers. swelling; fistulas. bone defect(s) and focal osteosclerosis. bacterial or fungal infections, most common in metaphyseal region of bones; paravertebral or psoas abscess in osteomyelitis of spine. bacterial or fungal osteomyelitis, with or without cavitation and fistulas. trauma, surgery, insertion of prosthesis, generalized ( ) or contiguous infection, inflammatory bowel disease ( ), diabetes mellitus,* and peripheral vascular diseases, deep vein thrombosis ( ). synonyms and related terms: aseptic necrosis of bone; avascular necrosis of bone; idiopathic osteonecrosis; perthes' dis-ease; postfracture osteonecrosis; renal transplant associated osteonecrosis. note: possible underlying conditions include chronic alcoholism,* decompression sickness,* diseases that have been treated with high doses of corticosteroids ( ), gaucher's disease,* human immunodeficiency virus infection* ( ), tuberculosis* ( ), sickle cell disease, * systemic lupus erythematosus,* tuberculosis* ( ), and bisphosphonate therapy ( ). other organs submit sample for biochemical study. for removal, prosthetic repair, and specimen preparation, see chapter . if osteonecrosis is suspected because one of the possible underlying conditions (see above) is present, prepare saw sections through femoral heads, medial femoral condyles, and heads of humeri. procedures depend on suspected underlying conditions, as listed above under "note." osteopetrosis synonymsand related terms: albers-schonberg disease; autosomal-dominantosteopetrosis; carbonic anhydrase-it deficiency; malignant infantile osteopetrosis ( ); marble bone disease. note: manifestations of renal tubular acidosis may have been a clinical complication. possible associated conditions: bone marrow transplantation* (for infantile osteopetrosis). anemia, recurrent infections, bleeding, and bruises may have resulted from myelophthisis associated with osteopetrosis. upper airway obstruction in malignant infantile osteopetrosis may have necessitated tracheostomy ( ). in heritable osteoporotic disorders of connective tissue (osteogenesis imperfecta,* marfan' s syndrome,* and others) are pre-sented separately. for"osteomalacia," see above. for "osteodystrophy," see under "failure, kidney." possible associated conditions: acromegaly;* chronic alco-holism;* chronic obstructive pulmonary disease; chronic kidney failure* ( ); cushing's syndrome;* debilitating disease (various kinds, often with immobilization); diabetes mellitus* ( ); epilepsy;* hyperthyroidism ( );* hypogonadism; malabsorption syndrome;* malnutrition;* primary biliary cirrhosis;*rheu-matoid arthritis;* scurvy;* steroid therapy or anticonvulsant medication. normal parathyroid glands in uncomplicated cases. hyperparathyroidism* ( ) (without osteitis fibrosa) may be present, however. features of osteitis fibrosa (osteoclastic osteoporosis; see "hyperparathyroidism") or osteomalacia* exclude the diagnosis of uncomplicated osteoporosis. osteoporosis, which may be localized, occurs in various neoplastic (e.g., mastocytosis) and inflammatory diseases. external examination brain and base of skull with middle ears examine external ear canal. for removal and specimen preparation, see chapter . culture any lesion appearing to be infectious. draining, foul-smelling greasy material associated with acquired cholesteatoma. bacterial or viral infection, with or without mastoid osteitis. neck abscess, sinus thrombosis ( ) and brain abscess* and meningitis* may complicate otitis media. otosclerosis ( , ) note: otosclerosis may be a measles-virus-associated disease ( ) and therefore, studies to rule out a past infection may be indicated. for removal and specimen preparation, see chapter . for current methods of temporal bone studies, see ref. ( ) . spongy bone in the capsule of the labyrinth. trabeculae of woven bone show pagetoid changes. if stapedectomy had been done, a prosthesis may be in place. brain is externally normal or mildly atrophic. characteristic is midbrain atrophy with aqueduct dilatation and depigmentation of the substantia nigra. neuronal loss with reactive gliosis, associated with neurofibrillary tangles (globose tangles). primarily affected are globus pallidus, subthalamic nucleus, red nucleus, substantia nigra, tectum, periaqueductal gray matter, and dentate nucleus (grumose degeneration). palsy, pseudobulbar (see "disease, motor neuron.") related terms: acute pancreatitis; alcoholic pancreatitis; chronic (or chronic fibrosing) pancreatitis; interstitial pancreatitis. note: some causes of pancreatitis such as adverse drug reactions (e.g., due to azathioprine, furosemide, or estrogens) cannot be diagnosed at autopsy. possible associated conditions: acute fatty liver of pregnancy; apolipoprotein cli deficiency; cystic fibrosis; *hyperparathyroidism;* kidney transplantation. * status post endoscopic retrograde cholangiopancreatography. synonyms and related terms: calcifying panniculitis; histiocytic cytophagic panniculitis; mesenteric panniculitis (mes-enteric lipodystrophy); "sclerema neonatorum." note: the term weber-christian disease described systemic panniculitis with fever, bleeding, pulmonary and pancreatic lesions, and other abnormalities also involving lungs and pancreas, among others. however, this condition is not a specific entity and thus, the name has become obsolete. the term histiocytic cytophagic panniculitis is more descriptive and preferred. leukemia* and subcutaneous t-celllymphoma* may closely simulate panniculitis. possible associated conditions: alphaj-antitrypsin deficiency;* dermatomyositis;* rheumatoid arthritis;* scleroderma and morphea; sjogren's syndrome;* systemic lupus erythematosus.* external examination, skin, subcutaneous tissue, and breasts chest heart lungs liver and spleen record extent and character of skin lesions. record size, location, and gross appearance of subcutaneous nodules. submit tissue samples for histologic study of grossly unaffected skin, skin lesions, and subcutaneous nodules. request verhoeff-van gieson, gram, and grocott's methenamine silver stains. request s-l protein stain. ascertain that cell infiltrates are not leukemic or lymphomatous ( ) . dimpling of skin; necroses; fistulas. panniculitis with subcutaneous nodules in trunk, breasts, and thighs ( ) . calcification may occur in breast tissue but also at other sites (e.g., in kidney failure*). pancreatic enzymeinduced fat necroses associated with severe pancreatitis. widespread hardening of fat tissue with rupture of fat cells and crystal formation in "sclerema neonatorum." focal traumatic panniculitis also may occur in newborns. s-ioo stain negative in panniculitis but positive in rosai-dorfman disease (sinus histiocytosis with massive lymphadenopathy). mediastinal panniculitis. pericarditis;* interstitial myocarditis. * pneumonia;* pleuritis. fat embolism. * features of alphal-antitrypsin deficiency* ( ) . fatty changes of liver; splenitis. intestinal mucosal erosions and ulcers, with or without perforation. massive gastrointestinal hemorrhage in histiocytic cytophagic panniculitis. blind intestinal loop ( ). for sampling and specimen preparation, see chapter . prepare samples for electron microscopy. vacuolar myopathy. ( ) . external examination, skin, and mouth record extent of skin lesions; photograph; submit multiple samples for histologic study, preferably of areas that are free of secondary infection. prepare sections for immunofluorescent staining. bullous and other lesions of scalp, eyelids, nose, axillae, umbilicus, inframammary areas, back, hands, groins, genitalia, anus, knees, and feet. similar lesions may occur in the mouth. acantholysis is suprabasal or near granular layer. igg deposits on the surfaces of keratinocytes. note: (i) collect all tissues that appear to be infected. collect inguinal, popliteal, axillary, and supraclavicular lymph nodes for aerobic culture. photograph enlarged lymph nodes, and prepare smears of fresh cut surfaces. submit consolidated areas for microbiologic study (see above under "note"). perfuse both lungs with formalin. submit samples of pulmonary tissue and bronchial lymph nodes for histologic study. submit samples of grossly abnormal organs, exudates, or drainage fluids for culture and gram stain. hemorrhagic necrosis; variable suppuration. severe hemorrhagic edema; pneumonia with lobular to lobar features. large areas of necrosis teeming with organisms and minimal to suppurative inflammation. plasmacytoma (see "myeloma, multiple.") platybasia note: possible causes or associated conditions include arnold-chiari malformation;* basilar impression* orinvagination; fusion ofatlas to the foramen magnum; klippel-feil syndrome;* malpositioning of odontoid process; osteitis deformans (paget's disease of bone*); osteogenesis imperfecta;* osteomalacia;* rickets; syringobulbia, syringomyelia. * skull and spine prepare roentgenograms of skull and cervical spine. flattening of the base of the skull (angle formed bythe planeoftheclivus andtheplane of the anterior fossa exceeds °). pleuritis (see "effusion(s) and exudate(s), pleural.") pleurodynia, epidemic synonyms: bornholm disease; devil's grip; epidemic myalgia; epidemic myositis. blood heart and pericardium submit samples for viral culture. sample for histologic study and for viral cultures (coxsackievirus a and b; echoviruses). if pericardia! fluid can be obtained, submit for viral culture also. cultures may be negative and search for viral rna by in situ hybridization may be indicated. chest organs other organs dissect thoracic duct and its tributaries (see chapter ). perfuse one or both lungs with formalin. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. submit samples of cystic lesions for histologic study. submit samples of cystic lesions for histologic study. gas in thoracic duct. emphysema* of lungs. necrotizing enterocolitis in premature infants. pyloric stenosis, colitis ( ), redundant sigmoid colon, ischemic bowel disease. mucosal pseudolipomatosis ( ) . usually, cysts are subserosal in adults and located between muscularis mucosae and muscularis propria in infants and children. mucosa may be inflamed. extraintestinal cysts, as listed above under "abdomen." cysts may also occur in vaginal mucosa. include bright-field and polarized light microscopy, transmission electron microscopy, scanning or transmission electron microscopy with energy dispersive x-ray analysis ( ), ion beam instrumentation, and secondary ion mass spectrometry. the last three methods are time-consuming, require much skill and expensive equipment, and do not lend themselves well to quantitation. ideally, bulk analysis and in situ microanalysis should be used in combination. analyses can be conducted in specialized laboratories at the following centers (for charges and other information, consult the appropriate laboratories): prepare chest roentgenogram. submit sample for microbiologic study. refrigerate sample for possible immunologic study. submit one lobe or samples of all lobes for bulk analysis and for in situ microanalysis (see above under "note"). microincineration may permit preliminary dust analysis. for demonstration of asbestos fibers, see chapter and ref. ( ) . submit one lobe or segment for microbiologic study. for pulmonary arteriography and bronchography, see chapter . prepare roentgenograms of entire lungs and slices. perfuse one lung (or both, if only routine studies are intended) with formalin. submit samples for histologic study of nodular dust lesions, diffuse fibrotic lesions, grossly uninvolved areas, bronchi, and bronchopulmonary lymph nodes. for preparation of paper-mounted sections, see chapter . prepare samples for transmission and scanning electron microscopic study. for removal, prosthetic repair, and specimen preparation, see chapter . alveolar rupture with dissection of air into the mediastinum in neonates with respiratory distress (see "syndrome, respiratory distress, of infant") and in adult patients ventilated on volume respirators. external examination prepare chest roentgenogram. photograph mediastinum. explore major veins and record compression in cases of tension pneumomediatinum. roentgenograms provide the best permanent record of a pneumomediastinum. air bubbles in mediastinal soft tissues. part ii / diseases and conditions abdomen and neck organs perfuse one lung with formalin. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. search for evidence of trauma of other lesions that may have allowed air to enter soft tissues. intubation injury (j); perforation or rupture of major bronchus, trachea, or esophagus. alveolar rupture, e.g., in asthma* ( ), may not be discernible. bronchiolitis obliterans ( ), interstitial pneumonia* ( ) and other lung conditions also may lead to pneumomediastinum. dissection of air from lesions in abdomen (e.g., retroperitoneal colonic perforation [ ] ) or in neck area. pneumonia, all types or type unspecified note: if the type of underlying infection is known, follow procedures suggested under the name of the infectious disease. if the etiologic agent of the pneumonia is unknown, proceed as follows: (l) collect all tissues that appear to be infected. and grocott's methenamine silver stains. ( ) special precautions may be required (see chapter ). ( ) serologic studies may be helpful once a specific etiologic agent is suspected. thus, collect serum at the time of autopsy or procure serum that was collected prior to death. ( ) this may be a reportable disease. related terms: acute interstitial pneumonia (hamman-rich syndrome); desquamative interstitial pneumonia (dip); idiopathic organizing pneumonia (or "bronchiolitis obliterans with patchy organizing pneumonia [boop]" or "cryptogenic organizing pneumonitis"); idiopathic pulmonary fibrosis; lymphoid interstitial pneumonitis (lip); nonspecific interstitial pneumonia (nsip) (or "cellular interstitial pneumonia" [eip]); usual interstitial pneumonia (uip); fibrosing alveolitis; pulmonary alveolitis; and many others. note: the conditions listed under "related terms" are histologic variants of idiopathic interstitial pneumonia. vip is synonymous with idiopathic pulmonary fibrosis (ipf) ( ). diffuse alveolar damage is the histologic finding in patients with the adult respiratory distress syndrome* (ards) causing interstitial and intra-alveolar fibrosis. this is an acute condition, referred to as acute interstitial pneumonia when it occurs as an idiopathic form of rapidly progressive interstitial pneumonia. possible associated conditions: acquired immunodeficiency syndrome* (aids) in patients with with lip or nonspecific interstitial pneumonia ( , ); trauma and shock in ards. secondary pulmonary fibrosis from inhalants (extrinsic allergic alveolitis or pneumonia; pneumoconiosis*), in drug-induced pneumonia, hypersensitivity pneumonitis (microgranulomatous hypersensitivity reaction of lung), rheumatoid arthritis,* sjogren's syndrome,* and other collagen-vascular diseases; primary biliary cirrhosis in patients with nonspecific interstitial pneumonia ( ). external examination postmortem chest roentgenograms provide the only reliable and permanent record of a pneumothorax and its main complication, mediastinal shift due to a tension pneumothorax (fig. - ) . if roentgenograms cannot be prepared, a reasonably reliable diagnosis still can be made if the prosector inserts a needle through the lateral chest wall. the needle should be connected to a water-filled flask. ifa pneumothorax is present, gas bubbles appear in the flask as shown in fig. - . one can also expose the intact parietal pleura and observe if the lung tissue is separated from the parietal pleura by gas. incising the thorax at the base of a water-filled skin pocket is the least reliable method. infants can be totally submerged under water before the chest cavity is incised. however, care must be taken not to make an accidental incision into the underlying lung tissue. poisoning, all types or type unspecified note: if a specific substance is suspected-for instance, arsenic or ethylene glycol-follow procedures described under the appropriate entry. similar entries can be found for poisons whose general character or source is known-for instance, gas or mushroom poisoning. for some substances, the appropriate entry can be found under "abuse,...," "death,...," or "dependence, ..." in all instances, routine sampling of toxicologic material should be done as described in chapter . if no specific substances can be incriminated, the toxicologist must be provided with all available clinical information, as shown in chapter . in most cases of fatal poisoning, the coroner or medical examiner must be notified. poisoning, alkaloid note: see under specific name of alkaloid-for instance, "dependence, cocaine," "poisoning, atropine," "poisoning, digitalis," or "poisoning, strychnine." only a fraction of this large group of plant poisons has been listed. whether the specific name of the alkaloid is known or unknown, complete toxicologic sampling is recommended. poisoning, antimony ote: toxicologic material should be submitted for anal ysis, as suggested under "poisoning, arsenic." iatrogenic antimony toxicity may occur after treatment with antimony compounds for conditions such as filariasis, fungal infections, and schistosomiasis.* external examination and eyes pharynx and gastrointestinal tract heart, liver, and kidneys record skin changes and eye abnormalities. for toxicologic sampling of contents, see chapter . submit tissue samples for histologic study. request frozen sections for sudan stain. if exposure was from du t in smelting work, dermatitis and conjunctivitis may be present. severe gastroenteritis in acute poisoning. if victim drank antimony trichloride, ulcerative pharyngitis and gastritis may be present. fatty changes of myocardium and hepatic and renal parenchyma. poisoning, arsenic note: toxicologic material will be contaminated by bringing it in contact with fluids. keratinized tissues take up arsenic from solutions. put plastic bags over hands of victim. if exhumed bodies are investigated for arsenic poisoning, include material from surrounding soil and coffin along with tissues submitted for chemical analysis. interpretation of toxicologic findings: after fatal poisoning, arsenic concentrations in the liver tend to exceed . - . mg/ioo g wet tissue. in acute poisoning, arsenic concentrations in hair may reach ! g/g ( ) and in nails ! g/g. organs collect all contents, particularly in accidental poisoning in children. body dry and warm after death. mydriasis. iatrogenic atrioventricular block ( ) . fruits of atropa belladonna or seeds of datura stramonium may be found. no characteristic findings. poisoning, barbiturate(s) note: this type of poisoning has become uncommon. barbiturates may cause sudden death. in all instances, concomitant alcohol intoxication* must be ruled out. standard toxicologic sampling is sufficient. blood bile urine esophagus and stomach liver and brain procedures submit samples of blood from portal vein and peripheral veins or heart for toxicologic study. refrigerate for possible toxicologic study. record total volume and ph value. request tests for protein, glucose, and ketones; request drug screen. submit all contents and record their character. analyze for barbiturates and alcohol. submit samples for toxicologic study. evidence of alcohol intoxication.* poisoning by other addictive drugs. gritty residues of unabsorbed tablets, powder, or capsules. mucosal corrosion, ulceration, and discoloration from capsules may occur. concentration of barbiturate in parenchyma important for interpretation. poisoning, bismuth note: accidental poisoning is common (industrial exposure or drugs with soluble bismuth compounds). search also for other heavy metals. acute kidney failure* may be the cause of death. external examination and oral cavity stomatitis with bluish black discoloration of gums; loose teeth; sticky white membranous patches in mouth and throat. jaundice. protein casts and tubular epithelial cells in sediment. gray or black mucosal membranes; swelling of mucosa; intestinal ulcers that may be perforated. hemosiderosis. fatty changes; hemosiderosis. fatty changes; renal tubular degeneration with amorphic basophilic deposits in epithelium of convoluted tubules. hemosiderosis. see above under "external examination." for removal and specimen preparation, see chapter . peripheral neuritis. synonyms: bromine poisoning; bromism. note: the lethal dose is about . g in children and i g in adults (ingested). after fatal methyl bromide poisoning, headspace gas chromatography revealed a subclavian blood concentration of . microgram/ml whereas inorganic bromide concentrations were micrograms/ml in the blood ( ). tissue concentrations were lower than those in the blood. toxicologic samples should include liver and kidneys. chemical bums on face and conjunctivitis indicate direct exposure; skin pustules over body and nodose bromoderma of the legs indicate bromism. blood is best suited for bromide determination. after ingestion of bromide, necrosis with brown discoloration of mucosa of upper gastrointestinal tract may be present. after inhalation of bromide, swelling and inflammation ofmucous membranes in upper and lower respiratory tracts may be present. there may be pulmonary edema. pneumonia occurs in bromism. organs to be analyzed for cd should have no contact with water or be contaminated with blood; they should be sealed in polyethylene bags. cd leaks into fixation fluid. postmortem blood concentrations are very high and no indicator of the antemortem values ( ). external examination and oral cavity blood urine gastrointestinal tract other organs procedures submit sample for toxicologic study. submit sample for determination of cadmium concentration. submit sample for toxicologic study and a portion of one lobe for microbiologic study. perfuse one lung with formalin. fix bowel as soon as possible. collect renal tissue for light microscopic and electron microscopic study. sample for toxicologic study. submit samples for histologic study also. poisoning, carbon monoxide note: if the victim had been in a fire, see also under "bums." carbon monoxide poisoning may rarely be responsible for automobile accidents. relatively low carboxyhemoglobin concentrations may contribute to death if there is concomitant poisoning-forinstance, with alcohol or drugs, particularly sedatives. anemia, atherosclerotic heart disease, and chronic pulmonary disease also increase sensitivity to carbon monoxide. ifblood had been withdrawn at time of hospital admissionfor instance, for crossmatching-submit this for carboxyhemoglobin determination. if no blood can be obtained, see under "heart, kidneys, and other organs." for quick-orienting qualitative tests, for quantitative methods of carbon monoxide determi-nation, and for interpretation of toxicologic findings, see below. request also determination of hemoglobin concentrations and of blood alcohol. request drug screen. for shipping of blood and tissues for carbon monoxide determination, see chapter . it should be noted that losses of up to % of the original saturation occurred when blood was kept in uncapped container at room temperature for yz wk or at °c for wk. external examination blood heart, kidneys, and other organs brain record color of fingernails, particularly in heavily pigmented persons in whom lividity is difficult to discern. record appearance of blood and submit sample of postmortem blood for toxicologic study. see also above under "note." if no blood can be obtained, prepare water extract of spleen, kidneys, or other organs. request determination of carbon monoxide content and of carbon monoxide-binding capacity of this mixture. submit tissue samples for histologic study. for removal and specimen preparation, see chapter . pink skin and fingernails; bullous edema of skin; decubital ulcers. blood tends to be cherry red. for interpretation of toxicologic findings, see below. necrosis of papillary muscles in the heart or myocardial infarction may occur. renal tubular degeneration may also be found. acute kidney failure* has been observed after rhabdomyolysis complicated by compartment syndrome ( ) . hemorrhagic necrosis of basal ganglia (lenticular nucleus in globus pallidus); diffuse petechial hemorrhages in white matter; cerebral edema. acute hydrocephalus in infants ( ) . many methods of carbon monoxide determination have been described. currently, carboxyhemoglobin is detected in most medical examiner toxicology laboratories by visible spectrophotometry or gas chromatography. in hospitals, carboxyhemoglobin is frequently detected and reported in the course of routine arterial blood gas analysis. pink discoloration of skin and organs usually indicates the presence of more than % carboxyhemoglobin (but rule out cyanide poisoning* and exposure to cold*). in a healthy, middle-aged person, a carboxyhemoglobin concentration greater than - % is usually fatal. if the victim was anemic or suffered from chronic lung disease, particularly emphysema* or atherosclerotic heart disease, the concentration may be lower. in association with alcohol, sedatives, and other drugs, carboxyhemoglobin levels may also be much lower and yet fatal. a heavy cigarette smoker may have a carboxyhemoglobin concentration of - %, and higher levels may occur in police officers and other persons exposed to automobile exhaust in dense traffic. if the victim survived the carbon monoxide poisoning for several hours, postmortem blood samples usually will fail to show the presence of carboxyhemoglobin. in these instances, blood taken at the time of admission to the hospital may still be available and of particular value. if the victim had spent h in fresh air before death, - % of the carbon monoxide will have been removed, and - % will have been removed during each subsequent hour. even though clearance may be complete, death may still occur-primarily from brain damage and infectious complications in prolonged coma. or delayed by only a few hours, particularly after inhalation of carbon tetrachloride. sudden death probably is caused by cardiac dysrrhythmia. alcohol concentrations should be determined in all cases or, if death was delayed, evidence of drinking at the time of exposure should be sought. alcohol considerably increases the hazards of carbon tetrachloride. note: see also under "bronchitis, acute chemical" and under "poisoning, gas." hydrochloric acid is sold by plumbing supply houses and pool supply companies as muriatic acid. it is a liquid. chlorine is a water-soluble gas. as supplied for pool sanitation, liquid chlorine is usually acidic. for convenience, chlorine and hydrochloric acid are discussed here together. prepare photographs of face. conjunctivitis and cyanosis in chlorine gas poisoning; burns of lips from hydrochloric acid. see also above under "larynx and trachea." photograph opened esophagus and stomach. sample for histologic study, particularly if there is doubt whether a perforation was antemortem or postmortem. for removal and specimen preparation, see chapter . severe pulmonary edema, bronchopneumonia, and swelling of mucous membranes in chlorine poisoning. arterial thrombosis may occur. pulmonary fibrosis may develop after prolonged survival. swelling and ulceration of mucous membranes in chlorine poisoning; acute laryngotracheitis. tracheoesophageal perforation. corrosion of mucosa with thickening, hemorrhage, and blackish discoloration after ingestion of hydrochloric acid. antemortem and postmortem perforation may occur. glomerular capillary thromboses. hemorrhages in white matter ( ). poisoning, cyanide synonym: hydrocyanic acid (hydrogen cyanide) poisoning. note: hydrocyanic acid (hydrogen cyanide, hcn) is a water-soluble gas. its salts, sodium cyanide and potassium cyanide are sold as "eggs" to the jewelry industry. hydrocyanic acid is formed when cyanide salts are dissolved in acidic solutions. containers from which the poison might have been ingested or inhaled should also be submitted for toxicologic examination. for cyanide screening tests in the autopsy room and for interpretation offindings, see below. caution: stomach may still contain cyanide gas, formed by acidic reaction of cyanide salt. it may be best to open the stomach under a hood ( ). the odor is quite characteristic for cyanide poisoning but most persons are unable to smell this odor. it is helpful to know in advance if any person in an office or laboratory can smell cyanide. forensic pathologists who can smell the compound state that it has its own specific odor, which differs from the often quoted smell of bitter almonds. autopsies also can be done in a negatively pressured isolation room ( ). for odor, see above under "note." bright red skin color is not always present. corrosion around mouth and in oral cavity may be found after ingestion of potassium or sodium cyanide. blood is fluid and sometimes bright red. if potassium or sodium cyanide was ingested, brown-red mucosal corrosion may be present in stomach or in upper digestive tract. pulmonary edema. for odor, see above under "note." if death was not instantaneous, there may be hyaline thrombi in small blood vessels, minute hemorrhages, and necroses of lenticular nuclei. this test can be used for blood and gastric contents ( ) . dip squares of filter paper in a small amount ofsaturated picric acid. let these squares dry until barely moist. place a drop ofthe material to be tested-e.g., blood or gastric contents--on a piece of paper. let material dry for a moment, and then place one drop of % sodium carbonate in the center of the material to be tested. ifcyanide is present, a reddish purple color will chromatograph out from the material. the higher the concentration of cyanide the more blue the color will be. it is possible to recognize whole blood because the blood turns a rather dark brown and the reddish to purple color is clearly visible. high concentrations of sulfide interfere by giving a false-positive test. another screening test is done as follows ( ) . dip filter paper into normal blood. then treat the paper with potassium chlorate, whereupon brown methemoglobin forms. place this preparation into the fluid suspected of containing cyanide (e.g., blood, gastric contents, pulmonary edema fluid). if bright red cyanmethemoglobin forms, the reaction is positive. if the concentration of cyanide in the stomach is high and the concentration in the lungs is low, cyanide was ingested. alternatively, if the pulmonary cyanide concentration is high and the concentrtaion in the gastric contents is low, hydrogen cyanide most likely was inhaled. occasionally, minimal cyanide levels will be present in decomposed bodies. related term: digoxin toxicity. note: certain drugs may interfere with correct digitalis determination. blood heart vitreous procedures submit sample of peripheral blood for digoxin radioimmunoassay. freeze fresh myocardium for digitalis extraction. submit sample for digoxin radioimmunoassay. poisoning, drug(s) (see "dependence, drug(s), all types or type unspecified" or under "poisoning,•••" followed by specific name of drug.) poisoning, ethanol (ethyl alcohol) (see "alcoholism and alcohol intoxication," "cardiomyopathy, alcoholic," "disease, alcoholic liver," "syndrome, fetal alcoholic," and syndrome, wernicke-korsakorr.") poisoning, ethylene glycol related term: antifreeze poisoning. note: pulmonary and cerebral manifestations are the main findings in acute poisoning, and renal tubular necrosis is the primary finding in chronic poisoning. for general toxicologic sampling, see chapter . calcium oxalate crystals can be demonstrated in routine histologic sections but also in scanning electron micrographs of thick deparaffinized sections. eyes blood urine for removal and specimen preparation, see chapter . in acute cases, submit sample for ethylene glycol determination; in chronic cases, request determination of calcium concentrations. prepare sediment. papilledema; optic nerve atrophy. ethylene glycol in serum (i) . protein casts; calcium oxalate crystals ( ) . crystals are light yellow and birefringent, arranged as sheaves, rhomboids, or prisms. poisoning, food related terms: bacillary dysentery* (shigella food poisoning); botulism;* clostridium perfringens food poisoning; favism; mushroom poisoning;* salmonella food poisoning; staphylococcal food poisoning. note: if cause of food poisoning is unknown, submit suspected food for aerobic and anaerobic cultures, gram stain of smears, and routine toxicologic study. this should include tests for heavy metals (antimony, cadmium, and lead) that may have leaked from old cooking utensils. test for the presence of staphylococcal enterotoxin are done only in specialized laboratories. if botulism is suspected, follow procedures described under that heading. mushroom poisoning also is listed as a separate entity. if salmonella food poisoning is suspected, see under "fever, typhoid." see also under "enteritis" or "enterocolitis" or under another specific heading such as "dysentery, bacillary." obtain sufficient material for microbiologic and histologic study to identify organisms such as chlamydia, clostridium (type f strains), salmonella, shigella, verotoxic e. coli, yersinia, and others. external examination gastrointestinal tract submit contents for aerobic and anaerobic cultures (see above under "note"); prepare smears of contents for gram stain. submit samples for histologic study. debilitated states; patients in extremes of life. enteritis or enterocolitis. poisoning, gas note: anesthesia-associated death, * carbon monoxide poisoning,* and sniffing and spray death* are presented under the appropriate headings. procedures discussed here deal with other volatile substances, including chemical irritants such as ammonia (nh ), chlorine or hydrochloric acid poisoning (see also under that heading); methylene chloride, phosgene (coci ), sulfurous acid (h s ), or sulfur dioxide (s ) ' gases from body cavities, heart chambers, or blood vessels can be removed as described under "embolism, air." gases can also be trapped with a rubber dam after cutting organs under water. samples from various organs should be shipped in hermetically sealed nonplastic containers or in analyzing solutions. external examination, and oral cavity blood larynx and trachea record extent of chemical bums. submit sample for gas analysis. in many instances, inhaled gases can be demonstrated chromatographically in gas from head space above sealed blood specimen. chemical bums in and around mouth or conjunctivas. chemical bums. other organs if gas was inhaled and is to be analyzed, submit intact lungs with bronchi ligated in airtight, nonplastic container to laboratory that can conduct gas analysis. if survival was short, formalin perfusion of lungs is not recommended; it may cause artifactual ballooning and internal ruptures of organ. submit samples of tissue for routine histologic study. see above under "note." chemical pneumonia; pulmonary edema. after longer survival, obliterating fibrous bronchiolitis, chronic bronchitis, and saccular bronchiectasis* may occur. poisoning, glycol (see "poisoning, ethylene glycol.") poisoning, halogen (see "fluorosis," "poisoning, bromide," "poisoning, chlorine or hydrochloric acid," "poisoning, gas?, and "poisoning, iodine!') poisoning, heavy metal (see "poisoning, antimony," "poisoning, arsenic," "poisoning, cadmium," "poisoning, lead," poisoning, mercury," "poisoning, thallium!') poisoning, insecticide (see "poisoning, organophosphate(s)*) poisoning, iodine related terms: lugol's solution; tincture of iodine. for toxicologic sampling, see chapter . external examination and eyes urine, blood, and parenchymal organs lungs and upper respiratory tract stomach record color of skin and extent of corrosive lesions. submit samples for toxicologic study. submit samples for histologic study. submit contents for toxicologic study; photograph mucosa; prepare histologic sections. see above under "stomach." perioral corrosive lesions; yellow discoloration of skin; conjunctivitis after exposure to vapors. acute inflammation of respiratory tract after inhalation of vapors. corrosive gastritis; if starch was used as antidote, gastric lining will be bluish. histologically, well-preserved mucosa is present because of in vivo fixation. mucosa may show same changes as stomach. swelling of tubular epithelium. synonyms: propanol; rubbing alcohol. procedures see under "alcoholism and alcohol intoxication." nonspecific autopsy findings: visceral congestion; pulmonary and cerebral edema. poisoning, lead note: lead-free syringes and lead-free polyethylene containers should be used. blood lead concentrations can be determined by inductively coupled plasma mass spectrometry ( ). for screening methods, see ref. ( ) . this is a reportable disease in some states. external examination, oral cavity, and hair bluish lead line at gingival margin in victims with poor oral hygiene. external examination, oral cavity, and hair for diagnosis of chronic plumbism, analysis of scalp hair may be useful. analysis is done by neutron activation (see also under "poisoning, arsenic"). remove samples with lead-free syringe (see above under "note") or -ml vacutainer tubes (becton, dickinson and company). do not add anti-coagulant or preservative. for coliection procedures, see above under "blood" and under "note." request also determination of coproporphyrin concentrations. submit sample for histologic study; submit remaining tissue for toxicologic analysis. submit with contents for toxicologic study, particularly in acute poisoning. submit sample from each kidney for histologic study; submit remaining tissue of both kidneys separately for toxicologic study. submit at least g of fresh bone for toxicologic study. for removal and specimen preparation, see chapter . poisoning, lsd (d-lysergic acid diethylamide) (see "abuse, hallucinogen(s).") poisoning, lye related terms: ammonium hydroxide poisoning; calcium oxide or quicklime poisoning; poisoning by alkaline corrosives; potassium hydroxide poisoning; sodium hydroxide poisoning. external examination and oral cavity blood neck organs, esophagus, trachea, and lungs record extent of oral, perioral, and other facial corrosive injuries. photograph lesions. prepare histologic sections of tissue from inside of lips or mouth. submit sample for toxicologic study. after removal of heart, remove neck organs with hypopharynx, esophagus, larynx, and trachea. leave stomach attached to esophagus. open pharynx and esophagus along posterior midline. in acute cases, formalin perfusion of lungs is not recommended; it may cause artifactual baliooning and internal ruptures of organ. lye bums on face and chest in acute cases; scars and manifestations of malnutrition* in chronic cases. sweliing, edema, and necrosis of mucous membranes in acute poisoning. fibrosis and strictures in chronic cases. bronchitis* and bronchopneumonia. submit specimen from pharynx for histologic study. for removal and specimen preparation, see chapter . submit sample of brain for toxicologic study. exfoliative dermatitis. blue line at gingival margin; hypertrophy of gum; acute and chronic gingivitis; exfoliation and loss of teeth ( ) . degeneration of myocardium. increased concentrations of mercury ( ) . congestion. erosive gastritis and colitis. increased concentrations of mercury ( ) . degeneration of proximal tubules; calcifications. chronic kidney failure* may be the cause of death. induration of mucosa. increased concentrations of mercury ( ) . cortical hemorrhages. cholinesterase activity will be low. pulmonary edema if poison was inhaled. airways may contain aspirated material. cholinesterase activity can be determined reliably even after decomposition and embalming. clinically, guillain-barre syndrome has been observed after poisoning with organophosphate. in acute poisoning, the cholinesterase levels may be % of the normal values (see below). the cholinesterase levels in the blood are not affected by the duration of the postmortem interval; measurement may be attempted even on decomposed or exhumed bodies. severe fatty changes; periportal necroses. fatty changes in myocardium, skeletal muscles, and other organs. for toxicologic sampling (gastric contents, urine, blood, brain, and other organs), see chapter . rigor mortis after fatal strychnine poisoning may occur very soon after death and may be very severe (opisthotonos); it may persist until decomposition sets in. congestion of viscera; no characteristic morphologic autopsy findings. acute pancreatitis has been observed ( ). high strychnine concentrations (also demonstrable in exhumed bodies [ ] ). poisoning, thallium note: for toxicologic sampling, see below and chapter . thallium is used as a rodenticide and pesticide, and has some industrial applications. retrobulbar neuritis. chapter and . submit brain for toxicologic study. submit sections of brain and optic nerves for histologic study. submit specimens for toxicologic study (take from lower extremity). for removal, prosthetic repair, and specimen osteomalacia;* osteomyelofibrosis. preparation of bones, see chapter . for preparation of sections and smears of bone marrow, see chapter . submit samples for toxicologic study. synonym: acute anterior poliomyelitis. note: the disease has been nearly eliminated in the usa but not in many other countries. ( ) collect all tissues that appear to be infected. ( for removal and specimen preparation, see chapter . in acute cases, submit portions of brain and spinal cord for viral culture. neurogenic atrophy of skeletal muscles in areas of paralysis. electrolyte disorder. * hypertensive heart disease; myocarditis.* aspiration or bronchopneumonia (or both); edema; atelectasis; embolism;* alveolar wall necrosis (acute or organizing diffuse alveolar damage) after oxygen toxicity. acute ulcers. acute gastric dilatation; acute gastroduodenal ulcers; gastrointestinal erosions and hemorrhages. dilatation of colon; perforation of cecum. urolithiasis and nephrolithiasis,* pyonephrosis and pyelonephritis. * phlebothrombosis of legs, most commonly on left side. necrosis of anterior hom cells of spinal cord, with neuronophagia and perivascular inflammatory reaction. old lesions show neuronal loss and gliosis. medulla ("bulbar polio") and other areas of brain stem, cerebellum, and cerebrum, particularly the motor cortex, may be affected in various degrees. part ii / diseases and conditions pregnancy note: in some instances, procedures described under "death, abortion-associated," "embolism, amniotic fluid," or "toxemia of pregnancy" may be indicated. synonym: werner syndrome. external examination and skin abdomen procedures record volume of intraperitoneal fluid; prepare smears; submit samples of peritoneum for histologic study. colloid carcinoma of stomach or colon. well-differentated adenocarcinoma of ovary or, rarely, of appendix; ruptured appendiceal mucinous adenoma is the most common cause of peritoneal adenomucinosis. pseudotumor cerebri synonym: benign intracranial hypertension; meningeal hydrops. note: this condition, which generally affects young, obese females, is characterized by symptoms and signs of increased intracranial pressure without a demonstrable cause. hence, intra-cranial mass lesions, infections, and related conditions should be excluded. such conditions include adrenal insufficiency;* guillain-barre syndrome* (increased colloid-osmotic pressure); hyperadrenalism; hypervitaminosis a* (e.g., after treatment of acne); hypoparathyroidism;* hypothroidism;* infectious mono-nucleosis;* lyme disease; pregnancy; sydenham's chorea; throm-bus ofthe lateral or superior sagittal sinus (otitic hydrocephalus); and wiskott-aldrich syndrome. submit samples of all accessible organs and tissues, with or without gross lesions. submit material for electron microscopy. for removal and specimen preparation, see chapter . for removal, prosthetic repair, and specimen preparation, see chapter . heart small bowel liver procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. fix the bowel as soon as possible. record weight. submit samples for histologic study. for removal, prosthetic repair, and specimen preparation, see chapter . aortitis involving ascending aorta and aortic valve with regurgitation; mitral valve and adjacent myocardium and conduction system also may be involved. sprue-like changes with loss of villi. fibrosis or cirrhosis and other abnormalities, particularly in patients who had been taking methotrexate. psoriatic arthritis and spondylitis. synonyms and related terms: allergic purpura; anaphylactoid purpura; hypersensitivity vasculitis. external examination and skin rabies note: (i) in most instances, an autopsy limited to the brain is sufficient to confirm the diagnosis. ( ) rabies virus is especially infectious and thus, universal precautions should be strictly followed (see chapter ) . avoid the use of scalpels whenever possible. the generation ofaerosols should be assidu- see above under "note." if a complete autopsy is done, sample heart, lungs, kidneys, pancreas, submaxillary salivary glands, and adrenal glands; freeze or refrigerate sampled tissues and submit for viral study (see below under "brain and spinal cord"). obtain serum for serologic studies. freeze or refrigerate cerebral tissue immediately after removal and submit frozen to special laboratory for fluorescent antibody staining. take these sections from hippocampus or brain stem. place the refrigerated tissues in % neutral glycerol saline solution for preservation. fix remaining brain and spinal cord tissue in % formalin and submit for histologic study. as an alternative to freezing of tissue, immunoperoxidase methods for detection of rabies viral antigens can now be applied to formalin-fixed tissue ( in almost all instances, the procedures described under "assault" and under "homicide" must also be followed. see also refs. ( ) and ( ) . for the evaluation of bite marks, photographs with and without scales and black and white film should be used. a forensic dentist is required to compare the victim's bite marks to the teeth of suspects. swabs of possible sperm or other fluids must be sent to the crime lab for proper evaluation. external examination other organs and tissues genital organs comb pubic hair over a towel and then pull sample; also, pull samples of hair from head. collect fingernail clippings and place in containers marked "right" and "left." collect blood, hair, fibers, and residues of urine or saliva and/or semen that may have stained the victim's clothing or that may be found on the skin of the victim. for study of stains and scrapings, see below. introduce sterile dry cotton swab into the posterior vault of the vagina or-preferablyinto the cervical canal. prepare smear on glass slide and send to crime laboratory for identification of sperm (see also above under "note" using a sterile cotton swab moistened with sterile saline, lift suspected dried spermatic fluid from hair or skin of external genitalia, perineum, buttocks, or other sites. stains on clothing will be extracted by forensic laboratory personnel. let specimens air dry in absence of sunlight and then place in paper bags or envelopes and seal. smears are made for the demonstration of spermatozoa, cytologic changes, bacteria, and other possible findings (some crime labs prefer to make their own smears from the swabs). other samples are used for the determination of acid phosphatase, as described below. usually, the survival time of morphologically recognizable spermatozoa in the vagina is less than h, but on occasion this may be much longer. thus, within the first h after coitus, % of cervical smears have been found to be positive for spermatozoa. at day , spermatozoa have been found in % of the smears. obviously, if the assailant had had a vasectomy, spermatozoa will not be found at any time. spermatozoa have been found in the vagina of some women several days or weeks after death. dried stains (see above) may give positive results after longer intervals. acid phosphatase activities greater than bodansky units (for method, see above) indicate that probably less than h have elapsed since the time of intercourse (i) . in another study, vaginal swab specimens showed acid phosphatase activities of more than , king if there is evidence of parotid involvement, other salivary glands should also be studied. parotid gland can be biopsied from scalp incision. submaxillary gland can be removed with floor of mouth. for sampling and specimen preparation, see chapter . for removal, prosthetic repair, and specimen preparation, see chapter . chronic meningitis; space-occupying lesions (submeningeal nodular granulomas). granulomatosis. iridocyclitis; chorioretinitis; papilledema; posterior uveitis; keratoconjunctivitis sicca; conjunctival follicles; cataracts. involvement of lacrimal glands and other orbital tissues ( ) . sarcoidosis of parotid gland is common. sarcoid neuropathy and sarcoid myopathy. cystic changes, primarily of small bones of hands and feet. sarcoidosis of joints ( ). synonyms and related terms: bilharziasis; schistosoma haematobium infection; schistosoma japonicum infection; schis-tosoma mansoni infection. note: unless specifically stated, the changes listed below refer to chronic schistosomiasis mansoni and japonica. ( ) collect all tissues that appear to be infected. ( ) request direct examination for schistosoma. the following procedures have been described and recommended ( ). for demonstration of schistosoma eggs, compress -mm tissue fragments-for instance, mucosa of the urinary bladder-between glass slides. if this gives negative results, digest a -g portion of tissue in potassium hydroxide. for the recovery of adult worms of schistosoma mansoni and schistosoma haematobium, remove the viscera en bloc and rinse with water. separate the intestines from the mesentery. subsequently, perfuse the portal vein system, the liver, and one lung with saline ( ) . then pass the perfusion fluid through a monofilament nylon cloth with an aperture size of m. submerge the cloth in water and examine with a dissecting microscope. fix worms in formalin solution. examine the intestinal mucosa directly. from the urinary bladder, ureters, and surrounding connective tissue, worms can be recovered as follows. inject water into the tissue until the tissue increases or times in thickness. then compress the tissue gently with a glass plate. cut slices . - . cm in thickness. compress these slices between the glass plate and the stage of a dissecting microscope and examine for the presence ofadult worms. many worms also will be present in the fluid expressed from the tissue as it is cut. for the counting of eggs in tissues, urine, and feces, see ref. ( ) . immunodiagnostic methods (elisa and imrnunoblot) also have been developed prepare skeletal roentgenograms. there are no diagnostic laboratory tests but it still is advisable to save a blood sample. record heart weight. test competence of valves (chapter ). open heart in line of blood flow. photograph and measure valvular lesions. prepare sections of valves, myocardium, conduction system (if there was a history of heart block), and ascending aorta. perfuse at least one lung with formalin. submit any consolidated area for microbiologic study. fix intestines as soon as possible. abnormalities as listed in right-hand column. sample for histologic study and request amyloid stains or renal parenchyma. prepare roentgenograms of spine, sacroiliac joints, symphysis ossium pubis, and manubriosternal, sternoclavicular, and humeroscapular joints. if spine cannot be removed in its entirety, it can be split in midline and one half can be removed, with costovertebral and costotransversal joints. hip joints should be exposed. histologic sections should include synovia and periarticular tissue. secondary and peripheral osteoarthritis* may be present. leukemia* developed in some patients who had had radiation treatment ( or earlier). acute anterior uveitis. ( ) request fungal culture. ( ) request grocott's methenamine silver stain. ( ) record weight of all organs (for expected weights, see part iii). submit samples of all major organs, including endocrine glands, lymphatic and fat tissue, bone, and bone marrow for histologic study. hunger edema; skin changes secondary to vitamin deficiencies. hypoproteinemia. atrophy; hemosiderosis of spleen. degree of atrophy varies from organ to organ; atrophy of fat tissue, lymphoid tissue, and gonads usually is most pronounced. severe infections, such as tuberculosis,* may be present without having been apparent clinically. steatohepatitis, nonalcoholic (nash) note: the morphologic findings in the liver are indistinguishable from those in alcoholic liver disease* ( ). follow autopsy procedures described under "disease, alcoholic liver." if the patient had received a liver transplant, procedures described under "transplantation, liver" should be followed also. possible associated conditions: celiac sprue (rare); diabetes mellitus* (type ); hyperlipidemia; malnutrition* from bulemia (rare); morbid obesity with liver failure particularly after episodes of rapid weight loss (e.g., after recent gastroplasty); lipodystrophy; mild obesity;* short bowel syndrome (rare); total parenteral nutrition. * stenosis, congenital valvular pulmonary related terms: isolated (pure, simple, or dome-shaped) pulmonary stenosis. note: the pulmonary valve is usually acommissural in isolated congenital pulmonary stenosis. with other coexistant congenital heart disease (such as tetralogy), the pulmonary valve is usually bicuspid and hypoplastic, but may be unicommissural or dys-plastic and tricuspid. heart and great vessels; peripheral pulmonary arteries brain procedures culture any grossly infected valve. for general dissection techniques, see chapter . record weight of heart, thickness of ventricles, and annular circumferences. for removal and specimen preparation, see chapter . infective endocarditis* of pulmonary valve and tricuspid valve; infective endarteritis at bifurcation of pulmonary trunk. it is preferable to have autopsies of fetuses and stillborns performed by pathologists experienced in perinatal pathology. if such personnel are not immediately available, the attending pathologist may nevertheless collect important information. if attempts to induce abortion appear to have caused the death of the mother, see "death, abortion-associated." the placenta should be immediately procured from the delivery room should it not arrive in the laboratory with the fetus. this will avoid the possibility of the placenta being discarded by the delivery room staff. no fetal autopsy is complete without a careful examination of the placenta (see part i, chapter ). pathologic changes of placenta that are causative of stillbirth are summarized in ref. . fascia lata or other aseptically obtained tissue should be collected for tissue culture for karyotype analysis. if the fetus is at all autolyzed, then the fascia lata cells may not grow in tissue culture. therefore, if the fetus shows any evidence of autolysis, tissue should be taken aseptically, from placenta immediately beneath the chorionic plate. a portion ofplacenta and liver should also be snap-frozen for possible molecular analysis. the initial stage of the autopsy should include photography and radiography, taking anterior-posterior and lateral views. the photographs will record the degree of maceration, which can be roughly correlated with the duration of fetal demise before delivery [ ] external measurements should include body weight, circumference ofhead, chest and abdomen, crown-rump length, crown-heel length, and foot length. these measurements are compared with tables of standards for normal fetuses (see part iii of this book). assessment of growth retardation may be based on these data. a careful external examination should search for abnormalities such as jaundice, bulging fontanel, cranial bone softening, hyper-or hypotelorism, choanal atresia, external ear anomalies, cleft lip, cleft palate, macroglossia, micrognathia, colobomata, cystic hygroma, shortened neck, contractures, omphalocele, gastroschisis, abnormal external genitalia, anal atresia, absent vagina, sacral pits, open neural tube defects, hemihypertrophy, syndactyly, clinodactyly, transverse palmar creases, or incomplete descent of testes. the organ bloc may be removed in the manner similar to the adult, using the technique of letulle (see chapter ) . if the thyroid gland is noted to be in its usual location and if it appears normal, then the tongue may be left in the body. in macerated fetuses, it is suggested that the organ bloc be fixed overnight in formalin solution prior to dissection. to aid adequate fixation, the following simple steps may be done: i) wash the organ bloc thoroughly prior to fixation; ) place multiple transverse cuts through the liver and lungs; ) dissect the posterior leaves of the diaphragm away from the adrenal glands and kidneys; ) bivalve the adrenal glands and kidneys in the coronal plane; ) instill formalin in the lumen of the intestine, using a syringe; and ) gently instill formalin into both ventricles of the heart, being careful to avoid the ventricular septum. the procedure for dissection of the organs is similar to that of the adult except: ) the venous and arterial connections of the heart, including the patency of the ductus arteriosus must be determined before the heart is removed; ) the esophagus must be opened posteriorly prior to its complete removal so that esophageal atresia or tracheo-esophageal fistula may be recognized and photographed prior to further dissection; ) the location of the appendix and of the testes should be recorded. until the intestine has been examined for stenosis or atresia, the mesentery should be left attached. the degree of autolysis as seen grossly and with histologic examination of both the fetus and the placenta can be used to estimate the duration of time between fetal death and delivery ( , , ) . trichrome stain is useful for better visualizing histologic features in severely autolyzed tissue. to remove the brain, benecke's technique of one of its modifications may be used. stillbirth vs livebirth. a decision has to be made whether the infant was born alive or was stillborn. the hydrostatic lung test, described in the previous edition, appears unreliable. the presence of gas in the lungs does not rule out stillbirth. after death, air can be introduced into the lungs, or putrefaction gases might be present. however, air artificially introduced after death will not distend the alveoli and can be squeezed out, whereas this does not seem to be the case after active ventilation. the distribution of fat in the fetal zone of the adrenal cortex may indicate whether intrauterine death was acute, more prolonged, or chronic ( ). this is particularly helpful if the stillborn baby is macerated. if the mother died also, see under "death, abortion-associated" strangulation note: in many instances, procedures described under "hom-icide" must also be followed. if a rope or some other material had been used (see also under "hanging"), leave ligature in place until autopsy can be done. see also under "hypoxia." toxicologic sampling, particularly for alcohol, should be done in all instances (chapter ). results than body fluids. ( ) universal precautions should be strictly followed. the generation of aerosols should be minimized. to sterilize tissue surfaces in preparation for culture, swab the surface with povidone iodine. avoid searing the tissue surface since this will produce an aerosol. keep no more than one scalpel in the dissecting area at anyone time. have an assistant available with clean, gloved hands to receive specimens in containers, so as to minimize the degree of contamination on the outside of the containers. for cleaning procedures and related information, see chapter . ( ) hiv-l in tissue may be demonstrated using polymerase chain reaction (pcr), immunohistochemistry, in situ hybridization, or immunofluorescence. ( ) for immunocytochemical and molecular studies, fix tissue in ethanol or carnoy's solution. the virus can be identified using pcr in most tissues, whether fresh, frozen, or fixed in ethanol. ( ) serologic tests as well as direct fluorescent antibody tests are avail-able for many of the expected infections. ( ) this is not a reportable disease. external examination and skin record body weight and evidence of lipodystrophy following aids medications. record and photograph any skin lesions. examine oral cavity. if the diagnosis is in doubt, samples can be submitted for enzyme immunoassay. cachexia. severe loss of subcutaneous fat in face and extremities, associated with large fat deposits on upper back and upper abdomen ("protease paunch"). cutaneous kaposi's sarcoma (l) . test may be positive for many weeks after death ( ) . syndrome, myelodysplastic synonyms and related terms: chronic myelomonocytic leukemia;* refractory anemia; refractory anemia with excess of blasts; refractory anemia with excess of blasts in transformation; refractory anemia with ringed sideroblasts; refractory dysmyelopoietic anemias. note: the myelodysplastic syndromes are represented by a heterogeneous group of normocytic anemias, often with neutropenia, thrombocytopenia, and monocytosis. for expected bone marrow changes, see above under "synonyms and related terms." autopsy procedures are similar to those recommended for most cases of leukemia, with particular attention paid to intercurrent infections and thrombocytopenic hemorrhages. in all instances, material should be collected using aseptic technique for tissue culture for chromosome analysis. common findings in these conditions are deletion of the long arm ofchromosome , deletion of chromosome or , or trisomy . syndrome, nephrotic note: see under name of suspected underlying condition, such as amyloidosis,* anaphylactoid purpura,*diabetes mellitus,* glomerulonephritis,* goodpasture's syndrome,* heavy metal poisoning, hemolytic uremic syndrome,* infective endo-carditis,* polyarteritis nodosa,* syphilis, * or systemic lupus erythematosus. * if accelerated hypertension or constrictive pericarditis is the suspected underlying condition, see under "hypertension (arterial), all types or type unspecified" or "pericarditis," respectively. in all instances, the renal veins and the inferior vena cava should be opened in situ. "en masse" removal of organs is recommended for this purpose. if thrombosis is found, record exact location and size of clot and submit sample of clot with wall of veins for histologic study. see also under "thrombosis, venous." coronary atherosclerosis and its complications seem to be increased in patients with the nephrotic syndrome. submit sample for bacteriologic, viral, and serologic study. record weight and submit samples for histologic study. if heart block was present, submit samples of conduction system for histologic study (chapter ). submit consolidated areas for microbiologic study. perfuse at least one lung with formalin. for removal of urethra, see chapter . procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for removal and specimen preparation, see chapter . consult roentgenograms (see above under "external examination, skin, and oral cavity"). keratoderma (keratosis blennorrhagica) with vasculitis ( ) of sole of feet, of palms, and of circumcised glans penis. arthritis* of knees, ankles, and metatarsal and midtarsal joints, with or without ankylosis. osteoporosis.* usually, sterile culture. pericarditis; * myocarditis.* aortic valve lesions. pleuritis and pneumonia.* pulmonary fibrosis involving upper lobes. erosions, papules, and plaques in urethral or bladder mucosa. enteritis. thrombophlebitis. conjunctivitis; multifocal choroiditis; acute anterior uveitis; iritis; keratitis. arthritis* (see above under "external examination, skin, and oral cavity") resembling rheumatoid arthritis. * nonspecific synovitis. for removal and specimen preparation, see chapter . syndrome, reye's note: hypoglycemia* may have been present, but that condition is difficult or impossible to confirm after death. an immediate autopsy is indicated (see below under "liver" and "pancreas"). vitreous blood urine heart lungs submit sample for sodium, chloride, and urea nitrogen determination. submit sample for microbiologic (viral) and serologic study, for ammonia and bilirubin determination, and for determination of salicylate levels if there is a history of treatment with this drug. obtain sample for biochemical and toxicologic analysis. record weight. request frozen sections for sudan stain. submit fresh tissue for bacterial and viral culture. request paraffin sections and frozen sections for fat stain (see above under "heart"). influenza;* varicella.* manifestations of liver failure. evidence of salicylate administration. assay for organic acids should rule out acylcoenzyme a dehydrogenase deficiency, and toxicity, e.g., of acetaminophen and valproic acid (l) . cardiomegaly; fatty changes of myocardium and patchy myocytolysis. viral pneumonia rarely present. acute interstitial pneumonia;* bronchitis;* hemorrhages. lipid-laden histiocytes in alveoli. record body weight and length, stature, and distribution of head, axillary, and pubic hair. record and prepare photographs of features of face and neck. prepare skeletal roentgenograms. submit samples of breast tissues for histologic study. these specimens should be collected using aseptic technique for tissue culture for chromosome analysis (see chapter ) . procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. submit for histologic study. submit samples of all portions for histologic study. if biliary atresia is suspected, follow procedures described under that heading. dissect kidneys and ureters in situ and record findings. submit samples of gonads or-if gonads cannot be identified-equivalent ridges on mesosalpinx for histologic study. also submit samples of endometrium, cervix, and vagina. record weight and submit sample for histologic study. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for removal and specimen preparation, see chapter . procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for sampling and specimen preparation, see chapter . peroxidase-labeled gastrin antibodies seem to react with cells in all gastrinomas. aberrant gastrinoma may occur at hilus of spleen. metastases are found in regional lymph nodes and liver. there may be manifestations of multiple endocrine neoplasia. * secondary syphilis. levaditi's stain or warthin-starry stain is recommended for paraffin sections, and labeled fluorescent antibody techniques are recommended for frozen sections. india ink preparations or the fontana-masson silver stain has been used for the study of fresh lesions, and electron microscopy has also been employed. ( ) in adult autopsies, no special precautions are indicated (see below under "liver"). ( ) serologic studies are available from local and state health department laboratories. ( ) this is a reportable disease. external examination record and prepare photographs of all abnormalities listed in right-hand column. prepare smears and sections of acute lesions; prepare sections of older skin lesions or anogenital mucosal lesions. hunterian chancre in primary syphilis; condylomata lata. noduloulcerative gummas and scarring in later stages. cerebrospinal fluid lymph nodes heart and aorta other organs and tissues prior to wk gestation, the destructive effects of syphilis may not be seen. gummata are rare in neonates. tabes dorsalis is also uncommon. serologic diagnosis is difficult in the neonate because of transplacental transfer of maternal igg antibodies. acquired syphilis (syphilis in adulthood) is presented above under a separate heading. ( ) collect all tissues that appear to be infected. ( ) culture methods are not available, but animal inoculation can be performed. consultation with a microbiology laboratory is recommended. ( ) special stains for treponema pallidum rarely are positive except with material from fresh lesions of primary or secondary syphilis and of syphilitic hepatitis of the newborn. levaditi's stain or warthin-starry stain is recommended for paraffin sections, and labeled fluorescent antibody tech-niques are recommended for frozen sections. india ink preparations or the fontana-masson silver stain has been used for the study of fresh lesions, and electron microscopy has also been employed. ( ) in neonates, special precautions are indicated (see below under "liver") ( ) serologic studies are available from local and state health department laboratories. ( ) this is a report· able disease. (i) collect all tissues that appear infected. ( ) request aerobic and anaerobic cultures. however, the presence of tetanus bacilli established in culture is not diagnostic, since spores of c. tetani frequently contaminate wounds. record body weight and length and appearance of wound(s); photograph and excise wound(s) for histologic study. record evidence of parenteral drug abuse, especially subcutaneous injection (i.e., "skin popping"). prepare chest roentgenogram. evidence of weight loss; subcutaneous abscesses. tetanus may occur in drug addicts. tension pneumothorax* after mechanical ventilation. submit sample for microbiologic study. submit any consolidated area for microbiologic study. bacterial meningitis must be ruled out. aspiration and bronchopneumonia; embolism;* atelectasis. tetany note: see under name ofsuspected underlying conditions, such as hyperparathyroidism,* malabsorption syndrome,* or vitamin deficiency. * respiratory or metabolic alkalosis* cannot be confirmed after death; determination of blood ph is not helpful because acidity increases rapidly after death. the concentration of serum phosphates also increases after death. synonym: large ventricular septal defect with pulmonary stenosis* or atresia.* note: the basic anomaly consists of subpulmonary stenosis, ventricular septal defect, overriding aorta, and secondary right ventricular hypertrophy. for general dissection techniques, see chapter . surgical interventions include modified blalock-taus-sig subclavian-to-pulmonary arterial shunt; complete repair with patch closure of ventricular septal defect, and reconstruction of right ventricular outflow tract (with a patch or with an extracardial conduit). possible associated conditions: origin of left pulmonary artery from aorta; minor abnormalities of the tricuspid valve; absent ductal artery ( %); atrial septal defect* (in %; pentalogy of fallot); bicuspid pulmonary valve;* dextroposition of aorta; double aortic arch; hypoplastic pulmonary arteries; patent ductal artery;* patent oval foramen; complete atrioven-tricular septal defect* (usually with down's syndrome*); persis-tent left superior vena cava; pulmonary valve atresia (see "atresia, pulmonary valve, with ventricular septal defect"); right aortic arch ( %); second ventricular septal defect; origin ofleft anterior descending (lad) or right coronary artery (rca) from contralateral aortic sinus or coronary artery ( %); syndrome with absent pulmonary valve and massively dilated pulmonary arteries (rare). chest cavity heart lungs other organs record course of superior vena cava and of its tributaries. record course of thoracic aorta and of its main branches. record origin of pulmonary arteries. if infective endocarditis is suspected, follow procedures described under that heading (chapter ). for coronary arteriography, see chapter . perfuse both lungs with formalin. request verhoeff-van gieson stain. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. persistent left superior vena cava. right aortic arch with or without right-sided ductal artery; double aortic arch; absent ductal artery. origin of the left pulmonary artery from descending aorta. for possible additional findings, see above under "note" and under "possible associated conditions." infective endocarditis* (usually of pulmonary or aortic valve). see also above under "possible associated conditions." marked right ventricular hypertrophy. right ventricular dilatation and fibrosis with late postoperative right-sided heart failure or sudden death due to arrhythmia. old in situ thrombosis of small pulmonary artery branches. paradoxic embolism; cerebral abscess. * thalassemia synonyms and related terms: congenital hemolytic anemia; alpha-thalassemia; beta-thalassemia major (cooley's anemia); beta thalassemia minor (beta-thalassemia trait). note: the changes described below are observed primarily in beta-thalassemia major. unless the cause of the renal vein thrombosis and the nature of the complicating or underlying renal disease are known, follow procedures described under "glomerulonephritis." procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. in infants, manifestations of dehydration. * pulmonary embolism. * tumor of the kidney* (renal cell carcinoma); aortic aneurysm; lymphadenopathy; other mass lesions. inferior vena cava thrombosis involving orifice or whole length of renal veins. tumor thrombus (e.g., from renal cell carcinoma). femoral vein thrombosis. rare venous malformations also may cause renal vein thrombosis (l) . hemorrhagic infartion. parenchymal renal disease with nephrotic syndrome,* including amyloidosis* and vasculitis* (these last conditions may be associated with renal vein thrombosis). acute gastroenteritis in infancy. hyperparathyroidism,* pregnancy,* trauma, and other conditions. thrombosis, venous note: for peripheral venous thrombosis, the autopsy procedures are essentially similar to those described under "phlebitis." see also under "hypertension, portal," "syndrome, budd-chiari," "thrombosis, cerebral venous sinus," "thrombosis, lateral sinus," and "thrombosis, renal vein." thymoma (see "'fumor of the thymus.") thyroiditis synonymsand related terms: chronic fibrosing thyroiditis (riedel's struma); chronic thyroiditis with transient thyrotoxicosis; hashimoto's thyroiditis; pyogenic thyroiditis; subacute (granulomatous, giant cell, de quervain's) thyroiditis. possibleassociatedconditions: withhashimoto's thyroiditisautoimmune (chronic) hepatitis,*megaloblastic anemia,*rheumatoid arthritis,*sjogren's syndrome,*and systemic lupus erythematosus.* with riedel's struma-retroperitoneal fibrosis,* sclerosing cholangitis,* sclerosing mediastinitis,*and other conditions with idiopathic fibrosis. with subacute thyroiditis-viral infection. submit sample for determination of protein concentration; record appearance of sediment. for removal and specimen preparation, see chapter . thromboses of small vessels. glomerulonephritis.* hemorrhagic necroses. proteinuria; abnormal sediment. abruptio placentae, small placenta with accelerated maturation of villi; infarcts; fibrinoid necrosis of decidual arterioles. thrombotic occlusion of small vessels with hemorrhagic necroses; anterior lobe of pituitary gland may contain necroses (see also "syndrome, sheehan's"). submit samples for histologic study. if infection is expected, submit material for microbiologic study. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. transposition, complete, of the great arteries note: the basic anomaly is the origin of the aorta from the right ventricle, and of the pulmonary artery from the left ventricle, with a shunt, and usually with a right anterior aorta. there are four major types: ( ) with an intact ventricular septum ( %), ( ) with a ventricular septal defect* ( %), ( ) with a ventricular septal defect and subvalvular pulmonary stenosis* ( %), and ( ) with an intact ventricular septum and subvalvular pulmonary stenosis* ( %). for general dissection techniques, see chapter . interventions include atrial septostomy or septectomy; mustard or senning atrial switch procedure; rastelli-type repair with a valved extracardiac conduit; and jatene arterial switch procedure with lecompte maneuver. possible associated conditions: abnormal origin ofcoronary arteries ( %); atrial septal defect* ( %); coarctation of the aorta;* interrution of the aortic arch;* juxtaposition of atrial appendages ( %); overriding aorta ( %); overriding pulmonary artery ( %); patent ductal artery;* patent oval foramen; subvalvular pulmonary stenosis* ( %); tubular hypoplasia ofthe aortic arch;* ventricular septal defect* ( %), often mal-alignment type ( %). synonyms: atrioventricular and ventriculoarterial discordance; l-transposition. note: the basic anomaly is a mirror-image ventricular inversion, with a left anterior aorta, and with blood flow from right atrium to left ventricle to pulmonary artery, and from left atrium to right ventricle to aorta. for general dissection techniques, see chapter . interventions include patch closure of the ventricular septal defect; relief of pulmonary stenosis; relief of tricuspid insufficiency; and insertion of pacemakers. possible associated conditions: anterior and posterior av nodes ( %), prone to develop complete heart block; dysplasia or epstein's malformation* ofleft-sided tricuspid valve ( %); mirror-image epicardial coronary artery distribution ( %); right-sided mitral valve anomalies; subvalvular pulmonary stenosis* ( %); ventricular septal defect* ( %). kinyoun's, or other acid-fast stains. polymerase chain reaction for mycobacterial dna may be helpful in the differential diagnosis ofgranulomas ( ). ( ) universal precautions should be strictly followed and aerolization should be avoided. ( ) reliable serologic studies are not available. a definite diagnosis requires isolation of the organism. ( ) this is a reportable disease. cavitary, fibrocalcific, miliary, bronchial, and other types of pulmonary tuberculosis; granulomas in hilar lymph nodes. most organs and tissues may be involved, including liver, pancreas, spleen, kidneys, adrenal glands and other endocrine glands, gonads, and lymph nodes. tuberculous meningitis; tuberculoma. iridocyclitis or panophthalmitis. tuberculous arthritis and synovitis (hips, spine, knee); tuberculous osteomyelitis (anterior aspect of vertebrae; metaphysis of long bones). part ii / diseases and conditions procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. chronic ulcerative colitis may be associated with carcinoma of bile ducts, typically arising in psc.* metastases common in regional lymph nodes and lungs. i for removal, prosthetic repair, and specimen preparation and decalcification procedures, see chapter . if osteoblastic metastases appear to be present, search for primary tumor in prostate and breast, carcinoid tumors and hodgkin's lymphoma. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. cachexia. evidence of hypercalcemia (particularly in presence of osteolytic metastases). metastases to bone (e.g., from carcinoma of prostate, breast, bronchi, thyroid gland, kidney, or urinary bladder) usually involve red bone marrow. therefore, distal extremities are rarely involved by metastatic tumors in adults. metastases, commonly in lungs. metastatic calcification in patients with hypercalcemia. eosinophilia. myopathy.* metastases in liver and regional lymph nodes. see also above under "possible associated conditions." malakoplakia (rare) ( ) . part ii / diseases and conditions other organs and peripheral arteries tumor(s). if tumor is within a heart chamber (usually a myxoma), record extent of mobility and capability of tumor to obstruct a valvular orifice. submit samples of tumor(s) for histologic study and, particularly if the tumor is of unknown type, for immunohistochemical study and electron microscopy (chapter ). procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. metastatic carcinoma or melanoma. secondary cardiac effects by tumors include carcinoid heart disease, amyloid heart disease in multiple myeloma, and lymphocytic myocarditis in pheochromocytoma. treatment effects such as adriamycin cardiotoxicity may be noted also. tumor (myxoma) emboli in pulmonary or peripheral vessels ( ) . multiple aneurysms* of cerebral and other arteries may rarely be associated with atrial myxomas. procedures depend on expected findings or grossly identified abnormalities as listed above and in right-hand column. part ii / diseases and conditions if there was evidence of endocrine activity (see above), snap-freeze portion of fresh tumor for hormone assay. perfuse lungs with formalin. sample neoplastic and non-neoplastic tissue for histologic study. procedures depend on expected findings or grossly identified abnormalities as listed above and in right-hand column. for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . for removal and specimen preparation, see chapter . see above under "possible associated conditions." note that hypoglycemia* generally cannot be confirmed at autopsy. nonbacterial thrombotic endocarditis.* carcinoma may be associated with asbestosis or other types of pneumoconiosis,* chronic bronchitis,* emphysema,* interstitial pneumonia,* and many other bronchopulmonary diseases. see above under "possible associated conditions." metastases (regional lymph nodes, liver, bones, brain, and many other sites) and metastatic calcification. migratory thrombophlebitis.* encephalomyelitis;* cerebellar corticoid degeneration;* subacute spinocerebellar degeneration. * crooke cell hyperplasia. myasthenic syndrome (eaton-lambert syndrome); myopathy;* dermatomyositis.* peripheral neuropathy. see above under "external examination and skin." bones (with red marrow) are common sites of metastases. record presence or absence of testes. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. thmor of the soft tissues the possible sites and characteristics of soft tissue tumors vary so much that no universally applicable autopsy techniques can be presented. in all instances, the size, weight, and location of the tumor(s) must be recorded and tissue must be sampled for histologic study. if the tumor had not been classified prior to death, samples should be snap-frozen for immunohistochemical study. other samples should be prepared for electron microscopic study. evidence of paraneoplastic syndromes (see below) may require additional procedures. possible associated conditions: only a few paraneoplastic syndromes or systemic complications can be presented here. for the type of soft tissue tumor that was associated with each condition, see title of reference. kasabach-merritt syndrome ( ) (thrombocytopenia, microangiopathic hemolytic anemia, and acute or chronic coagulopathy associated with a rapidly enlarging hemangioma); liver function abnormalities ( ); neurofibromatosis ( ); osteomalacia ( ). heart esophagus, stomach, and duodenum other organs and tissues t in most instances, detennination of vitreous sugar concentration and of blood group is not indicated. inspect valves and prepare photographs and sections of vegetations. leave esophagus and part of duodenum attached to stomach. submit samples of tumor and of grossly uninvolved stomach for histologic study. request pas stain and warthin-starry stain for identification of h. pylori. portions of endocrine gastric tumor should be snap-frozen for immunohistochemical study and possible hormone assay. record location of tumor metastases. other organs procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. for dissection of the thoracic duct (for search of tumor cell clusters), see chapter . tumor of the uterus (with cervix) possible associated conditions: acquired immunodeficiency syndrome* (aids) ( ) if peritonitis is present, submit exudate for bacteriologic study. record volume of exudate and location of perforation. see "disease, ischemic heart." other procedures depend on grossly identified abnormalities as listed in right-hand column. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. perfuse at least one lung with formalin. for celiac arteriography, see chapter . open stomach and duodenum in situ and record site of perforation or penetration. record measured or estimated volume of blood in gastrointestinal tract. rinse ulcer with saline to locate eroded vessel(s). pin stomach and duodenum on corckboard (serosa toward board) and fix specimen in formalin before sectioning. prepare histologic sections of ulcer(s) and of remainder of stomach. request warthin-starry stain for h. pylori. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. free air in abdomen suggests perforation of ulcer. peritonitis. * perforation of ulcer. coronary atherosclerosis and manifestations of coronary insufficiency are commonly associated with peptic ulcer disease. in rare instances, pericardial fistula may be present from ulcer in hiatus hernia. peptic ulcers may be associated with emphysema,* tuberculosis,* and other chronic pulmonary diseases. vasculitis (see "aortitis," "arteritis, .•.," "phlebitis," and "purpura,•••") ventricle, double inlet left ( ) synonyms: single functional ventricle; univentricular heart; univentricular atrioventricular connection; holmes heart. note: the basic anomaly is the connection of both atrioventricular valves to the left ventricle, often with transposed great arteries and a restrictive ventricular septal defect. there are four major types, based on the ventriculoarterial connection: ( ) with congenitally corrected transposition ( %); ( ) with complete transposition ( %); ( ) with normally related great arteries (holmes heart; %); and ( ) double outlet, persistent truncal artery, or pulmonary atresia ( %). for general dissection techniques, see chapter . possible associated conditions: bicuspid pulmonary valve; bilateral mirror-image mitral valves (without tricuspid morphology); subvalvular aortic stenosis,* often with hypoplasia, coarctation, or interruption of the aortic arch; subvalvular pulmonary stenosis;* dual av nodes and progressive heart block in patients with congenitally corrected transposition. ventricle, double outlet right synonym: origin of both great arteries from right ventricle; taussig-bing anomaly. note: the basic anomaly is the origin of the aorta and pulmonary artery primarily from the right ventricle, usually with a ventricular septal defect, and often with subpulmonary stenosis. for general dissection techniques, see chapter . possible associated conditions: complete atrioventricular septal defect (often with asplenia syndrome); muscular discontinuity between aortic and mitral valves; right ventricular infundibular stenosis; ventricular septal defect* that may be subaortic, subpulmonary, doubly committed, or remote. examine histologically with h&e and oil red a stains. hepatomegaly and splenomegaly, jaundice. deficiency of acid lipase ( ). foam cells in the lamina propria. hepatomegaly, severe steatosis with cholesterol clefts in hepatocytes and kupffer cells. fibrosis. splenomegaly with lipid-laden foam cells. symmetric enlargement with dystrophic calcifications, giving the adrenals a gritty texture. vacuolated cells of the inner zona fasciculata and entire zona reticularis with cholesterol clefts. foam cells may be found in the lungs, bone marrow, lymph nodes, vessel walls, as well as schwann and ganglion cells. new insights into lipoprotein assembly and vitamin e metabolism from a rare genetic disease angioid streaks associated with abetalipoproteinemia familial hypobetalipoproteinemia: genetics and metabolism aviation pathology scuba diving accidents: decompression sickness, air embolism aetiology and occurrence of diving injuries. a review of diving safety scuba decompression illness and diving fatalities in an overseas military community diving-related emergencies bodies found in water achalasia and squamous cell carcinoma of the esophagus: analysis of patients esophageal achalasia and adenocarcinoma in barrett's esophagus: a report of two cases and a review of the literature a technique for necropsy evaluation of stenosis of the foramen magnum and rostral spinal canal in osteochondrodysplasia mutation analysis of the men i gene in multiple endocrine neoplasia type i, familial acromegaly and familial isolated hyperparathyroidism. i increased incidence of neoplasia in females with acromegaly benign and malignant tumors in patients with acromegaly pathology of acromegaly ethanol in sequestered hematomas stages of acute alcoholic influence/intoxication electrolyte imbalance in alcoholic liver disease collection and storage of specimens for alcohol analysis analysis for alcohol in postmortem specimens disposition of alcohol in man medicolegal aspects of alcohol. garriott jc blood, urine and other tissue specimens for alcohol analysis relationship between postmortem blood and vitreous humor ethanol levels larson cpo alcohol: fact and fallacy gradwohl's legal medicine left ventricular hypertrophy is more prominent in patients with primary aldosteronism than in patients with other types of secondary hypertension intracranial aneurysm and hemorrhagic stroke in glucocorticoid-remediable aldosteronism association of adrenal medullae and cortical nodular hyperplasia: a report of two cases with clinical and morpho-functional consideration amyloidosis: recognition, confirmation, prognosis, and therapy electron microscopy of primary and secondary cutaneous amyloidosis and systemic amyloidosis ocular amyloidosis current status review: cerebral amyloid spinal cord compression by amyloid deposits the burden of "sticky" amyloid: typing challenges quantification of cerebral amyloid angiopathy and parenchymal amyloid plaques with congo red histochemical stain kidney involvement in takayasu arteritis pathogenesis of rheumatoid arthritis splenic involvement in rheumatic diseases aspergillus sinusitis in patients with aids: report of three cases and review invasive aspergillosis in systemic lupus erythematosus aspiration (see "obstruction, acute airway aspergillus endocarditis, myocarditis and pericarditis complicating necrotizing fasciitis. case report and subject review aspergillus-related lung disease biliary atresia and the polysplenia syndrome: its impact on final outcome biliary atresia nephromegaly and elevated hepatocyte growth factor in children with biliary atresia coccidiomycosis pneumonia in a nonendemic area associated with inftiximab codeine (see "dependence, drug[s], all types or type unspecified all types or type unspecified (see "enterocolitis, other types or type undetermined chronic ulcerative (see "disease, inflammatory bowei cytologic diagnosis of cryp -tococcus neofonnans in hiv-positive patients cryptococcosis as an opportunistic infection in immunodeficiency secondary to paracoccidioidomycosis hypereosinophilia in disseminated cryptococcal disease cryptococcosis-a review of autopsy cases from a -year period in a large hospital cryptosporidiosis in persons with hiv infection broncho-pulmonary cryptosporidiosis in four hiv-infected patients cryptosporidiosis and inflammatory bowel disease sclerosing cholangitis associated with chronic cryptosporidiosis in a child with a congenital immunodeficiency disorder cyanide (see "poisoning, cyanide cyst(s), choledochal choledochal cyst-dinical features and classification cyst(s), liver (see "disease, fibropolycystic, of the liver and biliary tract pulmonary related terms: congenital cystic adenomatoid malformation; congenital pulmonary lymphangiectasis; intralobular bronchopulmonary sequestration. references infection or calcification of cysts; pyelonephritis;* perinephric abscess. obstructive uropathy in recessive polycycstic renal disease, congenital hepatic fibrosis is present and cystic bile ducts may be present in dominant cases ( ). see above under "possible associated conditions the pathology ofhuman renal cystic disease cystic kidney: genetics, pathologic anatomy, clinical picture, and prenatal diagnosis hepatic fibrosis associated with hereditary cystinosis: a novel form of noncirrhotic portal hypertension ophthalmic manifestations and histopathology of infantile nephropathic cyctinosis: report of a case and review of the literature pathological investigations of deaths following surgery, anaesthesia and medical procedures clinical outcome versus post-mortem finding in thoracic surgery: a lo-year experience order from american registry of pathology sales office intra-alveolar pulmonary siderophages in sudden infant death: a marker for previous imposed suffocation a contribution to a possible differentiation between sids and asphyxiation sudden infantdeath syndrome (sids)-standardized investigations and classification: recommendations investigation of the sudden deth of infants: a multicenter analysis alpha l-antitrypsin deficiency-associated panniculitis: resolution with intravenous alpha i-antitrypsin administration and liver transplantation clinical manifestations of alpha l-antitrypsin deficiency risk of hepatobiliary disease in adults with severe alpha i-antitrypsin deficiency (pizz): is chronic viral hepatitis b or c an additional risk factor for cirrhosis and hepatocellular carcinoma? alpha i-antitrypsin deficiency and inflammatory bowel disease severe alphal-antitrypsin deficiency (piz homozygosity) with membranoproliferative glomerulonephritis and nephrotic syndrome, reversi-bleafterorthotopic livertransplantation klippel-feil syndrome associated with malfonned larynx. case report klippel-feil syndrome in association with a craniocervical dennoid cyst presenting as aseptic meningitis in an adult: case report klippel-feil syndrome accompanied by an aneurysm of the non-coronary sinus of valsalva the hereditary ataxias clinical and genetic characterizations of q-linked autosomal dominant spinocerebellar ataxia (ad-sca) and frequency analysis of ad-sca in the japanese population cocain-related medical problems. consecutive series of cases direct cocaine cardiotoxicity demonstrated by endomyocardial biopsy the pathology and etiology of cocaineinduced heart disease ischemic colitis related to cocaine abuse hepatic dysfunction accompanying acute cocaine intoxication high incidence of malignancies in patients with dennatomyositis and polymyositis: an li-year analysis partial lipodystrophy associated with juvenile dennatomyositis: report of two cases chronic pneumomediastinum and subcutaneous emphysema: association with dennatomyositis spontaneous esophageal rupture in adult dennatomyositis polyneuropathy in juvenile dermatomyositis combined glucose and lactate values in vitreous humor for postmortem diagnosis of diabetes mellitus diabetes is related to cerebral infarction but not to ad pathology in older persons hepatic lesions resembling alcoholic liver disease relationship between cardiomyopathy and liver disease in chronic alcoholism cyanamide-associated alcoholic liver disease: a sequential histologic evaluation caroli's disease: - experiences caroli 's disease and autosomal dominant polycystic kidney disease: a rare asso-ciation? spontaneous rupture of a bile duct and its endoscopic management in a patient with caroli's syndrome bartonella henselae endocarditis in an immunocompetent adult cat-scratch disease and bacillary angiomatosis attack, transient cerebral ischemic" and "infarction, cerebral!') cholesteryl ester storage disease: hepatopathology and effects of therapy with lovestatin clinical, biochemical and histological analysis of seven patients with cholesterol ester storage disease cutaneous manifestations of chronic granulomatous disease. a report of four cases and review of the literature aspergillus endocarditis in chronic granulomatous disease colitis complicating chronic granulomatous disease. a clinicopathological case report hypertrophic cranial pachymeningitis with spinal epidural granulomatous lesion ocular pathologic findings of chronic granulomatous disease of childhood review: creutzfeldt-jakob disease survival of creutzfeldt-jakob disease virus in formol-fixed brain tissue guidelines for high risk autopsy cases: special precautions for creutzfeldt-jakob disease tissue handling in suspected creutzfeldt-jakob disease and other human spongiforme encephalopathies (prion diseases) cerebrospinal fluid concentration of neuron-specific enolase in diagnosis of creutzfeldt-jakob disease autopsy case of sporadic creutzfeldt-jakob disease presenting with signs suggestive of brainstem and spinal cord involvement metastatic crohn's disease: a rare cutaneous manifestation crohn's disease with pulmonary involvement in a year old boy mesenteric fibromatosis asso-ciated with crohn's disease cholangiocarcinoma and crohn's disease the pancreas as a site of granulomatous inflammation in crohn's disease dermatomyositis associated with crohn's disease cardiocyte storage and hypertrophy as a sole manifestation of fabry's disease. report on a case simulating hypertrophic non-obstructive cardiomyopathy an atypical variant of fabry's disease with manifestations confined to the myocardium pulmonary involvement in fabry disease cerebrovascular complications offabry's disease high incidence of thrombosis in fabry's disease oral involvement in chronic graft versus host disease after allogenic bone marrow transplantation massive pericardial effusion complicating the course of chronic graft-versus-host disease (cgvhd) in a child with acute lymphoblastic leukemia following allogeneic bone marrow transplantation the histological spectrum of pulmonary graft-versushost disease in bone marrow transplant recipients bronchiectasis in bone marow transplantation oncocytic metaplasia of bile duct epithelium in hepatic gvhd immune-mediated myelopathy after allogeneic marrow transplantation gunther's (see ''porphyria, congenital erythropoietic heavy-chain synonyms and related terms: gamma heavy-chain inflammatory bowel disease (first of two parts) takayasu's arteritis associated with idiopathic ulcerative colitis cerebrovascular complications ofinflammatory bowel disease pathology of inflammatory bowel disease legionnaires' disease source of infection for sporadic legionnaires' disease: a review legionnaire's disease associated with macular rash; two cases severe skin loss after meningococcal septicemia: complications in treatment osteonecrosis following meningococcemia and disseminated intravascular coagulation in an adult: case report and review rhabdomyolysis during the subacute stage of meningococcal sepsis primary meningococcal arthritis in a child: case report and literature review chondrosarcoma as a complicating factor in paget's disease of bone lymphoma arising in paget's disease osteoarthritis and paget's disease sickle cell disease sickle cell disease and sudden death: a retrospective/prospective study of autopsy cases and literature review vascular lesions of the liver in sickle cell disease. a clinicopathologic study in living patients whipple's disease and "tropheryma whippelii periodic acid-schiff-negative granulomatous lymphadenopathy in patient with whipple's disease. localization of the whipple bacillus to noncaseating granulomas by electron microscopy serial imaging studies of cerebral whipple's disease: from onset to end the wilson's disease gene is a putative copper transporting p-type atpase similar to menke's gene the liver biopsy diagnosis of wilson's disease. methods in pathology hepatolenticular degeneration (wilson's disease) venous air embolism in homicidal blunt impact head trauma: case reports a practical device for demonstrating air embolism amniotic fluid . kulka w. laboratory methods and technical notes: a practical device for demonstrating air embolism proof of fatal air embolism venous air embolism from head and neck wounds hantavirus pulmonary syndrome: a clinical description of patients with a newly recognized disease liver involvement in hemorrhagic fever with renal syndrome hantavirus infection induces a typical myocarditis that may be responsible for myocardial depression and shock in hanta virus pulmonary syndrome lassa fever in the united states. investigation ofa case and new guidelines for management lassa fever: review of virology, immunopathogenesis, and algorithms for control and therapy early diagnosis of lassa fever by reverse transcription-pcr rheumatic pneumonia: reappearance of a previously recognized complication of rheumatic fever acute rheumatic fever and poststreptococcal acute glomerulonephritis caused by t serotype streptococcus scleritis, uveitis, and glaucoma in a patient with rheumatic fever comparison of clinical features and pathologic findings in fatal cases of typhoid fever during the initial and later stages of the disease typhoid fever complicated by acute renal failure and hepatitis: case reports and review hemophagocytosis and granulomas in the bone marrow of a child with down syndrome diseases involving intrahepatic bile ducts idiopathic eosinophilic esophagitis with stricture formation in a patient with longstanding eosinophilic gastroenteritis eosinophilic gastroenteritis presenting with colitis and cholangitis eosinophilic gastroenteritis presenting with acute pancreatitis idiopathic midline destructive disease: does it . mendenhallwmetai.lethalmidlinegranoloma-nasalnaturalkillerff-celi exist? nasal cocain abuse mimicking midline granuloma pulmonary lymphomatoid granulomatosis in acquired immunodeficiency syndrome: lesions with epstein-barr virus infection recurrent bilateral spontaneous pneumothoraces associated with pulmonary angiocentric immunoproliferative lesion lymphomatoid granulomatosis: pathogenesis, pathology and clinical implications wegener's granulomatosis: histological documentation of common and uncommon manifestations in patients necrotizing granulomatosis of the breast wegener's granulomatosis with gangrene of toes pathology of pulmonary granulomatous vasculitis. sarcoidosis gunshot (see "injury, firearm splenic involvement in rheumatic diseases wegener's granulomatosis, acute laryngotracheal airway obstruction and death in a -year-old female: case report and review of the literature antiendothelial antibodies in patients with wegener's granulomatosis: prevalence and correlation with disease activity and manifestations. i rheumatoi transfusion-transmitted disease references flattening, broadening, and possible fusion of villi. phlegmonous inflammation and edema, mainly in ileocecal region encephalitis.* leukopenia; thrombocytopenia; aplastic anemia ( ) (pancytopenia*) synovitis (arthritis*) hepatitis g virus infection in hepatitis c virus-positive patients co-infected or not with hepatitis b virus and/or human immunodeficiency virus hepatitis g and c coinfection in children tumor of the liver diaphragmatic related terms: congenital diaphragmatic hernia fulminant hepatitis in patients undergoing liver transplantation: evidence for anon-a, non-b, non-c marrow transplantation for hepatitis-associated aplastic anemia: a follow up oflong-term survivors langerhans cell histiocytosis research. past, present, and future an update on c onality, cytokines, and viral etiology in langerhans cell histiocytosis langerhans cell histiocytosis in adults pulmonary langerhans cell granulomatosis central nervous system disease in langerhans cell histiocytosis immunohistochemical analysis oflangerin in langerhans cell histocytosis and pulmonary inflammatory and infectious diseases pathological approach to the diagnosis of hydrocephalus thoracic lipomeningocele associated with diastematomyelia, tethered spinal cord, and hydrocephalus. case report infectious causes ofhydrops fetalis human parvovirus b infection associated with hydrops fetalis cardiac abnormalities associated with hydrops fetal is gmi-gangliosidosis presenting as nonimmune hydrops fetalis: a case report a case of recurrent infantile polycystic kidney associated with hydrops fetalis the pathologist and the hydropic placenta, fetus, or infant chronic diarrhea associated with thymoma and hypogammaglobulinemia (good's syndrome) systemic amyloidosis in a patient with hypogammaglobulinemia. i hcv infection in patients with primary defects of immunoglobulin production encepahomyelitis in primary hypogammaglobulinemia retinitis pigmentosaand hypogammaglobulinemia intestinal obstruction in the newborn with congenital syphilis meconium ileus and its equivalent as a risk factor for the development of cirrhosis: an autopsy study in cystic fibrosis biliary atresia and meconium ileus associated with nieman-pick disease postmortem cranial mri and autopsy correlation in suspected child abuse cytomegalovirus-associated pseudotumor simulating gastric malignancy in acquired immuno-deficiency syndrome: a case report with review of literature bone marrow fibrin ring granulomas and cytomegalovirus infection cutaneous reactions following herpes zoster infections: report of three cases and a review of the literature herpes zoster and internal malignancy posteriorhorn varicella-zoster virus myelitis death or injury caused by electrocution myocardial hemorrhagic necrosis in delayed death from electrocution safety in bullet recovery procedures: a study of the black talon bullet lightning injuries: prognostic signs of death lightning injuries adrenal insufficiency, recurrent bacteremia, and disseminated abscesses caused by nocardia asteroides in a patient with acquired immunodficiency syndrome ards and adrenal insufficiencyassociated with the antiphospholipid antibody syndrome adrenal insufficiency from bilateral adrenal hemorrhage after total knee replacement surgery non-hodgkin's lymphoma presenting with adrenal insufficiency and hypothyroidism: an autopsy case report a case ofprimary unilateral adrenal burkitt-like large cell lymphoma presenting as adrenal insufficiency effect of growth hormone on fatty liver in panhypopituitarism increased fracture frequency in adult patients with hypopituitarism and gh deficiency pituitary abscess the liver: an atlas and text of ultrastructural pathology intubation (see "injury, intubation iodine (see "poisoning, iodine attack, transient cerebral ischemic" and "infarction, cerebral heart (see "disease, ischemic heart isomorism (see "syndrome, polysplenia and asplenia a study on performance of two serological assays for diagnosis of leprosy detection of mycobacterium leprae infection by pcr pathology of eye in leprosy references intervillous abscesses containing grampositive, non-acid-fast bacilli bacteria are predominantly intracellular. enterocolitis. listeria monocytogenes can be cultured from meconium. generalized lymphadenitis. disseminated abscesses and/or granulomas, particularly after transplacental infection listeria monocytogenes empyema in an hiv infected patient fatal listeria meningitis, endocarditis and pericarditis in a patient with haemochromatosis liver abscesses due to listeria monocytogenes yust . brainstem abscess and meningitis due to listeria monocytogenes in an adult with juvenile chronic arthritis pulmonary hypertension associated with systemic lupus erythematosus: predominantly thrombotic arteriopathy accompanied by plexiform lesions the liver in systemic lupus erythematosus: pathologic analysis of cases and review of japanese autopsy registry data chronic pancreatitis: a complication of systemic lupus erythematosus disseminated perivenous necrotizing encephalomyelitis in systemic lupus erythematosus: report of an autopsy case skeletal muscle lymphocytic vasculitis in systemic lupus erythematosus: relation to disease activity clinically occult avascular necrosis of the hip in systemic lupus erythematosus storage histiocytes and hemophagocytosis: a common finding in the bone marrow of patients with active systemic lupus erythematosus extensive medium vessel vasculitis with sle: an unsual association cutaneous manifestations of sexually transmitted ing cause of proctitis in men who have sex with men lymphogranuloma venereum as a cause cutaneous malakoplakia in a patient with acquired immunodeficiency syndrome (aids) extensive malakoplakia ofthe nasopharynx: management of a rare disease malakoplakia and colorectal adenocarcinoma coinfection is common in measles associated pneumonia giant cell pneumonia: light microscopy, immunohistochemical, and ultrastructural study of an autopsy case subacute sclerosing panencephalitis manifesting as viral retinitis: clinical and histopathologic findings intestinal neuronal dysplasia thoracic lipomeningocele associated with diastematomyelia, tethered spinal cord, and hydrocephalus. case report disease, meningococcal encephalitis, all types or type unspecified" and "meningitis mercury (see "poisoning, mercury with myelofibrosis synonym: idiopathic myelofibrosis acute cardiac tamponade associated with pericardial extramedullary hematopoiesis in agnogenic myeloid metaplasia methyl alcohol) (see "poisoning, methanol (methyl alcohol) thrombotic thrombocytopenic (see "purpura, thrombotic thrombocytopenic epstein-barr virus in inflammatory diseases of the liver and liver allografts: an in situ hybridization study fulminant hepatitis due to epstein-barr virus infection the mucopolysaccharidoses: a clinical review and guide to management biochemical diagnosis of mucopolysaccharidoses: experience of diagnoses in a -year period ( - ) mucormycosis synonym: phycomycosis; zygomycosis. note: diseases that may be complicated by mucormycosis include bums,* diabetes mellitus,* leukemia,* lymphoma,* and tuberculosis cardiac involvement in mucopolysaccharidosis: effects of allogeneic bone marrow transplantation mitral and aortic regurgitation in patients with mucopolysaccharidosis toxic epidermal necrolysis possibly linked to hyperacute graft-versus-host disease after allogeneic bone marrow transplantation pulmonary complications in toxic epidermal necro-iysis: a prospective clinical study references possible or expected findings pyelonephritis;* nephrocalcinosis; granulomas; tumor infiltrates; manifestations of the conditions listed below under "other organs renal stones in patients with rheumatoid arthritis nephrolithiasis as a presenting feature ofchronic sarcoidosis: a prospective study nephrolithiasis and risk of hypertension posterior lens opacities; retinal hamartomas. peripheral neuropathy with focal schwannomatous changes or onion-bulb-like schwann cell or perineural cell proliferation neurofibromatosis type i. in: pathology and genetics of tumours of the nervous system neurofibromatosis type . in: pathology and genetics oftumours ofthe nervous system neurological complications involving the central nervous system in neurofibromatosis type i primary cutaneous nocardia asteroides infection after heart transplantation native valve endocarditis due to nocardia-like organisms hepatobiliary complications of total parenteral nutrition total parenteral nutrition: a histopathologic analysis of the liver changes in children obesity and breast cancer risk nonalcoholic steatohepatitis obesity cardiomyopathy: pathogenesis and pathophysiology bennett cpo pachydennoperiostitis in childhood unilateral hypertrophic osteoarthropathy associated with aortofemoral graft infection gastroesophageal reflux and esophagitis-associated hypertrophic osteoarthropathy hypertrophicosteoarthropathy caused by lipoid pneumonia achondroplasia" and dyschondroplasia, oilier's osteogenesis imperfecta synonyms and related terms: lobstein's syndrome osteogenesis imperfecta congenita; type i, ii, and iii; osteogenesis imperfecta cystica hypercalcemia in osteogenesis imperfecta treated with pamidronate gastrointestinal problems in patients who have type-iii osteogenesis imperfecta osteomalacia related terms: osteoporosis;* renal osteodystrophy; rickets. note: many possible causes of osteomalacia may not be apparent at autopsy, e.g., sodium fluoride or diphosphonate toxicity or use of anticonvulsant drugs osteomalacia associated with adult fanconi syndrome: clinical and diagnostic features epstein-barr virus in b-celllymphomas associated with chronic suppurative inflammation osteomyelitis and osteonecrosis in inflammatory bowel disease references hyperlipidemia; hyperuricemia. fracture or traumatic dislocation of hip see above under "note osteomyelitis and osteonecrosis in inflammatory bowel disease osteonecrosis and human immunodeficiency virus infection report of fifteen cases. therapeutic recommendations malignant infantile osteopetrosis: otolaryngological complications and management renal tubular acidosis and osteopetrosis with carbonic anhydrase ii deficiency: pathogenesis of impaired acidification spondylolysis in children who have osteopetrosis osteopetrosis with arnold chiari malformation type and brain stem compression osteoporosis and atherosclerosis in chronic renal failure endocrine disorders and osteoporosis otosclerosis: a measles virus associated inflammatory disease correlations between pathologic changes in the stapes and conductive hearing loss in otosclerosis the aetiology of otosclerosis: a review of the literature etiologic links between chronic pancreatitis and pancreatic cancer hyperparathyroidism and pancreatitis during pregnancy pancytopenia related terms: agranulocytosis;* aplastic anemia; fanconi's anemia.* note: some causes of pancytopenia such as adverse drug reactions (e.g., due to antimetabolites, sulfa drugs thrombotic thrombocytopenic purpura/hemolytic uremic syndrome secondary to pancreatitis retinopathy as asys-temic complication of acute pancreatitis references mesenteric panniculitis necrotizing pancreatitis. * vasculitis with thrombi; adrenocortical infarctions. vasculitis with thrombi and infarctions. relapsing polychondritis ( ). see also above under "possible associated conditions subcutaneous panniculitic t-cell lymphoma is a tumor of cytotoxic t lymphocytes abecassism. alpha i-antitrypsindefi-ciencyassociated panniculitis: resolution with intravenous alpha l-anti-trypsin administration and liver transplantation blind loop syndrome: an unusual cause of panniculitis. j am acad paracoccidioidomycosis synonyms: paracoccidioides brasiliensis infection cutaneous panniculitis and relapsing polychondritis: two cases normal subcutaneous fat, necrosis of adipocytes and classification of the panniculitides paraneoplastic pemphigus a case of pemphigus vulgaris with esophageal involvement rheumatoid constrictive pericarditis (clinical conference) benign paroxysmal (see "fever, familial mediterranean pneumatosis intestinalis in pediatric acquired immunodeficiency syndrome pneumatosis cystoides intestinalis: an unusual complication of systemic amyloidosis pneumatosis intestinalis in children with primary combined immunodeficiency pneumatosis cystoides intestinalis with abdominal free air in a -year-old girl after allogeneic bone marrow transplantation pneumatosis intestinalis: a review pneumatosis coli: a proposed pathogenesis based on study of cases and review of the literature collagenous inorganic dust pneumoconiosis, diffuse type: aluminosis; asbestosis; chronic pulmonary berylliosis; talcosis; collagenous inorganic dust pneumoconiosis, nodular type: silicosis; noncollagenous inorganic dust pneumoconiosis (includes mixed-dust fibrosis): baritosis; china clay pneumoconiosis; chromite pneumoconiosis; coal worker's pneumoconiosis; fuller's earth pneumoconiosis; hematite or magnetite miner's lung; siderosis or welder's lung; stannosis; organic dust pneumoconiosis: byssinosis. note: in addition to the aforementioned classic forms of pneumoconiosis, many other airborne substances have been impli-cated in recent years, for example, cerium uncommon causes ofoccupational interstitial lung diseases rare earth (cerium oxide) pneumoconiosis: analytical scanning electron microscopy and literature review diagnostic criteria for chronic beryl ium disease (cbd) based on the uk registry - pneumocystis carinii (see "infection, pneumocystis carinii this condition is diagnosed by inspection, palpation, and roentgenography. tension pneumomediastinum may autopsy evaluation of asbestos exposure: retrospective study of cases with quantitation of ferruginous bodies in digested lung tissue atypical mycobacteriosis as a complication of talc pneumoconiosis silicosis, mixed dust pneumoconiosis, and lung cancer complications associated with the use ofthe esophageal-tracheal combitube pneumomediastinum: an unusual complication of asthma in a young man spontaneous pneumomediastinum in a patient with bronchiolitis obliterans after bone marrow references i. katzenstein a-l, myers j. idiopathic pulmonary fibrosis: clinical relevance of pathologic classification update on lymphoid interstitial pneumonitis lymphocytic interstitial pneumonitis and nonspecific interstitial pneumonitis histologic diagnosis of extrinsic allergic alveolitis role of electron microscopy in interstitial lung disease detennination of arsenic in hair using neutron activation arsenic intoxication associated with tubulointerstitial nephritis atrioventricular block after administration of atropine in patients follow-ing cardiac transplantation death following intentional methyl bromide poisoning: toxicological data and literature review pulmonary edema, alveolar wall damage, and interstitial pneumonia after acute inhalation. severe pulmonary fibrosis may develop in chronic cases. gastroenteritis after nonlethal food poisoning. degeneration of proximal tubules and proteinuria in acute poisoning degenerative changes of liver and myocardium problems in the analysis of cadmium in autopsied tissues elevated urinary cadmium concentrations in a patient with acute cadmium pneumonitis cadmium-associated renal disease the effects of storage conditions on the stability of carbon monoxide in postmortem blood acute renal failure, poisoning, carbon tetrachloride synonym: tetrachloromethane poisoning. note: toxicologic sampling of body fluids and organs should be done routinely in all cases. in many instances, however, death occurs i wk to d after exposure, and by this time no carbon tetrachloride is demonstrable. death may be sudden compartment syndrome, and systemic capillary leak syndrome complicating carbon monoxide poisoning southern je acute hydrocephalus following carbon monoxide poisoning prevention of occupational cyanide exposure in autopsy prosectors gradwohl's legal medicine references possible or expected findings digoxin values in digitalis toxicity are greater than ng/ml ( ) digoxin concentration may be higher or lower than concentration in serum, depending on how long before death drug was taken (i) digoxin concentrations in postmortem specimens after overdose and therapeutic use a fluorimetric determination ofmyocardial digoxin at autopsy, with identification of digitalis leaf, digitoxin and gitonin ethylene glycol poisoning: toxicokinetic and analytical factors affecting laboratory diagnosis ethylene glycol poisoning: case report of a record-high level and a review lead concentrations in human plasma, urine and whole blood trace metals (lead and cadmium screening) an emg case report oflead neuropathy years after a gunshot injury aminoaciduria and glycosuria following severe childhood lead poisoning risk of nervous system cancer among workers exposed to lead demonstration of mercury in the human brain and other organs years after metallic mercury exposure spontaneous exfoliation of teeth following severe elemental mercury poisoning: case report and histological investigation for mechanism. oral surg oral med oral pathoi acute chemical pancreatitis associated with nonfatal strychnine poisoning homicide by strychnine poisoning coronary arteritis, with or without aneurysms and infarctions, primarily in childhood. myocardial hypertrophy secondary to hypertension. * minimal or no involvement by polyarteritis nodosa; considerable involvement in other types ofnecrotizing vasculitis (see "arteritis, all types or type unspecified") with or without formation of aneurysms and infarctions, may occur in all organs. the liver may show bile duct injury and rarely, nodular regenerative hyperplasia ( ) more frequently involved in giant cell elastic arteritis.* polyarteritis of small muscular arteries, including vasa nervorum. arthritis* may rarely be present with swollen joints. infarctions;* subarachnoid hemorrhage; arteritis of cerebral arteries. papillitis; retinal hemorrhages polyarteritis nodosa-like vasculitis in human immunodeficiency virus infection the coexistence of familial mediterranian fever and polyarteritis nodosa: report of a case microscopic polyarteritis during polymyalgia rheumatica remission development of polyarteritis nodosa in the course of inactive systemic lupus erythematosus bone marrow pathology in relapsing polychonditis: high frequency of myelodysplastic syndrome relapsing polychondritis associated with subclinical sjo-gren's syndrome and phlegmon of the neck giant cell arteritis and polymyalgia rheumatica posterior scleritis and polymyalgia rheumatica polymyalgia rheumatica in patients with ankylosing spondylitis: a report of cases inflammatory changes of hip synovial structures in polymyalgia rheumatica polymyositis (see "dermatomyositis polyneuritis (see "syndrome, guillain-barre familial, and related syndromes related terms: cowden's disease (multiple hamartoma syndrome); familial colonic polyposis gardner's syndrome; non-polyposis syndrome (hereditary nonpolyposis colorectal cancer syndrome) sampling for histologic study depends on expected findings or grossly identified abnormalities as listed in right-hand column. (see also below under "soft tissues syndrome; mucocutaneous pigmentations (buccal, perioral, priorbital, distal extremities) in peutz-jeghers syndrome;* papules in face and oral mucosa in cowden's disease; tumors of skin and subcutis (see below under "soft tissues gardner's syndrome. osteomas or exostoses (mandible, calvara desmoid in familial adenomatous polyposis malignant potential in intestinal juvenile polyposis syndromes ampullary carcinoma in familial adenomatous polyposis adenoma of the common bile duct in gardner's syndrome may cause relapsing acute pancreatitis orbital osteoma in gardner's syndrome myelopathylmyelitis," and "syndrome, guillain-barre.") references possible or expected findings hypertrichosis; erythrodontia; scarring and mutilation of hands and face. hemolytic anemia. erythrocytes contain large amounts of uroporphyrin i; nonnoblasts and reticulocytes exhibit intense red fluorescence uroporphyrin i and coproporphyrin i in high concentrations. splenomegaly (may have been treated by splenectomy) correction of congenital erythropoietic porphyria by bone marrow transplantation successful cord blood stem cell transplantation for congenital erythropoietic porphyria (gunther's disease) congenital erythropoietic porphyria associated with nephrotic syndrome atypical red cell inclusions in congenital erythropoietic porphyria possible associated conditions: adverse drug reaction; chronic alcoholism;* chronic hepatitis c ( ); human immunodeficiency virus infection porphyria cutanea tarda and human immunodeficiency virus: two cases associated with hepatitis c porphyria cutanea tarda and antibodies to hepatitis c virus an unhappy triad: hemochromatosis, porphyria cutanea tarda and hepatocel ularcarcinoma-a case report variegate porphyria possible associated conditions: ebstein's malformation of tricuspid valve. references possible or expected findings chronic eczematous skin lesions or superficial scarring; nail changes. perivascular pas-positive hyaline ( ) brown protoporphyrin deposits with red to yellow birefringence with a maltese cross configuration in hepatocytes, kupffer cells, and bile canaliculi erythropoietic protoporphyria cytofluorometry as a diagnosis of protoporphyria recessive inheritance of erythropoietic protoporphyria with liver failure pseudogout synonym: calcium pyrophosphate dihydrate (cppd) deposition disease puncture grossly affected joints and submit sample of synovial fluid for crystal analysis under compensated polarized light ( ) references possible or expected findings punctate calcium deposits in kneejoints and, less commonly, in joints of hips, ankles, shoulders, or wrists or in symphysis ossium pubis and intervertebral disks. arthritis* with synovitis. crystals of calcium pyrophosphate dihydrate in periarticular tissue ( ) and synovial fluid. cartilage contains calcium salts of pyrophosphate, hydroxyapatite, and orthophosphate. alkaptonuria;* gout calcium pyrophosphatedihydratedeposition disease presenting as tumoral calcinosis (periarticularpseudogout) calcifications in dermis; calcifications of blood vessels. diaphragmatic hernia.* hypertensive cardiomegaly. characteristic plaques in pericardium and endocardium, with or without mitral valve involvement. coronary atherosclerosis may have been a causeofangina( ).coronarythrombosisand myocardial infarction may be present also. accelerated atherosclerosis; calcification of peripheral vessels. gastrointestinal hemorrhage;* peptic ulcer degeneration of bruch's membrane with retinal hemorrhages; sclerosis of choroid vessels calcification of elastic fibers in pseudoxanthoma elasticum new report of severe coronary artery disease in an eighteen-year-old girl with pseudoxanthoma e asticum. case report and review of the literature hivrelated thrombotic thrombocytopenic purpura: report of cases and a review of the literature beham-pyelonephritis synonyms and related terms: acute ascending pyelone angiotropic large cell lymphoma presenting as thrombotic micro-angiopathy (thrombotic thrombocytopenic purpura) splenic pathology in thrombotic thrombocytopenic purpura emphysematous pyelonephritis in diabetic patients nephrobronchialfistulaandlungabscessresulting from nephrolithiasis and pyelonephritis a case ofan enterorenal pyothorax (see "empyema, pleurai.") fistula and pyelonephritis with airin renal pelvis systemic amyloidosis secondary to pyonephrosis. resolution after nephrectomy medicolegal investigation of death the laboratory's role in detecting sexual assault toluidine blue in the detection at autopsy of perineal and anal lacerations in victimes of sexual abuse sarcoidosis presenting as heart disease sarcoidosis and its ocular manifestations rheumatic features of sarcoidosis cutaneous sarcoidosis: differential diagnosis schistosoma mansoni and s. haematobium infections in egypt. i. evaluation of techniques for recovery of worms and eggs at necropsy. am i a quantitative post-mortem study of schistosomiasis mansoni in man immunodiagnosis of schistosomiasis. screen with fast-elisa and confinn with imrnunoblot hepatic connective tissue changes in hepatosplenic schistosomiasis schistosomiasis in women: manifestations in the upper reproductive tract sclerosis, systemic amyotrophic lateral (see "disease, motor neuron diffuse (see "sclerosis, schilder's cerebrai multiple synonyms and related terms: acute and subacute variants: acute multiple sclerosis (marburg type), acute necrotizing myelopathy; concentric sclerosis (ba type); concentric lacunar leukoencephalopathy; encephalitis periaxialis diffusa (schilder's type; see also next entry silicone ganuloma in acral skin in a patient with silicone-gel implants and systemic sclerosis organizing diffuse alveolar damage associated with progressive systemic sclerosis bayle , fiessinger in. brain involvement in scleroderma: two autopsy cases skeletal muscle pathology in systemic sclerosis mechanisms of scleroderma-induced lung disease thberous sclerosis complex and subependymal giant cell astrocytoma bilateral temporal arachnoid cysts associated with tuberous sclerosis poisoning, alkaloid accident, diving [skin or scuba scurvy (see "deficiency, vitamin c shigellosis (see "dysentery, bacillary shock related terms: anaphylactic shock; bacteremic shock; many others. note: see also under name of suspected underlying condition, such as "bums intestinal histological and ultrastructura inflammatorychanges in spondyloarthropathy and rheumatoid arthritis the sacroiliac joint in the spondyloarthropathies granulomatous ileitis in a patient with ankylosing spondylitis sporotrichosis synonym: sporothrix (sporotrichum) schenckii infection. note: ( ) mckiernan . partial lipodystrophy in coeliac disease coeliac disease and lymphoma: current status review: nonalcoholic steatohepatitis alcoholic and non-alcoholic wernicke's encephalopathy. be alert to the preventable and treatable disease non-alcoholic fatty liver disease in the metabolic syndrome the placenta in stillbirth estimating the time of death in stillborn fetuses: i. histologic examination of fetal organs: an autopsy study of stillborns estimating the time of death in stillborn fetuses: ii. histologic evaluation ofthe placenta; a study of stillborns estimating the time ofdeath in stillborn fetuses: iii. external fetal examination; a study of stillborns fat distribution in the adrenal cortex as an indication of the mode of intrauterine death stimulant(s) (see "dependence, drug(s), all types or type undetermined cutaneous manifestations of the acquired immunodeficiency syndrome aids: cardiac findings from autopsies the liver in hiv infection neuropathology of the spinal cord in the acquired immunodeficiency syndrome postmortem enzyme immunoassay for human immunodeficiency virus bk virus-associated renal failure among hiv patients - or write to american registry ofpathology sales office pathology of acquired immunodeficiency syndrome (aids) in children thymus involution in the acquired immunodeficiency syndrome micrencephaly in children congenitally infected with human immunodeficiency virus-a gross-anatomical morphometric study a rapid method for detecting the predominant ashkenazi jewish mutation in the bloom's syndrome gene bloom syndrome: multiple retinopathies in a chromosome breakage disorder severe eczema in a patient with di-george's syndrome digeorge's syndrome orthe iii -iv pharyngeal pouch syndrome: pathology and a theory of pathogenesis basal ganglia calcification and psychosis in q . deletion syndrome down's synonyms and related terms: mongolism possible associated conditions: acute lymphocytic, my references epicanthal folds; cleft lip/palate; high arched palate; furrowed tongue; rhagades. depressed nasal bridge small, broad or fiat nose; slanted palpebral fissures; fiat occiput; brachycephaly; palmar "simian crease"; short fifth middle finger ventricular septal defect;* complete atrioventricular septal defect* ( ). duodenal stenosis and atresia; imperforate anus. microcephaly; poorly developed secondary gyri hypoplastic brain stem, medulla and cerebellum; polymicrogyria; neurofibrillary tangles; meningomyelocoele. acute lymphocytic leukemia; acute myelocytic leukemia; acute megakaryocytic leukemia cardiovascular disease in down syndrome possible or expected findings hyperelasticity of skin, bruises or scars, hemorrhages, and hyperpigmentation. lipomatous pseudotumors that may be calcified or ossified. normal or abnormal amounts and fragmentation of elastic tissue. abnormalities of collagen. dislocation of hip, shoulder, patella, radius, or clavicle. loose-end clavicles; spondylolisthesis mitral valve prolapse; aortic insufficiency.* aortic dissection, aneurysm, ectasia, occlusion ( ). various congenital anomalies. congenital anomalies of gastrointestinal and genitourinary tracts. bleeding from various organ sites vascular ehlers-danlos syndrome: imaging findings note: this syndrome belongs to a large family (with more photograph abnormal features as listed in right-hand column. record appearance of external genitalia. prepare skeletal roentgenograms. procure long bones and vertebral bodies short-limb dwarfism* with narrowing of the rib cage; polydactyly; dysplasia of fingernails; thin and sparse hair; premature eruption of teeth; defective dentition; eye abnormalities; upper lip bound down by multiple frenula. cryptorchidism; epispadias; hypospadias. chondrodysplasia with acromelic micromelia (shortening of the distal segment of the limb); fusion of capitate and hamate bones of wrist; defects of lateral aspect of proximal tibia see lesions listed above under "external examination empty sella synonyms and related terms: primary empty sella syndrome; secondary empty sella syndrome (e.g., after surgical removal or spontaneous infarction of pituitary adenoma) chorea, philic pneumonia: histopathologic features in nine cases. am rev resp hereditary ascariasis and hookworm chronic eosinophilic pneupophosphatemic vitamin d-resistant rickets; galactosemia;transport defect; tyrosinemia. * excludes fan- weight and external measurements; record and photograph abnormalities as listed in right-hand column. prepare skeletal roentgenograms. radiographs of long bones. submit for tissue culture for possible enzyme analysis. possible or expected findings redlblond hair, fair skin (diminished pigmentation); dehydration;* stigmata of hypothyroidism;* delay of sexual maturation. rickets; osteomalacia.* positive rheumatoid factor. various types of pneumonia after splenectomy, presence of accessory spleen may account for treatment failure. manifestations of portal hypertension.* lymphadenopathy of mediastinal and paraaortic lymph nodes. anemia;* neutropenia sinusoidal lymphocytosis of the liver in felty's syndrome with a review of the liver involvement in felty's syndrome splenic involvement in rheumatic diseases felty's and pseudo-felty's syndrome nodular regenerative hyperplasia of the liver in rheu-matic diseases: report of seven cases and review of the literature fetal alcohol syndrome: craniofacial and central nervous system manifestations goodpasture's syndrome anti-glomerular basement membrane glomerulonephritis: a morphologic study of cases gronblad-strandberg (see "pseudoxanthoma elasticum acute inflammatory polyradiculoneuropathy; guillain-barre-strohl syndrome hemolytic uremic syndrome/thrombotic thrombocytopenic purpura: pathophysiology and treatment cyclosporin-induced hemolytic uremic syndrome: factors that obscure its diagnosis enterohemorrhagic escherichia coli :h : an emerging pathogen hemolytic uremic syndrome as a presenting form of hiv infection hemolytic uremic syndrome in prostatic carcinoma diabetes secondary to genetic disorders. baillieres suprasellar tumors of maldevelopmental origin in klinefelter's syndrome. a report of two cases klippel-feu synonym: congenital fusion of cervical vertebrae primary yolk sac tumor of the prostate in a patient with klinefelter's syndrome extragonodal germ cell tumors are often associated with klinefelter syndrome organs and tissues procedures possible or expected findings external examination prepare roentgenograms of chest, neck, and head skull, spine, brain, and spinal cord myasthenia gravis disorders with dysraphia (see below), fusion of cervical vertebrae. congenital elevation of the scapula (sprengel's deformity) chiari malformation;* basilar impression;* meningomyelocele; platybasia;* spinal cord compression; weight and length; record and photograph abnormalities as listed in right-hand column. record weight and sample for histologic study. if renal transplantation ( ) had been carried out, see also under that heading. follow procedures described under "glomerulonephritis retinal dystrophy ( ) and other retinal changes the cardinal manifestations of bardet-biedl syndrome, a form of laurence-moon-biedl syndrome laurence-moon-biedl syndrome accompanied by congenital hepatic fibrosis pediatric renal transplantation in laurence-moon-biedl syn-drome possible or expected findings typical skeletal proportions. arachnodactyly; pectus excavatum genu recurvatum; dislocation of joints diaphragmatic hernia. * infective endocarditis. * atrial septal defect. * myxomatous transformation of mitral ring. mitral valve prolapse. aortic and pulmonary dilatation and valvular insufficiency.* aortic dissection* with dissection of adjacent vessels. ascending aortic aneurysm multiple cysts (see "cyst(s), pulmonary") aortopulmonary fistula: an uncommon complication in dystrophic aortic aneurysm peripartum acute myocardial infarction in marfan's syndrome aneurysm of the cervical internal carotid artery associated with marfan's syndrome--ease report noonan's syndrome in association with acute leukemia lymphatic dysplasia in a neonate with noonan's syndrome noonan syndrome: a clinical description emphasizing the cardiac findings cerebral arteriovenous malformation in noonan's syndrome clinical variability in a noonan syndrome family with a new ptpnii gene mutation peutz-jeghers syndrome bilateral large-cell calcifying sertoli cell tumor of the testes in peutz-jeghers syndrome: a case report ovarian tumors associated with the peutz-jeghers syndrome robin sequence and a deficiency of the left forearm in a girl with a deletion of chromosome g -gter congenital heart disease in the pierre robin syndrome glossoptosis-apnea syndrome chiari i malformation, caudal regression syndrome, and pierre robin syndrome: previously unreported combination risk factors for squamous cell carcinoma of the esophagus heterotaxy syndrome; ivemark's syndrome possible associated conditions: with asplenia: right isomerism (bilateral mirror-image right-sided symmetry) of heart, lungs, and abdominal viscera; common atrium; total anomalous pulmonary venous connection; absent coronary sinus; complete atrioventricular septal defect;* subpulmonary stenosis;* pulmonary valve atresia;* midline symmetric liver; malrotation of bowel; absent spleen. with polysplenia: left isomerism (bilateral mirror-image left-sided symmetry) ofheart and lungs, with variable sidedness ofabdominal viscera primary immunodeficiencies reiter's syndrome-like patternin aids-associated psoriasiformdermatitis reference pneumothorax;* pneumomediastinum;* pneumoperitoneum. septicemia. right ventricular hypertrophy and/or dilatation. intubation trauma and mural edema and hemorrhage in trachea; hyaline membrane disease intubation trauma. hemorrhages or other evidence of birth trauma; germinal matrix hemorrhages in premature infants; infarctions of subcortical white matter in term infants coalson . pathology of bronchopulmonary dysplasia pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia inborn errors of metabolism and reye's syndrome: differential diagnosis prevalence and non-specificity of microvesicular fatty changes liver histopathology in clinical reye syndrome ultrastructural study of intranuclear inclusions in the exo-crine pancreas in reye's syndrome neuropathologic findings in reye syndrome sanfilippo's (see "mucopolysaccharidosis scheie's (see "mucopolysaccharidosis segawa's (see "syndrome, dystonia severe acute respiratory (sars) note: this highly contagious illness, caused by a coronavirus (sars-cov)details, see biosafety level laboratory for autopsies of patients with severe acute respiratory syndrome: principles, practices, and prospects pathology and pathogenesis of severe acute respiratory syndrome pulmonary involvement in primary sjogren's syndrome polymyositis and sjogren's syndrome associated with bronchiolitis obliterans organizing pneumonia the gastrointestinal manifestations of sjogren's syndrome clinicopathological factors relating malignant lymphoma with sjogren's syndrome acute transverse myelopathy and cutaneous vasculopathy in primary sjogren's syndrome primary localized cutaneous amyloidosis with unusual clinical features in a patient with sjogren's syndrome disease, parkinson's erythema multiforme man related terms: armadillo disease; continuous muscle fiber activity; neuromyotonia autoimmune central nervous system paraneoplastic disorders: mechanisms, diagnosis, and therapeutic options sudden infant death (sids) (see "death, sudden unexpected, of infant.") syndrome, superior vena cava related term: superior vena cava obstruction continue dissection of veins into neck and axillas. record and photograph site of thrombosis or of compression by surrounding pathologic conditions. submit samples for histologic study. benign or malignant tumors; fibrosing mediastinitis;* postradiation fibrosis; infectious disease (tuberculosis,* histoplasmosis*); thoracic aortic aneurysm;* chronic constrictive pericarditis;* chest trauma; arteriovenous fistula between ascending aorta and superior vena cava toxic shock synonyms and related terms: staphylococcal scarlet fever. note: ( ) collect all tissues this is a reportable disease. premature aging; increased number of pigmented nevi; infantile sex organs; webbed neck; broad chest with wide spacing of nipples. short fourth metacarpal; abnormal epiphyseal fusions; osteochondrosis-like changes of spine bicuspid aortic valve;* coarctation of aorta. * rarely other anomalies ( ) decreased/absent follicles. intestinal telangiectases. biliary atresia. * horseshoe kidney; double ureters. streak gonads without germ cells or follicles hashimoto's thyroiditis. * manifestations of diabetes mellitus,* hypertension,* or thyrotoxicosis. neuroblastoma and related tumors ( ). keratoconus acute aortic dissection, aortic insufficiency, and a single coronary artery in a patient with turner's syndrome neuroblastoma and related tumors in turner's syndrome turner's syndrome associated with bilateral retinal detachments weil's (see "leptospirosis related terms: alcoholic wernicke's encephalopathy; korsakoff's psychosis; nonalcoholic wernicke's encephalopathy ( , ); and specimen preparation, see chapter . for selection of histologic samples, see right-hand column melissas . wernicke's encephalopathy after vertical banded gastroplasty for morbid obesity syndrome, respiratory distress, of infant.") syndrome, wiskott-aldrich (see "syndrome, primary immunodeficiency malformation, ebstein's" and "preexcitation, ventricular alcoholic and non-alcoholic wernicke's encephalopathy. be alert to the preventable and treatable disease cerebro-hepato-renal syndrome; infantile refsum's disease. * note: this congenital familial cholestatic syndrome results from impaired assembly of peroxisomes and has its main manifestations in the brain, liver, and kidneys postmortem findings and prenatal diagnosis ofzellwegersyndrome. case report alcoholism and alcohol intoxication" and "disease, alcoholic liver possible associated condition: multiple endocrine neoplasia primary cardiac gastrinoma causing zollinger-ellison syndrome localization, syphilis, acquired synonym: treponema pallidum infection. note: congenital syphilis is presented below under a separate heading and surgical management of gastrinoma fundic argyrophil carcinoid tumor in a patient with sporadic-type zollinger-ellison syndrome nonimmune hydrops fetalis due to congenital syphilis associated with negative intrapartum mater-nal serology screening syringomyelia synonyms and related term: hydromyelia; idiopathic syringomyelia; secondary syringomyelia; syringobulbia. possible associated conditions: with idiopathic syringomyelia-arnold chiari malformation, type i;* basilar impression;* . oppenheimer eh, dahms bb. congenital syphilis in the fetus and neonate congenital syphilis: detection of treponema pallidum in stillborns with secondary syringomyelia-intramedullary gliomas (ependymoma, pilocytic astrocytoma) and vascular tumors; spinal arachnoiditis and pachymeningitislisted in right-hand column. prepare roentgenograms of spine and joints. for removal and specimen preparation, see chapter . for histologic sampling, see right-hand column. hypertrophy of body parts. muscle atrophy of upper extremities and hands cervical spinal cord is swollen and tense, with cavitation (syrinx) containing clear fluid. wall of cavity consists of degenerated glial and neural elements, with marked gliosis. spinal cord parenchyma is markedly compressed. see also above under hydrops fetalis caused by alpha-thalassemia: an emerging health care problem limb defects in homozygous alpha-thalassemia: report of three cases poisoning, thallium thirst (see "dehydration thromboangiitis obliterans (see "disease, buerger's purpura, thrombotic thrombocytopenic" and "syndrome, hemolytic uremic the pathological manifestations of pulmonary toxoplasmosis in the acquired immunodeficiency syndrome a spectrum in the pathology of toxoplasmosis in patients with acquired immu transfusion (see "reaction to transfusion bone marrow note: if the patient had symptoms of acute or chronic graft-versus-host disease (gvhd), see "disease, graftversus-host morphology and diagnostics of human toxoplasmosis fatal myocardial coinfection by toxoplasma gondii and parvovirus b in an hiv patient if there is evidence of infection, submit material for microbiologic studies. if there is evidence of recurrent leukemia or lymphoma, sample as described under those headings. if the patient had symptoms of thrombotic thrombocytopenic purpura (tip) or hemolytic uremic syndrome (hus), see these headings. record average size. fix specimens in b-plus®. make touch preparations. request giemsa or wright stain. for preparation of sections and smears (imprints), see chapter . manifestations of graft-versus-host disease (e.g., scleroderma-like changes).* septicemia. interstitial pneumonia* and cytomegalovirus infection* or human herpesvirus infection ( ). bacterial, fungal bacterial or fungal infections in gvhd.* other manifestations of gvhd. * recurrent leukemia,* lymphoma* (including posttransplant, epstein-barr virus associated lymphoproliferative disorder). recurrent solid tumor, e.g., carcinoma of breast, lung (small cell carcinoma), ovary lymphomatous or leukemic infiltrates myeloid cells may be absent in marrow graft rejection or nonimmunologic marrow graft failure. references hematomas; hemorrhagic necroses; infarcts; bacterial or fungal infections leukoencephal-opathy; vascular siderocalcinosis; neuro-axonal spheroids ( ) human herpesvirus infection and associated pathogenesis following bone marrow transplantation pulmonary veno-occlusive disease in an adult following bone marrow transplantation. case report and review of the literature acute renal failure following bone mar-row transplantation transplantation-associated thrombotic thrombocytopenic purpura and hemolytic uremic syndrome thrombotic microangiopathies associated with drugs and bone marrow transplantation record status of all vascular anstomoses. if there are hemorrhages, record volume and site. record weight, ventricular thickness, and valve circumferences. take multiple sections from all areas of myocardium. cut coronary arteries in cross sections; request verhoeff-van gieson stain. procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. references cushingoid features (see "syndrome, cushing's") after steroid therapy. suture dehiscence. hemothorax or hemomediastinum in recent cases. left ventricular hypertrophy, from cyclosporine-related hypertension. acute transplant rejection (for grading, see ref. ). chronic transplant vasculopathy with coronary artery stenosis ( ) opportunistic infection ( ). pneumonia a working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart and lung rejection study group autopsy findings in cardiac transplant patients: a lo-year experience fulminant toxoplasmosis following heart transplantation banff schema for grading liver allograft rejection: an international consensus document record status of all vascular anstomoses. if there are hemorrhages, record volume and site. record lung weights separately. if lung infection is suspected, submit material for microbiologic possible or expected findings cushingoid features (see "syndrome, cushing's") after steroid therapy. suture dehiscence. hemothorax or hemomediastinum in recent cases. opportunistic infection (in patients with a single lung transplant, infections may involve references the nontransplant lung, as well). chronic bronchitis* and bronchiectasis.* acute rejection (rare); chronic airway rejection (obliterative bronchiolitis); chronic vascular rejection. (for grading of rejection, see ref .) post-transplant lymphoprolijerative disorder a working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart and lung rejection study group recurrent salmonella enteritidis and hepatic tuberculosis tularemia synonyms and related terms: francisella tularensis infection; pasteurella tularensis infection cerebral abscesses complicating tularemia meningitis endocrine (see "neoplasia, multiple endocrine any type note: ifthe tumor had been treated by surgery, irradiation, chemotherapy, or other means (the most common situation possible associated conditions: with adrenocortical adenoma-conn's syndrome with adrenocortical carci-noma-cushing's syndrome;* hypoglycemia;* virilization. with pheochromocytoma-cerebellarhemangioblastoma ery-throcytosis; hypercalcemia; hypertension,* multiple enarteries record body weight and abnormal features. photograph skin changes. record heart weight and thickness of ventricles. procure multiple sections of myocardium possible associated conditions.") focal myocarditis. * hypertensive cardiovascular disease (see "hypertension myocardial infarction without severe coronary artery disease (with pheochromocytoma) asystematicreviewoftheassociationbetween barrett's esophagus and colon neoplasm malakoplakia and colorectal adenocarcinoma an unusual case of colonic mixed adenoendocrine carcinoma: collision vs composite tumor. a case report and review of the literature submit lymph nodes for histologic study. remove neck organs with tongue together with esophagus. open pharynx and esophagus in posterior midline. if fistulas and abscesses are suspected, dissect esophagus but leave attached to mediastinum, stomach and diaphragm. record width of lumen of esophagus at various levels. photograph and record size and location of tumor; sample tumor and uninvolved esophagus for histologic study. request pas stain of uninvolved esophagus (sample from all levels). procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. references cachexia. eczematoid dermatitis with adult renal cell carcinoma. cushing's syndrome,* galactorrhea or feminization or masculinization in some cases of renal cell carcinoma. evidence of hyperalcemia. pulmonary tumor embolism after invasion of renal vein and inferior vena cava. acquired renal cystic disease prolactin, renin, and prostaglandins in some renal cell carcinomas. see above under "possible associated conditions wilms tumor associated with polycythemia: case report and review of the literature renal medullary carcinoma: a potential sickle cell nephropathy of children and adolescents acquired cystic kidney disease byler's disease, carcinoid syndrome,*cirrhosis* of any type (mostly nonbiliary), congenital hepatic fibrosis, * cushing's syndrome,* erythrocytosis, genetic hemochromatosis, * glycogen storage disease (type ),* hepatitis b or c virus infection, hereditary tyrosinemia (type i),* hypercalcemia, hypercholesterolemia, hypoglycemia,* neurofibromatosis,* osler-rendu-weber disease* (hereditary hemorrhagic telangiectasia), polycythemia,* porphyria (acute intermittent and porphyria cutanea tarda), * pseudohyperparathyroidism,* and thorium (thorotrast) deposition. with bile duct carcinoma (cholangiocarcinoma}-clonorchis sinensis or opisthorchis viverrini infection, fibropolycystic liver and biliary tract disease,* hepatolithiasis, hypercalcemia, pri-mary sclerosing cholangitis,* and thorium (thorotrast) deposition. with hepatoblastoma-alpha-fetoproteinemia, cardiac and renal malformations, cleft palate, diaphragmatic hernia,* down's syndrome,* familial colonic polyposis,* hemihypertrophy, nephroblastoma. with angiosarcoma-thorium (thorotrast) deposition. see also below under "possible or expected findings describe and photograph cut surfaces. sample tumor and nonneoplastic liver for histologic study. if thorium deposition is suspected, prepare roentgenograms of liver slices and submit samples for energy-dispersive x-ray microanalysis of paraffin sections. procedures depend on expected findings or grossly identified abnormalities as listed above and in right-hand column. cachexia. precocious puberty. feminization and gynecomastia in rare cases of hcc. spider angiomas; clubbing of fingers. see above under "possible associated conditions thorotrast storage may cause cirrhosis, hcc, bile duct carcinoma, or angiosarcoma. see above under "possible associated conditions tumor of the lung or bronchus possible associated conditions: abnormal concentrations of hormons or other metabolites in blood and tumor tissue (adrenocorticotropic, antidiuretic, growth hormone, parathyroid-like substances, or -hydroxyindolacetic acid); acanthosis nigricans s syndrome;*dermal hyperpigmentation; feminization; hyperglycemia; hypercalcemia; hypoglycemia;* hypokalemia; hyponatremia; precocious puberty. for syndromes affecting the brain, peripheral nerves or muscles, see below under "possible or expected findings organs and tissues procedures possible or expected findings external examination and skin record body weight. record abnormal features as listed in right-hand column; prepare photographs. prepare histologic sections of normal and grossly abnormal skin clubbing of fingers; spider angiomas. for other rare tumor-related changes, see above under "possible associated conditions thmor of the pancreas possible associated conditions: with carcinoma of the exocrine pancreas--cushing's syndrome;* diabetes mellitus* (rare); hypercalcemia; hyperglycemia with islet cell tumor-abnormal concentrations of hormons in blood and tumor tissue (see below under "pancreas"); features. dermal hyperpigmentation. for other rare tumor-related changes, see above under "possible associated conditions biliary obstruction caused by tumor in head of pancreas. islet cell tumor with adrenocorticotropic hormone, gastrin, glucagon, insulin, or other peptide hormones. see above under "possible associated conditions paraneoplastic pemphigus associated with pancreatic carcinoma pancreatic cystadenocarcinoma in peutz-jeghers syndrome testes may have been surgically removed to achieve androgen deprivation. urinary obstruction and hydronephrosis. * osteoblastic metastases hemolytic uremic syndrome in prostatic carcinoma thmor of the small intestine possible associated conditions: acquired immunode oncogenic osteomalacia associated with prostatic cancer gardner's syndrome) submit sample of non-neoplastic small bowel for histologic study. procedures depend on expected findings or grossly identified abnormalities as listed above. cachexia. mucocutaneous pigmentations associated with peutz-jeghers syndrome. * carcinoid tumor. lymphoma (see also above under "possible associated conditions"). familial polyposis or related syndrome. * celiac sprue. * crohn's disease. * see above under "possible associated conditions kasabach merritt syndrome in infants paraneoplastic hepatopathy associated with soft tissue sarcoma neurofibro-thmor of the spinal cord possible associated conditions: with angioma-cerebellar hemangioblastoma; segmental cutaneous vascular nevi; with matosis in children with soft tissue sarcoma tissues record abnormal features; photograph and submit nevi for histologic study. for removal and specimen preparation, see chapter . see also below under "vertebral column bone erosion and calcification of spinal canal. vertebral hemangiomas may be associated with arteriovenous malformation of spinal cord. manifestations of von hippel-lindau disease. * thmor of the stomach possible associated conditions: hypoglycemia;* megaloblastic anemia;* skin changes (as listed under "possible or expected findings)weight. record abnormal features; photograph and submit samples of normal and abnormal skin for histologic study. possible or expected findings cachexia; lower leg lymphoedema ( ) metastases common in liver, retroperitoneal and supraclavicular lymph nodes (virchow's node), ovaries (krukenberg tumor), and peritoneal cui de sac (blumer's shelf') carcinoma of the stomach with hyperkeratosis palmaris and plantaris and acanthosis of the esophagus gastric lymphoma ofmucosa-associated lymphoid tissue and helicobacter pylori gastric signet-ring cell carcinoma: unilateral lower extremity lymphoedema as the presenting feature thmor of the testis possible associated conditions: demyelinating neuropathy record location, size, and weight of both testes and of testicular tumor. submit samples of tumor and of ininvolved testis and epididymis for histologic study. snap-freeze tumor tissue for hormone assay. possible or expected findings cachexia. cryptorchism; hypospadia. gynecomastia. increased concentrations of alphafetoprotein and human chorionic gonadotropin testicular microlithiasis. secondary testicular tumors (metastases to the testes) are rare, except in association with leukemia* in children. references pulmonari embolism.* paraneoplastic diseases as listed above under "possible associated conditions chronic inflammatory demyelinating polyneuropathy (clop) associated with seminoma dermatomyositis associated with intratubular germ cell tumor and metastatic germ cell cancer testicular cancer in association with developmental renal anomalies and hypospadias submit samples of lymph nodes, spleen, peyer's plaques, and bone marrow for histologic study. other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. references cachexia. dermal hyperpigmentation. pemphigus foliaceus (rare). hypogammaglobulinemia and other immunoglobulin abnormalities. usually, thymoma presents as an infiltrating, anterior mediastinal mass that rarely metastasizes. carcinoid tumor, malignant lymphoma* (hodgkin's disease), and metastases from carcinomaofthe breast* and other tumors also may occur in this location herpes simplex*) and fungal infections (e.g., candidiasis*) due to thymoma-related immunodeficiency ( ). manifestations of paraneoplastic diseases and conditions as listed above under "possible associated conditions thrombotic thrombocytopenic purpura accompanied by transient pure red cell aplasia and thymoma glomerulonephritis associated with myasthenia gravis thymoma and cellular immune deficiency in an adolescent possible associated conditions: with papillary carcinoma-familial adenomatous polyposis thyroid carcinoma associated with familial adenomatous polyposis an association between acromegaly and thyroid carcinoma thyroid carcinoma causing fatal laryngeal obstruction prostate cancer metastasis to thyroid gland schistosomiasis and the risk of bladder cancer in human papilloma virus infection ( ) or herpesvirus infection ( ) with invasive cervical carcinoma. erythropoietin may be found in some tumors. thrombosis. * myopathy* may be associated with carcinoma of the cervix. cerebellar cortical degeneration* may be associated with carcinoma of the uterus. manifestations of uremia (see "failure, kidney") invasive cervical cancer in human immunodeficiency virus-infected and uninfected hospital patients association of risk factors for cervical cancer and human papilloma viruses in invasive cervical cancer association of herpesvirus infection with the development of genital cancer tyrosinemia type ii: a challenge for ophthalmologists and dermatologists hereditary tyrosinemia type i (chronic form): pathologic findings in the liver richner-hanhart syndrome (tyrosinemia ii): early diagnosis of an incomplete presentation with unusual findings acute pancreatitis-associated acute gastrointestinal mucosal lesions: incidence, characteristics, and clinical significance uncinariasis (see "ancylostomiasis uremia (see "failure, kidney from: handbook of autopsy practice urticaria pigmentosa of childhood (see ''mastocytosis, systemic acute aortic dissection;* aneurysma(s) of cerebral arteries; aortic insufficiency;* calcific aortic stenosis,* coarctation of the aorta;* infective endocarditis;* shone's syndrome; turner's syndrome. * organs and tissues procedures possible or expected findings heart if infective endocarditis is suspected, see chapter . open heart in cross-sections (see chapter ). photograph aortic valve and test valvular competence note: reye's syndrome* is a possible postviral complication of varicella that must be distinguished from varicella encephalitis. varicella may also cause exacerbation of tuberculosis.* (i) collect all tissues that appear infected bicuspid aortic valves are associated with aortic dilatation out of proportion to coexistent valvular lesions varicella-zoster virus infection in children with underlying immunodeficiency virus infection group a beta-hemolytic streptococcal epiglottitis as a complication of varicella infection optic neuritis: a rare complication of primary varicella infection hsv-l-induced acute retinal necrosis syndrome presenting with severe inflammatory orbitopathy, proptosis, and optic nerve involvement varicella with acute motor axonal neuropathy descending necrotizing mediastinitis in a child with chicken pox double outlet right ventricle with intact ventricular septum respiratory syncytial (see "pneumonia, all types or type unspecified vitamin a (see ''deficiency, vitamin a" and "hypervitaminosis a vitamin bt (thiamine) (see "syndrome, wernicke-korsakott vitamin bt (see "anemia, megaloblastic deficiency deficiency, vitamin d" and "hypervitaminosis d waldenstrom's macroglobulinemia (see ''macroglobulinemia, waldenstrom's.) waterhouse-friderichsen syndrome (see "disease, meningococcal wegener's granulomatosis (see "granulomatosis, wegener's werdnig-hottman disease (see "disease, motor neuron acid esterase activity in cultured skin fibroblasts and amniotic fluid cells using -methylumbelliferyl palmitate which may be secondarily infected by bacteria. dark field microscopy will identify the organism expected findings mucosal ulcers, resembling those of typhoid fever, primarily of distal ileum and colon. villous shortening, crypt hyperplasia with mucosal microabscesses. microabscesses in regional mesenteric lymph nodes zellweger's syndrome (see "syndrome mucormycosis" and procedures under "diabetes mellitus abscesses and granulomas may occur in all organs and tissues. granulomatous lesions in central nervous system ( ) and eyes ( ). fungal osteomyelitis* that may be multifocal, including sites such as metacarpals and metatarsals. external examination other procedures depend on expected findings or grossly identified abnormalities as listed in right-hand column. osteosclerosis of vertebrae, ribs, clavicles, pelvic bones, scapulae, skull, and metaphyseal ends of femur and humerus. osteomyelofibrosis or osteoreticulosis; rarely, panhyperplasia of bone marrow; increase of megakaryocytes.changes associated with chronic granulocytic leukemia* or with polycythemia vera* may imitate idiopathic myelofibrosis. widespread extramedullary hematopoiesis ( ) with splenomegaly. infectious diseases, including tuberculosis;* gouty arthritis; ascites and other manifestations of portal hypertension;* or hepatic vein thrombosis (budd-chiari syndrome*). cardiac tamponade ( ) . synonyms and related terms: hepatic porphyria; protoporphyrinogen oxidase deficiency ( ) .note: the manifestations of the disease closely resemble those in porphyria cutanea tarda and hereditary coproporphyria. measurements of porphyrins and porphyrin precursors are the only clearly distinguishing features. for autopsy procedures, see under "porphyria cutanea tarda." fibrin and platelet thrombi, with or without microaneurysms and purpura in kidneys, adrenal glands, pancreas, heart, and brain; lesions may also be present in liver; spleen ( ), lymph nodes, muscle, bone marrow, and synovium. fibrin thrombi can be demonstrated with labeled antihuman fibrin antibodies. lesions in precapillary arterioles. hypertrophic osteoarthropathy* (with pleural mesothelioma).pleural effusions. * asbestosis may be complicated by pleural mesothelioma. other organs and tissue sample bladder tumor and uninvolved urinary bladder for histologic study.cachexia. hydronephrosis* (obstructive uropathy) and hydroureter, usually caused by distal ureteral obstruction. recurrent nephrolithiasis* or pyelitis may have been present and is a risk factor for urinary bladder carcinoma.chronic urocystitis; infestation with schistosoma haematobium ( ) (uncommon in north america). manifestations of uremia (see "failure, kidney"). key: cord- - ij vbqp authors: drosten, christian; doerr, hans wilhelm; lim, wilina; stöhr, klaus; niedrig, matthias title: sars molecular detection external quality assurance date: - - journal: emerg infect dis doi: . /eid . sha: doc_id: cord_uid: ij vbqp inactivated severe acute respiratory syndrome–associated coronavirus samples were used for an external quality assurance study within the world health organization sars reference and verification network and other reference institutions. of participants, correctly detected virus in all samples > , rna copies per milliliter and none in negative samples. commercial test kits significantly improved the outcome. inactivated severe acute respiratory syndrome-associated coronavirus samples were used for an external quality assurance study within the world health organization sars reference and verification network and other reference institutions. of participants, correctly detected virus in all samples > , rna copies per milliliter and none in negative samples. commercial test kits significantly improved the outcome. s evere acute respiratory syndrome (sars) is an infectious interstitial pneumonia that causes death in a considerable portion of patients. the first epidemic of sars began in november in southern china, spread to all five continents, and was interrupted in july . it caused deaths among the , cases. two laboratory-associated infections and four new isolated cases have since occurred ( ) . sars is caused by a novel coronavirus (sars-cov) that is shed in patients' respiratory secretions after infection ( ) ( ) ( ) ( ) . immune response to sars-cov appears with a latency of up to weeks from infection, and the concentration of virus particles varies greatly between patients or types of clinical samples. thus achieving a reliable virologic diagnosis early after disease onset is difficult. highly sensitive methods for virus detection, such as reverse transcription-polymerase chain reaction (rt-pcr) are required to confirm sars in the acute phase and prevent transmission. molecular detection methods have been developed by several research laboratories, and the first commercial test kits have become available ( , ) . the performance of such tests, however, has only been evaluated in pilot feasibility studies. little data exist about the relative performance of different laboratories and methods. the world health organization (who) has made the comparing and standardizing of laboratory tests an issue of high priority in sars research ( ) . comparative testing of characterized samples is a direct way to identify weaknesses of single laboratories or certain methods. we present the results of the first external quality assurance study on sars-cov molecular detection. ninetythree institutions involved in laboratory diagnostics of sars were invited to participate in the study. invitees were members of the international who sars reference and verification laboratory network ( ) or national and regional sars reference laboratories. the study was announced as an external quality assurance study on diagnostic proficiency, which included certifying and publishing the results in a comparative and anonymous manner. fifty-eight laboratories from countries ( european, austral-asian, north and south american, and african) eventually enrolled in the study. four companies that produced commercial diagnostic test systems also participated but were evaluated separately because they do not fulfill public health duties. virus material was obtained from supernatants of vero cell cultures collected one day after infection with sars-cov strains frankfurt and hku- . the supernatants were heated to °c for h and γ irradiated with kgy. residual infectivity was excluded by vero cell cultures ( passages). aliquots of the inactivated virus stock solutions were lyophilized and redissolved, and the virus rna was quantified by two different noncommercial real-time rt-pcr assays ( , ) . virion integrity was confirmed by morphology by electron microscopy (data not shown). test samples for the study were generated by diluting the inactivated virus stock solutions in human fresh-frozen plasma testing negative for hiv- , hepatitis b virus, hepatitis c virus, and sars-cov by rt-pcr. aliquots of µl each were then lyophilized and shipped at ambient temperature to the participating laboratories. each participant received a coded panel of seven positive and three negative samples. virus-positive samples contained - , rna copies per milliliter after resuspending in µl of water. the participants were asked to analyze the material with the molecular methods they routinely use in suspected cases in humans. details about the methods were requested, such as the sources of rt-pcr primers and protocols, the type of extraction method used, and suppliers and types of commercial kits, if used. the following two criteria were chosen as minimum requirements for overall proficiency. first, laboratories had to correctly detect the four samples containing > , copies of viral rna per milliliter, a concentration well above the detection limit of published and commercial nucleic acid amplification tests (nat) for sars-cov, ( , , ( ) ( ) ( ) . second, no false-positive results were allowed with the negative samples. indeterminate results in positive samples were treated as negative and in negative samples were treated as positive since the application of nat usually does not involve indeterminate endpoints, and laboratories should be able to resolve unclear results by double testing with another amplification assay ( ) . before evaluating the performance of individual laboratories, we determined how many participants managed to detect virus in each sample ( table ). the concentrationdependent, cumulative positivity rates per sample corresponded exactly with the response rates calculated by a probit regression analysis, which is equivalent to a doseresponse model (figure, p < . ) . the model could predict for the average laboratory that % of all test results could be expected to be correctly positive when ( % confidence interval [ci] . - . ) copies of virus rna per milliliter of sample were present, and % with more than , ( % ci , - , ) copies per milliliter. good compliance with the model furthermore confirmed that all samples contained the expected concentration of rna upon reception by the participants and that no rna degradation had occurred even in samples containing low amounts of virus. applying the proficiency criteria, ( %) of laboratories passed the minimum requirements for successful participation. failure in three laboratories was due to lack of sensitivity, in three due to false-positive results, and in one due to both. thirteen of successful laboratories ( . % of all participants) could also detect the virus in all three weakly positive samples (< , copies/ml), and another missed only one positive sample. ten of the laboratories issued indeterminate results in one or more samples. whether common technical factors would influence the performance of laboratories was also assessed. we subjected cumulative results from low concentration samples (< , copies/ml) to analysis of variance (anova) analysis. the overall positivity rate in these samples was . % ( % ci . %- . %). seven technical factors ( table ) were used to characterize the test procedures each laboratory was using. only use of commercial rt-pcr test kits made a significant difference with regard to total sensitivity. this finding was in concordance with results of the four participating companies who manufacture these kits: all were % correct. fourteen of participants used commercial test kits. for noncommercial tests, whether laboratories developed primers themselves or adapted from other researchers did not make a difference. this finding might be due to availability of wellevaluated primers through a who internet resource during the outbreak ( ) . forty-two of the participants used at least one procedure listed on this site. we finally assessed whether laboratories belonging to the international who sars reference and verification network ( ) were more proficient in sars molecular detection than others. in the three samples containing labs participating in the study. this difference was not significant (p value = . , t-test). the results of this first external quality assurance study on sars-cov molecular detection are assuring. compared to an earlier study on molecular testing for filoviruses, lassa virus, and orthopoxviruses, using very similar proficiency criteria ( ), almost double the portion of participating laboratories completed the study successfully ( % vs. . %). on the other hand, this study only examined paramount issues like sensitivity and control of contamination. validation of other aspects, like cross-reactivity of primers or control of pcr inhibition, is the responsibility of each diagnostic laboratory. commercial tests clearly were the preferred way of achieving good diagnostic performance, possibly because sars-cov is a pathogen with which relatively few laboratories have had experience. however, developing and approving commercial tests is a lengthy process and high costs limit their application. other approaches have to be adopted for efficiently providing good diagnostic tools in immediate response to an infectious disease outbreak. who's strategy of disseminating essential information through a public internet resource before publication has proven successful. laboratories have willingly shared protocols and positive control material with other institutions, enabling qualified diagnostics within weeks after the primary description of the new virus. the benefit is proven by good overall results in this study. international strain collections should be complemented with noninfectious reference material of rare pathogens. until now, such material has been available only for highly prevalent agents like hiv- , herpes viruses, or hepatitis viruses. for sars-cov, reference material has been created in this study for the first time. all samples described can be obtained for a nonprofit charge through the who sars reference and verification laboratory network. new case of laboratory-confirmed sars in guangdong, china-update identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome koch's postulates fulfilled for sars virus evaluation of advanced reverse transcription-pcr assays and an alternative pcr target region for detection of severe acute respiratory syndrome-associated coronavirus quantitative analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome world health organization. who sars scientific research advisory committee concludes its first meeting world health organization. who sars international reference and verification laboratory network: policy and procedures in the interepidemic period real-time reverse transcription-polymerase chain reaction assay for sars-associated coronavirus real-time polymerase chain reaction for detecting sars coronavirus detection of sars coronavirus in patients with severe acute respiratory syndrome by conventional and real-time quantitative reverse transcription-pcr assays world health organization. alert, verification and public health management of sars in the post-outbreak period first international quality assurance study on the rapid detection of viral agents of bioterrorism key: cord- -n q o authors: chan, paul k. s.; lim, pak-leong; liu, esther y. m.; cheung, jo l. k.; leung, danny t. m.; sung, joseph j. y. title: antibody avidity maturation during severe acute respiratory syndrome–associated coronavirus infection date: - - journal: j infect dis doi: . / sha: doc_id: cord_uid: n q o the maturation of virus-specific immunoglobulin g avidity during severe acute respiratory syndrome–associated coronavirus infection was examined. the avidity indices were low (mean ± sd, . % ± . %) among serum samples collected ⩽ days after fever onset, intermediate (mean ± sd, . % ± . %) among samples collected between days and , and high (mean ± sd, . % ± . %) among samples collected after day . avidity indices of % and % could be considered as cutoff values for determination of recent (⩽ days) and past (> days) infection, respectively. measurement of antibody avidity can be used to differentiate primary infection from reexposure and to assess humoral responses to candidate vaccines and january ) suggests that the clinical presentation of disease and the transmission behavior of the reemerged sars-cov strain can be different from what was known before [ ] . when a sars outbreak occurs again, it is mandatory that a serological survey be conducted, to define the epidemiological character of the outbreak. since these outbreaks may happen in places where a proportion of the population was exposed to the virus during a previous outbreak of sars, a reliable method for differentiating between recent infection and past exposure is vital if a meaningful interpretation is to result from such investigations [ ] . the avidity (functional affinity) of an antibody is a measure of the overall strength of interaction between antibody and antigen. the avidity of virus-specific igg antibody is low during primary viral infection and increases with time [ ] [ ] [ ] . however, exceptions to this rule have been observed for some viruses [ , ] . here, we report the maturation pattern of anti-sars-cov nucleocapsid protein-specific igg antibody (hereafter, "anti-sars-cov igg antibody") avidity over the course of a -month period after primary infection and discuss the potential applications of our findings. patients, materials, and methods. sixty-one patients with sars were recruited into the present study; they ranged in age from to years (mean ‫ע‬ sd, . ‫ע‬ . years), and . % were female. these patients presented with acute-onset fever that progressed to pneumonia, which was otherwise unexplained. nine patients ( . %) required intensive care, all of whom eventually recovered. all patients fulfilled the us centers for disease control and prevention criteria for sars [ ] and had serological evidence of sars-cov infection, as determined by the anti-sars-cov igg antibody immunofluorescence assay described elsewhere [ ] . forty-one patients ( . %) seroconverted, and ( . %) developed a у -fold increase in antibody titer. a total of serum samples were available from the patients, of whom provided sample for the study. anti-sars-cov igg antibody avidity was measured by a step approach. the first step was to assess the concentrations of anti-sars-cov igg antibody in samples, so that samples that required further dilution for testing during the second step could be identified. on the basis of our serial-dilution experiments, samples with ods of . needed to be further diluted to provide a linear range for measurement of avidity during the second step. concentrations of anti-sars-cov igg antibody were measured by a recombinant nucleocapsid proteinbased enzyme immunoassay, elisars (iggene), in accor- dance with the manufacturer's instructions. briefly, samples were diluted to a concentration of : by mixing ml of serum with . ml of sample diluent. an -ml aliquot of the prediluted serum was added to an antigen-coated well. after incubation at room temperature (∼ Њc) for min, the wells were washed times with the washing buffer provided. antihuman igg antibodies conjugated with horseradish peroxidase were added and were incubated at room temperature for min. after a second washing step, , , , -tetramethylbenzidine was added as a substrate for color development. optical density was measured at nm. the second step was also based on the elisars assay but included a urea-elution procedure. briefly, samples were further diluted, if necessary, according to the results obtained during the first step. the neat or prediluted serum samples were mixed with sample diluent as described above. the sample mixtures were added in duplicate to antigen-coated wells. after the first incubation step, ml of urea was added to one of the wells, whereas the same volume of washing buffer was added to the other well, which served as a reference. on the basis of our initial optimization experiments using early and late samples (collected ! and days after fever onset, respectively), a soaking step at room temperature for min with mol/l urea diluted in washing buffer was found to be most suitable and, thus, was used in the present study. the ureasoaking step was followed by washing times with the washing buffer provided. the subsequent conjugate-addition and colordevelopment steps were conducted in accordance with the manufacturer's protocol. the antibody avidity index was calculated as od urea /od reference and is expressed as a percentage. samples collected р days after fever onset were also tested for anti-sars-cov igm antibody, so that igm antibody detection and igg antibody avidity measurement could be compared with respect to demonstrating a recent infection. anti-sars-cov igm antibody was also detected by the elisars assay. briefly, samples were treated by use of a rheumatoid factor removal kit (chemicon) and then mixed with sample diluent to a final concentration of : . a -ml aliquot of the diluted sample was added to an antigen-coated well and subjected to the incubation, wash, and color-development steps described above, except that anti-human igm antibody was used as the conjugate. anti-sars-cov igg antibody titers for paired serum sam- figure . changes in severe acute respiratory syndrome-associated coronavirus-specific igg antibody avidity in paired serum samples ples were measured by an in-house indirect immunofluorescence assay that has been described elsewhere [ ] . this was done so that the value of using changes in igg antibody titers and that in antibody avidity could be compared with respect to demonstrating a recent infection. for the serum samples, the optical-density values obtained during the first step ranged from . to . (mean ‫ע‬ sd, . ‫ע‬ . ); samples had an od of . and, thus, required further dilution for the second step. of these, required further dilution of : , and required further dilution of : , to achieve a reference od of р . before they were subjected to the second step for measurement of antibody avidity. figure shows the pattern of maturation of anti-sars-cov igg antibody avidity after infection. the avidity indices were low (mean ‫ע‬ sd, . % ‫ע‬ . %; range, . %- . %) among the samples collected р days after fever onset and increased to intermediate levels (mean ‫ע‬ sd, . % ‫ע‬ . %; range, . %- . %) among the samples collected between days and . the avidity indices were high (mean ‫ע‬ sd, . % ‫ע‬ . %; range, . %- . %) among the samples collected after day . of the samples collected р days after fever onset, only ( . %) were positive for anti-sars-cov igm antibody, as determined by the elisars assay. for the igm-positive samples, the avidity indices ranged from . % to . %, with a median of . % and an interquartile range of . %- . %. for the igm-negative samples, the avidity indices ranged from . % to . %, with a median of . % and an interquartile range of . %- . %. there was no significant difference in avidity level between these groups of samples (p p . , mann-whitney u test). all together, sample was available from patients, samples were available from patients, and samples were available from patients. the results for the patients with at least samples were further analyzed (the third samples from the patients with samples were not considered). their first samples were collected between days and (mean ‫ע‬ sd, . ‫ע‬ . days) after fever onset, and the time span between collection of the first and second samples ranged from to days (mean ‫ע‬ sd, . ‫ע‬ . days). of the paired samples, only ( . %) showed a significant (у -fold) increase in anti-sars-cov igg antibody titer (as determined by an in-house indirect immunofluorescence assay) from the first to the second sample, a result that could be regarded as evidence of recent infection. when the antibody avidity indices for the paired samples were analyzed, they all showed an increase in avidity index with time. the changes in avidity levels for the paired samples are shown by collection time interval in figure . discussion. our data show that anti-sars-cov igg antibody avidity is low during primary infection and increases with time in a unidirectional manner. on the basis of this phenomenon, measurement of antibody avidity can be used to resolve certain difficulties that may be encountered in assessment of sars-cov infection. first, it can be used to differentiate between primary infection and reexposure. although it was not possible to include patients who had been reexposed in the present study, on the basis of experience with other viral infections that have a similar pattern of antibody avidity maturation [ ] , it is reasonable to infer that patients reexposed to sars-cov will mount a humoral memory immune response that includes the production of antibodies with high avidity within a short period of time. second, the presence of antibodies with low avidity could provide alternative evidence for demonstrating a primary infection when the igm assay result is in doubt. this is important, given that viral serological testing based solely on the determination of the presence of igm can lead to false conclusions, because igm responses last for only a very short period of time and could be missed if serum samples are collected too early or too late [ ] . on the other hand, igm can persist for months or even years after primary infection and reappear during secondary infection [ ] . when interpreting our igm results, one should be aware that the igm assay used in the present study was based on the indirect enzyme immunoassay format, the sensitivity of which might be inferior to that of the igm capture format, and that our omission of the igg antibody removal step might have decreased the assay's sensitivity. nevertheless, our igm assay results for the samples collected р days after fever onset support the view that low antibody avidity could be a valuable alternative marker for defining primary infection, in particular when serum sample availability is limited in terms of collection time points. our data show that all samples with an avidity index of ! % were collected before day , whereas all samples with an avidity index of % were collected after day . avidity indices between % and % could be considered to represent "the maturation zone," in which the correlation between avidity and time since infection is less strong. third, our comparison of the avidity indices for the paired samples indicated that this approach could provide a helpful alternative to the use of increasing antibody concentration as serological evidence of recent infection. this is particularly important if convalescent samples are collected when antibody concentrations are no longer increasing, as was the case for most of our paired samples. other possible applications of measurement of antibody avidity to sars-cov infection include use of the technique to assess humoral responses to vaccine candidates and to discriminate between primary and secondary vaccine failures. world health organization. summary of probable sars cases with onset of illness from laboratory-acquired severe acute respiratory syndrome severe acute respiratory syndrome (sars) in taiwan, china update : review of probable and laboratory-confirmed sars cases in southern china investigation into china's recent sars outbreak yields important lessons for global public health changes in antibody avidity after virus infections: detection by an immunosorbent assay in which a mild protein-denaturing agent is employed avidity of igg in serodiagnosis of infectious diseases comparative evaluation of the use of immunoblots and of igg avidity assays as confirmatory tests for the diagnosis of acute ebv infections differential maturation of avidity of igg antibodies to gp , p and p following infection with hiv- the relative functional affinity of specific anti-core igg in different categories of hepatitis b virus infection severe acute respiratory syndrome immunofluorescence assay for serologic diagnosis of sars differentiation of primary from nonprimary genital herpes infections by a herpes simplex virus-specific immunoglobulin g avidity assay hepatitis a virus infection among the hemophilia population at the bonn hemophilia center serological diagnosis of tick-borne encephalitis by determination of antibodies of the igm class key: cord- -cnvxlv h authors: paskey, adrian c.; frey, kenneth g.; schroth, gary; gross, stephen; hamilton, theron; bishop-lilly, kimberly a. title: enrichment post-library preparation enhances the sensitivity of high-throughput sequencing-based detection and characterization of viruses from complex samples date: - - journal: bmc genomics doi: . /s - - - sha: doc_id: cord_uid: cnvxlv h background: sequencing-based detection and characterization of viruses in complex samples can suffer from lack of sensitivity due to a variety of factors including, but not limited to, low titer, small genome size, and contribution of host or environmental nucleic acids. hybridization-based target enrichment is one potential method for increasing the sensitivity of viral detection via high-throughput sequencing. results: this study expands upon two previously developed panels of virus enrichment probes (for filoviruses and for respiratory viruses) to include other viruses of biodefense and/or biosurveillance concern to the u.s. department of defense and various international public health agencies. the newly expanded and combined panel is tested using carefully constructed synthetic metagenomic samples that contain clinically relevant amounts of viral genetic material. target enrichment results in a dramatic increase in sensitivity for virus detection as compared to shotgun sequencing, yielding full, deeply covered viral genomes from materials with ct values suggesting that amplicon sequencing would be likely to fail. increased pooling to improve cost- and time-effectiveness does not negatively affect the ability to obtain full-length viral genomes, even in the case of co-infections, although as expected, it does decrease depth of coverage. conclusions: hybridization-based target enrichment is an effective solution to obtain full-length viral genomes for samples from which virus detection would fail via unbiased, shotgun sequencing or even via amplicon sequencing. as the development and testing of probe sets for viral target enrichment expands and continues, the application of this technique, in conjunction with deeper pooling strategies, could make high-throughput sequencing more economical for routine use in biosurveillance, biodefense and outbreak investigations. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. high-throughput sequencing (hts), also known as next-generation sequencing (ngs), has many advantages for pathogen detection as compared to traditional methods such as polymerase chain reaction (pcr), serological assays, and/or culture-based methods. metagenomic sequencing is the high-throughput sequencing of nucleic acid from complex samples rather than from purified microorganisms. metagenomic sequencing is much less biased than other methods and allows for the detection of fastidious or nonculturable organisms as well as multiple unrelated pathogens within a single sample [ ] . moreover, detection via hts is much less susceptible to false-negative results caused by antigenic drift or signature erosion. despite these advantages, one of the technical challenges encountered with respect to metagenomic sequencing is obtaining adequate depth and breadth of coverage from pathogens like rna viruses that i) typically have small genomes, and ii) are typically present at low titers amidst the background 'noise' of the host and commensals [ ] . genome size directly affects sensitivity of detection by hts because the sampling of sequence fragments within a sample depends on the prevalence of those fragments and organisms with larger genomes typically contribute more fragments, therefore being sampled more often than organisms with smaller genomes. in other words, organisms with larger genomes have the potential to contribute a larger proportion to the overall number of sequencing reads even when the plaque-forming units (pfu), or colony-forming units (cfu) in the case of bacteria, are equivalent to that of an organism with a smaller sized genome. although conventional shotgun sequencing allows for the detection of all domains of life, it rarely returns robust coverage of a small viral genome when taken from a very complex sample. a variety of possible strategies exist to enhance the sensitivity of hts for virus detection and characterization, including purification of specific viral fractions by physical methods such as filtration and ultracentrifugation [ ] , amplicon-based target enrichment, and hybridization-based target enrichment. purification of viral fractions is ideal in some cases, although it can be laborious, and for certain sized samples (for instance clinical samples of very limited volume) it may not be realistic. the use of hybridization-based target enrichment could be preferable to the aforementioned technologies because it has the potential to yield sequence data covering the entire genome of multiple viruses with just one sequencing reaction by using genome-wide probes designed against multiple viruses to specifically select for viral cdna prior to sequencing. amplicon sequencing of viral genomes is a technique that has been widely used, but it has some disadvantages, which were articulated by metsky et al. in a recent study of zika virus (zikv) [ ] . first, traditional amplicon sequencing typically requires technically challenging normalization and pooling of individual amplicons to cover the entire genome of one specific virus. however, recently a protocol was published for efficient amplicon primer design and multiplex amplicon generation in a single tube for sequencing in the minion or illumina platforms [ ] . although this method obviates the amplicon normalization and pooling steps and is effective for producing whole genome sequence data from a low titer zikv sample, this method has not been demonstrated for production of whole genome sequence data for multiple diverse viruses from a single complex sample. additionally, amplicon sequencing typically requires as much as cycles of pcr amplification [ ] [ ] [ ] , which can introduce sequence errors. furthermore, amplicon-based sequencing is vulnerable to false negative results caused by mutations in primer binding sites, as was recently demonstrated for dengue virus (denv) [ ] as well as false positive variant results possibly caused by low and/or uneven coverage [ ] . by contrast, the use of probes tiled along the entire length of a viral genome to hybridize and select for virus-specific fragments has the potential to produce less false-negative pathogen detection results by virtue of many more potential binding sites along an individual genome and resulting more uniform coverage. ebola virus (ebov) is one specific example of a pathogen for which false-negative pcr results can have devastating consequences and for which available pcr-based assay effectiveness has been shown to be affected by drift [ ] . therefore, a panel of -mer oligonucleotide probes designed against eight filovirus genomes was recently used for post-sequencing library enrichment in a htsbased study of a recent ebov outbreak in west africa and in an investigation of potential genetic variation of ebov in experimentally infected nonhuman primates [ ] [ ] [ ] . in this protocol, viral enrichment is coupled with the rna access kit, developed by illumina, inc. the technical advancements of the rna access kit had already enabled the sequencing of previously unsequencable materials such as those of low concentration and formalin-fixed paraffin-embedded (ffpe) tissue [ ] , and now this protocol has been employed not only for the detection and characterization of ebov from clinical samples but also for detection and characterization of respiratory viruses in clinical samples [ , ] . in general, hybridization-based viral target enrichment has been successfully employed to characterize viruses found within both contrived samples and clinical samples [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] . the performance of the respiratory virus panel (rvp) version of this method [ ] , which uses probes for common respiratory viruses in conjunction with the truseq rna access protocol, was recently investigated and it was demonstrated to work well overall when tested on human clinical samples [ ] . specifically, the authors reported successful enrichment for of human clinical samples tested. importantly, rt-pcr was conducted on those same samples and ct values of respiratory viruses in those clinical samples ranged from to [ ] , which provides a framework for beginning to assess the limits of detection of hybridization-based enrichment sequencing. herein, we extend this approach by i) expanding this viral probe panel to include viruses of biosurveillance and biodefense concern and ii) employing carefully constructed mock clinical samples to systematically assess this technique's performance in a variety of conditions, such as deeper multiplexing for cost effectiveness as well as more extensive co-infection scenarios. we demonstrate the sensitivity and reproducibility of hybridization-based viral enrichment sequencing despite virus divergence and we show that this sensitivity is maintained even with extensive multiplexing of samples to decrease cost. herein we demonstrate that within one reaction tube, this technique can even be used to detect and discriminate between multiple serotypes of a virus within a clinical sample or to detect and discriminate amongst multiple unrelated viruses that present similar clinical symptoms, and we demonstrate this performance at clinically relevant concentrations of virus. hybridization-based target enrichment enhances sensitivity of hts for detection of virus in complex environmental samples enhanced detection of viruses from various clinical sample types using filovirus-or respiratory virus-specific probes has recently been demonstrated [ , , ] . to expand the range of viruses that could be detected, in this study those two probe panels were combined with new probes for additional viruses that are of biosurveillance and biodefense concern, for a full panel targeting diverse viruses (table ). in order to test this newly expanded probe panel and to specifically assess the effect of hybridization-based viral enrichment on the sensitivity of hts for detection of a single virus within a complex environmental sample, commercial bat guano was spiked with increasing concentrations of influenza virus (ifv). spiked samples were split into two parts each, with each part being processed in parallel with unbiased, shotgun sequencing versus target enrichment sequencing using an expanded panel of probes. as expected, a dose-dependent effect in the proportion of sequencing reads derived from ifv was observed as the number of spiked genome copies increased ( fig. a and additional file : table s ), in both the unbiased shotgun sequence data as well as the virus enriched sequence data. however, in this context, hybridizationbased target enrichment resulted in approximately -to -fold more sensitivity for detection of ifv as compared to detection via unbiased, shotgun sequencing. at the lowest concentration tested ( genome equivalents (ge) per ml), only . % of sequencing reads produced by unbiased shotgun sequencing were derived from ifv ('on target' reads), whereas by stark contrast, the majority of reads produced by target enrichment sequencing ( . %) were derived from ifv. given the dramatic increase in sensitivity observed when complex samples were spiked with an individual virus's genetic material and subjected to target enrichment, we next sought to evaluate whether these effects would still be observed in the presence of an additional virus and at lower concentrations of ifv grna overall. therefore, ifv grna was spiked into total rna derived from middle eastern respiratory syndrome coronavirus (mers-cov) cell culture lysate at an overall lower range of increasing concentrations than ifv was spiked in the prior experiment. as before, the samples were aliquoted into two parts that were processed by each method. in these synthetic co-infection samples, target enrichment sequencing resulted in a simultaneous increase in sensitivity for both viruses as compared to unbiased, shotgun sequencing (fig. b) . as expected, a constant high proportion of reads mapping to mers-cov was observed and the proportion of reads mapping to ifv increased in a dose-dependent fashion with the number of genome equivalents spiked (additional file : table s ). we next tested the sensitivity for detection of three clinically-relevant viruses that can co-circulate in tropical regions, can present with similar symptoms, and can be difficult to detect at low titers [ ] . mock clinical samples were constructed containing combinations of zikv, chikv, and denv at loads that correlate with real clinical loads from human specimens. briefly, varying titers of zikv, dengue virus (denv- ), and chikungunya virus (chikv) were spiked into rna extracted from human serum, in duplicate, to create synthetic co-infection samples. negative control samples consisted solely of rna extracted from human serum. viruses were spiked-in at concentrations corresponding to ct values from standard curves generated via rt-qpcr. the targeted spike-in values were chosen based on reports in the literature for clinical samples containing each virus to mimic a realistic co-infection scenario [ ] [ ] [ ] [ ] . given that in the literature there is at least one report of clinical samples being probed singly rather than pooled and probed [ ] and it is not well known how pooling may affect virus detection levels, in this [ , ] experiment we also sought to evaluate whether probing singly or within a pool would affect our ability to identify virus. therefore, total rna extracted from these samples was probed singly ("pool of ") and also pooled in groups of four and with singly-spiked and mock-spiked serum samples consisting of the other components of the pool. the resulting sequence reads were mapped to the reference genomes for each of these three viruses. in all cases, the three co-infecting viruses were able to be detected in each sample at relatively consistent proportions regardless of the number of samples within a pool (fig. a) . even denv- was detectable within each sample it was spiked, despite the low concentration of viral rna (estimated genome equivalents per ml). although each targeted virus was represented by enough sequencing reads to be easily detected, there were differences in the depth and breadth of genome coverage observed. whereas the full chikv genome was recovered from all spiked samples at a very high depth of coverage (fig. c) , in the case of denv- , an average of . % of the genome was recovered from co-infected samples (fig. d ) and the average zikv linear genome coverage was lower, at . % (fig. b) . these patterns in coverage were similar for both replicates (additional file : figure s ). overall, the proportion of reads mapping to a given virus was consistent and reproducible regardless of whether a sample was probed singly or probed within a group of four or . in addition to evaluating whether sensitivity and reproducibility are maintained despite multiplexing in pools of four and , we also sought to evaluate the probe panel's performance in the context of strain-level, and even species-level, genetic variation as well as differing concentrations of viral genetic material. specifically, synthetic clinical samples were also constructed to contain a different strain of zikv than the strain the probes were designed against (strain r rather than strain mr , which is the strain whose reference genome was used for probe design; fig. a and b) and a different species of human adenovirus (hadv) than the probes were designed to target (hadv- , a member of species d; as opposed to species c, b, and e, which the probes target specifically; fig. g and h) . these samples were also constructed to include biological replicates and were probed singly or in pools of four or . in all cases, the spiked-in virus was detectable, although there was some variation in depth of coverage among multiplexed samples. as might be expected, samples that were multiplexed in sets of yielded the lowest depth of coverage compared to samples that were multiplexed in sets of four or probed singly (fig. b, d, f ) . the target genomes were completely covered in the majority of on-target samples. the exception to this rich, consistent coverage included both strains of zikv, which, although both were detectable, did not achieve % linear coverage and exhibited lower depth of coverage than the other viruses that were spiked in at similar levels (fig. b) . it should be noted that in this experiment, both purified rna as well as total nucleic acid samples containing hadv- and hadv- genetic material were processed and sequenced (fig. g and h) . the total nucleic acid samples were processed without a dnase step to allow for potential detection of both the dna viral genome as previously used in [ ] [ ] [ ] well as viral transcripts. in the case of both hadv- (species e) and hadv- (species d, not targeted specifically by probes), the vast majority of the resulting sequencing reads were virus-specific and the reads were well-distributed along the length of the genome in coding regions, and in the case of the total nucleic acid samples, noncoding regions as well (fig. h) , a phenomenon that was consistent between replicates figure s ). it can be difficult to detect denv- and denv- in clinical samples when the ct value crosses above [ ] . therefore, spiked samples were created using two serotypes of denv with ct values corresponding to low titer, and the samples were subjected to hybridization-based enrichment and sequencing. the resulting sequence reads were found to cover the entirety of each target genome, even for the samples corresponding to ct value (estimated genome equivalents per ml). a dosedependent response was observed in the percentage of denv-specific reads as the ct value decreased (fig. a) . for each serotype, the depth of coverage was greater than x even when the ct value crossed (fig. b and c) . as expected, the remaining reads that did not map to denv- or denv- were derived from human genes in spiked-in human serum rna extract that were pulled down by the control probes, as well as the sequencing control library for phix. a major challenge faced in virus detection as well as virus sequencing is the difficulty to detect divergent strains of viruses typically present at low titers amongst a robust host or environmental background. small viral genomes present at low concentrations are effectively drowned out by signal from host nucleic acid and from commensal microorganisms. a variety of methods have been employed to increase the viral signal in highthroughput sequence data, including amplicon sequencing, but for viruses like denv, with its genome of less than kb in size, even amplicon sequencing is regarded as an inefficient approach for samples with ct values of or higher [ ] . such limitations have been of particular concern for u.s. department of defense (dod) laboratories tasked with biosurveillance and biodefense activities in regions with limited material resources and human we demonstrate here that hybridization-based viral target enrichment yields robust coverage of small genomes from clinical samples, even yielding full-length, deeply covered genomes at concentrations whereby fig. detection of close relative viruses irrespective of extensive multiplexing. sequencing libraries made from serum samples spiked with zikv, denv, chikv and/or hadv were prepared in duplicate and probed singly or probed in pools of four or . a, c, e, g the percentage of reads that map to each strain of spiked-in zikv, denv, chikv, and hadv, respectively. each co-infected sample is denoted with an asterisk (*). estimated genome equivalents per ml as extrapolated from rt-qpcr standard curves are listed along the top of each graph. the standard error of the mean of two replicates is shown. b, d, f, h coverage plot for replicate one of each zikv-, denv- -, chikv-, and hadv-containing sample, respectively current amplicon sequencing protocols may be expected to fail. moreover, we demonstrate that hybridizationbased target enrichment can allow for not only detection, but also genetic characterization such as strainlevel discrimination, even at very low concentrations of virus. the capability to detect and discriminate between multiple serotypes of a virus within a complex sample at clinically relevant concentrations by using this enrichment method increases the utility of high throughput sequencing for biosurveillance and for infectious disease diagnostics. for both biosurveillance and clinical sequencing, assay cost and time are important considerations. we have demonstrated that more extensive pooling and multiplexing can be performed to reduce cost and time without sacrificing the assay's ability to detect at least two strains of related virus and a variety of unrelated viruses in one sequencing reaction. to date, the published viral target enrichment studies vary in focus and include characterization of ebov during a recent outbreak in west africa [ , ] and detection of multiple viral families within clinical samples [ , ] . while the probes employed in the studies published to date vary in length from -to -mers, enrichment methods also can differ by the number of probes and target viruses included in a set. an additional potential protocol difference is the number of samples pooled, which ranges from a single sample to [ , , ] . the current recommendation by illumina for viral enrichment is to pool four samples [ ] . the experiments described here systematically test enrichment of a single library as well as pools of four or libraries and include a variety of titers of as many as three viruses within a single sample and as many as samples within an enriched pool. for all conditions, even with more extensive pooling and multiplexing, we observed a dose-dependent response to varying ct values even in co-infected clinical samples. a dose dependent response was also observed by o'flaherty et al. in two co-infected samples containing respiratory syncytial virus and human coronavirus oc spiked-in each at ct or [ ] . interestingly, although there was the expected dose-dependent effect on the proportion of fig. recovery of full denv genome at titers below limit of detection by conventional shotgun hts or amplicon-based sequencing. denv- and denv- rna were spiked into human serum rna at a range of ge corresponding to ct - , in duplicate, and libraries were prepared using target enrichment in pools of four. corresponding estimated genome equivalents per ml as extrapolated from a standard rt-qpcr curve are listed below the axis. mock samples consisted of human serum rna extract only. a the proportion of total reads that map to denv- or denv- at each ct value. error bars show standard error of two replicates. b-c the proportion of reads that map to denv- or denv- , respectively, at each spike-in level. bubble size corresponds to depth of coverage of the viral genome (average of two replicates) sequencing reads derived from ifv as the concentration of spiked ifv grna increased, there was a slight decrease in the proportion of mers-cov-derived sequencing reads in samples at the upper end of the ifv grna concentration range. this was not expected given that mers-cov was present in each replicate at a constant, high level. we hypothesize that this may be due to saturation of the streptavidin beads used to capture probe-cdna hybridized fragments. further experimentation will be required to test that hypothesis. efficacy of probes varies by homology to the viral target, as evidenced by our results and those in the literature [ , ] . for example, the reference sequence used to design the probe set for denv- exhibits % nucleotide identity over % of the length of the closest sequenced reference for the denv- strain that was spiked. it is possible that this overlap, which is not shared by the denv- probes and denv- spike-in, contributed to an overrepresentation of denv- reads in the experiments presented in figs. and . additionally, by comparison to the other richly covered strains of virus tested in multiplexed samples, there was an underwhelming coverage for both strains of zikv. it is possible that the quality of rna from these viruses was less than the other rna spike-ins, or that the probe panel for zikv is less efficacious when used in combination with the entirety of the probe set. the probes for zikv were synthesized and added later after all the other probes were combined (in response to the recent outbreak) and therefore it is possible that the comparatively lower performance of the zikv probes is due to a difference in quantitation of the zikv probe set. we observed that hadv- (species d) genetic material was efficiently enriched even though the only adenoviruses used to design the probe panel were species b, c, and e. this experiment indicates that the protocol works as well for this particular dna virus as it does for rna viruses. limits of detection may vary by viral target, which may explain why previously published experiments showed differences between dna and rna viruses [ ] . nucleotide identity between human adenovirus species d (the species to which hadv- belongs) and the other human adenovirus species hadv-a, b, c, and e was reported by kaneko et al. to range from . to . % [ ] . in our study, the probe panel containing probes for species hadv-b, c, and e effectively enriched for the entirety of the hadv-d genome. this suggests that when using long ( -mer) probes designed against several species of virus, related non-targeted species may also be enriched without being specifically included in the panel, if the nucleotide identity among them is at least - %, and if multiple related species are targeted by the probes in the panel (in this case three species). this cross-reactivity for related human pathogens could prove to be a useful feature, by allowing for enrichment of more relevant viruses without added cost spent to increase the number of probes. our findings demonstrate that breadth of coverage does not suffer from extensive pooling but that deeper depth of coverage is gained by limiting the number of samples pooled. extensive pooling makes hybridizationbased enrichment sequencing more economical. viral target enrichment could be applied as an economical approach to sequencing viruses known to mutate quickly and therefore evade other assays, fastidious organisms, or complex samples of limited volume. for example, this method could be prescribed to a scenario in which multiple serotypes of a virus such as denv are expected to be present in a sample but detection is prohibited via conventional methods such as amplicon sequencing due to low titers. viral target enrichment designed for a broad panel of targets could also be useful to the infectious disease field by enabling detection of low-titer viruses present in clinical samples taken from patients suffering from symptoms of unknown etiology. another applicable use of a broad probe panel could be to perform environmental sampling. the aforementioned applications often involve complex samples of limited volume, for which this method is ideal. an important caveat to this approach is that while viral target enrichment is an economical method by which to reduce background noise in a metagenomic sample, probe design requires prior knowledge of the closest-sequenced genome for each viral target. amplicon sequencing may be the best approach for previously known samples and unbiased whole shotgun sequencing may be more appropriate for a virus-rich sample. none of these approaches obviates the use of amplification by polymerase chain reaction or the potential introduction of sequence errors, and so standard quality analyses by computational methods should always be employed. as the development and testing of probe sets for viral target enrichment expands and continues, the application of this technique could make hts more economical for routine use in force health protection activities including biosurveillance, biodefense and outbreak investigations. extract from particles (new guinea c; atcc, manassas, va) were spiked into relevant matrices to construct contrived metagenomic samples for testing. to prepare guano samples five-gram quantities of commercial jamaican bat guano (planet natural; bozeman, mt) were placed in ml of sterile-filtered hank's balanced salt solution (hbss), vortexed to mix, centrifuged at , x g for min, and filtered sequentially through . μm and . μm filters prior to spiking with ifv particles. post-addition of ifv, samples were centrifuged at , x g for three hours at °c to concentrate spiked and native virus particles, supernatant was removed, and total rna was extracted from the pellet using the qiaampviral rna isolation kit (qiagen; valencia, ca). after elution in μl buffer ave, a second elution using μl of the eluate was performed. to prepare cell culture matrix samples, a nucleic acid spiking approach was used. in this case, decreasing amounts of ifv rna were spiked into a constant mass of total rna that had been extracted from vero cells infected with mers-cov. aliquots of the same vero cell culture were used for each sample. genome equivalents of ifv spiked into samples were calculated based on rna mass extracted from virus particles and genome size. for serum samples, rna was extracted from human serum (bioivt, westbury, ny) and mixed with viral rna. total nucleic acid was extracted from adenovirus particles using the qiagen qiamp minelute virus spin kit, omitting carrier rna. the samples were eluted in μl buffer ave. viral rna was extracted from virus particles and human serum using the qiaampviral rna isolation kit as described above. the qubit double stranded dna broad-range assay kit and the qubit rna broad-range assay kit (thermo fisher scientific; waltham, ma) were used to assay extracts. for quantitative reverse transcription pcr, superscrip-tiii rt/platinum taq mix (thermo fisher scientific; waltham, ma), dntp mix, mgso , rox reference dye (thermo fisher scientific; waltham, ma), and the primers and probes listed in table were used to assay in the cfx connect real-time pcr detection system (bio-rad; hercules, ca) using the following conditions: °c for min, °c for two minutes, and cycles of °c for s and °c for one minute. standard curves for denv- , denv- , chikv and zikv were generated from titrated viral rna extract. for virus enriched sequencing, truseq rna access libraries were created as per manufacturer's protocol (illumina; san diego, ca), with the following two modifications: i) rather than the standard cex oligonucleotides that are designed for enrichment of human genes, a custom pool of oligonucleotides was used that includes probes along the entire genome length of viruses (table ) as well as probes specific for several human house-keeping genes, and ii) in the second pcr amplification, cycles were used rather than ten. samples were probed singly or in pools of four or and multiplexed for sequencing on the illumina miseq platform using v chemistry, x bp read lengths. for conventional hts (shotgun) sequencing, truseq libraries were pooled and sequenced on the miseq platform using v chemistry, x bp read lengths. for the guano samples, these consisted of an aliquot of each of the truseq rna access libraries from the step prior to virus enrichment. for the mers-cov-vero cell matrix samples, these consisted of shotgun libraries made with the truseq ribozero gold library preparation kit (illumina; san diego, ca). a composite panel of -mer dna probes was assembled using a previously described panel for respiratory viruses [ , , ] , a previously described panel for filoviruses [ ] [ ] [ ] , plus an additional panel of newly designed probes for viruses of biosurveillance and biodefense concern, for a total of , probes. the methods employed for capture oligo design were next-generation sequencing for pathogen detection and identification laboratory procedures to generate viral metagenomes zika virus evolution and spread in the americas multiplex pcr method for minion and illumina sequencing of zika and other virus genomes directly from clinical samples targeted full-genome amplification and sequencing of dengue virus types - from south america full-genome amplification and sequencing of zika viruses using a targeted amplification approach evaluation of hybridization capture versus amplicon-based methods for whole-exome sequencing evaluation of signature erosion in ebola virus due to genomic drift and its impact on the performance of diagnostic assays reduced evolutionary rate in reemerged ebola virus transmission chains molecular evidence of sexual transmission of ebola virus fully human immunoglobulin g from transchromosomic bovines treats nonhuman primates infected with ebola virus makona isolate illumina: truseq rna access library prep guide comprehensive viral enrichment enables sensitive respiratory virus genomic identification and analysis by next generation sequencing targeted sequencing of respiratory viruses in clinical specimens for pathogen identification and genome-wide analysis capturing diverse microbial sequence with comprehensive and scalable probe design precision surveillance for viral respiratory pathogens: virome capture sequencing for the detection and genomic characterization of severe acute respiratory infection in uganda enhanced virome sequencing using targeted sequence capture a new comprehensive method for detection of livestock-related pathogenic viruses using a target enrichment system norovirus whole-genome sequencing by sureselect target enrichment: a robust and sensitive method complete genome sequence of a ki polyomavirus isolated from an otherwise healthy child with severe lower respiratory tract infection zika, chikungunya and dengue: the causes and threats of new and re-emerging arboviral diseases detection of dengue virus replication in peripheral blood mononuclear cells from dengue virus type -infected patients by a reverse transcription-realtime pcr assay assay optimization for molecular detection of zika virus chikungunya fever in travelers returning to europe from the indian ocean region genetic and serologic properties of zika virus associated with an epidemic, yap state, micronesia analysis of the complete genome sequence of epidemic keratoconjunctivitis-related human adenovirus type , , and a novel serotype enabling the democratization of the genomics revolution with a fully integrated web-based bioinformatics platform basic local alignment search tool ggplot : elegant graphics for data analysis chikungunya virus fusion properties elucidated by single-particle and bulk approaches development and evaluation of serotype-and group-specific fluorogenic reverse transcriptase pcr (taqman) assays for dengue virus suppression of chikungunya virus replication and differential innate responses of human peripheral blood mononuclear cells during co-infection with dengue virus characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking comparison of three next-generation sequencing platforms for metagenomic sequencing and identification of pathogens in blood quantitative realtime pcr detection of zika virus and evaluation with field-caught mosquitoes the authors would like to thank lcdr gabriel defang from nmrc for providing samples of mers-cov vero cell culture matrix and ltc richard jarman of wrair for providing chikv rna. this work was funded by u.s. navy, office of naval research, in-house laboratory independent research (ilir) program and wun a . the funding bodies had no role in the design of this study nor in the collection, analysis, or interpretation of data, nor in the writing of this manuscript. the datasets supporting the conclusions of this article are available in national center for biotechnology information (ncbi) sequence read archive (sra) as prjna . the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. essentially as described in o'flaherty at al [ ] , although the design varied somewhat across the target viral genomes. for instance, in the case of the previously described respiratory virus panel, the design was focused on coding regions [ ] . in general, genomes were tiled with capture oligos in a way so as to avoid low-complexity sequences and repetitive sequences. probe spacing and overlap vary per virus due to attempts to design probes that cover multiple related virus strains resulting in overlapping tiled design around more variable regions, whereas regions more conserved among multiple strains resulted in probes more or less tiled end-to-end. all probes were biotinylated on the ′ end. sequences of viral capture probes are provided in additional file . quality control, de novo assembly, taxonomic classification, and reference-based analyses were conducted using edge bioinformatic software v . [ ] with default parameters and host removal of human reference grch and also with clc genomics workbench v (qiagen bioinformatics; redwood city, ca). the reference mapping parameters in clc were modified from defalt settings to . length fraction and . similarity fraction with global alignment and random mapping of non-specific matches. blast [ ] was also used to further investigate specific datasets. the ggplot r package was used to generate depth of coverage plots [ ] . additional file : table s . number and proportion of reads mapped to ifv at spiked-in genome equivalents of , , , , and , given preparation by hybridization-based target enrichment or shotgun sequencing. (docx kb) additional file : table s . number and proportion of reads mapped to ifv or mers-cov at ifv spiked-in genome equivalents of , , , and , and a constant, high level of mers-cov genomic material given preparation by hybridization-based target enrichment or shotgun sequencing. (docx kb) additional file : figure s . coverage plots demonstrating the number of reads that mapped to zikv, chikv, and denv, respectively, as well as the distributionof those reads along the length of each genome. replicates shown in the views expressed in this manuscript are those of the authors and do not necessarily reflect the official policy or position of the department of the navy, the department of defense, nor the u.s. government. th is a military service member; kgf, and kab-l are employees of the u.s. government. this work was prepared as part of their official duties. title u.s.c. § provides that 'copyright protection under this title is not available for any work of the united states government.' title u.s.c. § defines a u.s. government work as a work prepared by a military service member or employee of the u.s. government as part of that person's official duties.ethics approval and consent to participate not applicable consent for publication not applicable key: cord- -seqwx x authors: battey, cj; ralph, peter l; kern, andrew d title: predicting geographic location from genetic variation with deep neural networks date: - - journal: elife doi: . /elife. sha: doc_id: cord_uid: seqwx x most organisms are more closely related to nearby than distant members of their species, creating spatial autocorrelations in genetic data. this allows us to predict the location of origin of a genetic sample by comparing it to a set of samples of known geographic origin. here, we describe a deep learning method, which we call locator, to accomplish this task faster and more accurately than existing approaches. in simulations, locator infers sample location to within . generations of dispersal and runs at least an order of magnitude faster than a recent model-based approach. we leverage locator’s computational efficiency to predict locations separately in windows across the genome, which allows us to both quantify uncertainty and describe the mosaic ancestry and patterns of geographic mixing that characterize many populations. applied to whole-genome sequence data from plasmodium parasites, anopheles mosquitoes, and global human populations, this approach yields median test errors of . km, . km, and km, respectively. in natural populations, local mate selection and dispersal create correlations between geographic location and genetic variation -each individual's genome is a mosaic of material inherited from recent ancestors that are usually geographically nearby. given a set of genotyped individuals of known geographic provenance, it is therefore possible to predict the location of new samples from genetic information alone (guillot et al., ; yang et al., ; wasser et al., ; rañola et al., ; bhaskar et al., ; baran et al., ) . this task has forensic applicationsfor example, estimating the location of trafficked elephant ivory as in wasser et al., -and also offers a way to analyze variation in geographic ancestry without assuming the existence of discrete ancestral populations. the most common approaches to estimating sample locations are based on unsupervised genotype clustering or dimensionality reduction techniques. genetic data from samples of both known and unknown origin are jointly analyzed, and unknown samples are assigned to the location of known individuals with which they share a genotype cluster or region of pc space (breidenbach, ; battey et al., ; cong et al., ) . however, these methods require an additional mapping from genotype clusters or pc space to geography and can produce nonsensical results if unknown samples are hybrids or do not originate from any of the sampled reference populations. existing methods for estimating sample location that explicitly model continuous landscapes use a two-step procedure. a smoothed map describing variation in allele frequencies over space is first estimated for each allele based on the genotypes of individuals with known locations, and locations of new samples are then predicted by maximizing the likelihood of observing a given combination of alleles at the predicted location. in methods like spasiba (guillot et al., ) and scat (wasser et al., ) , allele frequency surfaces are estimated by fitting parameters of a gaussian function of set form (but see rañola et al., for an alternate approach based on smoothing techniques from image analysis). since all such methods use relatedness to other contemporary samples, any information about the location of a new sample necessarily comes from ancestors shared with the reference set. as illustrated in figure , we expect a priori, that the genealogical relationships among a set of samples (and therefore the spatial location of ancestors) will vary along the genome. this means that a complete look at geographic ancestry would include not just a point estimate of spatial location, but an estimate of uncertainty that accounts for the partially correlated genealogies of recombining chromosomes. in the past few years, there has been a explosion in the use of supervised machine learning for population genetics for a number of tasks, including detecting selection (schrider and kern, ; mughal and degiorgio, ; sugden et al., ) , inferring admixture durvasula and sankararaman, ) , and performing demographic model selection (pudlo et al., ; villanea and schraiber, ) . applications to population genetics increasingly make use of the latest generation of machine learning tools: deep neural networks (a.k.a. 'deep learning') (sheehan and song, ; kern and schrider, ; chan et al., ; flagel et al., ; figure . conceptual schematic of our approach. regions of the genome reflect correlated sets of genealogical relationships (a), each of which represents a set of ancestors with varying spatial positions back in time. we extract genotypes from windows across the genome (b), and train a deep neural network to approximate the relationship between genotypes and locations using euclidean distance as the loss function (c). we can then use the trained network to predict the location of new genotypes held out from the training routine (d). adrion et al., ) . a significant feature of neural networks is that they allow the input of raw genotype information, as we perform below, without initial compression into summary statistics. in this paper, we introduce locator, a highly efficient deep learning method for the prediction of geographic origin of individuals from unphased genotype data. locator uses deep neural networks to perform prediction directly from genotypes, but without assuming any explicit model of how genotypes vary over the landscape. moreover, unlike many modern supervised machine learning methods in population genetics, (e.g. kern and schrider, ) our training set need not be obtained via simulation. we assume only that there is some function relating geographic locations to the probability of observing a given combination of alleles, and use a deep, fully connected neural network to approximate this mapping for a set of genotyped individuals with known locations. the trained network is then evaluated against a set of known individuals held out from the training routine and used to predict the geographic location of new samples based on their genotypes. applied separately to windows across the genome, locator also estimates uncertainty in individual-level predictions and can reveal portions of an individual's genome enriched for ancestry from specific geographic areas. for the empirical population genomic data we analyze here, locator achieves state-of-the-art accuracy an order of magnitude faster than competing methods. here, we describe the implementation, test on simulated data, and demonstrate its use in empirical data by estimating sampling locations for anopheles mosquitoes in africa from the ag g project (the anopheles gambiae genomes consortium, ) , p. falciparum parasites from asia, africa, and the americas from the p. falciparum community project (pearson et al., ) , and global human populations from the human genome diversity project (hgdp; bergströ m et al., ). we first evaluated locator's performance in simulations of populations evolving in continuous space with varying rates of dispersal -an idealized setting in which all alleles should vary smoothly over the map. in figure we show that validation error increases along with the dispersal rate of the population. interestingly, error is roughly constant when measured in terms of the mean per-generation dispersal rate, ranging from . to . generations of dispersal given our largest training dataset ( samples, , snps; figure -figure supplement ; supplementary file ). this suggests that error primarily reflects the underlying biological processes of dispersal and mate selection rather than simple noise from model fitting. increasing the number of training samples or the number of snps improves accuracy for all simulations ( figure b ). however, we observed diminishing returns when using over , snps or over training samples. median error for all simulations was also below generations of dispersal for all but the least-dispersive simulation using just training samples; suggesting that even relatively small training datasets can allow inference of broad-scale spatial locations. we discuss theoretical limits on the accuracy of genetic location estimation in appendix . we were interested to compare the performance of locator to that of spasiba (guillot et al., ) , the current state-of-the-art method for geographic prediction of genotype data (figure ; supplementary file ). however, we were unable to succesfully run spasiba with , or more snps from a simulated dataset or on simulations with dispersal rates of . or . map units/generation, due to out-of-memory errors on a -bit system with gb of ram. we could, however, compare at smaller numbers of snps and reduced dispersal. at a mean dispersal distance of . map units spasiba's median test error was slightly lower when run on snps (wilcoxon test, p= . ) but results were similar at or , snps. (wilcoxon test, p ¼ : and . ). however, locator is much faster -training on , snps in less than two minutes while spasiba requires around six and a half hours ( figure ). these long run times are caused in part by the large number of training localities in our simulated data, because spasiba's run time scales with the product of the number of genetic variants and the number of training localities (guillot et al., ) . while the simulations conform well to modeling assumptions of most methods, we can also compare performance on empirical data. by way of example, we applied locator and spasiba to subsets of snps from the first five million base pairs of chromosome l from the ag g dataset (miles and harding, ; figure ). locator achieves much lower mean error on all runs with more than snps, and runs from . x to x faster, depending on the number of snps. two factors likely explain this improved accuracy: locator can handle abrupt changes in allele frequencies across the landscape better than spasiba's geostatistical model, and the limited number of sampling localities in the ag g dataset may act as a prior encouraging locator to assign samples to specific sampling sites. maps of predictions from both methods are shown in figure -figure supplement . extrapolating from these run times, running a windowed whole-genome analysis of anopheles in spasiba would require roughly days of computation on an -cpu system for model training alone, versus . hr on one gpu for locator. by running locator in windows across the genome we aim to integrate over error associated with the model training procedure while also representing the inherent uncertainty caused by spatial drift of ancestral lineages backwards in time (kelleher et al., ) . this produces a cloud of predicted locations distributed around the true sample location (figure ) . for individuals near the center of the landscape, these clouds are roughly symmetrical, as expected from our model. predictions for individuals close to the edge of the landscape appear slightly asymmetrical and are bounded by the true landscape edges, suggesting that our networks have learned the rough shape of the sampled range. the true location was within the % contour of a d-kernel density surface estimated from the set of per-window predictions for all test samples, demonstrating that this distribution is indeed centered on the true location. we also tested the alternate approach of bootstrapping over a single set of snps, which could be useful for smaller datasets or those lacking a reference alignment. results for this method are discussed in figure -figure supplement . windowed analyses for the three empirical systems we studied are shown in the bottom panels of figures , , . we discuss the implications of these predictions for each species in the following sections, but in general we find that the windowed analysis provides a good depiction of uncertainty in a sample's location -either surrounding a single location for samples with low error, or distributed across a wide region including multiple training localities for samples with high error. in several cases, predicted locations also project in the direction of known historic migrations (as in human data), or are split among localities shown in previous analyses to experience high gene flow (as in anopheles). we summarize genome-wide window predictions in two ways: ) by taking a kernel density estimate of the predictions and then finding the point of maximum density, and ) by computing the centroid of the windowed predictions. these estimates are similar in spirit to ensemble prediction methods (ho and forests, ; breiman, ) , but should not be interpreted as true statistical confidence intervals. at least in the context of windowed analyses, differences in predictions among windows appears to primarily reflect variation in ancestry rather than uncertainty in the inference itself, so we suggest the intervals returned by locator's kernel density estimation are best interpreted as representing areas from which a given proportion of the genome is likely to have originated. in general, we found that the maximum kernel density estimator has lower error, but tends to show classification behavior more than the centroid estimator -snapping to a single training locality rather than interpolating between sets of localities for samples with variable window predictions. we next turn our attention to the application of locator to empirical population genomic datasets. in figure , we show predicted and true locations for individuals from the ag g dataset of anopheles gambiae and a. coluzzii, estimated in mbp windows across the genome. the location with highest kernel density across all windows had a median error of . km, and the centroid of the per-window predictions had a median error of km (supplementary file ). significant prediction error occurs only between sites in cameroon, burkina faso, and the republic of guinea -localities which were also assigned to a single ancestry cluster in the admixture analysis in miles and harding, . however uncertainty for these samples was relatively well described by visualizing the spread of per-window predictions, with predicted locations generally lying between sets of localities. the true locality was within the % interval of the kernel density across all windows for all samples. in a windowed analysis of p. falciparum, locator's median error is . km using the maximum kernel density and . km using the geographic centroid of window predictions ( figure ; supplementary file ). mean predicted locations across all windows consistently separate populations in the americas, west africa, east africa, southeast asia, and papua new guinea; consistent with the major population subdivisions described via pca in pearson et al., . we also see the geographic centroid of per-window predictions for each individual is shown in black points, and lines connect predicted to true locations. sample localities are colored by the mean test error with size scaled to the number of training samples. bottom -uncertainty from predictions in mbp windows. contours show the %, %, and % quantiles of a two-dimensional kernel density across windows. figure continued on next page good discrimination within clusters, particularly in southeast asia where the average test error is less than km for all but two localities. error is highest in west africa, where mean predictions tend towards the center of a set of regional collecting localities ( figure ). these patterns are consistent with previous findings of fine-scale spatial structure in p. falciparum in cambodia (miotto et al., ) and low levels of relative genetic differentiation (as measured by f st ) in africa (pearson et al., ) . rates of mixed-strain infection are elevated in west africa relative to southeast asia (zhu et al., ; pearson et al., ) , which we hypothesized could explain the higher prediction error in this region. to test this effect, we plotted locator's centroid prediction error as a function of within-host diversity (f ws ; auburn et al., ) . f ws measures the proportion of population genetic diversity present in individual hosts, with a value of representing maximum within-host diversity and one minimum within-host diversity. if mixed-strain infections explain outliers of prediction error, we would expect that samples with the highest prediction error had low f ws . instead we found a weak positive relationship (figure -figure supplement ), with the highest prediction errors seen in samples with maximum f ws (i.e. minimum infection diversity). test error then likely reflects low levels of differentiation within plasmodium lineages in west africa rather than local prevalence of mixedstrain infections. again we found that visualizing per-window predictions reflects expected patterns of uncertainty in samples with high mean prediction error. for example, sample qm -c was collected in madagascar and has a mean predicted location in mozambique, but the spread of per-window predictions indicates a % interval that includes the true locality ( figure , bottom right). the good performance we observed on this dataset also highlights a strength of locator's model-free approach. recall that the sequencing strategy of preparing libraries from human blood samples suggests variant calls represent binned allele frequencies across the population of plasmodium in a human blood sample rather than snps in a single plasmodium individual. from the perspective of the network, however, the input genotypes are simply a set of normalized vectors, and the network can approximate the relationship between these vectors and the spatial location of training samples regardless of the generative process. for humans in the hgdp dataset, the location with highest kernel density across all windows has a median test error of km, and the centroid of window predictions has a median error of . km (figure , supplementary file ). visualizing the geographic distribution of predictions across the genome shows that predictions tend to cluster around the true reported sampling location, but also extend toward other sampling locations in a manner that reflects known patterns of human migration. for example, the two largest individual errors in our analysis are found in south african bantu individuals and xibo people from western china. predicted locations of south african bantu people project towards the historic source of bantu migrations in west africa (de filippo et al., ) , with some regions of the genome also projecting in the direction of east african bantu populations (figure , sample hgdp ). in the case of xibo people from western china locator consistently predicts locations in manchuria, central china, and southern sibera -significantly east of the true sample location. this may reflect the known movement of this population, which historically originated in manchuria and was resettled in western china during the th century (gorelova, ; zikmundová, ; figure , sample hgdp ). a sample of individual-level predictions is included in figure . to test whether outlier geographic predictions reflect error in the model fitting procedure versus true variation in ancestry in a given region of the genome, we ran principal component analyses on windows for which a maya individual (sample hgdp ) has predicted locations in europe and africa. in these windows, the maya sample clusters with other individuals from the regions predicted by locator -western europe and africa, respectively -rather than with other individuals from the americas (figure -figure supplement ) . this suggests outlier predictions primarily reflect variation in ancestry in different regions of the genome, rather than stochastic error in model fitting. locator's prediction accuracy varied widely along the human genome. to assess the sources of this variation, we first examined how recombination rate interacts with the accuracy of locator predictions generated from different regions of the genome. a priori we might expect recombination rate to affect accuracy because in regions of the genome with higher recombination, there are a greater number of distinct genealogies, and hence a given sample has inherited from a larger subset of the possible ancestors. test error was estimated as the distance in kilometers from the true sampling location to the geographic centroid of the cloud of per-window predictions, and is shown in figure plotted against local recombination rates from the hapmap genetic map (international hapmap consortium, ) . we find a relatively strong negative correlation (p< : , r ¼ : ) -windows with the lowest recombination rates in general have the highest prediction error. this is consistent with our expectation that regions of the genome representing a greater number of marginal genealogies will yield more accurate predictions of a sample's location. this finding suggests recombination-based distances may be a better basis for establishing window bounds. we tested this by replicating our analysis of hgdp samples using a -centimorgan window size, which yields approximately the same number of windows across the genome. we found the distribution of centroid prediction errors across windowing methods was very similar ( examining plots of window error along the genome suggests much of this effect is caused by high error in windows containing a centromere when using physical distance-based windows ( figure -figure supplement , figure -figure supplement ) . centromere presence explains . % of variance in window prediction error using mb windows, but just . % of variance with centimorgan-based windows. thus, we recommend researchers interested in analyzing individual window predictions use recombination-based windows when possible, but find that using fixed windows is sufficient for estimating individual-level locations. last, to examine how predictions vary near a well-known geographically differentiated region of the genome, we trained a locator model using snps from within kb of the edar gene. the rs snp is a a ! g mutation that created a derived allele which has reached high frequency in east asian and north american populations but is rare elsewhere (bryk et al., ) . this variant is thought to be associated with traits including hair thickness (fujimoto et al., ) , and the edar region has been proposed as a site of recent positive selection in several analyses (voight and kudaravalli, ; tang et al., ; williamson et al., ) . we focused on predictions for samples from central asia, in the middle of the east-west cline in rs allele frequencies across eurasia. in this analysis, the direction of prediction error generally followed the genotype at rs individuals with a g allele tend to predict east of their true location (figure ) . this was particularly clear in heterozygous populations. for example, homozygous g/g xibo and hazara individuals predict east of their true location, while one homozygous a/a uyghur sample predicts well west of the sampling site. of heterozygous samples also predict east of their true location, and five other a/ a central/south asian samples predict significantly west. the discrepancies between sampling and predicted locations likely in fact represent signal: haplotypes carrying a g allele at this locus likely have more 'close' relatives in eastern than western eurasia. these patterns are magnified versions of the trends seen in windowed analyses, suggesting that strong differentiation in this genomic region biases location predictions away from the center of the geographic cline. to understand how locator predictions vary when a sample's true locality is not included in the training set, we ran analyses on a single window of the anopheles data at two scales -first dropping only sites from a specific sampling location, and then dropping all sites from a given country (figure -figure supplements - ) . prediction error is much higher for individuals from regions excluded from training -increasing from a median of km when training and test samples are randomly split to km when excluding individual localities, and km when excluding whole countries. in most cases, predicted locations appear to project toward the nearest locality included in the training set ( figure -figure supplement ) . this is particularly the case when populations at the edge of the map are excluded. locator networks appear to learn something about the boundaries of the landscape based on the distribution of training points, and show a tendency to project towards the middle of the landscape when given a small number of snps (e.g. the top right panel of figure a ), a trivial optimization of the loss function. we also see evidence of locator learning some nonlinear aspects of population structure in the sample. for example, when angolan a. coluzzii are excluded from the training set many of their predicted locations project toward the a. coluzzii sample localities in burkina faso rather than the much closer sampling localities for a. gambiae in cameroon and gabon. in general, we find that locator can interpolate unsampled localities relatively well when genetic differentiation is smooth over the landscape (as among a. gambiae localities in west africa), but does not extrapolate outside the bounds of the training set. sampling the full landscape, or at least a sufficient portion thereof, is thus an important consideration in running our method. the correlation of genealogy and geography leaves genetic signals of ancestral location across the genome that one can leverage for practical inference. for instance, tracking the migratory routes of disease vectors such as anopheles (huestis et al., ) could in principle be achieved if one could accurately predict origin from dna sequence data. similarly, establishing the location of origin from biological samples is critical to anti-poaching conservation efforts (wasser et al., ) , and efforts to map transmission during the ongoing sars-cov- pandemic have been informed by analysis of geographically restricted genetic variants (see e.g., https://nextstrain.org/ncov/global). in this report, we present a new tool, locator, which uses a deep neural network to predict the geographic location of a sample on the basis of its genotype. we show that locator is highly accurate, computationally efficient, and can scale to thousands of genomes. in simulations we found that our method achieves similar accuracy as a state-of-the-art modelbased approach, spasiba (guillot et al., ) , and does so at least an order of magnitude faster. we show that the accuracy of our estimator is naturally measured in terms of the dispersal rate of the population and that predictions from locator are consistently within - generations of mean dispersal across a wide range of dispersal distances (figure , figure -figure supplement ) . however, the greatest increase in accuracy relative to spasiba was in empirical data ( figure ) . this seems to reflect two aspects of our approach that highlight its strengths and weaknesses: as a process-agnostic model locator can easily handle situations in which allele frequencies do not vary smoothly over the landscape. however, the spatial concentration of the anopheles samples may act as a strong prior that incentivizes the network to predict sample locations near sampling localities (figure , figure ), thus acting more as a classifier than a continuous predictor. thus, sampling should be taken into account when interpreting locator's output, and when possible users should avoid highly clustered sampling regimes. locator's computational efficiency makes it practical to estimate uncertainty through resampling approaches like windowed analysis or bootstrapping over the complete genotype matrix. the full windowed analysis of the hgdp data took roughly hr to run on a single gpu, and windowed analysis of all complete plasmodium genomes took just hr. thus, training locator models for biobank-scale datasets including whole genomes of tens or hundreds of thousands of samples is well within reach, particularly if windows can be run on separate gpus. this allows us to estimate uncertainty in predicted locations due both to our prediction methodology as well as biology; with repeated training runs integrating over error associated with network training and prediction and the windowed analysis allowing us to predict geographic origins for regions of the genome reflecting distinct sets of genealogical relationships. disentangling these sources of error is challenging, but analysis of human data for which we have strong prior knowledge of recent population movements suggests that much of the variation in genome-wide prediction we see reflects historic patterns of migration rather than simple prediction error. for example, genomes from hazara individuals in central asia return predicted locations extending from central asia to mongolia (figure bottom, sample hgdp ) , which is consistent with historic records (qamar et al., ) , previous analysis of y chromosome data (zerjal et al., ) , and identity-by-descent tract sharing (lawson et al., ) all of which find evidence of recent shared ancestry between mongolian and hazara individuals. similarly some maya individuals found to have a small proportion of european ancestry in previous analyses rosenberg et al., have predicted locations extending from central mexico across the atlantic to europe and west africa in windowed locator analysis (figure bottom, sample hgdp ), and these signals are replicated in principal components analysis (figure -figure supplement ) . the correspondence between our explicitly geographic method and unsupervised clustering or dimensionality reduction methods highlights the implicit prior assumption of genetic-geographic correlation often made when interpreting the output of structure or pca. rather than mapping population structure to geography as a post-hoc interpretation, locator and other continuous assignment methods directly incorporate space in the model. this also points to a critical consideration in running any form of supervised population clustering. information about population structure comes only from the relative relationships among training and test samples, and interpretations can only be made relative to the set of training samples used. in the case of the hgdp panel, samples were intentionally selected to cover what were thought to be distinctive populations reflecting a vaguely pre-modern distribution of human genetic diversity (harry and marks, ) , and so would probably not be a good reference set for random individuals drawn from areas with recent histories of large population movements such as the united states. here, we have shown that our method, locator, is fast, accurate, and scales well to large samples. however, we see several next steps that could improve the approach. first, our current implementation uses only diploid genotypes and does not pass the network any direct information about haplotype structure. incorporating snp position information and phased haploid sequences would likely increase inferential power, as in the case of unsupervised clustering (lawson et al., ) . running this method on phased, haploid genomes could also in theory allow us to predict parental locations individually for loci with ancestry from different geographic regions. second, our network currently uses a simple fully connected architecture; it could be that other network architectures such as recurrent neural networks might be better suited for this task (e.g. adrion et al., ) . indeed the application of deep learning to population genetics is still in its infancy and we imagine much progress will be made in the coming years along these lines. a central issue in this task will be understanding the limits of process-agnostic inference: when and how can we best use machine learning approaches in population genetics? locator is essentially a flexible regressor designed without direct reference to genealogical or genetic process, so the exact design of the network or the arrangement of the weights and biases in a trained model gives little insight into the underlying mechanisms driving its inferences. these are instead best assessed by carefully studying the natural history of the focal organism, and by interpreting model output with population genetic theory and simulations. we suggest machine learning methods be seen as tools to answer narrowly defined questions, used as a complement to process-based statistical models that give deeper insight into the mechanisms generating the data being analyzed. in principle, a completely process-based inference method that accurately modeled spatial population dynamics might provide the most accurate and best-calibrated inference of location and/or ancestry. however, the problem of modeling spatial populations is not solved, and all existing methods are based on assumptions (e.g. an unchanged species range on a uniform landscape [wright, ] ) that may not hold in practice. for this reason, machine learning approaches might outperform methods based on process. beyond the present application we believe a powerful approach will be to combine process-based population genetic models with the generalization abilities of deep learning -analogous to natural language processing methods that supplement neural network word embeddings with syntax models (strubell and mccallum, ) . this could allow us to leverage theory where assumptions are well-founded while turning to robust generalized optimization techniques when they are not. locator transforms input data in vcf or zarr format to vectors of allele counts per individual using the scikit-allel (miles and harding, ) and numpy (van der walt et al., ) libraries. sites with missing data are replaced with two draws from a binomial distribution with probability equal to the frequency of the derived allele across all individuals -a discrete version of the common practice of assigning missing data as the mean allele frequency in genotype pcas (e.g. the default settings for pca in the r package adegenet [jombart, ] ). we provide functions for filtering snps based on minor allele count, and by default remove singleton sites from the alignment prior to model fitting. the geographical x and y coordinates are scaled to have mean and variance one prior to training, while allele counts are scaled prior to model fitting by a batch normalization layer within the network. batch normalization z-normalizes activations of a neural network during training to reduce shifts in the distribution of parameter values across batches, which allows faster learning rates and sometimes reduces overfitting (ioffe and szegedy, ) . locator selects user-defined fraction of the samples with known locations to use in training the model (the default is . ); remaining samples with known locations are kept aside as 'validation' samples. the validation set is used to tune the learning rate of the optimizer and set the stopping time of model training, but does not directly contribute to the loss used to fit model parameters. throughout this manuscript, we use 'validation loss' to refer to error estimated on the validation set, and 'test error' to refer to error calculated on a set of samples entirely held out from the model training procedure. for datasets with small sample sizes the random train/test split may lead to some regions being under-or overrepresented in the training sample. to mitigate this we suggest fitting multiple models with different random seeds, yielding an ensemble of models trained on different subsets of the original dataset. predictions from this ensemble can then be summarized in the same way as windows or bootstrap samples (see below). an example of this approach is included in the locator documentation (https://github.com/kern-lab/locator). we use the unphased, diploid genotype vector of each individual as input to the network, whose target output is the two-dimensional coordinates of that individual in space. locator uses a deep neural network consisting of a stack of fully connected 'dense' layers, implemented using the keras (chollet, ) frontend to tensorflow (abadi et al., ) . roughly speaking, the network is trained to estimate a nonlinear function mapping genotypes to locations using gradient-based optimization. models start with randomized initial parameters and are fit to data by looping through the training set and iteratively adjusting the weights and biases of the network. we use an early stopping function to monitor loss during training and under default settings stop training runs when validation loss has not improved for epochs. we also use a learning rate scheduler to decrease the learning rate of the optimizer when validation loss stops improving, which we found to be effective in preventing the trajectories of training and validation loss from diverging. the program also outputs a plot of training and validation loss after each training run (figure -figure supplement ) . locator's architecture uses a batch normalization layer followed by a sequence of fully connected layers with a dropout layer in the middle of the network (figure ) . the 'dropout' layer sets a random selection of weights to zero during each training step, which helps prevent overfitting (srivastava et al., ) . our implementation allows users to adjust the shape of the network, but current default settings use dense layers of nodes each with 'elu' activations (clevert et al., ) and a % dropout after the fifth layer. we describe performance under varying network width and depth in figure -figure supplement . in general, we found that all networks with over four layers perform similarly. we use the adam optimizer (kingma and ba, ) with euclidean distance as a loss function: individuals are born at a single location, but have inherited their genomes as a mosaic from ancestors spreading geographically into the past (as discussed in, for instance, wright, ; kelleher et al., ; bradburd and ralph, ) . any signal our method hopes to extract from the data must be due to geographic signal of recent ancestors shared between the test and training datasets. this suggests that any analogous method must quantify, roughly, 'which modern day populations are most similar to this genome?". the spatial spread of genetic relatedness both back in time from an individual's to its ancestors' locations and forward in time from ancestors to the present-day location of training samples means that even a perfect inference algorithm should have significant uncertainty associated with any predicted location from genetic data, and the magnitude of uncertainty should be in part a function of the dispersal rate of the population. in particular, no such method can infer locations more accurately than the mean dispersal distance, because in most cases an individual's genome is not informative about where they live relative to their parents. besides this fundamental limit to uncertainty, error in georeferencing of training samples and in model fitting will introduce additional prediction uncertainty. we use a windowed analysis across the genome to describe this uncertainty, which is possible thanks to locator's computational efficiency. genealogical relatedness on each contiguous stretch of genome can be described by a sequence of genealogical trees, separated by ancestral recombination events. by running locator on a particular window of the genome, we restrict inference to a subset of these marginal trees, and hence to a subset of the genetic relationships between test and training samples. predictions from different regions of the genome can then be visualized as a cloud of points, and the distribution of these points in space gives us a rough idea of the uncertainty associated with an individual-level prediction. because windowed analyses involve repeated training runs from randomized starting parameters, they also help us to integrate over uncertainty associated with the model fitting process. some datasets lack the size or reference alignments necessary to conduct windowed analyses. in this case, we recommend uncertainty be assessed by training replicate models on bootstrapped samples drawn from a single set of unlinked snps (that is, resampling snps with replacement). although this procedure does not reduce the number of marginal trees represented in the data, it does allow us to assess uncertainty associated with model training and prediction. in both cases, we summarize uncertainty in predicted locations by estimating a two-dimensional kernel density surface over a set of predicted locations, and provide plotting scripts to visualize the %, %, and % quantiles in geographic space (see figures - for examples). the location of an individual can then be predicted as either the location with highest kernel density (the modal prediction) or the geographic center of the cloud of predictions (the mean prediction). we tested this approach in simulated data and in all empirical datasets. to explore factors affecting the accuracy of predicted locations generated from different regions of the genome, we also examined the relationship between recombination rate and test error from windowed locator runs on human data from the hgdp panel (bergströ m et al., ) . recombination rates for each window were estimated by averaging per-base rates from the hapmap project (international hapmap consortium, ) . we first evaluated our method on genotypes from populations simulated by slim v (haller and messer, ) , using the model of continuous space described in battey, . we simulated a  unit square landscape with expected density (d) of individuals per unit area, resulting in census sizes of around , . we varied the mean parent-offspring dispersal distance s across simulations from . to , to create populations with varying levels of isolation by distance. in terms of wright's 'neighborhood size' (wright, ) , defined as n loc ¼ ps d, this yields populations with neighborhood sizes from to . each diploid individual carried two copies of a bp chromosome on which mutations and recombinations occured at a rate of - per bp per generation. simulations were run until all extant individuals shared a single common ancestor within the simulation at all locations on the genome (i.e., the tree sequence had coalesced). individuals were randomly sampled from the final generation of each simulation for use in model fitting. we selected individuals from each simulation as a validation set and ran locator while varying the number of training samples from to and the number of snps from to , . the snps used were a subset sampled from the full genotype matrix without replacement and thus mimic the semi-random distribution of genome-wide snps generated by reduced-representation sequencing approaches like radseq (etter et al., ) . to compare performance with an existing model-based approach, we also ran spasiba (guillot et al., ) on the simulation with s ¼ : using training samples and varying the number of snps from to , . locator was run on a cuda-enabled gpu and spasiba was run on cpu cores. last, we ran a windowed analysis on the s ¼ : (neighborhood size » ) simulation in locator using a mbp window size (each window then contains » snps). we applied locator to three whole-genome resequencing datasets of geographically widespread samples: ( ) mosquitoes from the anopheles gambiae/coluzzii species complex collected across sub-saharan africa (anopheles gambiae genomes consortium et al., ) , ( ) samples of the malaria parasite plasmodium falciparum sequenced from human blood samples collected across papua new guinea, southeast asia, sub-saharan africa, and northern south america (pearson et al., ) and ( ) whole-genome data for human populations from the human genome diversity project (bergströ m et al., ) . genotype calls for the anopheles dataset are available at https://www.malariagen.net/data/ag g-phase -ar , for p. falciparum at https://www. malariagen.net/resource/ , and for human data at ftp://ngs.sanger.ac.uk/production/hgdp. we used vcf files as provided with no further postprocessing. the plasmodium falciparum dataset is unusual relative to our other empirical examples in that sequencing libraries were prepared from blood samples without filtering for coinfections or isolating individual plasmodium. sequence reads returned from short read sequencing then reflect the population of plasmodium present in a human blood sample, or even multiple lineages of parasite if an individual is co-infected with multiple strains (zhu et al., ) , rather than individual plasmodium. the vcfs we analyzed were prepared by aligning illumina short read sequences to the plasmodium falciparum reference genome prepared by the pf k project (pf k consortium, ; https://www. malariagen.net/data/pf k- ), then calling snps in gatk (mckenna et al., ) . variant calls then represent the pool of mutations present in the infecting plasmodium population rather than snps in a single individual. we used only field-collected samples from the 'analysis' set, as described in pearson et al., . for the anopheles dataset, we ran locator in mbp windows across the genome with a randomly selected % of individuals held out as a test set. we also ran spasiba on subsets sampled from the first five million base pairs of chromosome l while varying the number of snps from to , . for the p. falciparum dataset, we used kb windows and held out % of samples from each collection locality as a test set. last, for humans we used mbp windows and selected three individuals from each hgdp population to hold out as a test set. window sizes in each case were chosen to include roughly , - , snps per window. all empirical analyses were run with default settings (  network size, patience , % dropout, a random % of training samples used for validation). we also tested locator's performance with empirical data when the true location is not represented in the training sample. to do this, we ran a series of models on , snps randomly selected from the first mbp of chromosome l in the anopheles data. for each run, we held out all samples from a given sampling locality from the training set, then predicted the locations of these individuals using the trained model. we also tested this approach while holding out all samples collected in a given country, which eliminates even nearby localities from the training set. locator is implemented as a command-line program written in python: www.github.com/kern-lab/ locator. snp calls for the anopheles dataset are available at https://www.malariagen.net/data/ ag g-phase -ar , for p. falciparum at https://www.malariagen.net/resource/ , and for the hgdp at ftp://ngs.sanger.ac.uk/production/hgdp. code to run continuous-space simulations can be found at https://github.com/kern-lab/spaceness/blob/master/slim_recipes/spaceness.slim (battey, ). this publication uses data from the malariagen plasmodium falciparum community project as described in pearson et al., . statistical analyses and many plots were produced in r (r development core team, ). . supplementary file . mean and median prediction error for locator and spasiba run on simulations and anopheles data as shown in figure . error is in terms of map units for simulated data (total landscape width = ). . supplementary file . test error for windowed analyses of empirical datasets using the location with highest kernel density and the centroid of per-window predictions, as median ( % interval). . locator is implemented as a command-line program written in python: www.github.com/kern-lab/ locator. snp calls for the anopheles dataset are available at https://www.malariagen.net/data/ ag g-phase -ar , for p. falciparum at https://www.malariagen.net/resource/ , and for the hgdp at ftp://ngs.sanger.ac.uk/production/hgdp. code to run continuous-space simulations can be found at https://github.com/kern-lab/spaceness/blob/master/slim_recipes/spaceness.slim. this publication uses data from the malariagen plasmodium falciparum community project as described in pearson et al. ( ) . statistical analyses and many plots were produced in r (r core team, ). the following previously published datasets were used: suppose that we know the spatial locations of some relatives of a given individual, and want to predict the location of that focal individual. this is a best-case scenario for our actual problem, as in fact we would have to infer the degrees of relatedness of the reference set to the focal individual, but the calculations are useful in establishing a lower bound on the resolution of inference. suppose furthermore that the displacement in spatial position along each parent-child relationship has mean zero and variance s , so that the net distance traveled along any path along k links in the pedigree has mean zero and variance ks . given the location of n relatives of a focal individual, a simple estimator of that individual's spatial location is simply the average of their locations. how well does this do? we can associate each link between parent p and child c in the pedigree with the displacement between them, x pc ¼ Àx cp ; we have assumed that ½x cp ¼ s for each. suppose that the i th relative can be reached by traversing relatives r i ; . . . ; r iki , and so their location relative to the focal individual is y i ¼ x ri ;ri þ Á Á Á þ x r iðk i À Þ ;rik i . to compute the variance of our estimator, y ¼ p n i¼ y i =n, let n cp be the number of i for which x cp appears in the sum for y i , so that y ¼ p cp n cp x cp =n. then, simply, ½ y ¼ p cp ðn cp =nÞ x cp . for instance, if those relatives are all k ancestors k generations ago (i.e., the great kÀ -grandparents) of the focal individual, then each of the ' links between the ' th and ð' À Þ th generations are traversed by kÀ' of the paths, and so ' kÀ' k s ¼ ð À Àk Þs : clearly, with less full pedigree coverage and more distant relatives, the error would become worse, but it does not depend strongly on the degree of relatedness used: in general, using a few close or many distant relatives should give an estimate of location within some moderate factor of s. tensorflow: large-scale machine learning on heterogeneous systems predicting the landscape of recombination using deep learning sequencing and data production, web application development, project coordination characterization of within-host plasmodium falciparum diversity using next-generation sequence data enhanced localization of genetic samples through linkage-disequilibrium correction a migratory divide in the painted bunting (passerina ciris) space is the place: effects of continuous spatial structure on analysis of population genetic data insights into human genetic variation and population history from diverse genomes novel probabilistic models of spatial genetic ancestry with applications to stratification correction in genome-wide association studies spatial population genetics: it's about time assignment of frost tolerant coast redwood trees of unknown origin to populations within their natural range using nuclear and chloroplast microsatellite genetic markers bagging predictors positive selection in east asians for an edar allele that enhances nf-kappab activation a likelihood-free inference framework for population genetic data using exchangeable neural networks fast and accurate deep network learning by exponential linear units (elus) genomics reveals the origins of ancient specimens bringing together linguistic and genetic evidence to test the bantu expansion a statistical model for reference-free inference of archaic local ancestry snp discovery and genotyping for evolutionary genetics using rad sequencing the unreasonable effectiveness of convolutional neural networks in population genetic inference a scan for genetic determinants of human hair morphology: edar is associated with asian hair thickness manchu grammar accurate continuous geographic assignment from low-to high-density snp data slim : forward genetic simulations beyond the wright-fisher model human population genetics versus the hgdp proceedings of rd international conference on document analysis and recognition windborne long-distance migration of malaria mosquitoes in the sahel the international hapmap project batch normalization: accelerating deep network training by reducing internal covariate shift adegenet: a r package for the multivariate analysis of genetic markers spread of pedigree versus genetic ancestry in spatially distributed populations diplos/hic: an updated approach to classifying selective sweeps adam: a method for stochastic optimization inference of population structure using dense haplotype data the genome analysis toolkit: a mapreduce framework for analyzing next-generation dna sequencing data cggh/scikit-allel multiple populations of artemisinin-resistant plasmodium falciparum in cambodia localizing and classifying adaptive targets with trend filtered regression an open dataset of plasmodium falciparum genome variation in , worldwide samples the pf k project reliable abc model choice via random forests y-chromosomal dna variation in pakistan r: a language and environment for statistical computing fast spatial ancestry via flexible allele frequency surfaces genetic structure of human populations supervised machine learning reveals introgressed loci in the genomes of drosophila simulans and d. sechellia s/hic: robust identification of soft and hard sweeps using machine learning deep learning for population genetic inference dropout: a simple way to prevent neural networks from overfitting syntax helps elmo understand semantics: is syntax still relevant in a deep neural architecture for srl? arxiv localization of adaptive variants in human genomes using averaged one-dependence estimation a new approach for using genome scans to detect recent positive selection in the human genome the anopheles gambiae genomes consortium. . ag g phase ar data release. malariagen. identifier: ag g-phase -ar the numpy array: a structure for efficient numerical computation multiple episodes of interbreeding between neanderthal and modern humans a map of recent positive selection in the human genome assigning african elephant dna to geographic region of origin: applications to the ivory trade localizing recent adaptive evolution in the human genome isolation by distance isolation by distance under diverse systems of mating a model-based approach for analysis of spatial structure in genetic data the genetic legacy of the mongols pf k project. . the origins and relatedness structure of mixed infections vary with local prevalence of p. falciparum malaria spoken sibe: morphology of the inflected parts of speech we thank members of the kern-ralph co-lab, daniel schrider, matthew hahn, and ethan linck for comments and suggestions on this work, and mara lawniczak for the suggestion to look at the plasmodium dataset. comments from reviewers and editors at elife significantly improved the final version of this study. cjb and adk were funded by nih award r gm . plr was funded in part by an i award from the university of oregon. appendix key: cord- - vzwc d authors: nan title: proceedings of scanning /seems charleston, south carolina, usa date: - - journal: scanning doi: . /sca. sha: doc_id: cord_uid: vzwc d nan the widespread application of monte carlo electron trajectory modeling has never been realized in the electron microscopy community because of the computation-intensive nature of the monte carlo algorithm (e.g., many hours of computer time were required to run one simulation which might improve one's ability to interpret data if sufficient statistics were obtained). massively parallel supercomputers with multiple-instruction multiple-data (mimd) architecture are a good platform for monte carlo electron trajectory codes without the complications of vectorization of the computer code required on vector supercomputers (e.g., cray-ymp). the nist monte carlo electron trajectory simulation code has been adapted to run on a massively parallel computer. for the first time, the increased speed of the calculations has made monte carlo calculations a real-time tool for data interpretation. the increased speed achieved by the parallelization of the monte carlo code results in the ability to model small probability events due to the massively parallel monte carlo (mpmc) code's ability to simulate large numbers of electron trajectories quickly. applications include both thin-film and bulk x-ray microanalysis and examples of contrast in backscattered electron images obtained in the sem. this work was supported by the united states department of energy under contract #de-ac - al . in electron probe x-ray microanalysis, "secondary fluorescence" refers to the emission of characteristic x-rays following photoelectric absorption of the primary x-rays generated by the electron beam. while generally a minor contribution to the total x-ray emission, secondary x-ray fluorescence can become an important issue, when the spatial resolution of analysis is of interest, because of the significantly greater range of x-rays than of electrons. thus, for a nickel- % iron alloy excited with a kev electron beam, the range of excitation of the primary radiation will be a hemispheric volume with a diameter of about one micrometer, while the range of secondary fluorescence of iron k-shell radiation by nickel k-shell radiation will be a hemispheric volume with a diameter of approximately micrometers for % of the total secondary radiation. such long-range production of secondary radiation is important when the measurement of trace element distributions near phase boundaries is attempted in situations where an analyte appears at high concentration across the boundary. monte carlo electron trajectory simulation provides a powerful tool for the calculation of the three-dimensional distribution of the primary radiation. to calculate the spatial distribution of the secondary fluorescence due to the absorption of the primary radiation, a second calculation is needed that integrates the x-ray absorption equation in three dimensions. the resulting hybrid calculation can give the total secondary radiation induced for a given structure, such as a planar interface between two materials. the transport phenomenon of incident electrons in solid materials is an important subject in microscopy, microanalysis, and microlithography. it has been treated by either an analytical method, the transport equation, or monte carlo simulation. because of the flexibility of modeling, monte carlo simulation has been developed extensively. various models for monte carlo simulation proposed so far are classified into four groups from the aspect as to how inelastic interactions are treated. the most simple model is the one which uses the inelastic mean free path. this model is applied, for example, to spectroscopic analyses of elastically reflected electrons, auger electrons, and photoelectrons. the most popular model is the single scattering model in which the step length is taking the free path of elastic scattering of electrons, and an energy loss during their traveling is calculated by the continuous slowing-down approximation of bethe, [de/ds] bethe . the most complicated model is the direct simulation model in which all inelastic interactions are taken into consideration as the discrete process. this model requires all differential cross sections for inelastic scattering and, thus, a long computational time. the last one, a compromise model, is the hybrid model which introduces partially the discrete process of inelastic scattering. in the model the continuous energy loss [de/ds] ( ) where [de/ds] dis includes both energy losses due to core electron ionization and valence electron excitation. a variety of the cross sections has been used for inelastic scattering. the fast secondary production model uses the moller equation for the cross section, assuming that all atomic electrons are free (murata et al. ). the authors have published a hybrid model by using the differential inelastic cross sections of vriens for core electron ionization and the moller cross section for free electron excitation (murata et al. ). the mott cross section is used for elastic scattering. typical results for backscattering have shown fairly good agreement with experimental data. the present paper gives a brief survey of monte carlo modeling and discusses the validity of the hybrid model mentioned above. figure shows a typical example of the calculated depth distribution of generated x-rays in an au target in comparison with the experimental result of castaing and descamps ( ) . agreement is good. also shown is the result without the discrete processes of inelastic scattering. an appreciable deviation is seen in the vicinity of the peak. however, the effect of the energy straggling is not so significant. we have also done calculations for other x-rays and have obtained reasonable agreement with experimental data. the effect of the energy straggling will appear more significantly in electron scattering in a thin film. figure shows the energy distribution of transmitted electrons, with a comparison between two monte carlo results and the experimental data of shimizu et al. ( ) . the agreement is not as good; it seems that the disagreement comes from insufficient discrete processes because we still keep the continuous energy loss process. we also propose a new model to improve the discrepancy in figure , caused by energy straggling. the straggling is impor-tant in thin-film analysis, especially at initial energies near the ionization energy for x-ray production. discussions of ways to improve monte carlo simulations have usually concentrated on topics such as the effect of the choice of scattering cross section, or the appropriate model for electron stopping power at low energies. much less attention has been given to considering whether or not we actually have sufficient experimental data to make it possible to demonstrate by a comparison of simulation and experiment that one model, or a portion of that model, actually performs better than another alternative. the earliest experimental data on electron-solid interactions was published years ago (by starke in germany in , and by campbell-swinton in england in ), and numerous workers since then have contributed to the literature on this topic. unfortunately, there never seems to have been any attempt to collect and collate all of this material, and consequently workers seeking data such as the variation of the secondary electron yield with energy for silicon have been forced to conduct a random search of the literature to find what values are available. it is not surprising that such a search typically finishes as soon as a set of data plausibly matching the simulated values is found. in order to try and remedy this deficiency, a systematic search of published data has been carried out to generate material for a rudimentary database, with the hope that this will provide some of the necessary numbers against which monte carlo simulations can be tested. in its current form the database, derived from separate references, is divided into four segments arranged by atomic number (or compound) and comprising secondary electron yields, backscattered electron yields, x-ray ionization cross sections, and electron stopping power. only experimental values are included; thus interpolated, extrapolated, or normalized data sets, or values not specifically indicated by the author to be experimental, have been removed. this restriction unfortunately eliminates most of the voluminous secondary electron data, since such published values have invariably been normalized in order to facilitate fitting to a yield curve equation. no attempt has been made to judge the quality of any of the data since any such assessment would be premature until a sufficient number of independent values are available to permit obviously erroneous, or possibly dubious, results to be safely identified and eliminated. the database is available on request from the author both in printed form, or as a set of cricketgraph ™ files for the apple macintosh. while the quantity and quality of data available for a given element vary widely, they are mostly sparse and scattered. for about half the elements in the periodic table, no experimental values ever appear to have been published, and data are conspicuously absent for even the most common alloys and compounds. even for an element such as silicon ( fig. ) , the scatter in the data is too large to make it possible to be certain to better than a factor of what the se yield is at some energies. thus, unless a major source of quantitative results has been overlooked, it is fair to conclude that the experimental data presently available are not good enough to allow the merits of competing monte carlo models to be judged, or even to permit specific numerical comparisons (e.g., the backscattered yield of carbon, copper, and gold at kev) to be made with any satisfactory level of certainty. on the positive side, however, the database does give some indication of interesting trends (e.g., the variation of secondary yield with atomic number) that have not been easily accessible previously. school of electrical engineering and the national nanofabrication facility, cornell university, ithaca, new york, usa two simulation programs have been developed at cornell, seel [ ] [ ] [ ] (for simulation of electron energy loss) and pyra-mid. seel is a two-dimensional monte carlo program for the simulation of electron trajectories in complex, multi-material nanostructures; the program incorporates energy loss models that include quantum mechanical cross sections for energies down to < ev. the second program, pyramid, , is a very fast electron beam proximity correction program that can perform proximity correction (electron scattering correction) for complex ultra-large-scale integrated circuit patterns with nm minimum feature sizes (mfss). pyramid uses seel to generate the point spread function or radial exposure distribution (red) as the input. seel simulations have been tested against numerous published electron energy loss data and pyramid has been tested on ulsi density ( nm mfs) exposures using . µm pmma. simulation results are compared with experimental data to evaluate pyramid's performance. a more recent application of seel is the evaluation of signal-noise ratio (snr) for time-resolved microscopy of microelectromechanical structures (mems) . for this application, we are interested in estimating the snr for high-speed video recording , of moving high-aspect-ratio mems oscillating > mhz, particularly applications of nanoelectromechanical structures for metrology and time-resolved characterization. iv- scanning vol. , supplement iv ( ) simulation of image formation and detection systems in the sem is a vital link in performing image analysis to obtain precise measurements, to provide the necessary connection between image parameters and structural dimensions, and to reflect important microscope beam and detector parameters. monte carlo methods allow a wide degree of freedom in specifying simulation conditions for sample composition and geometry. published examples often limit the simulation to two-dimensional structures, with a zero-diameter electron beam and all secondary or backscattered electrons (bse) collected. a more useful and realistic approach will take into account the effect of beam diameter, and detector geometry and gain characteristics. these effects for simulated bse images for three-dimensional patterned structures of carbon on a silicon substrate are shown in figure . the monte carlo simulation used here is based on a single-scattering procedure with the rutherford scattering cross section and the bethe energy loss formula modified for low-energy primary electrons. the program is a further development of a single-scattering monte carlo program (in pascal) widely distributed by david joy and modified later by russ et al. the program is written in c language and runs on a sun workstation. the program can scan the beam position in x and y directions over an arbitrary multielement and multilayer sample with topographical features to produce images. using the monte carlo procedure, it is possible to extract information about the position, energy, and direction of the electron at each scattering point. this allows tracking bses as a function of angle. if the dependence of detector gain on electron energy is added, then the details of signal formation can be modeled. the effect of electron beam shape and diameter on the image can be added by convolution after an ideal image is generated. figure shows the matrix representing a gaussian electron beam used in this work. an example of a simulated bse-sem image is shown in figure for a structure with carbon features on a silicon substrate. fig. . contour plot of gaussian shape electron beam with standard deviation of Å was used to convolute the data obtained for a zero-width electron beam. fig. . bse image simulated using a zero-diameter electron beam (left) and after convolution using the gaussian beam width shown in figure (right). the simulation was performed for kev primary electrons with trajectories for each point. the image represents × pixels. monte carlo simulations of electron scattering in a target normally use one of two elastic cross sections, either the screened rutherford cross section or tabulated partial wave expansions of the mott cross section. the screened rutherford cross section gives acceptable results for high energies and low atomic numbers, but mott cross sections are required for low to medium incident energies ( . - kev) and high atomic number targets. however, computations tend to be slow using tabulated data due to the need to interpolate between data points. empirical equations for the total and differential electron/atom elastic scattering cross sections have been found that can be substituted for tabulated mott cross sections in predicting backscattering coefficients. the total elastic mott scattering cross section is fitted by similar form to the screened rutherford cross section but contains three terms in energy in the denominator. the empirical total elastic scattering cross section is valid for atomic numbers up to and for energies from ev to kev: ( ) the fit to the differential mott cross sections is decomposed into two parts, one part being of the same mathematical form as the screened rutherford cross section (σ r ), and the second part being an isotropic distribution (σ i ). the screened rutherford part of the differential scattering cross section is first fitted to the half angle of the mott cross sections. this fit of the differential screened rutherford is in turn reduced to a fit of the screening parameter alone over energy and atomic number. in marked contrast to the screened rutherford cross section, the tabulated mott cross sections show only a small overall downward trend in half angle with increasing atomic number(z). implying an average rutherford screening parameter for all z, with e the electron energy, of: the ratio of the total cross sections (σ r /σ i ) between the screened rutherford part of the differential scattering cross section and the isotropic part of the distribution is fitted to the backscattering coefficients calculated directly from tabulated mott cross sections. the ratio of rutherford to isotropic cross sections is: ( ) figure shows a comparison of the calculated backscattering factors using the present empirical fit (solid lines) with those calculated using mott cross sections. the fit for al, cu, and au is good over the entire energy range. the fit for ag is moderate and the fit for c is high. however, most deviations are similar to differences because of the use of different atomic models in the mott cross sections and are acceptable. there are two major reasons why the simple monotonic eqs. - work well. first, the scattering of the electrons in a solid is a multiple scattering process. thus, many of the complex quantum interference effects are averaged out. second, the elastic backscattering is monotonic with atomic number. these two factors serve to smooth out the effects of the complex multidimensional cross sectional surface that is being fitted over z, e, and θ. reference iv- scanning vol. , supplement iv ( ) σ rutherford σ isotropic = e − z / z + z × e σ t = . × − z . (e + . z . e . + . z / e . ) a scanning interference microscope may be constructed by allowing light which has probed the object and light which has not probed the object (the reference beam) to interfere on a suitable detector. if the detector is large in extent, a conventional scanning interference microscope results, whereas if a point detector is used we have a confocal scanning interference microscope. the image in both cases may be regarded as a superposition of three terms. the first represents a normal conventional or confocal image depending on the kind of interferometer used. the second we will call an interference term image, whereas the third represents a constant background. it is possible to design a system in which the interferometer term image is identical for both conventional and confocal scanning systems in all respects, including optical sectioning. a simple scheme permits the interference term image to be detected separately and then processed in a variety of ways. let us consider two specific geometries. the first is an almost common path confocal system based on a single mode optical fibre. the interferometer in this case is confocal. simple image processing then permits surface profilometry to be performed. the second implementation is a conventional scanning interference system. the interference term image from this conventional image may again be isolated and processed to give a confocal image. in biology, the description and precise representation of microstructural forms is of increasing importance for threedimensional ( -d) computer image understanding, in particular to enhance -d visualization and analysis. the intermediate level of computer vision, located between the bottom layer (signals) and the top layer (model) is best suited as a starting point to improve multidimensional image understanding. we implement the concept of -d topology embedded in the bottom-up structure of data processing to enhance subsequent rendering and specific quantitative analysis. segmented and contoured serial section images require sophisticated algorithms to generate correct surface-rendered views. we have, therefore, developed a method to evaluate connectivities between sections, based on bijective correspondance analysis of the center of gravities of contours. these connections indicate nods and branches of -d structures and can be interactively edited in exploded views of the contour stack. because of the strong data reduction, such procedure can be implemented even in computer graphic systems with entry graphics. the topological skeletons are the backbone to render correctly biological structures of free forms, in particular those featuring frequent branching patterns. examples where such an analysis is successful include microvessel networks, dendritic trees, lung anatomy, or dental root canals (baumann et al. ) . moreover, the automatic labeling of connected structures gives rise to the analysis of topological criteria, which was only available in stereological methods until now. this includes criteria such as connectivity and branching angle, or higher representations of branching schemes. in return, topological connectivities can be used to improve the segmentation of images in the top down-process of data processing, or it can serve as embedded analytical graphics to improve volume renditions. the mammalian central nervous system (cns) contains at least ten times more glial cells than neurons. the three major populations of cns glia are astrocytes, oligodendrocytes, and microglia. astrocytes and oligodendrocytes together are often referred to as macroglial cells and arise from primitive neuroectodermal precursor cells, while microglia are derived from the mesodermal germ layer. it is thought that common precursor cells, known as o- a progenitors, can differentiate into either astrocytes or oligodendrocytes during gliogenesis. in contrast, the origin of microglial cells remains enigmatic, although there is substantial evidence that microglial cells arise from primitive hematopoietic stem cells that gain access to the cns at a very early stage of development. much of our knowledge about glia and glial cell function has been derived from histochemical and electron microscopic studies. astrocytes can be visualized reliably using immunohistochemical methods with antibodies against the glial fibrillary acidic protein (gfap). gfap is an abundant constituent of intermediate filaments which can seen ultrastructurally in most astrocytes. in addition, astrocytes also contain enzymes, such as nadph diaphorase and glucose- -phosphatase, which are readily detected by enzyme histochemical methods. we have focused much of our attention on developing methods for detecting oligodendrocytes and microglial cells in sections of rat brain. lectin histochemistry has been a particularly useful tool in studying these types of cns glial cells. using lectins i and ii from griffonia simplicifolia seeds with carbohydrate specificities against α-d-galacatose and nacetyl-d-galactosamine (glcnac), we were able to demonstrate selective labeling of microglia and oligodendrocytes, respectively. the b -isolectin from griffonia simplicifolia coupled to horseradish peroxidase (gs i-b -hrp) can be used to detect a membrane-bound glycoprotein on the microglial cell surface at all stages of cns development ranging from the early embryonic age to adulthood. in contrast, oligodendrocytes were found to express glcnac-containing glycoproteins in the perinuclear cytoplasm using biotinylated gsl ii. the perinuclear staining was determined ultrastructurally to be associated with golgi complexes. biochemical analyses using tricine/sds-polyacrylamide gel electrophoresis and western blotting with gsl ii showed the glcnac-containing glycoproteins to be insoluble, with molecular masses ranging from to kd. having available specific markers for the three major glial cell groups, we were able to combine lectin histochemistry with immunohistochemistry to perform double-labeling studies demonstrating specificity of each stain for a given glial cell type. following the study of glia in the normal cns, we went on to investigate glial cell reactions that occur as a consequence of nervous system injury or disease. it became immediately ap-parent that microglial cells were the major glial cell type responding to neuron injury. microglia not only proliferate and change their morphology in response to cns damage, but they can also vary their membrane phenotype by expressing new molecules on their surface. we found that antigens of the major histocompatibility complex (mhc), which are largely absent from the normal cns, are expressed de novo on microglia and related perivascular cells responding to neuron injury. our studies, which have included various neuropathologic conditions including stroke and brain tumors, have shown that the expression of mhc antigens, as well as other related immunomolecules, is always restricted to cells of the microglial lineage. these findings strongly suggest a role for microglia as indigenous immunocompetent cells of the cns. jeremiah r. lowney national institute of standards and technology, gaithersburg, maryland, usa a scanning electron microscope (sem) can be used to measure the dimensions of the microlithographic features of integrated circuits. however, without a good model of the electron-beam/specimen interaction, accurate edge location cannot be obtained. a monte carlo code has been developed to model the interaction of an electron beam with lines lithographically produced on a multilayer substrate. the purpose of the code is to enable one to extract the edge position of a line from sem measurements. it is based on prior codes developed at nist but with a new formulation for the atomic scattering cross sections and the inclusion of a method to simulate edge roughness or rounding. the code is currently able to model transmitted and backscattered electrons, and the results from the code have been applied to the analysis of electron transmission through gold lines on a thin silicon substrate, such as used in an x-ray lithographic mask. there is provision for both transmitted and backscattered electron detectors. by comparing the predictions of the code with measured data, it is possible to obtain edge positions to the order of nm, which is needed for the advanced lithography projected for the year . the uncertainty of these measurements is limited by the sample geometry and surface roughness and not by the measurement process. much of the improved code is devoted to the treatment of boundary crossings by the electrons. the present code allows for a substrate of at most three layers and one or two identical lines with a trapezoidal cross section on top. there is also provision for a symmetrical jog (i.e., a discontinuous change in the width of the trapezoid) along the edges to simulate edge roughness and rounding. the three layers that form the substrate, which are typical of an x-ray mask membrane, are silicon, polyamide, and chromium. the lithographically produced lines are gold, and the chromium improves adherence of the lines to the substrate. the code is easily modified to model other media by simply changing the atomic parameters in the input subroutine. the x-ray lithography mask, which has been used as a test sample, is a very good model system for the development of accurate sem standards because it provides a measurement of both transmitted and backscattered electrons for comparison with the predictions of theoretical models. a plateau in the transmitted and backscattered electron signal occurs as the beam traverses the sloping edge of the line trapezoid. this effect can be blurred by edge roughness, and the effects on the plateau of various widths and heights for the jog can be shown as well as the effects of rounding at the bottom of the lines and the calculated noise level for various numbers of trajectories. direct comparisons with measured transmission through an x-ray mask of gold on a silicon membrane can be used to demonstrate the determination of the edge of the gold line. figure shows the transmission (measured downward) along the axis of a gold line indicating the plateaus in the data near the middle of the edges. figure shows a simulation of the data with the plateaus at nearly the same location along the edges as in the data. this work shows how high-resolution metrology of the features produced by advanced lithography can be obtained with an sem. extensions of this code to modeling secondary signals as well as the effects of charging are planned. at philips research, a monte carlo program for electron microscopy is developed, which simulates electron-solid state interactions in the energy range . - kev. it is based on the program published by l. reimer (scanning , , ) . in particular, to ensure proper operation in the low-voltage region, mott cross sections for elastic scattering are calculated by numerically solving the dirac equation. the model for inelastic scattering treats inner shell ionizations as discrete events, described by a scaled gryzinski formula, whereas the effect of valence and/or conduction electrons is incorporated as a continuous bethe loss. this model, which differs from similar models presented in the literature, provides good fits to experimental stopping power data. the program is extended to include multilayer specimens, each layer composed of multiple elements. one of our objectives is the modeling of cd linewidth measurements in a high-resolution low-voltage scanning electron microscope. our monte carlo program can generate top-view backscattered electron (bse) and secondary electron (se) video profiles of lines with variable slope and pitch. we use the se generation model of d.c. joy (j microsc, , , ) , which is extended to account for the regions near the corners of the line profile. furthermore, the recollection of se by the sample is accounted for. as a result, realistic pure se profiles are generated. the primary beam parameters include the probe size and the depth of focus. determination of the top and bottom linewidths via the commonly used heuristic algorithms reveals the systematic errors of these methods. a simple example is the probe size dependence of the peak-to-peak width, which can be related to the strong asymmetry of these peaks. we expect that improved algorithms can be developed, which use a modest database acquired by monte carlo simulation. a scanning electron microscope (sem) fitted with a helium-neon laser interferometer is used to measure the widths of features on photomasks. in this way the magnification of the sem can be known very precisely. algorithms yielding good measurement repeatability, which use the back-scattered electron (bse) signal, have been developed (nunn , nunn and turner ) but in order to be able to relate measurements made on the image to the physical dimensions of the artefact, it has been necessary to model the image formation process. the essential details of the "plural scattering" model used in this work have been described by d.c. joy ( ) . typical geometries and materials of photomasks have been modelled along with a range of accelerating voltages and beam diameters to observe how the image is affected by the different parameters. more important, the modelled image intensity profiles are studied to relate the position of the physical foot of the sloping edges of actual physical lines to the broadened image intensity profile of the edges. although the "single scattering" model, also described by joy ( ) , is more rigorous and more applicable to the type of samples used in this work, it was not used in the first instance because of the much greater computer time required. results from the modelled profiles suggest that the position of the % thresholds of the bse image intensity profiles are located a few tens of nanometres away from the physical position of the edge. the exact offset depends on the angle between the sloping sides and the horizontal. the modelled offset increases from approximately nm for a vertical edge to nm for an edge angled at ˚ to the horizontal. the effect of the bse detector size and geometry on the signal detected has also been modelled. the model shows that for best performance the detectors should collect the largest possible solid angle, but that as long as the detector is placed symmetrically, the only adverse effect of a smaller detector on the image is a loss of signal to noise. measurement comparisons between optical microscopy and the sem corrected by the "plural scattering" modelled offsets have been encouraging (nunn and turner ) ; nevertheless, if time permits, a more rigorous study using more appropriate models should be pursued. audio enthusiasts carefully read the test reports when looking for their new equipment. however, usually no one tries to find out the frequency response, distortion or signal-to-noise ratio of a very expensive, complex system such as a scanning electron microscope (sem). sems were once used mainly as image-gathering devices, and in spite of certain obvious and sometimes serious problems they served as acceptable and effective instruments for many applications. these "image grade" microscopes are now being replaced with much better quality "measurement grade" instruments. the guaranteed resolution is no longer enough to ensure good performance and other factors must be considered as well. even though these newer systems have field emission of lab electron guns, the main advantage is in the built-in, computer controlled, digital image and data-processing capabilities. there are several alternatives to upgrading an existing microscope with an external, usually desktop computer-based measuring system. soon the sem, similar to a scanner or camera, will just be a part of an imaging network, and automatic image enhancement and processing will be commonplace. this presentation deals with a short description of the electrical properties of different components of an sem; the beam scanning circuitry, the video signal chain, sampling, analog to digital (ad) and digital to analog (da) conversion. it will relate their possible influences to the detected and observed signal to improve the imaging. this will also make possible better comparisons of measured data to the computer-modeled data. essentially, the main parts of an sem essentially are the electron source, electromagnetic coils, dc power supplies, (scan) generators, detectors, amplifiers and, finally, the displays. the quality of an sem image depends on the characteristics of the primary electron beam, the correctness of the beam scanning, signal detection, and the fidelity of the video signal chain. the correctness of the beam scanning not only means good linearity and proper amplitude and direction of the deflection of the primary and displaying electron beam, but the displaying or data assigning has to be in correct synchrony also. the deflection coils are fundamentally nonlinear and inherently have hysteresis; hence, correcting circuitry is indispensable. the total distortion caused by improper magnification, nonlinearity, and hysteresis can be greater than % and it is difficult to keep it below % . the fidelity of the video signal chain depends on its transfer function, noise, and distortion figure. the main parts of the video chain of a modern sem are the detector, amplifier, ad converter, computer memory (disk, imaging network), da converter, amplifier, and the display or printer. all of these parts make their contribution and form the overall characteristics of the video chain. to produce good quality images at a certain scan rate, the bandwidth of the video chain must be high enough to show the finest possible details of the image. the transfer function that describes the frequency or bandwidth and phase characteristics of a detector or an amplifier is very useful to characterize the video chain of an sem. for good quality tv frequency imaging, - mhz bandwidth is required, but for slow-speed image collection this bandwidth can be surprisingly low, that is, - khz. noisy signal means loss or lack of information. at a certain noise level, details that otherwise would be in the video signal cannot be seen. the visual effects of noise are closely tied to the resolution, appearance, contrast, brightness, and other aspects of the image, as well as intensity distribution and other properties of the noise itself. a db ( - ), signal-to-noise ratio gives a reasonably clean image, but more noise degrades the ability to differentiate two areas of different brightness by eye. to turn an analog signal to digital data, ad conversion is needed. usually there is a sampling and hold circuitry that keeps the analog signal unchanged for the time of conversion. depending on the design, this results in smaller or larger signal loss because just a fraction of the analog signal is collected due to the relatively short sampling and long conversion time. well-designed circuitry, for example, a gated integrator, can improve the signal-to-noise ratio, but the quantization process in the ad converter itself introduces noise also. modeling data with monte carlo or other techniques has to include the shortcomings of the real, nonideal measuring tool, that is, the sem with all its associated components. by knowing the transfer function, noise, and distortion figure in digital form, it is relatively easy to obtain more accurate comparison of the measured and calculated signal (fig. the calculation of image contrast in the scanning electron microscope (sem) can be done using monte carlo techniques if the electron trajectories can be calculated through the composition profiles in the specimen. this has been done for the case of a discrete one-dimensional composition variation in the incident beam direction , but most of the programs that are available are designed to calculate signal intensities from samples of uniform atomic number and density (e.g., reimer and stelter ) . nevertheless, the calculation of electron trajectories in a three-dimensional heterogeneous microstructure has not been reported. the basic idea behind a monte carlo calculation is that an electron, in penetrating a solid, will undergo a series of predictable elastic collisions (electron-atom) which change the direction and inelastic collision (electron-electron) which change the energy. in figure , which is the example of a calculation in a lead tin eutectic with somewhat unrealistic geometry, the electron enters the sample and is scattered to a new coordinate location. since the directional change and distance scattered are sensitive to the variations of atomic number, atomic weight, and density, the electron might travel to location in the block of the lead-rich position of the pbsn eutectic, scatter into the position , and then go on to position of the tin-rich matrix. thus, depending upon the details of the microstructure, that is, the size and location of the second phase, the image will appear different. the anticipated backscattered intensities from this rather unrealistic microstructure are shown, for example, in figure as the block varies both in size and location below the sample surface. we clearly see that the backscattered signal in this case is much more sensitive to the position in the specimen of the sec- iv- scanning vol. , supplement iv ( ) fig . the geometry of the sample and a schematic of the electron trajectory for the calculation. intensity ond phase rather than the size distribution, but for more realistic microstructural geometries such a calculation would be useful in determining the presence of alternative phases in a complex assemblage. terference microscopy - is based on the observation that a simple single-arm interferometer can be constructed by allowing light from a laser to be back-reflected from a target and to reenter the laser resonant cavity to produce a modulation of the laser light-intensity. [ ] [ ] [ ] the resultant light modulation is dependent on the optical phase (optical path length traveled) and amplitude of the reentry light. if the target is moved over several wavelengths, the laser light intensity displays a cos(z) dependence; for a mirror target, the light modulation index (i-i o /i+i o ) can be as great as . . these observations were denoted as laser-feedback interferometry (lfi); this phenomenon has been characterized and analyzed several times in the past years. [ ] [ ] [ ] [ ] [ ] [ ] [ ] the theory of lfi , , provides an understanding of the harmonic content of lfi signals; they follow a bessel-function dependence, a property that is useful in designing electronic feedback circuits necessary to produce a practical laser-feedback microscope. lfi can employ either diode or gas lasers as the interactive laser source/detector. using a he-ne laser with a high-reflectivity output coupler mirror (≤ %), it is possible to measure surface profiles of highly-reflecting surfaces (e.g., metals or high index-of-refraction materials such as silicon) with axial detail as small as nm and surface vibrations up to - mhz can be measured down to picometer amplitudes. in addition, weakly backscattering materials (e.g., biological cell surfaces and cell components) give sufficient signals to provide a useful method of high-resolution imaging in biology. a versatile laser-feedback microscope (phoebe) has been constructed having the following properties. the he-ne ( . nm) laser incorporated is a milliwatt, linearly-polarized output unit with special mounting to minimize output mirror movement. access to the low-power rear laser beam allows the laser intensity to be measured directly by a silicon photodetector. the main beam is expanded to fill the back aperture of any type of microscope objective (air-, water-, or oil-immersion) suitable for the microscopic examination being undertaken. an x,y scanning stage moves the sample under the laser beam in a raster fashion; pixel × pixel image frames are obtained in < s. axial (z) motion, furnished by a tubular piezoelectric transducer, provides both a modulation signal input for the electronic feedback circuit ( khz) and repositions the sample at each point. the electronic feedback circuit maintains a fixed distance between the output coupler mirror of the laser and a point in the object being imaged. the correction (output) signal of the feedback circuit then gives a measure of the surface topography of the sample. concomitantly, the laser intensity modulation gives a measure of the surface reflectivity of each point on the sample. these two images are digitized and stored in the computer memory. all microscope control functions have been consolidated in a dedicated small computer allowing samples to be run without interruption by the resetting or realignment of the microscope. phoebe scans for biological use range from µm × µm to µm × µm. when an air objective is used, surface topography images display a quantitative measure of height; for fluid-immersion objectives, the measured heights are shortened by a factor dependent on the index-of-refraction of the fluid (water or oil). the confocal property of this scanning microscope is due to the requirement that only backscattered light reentering the cavity-resonator mode (tem oo ) of the laser is effective; the through-focus response of lfm verifies this property in comparison with a confocal pinhole placed at the focus of the laserbeam expanding lens. the lateral resolution of phoebe is nm as determined by imaging silicon-based resolution standards. an important result of measurements with the phoebe lfm is that high-contrast images can be obtained from biological samples placed on a plastic or glass substrate (e.g., a microscope slide or petri dish). in contrast to images of samples with sharply defined index-of-refraction boundaries, what is measured in this case is a point-by-point optical path length difference with the substrate furnishing the majority of the back-reflected, lfi-measured light. other imaging modes include the use of interferometric optical sectioning and the buildup of three-dimensional structures from a series of twodimensional sections. because of the coherence requirement of lfm, the use of fluorescent labels and the detection of fluorescence is precluded; however, other reflective labels may be employed to gain back some of the advantages of that method. - , - ( ) . sarid d, iams d, weissenberger v, bell ls: compact scanningforce microscope using a laser diode. opt lett , lett , - lett , ( university of california, irvine, california, usa nmr microscopy is one of the newly emerging tools for the high resolution three-dimensional ( -d) imaging of live animals and plants for biological as well as medical research. more recent applications and developments include such things as porous materials and microflow, essential for oil research. one of the main interests of nmr microscopy, however, lies in the fact that the method is truly a noninvasive -d high resolution imaging tool with which µm resolution can be achieved. however, nmr imaging, especially nmr microscopy, has a number of formidable difficulties, namely, small signal-tonoise ratio due to the inherently small object size, diffusion and bandwidth limitations, and other inhomogeneity effects such as chemical shifts and susceptibility. among others, diffusion problems due to the random brownian motion of (water) molecules appear to be one of the fundamental physical limitations to nmr imaging, especially in high-resolution microscopy where these molecular diffusion distances are close to the resolution limits. although the diffusion effect in nmr has been studied extensively, the effect on nmr microscopy has not as yet been observed experimentally due to the current limitations of the nmr microscope. theory, however, suggests substantial resolution broadening in nmr microscopy if molecular self-diffusion prevails, especially when we deal with molecules with large diffusion coefficients. resolution limits on nmr microscopy especially include diffusion limits, namely, effects of phase variation due to molecular self-diffusion during data acquisition, effects of signal attenuation and related line broadening, and some diffusion effects of molecules which, when confined in boundaries or walls, exhibit anomalies in resolution observation in nmr microscopy. the first two are based on free-water molecules, while the third is based on the model of water molecules confined by boundaries, which have significant physical consequences. diffusion effects limit resolution in microscopic imaging, as does the phase factor which is related to the finite bandwidth of the imaging instrumentation: that is, the available gradient strength and acquisition time, both of which are intimately related to the diffusion effect. an x-ray microtomographic system is being developed at our laboratories amil & arts. a generalized feldkamp cone-beam reconstruction algorithm was already developed for our system (wang et al. ) . the generalized algorithm is approximate, but quite accurate and computationally efficient. under some feasible conditions, the generalized algorithm produces exact volumetric reconstruction for longitudinal invariant specimens and exact transaxial reconstruction for a point source contained in that transaxial plane. in the generalized feldkamp cone-beam reconstruction, a transaxial slice is reconstructed using projection data collected from a scanning turn of ˚ angular range. in fan-beam reconstruction, there actually are two complete sets of projection data over a full-scan range. exact reconstruction can be achieved using only projection data corresponding to a half-scan. in our cone-beam system configuration, it can be appreciated that there are "approximate redundancies" in data acquired along geometric rays that would be identical after projection onto a transaxial plane. there are various weight functions for half-scan fan-beam reconstruction. with gullberg and zeng's weight function, a half-scan generalized feldkamp cone-beam algorithm is obtained for less longitudinal blurring: ( ) where ( ) ∆ is an additional angle for a smooth transition between essential and duplicated radon regions, and φ(z) is an offset specific to the longitudinal coordinate z. optionally, cone-beam projection values associated with appropriate pairs of opposite rays can be lineraly interpolated to synthesize needed fan-beam projections for transaxial reconstruction. at an additional computational cost, the interpolation-based cone-beam algorithm allows exact reconstruction if the longitudinal specimen variation is linear. numerical simulation results demonstrate the feasibility of our extended algorithms. wang g, lin th, cheng pc, shinozaki dm: a general cone-beam reconstruction algorithm. ieee trans med imag ( ), - ( ) biological specimens can be preserved by rapid freezing, a process which takes just a few milliseconds and is termed "cryofixation." it is an alternative to chemical fixatives which may take many minutes to penetrate a specimen and even longer to fully stabilise cellular components, thereby compromising their ultrastructural and chemical integrity. when cryofixation is performed properly, the water molecules in the specimen do not have time to form ice crystals and the specimen is preserved in a near life-like state. the time-scale of cryofixation means that short-lived phenomena can be captured and preserved. when this aspect is combined with electrical or chemical stimulation in a controlled experiment, then cellular processes can be studied by freezing the experiment at predetermined time intervals, so that a series of transient steps are preserved. besides electrical and chemical stimulation, other methods have been combined with cryofixation; these have involved electrophoresis, electroporation, temperature-jump, and flash photolysis. electrically stimulated muscle was slam-frozen by sjöström et al. ( ) and van harreveld et al. ( ) . their methods held specimens in defined physiologic states: true time-resolved freezing was introduced by heuser et al. ( ) , who showed that ultrastructural differences could be observed in synaptic events at neuromuscular junctions between and ms after stimulation. chemical stimulation is effectively performed by a chemical flow method, involving the rapid mixing of small specimens with a stimulant from different syringes and flowing them for a predetermined time along a reaction tube before spraying the reactants into a coolant, thus quenching the reaction (knoll et al. , rand et al. . electrophoresis involves the movement of particles in an electrical field and was used with freeze fracture to study the diffusion rate of intramembrane particles (sowers and hackenbrock ) . electroporation uses a radio frequency field to induce transient pores in cell membranes (chang and reese ) . optical stimulation has been used to cause a temperature jump to investigate temperature effects, for example, a ˚c jump after ms exposure to a xenon lamp, on the ultrastructure of lipid specimens (chestnut et al. ) . flash photolysis methods have been used to release caged photolabile chemicals in the study of the dynamics of the actomysin cycle (funatsu et al. , ménétret et al. . when the available stimulation methods are considered with the available freezing methods (namely, slam, plunge, jet, and microdroplet-spray freezing), then it becomes clear that there is promising scope for the study of dynamic cellular processes using electron microscopy. the combined methodology integrates the temporal resolution of rapid freezing with the spatial resolution of the electron microscope. chang dc, reese ts: changes in membrane structure induced by electroporation as revealed by rapid-freezing electron microscopy. biophys j , - ( - ( ) universität des saarlandes, homburg-saar, germany contrary to standard preparation at ambient temperature, there is some confusion on what is optimal in cryopreparation of biological specimens for subsequent electron microscopy (em) investigation. this results mainly from the diversity of methods as well as instruments described and the rapid development of these techniques in the preceding years. since the big advantages of cryopreparation in various fields are often hidden by the choice of unsuitable or even antiquated techniques, it seems to be justified to report about the recent developments in this field and to classify different methods for different purposes. this tutorial is based mainly on experience in transmission electron microscopy (tem). nevertheless, most of the information may also be useful for scanning electron microscopy, since a proper preparation has the same importance in both fields. without doubt, cf is the most important first step in most cryopreparations with a big variety of different techniques. the simplest procedure (plunging or immersion cryofixation = icf) is best suited for vitrification of thin suspension layers ("bare grid" or "ice embedding") for subsequent cryo-tem in the frozen hydrated state. high pressure freezing (hpf) reduces ice segregation in suitable specimens (e.g., thin plant leaves or rigid tissues such as cartilage). the main problem of hpf in soft tissues results from the indispensable dividing of the specimens into small pieces. impact freezing on a metal mirror (mmf) is well suited for suspensions such as hpf or double jet (djf), but mmf has an advantage for soft tissues, since it is suitable for larger specimens, that is, tissue slices > mm . as far as liquid cryogens are concerned (icf, djf), ln or partially frozen n -slush are not to be recommended; ethane gives the best results. propane is well suited and less expensive for routine applications. as an alternative to cryosectioning, these more time-consuming preparations gain continuously in importance for cytochemistry. even element analysis is possible in certain cases. only for simple morphology or morphometry additives (e.g., oso , uo -acetate, aldehydes) are short drying times in freeze substitution (fs) or freeze drying (fd) possible. proper drying by fs or fd without additives seems only possible for small objects (diameter < . mm) in periods between - days at − ˚ to − ˚c. otherwise, severe artifacts by thermal collapse phenomena and redistribution effects result. a suitable instrumentation is of great importance for this long drying time. the results are excellent and the advantages are striking as long as sufficiently long drying times are employed. in addition, low-temperature embedding (lte) in special acrylics improves the results. care has to be taken in handling these monomeric resins, since some of them are strong allergens. sugar protected specimens are easy to section on the dry knife at − to − (− )˚c for subsequent histochemistry of macromolecular components. overall morphologic preservation is rather poor in comparison with fs/fd/lte. larger areas are mostly not obtainable, but speedy work and results within hours instead of days or weeks are possible. sectioning can be considerably improved by the use of cryo-diamond knives together with an ionizer (useless without antistatic tool). sectioning below − ˚c down to − ˚c is possible with cryo-diamond knives on an ionizer, if the specimen is well frozen (no ice segregation) and the cutting area << . × . mm . trimming with a diamond trimming tool is advantageous. good preconditions are given after hpf or mmf. the advantage of mmf is the mirror-like, well-frozen surface, which can be easily trimmed and orientated to the knife edge. cryotransfer to the tem/stem or proper fd for fedx pose no severe problems (fd for > h at − ˚c). most of the different actual cryomethods (except fresh frozen cryosectioning < − ˚c for cryo-tem/stem) allow routine work, if the most suitable method is carefully selected and modern instrumentation is available. in all cases, the additional effort and investigation are definitely justified by better and more reliable results (literature and reprints on request). paul walther, renÉ hermann, martin mÜller laboratory of em , department of cell biology, eth zurich, zurich, switzerland the reason for using low temperatures for preparation and microscopy is the decreased mobility of atoms and molecules reducing the danger of artefact formation. for scanning electron microscopy (sem) cryotechniques have the following advantages. . cryofixation (rapid freezing) is the fastest way to immobilize a biological sample at a defined physiologic state. thereby, all processes in a cell are arrested within milliseconds. chemical fixation, in contrast, takes seconds or minutes to act, leading to unpredictable osmotic effects and redistribution of cellular compounds. . a frozen biological sample behaves like any other bulk specimen, and redistribution of substances is almost excluded. inner structures can be made amenable to the electron beam by cryofracturing or cryosectioning. . the conductive metal layers have a finer grain size when applied to cold samples, because diffusion of the metal atoms on the surface is reduced. . the frozen sample is analyzed in the sem by use of a cold stage. this prevents volume changes due to drying artifacts. in addition, hydrocarbon contamination due to irradiation by the electron beam is greatly reduced at cold temperatures. for most applications in biology, cryotechniques are only used for some but not for all preparation steps; for example, samples are cryofixed and then dehydrated by either freezedrying or freeze-substitution and afterward stored and imaged at ambient temperature. for high resolution sem, it is advantageous to coat also dry samples at cold temperatures in order to obtain a finer grain size and to observe the samples at cold temperatures in the sem to reduce hydrocarbon contamination. ( ) a major limitation of cryotechniques is distortion of the ultrastructure due to ice crystal formation during freezing. high pressure freezing allows for direct cryofixation of living samples within minimal or no ice crystal artefacts up to a thickness of several µm. ( ) the problems of water vapour contaminants that condense on the sample during preparation and microscopy were well investigated for the tem freeze-etching technique in the s and s. during the last years, this knowledge has been adapted for the construction of cryo preparation systems for the low-temperature sem. ( ) drying artefacts such as shrinkage are omitted by observing fully hydrated frozen samples. on the other hand, the ice covers many structures of interest and therefore often needs to be partially removed, either by freeze-drying or by freezesubstitution. however, removal of water bears the risk of drying artefacts. ( ) hydrated samples are extremely sensitive to the electron beam. the high surface-to-volume ratio inherent in particulate samples, and the fact that we already live in a sea of possibly contaminating particles, necessitates special care and understanding in particle preparation. selection of reagents and handling practices can be critical. for instance, in a hypothetical case, a particle analyst reports that his sample consists of approximately five major inorganic phases. the first has a rounded morphology with crystalline overgrowths, the light element content of the second is depleted, the crystalline structure of the next is damaged, the fourth occurs at a % volume level, and the fifth consists of glass shards. in this example, these results are essentially worthless. phase one was soluble in a liquid used in the preparation process and then reprecipitated upon evaporation. the light elements of the second were leached because of the use of the same solvent. the third was attacked by hf formation during ultrasonification in a fluorinated solvent. the fourth was originally present at a % volume level, but was preferentially attracted to the sides of the storage container prior to sample preparation, biasing the sampling; and the fifth is not part of the original sample, but comes from the ground glass neck of one of the reagent bottles. decisions on reagents and methods are crucial but unfortunately cannot satisfy every concern. for instance, reagents chosen for inorganic preparation should be nonpolar when easily leached elements such as boron or lithium are of interest, or if surface oxidation or ionic dissolution is a concern. but nonpolar solvents can increase particle agglomeration problems due to the lack of charge dissipation. therefore, wise choices in preparation methods are strongly tied to the objectives of the analyst, and sometimes multiple methods performed in parallel with additional analyses may be required to obtain truly representative results. contamination often is a concern, especially when performing high resolution work on a trace particle constituent. suspended urban air particulate is typically - µg/m , mostly in the . - . µm range and usually with less than particles per cubic centimeter above µm. supermicrometer-sized par-ticles can constitute µg/m . these particles may settle out gravitationally, electrostatically, or may simply happen to intercept the surface of the sample mount or sample particles. a large background can accumulate on unprotected substrates. the use of easily charged containers such as polystyrene disposable petri dishes or polyethylene centrifugation cones can further complicate dust collection or an additional problem, sample losses, due to electrostatic forces. the use of light microscopes for particle preparation makes possible a variety of preparation methods including micromanipulation. uses include ensuring even particle distributions on mounts, precise particle positioning, particle size reduction, washing unwanted films or residues off of particles, and many more. a light microscope can also be used to make some helpful observations such as specific gravity, population densities, refractive index, morphology, size, solubility, film thickness, and color. "micro world" effects often have to be provided for when performing preparation on a microscale with a microscope. for example, the polarity of solvents sometimes must be sacrificed for lower evaporation rates. reduction in evaporation rate can be aided by the use of small transparent covers, air movement shields, or by the choice of a combination of substrate and solvent that have poor affinity for one another (the resulting bead-ing-up action decreases the surface-to-volume ratio). the insertion of a "reservoir object" such as a probe tip at °"holding" a liquid droplet against the substrate, or a coverslip from the edge of which a supply of underlying liquid can be obtained, are examples of creative procedures that may be used (fig. ) .recommended polar solvents include heptane and cyclohexane. these avoid hfc environmental concerns, evaporate readily, and avoid halogen interactions. isoamyl acetate, flexible collodion, and formvar in ethylene dichloride are preferred micromanipulation and mounting media. microscale fume "hoods" protect microscope optical coatings and personnel. for containers, glass or static dissipator-coated plastics are preferred to ordinary uncoated plastics. if clean rooms are not available, static dissipators (physical or chemical), laminar flow work benches, single hepa-filter curtained areas, and modified work practices are inexpensive solutions. syringe filters help provide easy to handle point-of-use contamination control for reagents. recommended micromanipulation probes include tungsten and specific animal hairs. intermediate substrate surfaces may employ temporary teflon or paraffin coatings. other procedures include ashing, centrifugation, filtration, ultrasonification, and the exploitation of effects observed during micromanipulation. an approach for the indirect visualization of biological material in transmission electron microscopy (tem) is the freezeetch or freeze-fracture/replica method. the preparations steps ( fig. ) of this purely physical method include: (a) fast fixation and stabilization by quick freezing (in ms vs. min necessary for conventional chemical fixation); (b) creation of clean fracture faces with a fracturing cryotome (high vacuum required); (c) replication with electron beam evaporation (ebe, high vacuum required); (d) three-dimensional imaging of fracture faces and surfaces (with structural resolution of - nm); (e) high stability of the pt-c replicas in the electron beam (selection of relevant details) and possible long-term storage (weeks to months). the important drawbacks are that the replicas have to be cleaned (subsequent changing of several cleaning solutions over hours or days), picked up (disintegration of highly structured replicas happens frequently), and can be viewed only as a result of one single fracturing event. specimens providing iv- scanning vol. , supplement iv ( ) fig highly redundant structures and relatively smooth fractures, such as cell suspensions or o/w emulsions, were investigated using freeze fracture/replication and ambient temperature transmission electron microscopy (at-tem). freeze-drying is comparable to freeze-etching ( fig. ) , but with the important difference that all the water is removed from the specimen by sublimation. in tem, scanning electron microscopy (sem), or scanning tunnelling microscopy (stm), small cell components or particles, such as viruses, can be imaged together with the replication layer on top. in freeze-fracturing for low-temperature sem (lt-sem), most advantages of tem cryopreparation are kept. the preparation includes: (a) quick freezing; (b) fracturing under high vacuum conditions (c) coating with a planar magnetron sputter source (pms, ar as working gas); (d) -d imaging and observation of changed conditions (in situ etching); (e) high signal/noise ratio of pt or w coatings for secondary electron imaging and low yield from cr for backscattered electron detection of immuno-gold labelled specimens. the specimens can be imaged directly eventually after multiple fracturing (i.e., searching of distinct structures) in a different situation (i.e., fully hydrated or partly freeze-dried, coated or uncoated), in a "close to nature state." however, new problems have to be solved in order to get thin reproducible, conductive coatings, without superimposition of specimen and coating film structures and direct imaging, reducing contamination and beam damage, or long-term low temperature storage. moreover, the coating film thickness, the ice in the frozen bulk specimen, and, most important of all, the type of sem are now limiting the structural resolution. the biological relevant details of results obtained with low-temperature sem in a conventional fieldemission sem can be compared with those achieved with routine freeze fracture/replication and tem. according to honig and hook, the physics of water in high vacuum play an ultimate role in the application of these techniques (fig. ) ; therefore, only this knowledge in combination with cold stages and high vacuum technology enable the application of state of the art preparations of biological material for electron microscopy. the cryo-jet freezing technique provides a means for the in situ preservation of diffusible ions in mouse spinal cord explants after trauma. using the technique, the effects of trauma, in particular the intracellular shifts of calcium and other diffusible ions, can be analyzed and recorded by qualitative and quantitative electron probe microanalysis (epma). chemical fixation must be avoided if one intends to attempt the epma localization of diffusible substances in cells and tissues. the goal of the cryo-jet method is to confine axonal components and chemical elements to a biologically natural position. trau- matized mouse embryo cord explants were compared with the intact rat spinal cord trauma model, using epma studies of perturbations of calcium and other ions that are reported to be located in the axonal cytoskeleton. icr mouse embryo spinal cord explants were grown to - -day maturation in maximow chambers. cords that survived stringent elimination examination criteria and exhibited minimal degeneration or spontaneous necrosis were randomly divided into four groups. group i served as experimental controls; group ii was traumatized by dropping a mg weight through a . cm glass pipette positioned directly over the culture and cryojetted h after trauma. representative explants from i and ii were freeze-substituted and embedded in epoxy for comparison. by light microscopic examination of the whole mount, living culture, the site of impact could be determined within - min. the impact zone became markedly dense when compared with the surrounding, uninjured tissue. toluidine blue of the freeze-substituted sections revealed the site of impact on the surface and path of the trauma damage through the explant. control cultures with spontaneous necrosis could be distinguished from the experimental group, because the traumatic necrosis was confluent and often even wedge-shaped. within the traumatized zone, nerve fiber alterations similar to those observed in in vivo spinal cord trauma and calcium toxicity could be found, that is, granular degeneration of axoplasm, pleomorphic spheroids, tubulovesicular profiles, and myelin showing adaxonal, periaxonal, and intramyelinic vacuolization. light microscopy sections from the freeze-substituted explants were noted to be well preserved at the surface and into the culture for - µm (fig. ) ; however, some ice crystal damage was noted deep within the body of the culture. previous studies that compared metal mirror and plunge freezing results with the propane jet methodology reported here confirmed that propane jetting is the method of choice for preserving organotypic spinal cord tissue cultures. x-ray microanalysis of cryosections of the cytoskeletal tubules, cross bridge structures, mitochondria, and the myelin sheath of in vitro spinal cord explants, compared with the in vivo model of spinal cord trauma, has yielded unique data concerning the ionic changes that occur as a result of trauma or experimental manipulation. after trauma, populations of axons were found that could be distinguished by their elemental compositions. one group was similar in elemental composition to controls, although the morphology was dissimilar, while a second group had - % lower k and - -fold elevated ca (fig. ) . the response of axons and the associated myelin was paired, that is, loss of k and gain of na within the axon was always accompanied by comparable changes in the myelin and vice versa. the morphologic appearance of axons was not a predictor of the elemental composition. the cardiomyopathic (cm) hamster manifests cardiac dysfunction from an early age, with the disease ultimately progressing to congestive heart failure. on the basis of significant elevation in the ca + content of the cm hearts, as well as significantly increased ca + in the mitochondrial (mt) fractions and an increased density of voltage-sensitive ca + channels, previous studies suggested a ca + overload in the cm hearts (sen et al. ). evidence has also been presented for an increased sensitivity of cardiac muscle cells of cm hamster hearts to an external ca + "stress," that is, any manipulation which increases ca + influx into the cells (hano and lakatta , sen et al. ). on the other hand, ultrastructural studies have shown localized focal lesions from an early age, believed to be due to local cell injury. the cardiac myocytes within these areas suffer irreversible damage, becoming ca + overloaded as demonstrated by measurements of mt and a-band (ab) ca + content by electron probe microanalysis (epma) in a previous study from our laboratory (bond et al. ). this study also demonstrated that the vast majority of cardiac myocytes throughout the cm hearts not only had a normal ultrastructural appearance, but also showed no elevation of subcellular ca + content. an alternative explanation for impaired contractile function may be a decreased amount or availability of ca + stored in the sarcoplasmic reticulum (sr) for stimulated release and activation of contraction. to investigate this question, we have utilized epma to measure ca + content directly in the junctional sr (jsr), as well as in mt and ab of the cm hamster heart, either under control conditions or, alternatively, after pretreatment with the ca + channel agonist, bay k , in order to increase ca + influx into the cardiac muscle cells. isolated papillary muscles from normal and cm hearts at days of age were stimulated to contract electrically, and parameters of isometric contraction were recorded at l max . muscles were randomly assigned to one of three protocols: ( ) eleven cm and normal papillary muscles were used to construct dose/response curves to the ca + channel agonist, bay k. after stabilization at l max , cumulative doses of bay k ( - m to - m) were added to the muscle bath. all contractile parameters were recorded from the muscle at each dose. ( ) for epma, five cm muscles were frozen, after stabilization at l max , at peak +dt/dt and five muscles during relaxation. ( ) ten cm papillary muscles were incubated with single dose of - m bay k in order to elicit a maximal inotropic effect. once the contractile response to drug addition had stabilized, the muscles were frozen either at peak +dt/dt (n= ) or during relaxation (n= ). ultrathin cryosections were cut from the surface of the frozen muscles and freeze-dried overnight. subcellular ca + content (ab, mt, and jsr) of cm muscles was measured by epma in stem mode in a philips cm scanning transmission electron microscope (fig. ) . measurements of baseline contractile function revealed a significant decrease in develop tension (dt) (from . ± . g in cm muscles to . ± . g in normals), +dt/dt ( . ± . g/s to . ± . g/s) and a decrease in −dt/dt ( . ± . g/s to . ± . g/s) in cm versus normal hamster. there was no significant difference in values of resting tension (rt). the inotropic response to increasing doses of bay k was markedly blunted in the cm muscles compared with controls, suggesting that even when ca + entry into the cardiac muscle cells is increased, force development is still impaired. a comparison of elemental content (na, mg, p, s, cl, k, and ca) of ab and mt between experimental groups revealed no statistically significant differences. in addition, no differences in elemental composition of ab and mt were observed compared with our previous measurement on normal hamsters frozen during contraction and relaxation (moravec and bond ) . the amount of ca + stored in the sr of the cm muscles that were rapidly frozen during relaxation (in absence of bay k) was . ± . mmol/kg dry weight ( fig. ) (left panel, rel); however treatment with the ca + channel agonist bay k significantly increased the size of the store to . ± . mmol/kg dry weight (right panel, rel). thus we conclude that ca + uptake into the sr of cm muscles was enhanced as a result of bay k treatment. the contractile data show that the amount of ca + that can be released from the jsr of cm muscles during a cardiac twitch is very small (equivalent to the difference between the relaxed and contracted values) in "control" (untreated cm hamsters, left panel); however, treatment with bay k increases the sr ca + load in the relaxed muscle, with little demonstrable effect on the amount of ca + remaining in the sr at peak +dt/dt (right panel, cont), resulting in a significant increase in the amount of releasable ca + in the sr store. the total ca + content measured in the relaxed bay k treated muscles was, nevertheless, considerably less than previously measured in normal papillary muscles rapidly frozen during relaxation. in summary, these data suggest that ( ) a ca + deficit, as opposed to a ca + excess or ca + overload, may be an important factor contributing to the cardiac dysfunction in cm hamsters, and ( ) that, specifically, impaired ca + regulation by the sr may result in this ca + deficit. the application of automated scanning electron microscopy (sem) to the analysis of particulate populations is in many ways a unique application of microanalytical instrumentation. when applied to the analysis of large numbers of particles, the sem is used as both a microanalytical and a macroanalytical instrument. as a microanalytical instrument, the sem provides single-particle compositional and morphologic information that is not available from the conventional macroanalytical techniques used in particle analysis such as atomic absorption and instrumental neutron activation analysis. as a macroanalytical instrument, the sem, when used for automated particle analysis, provides information from which population characteristics can be inferred. for example, automated sem information often is used to determine the percent of an aerosol that originates from a given source by extrapolating the single-particle results (e.g., the number of particles containing major fe) to the entire particle population, that is, the air filter. this value is then extrapolated to the sampled air volume. while providing the analyst with a wealth of information, the dual role of the sem in automated particle analysis has several limitations that must be considered when doing an analysis. these limitations involve, among other things, the spatial dispersion of the particle sample; particle size distribution; analytical parameters such as magnification, accelerating voltage and electron dose; and the algorithms for determining particle composition and particle groupings. at nist we have been conducting a series of experiments to study the limitations associated with the quantitative elemental analysis of particles during an automated run. specifically, we have been evaluating the relationship of measured x-ray intensity to the accuracy, precision, and detection limits of automated x-ray analyses. analytical accuracy, precision, and detection will have a pronounced effect on the ability to separate particles with similar but different elemental compositions into groups. for this experiment, we developed an analytical glass series containing six glasses with varying amounts of uranium and lead ( table i) . samples of the different glasses were prepared as bulk-polished specimens and as particles. the results from the analyses of the bulk samples represented the "best case" that could be expected under a given set of analytical conditions since there were no particle effects involved. all analyses were done on an electron probe and a sem at kev and na beam current. dead times for the bulk and particle analyses were between and %. two separate counting times were selected for the experiment , s and s. these times resulted in the x-ray peak intensities for pb and u m xrays that are shown in table ii . glass k- was used as the standard for the quantitative analyses of the different runs. for the particles, both bulk and particle forms of k- were used as standards. the concentration of oxygen was determined by stiochiometry. the results of the experimental runs on the bulk and particle forms of the glasses are shown in figure a and b as plots of the u versus pb concentrations in wt.% (normalized to % total analysis) for the s data. the bulk plot shows a complete separation of the six different glasses, while the particle results show an overlap of adjacent glasses even at the s count time. the s data show a much stronger overlap between adjacent glasses for both bulk and particle morphologies. the analytical data were also processed with a clusteranalysis algorithm to determine the average silhouette width (asw) for both bulk and particle forms of the glasses at the different counting times, (fig. ) (kaufman and rousseeuw ). the asw is a robust measure of the cluster strength with a value approaching representing the maximum association among cluster members. since there are six glasses in the series, the asw for six clusters should be the highest. of all the different runs on both bulk glasses and particles, only the s data on the bulk glasses has the highest asw for six clusters. the results of this study indicate that the uncertainties associated with the shorter counting times in asem analysis may severely limit the ability to distinguish correctly between similar groups of materials. in addition, the uncertainties associated with particle analyses are considerably greater than those from bulk analyses due to absorption and mass effects. these greater uncertainties for particles underscore the need to define the strengths and limitations of asem analysis and to design automated methods that will maximize the information that can be obtained from a sample. botany department, university of georgia, athens, georgia, usa the monoblepharidales (chytridiomycetes) produce asexual motile reproductive structures known as zoospores through the cleavage of cytoplasm in the zoosporangium. although different aspects of zoosporogenesis have been studied in a number of zoosporic fungi, little is known about the events of zoospore formation in the sporangium of the monoblepharidales, or the chytridiomycetes in general. earlier studies of other zoosporic fungi proposed various spore formation events that have been challenged recently (hyde et al. ) , especially with respect to the relationship of vesicles, golgi, and cleavage furrows. hyde et al. ( ) concluded that all eukaryotic cleavage events may need reinvestigation. we are studying the various aspects of zoosporogenesis in monoblepharella using both chemical fixation and cryofixation methods to determine whether the cleavage events in this chytridiomycete are similar to those found in the study by hyde et al. ( ) . questions that remain include how the nuclei accumulate in the sporangium, how the cytoplasmic domains are established, and how each zoospore obtains its usual complement of cellular components. mycelia were induced to form sporangia by transferring the cultures to distilled water. sporangia were chemically fixed at different stages of development using a sequential aldehyde/ osmium fixation protocol. the resulting tissue was embedded in araldite/embed . freeze substitution fixation was also used for comparison of vesicle and cleavage furrow profiles. when the tissue was to be used for cytochemical localizations, osmium was omitted and lr white was used as the embedding medium for both fixation protocols. various dyes, lectins, and antibodies were used to localize organelles within the sporangium. nuclei were close to the plasma membrane as they moved from the subtending hyphal strand to the swelling tip of the developing sporangium. the centrioles were closely associated with the nuclei and oriented toward the periphery of the sporangium. nuclei then moved a short distance from the centriole toward the center of the sporangium. microtubules, originating from the centriolar region ( fig. ), were seen around the nucleus and extended into the cytoplasm, possibly delimiting the boundary of the forming zoospore. membranes forming the cleavage furrows appeared around the forming flagella near the kinetosome (fig. ) and also at specific sites along the periphery of the sporangium. the furrows continue to expand at both sites as extending sheets of membrane. dictyosomes with large numbers of vesicles were closely associated with the nuclei, suggesting a source of the membrane needed for furrow extension. er was first seen in large sheets in the central region of the sporangium. later the strands of er surrounded the nuclei prior to ribosomal aggregation. iv- scanning vol. , supplement iv ( ) fig. nucleus (n) with associated centriole (arrow) and microtubules (arrowheads) at periphery of sporangium. fig. developing cleavage furrow and flagellum (f). note coated region of furrow (arrowhead) and radiating microtubules (arrows). hyde gj, lancelle s, hepler pk, hardham ar: freeze substitution reveals a new model for sporangial cleavage in phytophthora, a result with implications for cytokinesis in other eukaryotes. j cell sci , ( ) center of ultrastructural research, barrow hall, university of georgia, athens, georgia, usa cryptocaryon irritans, a parasitic ciliate of many species of seawater fishes, has a complex life cycle consisting of feeding, resting, dividing, and infective stages. the disease, termed white spot disease, can cause extreme fish loss in marine aquaria and mariculture environments. cryptocaryon irritans attaches to the epidermis of the fish and feeds on epidermal cells. this form of the ciliate termed the "trophont" appears as large white spots on the fish. the trophonts grow in size and eventually leave the host. the free-living trophont settles down to the substrate and secretes a cyst wall. while in this resting stage known as the "tomont," the cell undergoes a series of unequal palintomic divisions to form daughter cells called "tomites." the cyst wall ruptures and free swimming ciliates known as "theronts" are released. the theronts represent the infective stage as they search for new hosts. different procedures were used to prepare tomite, theront, tomont, trophont, and cyst stages. organisms were fixed with glutaraldehyde and osmium tetroxide for transmission electron microscopy (tem). parduc's fixation (oso +saturated hgcl) was applied for the scanning electron microscopic (sem) work. the parasite's cytoskelatal framework was stained by using indirect immunofluorescence technique. for this purpose monoclonal anti-α-tubulin was used as a primary antibody, and rhodamine-labeled goat antimouse igg as a secondary antibody. fluorescently labeled cells were examined by laser scanning confocal microscopy. the trophont has an elongate body shape with a broad anterior end, a tapered posterior end, and is completely covered with somatic cilia. the cytostome is apically located and trophonts often were observed moving with their mouth part leading. kineties were arranged in a parallel fashion along the longitudinal axis of the cell and terminated in a ring around the cytostome. there is no oral membrane at the oral region. there are cirri-like structures around the oral opening as described by cheung et al. ( ) . these cilia are shorter ( - µm) and wider than somatic cilia. the cytopharynx is surrounded by ridges or oral ribs (nonciliated lining). large bundles of microtubules support the oral region. the theronts are oval to teardrop-shaped and completely covered with cilia. they have a ventral mouth with a slit-type structure in the middle. the cytostome covers almost / - / of the body. the cirri-like structures are found around the mouth and are similar to those in trophonts. the oral ribs are present but they are layered. based on sem and tem results, it was found that there were some structural differences in the cytostomes of trophonts and theronts. the mouth probably does not become functional until the theront has entered a host. the short and stiff cilia seem to be used for burrowing and the gathering of food particles. the role of the pellicle and cyto-plasm is discussed in relation to penetration into fish epidermis. we found that there are mucocysts in both trophont and theront, but not in tomont. theront mucocysts are concentrated around the slit type structure of the mouth. the secretion of mucocysts might contain enzymes that help the theront to penetrate into fish epidermis. they also might aid trophonts in feeding on fish tissue and have a function in cyst wall formation at the later stage. penetration into the fish tissue probably is started by mucocyst secretion that either enables the parasite to stick to the tissues or to help it enter the tissue by enzymatic reaction over the irritated areas. the theront may then utilize its relatively stiff oral cilia for burrowing into the these irritated areas. after burrowing into the epidermis of fish, the theront develops its oral apparatus and increases its size. trophonts eventually leave the host when they reach a certain size ( - µm). this study is supported in part by the national aquarium, baltimore, md. over the years that the fbi has practiced sem/edxa, the technology has grown in importance to be considered an essential tool for investigative forensic exams. because of the wide variety of applications, there is a corresponding vast array of preparation methods and analytical techniques. analytic techniques and sample preparation methods are inextricably linked, and although many are routinely applied, often only imaginative approaches serve to fulfill the desired analytic result. the scanning electron microscope (sem) practitioner is expected to be knowledgeable about the applications and proficient at the methods of preparation in order to utilize the sem to its greatest advantage. the main types of analysis practiced at the fbi include ( ) visualization of structure, including surface features and internal structure, ( ) inorganic elemental characterization, ( ) particle analysis, and most recently ( ) the use of a compositional data base for identification and association. sem is a powerful complement to light microscopy (lm) for low magnification morphology characterization and is unsurpassed for applications requiring greater depth of field than are available with lm. toolmark and fracture exams can be enhanced by stereo analysis. preparation can be minimal, and several electronic signals (bei, sei) are available. the preparation of cross sections permits the study and comparison of heterogeneous materials. embedment usually is necessary to support the object during cross sectioning. hard materials generally are polished by an adaption of metallurgical polishing methods, and soft materials generally are microtomed. sectioning often is possible by manual methods, without the use of a microtome. this method can reveal complex structures such as plating layers and document laminates. the most routinely applied application of edxa is the qualitative exam. it is part of the inorganic analysis scheme for material characterization. the qualitative exam frequently is combined with elemental distribution mapping to provide "compositional pictures." particle populations often are indicative of an environment and can be used to associate an item or individual with an activ-ity. too small for individual manipulation, they are most easily sampled by adhesive lift. additional methods involving separation and concentration often are effective. compositional characteristics of materials are stored in a database to permit comparison and identification of a questioned material. standard spectra are collected and a specific peak for each element is integrated above background and ratioed to the sum peaks from all elements. this value representing % x-ray counts is stored in a "periodic table" data base including standard information. in addition, the original spectrum is stored on disk and a hard copy is filed. the standard files include metal alloys, building materials, paints, tape adhesives, fingerprint powders, and cosmetics. the reference list can be queried for comparison to an unknown for alloy matching, identification of an unknown material, or manufacturer identification. the effectiveness of this method depends upon the compositional uniqueness of the material and the variation of composition within the class of materials to which it belongs. this project is in its infancy, with only several hundred entries to date. data entry currently is manual, although software currently is being developed to extract required data from spectra automatically and to export it to the database. the need for a vehicle for information exchange has been expressed within the community of sem users in crime labs. since an electronic medium such as internet was not feasible because most forensic laboratories are not electronically linked, a "newsletter" was produced to link laboratories involved with sem in forensic science and related areas, as well as individuals in industry and academia. timely and informal, it augments the professional publications and attempts to bring practical methods, reprints from obscure journals, translations from foreign publications, and questions/answers directly to the user. hamilton county coroner's laboratory, cincinnati, ohio, usa many examinations in the crime lab involve comparing questioned material from a suspect to known material from a victim. by characterizing the material it may be possible to establish a link between the suspect and victim. our trace evidence section characterizes materials by using a combination of analytical instruments. the infrared microspectrometer is used to determine the organic constituents, and the scanning electron microscope-energy dispersive x-ray spectrometer (sem/edxa) is used to analyze the inorganic composition. this approach is routinely applied to paint particles because paint formulations include both organic and inorganic constituents. analysis by sem/edxa is very valuable when more than one layer is present in the particle. the instrument can then be used in line scan mode to analyze each layer individually without separation. in addition to comparisons, the sem/edxa is used to identify materials. in bombing cases it may be difficult to identify the explosive as well as other components. large amounts of potassium and sulfur in residues from a pipe bomb indicate black powder as an explosive. if chlorine is also present then "pyrodex," a black powder substitute, may have been used. other applications, such as matching small fractures, make use of the superb imaging capabilities of the sem. the capability of maintaining excellent depth of field at high magnifications is particularly important when matching the ends of wires that have been pulled apart. this is exactly what was done in a case of tape players that were jerked out of victims' vehicles. small fractured surfaces also have been encountered in the investigation of hit-and-run accidents when pieces of chrome trim were knocked loose and left at the scene. clearly modern instrumental means of analysis, such as the sem/edxa, are critical to the work of the forensic scientist. the increased sensitivity of the instrumentation, however, may raise questions as to the relevance of the evidence found. if a single, very small paint particle is found on the jeans of a pedestrian struck in a hit-and-run accident, could the particle be from the striking vehicle, or merely from the roadway debris at the scene? such questions indicate that increases in instrument sensitivity require sensitivity on the part of the analyst to questions of contamination and weight of evidence. research division, office of laboratories and scientific services, u.s. customs service, washington, d.c., usa the u.s. customs service laboratory system provides a variety of analytic services to control the commerce and assure enforcement of numerous regulations at the border. any item which is imported into the united states may need to be analyzed to determine the answer to any number of questions. what is the item? is the item correctly described? does the item infringe upon a u.s. patent? the scanning electron microscope (sem) and eds x-ray system can be utilized to answer some of the questions which arise. several examples follow. a u.s. company holds a patent on a feature incorporated into an electronically programmable read-only memory (eprom) cell. they allege to the international trade commission (itc) that another company is incorporating this patented feature in their eproms without the patent holder's permission. they win their case and the itc issues an exclusion order. it is at this point that the u.s. customs service becomes involved. we are charged with enforcement of the exclusion order. the incorporated feature is exceedingly small on a visible scale. how will we know which shipments of eproms should be excluded from entry into the u.s.? now the ability of the sem to produce images easily at very high magnifications comes into play. with the help of the sem, the laboratories are able to determine if the infringing feature is present or not. another u.s. company holds a patent for a denim (textile) finishing process. they bring a complaint before the itc that their patent is being infringed upon. the complaint is upheld by the itc and an exclusion order is issued. the u.s. customs service laboratory system must now develop a method to differentiate among various finishing processes in use. research at the headquarters laboratory found that a combination of imaging with a stereomicroscope and an sem could make the differentiation. the sem samples were au-pd sputtercoated with a hummer vi a sputtering system (anatech ltd.). the sputtercoated denim samples clearly showed distinctive features not easily seen with an optical microscope. a sample purported to be eelskin was submitted to the headquarters laboratory for conformation of its identity. it was thought that the product might be embossed plastic or possibly leather of mammalian origin. the top surface of the sample was au-pd sputtercoated and then examined with the sem. the features displayed by this examination were convincing evidence that the sample was indeed leather. a cross section of the sample was then prepared and au-pd sputtercoated. the sem images showed cell patterns consistent with reptilian or marine origin. another sample arrived courtesy of a foreign customs service. their inspectors had seized a large statue of a roman gladiator and his horse and chariot. the original reason for suspicion was that the declared value for the statue was much higher than one would expect for an object of this type; it appeared to be a cheap plastic statue. it had been dismantled and tested for the presence of drugs. the results were negative. using our princeton gamma-tech eds x-ray system, an elemental analysis of the exterior covering of the portion of the statue we received was performed. it showed the presence of large amounts of silver. this would explain the high declared value of the statue. when the u.s. customs service ran tests on narcotics particle detection systems, it was noted that sampling for heroin was more difficult than sampling for cocaine. a brief study of samples of each narcotic using the sem showed that in general the average particle size for heroin is less than that of cocaine. this may help to account for the sampling difficulty for heroin. allan n. walters u.s. postal inspection service, forensic laboratory, dulles, virginia, usa currently, the two main applications of sem/edax are explosive residue analysis and alloy quantitation. most explosives encountered are from improvised explosive devices (ieds) and commonly are low explosives such as black powder, smokeless powder, pyrodex, and flash powder. pyrodex and smokeless powder are commercially manufactured, while black and flash powder may be either of commercial or improvised (homemade) manufacture. the following table illustrates the composition of the common low explosives: device components are placed in the sem, and edax is performed before disturbing the residue. the resulting elemental profile is then used to guide further analyses with other instrumentation such as xrd, ftir, hplc, tlc, and chemical spot tests. sputter coating of samples is not performed so as to avoid modifying the sample and interfering with subsequent analyses and examinations. quantitative analysis is performed on alloys which are of interest to the u.s. postal service engineering and development center and is required to ensure that the materials meet the re-quired specifications. samples have included lock bodies, lock springs, keys, and lock tumblers. reverse engineering using quantitative analysis of current collectors for mobile electrification systems (mail sorting machines) has been conducted. quantitations are performed using a standardless method. scanned probe microscopy has evolved significantly over the last years. beginning with the first commercial scanning tunneling microscopes (stm) and continuing through the sophisticated "multifunction" microscopes of today, the "probe" has been one of the most critical and, in some instances, the least understood component of these systems. depending on the type and geometry of the sample, the properties of a probe can be optimized to reduce imaging artifacts. examples of this will be given using two well-known techniques, scanning tunneling microscopy (stm) and atomic force microscopy (afm). also, new innovations in probes for these two techniques (and others) will be presented. scanning tunneling microscopy, as first introduced, used a probe constructed of a chemically sharpened tungsten wire. later, it was found that a suitable probe could be formed from a mechanically sharpened wire (usually pt/ir). both of these probes proved that they could produce self-consistent images under specific sample and environmental conditions. however, problems arose with the tungsten probe while imaging in air because of the formation of native oxide. in addition, the mechanically formed probe tended to produce significant imaging artifacts if used on samples with topography > nm. in many cases the distortion produced by these artifacts made the data uninterpretable. a solution to these problems was to use a chemically etched probe (to control the shape) made from an inert material with physical properties suitable for the samples involved. musselman et al. developed the techniques necessary to chemically etch pt/ir wire into tips with a controlled geometry. these probes were capable of imaging surface structures greater than µm peak-to-valley depths with significantly reduced tip-related artifacts. this controlled geometry shape also made it more advantageous for use as a coated tip for electrochemical imaging. still, the aspect ratio of this particular probe was not suitable when imaging high-aspect ratio features such as pits in optical discs, contact vias in ics, fracture surfaces, etc. to obtain images from these types of structures, it was necessary to "machine" (in a controlled way) the probe described above. by using a focused ion beam (fib) as a machining tool, it was possible to create a probe to image the above features ( fig. ) . this fib "nanomachining" technique has opened up many possibilities for specialty probes. the majority of atomic force microscopy still utilizes the basic silicon nitride (si n ) triangular cantilever and pyramidal probe combination. the base of the pyramid is on the order of µm with the sidewalls extending upward at an approximate o angle to the apex. again, for samples with features < nm, such as mica, these probes have provided very good image repeatability. in general, however, for structures much greater than nm, a convolution of the probe and the sample surface will again occur. these probes can be modified, using the fib technique, to increase their aspect ratio significantly. because of their "hollow" design, these modified probes are still limited to topographies of ≤ . µm. most of the advances in probe manufacturing (on a wafer level) have come about by utilizing silicon as the probe material. several silicon probe types are now available, which provide significantly sharper tips with aspect ratios on the order of to . for imaging structures of higher aspect ratios, such as vias or deep trenches, longer and thinner probes are needed. these are made possible by using a combination of fib and electron beam techniques to "grow" a thin probe using an existing si n pyramidal probe as the base (fig. ). these probes are available in lengths up to µm with aspect ratios of ≥ to . utilizing a combination of fib milling of existing structures and electron beam growth, probes with lengths of - µm are feasible. an obvious problem with probes of this type (and with any long, thin structure) is "flexing" during imaging. analysis of one such probes' physical characteristics has shown that the elastic modulus is relatively low (e~ . gpa), while the coefficient of friction on most surfaces is extremely low (µ< . ). thus, probes of this type should be kept as short as possible while exceeding the maximum peak-to-valley distance to be imaged. other scanned probe techniques now in the prototype phase include thermal, magnetic force, near field optical, and probes capable of imaging undercut sidewalls. probes for all of these methods have been shown to be feasible, although manufacturing techniques to provide large quantities, reliably and repeatably, have yet to be developed. when imaging with an atomic force microscope (afm), the image resolution is a complex function of the relative tip and sample geometries. when imaging or measuring high-aspect ratio features, sharp and slender tips offer the possibility of probing down into extremely small topographical features. the most commonly used contact mode afm tips are batchfabricated si n thin-film cantilevers with an integrated pyramidal structure used as the tip. it has been shown that microtips, which are fabricated by electron beam-induced growth of carbonaceous material on the apex of the pyramid, can reduce the artifacts associated with integrated pyramidal afm tips. graph of a whisker of electron beam-grown contamination, or microtip, grown on the apex of an integrated pyramid, is shown in figure . an obvious problem with the use of a long slender microtip is the lateral deflection of the microtip as it is scanned across the sample surface. the proper use and interpretation of artifacts associated with electron beam-grown microtips demands an understanding of the mechanics of microtip deflection. it has been observed that long, slender microtips scanned over flat surfaces (rms roughness of < nm) produce hysteresis in the fast-scan direction. a model has been developed to explain the observed hysteresis loop in terms of a mechanical cantilever beam deflecting because of lateral frictional forces induced by repulsive imaging forces. this model is shown schematically in figure . by applying cantilever beam mechanics to the model of microtip deflection, a method of calculating the elastic modulus of microtip material has been developed. to find the elastic modulus of microtip material, a series of experimentally determined microtip deflection distances and the respective microtip lengths are required. microtip deflection distances have been experimentally determined for different microtip lengths on two different flat surfaces; fusion deposited boro-silicate glass and polished silicon. the elastic modulus of the microtip material has been determined from the deflection data to be approximately . gpa. once the elastic modulus has been determined, the coefficient of friction between microtip material and a sample sur-face can be calculated. the coefficient of friction between a microtip and the sample surface will indicate if the sample material is suitable for afm imaging with electron beam-grown microtips. the elastic modulus of microtip material and the coefficient of friction data lead to a better understanding not only of microtips but of electron beam-induced contamination in general. the low elastic modulus rules out the possibility of the material being diamond-like and suggests a polymeric material. the use of microtips can greatly improve image resolution; however, it is important to note that since microtip deflection increases with increasing microtip length, the microtip used to image a sample surface should be as short as possible while remaining long enough to image the largest peak-valley structure on the sample surface. the magnitude of observed microtip deflection should be reduced substantially by the use of ac mode microscopes where surface friction is less of a concern. because of their ability to achieve high resolution simultaneously in all three dimensions in a wide range of ambient conditions, scanning probe microscopes are promising candidates for performing measurements of surface topography. crosssectional and perspective views can be generated, nondestructively, at any location once an image has been acquired. surface topography measurements fall into two basic classes: position (or pitch) measurement and size (or critical dimension) measurement. the ability of a microscope to perform position measurement depends more on the quality of its design and construction than on the fundamental interaction of probing beam or stylus with the sample. size measurement, on the other hand, depends strongly on the probe-sample interaction. modern manufacturing, especially semiconductor lithography, often produces high-aspect ratio, submicron structures whose size and shape must be known with tiny uncertainties. surprisingly, stylus profilometers in the guise of scanning probe microscopes can perform some of these measurements at a level unmatched by any other type of microscope. to obtain size and shape measurements in semiconductor manufacture, we have developed a scanning probe microscope with several refinements, depicted in the figure. we use capacitance-based sensors for probe force sensing iv- scanning vol. , supplement iv ( ) cantilever and microtip at rest and for probe position measurement. , many of the samples that we scan are at least partially electrically insulating. for this reason, our microscope is used primarily as a scanning force microscope, although it is capable of operating as a tunneling microscope also. our force sensor employs a small silicon beam that pivots in one dimension about a pair of magnetically constrained ball bearings. the beam forms a pair of capacitors that both sense the position of the beam and maintain its balance with a suitable servo loop. this force-balance technique allows high-force sensitivity without sacrificing the stiffness required to resist surface forces. capacitors are also used to measure the position of the probe tip. the piezoceramic tube used as a scan actuator exhibits strong hysteresis and creep, so the drive voltage is an unreliable measure of probe position. the capacitors monitor the probe position in all three dimensions during the scan, and these data are collected along with the topograph. the most important factor determining the quality of the measurements is the shape of the probe tip. geometry alone makes the probe-sample interaction strongly nonlinear. in surface roughness measurements, a blunt probe can severely limit the range of spatial frequencies that can be detected. in scans of high-aspect ratio features, the probe shape determines what parts of the features can be measured. when scanning deep trenches and holes on a patterned surface, we use either a conical probe or a cylindrical probe, depending of what part of the feature is most important. the conical probes are made using focused ion beam sputtering of iridium. a chemical etch is used to form the cylindrical probes. if the probe shape is well known, then it is possible to determine what parts of a scan were distorted by the probe tip and, in some cases, this distortion can be removed. , the probe microscope itself can be used to determine the probe tip shape if a suitable structure is available for probe characterization. since the pioneering work of binnig and rohrer in the early s on scanning tunneling microscopy (stm), the stm has evolved into a powerful tool for spectroscopy, metrology, electrochemistry, and nanolithography. many other instruments have also evolved from the stm technology under the family of scanning probe microscopes (spms) with applications in atomic force, electric potentiometry, and magnetic force imaging. in the magnetic force microscopy (mfm) mode, the technique has been applied to the imaging of magnetic bit patterns in recording media (grütter et al. , mamin et al. ) and the mapping of static and dynamic magnetic fields of recording heads (martin and wickramasinghe ) . mfm is typically performed in the noncontact atomic force microscopy (afm) mode using a silicon cantilever which is coated with a thin film magnetic material, usually co, ni-fe alloy, or co-pt-cr alloy. the force exerted on the magnetic tip by stray fields from the sample causes the deflection of the cantilever which is subsequently measured. using a novel variation of the stm technique with a flexible iron tip, rice and moreland ( ) have also performed mfm imaging on magnetic bit patterns on a hard disk in a tunneling-stabilized mfm (tsmfm) mode. standard mfm images, however, reflect both topographic and magnetic information, with the relative strengths of each signal depending on the tip-to-sample spacing. using a differential interferometric technique, schönenberger et al. ( ) have shown that the topographic and magnetic information can be reasonably separated. in this abstract, results of some applications of the spm for topographical and magnetic force imaging of magnetic materials are presented. as the critical dimensions of magnetic devices are getting smaller, the surface topography and magnetic morphology of the recording head and media are becoming increasingly important with respect to optimization for best performance. the ability of the spm to obtain submicron topographical and magnetic information makes this an invaluable metrology and failure analysis tool for the magnetic recording industry. compared with other techniques for highresolution magnetic imaging, such as lorentz microscopy, electron holography, scanning electron microscopy with polarization analysis, mfm has the advantages of ease of sample preparation and the ability to operate in air. typical resolutions of - nm are obtainable in the mfm mode, although resolutions of - nm have also been achieved with considerable effort. in our work, topographical imaging was performed in the contact mode using a v-shape silicon nitride cantilever of length l = µm, width w = µm, thickness t = µm and force constant k = . n/m. the sensing tip at the end of the cantilever is pyramidal in shape, with a × µm square base and : aspect ratio. the tip radius is typically smaller than Å. for magnetic force imaging, the instrument was operated in the noncontact amplitude resonance mode by oscillating the cantilever and measuring changes in its resonant frequency using either phase or amplitude detection. this method of detection is responsive to the force gradient. for each point of the recorded image, the cantilever was first brought to a large tipto-sample distance of nm or greater to acquire the magnetic force image and subsequently moved close to the sample surface to acquire the topography image. this allows the simultaneous acquisition of topography and magnetic force images. a - v specimen bias was also applied to provide a linearizing and stabilizing force to the servo feedback loop. the cantilever used for the mfm imaging is a diving boardshape probe made of ( ) silicon. the geometry of the cantilever are l = µm, w = µm, t = µm and k = n/m. the sensing tip is conical in shape with a height of about µm, an aspect ratio of : , and a tip radius of < Å. the cantilever, which was sputter-coated with a Å thin-film cobalt material and magnetized inside a - gauss solenoid field, has a typical resonant frequency of khz. figure shows a three-dimensional topographic profile of a defective thin-film recording head, in which the pole tips protrude slightly from the surface, using the contact afm mode. the dimensions of the two rectangular pole tips are about µm × µm and µm × µm. the gap width was measured to be about . µm. dirt particles can also be seen and these are due to the cleaning process during sample preparation whereby the head was lightly swabbed with a cotton tip soaked in methanol. figure shows the magnetic force image of the surface of a recorded computer hard disk. the bright and dark lines represent regions of different magnetization or bits on the hard disk surface. the separation between each pair of bright-dark lines was measured to be about . µm and the track width is about µm. the striations in the topographic image (not shown), representing the texture lines, are approximately perpendicular to the recorded bit patterns. the line profile of the bit patterns in figure (not shown) is very similar to the calculated force gradient contours by mamin et al. ( ) in which both the horizontal and vertical magnetization components of the tip are are being sensed. iv- scanning vol. , supplement iv ( ) fig. three-dimensional topographic profile of a defective thin-film recording head using contact afm, in which the pole tips protrude slightly from the surface. in a good recording head, the pole-tips are supposed to be slightly recessed from the surrounding region. image resolution in traditional far-field microscopy is limited by diffraction caused by the primary aperture in the optical system of the microscope being used. in practice, diffraction limits lateral resolution to approximately λ/ , or about . micron for optical microscopes. to image smaller cellular structures and biological materials which are smaller than . micron, biologists have used microscopies which "illuminate" the specimen with radiation of smaller wavelength, such as an electron beam (i.e., using an electron microscope). the nearfield scanning optical microscope (nsom) breaks the diffraction limit to lateral resolution by illuminating (or collecting light from) the specimen through a sub-wavelength aperture which is held a small fraction of a wavelength above the surface of the specimen. using this nearfield technique, resolution far better than λ/ has been demonstrated on biological specimens and resolution better than λ/ has been demonstrated on standards (betzig and trautman ) . with nsom we have recently obtained optical images of tobacco mosaic viruses ( nm diameter) and of photoreceptor rod outer segments at a sub-wavelength resolution. betzig e, trautman jk: near-field optics; microscopy spectroscopy and surface modification beyond the diffraction limit. science , in june , the initial incident involving a report of a syringe found in a canned pepsi ® product received nationwide publicity. during the following months, law enforcement authorities investigated hundreds of additional claims of tampering from nearly every state in the united states. more than cases involving foreign objects allegedly found in soft drink containers were processed by the national forensic chemistry center (nfcc). to date, the forensic and law enforcement efforts of the fda have resulted in numerous arrests and convictions for charges related to product tampering and felonious reporting of product tampering. scanning electron microscopy (sem) has been valuable, and in several cases crucial, in providing conclusive evidence of fraudulent reports of product tampering. the items reportedly found inside of suspect cans were primarily hypodermic syringes (with and without needles), but other reported objects included nails, screws, bullets, pins, sewing needles, tacks, glass and plastic shards, and rodent carcasses. since most of the cases involved syringes, primarily insulin-type syringes, the first analyses were designed to determine if the syringes were contaminated and if they contained human blood, tissue, drug, or insulin residue. syringe/needle rinses were examined by a number of techniques including sem, which was used to find any intact blood cells and/or tissue. energy dispersive x-ray analysis (edxa) was performed on residue preparations. edxa was able to detect a small k αl peak for zinc in several syringe rinses which was consistent with edxa analysis of dried residues containing some types of insulin. type identification of insulin from syringe rinsing was performed by lc/mass spectrometry in a method developed at nfcc. in another case, a broken pin was reportedly found in a soft drink can. a cross-sectional analysis of the suspect pin by edxa showed the pin was a nickel-plated, iron-core straight pin. the absence of observed iron corrosion provided evidence that the object had not been in contact with the soft drink from the time the canned product was produced until the time the pin was allegedly discovered. the most common question requested by investigators was "how long had the syringe been in the soft drink can?" to answer part of that question, a number of experiments were conducted at nfcc. one sem/edxa method investigated the corrosion of the aluminum crimp (found on some syringe needles) submerged in diet cola. new aluminum crimp needles were sealed in diet cola and removed at -h intervals. stereoscopic light microscopy was initially used to examine each needle crimp. it was discovered that the submersion produced a brown to black "corrosion flower" in less than h of soaking. of specific interest, the submersion repeatedly produced a single point of corrosion on the crimp. the single point of corrosion suggested possible electrolysis of the aluminum crimp in the soft drink. this corrosion point continued to enlarge and penetrate more deeply into the aluminum crimp over time. backscattered electron (bse) imaging and x-ray mapping were used to highlight the region of corrosion in the sem. figure shows the secondary and backscattered electron image (sei and bei) of an aluminum crimp on a syringe needle after h submersion in diet cola. the sei shows the corrosion of the aluminum crimp as pitting in the crimp surface. the bei shows the corrosion area as a darker region caused by surface oxides blocking bse generation from the aluminum below. the study also demonstrated that the corrosion of the aluminum crimp was dependent upon the amount of time the crimp was submerged in the pressurized soft drink container. figure shows a point of corrosion on an aluminum crimp after weeks of submersion. at this time interval, several points of corrosion often developed. the pitting of the aluminum mater-ial immediately beneath the corrosion was extensive. sem/edxa analyses have shown that the aluminum crimp on syringes submerged in diet cola produced a characteristic single point of corrosion after only h. the corrosion resulted in damage to the crimp surface and the production of a metal oxide. the location of the oxide was identified and plotted by x-ray mapping for oxygen (via edxa). the analysis of samples related to the pepsi "tampering" cases of are continuing to date. in many cases, the circumstances and sample condition are unique and generated specific questions which require further forensic research using additional time-related studies, multielement x-ray mapping, and/or image analysis. confocal microscopy can be used to investigate the properties of rough surfaces. based on the kirchhoff approximation (sheppard et al. a,b) , the three-dimensional ( -d) confocal image can be modelled using the -d coherent transfer function (ctf). the form of the -d ctf for confocal reflection and transmission have been derived using a high-angle scalar theory (sheppard et al. ) . in both cases the ctfs can be expressed analytically. using these allows the profile of the rough surface to be reconstructed. in many cases we need to know only the statistics of the rough surface, rather than the actual profile. ways in which the statistical properties can be extracted directly have therefore also been considered (sheppard iv- scanning vol. , supplement iv ( ) fig . a. castenholz in previous in vivo studies based on vital microscopic and fluorescence microscopic techniques it was possible to observe lymph flow in initial lymphatics and regional lymph nodes. [ ] [ ] [ ] [ ] as an expression of immunological mechanisms, the studies carried out in the rat tongue under various issues showed some remarkable phenomena such as cell traffic along lymphatic pathways and phagocytotic activity of the lymphatic endothelium. since these phenomena could not be defined with traditional fluorescence microscopy on the cytological level, confocal laser scanning microscopy (clsm) was applied to the living tissue and fixed specimens). moreover, in morphologic studies on the lymphatic and blood vascular system, clsm has proved a very suitable tool also for the representation of resin-injected tissue, which is commonly processed as corrosion casts for scanning electron microscopy. all these approaches, also including the application of special fluorescent markers, should be outlined here. in vivo studies with the clsm have been designed for the functional morphologic analysis under normal conditions and in a state of inflammation and experimental edema. after staining the lymphatic endothelium with dimethyl-carboxfluorescein, clsm revealed a distinct pattern of the so-labelled initial lymphatics consisting of bright (cytoplasm) and dark zones (nuclear portions). for labelling tissue macrophages passing the lymphatic pathways and other phagocytotic cells, fluorescent microbeads (latex standard particles and liposomes) were applied by interstitial injection. thus, clsm enabled a certain identification of moving particle-laden cells and also gave evidence of phagocytotic activity of the endothelium of initial lymphatics. in long-term experiments, phago-cytosis of cells lining the sinuses of lymph nodes could be clearly recognized. tissue (tongue, skin) and lymph nodes from different sites of the rat were examined in the clsm as unfixed or fixed (glutardialdehyde) thick sections. cells labelled with fluorescent microbeads or stained with fluorescent dyes thus could be easily detected and identified. thereby, it was possible to determine the location of single beads at the endothelial surface or within the endothelial cytoplasm (fig. ). if the tissue was conventionally stained with hematoxilin eosin or acridine orange, optimum information could be obtained from unlabelled structures in these specimens as well. by means of the two-detection system, clsm also was able to distinguish two or more different fractions of microbeads incorporated by macro-phages ( fig. ) or lying in tissue spaces as free elements. in hemal lymph nodes of rats, erythrophagocytosis related to sinus macrophages was successfully represented by the clsm after vital staining of erythrocytes with the fluorescent celllinker pkh . in this approach, mercox (acrylate) stained with rhodamin and fluorescent yellow was used to create high fluorescence in the lumen of blood or lymphatic vessels. if the resin was injected into the tissue, the interstitial spaces were filled up by resin. clsm of sections from such resin-injected specimens enabled exact distinction between casts related to the blood or lymphatic system and those of the interstitial spaces, when two differently stained resins were simultaneously injected from the arterial system and into the interstice. clsm of resin-injected specimens was also applied as a suitable method for the control of corrosion casts of sem. proceedings of scanning /seems iv- today, clsm has become an established tool in many fields of biological sciences. because of its abilities to produce images with optimum resolution and clarity, the application in experimental lymphology is striking as well. some experiments in techniques related to in vivo experiments, supra vital and fixed tissue, and resin-injected specimens have been reported here. these approaches, also comprising the application of new fluorescent markers for living cells, may demonstrate how wide the spectrum of application is spanned for clsm in experimental lymphology and immune research. this study was supported by a grant of dfg (deutsche forschungsgemeinschaft, bonn) daniel chin, ph.d agouron institute, la jolla, california, usa the regulated metabolism and distribution of nucleic acids is required for endogenous antisense or rna regulatory systems. recent interest has focused upon using exogenous agents as antisense therapeutics to treat viral infections or metastatic diseases. both endogenous and therapeutic antisense regulation of gene expression requires the formation of a hybrid between the antisense molecule and the message or gene sequence. it is not surprising that detection of such hybrids in living cells has not been reported to date. we have used fluorescence resonance energy transfer (fret) to study hybrid formation and dissociation after microinjection of oligonucleotides (odns) into living cells. two systems were examined: one system characterized the kinetics of hybrid dissociation of two synthetic odns while a second system examined the distribution of hybrid complexes formed by hybridization of injected odns with endogenous mrna. in the first system, a -mer phosphodiester odn (+pd) was synthesized and labeled with a ′ rhodamine (+pd-r). the complementary, antisense ′-fluorescein labeled phosphorothioate odn (-pt-f) was specifically quenched by addition of the +pd-r, as detected by both absorbance and fret in solution. rapid and specific hybridization between the odns occurred at µm within min and the preformed hybrid slowly dissociated (t / ≈ h) in the presence of a -fold excess of the unlabeled odn. upon microinjection into the cytoplasm of cells, preformed fluorescent hybrids dissociated with a halftime of min, which is attributed to the degradation of the phosphodiester. formation of the hybrid from sequentially injected odns was detected by fret transiently in the cytoplasm and later in the cell nucleus, where nearly all injected odns accumulate. diffuse, specific nucleoplasmic fret could be distinguished from nonspecific fret in punctate nuclear structures which may result from concentration effects similar to fret seen in late endosomes or lysosomes between endocytosed, inert fitc-dextran and +pt-r odn. a second set of experiments with two adjacent -mer pt odns complementary to mouse b-actin mrna was performed in mouse t fibroblasts or n neuroblastomas. the upstream odn was ′ labeled with fitc and the downstream odn was ′ labeled with rhodamine. when hybridized to synthetic actin mrna or a complementary -mer pd odn, quenching of fitc and fret was observed in solution. injection studies in t cells showed transient odn accumulation and fret in filopodia and extended processes. in n neuroblastomas, fitc quenching was dependent upon cytoplasmic compartmentalization. after min, both odns accumulated into the nucleus. in summary, these experiments suggest that antisense odns can hybridize to intracellular targets in both the cytoplasm and the nucleus. the goal of this work was to increase the sensitivity of the electron backscattering pattern (ebsp) technique by introducing an electron energy filter to increase contrast and improve pattern visibility. energy filtering previously has been shown to increase contrast in selected area channelling patterns (joy ) . energy filtering differs from arithmetic background subtraction in signal processing, because filtering before detection has a physical basis and eliminates the unwanted background without increasing the noise component in the signal. the electrostatic retarding filter used in this experiment was constructed as a cone with five coaxial electrodes. the primary purpose of the electrodes was to create symmetric fields that would not distort the pattern, yet allow only backscattered electrons with the least energy loss to contribute to the pattern. analysis (courtesy e. munro, micro electron beam software, ltd.) of the electron trajectories indicated that for kev electrons, filtering voltages of up to kv could be used before chromatic aberrations became unacceptable. the angular field-of-view of the filter was approximately o . another feature of the detector is the use of a microchannel plate as the initial sensing component. microchannel plates have high gain and are very sensitive to the low-energy electrons that pass through the retarding field filter. in a configuration similar to that of venables ( ) , the output of the microchannel plates was proximity-focussed to a phosphor screen that displayed the microdiffraction patterns. the gain of the assembly exceeded a simple phosphor by more than times. the light output of the assembly was focused on a kodak megaplus . ccd camera using either a macrolens alone or a hybrid fiber optic-lens combination. no vignetting was observed across the field. the digital output of the camera was displayed and analyzed on a macintosh iix using nih image. results were obtained on a variety of materials including single crystal silicon, al-sic metal matrix material, alumina, aluminum foil, and aluminum used in aluminum cans. the high voltage design limited the maximum retarding potential to − kv so that meaningful data were obtained with incident electron beam energies between and kev. filter potentials above % of the primary beam accelerating voltage increased the kikuchi band contrast relative to the background. figure contains a very low contrast pattern for alumina obtained with essentially no filtering. the effect of filtering to within kv of the beam accelerating voltage is shown in figure for the same specimen. the best patterns were obtained when the filter voltages were between and % of the accelerating potential. filtering above % degraded pattern resolution while less filtering usually produced less contrast. up to four-fold improvements in the contrast ratio were achieved on some specimens. the detector also had the ability to obtain energy-filtered backscattered electron images in sem raster mode. one advantage of this configuration is that the sem characterization of a given feature and the ebsp data obtained from it can be obtained from the same perspective. another is that filtered im-ages appeared to have improved surface detail and greater crystallographic contrast. venables ( ) showed that a detector of this kind could be used for "dark field" sem imaging. this work demonstrated the feasibility of employing an energy-filtering detector with high gain to increase the sensitivity of microdiffraction measurements in the sem. further work is planned to optimize the filter and improve pattern quality and the range of filtering voltages. the authors gratefully acknowledge the contributions of munro electron beam software, ltd. and of mark vaudin at nist, as well as the support by the national science foundation under award number iii- , and the department of commerce under contract number -dkna- - . submicron-sized elements are of great interest in physics and technology. there exists a variety of methods to produce them by using conventional microelectronics technology as well as new methods for deposition of such elements directly from the desired material onto a substrate. the current onestage maskless techniques, for example, laser-induced cvd or laser-induced etching , do not ensure resolution below nm because of their physical limitations imposed by the laser wavelength. focused ion beams, which generally allow the formation of submicron-sized elements, often are unacceptable as they can cause radiation damage in the material. moreover, the cost of the necessary equipment is high. these circumstances stimulate the development of techniques for direct formation of submicron-sized patterns with the necessary geometry from an arbitrarily chosen material directly at substrates based on electron-beam-induced cvd . the cvd experiments were performed in a temscan jem- cx electron microscope at an accelerating voltage of kev and a beam diameter ~ nm. the electron microscope was equipped with a specially designed pressure cell and an oil-free system for pumping and reagent vapor inlet. this allows to maintain a well-defined atmosphere of chemical compounds (metal carbonyls and halides, freons) around the specimen. two electronically controlled needle valves are capable of supplying two different reagents simultaneously into the specimen holder. the sample itself is placed inside a heater which provides temperatures up to ˚c required for the local electron-beam-induced etching. contacts are made to the sample, which allow in situ measurement of its electrical characteristics. the holder is supplied with two differential apertures (through which the electron beam passes) placed above and beneath the sample. these apertures keep the gas pressure around the sample up to pa without breaking the vacuum in the microscope column. w(co) and rez(co) were used as reagents. structure and composition analysis both were performed in a jem- fx electron microscope equipped with a link an- / s system for edx analysis. self-supporting rods of - nm in width, containing tungsten or rhenium, respectively, were grown up to nm long at a speed of the electron-beam movement - nm/s and at a vapor pressure near the sample of . pa. provided a constant speed of the beam is maintained, the thickness of the produced rods was found to be inversely proportional to the beam speed but decreasing towards the rod's end. this result is similar to that obtained by electron-beam-induced fabrication of self-supporting carbon-containing rods electron microscopic observation of the inner structure of these self-supporting metal-containing rods revealed many individual fibers mutually aligned in parallel. thus, the mor- iv- scanning vol. , supplement iv ( ) fig. tem image (a) and sad pattern (b) from a self-supporting tungsten containing rod section after annealing in vacuum at ˚c for min. the accelerating voltage is kv, the diffraction length on the (b) is cm. phology of these rods is similar to that of the well-known carbon rods . this observation suggests similar formation mechanisms independent from the rod material. selected area diffraction (sad) patterns show the prepared rods as being amorphous. in situ annealing of the tungsten-containing rods at ˚c for min in the heating holder inside the microscope column transforms the amorphous structure into a nanocrystalline one (fig. ) . sad patterns reveal the presence of a set of different phases: in addition to pure crystalline tungsten there are various tungsten-containing compounds. among them, in particular, two new cubic phases with lattice constants ± pm and ± pm could be identified. after annealing chemical and phase compositions of the rods grown from w(co) and rez(co) were found to differ depending on the conditions of their formation (reagent vapor pressure, electron current, beam speed). the rods of nano-sized width, grown from rez(co) are considered to be promising as tips for scanning tunneling microscopes because of their chemical stability. an important requirement for developing high-speed highpower devices is fabrication of thermally stable ohmic contacts with low resistance and smooth interfaces. metal semiconductor interface inhomogeneities, such as lateral interface phases and spiking protrusions, lead to nonuniform current flow and consumption of the gaas substrate. this type of interface morphology is not acceptable for device applications where a large electric field or shallow contact is required. in particular, devices such as a heterojunction bipolar transistor (hbt) cannot tolerate contacts with lateral and vertical interface inhomogeneities. recently, we have introduced a novel metallization scheme, pt/ti/ge/pd, which yields thermally stable and low resistance ohmic contacts to both n and p + -gaas (han et. al. ) .to insure processing reproducibility and contact reliability, one must identify and understand the mechanisms responsible for this contact's electrical per-formance. this study employed cross-sectional transmission electron microscopy (tem), auger electron spectroscopy (aes), and electrical measurements (transmission line mode) to investigate the structural, chemical, and electrical properties of this contact. the interface morphology, phase composition, and elemental diffusion were examined and correlated with the measured contact resistances at annealing temperatures which yielded the best slight degradation and the worst electrical performance. annealing at ˚c yielded the lowest specific contact resistance, ~ . × - Ω-cm . the metal-semiconductor interface was planar and structurally abrupt. the pd and ge reacted to form a pdge phase. directly beneath the pdge was a thin, discontinuous, ga-rich pd-ga-as ternary phase. the presence of this ga-rich ternary compound has important implications for contact formation on the n-gaas substrate. formation of this interface phase creates excess ga vacancies in the gaas substrate. upon heating, ge diffuses into the gaas, occupying ga vacancies at dopant levels, to form an n + surface layer, thus allowing considerable tunneling at the metal-semiconductor interface. the existence of the ga vacancies is crucial, because otherwise ge would not readily diffuse into the n-gaas to form this n + -gaas layer. for the p + -gaas, this ge indiffusion occurs also, but the ge concentration level is not such that it has a detrimental effect, that is, it does not approach that of carbon (the p-type dopant) ~ × cm - . the ti/pt layers remained stable, hence no surface degradation was present. annealing at ˚c resulted in a slightly higher specific contact resistance, ~ . × - Ω-cm . there was significant elemental diffusion within the contact metal and minor elemental diffusion into the substrate. the interface exhibited a roughness on the order of nm and possessed large areas where spiking single-phased pdgega protrusions spatially dominated the metal-semiconductor interface region. the nonplanar nature of the interface lends itself to several explanations: ( ) nonuniformity in the heating associated with the annealing process, ( ) material defects or pitting in the gaas substrate such that the contact metallization covered and filled the pitted area as it would a flat gaas surface, and/or ( ) new phases being formed as a result of the ti layer beginning to break down as a diffusion barrier. apparently, this interface nonuniformity (spiking) does not cause significant deterioration in the specific contact resistance, since the contact resistance maintains reasonable electrical integrity. it is speculated that the compositional uniformity of the protrusion phase, which is close to that of the pdge phase formed during the ˚c anneal, is responsible for this behavior. annealing at ˚c proved to have a detrimental effect on the specific contact resistance, ~ - Ω-cm . this degradation was accompanied by strong chemical intermixing between the contact and the substrate, resulting in laterally continuous and vertically deep (> nm) multiphased protrusions spiking into the gaas substrate. the surface of this contact possesses surface anomalies (~ . µm in size) with a density of ~ %. the anomalies are somewhat enriched in pt and as and depleted in ge. this nonuniform surface morphology demonstrates that pt no longer represents a smooth surface for bonding. our results demonstrate that annealing temperatures between - ˚c are of practical interest for hbt device processing. the thin base region ( Å) and narrow distance between the contact and emitter ( . - . µm) make the ˚c anneal impractical for the hbt. specifically, the composition, extent, and magnitude of the interface spiking would be totally detrimental to this device design, that is, spiking areas would penetrate through the base region, into the collector, and short the device. the present paper is a review of diagnostic availabilities as well as of physical data taken by color cathodoluminescence scanning electron microscopy (ccl-sem) used for investigation of structural, polytypic, and impurity inhomogeneities of sic crystals, epitaxial layers, and devices (saparin and obyden ) . as examined objects, the "alpha" and "beta" sic crystals, different polytypes ( h, h, c, r) sic epitaxial layers and devices have been studied. the epitaxial layers were grown by the sublimation "sandwich" method (vodakov et al. ) in the vacuum or ar-media under temperatures between and ˚c. from results of experimental studies it is possible to enumerate the following that are impossible to prove by other techniques: ( ) space distribution of impurities; ( ) static and dynamic characterization of polytype transformation. multitransformation of polytypes during the growth process was observed by way r → c → h. it was concluded that the growth conditions of epitaxial layers and surface perfection of the substrate markedly influence polytype variations. ( ) space distribution of radiation defects on dependence of annealing temperature; ( ) action of mechanical defects on the spatial distribution of luminescence centers; ( ) availability to observe the transient layers in sic devices; ( ) -d analysis of epitaxial layers with polytype transformation. saparin gv, obyden sk: colour display of video information in scanning electron microscopy: principles and applications to physics, geology, soil science, biology, and medicine. scanning , - the examination of chemical vapour deposition (cvd) diamond films by the scanning electron microscope (sem) (secondary electron mode with spatial resolution - nm) shows that the morphologies of films are strongly affected by the synthesis conditions, especially the substrate temperature, the methane concentration, total pressure in the deposition chamber, etc. studies demonstrate that the changes of ch for concentrations in the range of . - % vol. create the shape variation of crystals determined by the ratio of the apparent growth rates of the ( ) and ( ) faces r( )/r( ) in cubo-octaedrons interval from . - . . the exact cubooctahedral shape observed very frequently is determined by the value of ratio . and preferable orientation of the ( )-or ( ) faces. thus, the secondary electron mode of the sem is a very useful diagnostic technique for diamond films. less frequently researchers use the second diagnostic availability of the sem (saparin and obyden ) : cathodoluminescence (cl) mode (spatial resolution . - . mkm) with monochromatic and panchromatic (real color) images and local cl spectra. diagnostic possibilities of this mode identify the quality of diamond films in comparison with natural diamond. the cl spectra of undoped epitaxial films show the dependence on the face upon which the epitaxy was done. cl discriminates between different types of diamond. there are distinct spectral differences between natural, synthetic diamonds and cvd films. the cl emission of the a-band was associated with donor-acceptor pair recombination (blue and green regions); vacancy-rich material emits in the red spectral region ( - nm). we would like to note that the cl spectra of natural diamonds (types ia, ib, iia, and iib) lie in the range of - nm. variations in these cl spectra were attributed to differences in the defect structures formed during the growth of material. cl images and local spectra allow one to recognize, for doped and undoped films, the small amounts of impurities, such as al( ppm) and b( . ppm) and nitrogen remaining in the gas, which could be a possible source of donors. thus, the sem investigations of morphology, crystallinity, and cl emission of films led to the correlative estima-tion of diamond film quality in comparison with the natural diamond as standard. the etching process of a crystal surface during its dissolution is well-known. the process of etch pits formation, as a result of crystals dissolution, usually is explained by scientists by the presence of dislocations inside the crystal structure. however, the same process occurring near the crystal edges is studied less, although such investigations can give additional scientific knowledge for a better understanding of the dissolution mechanisms of solids. this brief communication continues the series of investigations (dorozhkin , dorozhkin et al. the results show that here can be various surface structures taking place under geometric interaction among the growing etch pits and dissolving fap crystal surfaces. as the fap has a hexagonal crystal structure, the growing etch pits mainly have a hexagonal shape also (dorozhkin , dorozhkin et al. ). on the other hand, the investigated fap crystals were obtained from natural fap-containing rock after its disintegration and concentration stages. therefore, the crystals had a very irregular shape and a lot of dislocations inside. figure shows an example of a typical hexagonal etch pit having only three faces instead of the necessary six; three lacking faces (left side) have already been dissolved by the acid. a very similar situation is presented in figure . this is another example of a former hexagonal etch pit having only three faces (center). it is easy to restore events which occurred with the pits some minutes before. the pits began to form and grow from the moment when the outputs of dislocations on the fap crystal surface began to interact with the acid solution. as the pits are always faced only by fast dissolving faces, their dissolution rate is greater than the one for the crystal surface in common. on the other hand, the growing of etch pits and the fap crystal surface dissolution are always occurring simultaneously. as a result, a layer of fap substance between the growing pit, which is situated close to any vicinal dissolving crystal face, and the vicinal crystal face itself were getting increasingly thinner. finally, this layer disappeared completely, resulting in the pits' formation having only three faces (figs. , ) . if one had dissolved the fap crystal for a few more seconds, its surface would have reached the bottom of the pits and they would have disappeared entirely. figure shows an example of interaction between etch pits with an irregular shape and a surface of the fap crystal. for this purpose a very thin and sharp spallation fragment of the fap crystal was chosen which was then etched by phosphoric acid solution. inasmuch as the spallation fragment was equal to a random and unknown crystallographic face, the irregular etch pits were obtained. it is easy to see four different states of such pits (fig. ) : they point to a pit which ( ) ( ) is only running through the fap crystal but is situated relatively far from the dissolving crystal face. finally it should be noted that all the above-mentioned cases of interaction among pits and crystal surface may only take place when directions of dislocations inside the dissolving crystals are close to lines parallel to any nearest vicinal crystal face. k. habib and p. g. caceres* materials applications department; *central analytical laboratory kisr, safat, kuwait a fundamental study of co-based metallic glasses has been conducted. the study focused on understanding the changes of the properties and structures of an fe-b-si glass as a function of co, co-ni, co-mn, and co-ni-mo additions. the separate addition of co, co-ni,co-mn, and co-ni-mo elements was successful in a way four new metallic glasses were produced. the compositions of the new glasses are fe co b si, co fe ni b si , co fe mn b si , and co fe ni mo b si . consequently, an evaluation of the physical and magnetic properties was determined. furthermore, the internal and surface structures of the glasses have been characterized by a transmission electron microscope and a scanning tunneling microscope, respectively. a comparison between the internal and surface structures of the glasses was carried out on both amorphous and crystallined forms. as a result, a correlation between the properties and structures of the glasses is established. for instance, figures a and a show surface structures of the co fe ni mo b si metallic glass in the amorphous and annealed crystalline forms, respectively. on the other hand, figures b and b show the corresponding x-ray diffraction patterns of the amorphous (fig. a) and the annealed structures (fig. a) , respectively. figures a and a are basically three dimensional line plots of the surface profile. it is clear that the amorphous structure (fig. a) represents a complete rough surface along nm × nm scanning area. in contrary, the annealed structure (fig. a) exhibits a surface profile with less surface roughness than the annealed structure. this observation is in agreement with work done by the author on other metallic glasses cited elsewhere (habib et al. ). the current status of external funding for most academic and research facilities throughout this country is meager at best. many institutions are being forced to seek financial support from sources other than the conventional governmental agencies, private funding, or contractual agreements. this facility has, for the past years, derived the bulk of its operational budget through third-party payments for services rendered as hospital charges for diagnostic pathology services. while we have been fairly successful in maintaining a relatively consistent level of service, the overall cost of this operation continues to rise. as cost containment became the buzz word and hospital admissions declined, the number of requests for diagnostic procedures also began to decline. this pattern was observed not only in the electron microscopy (em) lab but in many other specialized laboratories throughout the hospital and our affiliated clinics. as the health care debate accelerated and new concepts such as hmos, emergency care clinics, managed competition, and regional alliances all geared to assure "more health care for the dollar came into being," it became apparent that an entire new concept was needed to provide high-quality diagnostic services for these newer group practices and smaller clinics that were being created to meet these new demands. the diagnostic referral service * has been established in the department of pathology, medical college of georgia, to offer a range of state-of-the-art diagnostic services to external referral sources, including private practitioners, group practices, pathology laboratories, and hospitals. these specialized services are designed to supplement other routine analyses and assist in the diagnosis, prognosis, and clinical management of patients with a wide range of diseases. the laboratories involved are fully cap-accredited, and all diagnoses are evaluated by a pathologist certified by the american board of pathology. the following is a listing of the laboratories contributing to this endeavor and a brief summary of some of the diagnostic offerings. this laboratory provides cytogenetic analyses to identify specific chromosomal abnormalities. these are useful not only in establishing a diagnosis of malignancy, but also in classifying certain malignant disorders, monitoring remission and progression, deciding on treatment regimen, and estimating prognosis. chromosomal analysis can be conducted on bone marrow aspirates, peripheral blood, and lymph node biopsies. this laboratory offers standardized transmission electron microscopic analysis of biopsies from any organ or tumor, as well as specialized procedures for examination of cell suspensions such as bone marrow and lung aspirates and blood samples. em analysis is particularly useful in conjunction with light and/or immunohistochemical microscopy for the diagnosis of tumors which cannot be classified by conventional light microscopy. working in close collaboration with the histology laboratory, the immunohistochemical laboratory, and the renal biopsy service, the em laboratory provides standardized, reproducible ultrastructural analysis which allows a consistent comprehensive approach to the interpretation of biopsy pathology. immunophenotyping by flow cytometry details the presence or absence of surface antigen markers of cellular maturation which define cellular subsets present in specific forms of leukemia and lymphomas. this technique provides precise information to aid in the diagnosis, prognosis, and clinical management of patients thought to have varying forms of lymphoproliferative disorders. in addition, quantitative dna ploidy and cell cycle analysis in combination with standard histopathologic and cytochemical methods provides the most comprehensive assessment of clinicopathologic status, tumor aggressiveness, and the likelihood of disease progression for patients with breast, colon, and ovarian cancer. this facility performs state-of-the-art diagnostic immunohistochemical and in situ hybridization techniques for the morphologic analysis of cellular and molecular events. these techniques are adjuncts to histopathologic examination and can be applied to the study of neoplastic, infectious, and other diseases. the laboratory offers more than immunohistochemical markers as special decision-making tests to resolve differential diagnoses. special histochemical stains and molecular probes for colorimetric in situ hybridization are also provided by this laboratory. this laboratory offers a full service for examination and consultation on kidney biopsies involving a wide range of disease processes. in addition to routine histopathology, all renal biopsies are examined by immunofluorescence for the identification of immunoglobulins, albumin, and c deposits, and by transmission electron microscopy for the detection of early or submicroscopic abnormalities. this laboratory presently offers complete dau screening and gc/ms confirmation for pre-employment/employee drug testing. upon completion of certification as a forensic urine drug lab, this will be the only such certified lab in this area. department of pathology, duke university and va medical centers, durham, north carolina, usa microprobe analysis in biomedicine is usually done on an electron microscope (em) equipped with an energy-dispersive x-ray detector (edx). this type of analysis is commonly referred to as electron probe microanalysis (epma). there are also other fundamentally different new techniques for microprobe analysis which involve the use of laser or ion beams. however, these are not yet commonly employed for diagnostic studies (ingram et al. ) . whereas biomedical epma primarily used to be a research tool, that is no longer the case. epma findings now often have diagnostic, therapeutic, and/or legal significance for the patient (baker et al. , shelburne ). in addition, since much of the current work involves conditions such as the pneumoconioses, the findings frequently have public health and/or industrial medicine implications as well as implications for single patients (shelburne et al. ) . currently the most commonly studied clinical conditions include the pneumoconioses, especially asbestosis and related conditions, "hard metal" pulmonary fibrosis, and other min-eral-induced pneumoconioses (roggli and shelburne ). a second major application is the use of this technology for the analysis of stones, particularly renal stones. microprobe analysis can be more sensitive than x-ray diffraction or chemical techniques, particularly for the identification of small components of complex stones. another major application is the use of microprobe analysis to identify unexplained pigments or deposits and to study unexplained granulomas (kupke et al. , pickett et al. . as is evident from the foregoing discussion, most applications involve the study of insoluble particulates. accordingly, conventional histologic processing with chemical fixation and paraffin or plastic embedding is acceptable. an obvious limitation of this approach is that electrolytes cannot be studied. currently we are exploring the feasibility of utilizing flash freezing techniques to permit studies on electrolytes utilizing cryoultramicrotomy. one way to gauge the usefulness of this technology is to study the use of microprobe analysis in a single hospital system, that of the veterans administration medical centers. currently there are va medical centers in the united states. within these hospitals there are diagnostic electron microscopy laboratories. of these, only five currently utilize epma as a diagnostic technique. at each of these laboratories, conventional transmission electron microscopic studies are the predominant type of analysis. epma studies constitute less than % of our electron microscopy laboratory workload. nevertheless, as the chemical information available from epma is better understood by clinicians, and as cryotechniques are shown to be useful, we anticipate increased usage. in conducting these studies, it is important to be aware of several artifacts that are common problems. the major types are those caused by poor specimen fixation. not only does traditional chemical fixation remove electrolytes from the tissue, it is common in electron microscopy laboratories to add heavy metals such as osmium, uranium, and lead. these elements may produce peaks in the final spectrum that can obscure important elements of physiologic significance. for example, the m-alpha line for lead obscures the k-alpha line for sulfur. in conducting an epma study, it is important to identify all peaks obtained so that the investigator is not mislead by a contaminant. in addition, it is important to utilize several controls. the investigator should not only probe the feature of interest, but also the cytoplasm adjacent to that feature and the blank stub. only in this manner can artifacts contributed by, for example, metal in the microscope column be understood and eliminated from consideration. all living things are infected/affected by viruses. whether the subject is a tissue culture, an animal being used in research, or a human, it behaves differently when infected with a virus. in research subjects virus identification is important to prevent erroneous data due to the presence of a foreign organism. in the case of human viral illness, it is increasingly important to identify pathogens so that appropriate viral therapy can be initiated. several antiviral agents are already on the market, and many more are presently in clinical trials. advantages of using electron microscopy (em) in viral diagnosis are that it is rapid; specific standards and reagents are unnecessary; and infectious particles are not required. disadvantages include the facts that a fairly high concentration of virions must be present in liquid samples to visualize them, and that solid tissues may have focal infections that must be included in the sampling. identification of viruses by direct em is performed by two techniques: negative staining of liquid samples and thin sectioning of tissues, cells, and tissue cultures. negative stains most used in virology are phosphotungstic acid (pta) and uranyl acetate, although many others have been described. for thin sectioning, any fixation method used successfully for em of tissue will preserve viruses; this includes some form of glutaraldehyde and osmium fixation, usually followed by uranyl acetate. detailed preparatory techniques have been described. in negative stains, morphology questions used in identification are: is the virus naked (always icosahedral) or enveloped (pleomorphic); if naked, what is its size, and does it have distinguishing capsid (outer) markings; if enveloped, does it have visible spikes or fuzz around the outside; what is its size, and is the nucleocapsid (the core) visible in particles that have been penetrated by stain; what is the shape of the nucleocapsid, if visible? naked human viruses are all icosahedral; these pathogens fall into three size ranges: - nm, - nm, and - nm. the small viruses may or may not have surface substructure; those that do not are not morphologically distinguishable and have been referred to as small round viruses (srv) (fig. a) if smooth, or small round structured viruses (srsv) if rough. others may have characteristic surface markings that permit precise morphologic identification. the medium-sized (fig. b) and large (fig. c, d) viruses are identifiable. enveloped viruses (those that have a pliable covering) are harder to identify, especially if mixed together with cellular debris. if they have surface fuzz or spikes (fig. e) , they are more readily distinguished. the genetic material inside is sometimes packaged into a distinct form such as an icosahedron, similar to the naked viruses (fig. a-d) or helical filament (fig. f ). if the negative stain penetrates the membrane, this nucleocapsid may be recognizable. however, some viruses do not have a morphologically characteristic nucleocapsid. in thin sections of infected cells, dna viruses are usually seen in the nucleus ( fig. a, b) where they are constructed, and rna viruses are usually found in the cytoplasm where they are formed (fig. c, d) , but there are exceptions. enveloped viruses can be seen associated with or budding through cell membranes; the membrane type is a further clue to identity. finally, the shape of the nucleocapsid within enveloped viruses is a key. possibilities are icosahedral (round in sections, fig. a, b) , helical or filamentous (like worms in sections, fig. c ), complex (pox viruses), or morphologically nondescript. recognition of viral particles and differentiation from cellular components and debris is paramount. once the presence of a virus has been determined, one may consult an atlas to confirm identification. [ ] [ ] [ ] [ ] [ ] for specific concentration or identification of viruses, some antiviral antibodies are available. these reagents can be used to aggregate, to coat, or to gold-label viruses. use of antibodies requires an a priori hint of the identity of the potential pathogen for selection of the proper reagent. charles d. humphrey, cynthia s. goldsmith, luanne h. division of viral and rickettsial diseases, cdc, atlanta, georgia, usa an unexplained acute pulmonary illness resulting in the death of previously healthy individuals was recognized in may . the cause of the illness was quickly identified serologically, pathologically, and genetically as a close relative of prospect hill virus (a rodent-transmitted hantavirus). recently, we isolated the virus from trapped rodents near the homes of patients, cultivated it in vero e cells, and determined that it was identical genetically and morphologically to the causative infectious agent. preparations for electron microscopy (em) were made by extracting, clarifying, and concentrating the virus from unfixed and . % glutaraldehyde-fixed, supernatant fluids of infected vero e cells . uninfected supernatants were prepared similarly as controls. concentrated virus suspensions were applied to glow-discharge treated formvar-carbon grids, blotted, and stained with . % uranyl acetate (ua) or with % phosphotungstic acid (pta), ph . . infected and noninfected cells were prepared for thin section by washing with . m phosphate buffer (po ) ph . , fixing in situ with . % glutaraldehyde in po , scraping, and pelleting. cell pellets were postfixed en bloc in po buffered % osmium tetroxide, stained in . % ua, dehydrated, infiltrated, and embedded in epon-substitute araldite. thin sections were stained with ua and in lead citrate. the extracted negatively stained virus resembled a hantavirus. the nm - + nm viruses were spherical, generally nonelongated particles with typical hantavirus grid-like surface features to which surface projections were attached (fig. ) . iv- scanning vol. , supplement iv ( ) fig our previous studies have demonstrated that application of scanning electron microscopy (sem) imaging for examination of transplanted hearts gave new possibilities for evaluation and early detection of acute rejection (jakobczak et al. ) . continuation of our studies confirmed the benefits of sem for diagnostics and basic investigations in cardiac transplantations. the aim of this study was to investigate coronary vessels during acute rejection in experimentally transplanted hearts. allogeneic heterotopic heart transplants were performed in ether anaesthetized rats, using microsurgical techniques according to the ono-lindsey method. male rats of the inbred long-evans strain were used as recipients, and male inbred sprague-dawley rats served as donors. the anastomoses-the donor aorta to the recipient abdominal aorta and the donor pulmonary artery to the inferior vena cava of the recipient (end to the side)-were performed under the operating microscope (wild m ) with - microsutures (davis-geck). cardiac allograft survival was estimated daily by electrocardiogram and palpation of ventricular contractions. rejection was considered complete at the time of cessation of a palpable cardiac beat. rejection was confirmed with laparotomy and histologic examination. the animals did not receive immunosuppression. allograft survival ranged from to days. transplanted hearts were perfused with . % nacl and . % glutaraldehyde solution in . m sodium cacodylate buffer ( mosm; ph . ). hearts fixed in glutaraldehyde were cut into thin sections, mm thick slides orientated perpendicularly to interventricular and interatrial septums. slides were treated with % osmium tetroxide for h at room temperature. then the specimens were dehydrated in an ascending series of ethanol solutions, ending in rinses in % ethanol and in pure acetone. thereafter, slides were criticalpoint dried using co , mounted on aluminum stubs with conductive silver paint and coated with a thin layer of gold. for examination a jeol jsm c sem was used. microcorrosion casts of the coronary arteries and veins and myocardial microvasculature of transplanted hearts were prepared by infusion of methacrylate casting medium (mercox). following infusion of approximately ml of casting material for one heart, the preparations were left for min to allow polymerization to occur. then, the tissue was macerated, leav- empty nm particles were often seen. virus particles were seen by thin section em as shells coated with barely distinct surface projections and enclosing hair-like strands of nucleocapsid material (fig. ) . particle size ( - nm) was smaller than that seen by negative stain em. spherical particles with elongated tubules (fig. ) were often observed by both negative stain and thin-section em. we conclude that deer mice trapped near the homes of humans with unexplained acute pulmonary illness harbor hantaviruses that likely are the causative agent for the human illness. ing a microcorrosion cast. dried preparations were mounted on stubs, coated with gold, and examined with sem (murakami , potter et al. . investigations of the coronary vessels of the right and left ventricular walls, septum, and the apex region were performed. tissue fragments were taken from various heart regions each day during the first postoperative week for studies with sem, and remnant tissues were used for histologic procedure. grading of the severity of cardiac graft rejection was based upon the stanford classification. in addition, the coronary vessels of transplanted hearts (of the other experimental group) were reproduced with a casting medium and examined with sem. the observations were compared with views of nontransplanted and syngeneically transplanted control hearts. the applied vascular casting method enables one to study the three-dimensional architecture of the vascular network of the rejecting myocardium. besides the large areas with minimal damage and readily distinguishable impressions made in the cast material by endothelial nuclei, there were the regions where various signals of vascular pathology were present: changes of diameter of coronary arteries and veins; unusual size and shape of the vessels; indentations on the cast surface caused by adhesion of blood cells to the vessel wall; characteristic occlusion of the vessels; irregularity of capillary network with changes in diameter and cast surface; capillary destruction with accompanying extrusion of casting material into the interstitial tissue. the observed vasculopathies varied in mild, moderate, and severe rejection and in several areas of the hearts. studies of coronary vessels of transplanted hearts using sem imaging have made significant advances in the understanding of rejection vasculopathy possible. sem observations of sectioned transplanted hearts correlate with results obtained by application of the microcorrosion casting technique. data showed that the highest rate of acute rejection occurs in the interventricular septum, and the development of rejection in various heart regions differs significantly. observed differences in localization and dynamics of development of vasculopathies in the septum and left and right ventricular walls of transplanted hearts confirm the unequal character of rejection. sem imaging is a valuable method for investigation of vascular pathology during acute rejection in transplanted hearts. the authors are very grateful to prof. a. miodonski, director of the sem laboratory of ent dept. of the n. copernicus academy of medicine, cracow, for the enabling of sem observations. preservation of the ureteral blood supply in kidney transplant surgery and ureteral reconstruction is of paramount importance in preventing urological complications of the ureter. the incidence of complications in ureteral surgery has declined in recent years, in part due to greater understanding of the ureteral blood supply and improved surgical technique. however, the ureteral vasculature has been described primarily at the gross level and only recently received attention at the microvascular level. because of its critical role in the success of renal transplants and its potential vulnerability to surgical trauma, the ureteral vasculature merits further investigation. we studied the microvascular anatomy of the ureteral vasculature (uv) in male new zealand rabbits using transmission electron microscopy (tem) and scanning electron microscopy (sem) of vascular corrosion casts and alkalitreated tissue samples. the uv was perfuse-fixed with buffered glutaraldehyde via the abdominal aorta, washed in buffer, and treated with alkali according to the method of takahashi-iwanaga for sem. for tem, fixed tissue was embedded in epon, sectioned, and stained with uranium and lead. for vascular corrosion casts, the uv was washed free of blood with buffered saline and filled with plastic resin (mercox•/methylmethacrylate, / ) at physiologic temperature and pressure. resin-filled tissues were digested with % koh, washed in water, critical-point dried, mounted on stubs, and sputtercoated with gold palladium for sem examination. the blood supply to the male rabbit ureter originates primarily from ureteric branches of the renal artery, the testicular artery, and the vesicular artery. these intrinsic vessels run within the wall of the adventitia the full length of the ureter. perforating arterioles and venules pass through the muscle wall and divide further in the lamina propria to supply a dense plexus of continuous capillaries in the mucosa. although numerous arterioles and venules populate the lamina propria, the ureteral musculature does not possess a rich capillary bed. the capillary plexus is positioned between the transitional epithelium and the lamina propria and uniformly extends the entire circumference of the ureter. capillary orientation occasionally follows the longitudinal axis of the ureter, but is primarily plexiform and exhibits multiple "y" and "t" shaped anastomoses (fig. ) . at the points of junction with the underlying arterioles and venules of the lamina propria, the capillaries commonly exhibit "kinks" and "bends." alkali treatment revealed that the capillary bed is supported by a dense, fibrous network of collagen (fig. ) . pericytes were occasionally associated with the capillary network. intercapillary distance ranged from - µm and capillary diameters typically were - µm. venous valves were not observed in the ureter. the combination of vascular corrosion casts, alkali treat-ment, and tem provides a clear and comprehensive perspective of the microvasculature of the rabbit ureter. the limited number of intrinsic vessels supplying the ureter wall and capillary bed emphasize the importance of understanding the blood flow in this organ in related surgical procedures. rabbits were perfused with glutaraldehyde and mercox ® via the abdominal aorta and subsequently processed for preparation of vascular replicas according to a previously described method. the angioarchitecture of the tibia was studied with use of the scanning electron microscope. at low magnification the pattern of vascularisation to the tibia could be established. they are richly supplied with arteries which pass into the bone substance proper from the periosteum. the main supply is from the nutrient arteries, which divide longitudinally in a dichotomous manner after passing through a foramina in the compact bone, major branches running both proximally and distally. this is the general case in all long bones. these branches reach the epiphysis and diaphysis of the tibia. in addition, separate arterial entities supply specific regions of the tibia: i. periostal vessels, ii. epiphyseal vessels, and iii. diaphyseal vessels. arteries and veins can be readily distinguished due to their characteristic nuclear impression present on the surface of the replicas. arterial nuclear impressions are oriented longitudinally to the long axis of the vessel, whereas in veins they are situated circumferentially (miodonski et al. ) . for further details about methods, see syed ali et al. . at higher magnification it was possible to demonstrate the microangioarchitecture of the epiphyseal region, which consisted of a very fine meshwork of capillaries arising from a main epiphyseal artery. a connection between arteries of the epiphysis was observed and consisted of medium-sized arteries. the capillaries of the epiphyseal meshwork show a rich anastomosing network building open, sinus-like dilatations. capillaries are also richly present in the areas of the growth plate and stressed areas of the epiphysis of the tibia. the so-called sinuses are situated under the cartilaginous plate. the venous drainage takes place through the vessels running parallel to the corresponding arteries. the animals were premedicated with chloroform and were then anesthetized with ketanest ( . ml/kg body weight) via intraperitoneal injection. after examining the abdominal reflex the rabbits were fixed on an operating table. all preparations were carried out at room temperature. the abdomen was shaved and opened cranially at the xyphoid process down to the pubic symphysis. after gently displacing the intestines and mesenterial convolutes, the abdominal aorta and the inferior vena cava were exposed very carefully caudal to the kidney vessels. a knob cannula was inserted through a small incision in the abdominal aorta and held in place by a ligation around the cannula distal to the point of incision. these procedures should be performed very quickly, otherwise the blood pressure will fall. the hindlimb vessels were washed through the cannula with an isotonic nacl solution containing heparin ( i.e.) and sodium nitroprusside ( mg/l) in ml. the inferior vena cava was opened with an incision as exit for fixing fluid and clamped when required. soon thereafter, . % glutaraldehyde in phosphate buffer ( . m, ph . ) was passed through the same cannula. after completing the perfusion, about ml mercox (methacrylate) was applied through the cannula and observed until the mercox came out of the inferior vena cava. it was then clamped to avoid unnecessary backflow. the animals were left overnight at room temperature and then placed in a water bath for h for rapid polymerization. the bones were dissected free from other tissues and left in a % koh solution, which was changed every day. the specimens were washed with distilled water very carefully; it is very important to avoid every kind of mechanical disturbance. the solution and water can be changed with the help of a small water pump. the best results were achieved with an alternative change of % koh and % trichlor tetra acetic acid solutions. after complete maceration, the preparation was checked under a stereo microscope to eliminate all rest tissue. it was then frozen at − °c and freeze-dried. the preparation was contrasted with % os o vapour in a desiccator for about h to increase the contrast in the microscope. the specimens were fixed on scanning stubs with conducting silver and sputtered with gold ( min, - ma). they were then viewed in a psem (philips) microscope and photographed with kodak film. iv- scanning vol. , supplement iv ( ) fig. low magnification view of a rabbit tibia from the epiphysial zone with its specific end capillaries arrangement, the proliferation zone, and the connecting capillaries between the epiphysis and metaphysis ( apart from the ability to capture three-dimensional ( -d) images of microscopic structures, a confocal laser scanning microscope (clsm) equipped with multiple detectors allows one to add an extra dimension to the data acquisition process. a typical example are the ongoing studies in our laboratories. in this paper, we discuss the channel spill-over problem associated with acquiring two-color clsm images and present image processing techniques that analyze multichannel image data. for double labeling of dna replication sites, mouse t cells, exponentially grown on cover slips or synchronized at specific times in -phase, were pulsed for brief times ( - min) with cldu (chlorodeoxyuridune). the pulsed cells were then fixed and processed for fluorescence microscopy using monoclonal antibodies, appropriate extraction conditions, and fluorochrome-conjugated secondary antibodies which enabled differential recognition of sites of cldu versus idu incorporation into newly replicated dna. under the conditions used, there is no measurable cross reaction between the antibodies for cldu-versus idu-labeled replication sites. optical sections of labeled cells were collected with an olympus lsm gb- clsm with a m w ar ion laser. the microscope is operated in high resolution mode ( × pixels) with three fluorescence channels and a transmission channel. the microscope is controlled by a - mhz computer with mb ram and . gb hard drive. the digital confocal optical sections were transferred via ethernet to a dedicated sun sparc / with mb memory for further analysis. during image capture, a combination of band-pass and high-pass filters are used to mask unwanted emission at each detector. a major problem with the dyes used is their overlapping emission spectra. this seemed to contribute a significant amount of spill-over from the green channel (fitc) to the red channel (texas red or rhodamine) and only an insignificant amount in the other direction. because of the unidirectional nature of the spill-over between red and green channels, we were able to apply a correction factor to the red channel based on the green-channel intensity. to obtain the correction coefficients, a set of calibration images were obtained by using a sample which is identically labeled in both color fluorescent dyes and captured at different gain and offset parameters. for each of these images, the mean spill-over value in to the red channel (r i ) is calculated for each pixel value (i) in the green channel as follows: ( ) where i r (x,y,x) and i g (x,y,z) denotes the image intensities of the red and green channels, respectively, and n i indicates the number of pixels in the green channel having value i. a third order polynomial is fitted to the data set (<i, r i >,i= , , ... ) and the corresponding coefficients (a o a ,a ,a ) are estimated using the least square error criteria. the correction factor for each pixel of the red channel is then computed as follows and subtracted from the original intensity of the red channel to obtain the corrected image. ∆i r (x,y,z)= a o + a i g (x,y,z) + a i g (x,y,z) + a i g (x,y,z) ( ) figure shows the channel spill-over for different filter sets. the corrected images were analyzed using a set of image processing routines developed at our laboratory to obtain the boundary of replication sites in individual channels. these boundary data are then used to detect the overlapping particles between channels, and the overlap area is calculated for each overlapping particle. the results are presented in several forms including a two-dimensional histogram of particle volume and overlap percentage. the image processing routines are able to generate boundary data of a × × image with particles in approximately mins and the overlap estimation using boundary data takes approximately s. currently we are working on visualization techniques of raw and processed multichannel -d images. these fringes, these dark, granular, linear structures shed dark particles which then fused with opposite lines. a stream of particles trafficking between cells in contract, particularly at overlaps, was noticed. this pointed to a rather intense exchange of very small particles between cells of the same origin on contact. we conclude that interference reflection mode, video-rate, laser scanning confocal microscopy is a useful tool for intravital analysis of the intracellular structural dynamics in relationship to cell type, function and pathophysiological state. department of plant pathology and department of botany, university of georgia, athens, georgia, usa powdery mildew diseases of plants are caused by a group of obligately parasitic fungi belonging to the order erysiphales, class ascomycetes. the somatic hyphae of most of these fungi grow exclusively on the surfaces of infected plant organs, most typically leaves. these hyphae form specialized structures known as appressoria that attach to the host surface. each appressorium gives rise to a tiny penetration peg that penetrates the wall of the underlying epidermal cell and invaginates the host cell plasma membrane. the penetration peg then develops into a specialized structure known as a haustorium that absorbs nutrients from the host cell. in this study transmission electron microscopy was used to examine the haustoria of the powdery mildew fungus erysiphe lagerstromemiae and the relationship of these structures to epidermal cells of infected leaves of crape-myrtle (lagerstromemia indica). the haustorium of e. lagerstromemiae (fig. ) possesses a slender neck region and an expanded body that contains a single prominent nucleus. much of the neck is surrounded by a collar of host cell wall material. a single septum with a tiny central pore with which woronin bodies are associated is present in the distal portion of the neck near the haustorial body. the haustorial body is divided into numerous small, coiled branches. the entire haustorial apparatus is separated from the host cell cytoplasm by an extrahaustorial membrane (figs. , ) . though continuous with the host cell plasma membrane, the extrahaustorial membrane is much thicker than the plasma membrane and is highly convoluted in certain regions. the haustorial branches are separated from the extrahaustorial membrane by a region known as the extrahaustorial matrix. at this point it is unclear whether the finely fibrillar material comprising this matrix originates from the fungus, the host cell or both. although the haustorial apparatus of e. lagerstromemiae occupies a considerable portion of the overall volume of an invaded cell, the host cell organelles appear normal. however, numerous golgi bodies and many structures resembling microbodies are concentrated in the host cytoplasm very near the extrahaustorial membrane (fig. ) . pavel vesely and alan boyde* institute of molecular genetics, academy of sciences of the czech republic, prague, czech republic; *department of anatomy and developmental biology, university college, london, u.k. video-rate laser scanning confocal microscopy in the reflection interference mode (irm) enables the visualisation of fine intracellular structures in living cells in vitro and the observation of rapid changes of shapes, the trafficking of very small particles, and exchange of material. the dynamics of this intracellular activity can be established by optical sectioning with objectives of high magnification and numerical aperture, from within the bottom level of the cell contact to the substrate through to the top cell surface: these levels may be up to µm apart for large and/or tall cells. in the present studies, three video rate confocal microscope systems (lasertec lm , noran odyssey and biorad dvc ) were used to compare the ultramicroanatomy and dynamics of motion of intracellular structures in established cell lines (k , k , t , and a on). k cells are clonal descendents of spontaneously in vitro transformed lew/cub rat fibroblasts which give rise to low metastatic sarcomas in vivo: k cells are rous sarcoma virus transformants of the k line and both t and a on cells developed from k by neoplastic progression to a higher metastatic capacity. previously observed differences in the incidence of cells with rapidly oscillating and trafficking particles between k and the other cell lines were analysed at improved resolution. using the noran odyssey system, with zoom factor and a / . nikon lens, this phenomenon was observed in almost all the cells of all the cell lines compared. this finding provided an explanation of the previously described difference between k and the other cells which was obtained using a nikon / . wi lens (but not with an olympus / . wi using the lasertec system, zoom factor ) from the level of the cell periphery adjacent to the culture surface only. the present observations indicate that there may be a difference in the extent of this type of intracellular activity towards the periphery of the cell. more accurate mechanical targetting of the optical probe will be needed before it will be possible to measure this effect. at the free, top surface of some k cells, small pinocytic opening and closing was seen. similar images of opening and closing near the bottom sides of some cells in all cell lines were evidently produced by vertical oscillatory movements of particles in small clusters. when a . mm pinhole diameter (odyssey) was used to improve confocality, the interference fringes were observed to move, during focussing, along the steep slope of the high bodies of the k cells, optically transforming granular, linear, or randomly oriented structures inside the cell into concentric lines. above and below florida institute of technology, department of biological sciences, melbourne, florida, usa acid phosphatase (ap) is well known as one of the representative lysosomal enzymes. ap activity is recognized at the light microscopic level as intensely stained granules. although the localization and distribution of ap activity in the mammalian inner ear were researched by several investigators, we found only one report about the occurrence of ap in the avian inner ear (marmo ) . the objective of the present study is to demonstrate the occurrence of ap activity in the membranous labyrinth of the chick's inner ear, using the simultaneous coupling azo dye method (barka and anderson ) , and to discuss the significance of these findings. after newly hatched chicks were sacrificed, their ears were fixed for h in . % glutaraldehyde (ga) or . % paraformaldehyde (pfa). the membranous labyrinths were dissected out, dehydrated in either a graded series of acetone (used only for . % ga fixation specimens) or n,n-dimethylformamide (dmf), and embedded in jb- (polysciences, warrington, penna.), a methacrylate plastic. these sections were incubated for or h at °c in an incubation medium (ph . ) containing naphthol as-bi phosphate as substrate and hexazonium pararosaniline as a coupler that used the azo-coupling method. in some instances, intact, fixed whole specimens were incubated in an aliquot of the same incubation medium as that of sectioned specimens and were then dehydrated with dmf, embedded in jb- or lr white, and sectioned. for control specimens, either a substrate-deficient medium or an incubation medium containing cupric sulfate ( . m) was used. methyl green was used as a nuclear stain. intense ap activity, as represented by dense dye deposits, was detectable in the supranuclear area of almost all hair cells in the basilar papilla and vestibular sensory hair cells (fig. ). this result was in agreement with the finding of marmo ( ) . there were no differences in the distribution pattern of ap activity of the hair cells from the distal to the proximal region in the basilar papilla. in particular, hair cells of lagenar macula were most often characterized by intense ap activity in the subcuticular area (fig. ) . the functional significance of high levels of ap in the hair cell is still a matter of conjecture. for example, ishii and balogh ( ) demonstrated no morphologic evidence for phagocytic or secretory activities in hair cells, although their electron micrographs of hair cells reveal lipofuscin-like substances within lysosomes. it has long been contended that nonsensory hair cells such as stria vascularis, external spiral sulcus cells, and those around the macula and cristae ampullares help to regulate the ionic composition of endolymph (kimura et al. , kikuchi and hilding ) . in the present study, the columnar cells and the cells of the tegmentum vasculosum showed moderate to strong ap activity, and the transitional epithelia of the cristae ampullares also showed strong ap activity . the dark cells help to regulate the ionic composition of endolymph and perilymph (kimura ). in the present study, intense ap activity of dark cells was concentrated in the supranuclear area or diffusely in the cytoplasm. these results show that ap activity is highest in cells with highest levels of transport activities on the production of endolymph (marmo ) , although the metabolic linkage between ap and the transport enzymes is uncertain. the wall cells and supporting cells of the vestibular labyrinth showed no enzyme reaction. the statoacoustic and vestibular ganglion cells showed various degrees of ap activity. the sections of statoacoustic and vestibular ganglion cells of specimens that had been embedded in lr white displayed more intense ap activity than those sections from specimens embedded in jb- . ap activity was also stronger in specimens fixed in . % pfa than in . % ga. the syncytiotrophoblast layer covers the fetal villous tree which is in direct contact with maternal blood. it contains many microvilli, resembling a carpet surface, which are responsible for the absorption, excretion, and synthesis of many key hormones which are important for fetal development, secretion, and exchange of gases; of course, many earlier scanning electron microscopy (sem) studies of normal and pathologic placental villi have been described and many investigations on the histology, ultrastructure, and three-dimensional features of human placental villi have been carried out. [ ] [ ] [ ] [ ] technical handicaps causing structural deformations on the placental villi have been indicated. all of these studies depended on the normal development of placental villi but were not found to be comparable with tubal pregnancies. the purpose of this study is to describe the development of the human placental villous trees emerging from chorionic plate during the early periods of uterinal and tubal pregnancies, and to compare their three-dimensional structures using sem technique. iv- scanning vol. , supplement iv ( ) fig samples of human placentas have been studied. six early specimens obtained by curettage or by hysterectomy have been staged as described in our previous study. four additional specimens dated according to anamnestic data in comparison with the measurement of fetal weight and length at , , , and weeks p.c. were obtained from clinically normal pregnancies interrupted by legal curettage or by hysterectomy, and two samples aged and weeks p.c. from tubal ectopic pregnancies. all specimens were fixed and prepared for sem. the specimens were dehydrated in ascending concentrations of ethanol, critical-point (bio-red e ) and air dried, and coated with a layer of gold particles using a bio-red sc super coater. observations were made with a jeol-jem sem. ectopic tubal placental villi trees are not as well developed as normal uterinal villi trees. villi formation and ramification were very rare depending on the physiologic causes. some villi shoots were thinned gradually from base to its apex, and at the top these developing villi showed very interesting properties; they were curved, thinned, and crossed each other (fig. ) . the predominant villi types were immature, intermediate, and mesenchymal villi. [ ] [ ] [ ] the surface of these villi trees was completely covered by small and deformative curved microvilli and by fibrinoid particles compared with the normal uterinal villi trees. the trophoblastic shell of some villi was peeled off and degenerated. developmental retardation of placental villus trees is clearly seen. some villi were curved and crossing, and gradually thinned to the apex; on the villi surface, many wrinkles and furrows resembling very old skin were observed (fig. ) . it is well known that the placental development shows a parallel regulation due to the desidual properties. cellular and extracellular interactions take place between uterine and trophoblast during initial stages of placentation. , - khong and robertson suggested that the deficient decidua is the main reason why tubal pregnancies do not reach to term. the findings they describe may be explained as being due to absent decidua rather than being its cause. moreover, another reason for the usual failure of ectopic pregnancies to reach term is placenta accreta which becomes placenta percreta with bleeding. according to our sem observations, in tubal pregnancies the formation and development of placental villi trees were found in some patterns observed in uterinal placental samples. villi formation patterns-buds, tendrils, and shoots-are very rare on the surface of villi trees (fig. ) . placental villi shoots do not show uniform composition compared with to the uterinal villi samples. these are very thinned to the apex of villi, and are compressed in a common configuration, resembling a racket which does not appear as an alive condition. these observations are very interesting and original. the university of iowa central electron microscopy research facility (cemrf) was established in to support all faculty, staff, and students needing this technology. initially, the cemrf operated with one transmission electron microscope (tem), one scanning electron microscope (sem), and three staff members, and it supported about projects annually. during the past years, all instrumentation predating has been replaced and now includes two tems, two sems, two eds systems, a scanning probe microscope, a laser scanning confocal microscope, and all necessary supporting equipment. the facility presently consists of staff members and supports over projects yearly from departments in colleges and industrial laboratories. one of the unique strengths of the cemrf is that both biomedical and physical scientists use the facility. the development of the university of iowa cemrf was made possible due, in large part, to the central administration's support of equipment acquisition over the past decade. of the $ , , invested in equipment, more than % was provided by the graduate college and the vice president for research, with the other % obtained through equipment grants and user fees. of the operating expenses for the facility, % are recovered from a large and well-funded group of investigators who are charged on an at-need, fee-for-service, first-come, first-served basis. faculty recognize that they have a facility available to them that provides immediate access to state-of-the-art equipment and techniques, as well as one that provides training and supervision. in addition to supporting research, the cemrf offers four formal courses, as well as assisting with sections of eight other classes. the facility annually organizes at least two workshops and provides about tours for visiting scientists, faculty and student recruitment, high school students, business groups, and politicians. the facility also serves as the business office for the iowa microscopy society. concurrent with the development and success of the cemrf, several departments voluntarily divested themselves of their own electron microscopy (em) equipment ( ems on campus in compared with ems in ). this represents a savings of more than $ , annually ( dollars), as well as the release of or more rooms for other use. in addition, the interaction between the remaining six em laboratories is more positive as a result of this downsizing and centralization. the availability of user-friendly quality equipment in the cemrf assures that em is now accessible to all university faculty, staff, and students. in addition, with many investigators sharing instruments, it is easier to justify acquisition of new equipment for the cemrf. accurate dimensional metrology of the submicrometer gold absorber structures of x-ray masks can be accomplished in the scanning electron microscope (sem) with the use of electron beam modeling (postek ) . accurate metrology is possible because the x-ray masks present a unique measurement object from most other semiconductor structures viewed in the sem. this occurs because the silicon support membrane is x-ray transparent by design. this characteristic can be used as a distinct advantage in electron beam-based mask metrology since, depending upon the incident electron beam energies, substrate composition, and substrate thickness, the membrane can also be essentially electron-transparent. the areas of the mask where the absorber structures are located are essentially x-rayopaque, as well as electron-opaque. viewing the sample from a perspective below the mask by placing an electron detector beneath the mask provides excellent electron signal contrast between the absorber structure and the base membrane. the present technique utilizes a broad acceptance angle detector which is different in concept from other transmission electron (te) detectors used in the sem. in this case, the broad angle is used to detect as many of the transmitted electrons as possible (i.e., whether scattered or not) that have an energy above some predetermined threshold which is usually several kiloelectron volts. then, the electrons are physically filtered both by the signal threshold characteristics of the detector and an by an electron energy filter in front of the detector. energy filtering of the transmitted electrons excludes the highly scattered and thus lower-energy electrons from entrance into the detector. this greatly improves the contrast level over the conventional transmitted-electron detection mode for this type of application, and greatly simplifies the required electron beam/sample interaction modeling necessary for edge determination. it is, in fact, this change in electron detection philosophy that makes the present te approach so attractive for dimensional metrology and inspection of x-ray masks. monte carlo modeling of the transmitted electron signal was used extensively to support this work in order to determine the optimum electron detector position and characteristics, as well as to determine the position of the edge in the image profile. the monte carlo modeling is more accurate in this work, in contrast to the secondary electron detection mode, because in the transmitted electron detection mode the modeling of the electron beam/specimen interaction becomes far less difficult than in the modeling of typical secondary electron images of other opaque objects. the generated low-energy secondary electrons (which are complex to model) are excluded from the detector and, therefore, do not need to be included in the calculation. combining all of these factors provides a modeled signal that is extremely sensitive to wall slope. wall slope variation can result in large differences in the modeled profiles. the comparison between the data from the theoretically modeled electron beam interaction and actual fitted experimental data is shown in figure for a wall angle of ˚. the theoretical profiles were shown to agree extremely well with experiment, particularly with regard to the wall slope characteristics of the structure obtained from the sem images and video profiles. a plateau in the transmission is seen in the modeled profile as the beam traverses the edge, which can be used to identify the loca-tion of the edge of the absorber line and thus allows accurate measurements to be made. this work provides an approach to improved x-ray mask linewidth metrology and a more precise edge location algorithm for measurement of feature sizes on x-ray masks in commercial instrumentation. the transmitted electron detection mode is also useful in both mask inspection and mask repair, because the high contrast of the image allows for rapid determination of mask defects and high-density contamination particle detection because the transmitted electrons simulate transmitted x-rays. this work represents the first time that electron beam modeling has been used to determine the accurate edge location in an sem image. this also represents an initial step toward the first sem-based accurate linewidth measurement standard from nist, as well as providing a viable metrology for linewidth measurement instruments of x-ray masks for the x-ray lithography community. postek using monte carlo models that take into account the gaussian beam width of the incident electron beam, a noticeable broadening of the linewidth measurements is simulated for aluminium linewidths under glass. this simulated effect correlates closely with measurements of linewidth using energy dispersive x-ray analysis of an aluminium ka line under glassivation. detailed simulation of a full linewidth measurement has been performed using a point source and gaussian incident electron beam for the multiple scattering and single scattering models. the modeling effort was undertaken to better understand the effect that incident electron beam width would have on the accuracy of scanning electron microscope measurements. the multiple scattering (ms) model was adapted from pas-cal code for a single material to simulate layered materials, specifically, sio over aluminium. further, application-specific code was added to simulate an electron beam scanning across a linewidth on a fabricated microelectronic circuit. the linewidth selected for these simulations was one micrometer and one micrometer thick with a selectable sio thickness for the overcoat. in this case, the sio thickness was . micrometers. the single scattering (ss) model was adapted also from available pascal code for layered materials with the same application-specific code used in the multiple scattering model. the differences in the results from the two models used for the simulation are shown in figures and . the results for the ms and ss models at k trajectories are shown in table i . the gaussian beam width was selected to be . micrometers. the gaussian beam noticeably broadens linewidth measurement over point source, based on the results in table i . the linewidth measurement was taken at fwhm by extrapolation between points. the large differences between the ss and ms models is under further study. the calculation of the image contrast from samples with surface topography can be done using monte carlo techniques as long as the electron trajectories can be calculated through a surface profile. the image simulations that are described here were done using the same methodology that has been applied to the calculation of the electron backscattered signal from samples where the composition variation is taken into account (ly and howitt and these proceedings) . the design of the program is such that the scattering cross sections are reassessed as the electron passes from one region of the specimen into another, which in this case includes free space. the program takes longer to run than that for a homogeneous specimen with a flat surface because the parameters, such as the atomic number and atomic weight and density, need to be constantly updated at each point in the calculation. depending upon the image resolution that is required, we divide the three-dimensional specimen into blocks of either specimen or vacuum. the modifications to the conventional monte carlo approach to such calculations include not only the specimen geometry but also the determination of the energy of the electron that is backscattered and the direction in which it travels. in this way the signals at the backscattered and secondary detectors can be distinguished because almost all the low-energy electrons find their way to the secondary electron detector, whereas only those in the line of sight to the backscattered detector contribute to this signal. in most practical situations, where the geometry of the specimen is difficult to predict, it is useful to have specimens of standard shape, such as a sphere cone or box, to compare directly with the images. the calculation of the image at the secondary electron detector for spheres of the same size but of different atomic weight is shown as an example in figure . the spheres are assumed to be on a graphite substrate and the electron-beam energy is kev. the signals are displayed as they would appear in a micrograph, that is, in an intensity scan across the image and as a two-dimensional profile of the image intensity. we have also found that pseudo color images, where the various gray levels are replaced with different colors, are very useful when it comes to comparing calculated and experimental images. calculated secondary electron images from nm spheres of silicon, titanium, nickel, and molybdenum on a carbon substrate at an electron beam energy of kev. the signals are displayed as they would appear in a micrograph, that is, in an intensity scan across the image and as a two-dimensional profile of the image intensity. pierre hovington, dominique drouin, raynald gauvin, david c. joy * department of mechanical engineering, university de sherbrooke, sherbrooke, québec, canada; * em facility, university of tennessee, knoxville, tennessee, usa quantitative analysis of resolution < nm can be achieved at low accelerating voltages. however, to exploit all the emitted signals (x-rays, secondary, and backscattered images) fully, specialized programs have to be used. in this paper we present some results that can be achieved with such programs. it is shown that our single scattering monte carlo program can effectively predict the effect of surface oxidation on aluminum (al), model the backscattered coefficient even at very low energy (< kv) and accurately predict the backscattered profile around a spherical inclusion. at low voltages, mott elastic cross section has to be used. the tabulated mott scattering cross section of czyzewski et al. ( ) , combined with the energy loss of joy and luo ( ) , is used as the physical basis of the program. hence, the program can be used accurately for simulation even at very low energy (e < kv) (fig. ) . numerical experiments can be made with one or several regions of different composition and shapes. the regions can be defined as horizontal (i.e., layered samples) or vertical (i.e., grain boundary). in addition, based on the results of gauvin et al. ( ) , spherical inclusions can also be modeled. the program is written in c language and makes use of a fully graphic environment both for input and output of data. in figure , we present a typical output of the program of a simulation on a multilayer electronic component [a nm ga ( % at ) al ( % at) as ( % at) on a gaas substrat] covered with nm of contaminated carbon. the effect of the backscattered electrons on the resolution can be clearly determined since their paths are marked with a different color. in addition, at the end of the simulation, the interaction volume ranges at , , , and % are plotted. to increase the range of applicability of our program, backscattered, secondary, and x-ray images (k, l, and m lines) can be generated. generated and experimental images can then be used for metrology and microanalysis. we present in figure a backscattered linescan of a nm diameter beam (eo = kv) with a hypothetical nm diameter hemispherical mns inclusion in a fe matrix. it is important to note in figure that the difference between the "screen dimension" and the "real dimension" is over % . in figure , we present an experimental backscattered profile taken across an mns inclusion in steel at kv. we note the similarity of the theoretical and the simulated backscattered profile (slight decrease between the center and the end of the inclusion and the presence of peaks at the interface between the matrix and the inclusion). iv- scanning vol. , supplement iv ( ) fig . when low voltages are used, the geometry and composition of the surfaces greatly influence the resulting signals. in figure , we present an experimental spectrum from a pure aluminum (al) sample taken at kv with a windowless eds detector and a theoretical generated spectrum (hovington et al. ) . the difference between the two spectra is mainly due to the presence of oxide (al o ) at the surface of the sample. in figure , we compute the j(rz) curves for an oxide thickness of and nm and for a nonoxidized sample. the decrease of the al k intensity for the and nm compared with the nonoxidized sample is and %, respectively. the experimental to theoretical ratio found on the spectra (fig. ) is approximately %, indicating that the oxide layer is < nm thick. it is important to note that because the standard and the unknown may not have the same thickness of al o , the use of theoretical stan-dards may become critical in quantitative x-ray microanalysis at low voltages. material investigation methods based on the interaction of the electron beam with a solid requires detailed information about the electron distribution. among the various approaches which allow one to calculate the electron distribution, the monte carlo simulation seems to be most suitable. this is due to the fact that the monte carlo method generally can be applied to any target. the main problem of this approach is the accuracy of the electron distribution approximation. one important criterion of the quality of the approximation is the coincidence of the calculated and experimentally observed x-rays in depth distribution. note that the correct calculation of this function plays the essential role in epma data interpretation. as have been shown in the monte carlo process developed by murata et al., which is based on the mott cross section for elastic and on a knock-on model for inelastic interactions, it provides a rather good agreement between the calculated and experimental φ(ρz) function for cdlα line in al and au targets at the electron beam energy e = kev. but a more careful comparison of φ(ρz), obtained according to this model with the experimental data for sikα in al, ni, ag, and au at e = , , kev, respectively, and cukα at e = , kev, reveals some discrepancy, especially in the tail part. this discrepancy increases when the atomic number grows and the electron beam energy decreases. the origin of this discrepancy can be connected with the errors of the approximation of the differential cross sections for elastic and inelastic interactions, as well as with the ionization cross section. as the mott formula for elastic cross section uses the model of the atomic potential, then the approximation of this potential can influence the results of the scattering. to avoid the errors connected with the choice of the atomic potential approximation, we have used the elastic differential cross section obtained by riley et al. with the help of the static approximation theory (sat). the main features of the developed monte carlo program are as follows: . the elastic interaction is calculated according to the sat cross-section . the energy dissipation process is described by the knockon model. the bethe formula for de/ds is used in the high-energy region (e > . j i ). in the low-energy region (e < . j i ), the equation used in [ ] is exploited instead of the bethe law . the fast secondary electrons produced by electron-electron interactions in an inelastic collision are taken into account . the mean ionization potential is chosen according to the berger and seltzer formula . to calculate the ionization cross section, the formula of gryzinski is used. we have calculated the energy and angular spectra of the electrons transmitted through the films of the various compositions and thicknesses. comparison of these results with the data obtained with the help of the mott cross section shows that the use of the sat approximation for the elastic interaction does not influence essentially the electron distribution and cannot improve the accuracy of its determination. the comparison of φ(ρz) functions which are found in accordance with these two models confirms this conclusion. therefore, one can state that the details of the mott cross section together with the errors in atomic potential approximation cannot influence the electron distribution in targets. the factor which can affect the φ(ρz) in targets independently of electron distribution is the ionization cross section. to take into account this factor, we have used the experimental results obtained by long et al. during differentiation of the heart there appears to be a sequential pattern to the formation of individual muscle fibers. phenotypic changes result in the expression, synthesis, and organization of complex proteins to form the terminally differentiated myocytes. the formation of the basic pattern of myofibers ultimately determines the physiologic performance of the final form of the heart. pattern formation involves both intracellular events associated with myofibrillogenesis and extracellular events associated with cell:cell and cell:matrix interactions. this basic pattern is repeated to form complex layers which results in the final form of the heart. it is essential to understand the sequence of expression associated with these patterns of fibers in order to understand errors that may result in congenital defects in heart formation. in this study time pregnant ( . - . days of gestation, ed) sprague dawley rats were obtained from harlan sprague dawley laboratories (indianapolis, ind.). animals were anesthetized with % chloral hydrate in normal saline solution and the individual embryos, including uterine tube, were removed and placed in . m phosphate buffered saline solution containing . % azide (pbs-a) at °c. the embryos were removed from the uterine tube under a dissecting microscope and fixed for - h in % paraformaldehyde in . m hepes (n- -hydroxyethylpiperazine-n'- -ethanesulfonic acid) at ph . . the embryos were scored and gestational ages were assigned. the embryos ( . - . days of gestation) were stained using a modification of the procedure described by tokuyasu and maher ( ) . after fixation, the embryos were placed in pbs-a for h and immersed in . % triton x- in pbs-a for h. after washing for an additional hour in pbs-a, the embryos were incubated in units/ml bovine testicular hyaluronidase for min, washed in . % triton x- with . m glycine in pbs-a for h and immersed in % bovine serum albumin (bsa) in pbs-a for h. the individual embryos were incubated overnight with rhodamine-labelled phalloidin from molecular probe, eugene, ore. ( µl of stock solution/embryo), or control buffer. following incubation, the embryos were washed in pbs-a at °c for h and immersed in % bsa for h. hearts from . - . day gestation embryos were removed and fixed as above. after fixation, the embryos were encased in % polyacrylamide gel using a modification of the procedure described by hansen and dryer ( ) . after fixa-tion, the hearts were washed in pbs-a for h and immersed in . % triton x- in pbs-a for / h. the hearts were embedded individually in blocks containing % polyacrylamide with . % bis-diamine which was polymerized with % ammonium persulfate. the polyacrylamide blocks containing the hearts were sectioned at µ with an oxford vibratome model g before being stained as described above. the tissue was examined using a bio-rad mrc- confocal laser scanning microscope (biorad, cambridge, mass.) using × (na= . ) and × (na= . ) objectives. at ed . to . , the cells of the myocardium are round and tightly packed with no discernable pattern. the first indication of cell orientation into fiber patterns occurred in the outflow tract and ventricle. the outflow tract myofibers developed circumferentially and maintained this pattern throughout the study time. the ventricular myofibers also developed circumferentially; however, they gradually changed into a net-like pattern ( fig. ) followed by a thickening of the ventricular wall and protrusion of trabeculae. with continued maturation, the ventricular trabeculae appeared to coalesce especially in the re-gion of the muscular interventricular septum (fig. ) . the myofiber pattern developed later in the atria. the atria expressed the same early pattern seen in the ventricles; however, they retained the net-like appearance throughout development. the results indicate that there are region-specific differential changes in the orientation of myocytes that result in the unique myofiber patterns of the heart. these regional pattern changes may be correlated with mechanical forces and hemodynamic alterations during development. the preparation of thick, optically clear sections of fragile tissue structures greatly aids the power of confocal laser scanning microscopy in imaging three-dimensional structures. hale and matsumoto ( ) have presented confocal images of agarose-embedded, vibratome-sectioned tissues; earlier workers have embedded soft tissues in polyacrylamide gels for frozen sectioning and lectin or immunohistochemical staining of structures in - micron sections (bronson et al. , hausen and dreyer , johnson and blanks . we report here conditions for embedding, sectioning, and staining embryos in polyacrylamide gels for a variety of confocal imaging techniques. in general, infiltration of - mm specimens for - h in a mixture of - % acrylamide monomer ( part bisacrylamide cross linker to parts monomer) yields, upon polymerization (with . volume of % ammonium persulfate), blocks that cut easily by vibratome between - microns. these conditions work well for tissues previously stained (with fluorescent probes or dii tattoos) or for staining gel sections water-soluble fluorochromes with low molecular weight (e.g., propidium iodide, phalloidin). for immunostaining after sectioning, the acrylamide concentration must be reduced to - % acrylamide to facilitate access of immunoglobulins to antigenic sites, and the gel must be supplemented with % agarose to aid sectioning and handling. illustrated below are confocal images from acrylamide sections of a stage chick embryo, fluorescence-immunostained in whole mount for cadherins. this specimen was fixed in % dmso/ % methanol (dent and klymkowsky ) and incubated overnight with a : dilution of commercial antiserum against a synthetic peptide common to all known cadherins (sigma, pan cadherin, #c ). rinsing preceded overnight reaction with tritc-conjugated secondary antibody, brief formaldehyde fixation, and embedment in % acrylamide. a survey ventral view was taken before transverse vibratome sectioning across the entire embryo at micron intervals, yielding eight sections that were spanning the thoracic region and were mounted in % glycerol for closer inspection. figure illustrates a stereo pair of projections from a series of confocal images across the atrioventricular junction. this gel-embedding and vibratome-sectioning method yields abundant, optically clear, and easily handled sections for confocal examination of fluorescent structures in water-miscible media. greater detail concerning procedures and technical problems with this technique are provided elsewhere (germroth et al. in press) . department of anatomy and cell biology, suny health science center, syracuse, new york, usa endothelial cells arise in the early embryo from precurser cells called angioblasts. in the quail embryo, the emergence of these cells can be observed as an epithelial to mesenchymal conversion of cells from the mesoderm which may be observed by scanning electron microscopy (sem) after removal of the endoderm or by immunolabelling the whole embryo or sections with a monoclonal antibody (qh- ) which labels angioblasts. the process of angioblast migration and assembly into the solid cords, which are the rudiments of the earliest blood vessels, is called vasculogenesis. simple embryological manipulations have been used to distinguish the role of cell migration in the early vessel rudiments. the dorsal aortae arise ventrolateral to the forming somites, and inserting blockages revealed that little cell migration is involved in their formation. the endocardial cells which form the endothelial cell lining of the heart, in contrast, undergo an extensive migration from more lateral regions of the embryo which has been studied by blockages and the construction of quail/chick chimeras. iv- scanning vol. , supplement iv ( ) the first rudiments of veins in the embryo are the cardinal veins. these form beneath the ectoderm of the embryo in an area with few angioblasts. the early stages of cardinal vein formation have been studied by using immunogold-labelled secondary antibodies after qh- labelling and imaging in the sem backscatter mode after silver enhancement. these studies revealed that small groups of angioblasts appear over the lateral mesoderm, possibly by migration from the angioblastrich mesoderm adjacent to the endoderm, and then migrate medially to assemble into a solid cord of cells in association with the developing wolffian duct. the ability of scanning probe technology to image atomic topography of a surface, to manipulate individual atoms, and even to probe the internal structure of a molecule confirms the significance of these super-resolution microscopies used at the nanometer-scale analysis of molecular systems. so far, a number of interactions between the sample and the probe tip gives access to a variety of local properties (wickramasinghe ) . the latest applications of the local probe microscopies on organic molecules include photoemission, tunneling potentiometry, electrostatic, elastic and tribo logic properties, or near-field thermal measurements and as many other phenomena on which various contrast formation mechanisms are based. topics include imaging of individual biomolecules, highly ordered molecular assemblies, and polymeric materials under various environments. the development of additional imaging techniques,based on near-field electromagnetic interaction between the probe tip and the surface (betzig and trautman ) , or even by means of magnetic resonance (rugar et al. ) , indicates that new technologies with subnanometer spatial resolution could be achieved in principle. recent advances in near-field optical microscopy (nsom) confirmed spatial resolution in the range of - nm but still limited by the diameter and optical penetration depth of the aperture. here, we demonstrated a new concept for an aperture near-field scanning optical microscope which combines force microscopy and optical scattering for imaging the sample at subwavelength resolution without the use of an aperture. the end of a silicon tip is illuminated in transmission mode by a laser beam through a transparent substrate. both the tip and the sample undergo perpendicular motion, each at a different frequency, with amplitudes chosen comparable to the desired measurement resolution. the scattered light from the tip and the surface is detected at the difference frequency for imaging and sample at sub-wavelength resolution without the use of an aperture. we describe the novel experimental scheme and present the most recent results obtained from our system. the resolution demonstrated to date is nm using helium-neon wavelength and optical line scans are shown in figure . finally, a consideration of the basic theory demonstrates that much higher resolution can be easily anticipated. these preliminary results firmly establish the great potential of this new near-field optical microscopy for biological research. high-resolution scanning electron microscopy (sem) studies of enamel crystals from remineralized enamel have provided clues as to the changes in crystal diameters within specific zones of artificially created carious-like lesions. , these data have supported the evidence obtained through polarized light microscopy of zones of demineralized and remineralized enamel. the dark zone, viewed in polarized light, has been identified as the zone of remineralization. these initial studies were conducted using sputtered coatings of gold/palladium (au/pd). high-resolution images of the crystals within the zone of remineralization revealed large crystallites in excess of µm, often with what appeared to be remineralized "nodules." however, it was difficult to determine whether these were one crystal, or two crystals which had fused during the remineralization phase. a study was conducted using an ultrathin coating of sputtered chromium (cr) film and se-i imaging to provide topographic contrasts of crystal surfaces. since the majority of the se-i information should be produced close to the surface, the resultant image is specimen-surface specific. , artificial caries-like lesions were prepared in noncarious human teeth (second and third molars) and sectioned into µm sections which were then viewed in polarized light to identify the dark zone of remineralization. the sections were then fractured through the dark zone producing a spicule of enamel with an edge of fractured enamel free from sectioning artifacts. each specimen was then mounted onto supports with silver paste with its fractured edge facing up. measurements were then made to determine the location of the dark zone so that it would be easily located within the sem. specimens were degassed and then sputtercoated with nm au/pd and nm cr. , specimens were imaged at high magnification in the se-i mode by placement on the condenser/objective (c/o) lens stage of an isi ds- /lab sem at kv and a hitachi s- cold cathode field emission (fe) sem operated at kv. high magnification se-i images (s- fe sem) of au/pd coated enamel crystal surfaces within the dark zone, revealed particulate features decorated by metal giving it a continuous granular appearance (fig. ) . analysis of a specimen coated with a . nm cr film revealed crystal surfaces with well defined particulate features that were clearly delineated and separate from one another. this study documents the effect au/pd coating has upon the examination and evaluation of enamel crystals at high magnifications. results obtained document that an ultrathin coating of sputtered chromium film of - nm does not produce the coating artifacts found with conventional sputtered au/pd. an ultrathin coating of cr, together with the use of se-i imaging, allows surface features to be more accurately imaged and measured. iv- scanning vol. , supplement iv ( ) fig . previous results have shown that the ability of intercalative dyes to modulate the antiviral activity of poly r(a-u) was related to the groove through which the dyes intercalated into the poly r(a-u). when poly r(a-u) was combined with the minor groove intercalating dyes such as ethidium bromide (eb) or the minor/major groove intercalating dyes, optimum enhancement of antiviral activity was observed at a dye/ribonucleotide ratio of / . no enhancement of antiviral activity was observed when poly r(a-u) was combined with the major groove intercalating dyes. when eb was combined with poly r(a-u) and then added to human foreskin fibroblast (hsf), the % effective dose of the poly r(a-u) was -fold lower. the results of additional studies demonstrated that the enhanced antiviral activity was not due to superinduction of interferon, direct viral inactivation, or host cell cytotoxicity. phase contrast, fluorescence, and confocal micrographs of hsf cells following a -h exposure to µm eb alone or a µm eb/ µm poly r(a-u) combination stained the nucleolus, but not the chromatin. negatively stained transmission electron microscopy (tem) preparations (fig. a) and replicas (fig. b) (fig. a,b) which may be associated with the enhanced antiviral activity of this combination. under the conditions employed in this study, poly r(a-u) exhibits an elongated conformation ( - nm in length) that possesses a number of hairpin loops as well as single-stranded and double-stranded domains (fig. a,b) . the double-stranded domains are found predominantly at the base of nm hairpin loops. in contrast to the poly r(a-u) alone, micrographs of the eb/poly r(a-u) combination illustrate the presence of condensed structures with diameters ranging from to nm. results from scanning force microscopy corroborate the results of both tem preparations. tem of unstained and uranyl-stained eb/poly r(a-u)-treated hsf cells illustrate the endocytosis of electrondense material into acidic compartments of the hsf cells. subsequently, the electron-dense material escaped from the acidic compartment and formed electron-dense bodies with dimensions that closely approximate the dimensions of the eb/poly r(a-u) combination visualized in the negative staining preparations. these electron-dense bodies are detected near the nuclear membrane and in the nucleolus. the nucleolus of an unstained, eb/poly r(a-u)-treated hsf cell demonstrates the segregation of the nucleolar components so that the fine fibrillar component, which comprises the nucleolar organizer region, is being peripheral to a dense granular material occupying the major central area of the nucleolus. the results of the current study and our previous work suggest that the elevated antiviral activity of the eb/poly r(a-u) combination may be related to the ability of the eb to complex with poly r(a-u) and condense it into a conformation with dimensions that can be accommodated by endocytotic vesicles. exit from the acidic compartment is promoted by unbound eb that induces endosomal or lysosomal swelling. subsequently, changes in the topology and surface charge of the poly r(a-u) induced by the eb allow increased access to the nucleolus which results in the modulation of additional cellular processes, especially rrna synthesis and processing. aromatic constituents in plant cell walls are associated with other wall components and affect strength and other characteristics of plant cell walls. ultraviolet (uv) absorption microspectrophotometry has been useful in characterizing aromatics within specific cell types in plant organs. , , this technique was applied to walls of bran and endosperm cells in a series of hard and soft wheats. kernels from hard and soft cultivars were fixed in glutaraldehyde ( % in . m cacodylate buffer at ph . ), and sections were cut with a microtome at µm thickness. the sections were mounted on quartz slides in glycerin and covered with a quartz cover slip. cell types in the sections were analyzed for uv absorption using a computer-controlled zeiss umsp microspectrophotometry system. transmitted illumination was provided by a xenon lamp (xbo w) with a connecting grating monochromator using a bandwidth of nm. a -× quartz objective lens with a final aperture of . µm, which was delimited within about onethird of the area of a field-limiting diaphragm to reduce stray light, was positioned over walls of selected cell types. absorbance of transmitted uv illumination was recorded over a range from - nm at -nm increments. the system was standardized at µm. spectra were collected, displayed, and evaluated using the zeiss lambda scan software. uv absorption maxima or distinct shoulders occurred near , , and nm. while the absorption near nm is not defined, absorption near nm is believed to be due to lignin (i.e., polymerized phenolic constituents) and that near likely is due to ester-linked ferulic acid. synthetic lignin, from polymerization of coniferyl alcohol with horseradish peroxidase, has a strong absorption at nm and no absorption at higher nm. further, ferulic acid linked to arabinoxylans, which has been isolated from bermudagrass cell walls, gave a shoulder near nm and a strong max at nm. other work, using the addition of cinnamic acids to milled lignins or removal of phenolic esters by alkali treatment, supports the above spectral interpretations. the kernel of wheat consists of several distinct cell types (fig. ) . spectra were obtained of walls of these various cell types (fig. ) . epidermal and parenchymal walls gave the highest absorption and both had spectra indicative of lignin (near nm) and also of ester-linked ferulic acid (near nm). nucellar epidermis walls have spectral patterns similar to the cell types above, but absorption was much less. aleurone walls gave spectral patterns indicative of mostly ester-linked ferulic acid and less lignin than previous cell types; the side walls gave a considerably higher absorbance than upper or lower walls. endosperm cell walls, which enclose the starch/protein matrix, were totally lacking in uv absorption maxima, indicating no aromatic constituents in these walls. variations occurred in uv absorption and spectral patterns for walls of various cell types in wheat kernels. however, no consistent differences occurred among the various kernels that might relate to hard-or soft-rated wheats. iv- scanning vol. , supplement iv ( ) fig . food microscopists are continually searching for noninvasive methods of examining the internal structure of foods, especially those with delicate labile structures. x-radiation is of considerable potential because of the great penetrative power and the minimal need for sample preparation. examination can be carried out at atmospheric pressure, and there is no intrinsic reason why temperature-controlled or temperature-ramped studies are not possible. the term x-ray microscopy covers several techniques and can be defined as the use of x-rays to produce a magnified image, or to give information about specific microscopically identifiable parts of a specimen. our current equipment consist of an electron gun, two electromagnetic lenses (condenser and objective), and x-ray target. x-rays are generated from a µm spot on a tungsten target. the target usually is positioned so that the microfocused electron beam glances the side of the target. x-rays from the microfocal spot form a cone which diverges through a thin window. focusing is achieved by placing a grid with bar widths of and µm into the beam and adjusting the current in the condenser and objective lenses. the image is captured on a tv monitor. finally, test radiographs are recorded on x-ray sensitive film. the x-ray radiograph is a shadowgraph and, where the sample is thick, the overlaying shadows will be complicated to interpret. two methods can be used to help interpret the image. one method is to take stereo pairs of the sample and view these images with a suitable stereo viewer which will show a threedimensional image. the other method is tomography. in the projection x-ray microscope, the entire specimen is in focus at once with the same resolution, determined by the spot size, although different depths within the specimen are magnified by different amounts. three-dimensional images can be made by moving the specimen either by tilting or translating it between exposures in the divergent x-ray beam. these images are then viewed with a suitable stereo viewer which will show a three-dimensional image of the structure removing some of the confusing overlay of detail to be found. we have also obtained images from pieces of tissue subject to tensile elongation. in the design of our x-ray microscope there is considerable distance separating the x-ray film from the specimen, and this space allows the apparatus for tomography to be set up. tomography is a form of imaging whereby a section or "tome" can be taken through a specimen by using x-rays, and details above and below the sectional area of interest will be blurred out, giving a fairly clear image of this area. this technique is nondestructive to the sample. the method is similar to that de- scribed by lindegaard-andersen and thuesen, originally proposed by watson. in their method, the x-ray source is stationary and the subject and recording film are rotated synchronously. the x-ray beam strikes the film at near glancing incidence producing transaxial summation images. however, the associated point spread function has a /r radial dependency, which means that smearing of detail will have occurred. the results are sufficiently encouraging, nevertheless, to stimulate the search for improved contrast and resolution, with provision for three-dimensional mapping of internal structures. the images shown here are of food products containing large air cells, giving maximal contrast in the x-ray beam. we have obtained comparable success with water-filled cellular structures such as vegetable and fruit tissue. in the future, we anticipate further enhancement of contrast and resolution by the use of improved x-ray flux, image capture, and image processing. centre for food and animal research, agriculture and agri-food canada, ottawa, ontario, canada over the past years, electron microscopy (em) has proven to be a useful tool in assessing the effects of various physical and chemical treatments on the microstructure of egg components (related to the production of egg products, baking, behaviour in various food systems, nutrient bioavailability, etc.) much of this information has been acquired using transmission electron microscopy (tem) to view yolk samples which have been biochemically extracted after removal from the egg. the author studied microstructural changes in yolk during its in vivo digestion by the epidermal cells of the chick embryo yolk sac. yolk is digested within these cells, as signified by its presence in varying degrees of microstructural alteration, compartmentalized within cell organelles. a temporal sequence can be inferred with the degree of microstructural change observed. it was possible to follow the fate of yolk granules and low density lipoprotein (ldl) particles during digestion, because yolk granules retain their integrity during preparation for em. ldl particles were made visible by crosslinking action of the fixatives and by the use of a specific enhancement technique. initial work focused on the microstructure of native, undiluted yolk which had been fixed using conventional and novel fixatives. thin sections were examined by tem. these experiments allowed us to optimize fixation conditions and confirmed earlier results by other workers that yolk is composed of granules which have a subgranular structure and lack a boundary membrane. the granules sit in a suspension of closely packed ldl particles. yolk granules from incubated eggs were identical to those from unincubated eggs and showed no microstructural changes to indicate that digestion had taken place outside of the cell. after clarifying that yolk is digested intracellularly, the next phase of the study was the microstructural description of intracellular yolk and yolk sac epithelial cells during incubation, prepared using half-strength karnovsky's fixative and the imidazole-buffered osmium tetroxide protocol (ibo) of angermuller and fahimi ( ) which enhanced lipid staining. epithelial cells were examined systematically using em, from apex to base, to study the three processes associated with digestion: uptake, breakdown, and transport. using tem, granules and ldl particles were observed to enter the cells in uptake structures, the microplicae and coated pits, respectively. when samples were viewed using sem (fig. ) , these uptake structures appeared as fossae of different sizes, which have not previously been described in the literature. yolk granules and ldl particles were observed by tem, within the cells, in phagosomes, endosomes, and acid phosphatase-containing vacuoles (secondary lysosomes), components of an active intracellular digestion system as it is presently known. the secondary lysosomes contained yolk granules exhibiting various degrees of microstructural alteration (fig. ) . the microstructure of these intermediates in yolk digestion appears to be very similar to that appearing in the literature describing known biochemical manipulations of yolk ex ovo, and indicates that a re- iv- scanning vol. , supplement iv ( ) fig. sem of apices of yolk sac epithelial cells from an embryo incubated for days. yolk granules have entered the cell, and a fossa (f) is observed. mp = microplicae. lationship may exist among structural, functional, and biochemical information. the results of these individual experiments were used to propose a scheme of yolk digestion based on progressive microstructural changes of intracellular yolk. in addition, a transport system for yolk lipid and its digestion products to the embryo was microstructurally demonstrated using ibo. this transport system shared ultrastructural characteristics with that reported for lipid transport in intestinal cells, especially with respect to the formation and transport of chylomicronlike particles. laser scanning confocal microscopy and digital image processing were used to visualize the pattern of de novo blood vessel formation in the quail embryo. stage quail embryos were fixed on the yolk, excised, then immunolabeled with qh , an antibody marker for angioblasts and vascular endothelial cells . after incubation in fluorescein-conjugated secondary antibodies, the embryos were mounted in pbs/glycerol ( : ratio) under a # glass coverslip. specimens were examined on a biorad mrc confocal laser scanning device equipped with zeiss optics. a × objective lens was used to image the entire caudal half of each embryo during the laser scanning step. ten µm optical sections were acquired in a plane parallel to the embryonic axis (en face). the ten optical sections were then collapsed into one composite image using biorad's proprietary software (fig. ) . the image was imported into photoshop ™ software on a macintosh quadra . using this software, the single composite fluorescence image was processed with the "emboss" tool (fig. ) . other image processing routines such as edge tracing, sharpening, and contrast enhancement can also be applied with ease (not shown). in contrast to conventional microscopy, this procedure yields a map of the entire endothelial network of the quail embryo in sharp focus, and with a highly favorable signal-to-noise ratio (fig. ) . while some geometric distortion occurs when ten sections are collapsed into one imaginary plane, the flat- ( ) and ldl particles ( ) are found in separate organelles, and are also found together within the same organelle ( ) . myelin bodies (m) and lipid drops (l), products of intracellular digestion of yolk, are also observed. fig . this immunofluorescence image depicts a map of all the vasculature elements in the caudal half of a developing quail embryo (stage ). the wide vessels on each side of the embryonic axis are the dorsal aortae; lateral to the aortae are two fields of rapidly forming vascular networks. tened nature of the early avian embryo minimizes this problem. also, since the qh epitope (a carbohydrate) is present on multiple cell surface molecules, the image gives a reasonable approximation of vessel morphology and the protrusions of the vascular endothelial cells. the rendering of the data into a pseudo three-dimensional relief provides the observer with a more familiar visual format (fig. ). the type of image shown in figure facilitates comparison of endothelial sprouts and anastomotic foci during formation of the first vasculature in the quail embryo. based on images from embryos at progressively older developmental stages, we suggest that morphogenesis of the lateral anastomotic network occurs by mechanisms that involve angioblastic tractional structuring of the extracellular matrix (madri et al. , montesano et al. , stopack and harris . this hypothetical mechanism more recently has been elaborated upon by vernon and colleagues ( ) . according to the latter workers, cellular responses to morphogenic cues within the highly planar extracellular matrix underlie early vascular patterning. in addition, the digital imaging approach shown here offers an improved method of comparing normal vasculogenesis with experimentally produced malformations (drake et al. ) . ( . - . ). chemical composition and ph did not change during extended heating. the shmp sample was the firmest and the sc sample was initially the softest. the firmness of the sc and tsp samples increased with extended heating whereas the firmness of the dsp sample did not change. meltability decreased with extended heating in all samples, except the shmp sample which had the lowest meltability of all samples throughout the experiment. there were marked differences in the microstructure of the process cheese protein matrices after heating. osmiophilic areas gradually developed during heating and their incidence was related to the melting salt used. after h of heating, they were numerous but considerably smaller in the dsp sample ( fig. ) than in the tsp sample (fig. ) . the highest and the lowest incidence was in the shmp and the sc samples, respectively, which were the samples with the lowest and highest meltability. new york, usa mozzarella cheese contains parallel protein fibres created by stretching the curd in hot water during manufacture. the protein fibres are oriented parallel to the direction of extrusion and are separated by milkfat or whey (kaláb , taneya et al. . by varying manufacturing parameters and storage time, cheeses with different sensory/functional properties, as well as with different microstructural characteristics, can be produced (kiely et al. ). in the present study, the impact of varying the stretching temperature ( °c, °c, and °c) and storage time ( days, weeks, and weeks) at °c on the microstructure of mozzarella cheese was investigated using light microscopy (lm) and scanning electron microscopy (sem). for both lm and sem, samples were fixed in % glutaraldehyde in mm pipes containing mm cacl , before cryosectioning ( µm sections). sections were cut both parallel to the protein fibres (longitudinal) and across the fibres. for lm, sections were stained with ice-cold oil red o and methyl- ene blue. because the fat is not well fixed by aldehyde fixation, slides were held on a bed of ice during staining to prevent fat migration. for sem, the sections were stained with uranyl acetate in methanol, washed in methanol, and stained with lead citrate, dried, mounted on aluminum stubs, and gold-coated. this procedure removed most of the free fat and water/whey, allowing the association of the protein fibres to be clearly visualized. increased stretching temperature increased the size of the fat globules, which was particularly noticeable in cross sections. with °c stretching, the fat globules were relatively small and uniformly distributed across the section, while with °c and °c stretching there were almost two populations of globules: some small and some very large. in longitudinal sections, the protein strands formed during stretching at °c and °c were slender and smooth, while those formed during stretching at °c were thicker and less regular. little difference was seen with storage time in either longitudinal or cross sections for the three different stretching temperatures using lm. with sem, however, after removal of the fat, the ability of the fibres to hold together decreased with storage time. this is best seen in longitudinal sections, where the protein fibres are closely associated in cheese stored for days (fig. ) , and are able to withstand the stress of drying, while in cheese stored for weeks (fig. ) , the fibres have been weakened (presumably by proteolytic activity from coagulant and culture enzymes) and pulled apart as the section dried. indian dairy association, sector-iv, r.k. puram, new delhi, india milk is a canvas of seemingly silent molecular structure. there is a large variety of "resident molecules" with molecular harmony in milk which are of mammary gland origin. in general, the "structural matrix" of milk comprises three classes of biomolecules: molecules in suspension (casein micelles), molecules in solution (lactose, proteins, vitamins, and salts) , and molecules in emulsion (fat globules). because of its excellent nutritive composition, milk is ubiquitous and the most popular "ready-to-consume" food from time immemorial. today, in iv- scanning vol. , supplement iv ( ) fig . different parts of the world, milk from various species of animals is used for food. in the u.s., however, the cow furnishes virtually all of the available market milk, whereas in my country more than % of the total milk produced is of buffalo origin. hence, structural studies particularly on milk and milkfood products made from various animal species are more fascinating and challenging from a global angle. it is the local consumer habits that determine the final structural orientation of the product in the country of origin, especially in a country such as india where to % of milk produced is converted into a variety ( plus) of traditional milk products, using processes such as coagulation (heat and/or acid), desiccation, and formulation. hence, a presentation on the "structural style" of milkfoods which are developed on the basis of "environment-friendly green technology" in the tropical countries might generate a new dimensional need for future studies on milkfood products. a comprehensive literature scan of published papers in the areas of "food" and "food structure" during the last years reveals the following publication status: during the period to : . global: of the total number of , food articles published, , are related to "food structure." the number on "food microstructure" is . when i think of their contributions, i claim no merit for my presentation in this conference today. these data provide a "food-for-thought" why food structural studies do not receive the desired attention. we may ponder this issue. while preparing this presentation, the excellent review article by kalab on food structure and milk products was extensively consulted. keeping in view the existing and reported studies on the subject, the situation in the tropical countries, particularly regarding indian milk such as buffalo milk, deserves special attention. according to kalab the structure of milk and milkfoods determines their properties such as firmness, spreadability, elasticity, viscosity, and susceptibility to syneresis, which are globally recognized as texture. the understanding of the processes both conventional and traditional and their relationship between "structural style" and "textural mood" is important in product development. in the tropical countries, the preprocessing exposure of milk to the environment, that is, temperature, microbes, and humidity has to receive extra attention. the dynamic status and kinetics of molecular interaction in different species of milk should be a vital area of structural studies in asian countries. the structural studies on milk and milkfood are generally conducted by use of microscopy. the methods used are optical microscopy such as fluorescence microscopy and confocal scanning laser microscopy, and electron microscopy consisting of two major types: scanning electron microscopy (sem) and transmission electron microscopy (tem). these techniques meet the particular requirements of the study. the application and findings derived using sem and tem in various milk food systems have been extensively covered recently by kalab . repeating and reproducing the published data may not be necessary for the participants in this conference. however, a brief microcosm might be refreshing, and is hence documented below: . milk casein micelles ( - nm), being small, cannot be seen using a light microscope, but tem shows them as globules that are apparently composed of submicelles. . the casein micelles, if deprived of their stabilizing factor, aggregate and form a gel of a regular structural "crowd." . in the case of acid-gel, the micelles start disintegration as a result of the solubilisation of their structural partners such as calcium phosphate. . there is an interstructural and intrastructural relationship between the suspended casein micelles and soluble blactoglobulins. heat disturbs their stability equilibrium and their molecular relationship. . among the milkfoods, cultural milk products like yoghurt are highly influenced by heat in relation to their "set-style" and "stirred-style." the indian counterpart, "dahi," offers its advantage when made from buffalo milk because of its high curd tension due to high calcium level. this area deserves a close look using sem technique. . as far as cheese is concerned, because of its inherent multinatural processing steps using chymosin on one hand and starter culture on the other, it is an evergreen exploratory platform using either sem or tem. . dried milkfoods as spray-dried milk particles are globular of larger diameter fat-globules, however, they undergo some changes as revealed by sem studies. lactose plays a commanding role in a so-called instantisation process. while making a presentation before a "highly-structured" qualified scientist using sem and tem, it is tempting for me to present our own work of limited nature. in our laboratory, during the last decade, we carried out a sem study on buffalo milk and its casein. the micelles are of larger size and have higher-bound calcium unlike cow casein micelles. regarding the casein fractions, an elaborate study on the b-casein in relation to structure and function as affected by heat and enzymic cleavage were carried out. work relating to microstructure development on another indian milk product, namely "paneer," from buffalo and cow milk has been reported. more recently, while working on casein micelles in csiro dairy research laboratory in australia, the author made an original contribution when ascertaining the micellar integrity of casein micelles in milk. he used a very simple technology not dependent on sem or tem. it was on the "colour communication" property of molecules as an integrated system or in isolation. using this new technique, it is possible to declare the miscellar integrity and to identify milk from different animal species, based on the different miscellar structure-dependent light-reflecting phenomena. the time has come to develop a "global strategy" on milk structural studies with a view first to understand the local product and then to ensure marketing of "rightly-structured milkfoods" of the right type to meet the countries' marketing needs. starch is a major component of many foods and is commonly used to modify the texture of a system. starch gels are composites of swollen gelatinized granules embedded in a continuous amylose network. the rheological characteristics of the starch pastes or gels depend on the shape and swelling power of the granules, amount of amylose and amylopectin leached outside the granule, network entanglement, and interaction between the paste components. many food systems also include in their formulations a lipid phase, thus, starch-lipid interaction becomes a major concern in starch paste rheology. the interactions of triglycerides might be different from those of monoglycerides due to their amphiphilic character, dispersion states, and steric hindrances. triglycerides owe their characteristics to their fatty acid composition (chain length and insaturation) and distribution (davis et al. ) . the objectives of this work were to analyze the effect of different lipid phases on the swelling power of the starch granules, as well as to analyze the rheological behavior of the starch pastes (apparent viscosity and viscoelasticity). commercial corn starch (cs) (refinerías de maíz, argentina) was used as thickener. the lipid phases used were: ( ) shortening (sh) (molinos río de la plata s.a., argentina) containing . % of mono-and . % of polyunsaturated and . % of saturated fatty acids; ( ) sunflower oil (so) (molinos río de la plata s.a., argentina) containing . % of monoand % of polyunsaturated and % of saturated fatty acids, and ( ) commercial glycerol monostearate (gms) mivaplex (eastman kodak co., u.s.a.), which contained % of monoglycerides and % of diglycerides. seven percent starch suspensions with and without lipid phase ( % of sh or so or % of gms) were prepared using a modification of the method of eliasson ( ) . swelling power was calculated as the weight of sedimented gel divided by the original dry weight of starch. samples were placed on slides and micrographed in a leitz ortholux ii microscope with a photographic camera leitz vario orthomat (leitz, germany). the suspensions were gelatinized in a thermostatic bath at ± . °c under standardized conditions. a rotational viscometer haake rotovisko rv (germany) with a sensor mvip of concentric cylinders was used. the measurements were performed at °c and the transient shear stress curves (τ vs. time) were obtained at different constant shear rates (d) from to s − . apparent viscosities were calculated as τ/d ratio at d= s − . iv- scanning vol. , supplement iv ( ) table i shows the swelling power obtained from the starch suspensions with and without lipid phase. when either of both triglycerides, sh or so, was added, the swelling power values were higher than the value of the control (cs). the so, with the highest insaturation content, presented the higher swelling power. the addition of the monoglyceride (gms) led to the lowest value. the relative sizes of the swollen granules shown on the micrographs of figure confirm these observations. gms forms a helical inclusion complex with amylose, which might delay the transport of water into the granule and consequently decreases the swelling power (eliasson ) . the formation of inclusion complexes between the triglycerides and the amylose seems to be difficult because of the steric characteristics of these lipids. apparent viscosities correlated well with the swelling power (table i) ; the larger the swelling power the higher the viscosity obtained from the rheological curves at long shear times. bird-leider model (dickie and kokini ) was used to analyze the viscoelastic behavior and the shear time dependence of the different pastes: where n and m are the power law parameters and b and c are adjustable parameters related to the viscoelasticity and to the structure breakdown of the samples. a satisfactory goodness of fit was obtained (r min= . ) (fig. ) . when the swelling power increases, b parameter decreases (table i) . since viscoelasticity (b) depends mainly on the network entanglement (leached amylose) and the volume of the swollen granules, both factors should be considered to explain this behavior. because of the health risk and other environmental factors that airborne grain dust presents to the working population, our laboratory has initiated studies on the isolation, identification, and characterization of bacteria that possess multiple resistance to a series of antibiotics and other compounds (e.g., insecticides and pesticides) at the molecular level. samples of grain dust were collected from various grain elevators in the duluth-superior regions of the u.s. during the diverse growing seasons. each sample collected consisted of a heterogenous population of constituents that vary with encountered geographic, climatic, and handling differences. in addition, the geographical growth regions and the mechanism of storage of grains appear to be directly associated with the microbial flora and occurrence of toxic substances. scanning electron micrographs of the concentrated grain dusts were morphologically consistent with the observation of previous investigators. the dust from various plants consisted of a distinct assortment of particles; small husk fragments or pericarp (seed coat in case of flax) and "trichrome-like particles" were also present. numerous bacteria spores were seen at high magnification, particularly durum wheat and barley. light photomicrographs showed a heterogenous population of both gram negative and gram positive bacteria. the bacteria consisted of different shapes such as short rods, long rods, and cocci. transmission electron photomicrographs revealed the isolated strains to consist of one or more flagella attached to the membrane surfaces. at least three of the bacterial strains isolated were encapsulated. we exposed, selectively, twelve of the isolated bacterial strains to a variety of antibodies, pesticides, and insecticides. as a result, of the bacterial strains tested showed resistance to both ampicillin and bacitracin ( µl/mg). of the strains, showed resistance to insecticides (sevin) and the pesticides (enforcer) as high as µl/mg. three of the five strains that were resistant to both ampicillin and bacitracin were also resistant to the insecticide sevin at high concentrations ( µl/mg). these data suggest that bacteria found in grain dust may be directly or indirectly related to occupational health disorders. chlordiazepoxide (librium) is a commercial antianxiety agent, but its use has not been associated with any striking health improvements. nevertheless, millions of chlordiazepoxide (cdz) prescriptions have been written and the drug is widely employed clinically as a muscle relaxant, anticonvulsant, anxiolytic, and hypnotic. adverse effects vary from skin rash, nausea, headache, and impaired sexual functions to vertigo and lightheadedness, as well as complications from the drug's administration during pregnancy. the ciliated protozoan, tetrahymena pyriformis, has been of significant importance to research since its successful axenic culture years ago. the drug's tranquilizing effects on humans may possibly be elucidated by investigating cdz-induced impairment of the protozoan's growth and motility through modifying microtubular-directed ciliary function (bell et al. ) . here, uptake, recovery, and attempted localization of cdz, as well as its alterations of cellular ultrastructure are reported. thin layer chromatography of cdz (hoffman laroche, nutley, n.j.) in three different mobile phases revealed optimal cdz stability when dissolved and stored in isoamyl alcohol; benzene. after days of liquid culture, t. pyriformis removed % of the radioactivity from [ c] -cdz in the growth medium. liquid scintillation counting of cell washes during the -day time course suggested surface, nonspecifically-bound radioactivity. following days of culture, [ c] -cdz together with its metabolites and/or breakdown products were recovered from homogenates of tetrahymena. to detect the intracellular sites of cdz localization and possible action, both transmission electron microscopy and immunoelectron microscopy were performed. the former revealed that cdz disrupted cytoplas-buffer or cacodylate-buffered dopa revealed dopa/ppo reaction product but failed to reveal a definitive, substrate localization of the enzyme. instead, cytoplasmic morphologic distortions of buffered dopa-treated hyphae were apparent, mandating modification of the employed higher-plant, cytochemical procedure for localizing fungal ppo. thus, attempts to establish the route of ppo secretion to the growth medium utilizing liquid cultured hyphae may be of limited value as the sheath appears to be sloughed during shake culture. in this connection, c. versicolor grows upon a solid substratum when decaying timber in situ. support: doe-bctr program. the evaluation of surface topography is conventionally done with the scanning electron microscope (sem) by visually reconstructing stereo images in a stereo viewer. the monocular clues in an sem image, shown for example in the image in figure , can also be used to map the topography of a sample surface. the first attempt to identify the surface gradient from a secondary electron detector in this way was reported by suganuma ( ) who found that ( ) and described the inclination of the surface to the specimen plane, where a and b are the signal outputs from the two detectors and a n and b n are the signal outputs from the same material when it is perfectly horizontal. although it is possible to add additional detectors and to modify their signal outputs in a conventional electron microscope, the technique can also be implemented in a straightforward manner by using the differences in signal intensity from the two halves of a split backscattered detector. thus if a and b are the signal outputs from the two different halves of the detector, the backscattered signal, which is usually collected as topographic information (a − b) or composition information (a + b), can be multiplied together (a − b ) to produce the basis of the empirical relationship described by suganuma iv- scanning vol. , supplement iv ( ) fig . ( ) to fit the contrast variations. indeed, since the denominator in this equation is also a constant and there is invariably a flat spot somewhere in the image, it is easier to simply use the expression to try and describe the surface gradient. this function can then be scaled to an absolute magnitude of about to take maximum advantage of the contrast variations that exist. the simplest way to evaluate the surface topography without actually modifying the microscope is to process a digital image in a computer. such an image can be collected directly or a polaroid micrograph can simply be scanned into the machine. in either event the processing is fairly straightforward to accomplish the use of software such as the nih image program. the surface profile is obtained by integrating the surface gradient over the scan distance, and in figure we show, by way of an example, a faceted surface displayed as a conventional secondary image, as a topographic profile, and as a grey scale image indicating the faceted surfaces with the same orientation to the electron beam direction. suganuma t: measurement of surface topography using sem with two secondary electron detectors. j electron microsc ( ), - ( ) corn kernels of an unknown italian cultivar were surfacesterilized in full strength bleach ( . % sodium hypochlorite), rinsed in sterile distilled water, and germinated on difco potato dextrose agar (pda) under fluorescent light at room temperature. isolation of bacteria was made either directly from the rhizosphere of seedlings or from colonies produced on agar. bacteria were identified by their fatty acid profiles (microbial identification system, newark, del.) and diagnostic biochemical test (micro-id, durham, n.c.) . fusarium moniliforme used to test for bacterial antagonism were isolated from corn. experiments designed to infect seedling roots with the isolated bacterium were performed on corn kernels that were subjected to a double sterilization process in which the kernels were surface-sterilized with bleach and then subjected to a mild heat treatment to remove internal bacteria and fungi. this process produced sterile seedlings which remained so at least up to the to leaf stages of growth. the corn cultivars used were trucker's favorite, silver queen, reid's yellow dent, and an inbred cultivar, pr. samples were fixed in % glutaraldehyde in sodium cacodylate buffer and postfixed in % osmium tetroxide in buffer. the tissues were dehydrated in an ethanol series, critical-point dried and coated with gold-palladium or embedded in spurr's medium and sectioned. samples were examined using a philips scanning or jeol cx transmission electron microscope. the bacteria isolated from roots of the italian corn cultivar were gram-negative rods and were further characterized as being positive for voges-proskauer reaction, nitrate reductase, and ornithine decarboxylase and b-galactosidase. the isolate fermented arabinose and malonate. it was negative for phenylalanine deaminase, lysine decarboxylase, urease, h s, and indole production. it lacked the ability to utilize adonitol, inositol, and sorbitol, and was negative for esculin hydrolysis. these biochemical characteristics served to place this bacterium in the family enterobacteriaceae, the tribe klebsielleae, and it was identified as e. cloacae according to ewing ( ) . microscopic studies established that the bacterium was endophytically associated with the roots of the corn cultivar. scanning and transmission electron microscopy demonstrated that the bacterium was distributed uniformly over the corn root epidermis but was randomly distributed intercellularly in the root cortex and outer margin of the pericycle, usually adjacent to phloem cells. although there was a proliferation of bacterial cells in the intercellular spaces of roots (figs. , ) , there was no evidence of damage to host cells, decline in seed germination, nor seedling growth from e. cloacae infection during the week observation period. the presence of the bacterium on kernels of several corn cultivars enhanced the growth of corn seedlings and inhibited growth of f. moniliforme when the bacteria-infected kernels were germinated in fungal-amended soil. this indicated that e. cloacae is biologically associated with corn plants. the bacterium exhibited strong antagonism to several f. moniliforme isolates when grown on nutrient agar and pda plates. the fungus growth was inhibited and restricted to aerial growth on agar plates. the endophytic nature of e. cloacae in corn roots and its antagonism against isolates of f. moniliforme indicate that this bacterium has potential for the biocontrol of f. moniliforme, a corn pathogen. tracheal organ cultures have been widely used for the demonstration of the cell-and organ-destroying capacities of bacteria and viruses colonizing the respiratory tract. however, the assessment of the cell-damaging abilities of fish parasites, the gill organ culture appeared to be a suitable tool (stadtländer and kirchhoff ) . piscine gill epithelium represents the relevant target tissue for cytotoxicity studies of fish parasites, but this tissue is diffi- iv- scanning vol. , supplement iv ( ) fig cult to cultivate in vitro. excessive mucus production complicates the investigation with the light and electron microscope. this is even more difficult when infected specimens are investigated. here, cell debris due to released cells and cell fragments require special attention of the investigator to obtain interpretable electron micrographs. a fine balance is required between preservation of the status quo of the in vitro situation and the necessary procedures (multi-step preparation) for scanning electron microscopy (sem). in this abstract, we describe the detailed and improved preparation of piscine gill epithelium for sem. this will allow other investigators using piscine tissue for research to obtain scanning electron micrographs with high resolution at high magnification. gill filaments were obtained from rainbow trouts (salmo gairdneri, richardson) and were infected in vitro with mycoplasma mobile k, a wall-less prokaryote causing severe damage in gill organ culture (stadtländer and kirchhoff ) . each gill filament (noninfected and infected) was rinsed in several changes of . m cacodylate-trihydrate [cacodylic acid buffer (cb)] at ph . to free the tissue surface from mucilage, medium, and unattached mycoplasmas. the washing step turned out to be critical for obtaining satisfactory results. excessive washing led to complete removal of cell debris and did not reveal the same status quo of infection as seen with the light microscope during the in vitro cultivation of gill filaments. on the other hand, insufficient washing complicated the interpretation of electron micrographs, especially the identification of m. mobile on gill epithelium, due to excessive mucus and cell debris released from infected tissue. the pre-fixation was performed with . % glutaraldehyde (v/v) (in cb) for h at ˚c. the aldehyde was removed by careful rinsing in cb (three times for min). samples were not postfixed, but dehydrated in acetone covering a graded series of solutions (acetone/water) from through % (v/v). all specimens were critical-point dried (cpd) using carbon dioxide as the transitional fluid. cpd was performed in a critical point drying apparatus e (polaron equipment ltd., england) by filling the chamber with / of liquid carbon dioxide. the heating process was conducted in ˚c per min steps until the critical point was reached ( o c and bar). samples were immediately placed in an exsiccator containing anhydrous calcium chloride to avoid rehydration of samples by air contact. after being attached to aluminum mounting stubs using double-sided sticky tape, each specimen was coated with a layer of to nm of gold in a hummer v sputter coater (technics inc., alexandria, va., usa). samples were examined in an etec auto scan b (etec, calif., usa) operating at an accelerating voltage of . - kv. results were documented on a polaroid × land film (type , positive-negative). the method described above allows the detailed investigation of noninfected and infected piscine gill epithelium at high magnification with an sem (fig. , ) . good structural preservation of noninfected, apparently intact secondary lamellae (fig. ) and destroyed lamellae after infection with m. mobile (fig. ) document the usefulness of the described method for the study of the infectious process with this mycoplasma on fish tissue and will certainly also give convincing results in investigations with other fish pathogens. the arrows in figure indicate the presence of the flask-shaped mycoplasma on damaged piscine gill epithelium. bars represent figure : µm, figure : µm. for this study, the sem was superior to light microscopy (lm) and transmission electron microscopy (tem). lm does not demonstrate details in the damaged tissue and tem is much more laborious and time consuming. the mammalian bladder functions in urine storage and expulsion and is thus subject to alternating distension and contraction. blood flow in the bladder wall is compromised by distension (dunn , levin et al. , and acute and chronic overdistension result in ischemia and necrosis, respectively. the latter often leads to loss of mucosal integrity. yet, the vasculature of the bladder wall has rarely been studied (inoue and gabella ) except at the gross level. in this study we describe several unique features of the bladder microvascular anatomy using light microscopy (lm), transmission and scanning electron microscopy (tem and sem), vascular corrosion casting, and alkali digestion (takahashi-iwanaga ). twenty four male new zealand white rabbits were anticoagulated, anesthetized, and cannulated via the abdominal aorta. for routine lm, tem, and sem, the bladder vasculature was flushed with buffered saline at physiologic temperature and pressure, then perfuse-fixed with buffered glutaraldehyde; for corrosion casting, the vasculature was filled with resin (mercox/methylmethacrylate monomer, / ); and for alkali digestion, tissue was treated with n naoh at ˚c for min. thin sections were stained with lead and uranium, and casts and digested tissues were mounted on sem stubs with silver paste or colloidal carbon and viewed at - kv. the bladder is supplied by left and right vesicular arteries, branches of the internal ( %) or external ( %) iliac arteries. within the adventitia, the vesicular arteries send coiled branches dorsally and ventrally over the bladder surface. secondary arteries penetrate the muscularis to supply a rich capillary plexus (fig. ) closely apposed to and located in grooves within the base of the transitional epithelium (fig. ) . capillaries measure - µm in diameter and are often fenestrated and invested with pericytes. veins exhibit abundant valves primarily in the basal half of the bladder. the lamina propria of the bladder consists of very loose, flexible, collagenous connective tissue. unusual capillary "glomeruli," associated with accessory vessels paralleling the primary bladder vessels, are present in the adventitia on either side of the bladder wall. these glomeruli consist of one to four contiguous capillary spheres. these various methods provide a clear, three-dimensional view of the microvasculature of the rabbit bladder, reveal the very close association between the urothelium and the underlying capillary plexus, and describe the fine structure of the mucosal capillaries. several unique features of the bladder vasculature including capillary glomeruli require further char- iv- scanning vol. , supplement iv ( ) acterization. the latter may be associated with sensory ganglia related to pressure sensation, but their function has not been determined. these results form the basis for comparison of normal bladder vasculature with that of experimentally compromised vasculature. the epithelial cell layer lining the nasal turbinates of humans is functionally and histologically consistent with that of the lower airways. this anatomic site is easily sampled using an inexpensive, disposable curette, and the epithelium obtained can be evaluated for both clinical and experimental objectives. the original rationale for ultrastructural evaluation of clinical specimens of nasal epithelium in this facility was to document index ciliary lesions consistent with the diagnosis of primary ciliary dyskinesia (pcd) (figs. , ) among individuals considered at risk. patients referred for study encompassed both adult and pediatric populations and had lifelong histories of chronic sinusitis, bronchiectasis, and/or otitis media, but with normal immunoglobulin levels. several patients presenting with situs inversus, polysplenia, or infertility problems-clinical findings considered risk factors for pcd-also were evaluated. approximately nasal biopsies are submitted annually for evaluation, of which approximately % demonstrate impaired ciliary motion and ultrastructural abnormalities that could be the basis for altered ciliary motion and a diagnosis of pcd. this finding is consistent with the suspected prevalence of pcd in the general population although the rate appears higher among individuals of scandinavian descent, pacific islanders, and inbred populations. the diagnosis of pcd is confirmed by the electron microscopic documentation of any of three ultrastructural level lesions of airway cilia. these lesions are: ( ) dysmorphology of dynein arms, ( ) absent radial spokes, and ( ) microtubular transpositions. in our experience, dysmorphology of the dynein arms represents the major form of ciliary abnormality associated with pcd. missing radial spokes and microtubular transpositions have not been documented among any of our patients. these clinical studies also have provided a unique opportunity for defining a spectrum of ciliary abnormalities which distinguish the heritable primary ciliary abnormalities associated with pcd from acquired cil- iary abnormalities associated with chronic disease or acute injury due to infectious processes or exposure to irritant gases in the ambient air. in addition, other histopathologic features such as mucus cell hyperplasia have been encountered occasionally among children presenting with chronic respiratory disease and referred for evaluation. in summary, our experience shows that ultrastructural evaluation of nasal epithelium can provide a clinically significant perspective on respiratory health. this work was supported by scor grants hl , hl , and hl from the national heart, lung, and blood institute and by u. s. environmental protection agency cooperative agreement cr to philip a. bromberg. this is an abstract of a proposed presentation and does not necessarily reflect epa policy. using conventional light microscopy, silicone has been described as a translucent, clear, mucoid, refractile material which is often difficult to visualize. it is not birefringent, as is silica, by polarized light microscopy. silicone gel tends to form homogenous, rounded "globules," "vacuoles," or "droplets" unlike the angulated, sharp spicules observed with silica. in paraffin-embedded tissue sections, silicone gel often appears to be slightly out of the plane of focus. in addition, silicone gel "globules" occasionally drop out of standard µm histologic sections during tissue processing, leaving partially or totally vacant holes or "cysts." because silicone extravasation and deposition into surrounding fibrous breast capsules is difficult to visualize by standard light microscopy techniques, the electron microscopist must often "blindly" examine multiple fields/grids in a labor-intensive fashion. to decrease the time generally required for the positive identification of silicon by electron probe microanalysis (epma), alternative light microscopy techniques for preliminary screening purposes were investigated. six periprosthetic capsulectomy specimens, synovium from a previously reported silicone breast augmentation patient with arthritic pain and a silicone granuloma from another patient with a ruptured prosthesis were utilized in this study. sections were cut at , , , and µm and mounted on glass slides. in addition to standard permount mounting media, aqua-mount was stained with ink preparations: black stamp pad ink, india ink and aniline artist dyes-black, blue, brown in a : mixture for coverslip application. each series was stained with a battery of common histochemical stains. the sections were then viewed with a zeiss axioplan microscope utilizing conventional incidental light, polarized, non-koehler, phase contrast, and darkfield microscopy. a commercial silicone gel and silicone gel extracted from a previously implanted silicone breast prosthesis were smeared and examined unstained or stained with papanicolaou and "diff quik." silicone was noted to be refractile, nonpolarizable, and nonstainable. the results confirm the refractile, nonpolarizable, and nonstainable properties of silicone in histologic and cytologic preparations (fig. , scale bar = µm). decolorizing techniques (toluidine blue o, etc.) that were not completely differentiated, especially with thicker sections, occasionally demonstrated nonspecific dye "trapping" on the larger silicone globules. the relative ease of silicone localization was greatly increased in histologic specimens with non-koehler, phase contrast, and darkfield microscopy when compared with conventional light microscopy. although standard h&e staining was adequate for silicone localization in tissue sections, uniform dark staining with toluidine blue o increased the contrast between the stained tissue and the unstained refractile silicone. the contrast was also enhanced by aqua mount black stamp pad ink mounting media, with the silicone appearing milky white. in thicker sections, negative staining was slightly accentuated because of the increased concentration of stain as well as thicker, more refractile silicone globules, especially with darkfield microscopy. by utilizing these sensitive screening techniques, we were then able to sample paraffin-embedded tissue selectively, which dramatically decreased the time and effort for correlating confirmatory identification of silicon by epma (fig. ) iv- scanning vol. , supplement iv ( ) the majority of biological samples are high in water content. preparing tissues for scanning electron microscopy (sem) requires removal of this water and often produces severe structural distortions due to surface tension forces during phase transitions. this can result in bulk shrinkage artifact, surface cracking, curled cell borders, clumping or flattening of cilia, and collapse of surface vesicles. failure to postfix biological material with oso can lead to extraction of surface membrane lipids during solvent dehydration. it is likely that many shrinkage artifacts are related to incomplete or improper fixation and drying. critical-point drying (cpd) has become the standard procedure for most biological materials. although it produces a relatively intact end product, wollweber et al. have reported shrinkage of as much as % after cpd of glutaraldehyde and osmium-fixed macrophages and lymphocytes. the use of mordant techniques to complement or enhance oso and uranyl binding may aid in preserving fine structural details regardless of the drying method. numerous substitution/transition fluids have been introduced for both critical-point and direct-evaporative drying, some with more success than others. an advantage in using solvent drying techniques as alternatives to cpd is the ability to process large numbers of samples simultaneously. early attempts at direct drying of nonosmicated samples from ethanol and other solvents produced a generalized cell shrinkage and collapse. freon evaporative drying was shown to be a useful rapid drying technique by liepins and de harven. gamliel further refined the freon direct-drying technique to include the use of guanidine hcl as a bifunctional mordant, prior to osmification, to minimize shrinkage of leukocytes. direct drying from hexamethyldisilazane(hmds) was introduced by nation to dry insect tissue and has subsequently been used for drying various other tissues (adams et al. ) . peldri ii has since been shown to be an effective solvent drying medium. it is a solid at room temperature. a comparative study by bray et al. of peldri ii, hmds, and cpd, using both plant and animal tissues, has produced identical results with animal tissues but not plant tissues. the purpose of this study is to introduce acetonitrile as a potential solvent for direct evaporative drying. acetonitrile has been used as a less toxic propylene oxide replacement transition fluid for transmission electron microscopy (tem) dehydration and infiltration (edwards et al. ) . to date, no studies have been published indicating the of use of acetonitrile as both a dehydration and intermediate transition fluid for direct solvent-drying of tissue-cultured cells for sem. this study compares solvent drying of kb (hela) cells from freon , peldri ii, hmds, acetonitrile, and ethanol to critical pointdried cells grown on thermanox coverslips. the mordant (gtgo) technique of gamliel is included to illustrate its usefulness in shrinkage control. solvent-plasticware compatibility should always be tested before preparing cultured cells for sem; whenever possible, the use of glass is best. cells grown on thermanox coverslips were fixed in . % glutaraldehyde, . m cacodylate, . m sucrose, . mm cacl , ph . . the gtgo fixation protocol of gamliel was found to be the most useful one in preserving cell architecture (figs. , ) . in figure , cells were evaporative-dried under a mild aspirator vacuum after gtgo fixation and dehydration in acetonitrile ( %, %, %, %- ×, min each). as a comparison, cells in figure were identically fixed and criticalpoint dried after ethanol dehydration. the overall appearance of the cells from both treatments is similar at low magnification (figs. a, a) , with some shrinkage evident in either case. under these conditions, it appears that the cpd cells retain more fine structural details than those evaporative dried from an (figs. b, b) . the results of this study are encouraging and may lead to future studies to develop a simple fixation protocol which enables evaporative drying from solvents directly miscible with water such as acetonitrile. snow, which may occasionally cover up to % of the earth's land, supplies about one-third of the water that is used for irrigation and the growth of crops (gray ) . for this reason, estimating the amount of water in winter snow pack is an extremely important forecast activity that attempts to predict the amount of water that may be available for the following growing season. unfortunately these estimates can be easily iv- scanning vol. , supplement iv ( ) fig . confounded by the sizes and shapes of the snow crystals that comprise the snowpack. a snow crystal is a single frozen ice grain that generally results from a process known as nucleation in which atmospheric water vapor condenses on a solid particle or nucleus at temperatures below °c. when nucleation occurs, the water molecules form a hexagonal crystal lattice resulting from the specific orientation and binding that occurs between the oxygen and hydrogen atoms. depending on the temperature and moisture that prevails during formation and descent of snow crystals, nucleation may result in plates, stellar crystals, columns, needles, or dendrites-all of which are based on the hexagonal lattice structure. an individual snow crystal may range in size from µm to mm (gray ); aggregations of two or more of these crystals form a snowflake. the shapes of snow crystals have been extensively studied and photographed with the light microscope (bentley and humphreys , nakaya ) . although these studies have resulted in a classification system that currently recognizes distinct classes and over subclasses of snow crystals (hobbs ), detailed examinations have been hampered by the difficulty of working with a frozen specimen, which is susceptible to sublimation and melting, and by the limiting resolution of the light microscope. for these reasons, an attempt was made to determine whether snow crystals could be collected/stored and prepared for observation and recording in the low-temperature sem. attempts to allow snowflakes merely to settle on a precooled specimen holder were unsuccessful; the snowflakes tended to "bounce" off the holder and those that did alight did not remain attached during subsequent handling. a successful procedure consisted of placing a thin layer of methyl cellulose solution on a holder and precooling it to the prevailing outside temperature during a snow fall. next, snowflakes were allowed to settle on the surface of the methyl cellulose solution. after a few minutes, the holder was plunged into liquid nitrogen and transferred to the laboratory where it was retrieved from liquid nitrogen, mounted on the transfer rod of an oxford ct- hr cryosystem, moved into the prechamber for sputter coating with au/pd, and then inserted into a hitachi s- field emission scanning electron microscope (sem) equipped with a cold stage that was maintained at − °c. these procedures allowed us to observe several forms of the individual snow crystals as well as their nucleation centers. at low magnification, the specimens, which did not appear to be altered by the sputter coating, resembled those that had been previously photographed with the light microscope. the snow crystals were stable in the beam, did not sublime, and could be observed at magnifications of , × or more to reveal microcrystalline water deposits or rime on the surface of some the snow crystals. this procedure, which was used to collect specimens during several snow falls in beltsville, maryland during the - winter season, was also capable of preserving sleet, graupel, and hail. furthermore, storage holders were devised that allowed capture of the snowflakes and their storage in liquid nitrogen until the specimens could be processed for examination in the sem. finally, the specimen stage of the sem allowed specimen tilt so that stereo images of the snow crystals could be recorded (fig. ) . in conclusion, low-temperature sem is a viable technique for examining snow crystals at magnifications that far exceed the resolution of the light microscope. furthermore, the ability to collect and store samples enables investigators to accumulate samples from numerous locations or different time intervals so that detailed observations and comparisons can be done in a convenient and orderly manner. these were identified and returned into the corresponding reconstruction sections in an automatic tracing procedure, programmed and executed on vidas . (zeiss/kontron germany). in addition it is possible to obtain a plastic, -d-like impression by applying the rcm with oblique illumination. this is achieved by decentralization of stach's slide (central diaphragm). . two main problems in -d reconstruction of histologic specimens are the horizontal distortion during the preparation of serial thin-tissue slides and the following vertical readjustment (alignment). we have recently shown that optical sections can be obtained by rcm within thick tissue-slides. its confocal-like principle provides an alternative to mechanical slices. the preserved integrity of the examined object allows the precise movement within the optical axis in one (vertical) dimension, thus avoiding manual alignment. applying the rcm on histochemical and immunohistochemical stains, reflections can be observed within the tissue. the depth of penetration of the light beam amounts to approx. µm, equivalent to about optical tomolevels (fig. ) . using the example of neurons of the supraoptic nucleus of the rat, we demonstrate this new technique on chrome-alum haematoxylin stained neurosecretion. the reflections of the dye particles associated with neurosecretory granules allow the precise localization of these subcellular structures. the visualization of the neurosecretion and its distribution is more distinct and of sharper contrast than in bright-field microscopy. reflected light is like a binary signal and therefore generates a suitable prerequisite for automatic discrimination in greyscale image analysis. thus identified black and white negatives were reconstructed with the module rec d on vidas . . this paper introduces two extended applications of the rcm for -d reconstruction. the generated optical slices and isohypses allow qualitative and quantitative investigations of intracellular structures and surfaces. quantitative chemotaxis is of great interest in a broad field of cell research (e.g., receptor-ligand interaction, ionic channels, cytoskeletal and metabolic processes, embryogenesis) as well as in clinical studies (e.g., immune reaction, wound healing, infection, tumor invasion). the most accepted assay to measure chemotactic behaviour of cells is the boyden filter assay, in which cells move against a gradient of chemical or biological substances. the chemotactic response is analyzed by measuring either the distance travelled by the leading front of cells or by quantifying the number of cells on the lower surface of the filter. this method is laborious and limited by the observer's subjective errors. we have developed a computer-based image analysis system to estimate the three-dimensional distribution of the migrated cells inside the filter. using one-micron optical sectioning, we can determine the position, size, and shape of many individual cells. within min, the position of thousands of cells can be recorded and the migration profile in the filter can be determined. it is possible to distinguish between different cell populations by selecting particular cell parameters. the system consists of a nikon labophot- a microscope with video microscopy and programmable focus control, used in connection with a hasotec-image processor with fast data acquisition and user-friendly software (mswindows). the high throughput, the consistent accuracy, and the simple operation of the system optimize all aspects of routine chemotaxis analysis in basic research and clinical studies. the atomic force microscope (afm) is being used increasingly in the life-sciences field. with this increase in usage, a concomitant increase in the need for both better developed specimen preparation techniques and better defined operational parameters for the afm instrument has occurred. lifesciences afm methodology can be divided into three main areas: ( ) choosing the appropriate support substrate for the specimen, ( ) choosing the most appropriate immobilization techniques for attaching the specimen to the support substrate, and ( ) selection of the optimum instrument scan parameters to ensure reliable transfer of data from the specimen to the image. of central importance to life-science afm is the nature of the substrate to be used. the most reliable substrates are those with a well-documented surface, whose features are at least an order of magnitude smaller than the specimens of interest. furthermore, the material should be transparent so that other forms of microscopy can be used (i.e., light, fluorescence, near-field scanning optical, etc.) and the surface chemistry should be both well documented and susceptible to chemical derivitization. the two most common substrates to fit the above descriptions are glass and freshly cleaved mica. both of these surfaces can easily be manipulated to produce a wealth of reactive primary amines, either by coating the substrate with poly-l-lysine or by treatment with -aminopropyltriethoxy silane (aptes). an analysis of the modified and unmodified substrates show that treatment with either aptes or poly-l-lysine resulted in an increase in surface roughness (ra). based on the roughness information, we recommend modified mica as being suitable for biological material ranging from whole cells to dna, whereas modified glass is unsuitable for samples with heights < . nm. the red blood cell (rbc) cytoskeleton was examined by atomic force microscopy (afm). samples were placed on either glass or mica and imaged in air. no fixative or stain was used; this allowed modification of the samples between images so that molecular components could be identified. the meshlike structure, which is observed when the rbcs are lysed, is identified as a complex of the cytoskeletal integral proteinsspectrin, actin, and band . . the identification was accomplished by imaging the intact cytoskeleton, then treating the sample to selectively remove these proteins and re-imaging the sample. to support the identification of the cytoskeletal proteins further, images were obtained of samples which had been treated with detergent to remove the lipid membrane and leave the cytoskeleton behind. this is the first study of the intact rbc cytoskeleton which identifies specific proteins. more generally, it shows that afm is a useful tool for examining biological systems in their native state since sample preparation is simple and, once attached to the substrate, the sample can be treated in a variety of ways. we have reported some features about the structural and morphologic changes in nylon transformed from β-alanine single crystal and nylon polymerized from ε-amino-ncaproic acid one, which are members of the ω-amino acid family, through the solid-state polycondensation procedure. it was also found that p-aminohippuric acid [n-( -aminobenzoyl)glycine] single crystal could be converted into an aromatic polyamide crystal by heat treatment below its melting point. in this report, crystalline and morphologic structures of polyglycine produced from glycine (the simplest amino acid) single crystal were examined mainly by means of scanning electron microscopy (sem) and x-ray diffraction technique. the monomer solution was prepared by dissolving g of commercial glycine powder into ml of distilled water at °c. the monomer single crystal was precipitated at °c from the solution, which was transparent and prism-shaped. definitive cleavage planes were observed in the monomer crystal, which is found to be parallel to the a-c plane in the αform crystal of glycine. it has been reported that such cleavage is caused by the characteristic structure of α-form glycine crystal. the monomer single crystal was used as the original specimen for the polycondensation reaction, where the original specimen was annealed in decaline at and °c up to h. morphology of the polymerized material was observed by using a hitachi s- sem with accelerating voltage of kv after being coated with gold. x-ray photographs were taken by a flat camera mounted on an x-ray generator with ni-filtered cu-kα radiation, where the crystal took three orientational positions so that a-c (cleavage plane), a-b, and b-c planes made a right angle with the incident beam, respectively. figure shows two types of x-ray diffraction patterns from a specimen polymerized at °c for h with the a-c plane perpendicular (fig. la) and parallel (fig. lb) to the incident beam, where patterns reflected from the polymerized material were observed as well to have spots from the original monomer single crystal. in figure la , two strong arcs are observed, which are indexed as (inner reflection) and (outer reflection) from type-i modification of polyglycine crystal. a more complicated diffraction pattern is shown in figure lb , which seems to be a fiber diagram. almost all molecular chains in polyglycine-i crystal are considered to be normal to the a-c plane and parallel to the hydrogen bond in the original monomer single crystal. figure a shows a sem photograph for the surface of the original monomer crystal. lamination layers of lamellae are observed on the cleavage plane, edges of which are parallel to the b-axis of the monomer crystal. voids were observed in the grain boundary or crack region. a sem picture of the specimen polymerized at °c for h is shown in figure b , where the laminal materials are seen to overlap each other crosswise. such intersecting laminal structure seems to be responsible for the biaxial crystalline orientation observed in the x-ray diffraction pattern shown in figure la. in the case of the specimen polymerized at higher temperatures, the fibrillar structure was observed over the surface. figure c shows the results for the specimen annealed at °c for h. hiroshi toyoda, takashi itoh, hiroshi sakabe, takashi konishi department of polymer science, kyoto institute of technology, kyoto, japan it is well known that polytetrafluoroethylene (ptfe) is produced by emulsification polymerization in the form of powder or aqueous dispersion to be processed industrially. the dispersion-type material is mainly composed of fibrillar and/or lamellar crystallites, where the spherulite structure generally is unseen because of stiffness of the molecular chain. such morphologic feature is considered to be responsible for the remarkable repellent property of the polymer to liquid. the authors prepared thin films of ptfe from the aqueous dispersion to observe change in the fibrillar structure through heat treatment, considering that from such a viewpoint the fibrillar morphology in ptfe is the aspect that is most different from other polymeric materials. the polymerized ptfe used in this study was produced by dupont ltd. a glass plate was dipped into the solution and then withdrawn vertically. the thin film was prepared by drying the solution on the plate at room temperature. the heat treatment was performed at , , , , and °c for h in an air-drying oven. the samples were immediately quenched in ice water (cooling rate: , °c/min) or cooled in air at controlled rates ( , , l, . , and . °c/min). any serious oxidation and/or degradation effects were not observed even during annealing at °c. a scanning electron microscope (sem)(jeol, jsm- lv) was mainly used to observe the au-coated surface structure of the sample, when an accelerating voltage was set at kv to make the resolution of the sem high. composite materials were produced by coating ptfe on textile of glass-fiber and that of carbon fiber. after the coating and subsequent annealing at °c for min, the material was cooled to room temperature. the surface was found to be covered with balls of a diameter of about nm, composed of several hundred nodules of a diameter of about nm as shown in figure . in this sample, the degree of crystallinity of ptfe was about % and no fibrillar structure was observed. by increasing the annealing temperature to > °c, the fiber bun- iv- scanning vol. , supplement iv ( ) dles grew up. such phenomena may be explained by stiffness of the molecular chain including f instead of h, that is, the chain does not fold even if the molecular motion is stimulated at elevated temperatures. any sheaf-like or microspherulitic structure (a precursor of the spherulite), which is often observed in thermoplastic polymers such as polyethylene, did not appear in ptfe. when decreasing the cooling rate, the fibrils grow laterally and interfibrillar space tends to become larger. it is unknown how such space affects the physical properties of the material. thinner fibrils appeared, which connected the original thick ones, in the case of . °c/min cooling as shown in figure . such morphologic structure characteristic of ptfe seems to be responsible for the processing efficiency. schott glaswerke, department of instrumental analysis and mineralogy, mainz, germany fused-cast refractories of the zac-type (zirconia-alumina cast) are of great importance in the manufacturing of glass. especially glass-tanks for the melting of specialty glasses are built with these materials. zac refractories are produced by melting the raw materials at very high temperatures and subsequently casting them into suitable molds. on cooling, part of the material crystallizes, forming al o (corundum) and zro (zirconia, baddeleyite), where the zirconia crystals are also growing within or into the corundum crystals. besides the crystalline phases there also exists a glassy phase, composed mainly of sio , al o , and zro , and small amounts of alkali (na o, k o), tio , and fe o . this microstructure of the material must have an influence on the corrosion behavior when subjected to the aggressive melt in a glass tank. the aim of this work was to establish a method to characterize quantitatively the microstructure of these refractories. the inhomogeneous nature of the material can already be discerned in the light microscope; however, the resolution is not sufficient to measure the sometimes very small (< µm) crystal sizes of zro , not to mention the determination of a form factor! therefore, the electron microprobe (epma) was used to perform this work. the instrumentation used was a jeol jsm- scanning electron microscope, equipped with an optical microscope for reflected and transmitted light, motorized stage (x, y, z), and coupled with a combined eds/image analyzing system voy-ager from noran, which also controls the stage movement. the following parameters of the microstructure, for example, the geometric and chemical properties of the crystalline and glassy phases, were to be determined: maximum and minimum diameters, area of the individual phases and fractional area within the material, form factors and orientation to a reference plane, and the chemical composition of the glass phase. furthermore, it was necessary to distinguish between the zirconia within the corundum and the more isolated crystals. since the material is inhomogeneous, a rather large number of image fields had to be analyzed (at magnifications of × and higher the individual image field is very small!); as a consequence, the whole procedure had to be automated as much as possible. sample preparation: a flat and polished surface of a section of the material had to be produced. although the grinding and polishing was done with diamond wheels and paste, a certain amount of relief between the hard corundum and the "soft" glass is unavoidable. the first step in analyzing the microstructure is to recognize/discriminate the three phases in the material. the signal used for image formation and subsequent phase discrimination is the backscattered electron (bse) image; however, the abovementioned relief due to differences in hardness made the distinction between glass and corundum impossible, since (a) brightness differences between glass and corundum are not very strong in the first place, and (b) edge effects in the corundum crystals showed brightness values of the glass, and vice versa (discrimination, i.e., tranformation of a particular phase into a binary image, is based on such brightness differences). contrast enhancement or image filters were not sufficient for clear separation of these two phases. therefore, for the recognition of the glass phase, an element distribution for si (x-ray map) was used; combined with the image of zro , which is easily discriminated from other phases because of its high average atomic number/high backscatter signal, it yields the inverse of the corundum image. this sounds quite simple; however, since the resolution of the x-ray signal is considerably less than the bse signal, quite some effort had to be put into the treatment of the si-distribution to give correct areas and outlines of the glass phase. another rather complicated step was the recognition of the zirconia crystals grown within or into the corundum. crystals fully enclosed by the corundum presented no problem, but those growing from the edge into the al o were difficult to discriminate as to belonging to this particular structure. the problem here is to tell the machine what the eye and judgement of the operator consider to be part of this feature. an additional treatment of the glass phase (binary image filters) was necessary to solve this task. the binary image of the glass phase is used as a template for the determination of the chemical data of the glass; it provides the control for stepping the electron beam only over the desired areas while acquiring data with the energy dispersive spectrometer (eds). all image analysis procedures, including the storage of images and eds spectra together with the evaluation set-ups (selection of properties to be determined), were combined within a schedule. the stage control and automation software then connects the stage movement to predetermined points (or a number of points along a line) with the execution of the schedule at each of these points on the sample. after completion of the analysis run, which needs about hours for image fields and is therefore done unattended overnight, the large amount of stored data are statistically evaluated (for the zirconia particles alone, there are easily more than , data sets, consisting of seven measured or derived properties each!). this task is performed using the lotus - - calculation program. the chemical data are extracted from the stored spectra with the zaf-correction procedure to yield wt-% oxide data, and also statistically evaluated with the lotus program. although the aim of this work was only to set up an analysis procedure, the test runs on various samples have already shown differences in the microstructure as determined by the geometric and chemical properties of the individual phases. the application of this procedure to "real" samples will then establish correlations between the microstructure and the properties of the material with respect to their use in glass melting. approximately , tons of electric furnace flue dust accumulated in an industrial area in tifton, georgia. vehicles transporting the flue dust, classified as k hazardous waste, initially dumped the material in a warehouse. once the warehouse was full, the flue dust was dumped in an uncovered pile. run-off from the pile and wind-driven particles contaminated nearby industries, residential buildings, and soils over a period of many years. scanning electron microscopy-energy dispersive x-ray spectrometry (sem-eds) was used to compare the morphology and chemical composition of fly ash dust from the suspect pile ( fig. ) with samples collected from the surrounding buildings and soil. post-it notes (millette et al. ) , modified with a strip of conductive carbon tape, were used to collect dust that had accumulated in buildings surrounding the fly ash dump site. suspect dust particles were analyzed by sem-eds to compare with known dust particles from the fly ash pile. soil samples were sieved, with "fines" from the dry soil analyzed by sem-eds and compared with samples from the fly ash pile. particles similar in chemical composition and morphology were identified in most of the buildings sampled that surround the fly ash dump site. soil samples from areas surrounding the dump site were also found to contain fly ash iv- scanning vol. , supplement iv ( ) fig. backscattered electron image of fly ash spheres from the flue dust pile. scale bar = µm. particles similar in morphology and chemical composition to fly ash from the suspect pile. in conclusion, soil and dust samples taken from homes and outdoor areas surrounding the fly ash pile were found to contain particles similar in morphology and chemical composition to particles from the fly ash pile. scanning electron microscopes (sems) are widely used in detection and quantification of material microstructure and imperfections. results obtain with sems need a high expertise to be fully exploited. specialized programs such as a single-scattering monte carlo simulation can effectively predict the electron beam interaction with solids and thus help the quantification. one of the most powerful advantages of monte carlo simulation to help microscopists is to generate images. with high-speed computers we can now simulate images in a reasonable amount of time. in this paper we present images of spherical inclusion of mns in a fe matrix. it is shown that there exists a difference between the image dimension and the real dimension. also, it is shown that geometric effects can alter the resulting image. the monte carlo program used for low-energy simulation is described elsewhere . figures - show simulated image of mns inclusions (the dark center of the figures) in a fe matrix (lighter part). to build this image we need to simulate , primary electron trajectories and then calculate the associate backscattering coefficient for each pixel of the screen. the image will then need , simulations for a total time of approximately , cpu min on a risc workstation. because of the symmetry of the image we can use an better method. starting from the center of the inclusion we simulate points moving on a radius to the border. then we rotate this line on °and add random noise for the monte carlo simulation to obtain the full image. this image is coded in windows bitmap format and can then be converted to tiff or another format. for our simulation, we used a diameter of nm for the electron beam, at which value the incident beam current would be × − amps in our jeol sem. figure shows a spherical inclusion with a radius and at a depth of nm simulated with a beam energy of kev. figure shows the same inclusion but simulation at kev. figures and show the inclusion with a ratio depth/radius of . , simulated at and kev, respectively. in figure we can observe the discrepancy between the image dimension and the real dimension as a function of the ratio depth/radius. as was expected, the error increased with the depth/radius ratio. it is also interesting to note that increasing the energy of the incident electrons increases the image error. the geometry of the solids affect the backscattering coefficient. we can obseve a white border in figures - a similar problem occurs in the middle of the inclusion which appears lighter. it is interesting to see the same phenomena in figure for an image of a real inclusion. the photograph of the embedded particulates in a matrix taken by sem in backscattering mode should be analysed carefully because this method usually overestimates the dimension of such particulates. the measurement of strain, using lattice parameter changes, can be determined from channeling patterns in the scanning electron microscope (sem) using holz lines. the method used by kuzubowski, for example, in the [ ] orientation of silicon, utilizes the change of the height of the triangle from the intersections of the ( ) and two { } holz lines. more recently we have been investigating the channeling patterns for silicon to calibrate accurately the voltage in an sem and we have utilized a pin wheel pattern formed from the { } holz lines in [ ] pattern at . kev. at this voltage, the ( - - - ) and ( ) are very close to each other, within . degrees, and these two lines split as the voltage increases or decreases. the width of the splitting is quite sensitive to any voltage changes as are the { } intersections with these lines. an experimental electron channeling pattern (ecp) of these lines, along with a simulation are shown in figure . the voltage calibrated from the simulation is . kev as shown in figure c . these same holz lines are, of course, also appropriate for strain measurement since the variation in the voltage is equivalent to an isotropic strain field that would uniformly change the lattice spacings. the shifting of the holz lines in the electron channeling patterns due to strain are from both the change in the magnitude of bragg angle and the change in the orientation of the diffracting planes. the former corresponds to a change in the distance of reciprocal lattice point from the origin(i.e., d spacing) and the latter is due to the rotation of reciprocal lattice point about the origin. thus the total angular change can be written as where θ b is the bragg angle, ∆θ p is the rotation of the plane, g is the diffraction vector, λ is the electron wavelength, and | | represents the length of a vector. e is a matrix equal to ε + i where ε is the strain tensor and i a unit matrix. the approximation ~ is due to the small angle approximation for sin(∆θ b )~∆θ b and − ∆θ b ~ . the holz lines toward the center of the channeling patterns are more sensitive to lattice parameter variations because they have a larger value of g and the change of the bragg angle is in proportion to this magnitude. the angular iv- scanning vol. , supplement iv ( ) ∆θ = ∆θ b + ∆θ p~e g − g widths of these channeling lines are also smaller and so they are well defined. when the strain is not isotropic, the sensitivity is also determined by the orientation relationship between the strain ellipsoid and the g vector. thus the appropriate choice of the g vector can be used to maximize the second term in the equation. in figure a , for example, we show the simulation of a channeling pattern for a strain along only one direction (e.g., [ ] ) which breaks the four-fold symmetry of the [ ] pattern. it can be seen that the strain effects on the { } are all the same since all the planes in this family are symmetrical to the strain direction. however, the sensitivities to the strain in the { } are not all equal, ( ) being more sensitive than ( ). the comparison of the sensitivity can be seen from the simulation and from figure b , in which the angles of splitting are plotted as a function of the strain. for years lab has been the industry standard for thermionic emission cathode material. in , fei introduced ceb as an improved alternative to lab . ceb directly replaces lab and has certain distinct benefits. ceb has a lower volatility than lab , increasing the lifetime of the cathode. this reduces the frequency of cathode purchases and replacements. in addition, greater beam stability and faster startup are achievable from ceb 's greater resistance to contamination. however, to gain the benefits of ceb , it must be operated correctly. studies have shown that the operating characteristics of ceb , such as total emission current, are different from lab . proper operation of ceb comes from understanding the operating characteristics of ceb and how they interact with the system in which it is operating. to compare the operating characteristics of ceb and lab , we performed parametric studies in a jeol scanning electron microscope. the jeol is a self-biased system where the bias voltage (vb) is dependent upon the total emission current as vb = ie*rb, where rb is the bias resistor and ie is the total emission current. the results show that ceb operates at a lower total emission current than that of lab (fig. ) . therefore, for the same bias resistor settings, the bias voltage on ceb is less than that on lab (fig. ) . please note that the lower emission current of ceb does not imply a lower probe current. ceb has high transmission and has been shown to provide probe currents similar to lab . to optimize the operation of ceb in a self-biased system, the operating conditions must be optimized for a low emission current source. these operating conditions include the bias resistor value and wehnelt-to-tip spacing. we have found that increasing the bias resistor improves the performance and lifetime of ceb . other experiments show that adjustments to the wehnelt-to-crystal tip distance further improve the performance of ceb . the effects of adjusting the wehnelt-to-tip distance vary for different electron microscope systems. for independently biased systems, we recommend setting the filament temperature to k (the corresponding filament current is provided with the cathode) and subsequently adjusting the bias until the desired emission image is obtained. this is possible because the emission image of ceb is the same as the lab emission image. the wehnelt-to-tip distance should be set to the distance recommended for lab . in transmission electron microscopes (tems), the operating point is set by viewing the source image. because the emission image of ceb is similar to lab , the procedures for obtaining the lab operating point in tems still apply to ceb . however, the total emission current at the operating point will be lower than with lab . by following these guidelines, the operation of ceb can be optimized and with this optimized operation, the benefits from additional lifetime and increased stability over lab can be achieved. takeshi hatsuzawa, yoshihisa tanimura, kouji toyoda, makoto nara*, syuuji toyonaga*, shin-ya hara*, hirotaka iwasaki*, kazuhiko kondou* national research laboratory of metrology, miti tsukuba; *nikon corporation, tokyo, japan a compact laser interferometer with a piezo-driven scanner has been developed for metrological micro-linewidth measurement in regular scanning electron microscopes (sems). so far, special sems combined with various scanners and interferometers (postek , hatsuzawa et al. ) are necessary to perform absolute and precise measurements; however, this device solved the problem by miniaturizing a one-dimensional mechanical scanner and a multi-optical path interferometer. the arrangements of optical components are illustrated in figure . a mechanical scanner is constructed in the center of a mm diameter base disk. the square part, slit by using a electric discharging machine, is suspended by thin elastic suspensions at each corner. the table is driven by a piezo-electric actuator of a traveling length of micrometers. a he-ne laser beam is introduced on the disk by a single mode fiber through a collimator lens, and it is split and deflected by beam splitters and lenses so that two beams are facing each other and form a differential interferometer. at both ends of the table, right-angle prisms are facing each other so that the laser beam goes back and forth five times. by using the differential arrangement and optical-path multiplication technique, the resolution of the interferometer is improved ten times from the original michelson's arrangement. the reflected beams are surperimposed by a half-mirror to generate an interferometric signal, and it is detected by four photo diodes (pd -pd ) after changing its phase by ˚ through a half-and a quarter-wave plate arrangement. this detection method and an electrically operational processing enhance the resolution iv- scanning vol. , supplement iv ( ) times from its original. eventually, by using physical and electrical methods, the resolution of the compact interferometer is . nm (âλ/ ). to evaluate the performance of the compact interferometer, it was installed in the vacuum chamber of a regular sem (joel jsm- a) as shown in figure . the fiber and electric wires are introduced through two flanges next to the chamber. a software servosystem is constructed by using the interferometer and a piezo driver. according to the positional information read through the interferometer counter, a d/a converter commands the piezo driver to change table position precisely, allowing simultaneous sampling of the secondary electron intensity distribution. in the system, the resolution is improved to . nm (âλ/ ) by using the counter function. thus, a precise measurement system is realized for the absolute measurement of micro-linewidth in a regular sem. a maximum drift of the interferometer counter of nm/h was observed by fluctuations in room temperature, however, the influence of the drift can be neglected in actual measurements since a line-scan is finished within a dozen seconds. a comparison of measurements between the metrological sem (hatsuzawa et al. ) and the compact system was made by using silicon micro-line artifacts, ranging from . to . micrometer. the measured linewidths agree within a couple of nm in both measurements, although the measurement conditions are different in acceleration voltage, etc. the results show that the measurement system using the compact interferometer has the same performance as the metrological sem. this means that absolute and accurate measurements can be obtained everywhere by using the compact laser interferometer and a regular sem. this device can be applied to various types of scanning probing microscopes as well as to optical microscopes by improving the table mechanism and the arrangement of optics. institute of ecology and department of botany, university of georgia, athens, georgia recent studies of lake lanier, georgia, revealed a fungal epidemic on the planktonic alga, synedra acus. clonal isolates of the fungus were identified as zygorhizidium planktonicum (chytridiomycetes), an obligate parasite of freshwater diatoms. although frequently present in lakes and reservoirs of western europe, the occurrence of z. planktonicum in north america has not been previously confirmed. earlier studies have described the morphology of z. planktonicum on asterionella formosa; however, little is known of the fine structure and infection process on s. acus. as a basis for further investigations, morphology and mechanism of infection were characterized by scanning and transmission electron microscopy. these observations provided exceptional accounts of germinating spores and developing thalli. moreover, conjugation was characterized as the fusion of heterogametangia by means of an extended smooth-walled tube, emanated from the smaller "male" gametangium. upon attachment, the club-shaped conjugation tube adhered to a small region of the adjoining thallus. this point of contact became continuous with the maturing "female" gametangium which appeared smooth-walled and often highly vacuolate. ultrastructural examinations also illustrated a shared cytoplasm between conjugating gametangia and apparent migration of organelles; however, fusion of nuclei was not observed. the mechanism of infection on s. acus appeared identical to earlier descriptions of z. planktonicum on a. formosa (beakes et al. ) . following encystment, spores typically produced a single germ tube which grew over the frustule valve in an unwavering, linear fashion. penetration occurred between an overlapping region of the outer and inner frustule. at the point of intrusion, the rhizoid appeared slightly swollen, often further displacing the outer diatom wall. a program for monte carlo simulation of electron energy loss in nanostructures a monte carlo calculation of the backscattering coefficient for a multilayer sample monte carlo program for minicomputers using mott cross sections an improved method of measuring biological submicron motion and displacement using laser amplified motion detection and analysis friedlander sk: smoke, dust and haze, fundamentals of aerosol behavior subcommittee on airborne particles: airborne particles micromanipulators and micromanipulation moor h: recent progress in the freeze-etching technique optimization and application of jet-freezing platinum-iridium/carbon: a high-resolution shadowing material for tem, stm, and sem of biological macromolecular structures freeze-fracturing for conventional and field emission low-temperature scanning electron microscopy: the scanning cryo unit scu cryo-preparation and planar magnetron sputtering for low temperature scanning electron microscopy imaging of intramembranous particles in frozen hydrated cells (saccharomyces cerevisiae) vapor pressure data for some common gases. r.c.a. review an improved cryo-jet freezing method in vitro spinal cord trauma early post trauma changes in rat spinal cord: electron probe microanalysis surface studies by stm fabrication technique for tips with controlled geometry for scanning tunnelling microscopy scanning probe metrology low temperature thermal oxidation sharpening of microcast tips microfabrication of afm tips using focused ion and electron beam techniques dimensional metrology with scanning probe microscopes a rocking beam electrostatic balance for the measurement of small forces a scanning tunneling microscope with a capacitance-based position monitor scanning probe tips formed by focused ion beams envelope reconstruction of probe microscope images surface recovery in scanning probe microscopy probe characterization for scanning probe metrology comparison of diffraction techniques for the sem. scan electr microsc electron channelling in the sem a review of excimer laser projection lithography m: direct electron-beam patterning for nanolithography direct stem fabrication and characterization of selfsupporting carbon structures for nanoelectronics die entstehung einer vielzahl von kontaminationsfäden unter der electronen-mikrosonde transition from chemical etching to chemical polishing studied by the sem chemical preparation of dielectrics for studying their microtopography by the sem microprobe analysis in human pathology shelburne jd: preparation of biological tissue sections for correlative ion, electron and light microscopy negative staining: applications and methods detection and identification of viruses by electron microscopy diagnosis of viral infection by electron microscopy electron microscopy in diagnostic virology electron microscopy in viral diagnosis genetic identification of a hantavirus associated with an outbreak of acute respiratory illness isolation of muerto canyon virus, causative agent of hantavirus pulmonary syndrome distinction between bunyaviridae genera by surface structure and comparison with hantaan virus using negative stain electron microscopy anatomic complications of abdominal surgery with special reference to the ureter urological complications of renal transplantation can be prevented or controlled the microvasculature of the guinea pig ureter. a scanning electron microscopic investigation application of an naoh maceration method to a scanning electron microscopic observation of ito cells in the rat liver sem blood vessel cast-analysis microangioarchitecture of the islets of langerhans in the snakes, naja naja, vipera russelli and echis carinatus histochemical methods for acid phosphatase using hexazonium pararosanilin as coupler acid phosphatase activity in the inner ear the development of the stria vascularis in the mouse kimura rs: distribution, structure and function of dark cells in the vestibular labyrinth secretory epithelial linings in the ampullae of the guinea pig labyrinth la fosfatasi acida del labirinto membranoso dell'embrione di pollo durante lo sviluppo the development of human placental villous tree scanning electron microscopic observations on the surfaces of chorionic villi of young and mature placentas ultrastructure of the epithelium of the chorionic villi of the human placenta some new findings about hofbauer cells in the chorionic villi of the human placenta the fine structure of human placental villus as revealed by scanning electron microscopy monte carlo simulation with ebic a monte carlo calculation backscattering coefficients calculations of mott scattering cross section simulation of sem screen image by a monte carlo method quantitative x-ray microanalysis of spherical inclusions embedded in a matrix using a sem and monte carlo simulations a standard procedure for the modeling of the decrease in detection efficiency with time for low-energy eds spectra an empirical stopping power relationship for low-energy electrons measuring the backscattering coefficient and secondary electron yield inside a scanning electron microscope applications of a knock-on process monte carlo simulation based on the mott cross section to quantitative electron microprobe analysis x-ray production as a function of depth for low electron energies theoretical electron-atom elastic scattering cross section cross section for k-shell ionization by electron impact the use of polyacrylamide as an embedding medium for immunohistochemical studies or embryonic tissue immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos i: presence of immunofluorescent titin spots in premyofibril stages novel applications of acrylamide for cryosectioning of isolated cells, tissues, and arthropods whole-mount analyses of cytoskeletal reorganization and function during oogenesis and early embryogenesis in xenopus confocal microscopy of thick sections from acrylamide gel embedded embryos resolution of subcellular detail in thick tissue sections: immunohistochemical preparation and fluorescence confocal microscopy the use of polyacrylamide as an embedding medium for immunohistochemical studies of embryonic tissues application of acrylamide as an embedding medium in studies of lectin and antibody binding in the vertebrate retina developmental angiogenesis: quail embryonic vasculature embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia vasculogenesis and angiogenesis: two distinct morphogenetic mechanisms establish embryonic vascular pattern endothelial cell origin and migration in embryonic heart and cranial blood vessel development morphogenetic mechanisms in avian vascular development near-field optics: microscopy, spectroscopy and surface modification beyond the diffraction limit mechanical detection of magnetic resonance related scanning techniques morphology and diameters of crystallites in remineralized enamel the shape of enamel crystal within human enamel densitometric study of polarized light images from carious lesions conditions required for detection of specimen specific se- secondary electrons in an analytical sem a high resolution se- sem study of enamel crystal morphology high resolution topographic imaging of enamel crystal surfaces analysis of metal films suitable for high resolution se- microscopy antiviral activity of rnadye combinations microspectrophotometry and digestibility of alkali-treated walls in bermudagrass cell types simplified highly efficient apparatus for photographic transaxial x-ray tomography embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia in quail embryos antibodies to b -integrins cause alterations of aortic vasculogenesis, in vivo capillary endothelial cell cultures: phenotypic modulation by matrix components in vitro rapid organization of endothelial cells into capillary-like networks is promoted by collagen matrices connective tissue morphogenesis by fibroblast traction. i. tissue culture observations reorganization of basement membrane matrices by cellular traction promotes the formation of cellular networks in vitro consumption of various process cheese products has been steadily increasing in the u.s.a. and worldwide. these products are manufactured in various styles depending on composition and physical properties sodium citrate (sc), trisodium phosphate (tsp), disodium phosphate (dsp), and sodium hexametaphosphate (shmp) were used at . % levels as melting salts. after cooking, the samples were held at °c for up to h. they were removed after , , . , and h, cooled at °c, and stored for days, after which they were analyzed for moisture, fat, protein, ph, firmness, and meltability. for transmission electron microscopy, small samples ( mm ) were fixed in . % glutaraldehyde and % osmium tetroxide, embedded in spurr's low-viscosity medium, sectioned ( nm thick sections), stained with uranyl acetate and lead citrate, and examined at kv accelerating voltage the data in figure were converted to a pseudo three-dimensional relief map of the embryonic vessels using digital image processing software. it can be argued that this digital rendering, which contains apparent overhead illumination (and shadows below), provides a more comfortable format for assessing visual information. references caric m, kaláb m: processed cheese products textural properties and microstructure of process cheese food rework microstructure of processed cheese products milk gel structure. vi. cheese texture and microstructure effect of draw ph on the development of curd structure during the manufacture of mozzarella cheese structure and rheology of string cheese mozzarella cheese: impact of coagulant type on functional properties encyclopedia of food science & technology food texture and microstructure electron microscopic observations on the casein micelles of buffalo milk: a preliminary study development of microstructure in raw, fried, and fried cooked paneer made from buffalo, cow and mixed milks the role of casein micelles in changes in the colour of milk microstructural evaluation of model starch systems containing different types of oils use of the bird-leider equation in food rheology starch gelatinization in the presence of emulsifiers. a morphological study of wheat starch fixation analysis of tetrahymena pyriformis ultrastructure distribution of polyphenol oxidase in organelles of hyphae of the wood-deteriorating fungus, coriolus versicolor. biodeterioration research a study of the bladder blood flow during distention in rabbits a vascular network closely linked to the epithelium of the urinary bladder of the rat the effects of acute overdistention of the rabbit bladder applications of an naoh maceration method to a scanning electron microscopic observation of ito cells in the rat liver light microscopy techniques for the demonstration of silicone synovial metaplasia of a periprosthetic breast capsule demonstration of silicon in sites of connective tissue disease in patients with silicone-gel breast implants biological specimen preparation for sem by a method other than critical point drying comparison of hexamethyldisilazane (hmds), peldri ii, and critical point drying methods for scanning electron microscopy of biological specimens acetonitrile as a substitute for ethanol/propylene oxide in tissue processing for transmission electron microscopy: comparison of fine structure and lipid solubility in mouse liver, kidney, and intestine optimum conditions may allow air drying of soft biological specimens with minimum cell shrinkage and maximum preservation of surface features a rapid method for cell drying for scanning electron microscopy nation jl: a new method using hexamethyldisilazane for preparation of soft insect tissue for scanning electron microscopy the use of a simple method to avoid cell shrinkage during sem preparation handbook of snow: principles, processes, management and use hobbs pv: ice physics snow crystals: natural and artificial interference reflection microscopy in cell biology: methodology and applications rekonstruktion des oberflächenreliefs von erythrozyten mit hilfe der leitz-reflexionskontrast-einrichtung. leitz-mitt wiss tech vii/ reflection contrast microscopy within chrome-alum haematoxylin stained thick tissueslides reflexionskontrastmikroskopie in der immunhistochemie lichtmikroskopische untersuchungen zum einfluss von atrialem natriuretischem peptid (anp) am nucleus supraopticus scanning electron microscopy of post-it ™ notes used for environmental sampling development of a monte carlo program for low energy work measurement of small elastic strains in silicon using electron channeling patterns electron channeling patterns in the scanning electron microscope a metrological electron microscope system for microfeatures of very large scale integrated circuits scanning electron microscope-based metrological electron microscope system and new prototype of scanning electron microscope magnification standards comparative ultrastructural ontogeny of zoosporangia of zygorhizidium affluens and z. planktonicum, chytrid parasites of the diatom asterionella formosa iv- scanning heterogametangia attached by a fully developed conjugation tube (arrowhead) infection sites of diatom host showing intruding germ tubes (arrowheads) and developing thalli. scale bar = µm the author is grateful to dr. david howell for critically reviewing this manuscript and to ms. lara muffley for technical assistance.this research is supported in part by nih grant nddk r dk - . the authors gratefully acknowledge dr. steven armstrong for his assistance in animal preparation. mic integrity and induced mitochondrial hypertrophy without altering microtubular ultrastructure (fig. a, b) . to localize exogenously administered cdz, a polyclonal antibody to the drug conjugated to keyhole limpet hemocyanin (pierce, rockford, ill.) was prepared by immunizing new zealand rabbits and subsequent bleeds. the antibody titer was both detected and quantified by an indirect elisa assay (pierce elisa starter kit). the detected antibody was separated from other serum proteins by immunoaffinity chromatography utilizing pharmacia's mab trap g and then tagged with nm immunogold particles. transmission immunoelectron microscopy employing tagged antibody and glutaraldehyde/ paraformaldehyde-fixed, dmf dehydrated, and lowicryl-embedded cdz-treated and nontreated tetrahymena revealed no immunogold association with either microtubules or any other cytoplasmic organelle. this suggested that intracellular cdz was cytosolic and leached out during em processing. thus, cdz appears to impair growth and motility through an effect on general metabolism, for example, protein synthesis and/or respiration, rather than a direct action upon microtubules. however, isolation, purification, and characterization of microtubular proteins from tetrahymena cultured with and without cdz are required to substantiate this tentative conclusion. support: nsf-rimi grant no. rii- . coriolus versicolor, a white-rot, wood-decay basidiomycetous fungus, elaborates extracellular ligno-cellulolytic enzymes which possess marked industrial and agricultural applications. thus, we have been attempting to overproduce and enhance/regulate the secretion of these enzymes employing polyphenol oxidase (ppo) as a model enzyme. it catalyzes the conversion of o-diphenols (tree-generated resistance factors) to o-diquinones and oligomerizes syringic acid, alignin derivative. previously, we (moore et al. ) employed biochemistry and immunoelectron microscopy to map the route of ppo secretion through intracellular endomembrane and possible wall-associated components for hyphae cultured in defined liquid (biochemistry) and solid (microscopy) media. here, the ultrastructures of c. versicolor hyphae cultured in kirk and kelman's defined liquid or solid media are compared. in addition, ultrastructural cytochemistry to define further the intracellular route of ppo secretion to the growth medium in liquid cultured hyphae is described. hyphae of various culture ages ( - days) were prefixed min in . - . % glutaraldehyde buffered with . m cacodylate/cacodylic acid, ph . and after washing with buffer postfixed for h in buffered % s . for cytochemistry, prefixed hyphae were washed and treated with either cacodylate buffer or buffered mg ml - tlc pure, dihydroxyphenylalanine (dopa) on ice, followed by h at °c and then s postfixed. the hyphae were dehydrated through a graded acetone series and embedded in spurr's low epoxy resin. the ultrastructures of hyphae cultured in defined medium containing or lacking agar were similar (fig. a , b) except that hyphae grown upon agar possessed a sheath (hs) external to the cell wall. comparisons of numerous micrographs of aldehyde-fixed hyphae treated with cacodylate christian h. rickert and timm j. filler institute of anatomy, westfälische wilhelms-universität, münster, germany until today, quantification in cytochemistry has mainly been performed on supracellular level and by subjective estimation which makes it difficult to compare results of different investigations, even for standardized cytochemical procedures. reasons for this are the lack either or of calibrations or of relative reference points. greyscale image analysis principally allows the quantification of dye-density, but in practice stain intensities depend on many technical circumstances, that is, slide thickness, density of materials, light conditions, or costaining effects, and do not allow automatic identification because of weak grey-contrast. thus, greyscale image analysis for cytochemical quantification on subcellular level necessitates four prerequisites: ( ) applicability within tissue-slides combined with high resolving power, ( ) thin optical tomolevels to avoid superimposition of stained structures, ( ) clear distinction of structures (specificity), and ( ) high contrast. the reflection contrast microscope (rcm; leica germany) meets the above mentioned requirements. it combines effective suppression of aspecific reflected light with epi-illumination. because of its confocal-like principle, the rcm can be applied on thick tissue slides to obtain distinct optical sections. , at a magnification of ×, the depth of these sections amounts to approximately - µm, circumventing an accumulative effect as seen in transillumination. some cytochemical stains show specific reflections in the rcm, increasing the detection sensitivity to a level of objects nm in size. , reflections are like a binary signal (all-or-nothing principle) and deliver an intense contrast against a dark background, thus facilitating image processing by substituting the common density measurements with field measurement corresponding to areas of reflections.we designed an analysis program on vidas . (zeiss/kontron germany) for quantification of rcm images. one of the main parts of this application handles the greyvalue manipulation. apart from standard routines for image optimization and automatic region selection, the mean greyvalue of the whole image was determined. this was used as a reference parameter for the identification threshold in order to minimize the deviation of the identified area from the real area of reflection caused by inconsistencies of the light intensity.applying the rcm with consecutive image analysis on gomori-stained neurons of the supraoptic nucleus (son), we verified the validity of our measuring routine by employing it on a recognized system. a linear correlation between the process of neurosecretion and nuclear volume of the son is well known. switching from rcm to bright field, it is possible to obtain topographically identical images of the chosen tissue region. thus, we photographed reflecting neurosecretory granules in the former while karyometry was performed on the latter. the nuclei were classified by area and matched with the corresponding nucleus area of the same cell. a linear regression of these parameters was computed within a % confidence interval. our results in general confirm former findings about the measurable relationship between nuclear size and specific cell activity, surpassing earlier methods by increasing the sensitivity and decreasing the duration.this paper introduces the rcm combined with digital processing as a useful tool for quantification in the investigative gap between light and electron microscopy. the reflection contrast microscope (rcm; leica germany) is a light microscopic instrument, making reflections along interfaces visible by means of centrally polarized epi-illumination. these reflections cause interference patterns that are suitable for the analysis of superficies or detection of contact zones . we present two properties of this widely unknown technique allowing three-dimensional ( -d) reconstructions in the following manners: . applying the rcm on the surface of air-dried unstained erythrocytes, we made use of the phenomenon that neighbouring zones of equal altitude are joined by closed interference fringes; these can be interpreted as isohypses. to quantify the angle of declivity within a period of the resulting dark-light pattern, it is necessary to know the mean wavelength. the difference of layer thickness between two interference lines of equal tone is about nm for monochromatic light of λ= nm. the advantage of this technique is the simultaneous presentation of the profile of all the isohypses in just one picture (fig. ) . key: cord- -lq f qv authors: han, tae-hee; park, sang-hun; chung, ju-young; jeong, hyo-won; jung, jihun; lee, jae-in; hwang, young-ok; kim, il-young; lee, jip-ho; jung, kweon title: detection of pathogenic viruses in the ambient air in seoul, korea date: - - journal: food environ virol doi: . /s - - - sha: doc_id: cord_uid: lq f qv the possible transport of pathogenic microorganisms during asian dust events could be an important concern for health workers; however, this is still uncertain owing to a lack of supporting evidence. the present study aimed to investigate the presence of pathogenic microorganisms in air samples collected during the asian and non-asian dust periods. between march and september , air samples were collected at three weather observation stations in seoul using a high-volume air sampler. multiplex pcr was performed using the allplex™ respiratory and gastrointestinal panel assay kits to detect microorganisms. rt-pcr was performed for klassevirus, aichivirus, and human parechovirus (hpev) detection. in total, air samples were collected during the asian ( samples) and non-asian ( samples) dust events. during an asian dust event, only one human rhinovirus (hrv)-positive air sample was collected on april . during the non-asian dust period, hrv, hpev, norovirus (nov), enteroaggregative escherichia coli (eaec), enterotoxigenic e. coli (etec), and blastocystis hominis were detected in four, two, one, one, one, and one air samples, respectively. pathogenic viruses were mostly detected in ambient air samples during the non-asian dust period, which suggests a possible air-borne transmission of viral pathogens; however, the role of asian dust in epidemics caused by pathogenic viruses is unclear. asian dust is a meteorological phenomenon in which sand and particulate matter originating from the southeast of mongolia and northwest of china are transported by the wind to korea, taiwan, japan, and western usa. it is known to occur mostly in the spring, although it is sometimes observed throughout the year (griffin ; kim et al. ) . asian dust contains heavy metals (lead, mercury, or cadmium), air pollutants (no − , so − ), and various microorganisms that could elicit harmful effects on human health. increased microdust (pm ) and ultra-microdust (pm . ) levels during the asian dust period are associated with an increase in the daily mortality rates, cardiovascular diseases, cerebrovascular accidents, hospitalization by acute exacerbation of underlying pulmonary or allergic diseases (kwon et al. ; chen and yang ; kashima et al. ) , and carcinogenic effects (goldberg et al. ; weichental et al. ) . the possible role of african dust events in transporting the viruses causing influenza or foot and mouth disease to european areas has been reported (griffin et al. ) . recent studies regarding the detection of microorganisms in the ambient air during the asian dust period were mostly focused on the composition of bacterial communities (nishimura et al. ; yamaguchi et al. ; cha et al. ) . therefore, their role in transporting pathogenic viruses into the ambient air could not be clarified (park et al. ; chen et al. ) . the purpose of the present study was to detect pathogenic viruses in the ambient air in seoul during the asian and non-asian dust periods. air samples were collected at three meteorological observation stations located in the southern (yangjae-dong, latitude: n ° ′, longitude: e ° ′), eastern (gueuidong, latitude: n ° ʹ, longitude: ° ʹ), and northern (jongro-dong, latitude: ° ʹ, longitude: ° ʹ) parts of seoul. between march and september , ambient air samples were collected at the flow rates of - l/min using polycarbonate (pc) filters (skc inc., eighty-four, pa) with the pore size of . µm for h (verreault et al. ) . the size of the particulate matter (> . , > . , > . , > . , > . , and > . µm) was confirmed by a particle counter (arti hhpc- [hach, tokyo, japan]). information about asian dust was obtained from the open access database of the korean meteorological association (http:// web.kma.go.kr/info_open/publi c_data/guide page). the particulate matter attached to membrane filters was separated by mixing them with ml of phosphate buffered saline (pbs) and centrifuging at ×g. then, supernatants were stored in a refrigerator at − °c. rt-pcr was performed to detect respiratory viral pathogens [influenza a (if-a) and if-b, respiratory syncytial virus (rsv)-a and rsv-b, human adenoviruses, human metapneumovirus (hmpv), human coronavirus (hcov) e, hcov nl , hcov oc , parainfluenzavirus (piv)- , piv- , piv- , piv- , rv, human enterovirus, and human bocavirus] and bacterial respiratory pathogens (streptococcus pneumoniae, haemophilus influenzae, chlamydophila pneumoniae, legionella pneumophila, bordetella pertussis, bordetella parapertussis, and mycoplasma pneumoniae) using the allplex™ respiratory panel assay (seegene, korea). the allplex™ gastrointestinal panel assay (seegene, korea) was used to detect viral (rotavirus, nov-i, nov-ii, astrovirus, enteric adenovirus, sapovirus), bacterial [salmonella spp., shigella spp., vibrio spp., campylobacter spp., clostridium difficile toxin b, clostridium perfringens, yersinia enterocolitica, e. coli o :h , enterohemorrhagic e. coli (stx / ), enteropathogenic e. coli (eaea), etec (lt/st), eaec (aggr), aeromonas spp.], and protozoan (giardia lamblia, cryptosporidium spp., blastocystis hominis, dientamoeba fragilis, cyclospora cayetanensis) pathogens. additionally, conventional rt-pcr was used to detect other gastrointestinal viral pathogens such as hpev, klassevirus, nov-giv, and aichivirus, as described previously (han et al. (han et al. , a . semi-nested pcr was performed to confirm the genotypes of hrv, nov, gii, and hpev using primers based on the vp /vp (han et al. ), capsid (chung et al. ) , and vp genes (han et al. ). pcr products were purified with qiaquick (qiagen), sequenced in both directions using the bigdye terminator v . cycle sequencing kit (applied biosystems, foster city, ca, usa), and subsequently resolved by an abi xl autoanalyzer (applied biosystems). the resulting sequences were aligned by bioedit v . and a phylogenic tree was constructed using mega . software. the finally obtained sequences were registered at the gen-bank database as follows: hrv(mg -mg ), hpev(mg - ), and nov(mg ). between march and september , ambient air samples were collected in three meteorological stations located in seoul (yangjae-dong, jayang-dong, and pyeongchangdong). asian dust was observed for days (march , march , april , april , april , and april ). during the asian dust period, ambient air samples were collected at yangjae (six samples), pyeongchang (one sample), and jayang (one sample); air samples were collected (yangjae- , jayang- , pyeongchang- ) during the non-asian dust period. the monthly distribution of the collected air samples was as follows: samples were collected in march ( , , , , , , , and ) , samples in april ( , , , , , , , , , and ) , samples in may ( , , , and ), samples in june ( , , , and ), samples in july ( , , , and ), samples in august ( and ), and sample in september ( ). the daily average concentrations of pm were . and . µg/m , whereas the daily average concentrations of pm . were . and . µg/m in the asian and non-asian dust periods, respectively. hrv was detected in ambient air samples collected during the non-asian dust period on april (yangjae), june (yang jae), june (jongro), and july (yangjae), . however, only one air sample collected during the asian dust period on april , (table ) was positive for hrv. genotyping of hrv-positive samples was used to detect the presence of hrv-a and hrv-c in three and two samples, respectively (fig. ) . hpev was detected in two air samples collected during the non-asian dust period on march (yangjae) and march (yangjae), ; they were determined to be hpev- (fig. ) . a sample collected on june , (yangjae) was positive for nov, and it had the nov gii- genotype (fig. ) . rt-pcr was negative for the presence of klassevirus, aichivirus, and nov giv in all samples collected during the asian and non-asian dust periods. during the non-asian dust period, eaec and etec were detected in samples collected on march (yangjae) and march (jongro), respectively. b. hominis, one of the protozoan agents, was positive in a sample collected on april (yangjae). the respiratory panel assay (seegene, korea) detecting seven kinds of respiratory bacterial pathogens was performed, and all samples were negative for the pathogens. the present study is the first to detect and genotype the viral pathogens hrv, hpev, and nov in ambient air samples collected in seoul, korea. until now, the transmission of respiratory viruses by aerosol was mostly confirmed by studies on experimental animals, volunteers, and indoor samples collected from the hospital or office (griffin ; myatt et al. ) , although hrv or influenza viruses have been previously detected in outdoor ambient air samples (myatt et al. ) . the possible transmission of the hand, foot, and mouth disease virus to europe by african dust was suggested (griffin et al. ), but it was not confirmed in korea during the asian dust period (park et al. ) . in a recent study in taiwan, the influenza virus was detected in ambient air samples during the asian dust period (chen et al. ; myatt et al. ), which was not proven in a korean study (park et al. ). in the present study, the presence of hrv, one of the respiratory viral pathogens, was confirmed, for the first time, in ambient air samples in seoul, although the presence of the influenza virus could not be detected. however, it is unlikely that asian dust has an important role in epidemics caused by respiratory viral pathogens, because hrv was more frequently detected in non-asian dust samples than in asian dust samples. the rate of positive detection of important respiratory viruses is known to have a close association with climatic variables such as temperature and humidity (du prel et al. ). however, some limitations to this study exist, such as the short study period, small number of samples during the asian dust period, and restricted sampling area, which should be considered with regard to the results of this study. it has also to be considered that this study could only show monitoring results of viruses in the ambient air, which could not conclude the role of asian dust in spreading microbial agents. and it is difficult to prove the infectivity of detected viral sequences by biomolecular tests in the air. norovirus is one of the important viral pathogens causing acute gastroenteritis (age), which is mostly transmitted by the fecal to oral route, though it could also be transmitted by contact and airborne transmission (nasaroff ; nenonen et al. ; bonifait et al. ) . in the present study, nov was detected for the first time in ambient air samples in korea and had the genotype gii- , which is presently prevalent worldwide (chan et al. ) . although hpevs can be classified into several genotypes, hpev- and hpev- are the most clinically important genotypes. the hpev- infection is mostly represented by a mild respiratory or gastrointestinal infection, but sometimes becomes clinically severe, leading to sepsis-like illness and central nervous system infection in infants. hpev- is considered to be one of the most important viral pathogens to be differentiated, especially in neonates and young infants with a sepsis-like illness (han et al. ) . the present study is the first to report the detection of hpev- in ambient air samples during the non-asian period. the study tried to detect other viral gastrointestinal pathogens such as aichivirus, klassevirus, and nov-giv, which were proven to circulate in korea in previous studies, but all ambient air samples showed negative results for these viruses in pcr. the previous studies regarding bacteria in ambient air samples during the asian dust period mostly focused on delineating the bacterial community using the metagenomic method, which showed different results depending on the region and sampling time cha et al. ; park et al. ) . the present study aimed to investigate the presence of clinically important respiratory and gastrointestinal bacterial pathogens in ambient air samples using the multiplex rt-pcr panel assay, which was positive for eaec and etec in two ambient air samples during the non-asian dust period. in conclusion, this study was the first to confirm the presence of pathogenic viruses, including hrv, nov, and hpev in ambient air in korea. most of these pathogenic viruses were detected in ambient air samples during the non-asian dust period, which suggests that asian dust might not play a major role in epidemics caused by viral pathogens, although further studies are needed to confirm these findings. detection and quantification of airborne norovirus during outbreaks in healthcare facilities alterations in the airborne bacterial community during asian dust events occurring between global spread of norovirus gii ambient influenza and avian influenza virus during dust storm days and background days effects of asian dust storm events on daily hospital admissions for cardiovascular disease in taipei detection of gii- / b variant and recombinant noroviruses in children with acute gastroenteritis are meteorological parameters associated with acute respiratory tract infections? clinical infectious disease the association between the incidence of postmenopausal breast cancer and concentrations at street-level of nitrogen dioxide and ultrafine particles atmospheric movement of microorganisms in clouds of desert dust and implication for human health african desert dust in the caribbean atmosphere: microbiology and public health detection of human rhinovirus c in children with acute lower respiratory tract infections in south korea human parechovirus- infection in children detection of human parechoviruses in children with gastroenteritis in south korea detection of norovirus genogroup iv, klassevirus, and pepper mild mottle virus in sewage samples in south korea detection of aichi virus in south korea are people with a history of disease more susceptible to a short-term exposure to asian dust? a case-crossover study among the elderly in japan characteristics of asian dust transport based on synoptic meteorological analysis over korea effects of the asian dust events on daily mortality in seoul detection of airborne rhinovirus and its relation to outdoor air supply in office environments norovirus, gastroenteritis, and indoor environmental quality norovirus gii. detection in environmental samples from patient rooms during nosocomial outbreaks similarity of bacterial community structure between asian dust and its source determined by rrna genetargeted approaches investigation of bacterial effects of asian dust events through comparison with seasonal variability in outdoor airborne bacterial community detection of pathogenic viruses in the atmosphere during asian dust events in incheon city comparison of polycarbonate and polytetrafluoroethylene filters for sampling of airborne bacteriophages spatial variations in ambient ultrafine particle concentrations and the risk of incident prostate cancer: a case-control study abundance and community structure of bacteria on asian dust particles collected in beijing, china, during the asian dust season key: cord- -vfvnjmv authors: carpenito, l.; d'ercole, m.; porta, f.; di blasi, e.; doi, p.; fagara, g. redolfi; rey, r.; bulfamante, g. title: the autopsy at the time of sars-cov- : protocol and lessons date: - - journal: ann diagn pathol doi: . /j.anndiagpath. . sha: doc_id: cord_uid: vfvnjmv a new viral disease named covid- has recently turned into a pandemic. compared to a common viral pneumonia it may evolve in an atypical way, causing the rapid death of the patient. for over two centuries, autopsy has been recognized as a fundamental diagnostic technique, particularly for new or little-known diseases. to date, it is often considered obsolete giving the inadequacy to provide samples of a quality appropriate to the sophisticated diagnostic techniques available today. this is probably one of the reasons why during this pandemic autopsies were often requested only in few cases, late and discouraged, if not prohibited, by more than one nation. this is in contrast with our firm conviction: to understand the unknown we must look at it directly and with our own eyes. this has led us to implement an autopsy procedure that allows the beginning of the autopsy shortly after death (within – h) and its rapid execution, also including sampling for ultrastructural and molecular investigations. in our experience, the tissue sample collected for diagnosis and research were of quality similar to biopsy or surgical resections. this procedure was performed ensuring staff and environmental safety. we want to propose our experience, our main qualitative results and a few general considerations, hoping that they can be an incentive to use autopsy with a new procedure adjusted to match the diagnostic challenges of the third millennium. the autopsy has played an essential role in the classification and definition of the etiology and pathogenesis of diseases for over two centuries, since the pivotal work "de sedibus et causis morborum per anatomen indagatis" of g.b. morgagni (venezia, ). this role is still relevant, given the periodic appearance of new nosological entities and the resulting need to understand their characteristics, consequences on the human body and possible pharmacological treatment. the world health organization (who) reports that new viral diseases and consequent epidemics will continue to appear with relevant consequences for public health. when a new infectious and diffusive disease appears the autopsy provides, other than the usual clinical and scientific data, crucial epidemiological information useful for the proper planning of hygiene and public health programs and contributes also to the correct address of health care expenditure. the autopsy ascertains the cause of death both in hospitalized patients and in people who died without medical assistance, transported to hospital or in a morgue. last, but not least, the autopsy is a valid aid for training and education of clinicians themselves [ ] [ ] [ ] . in recent months we have witnessed the emergency of a new global threat, the sars-cov- , which produced a new disease that quickly turned into a pandemic. its epidemiology, pathogenesis and, therefore, therapeutic possibilities are still little known [ ] . in the current pandemic scenario of sars-cov- , the autopsy appears to be a crucial tool to clarify the virus target cells in human, the frameworks of organ damage and the biological mechanisms that lead to death or allow the patient to heal. the autopsy execution on a patient who died of sars-cov- meets two conflicting needs. first, the high infectivity and dangerousness of the virus that requires the adoption of rigorous but timeconsuming methods to guarantee the safety of the personnel carrying out the investigation and to prevent the spread of the virus outside the autopsy room. on the other hand, the need to carry out the autopsy as soon as possible after death and to perform it quickly, in order to have as little tissue damage as possible from post-mortem degenerative phenomena [ ] . the quality of the samples is j o u r n a l p r e -p r o o f essential for diagnostic and research activities, necessary to improve the standard of health care [ ] [ ] [ ] . the practical challenge with the sars-cov- emergency led us to significantly change our operational autopsy protocol, to obtain qualitative technical results that go beyond the limits of this disease and that can be useful in a much wider range of situations. this article reports our modus operandi and the main qualitative results obtained and discusses the findings and the horizons of autopsy in the third millennium. we performed ten autopsies on sars-cov- positive patients. the major aims that guided us are the following: . minimize the risks for the personnel who performed the autopsy; . reduce the probability of spreading the virus into the environment through leakage of unfiltered air from the anatomical and surrounding rooms or through blood or other biological liquids; . obtain samples for diagnosis and research of quality similar to biopsy or surgical samples; . make a concrete operative contribution for doctors who followed the patients in the hospitalization wards; . give precise informations on causes of death to the relatives of the deceased. the regulatory references framework in which we developed and applied our protocol was issued by the italian government's ministry of health and by the governor of the lombardy region [ ] [ ] . our hospital is equipped with a complex unit (cu) of infectious diseases. the autopsy room and the arrival/sampling/inclusion areas of the biological samples of the c.u. of pathological anatomy are designed with a safety level three (bsl , according to cdc) [ ] . the patient's cardiac death is immediately confirmed by continuous electrocardiographic monitoring that verifies the costant absence of cardiac activity for no less than minutes [ ] . this procedure is almost never done in italy; it is preferred to wait at least hours after the presumed death when the first putrefactive skin spots appear. if the patient died outside the hospital but was still transported to the hospital emergency room, the epa protocol is applied only if it has passed less than one hour since the presumed death. our protocol sets that an epa, complete of samples for electron microscopy and molecular investigations on rna and proteins, must start within three hours from the presumed patient's death. in our experience the effects of postmortem processes that arise after this time prevent a sufficient diagnostic quality for these types of examinations. when it is not possible to do an epa, the autopsy is still carried out but only routine histological, immunohistochemical and molecular investigations on dna are performed. after the instrumental ascertainment of death, the patient is transferred to the autopsy room. while authorization procedures for the autopsy are in progress, the doctor in charge of the deceased patient and the director of pathological anatomy discuss the clinical aspects of the case and any question j o u r n a l p r e -p r o o f that the autopsy must answer. the goal of this type of autopsy is not only the recognition of the cause of death or of pathologies already described in medical literature, but also the understanding of the etiopathogenetic mechanisms of this new disease and the confirmation of the adequacy of the therapies in use. this is made possible by the correlation between organ and cellular pathological findings and clinical aspects (symptoms, signs, laboratory and instrumental data). the epa is a procedure that needs to be meticulously planned before it begins, particularly if it is performed on a patient with a potential high risk of infectivity. nothing should be left to chance and, once the staff has entered the autopsy room, there must be no unforeseen incidents. the staff that performed the autopsy must be trained and tightly-knit to be able to make decisions quickly and to adapt their work to the needs of each specific case. in addition, the organs that need sampling also for special investigations must be planned first since these procedures lengthen the autopsy time. according to the rules of the cdc, "autopsies on decedents known or suspected to be covid- cases should be conducted in airborne infection isolation rooms (aiirs)". these types of rooms must be "at a negative pressure to surrounding areas", with a "a minimum of air changes per hour (ach) for existing structures and ach for renovated or new structures" and "have air exhausted directly outside or through a high efficiency particulate aerosol (hepa) filter" [ ] . the who's guidelines firmly suggest biosafety level (bsl ) for autopsies perdormed on patients died for sars-cov- [ ] [ ] . an autopsy room of bsl must be organized as a surgical room: it must have two differentiated paths, one for entrance ("dirty" one) and one for exit ("clean" one). it must be equipped with adequate instruments and clothing checked and guaranteed. in addition, effective cleaning and sanitization of both the instruments, the sector tables and the areas used must be constantly planned. personnel working in the room must have adequate personal protective equipment (ppe) [ ] and tools that can guarantee a fast surgical act. a modern autopsy cannot be performed with old and blunt instruments. in the room, during the autopsy, there must be abundant availability of alcohol at - volumes to sanitize instruments and surfaces [ ] , % buffered formalin, five containers for formalin fixed samples (three medium-size for organs of the right hemibody, left hemibody and median axis, two big-size for brain and heart taken in full), four numbered containers with glutaraldehyde for electron microscopy samples, numbered containers with % alcohol solution for samples for extraction of dna, numbered containers with rnalater solution for samples for extraction of rna and proteins and a transport bag to convey the containers with the samples outside the autopsy room. any type of freezing in isopentane / nitrogen liquid must be performed inside the autopsy room only if a dedicated freezer at - ° c / - ° c is not available. cell culture should be avoided in the absence of biohazard hoods for high biological risk. the autopsy staff must be composed of at least two subjects, maximum three (table ) : the first operator (the "dirty" one), the second operator (the "clean" one) and, possibly, a technician. in our case the dirty operator has always been a doctor specialized in autopsies. the technician must not replace the first operator in the evisceration and sampling of the organs because these maneuvers are essential for the correct medical diagnosis: the diagnosis is macroscopic and inspective (look, touch, smell), before histological or molecular. the body of the patient must be positioned completely inside the sector table to prevent blood or biological liquids from leaking onto the floor. in particular, it must be taken care that the head is not at the edge of the table (often overdrawn and not aspirated) to prevent the dripping of blood from the incision of the scalp, thus producing small drops that splash in the environment. to minimize the dispersion of blood and biological fluids, it is essential to always operate in the area of the autopsy table: the viscera removed from the body must be placed either on the iron section table, placed above the patient's thighs, or in a large tray with high steel edges resting on the patient's legs, during weighing, macroscopic examination and sampling of the viscera. the autopsy begins with a careful external inspection of the body. the length of the body and its state of nutrition must be detected, as the condition of the skin; any skin lesions and, in particular, those likely to be related to sars-cov- infection, should be sampled [ ] . the scalp incision is made with a single cut from one mastoid process to the other, passing through vertex. the skull opening must be immediately suspended if there is a smell of bone dust in the environment: this means that the suction is not effective and that the risk of environmental contamination is unacceptable. the evisceration of the brain, cerebellum and brainstem has to maintain their anatomical continuity. the block is weighed and samples are immediately taken for electron microscopy and for molecular investigations. after sampling for special surveys ( fig. .a-c) the brain is immediately placed suspended inside the container with formalin, to prevent it from deforming by touching the bottom ( fig. .d-f) . if there are no specific indications, the structures of the inner ear, located inside the petrous rock of the temporal bone, and the eyes are not removed, as the dura mater corresponding to the base and cranial vault. the "y" cut is preferred by us to the longitudinal one conducted from the chin to the pubis for the examination of the viscera of neck and trunk. the examination and evisceration of neck and trunk organs is performed with a mixture of techniques, also on the basis of the specific clinical questions of each individual case ( table ) . after the brain examination, heart and lungs are the organs most frequently evaluated and extensively sampled. a swab for molecular detection (pcr) of sars-cov- is carried out, inserting the swab in large intraparenchymal bronchi or directly into the lung parenchyma. when there is clinical evidence of cardiac arhythmias or sudden death, the heart is opened, dissected only in its lower third and fully placed in formalin to allow the study of its conduction system [ ] . cavities or blood vessels must be inspected for thrombus or clots that must be taken and measured. the next step is the evisceration and examination of liver and gallbladder in a single block; the liver parenchyma should be examined macroscopically with particular attention to the characteristics of the blood vessels contained in it. then spleen, kidneys and adrenal glands are individually eviscerated, mobilizing the segments of the gastrointestinal tract that cover them, incising the retroperitoneal soft tissues that surround some of them and dissecting the hilar structures. these organs should also be examined macroscopically, measured and weighed. the epiploon cavity is opened and the pancreas is inspected: in the absence of focal lesions a full-thickness section of the body, approximately cm in length, is removed, internally examined and sampled. in males, gonads are removed by herniating them in the abdominal cavity through the inguinal canal by traction on the spermatic cord. it is advisable to always carry out samples for electron microscopy and molecular investigations of brain, heart (right and left ventricle, interventricular septum and emerging tract of the aorta and pulmonary artery), lungs, liver (right and left lobe), spleen, kidneys, skeletal muscles ( - different muscles), blood and bone marrow (from the first-second rib). all of this in addition to samples for histology and immunohistochemistry. if possible, examine also gonads and pancreas. once the j o u r n a l p r e -p r o o f samples on the main parenchymatous organs have been obtained, the autopsy is completed by gutting in a single block the tongue in continuity with the viscera of the neck and the posterior mediastinum. also these viscera must be carefully examined macroscopically, opening the hollow ones and sampling them for histological and immunohistochemical examinations. two obstructive ligatures are performed, immediately after the treitz ligament and about cm from the ileocecal valve, to remove the small intestine by cutting it at the base of the mesentery; the intestine must be manually inspected along its entire length and, in the absence of focal lesions (which must be sampled for histological examination), two-three sections of a few centimeters are removed and fixed in formalin; in order to avoid the dispersion of feces, the section of the intestinal segments must be carried out by cutting the bowel between two adjacent ligatures for each section point. the large intestine is then mobilized by detaching it from the abdominal wall but without removing it. the course of ureters, bladder and, in females, uterus are inspected. if there are no focal lesions, they can be sampled in situ for histological and immunohistochemical analysis, but it is advisable not to open the bladder to avoid the spread of urine. if prostate samples must be performed, the bladder has to be opened and emptied by aspirating the content. after these procedures, the abdominal tract of the aorta and the large retroperitoneal vessels are inspected. finally, the stomach is opened in situ to examine, and possibly sample, the mucous surface. in our experience the rigorous application of the protocol described for the execution of complete autopsies did not produce accidents or negative consequences on the staff. to date, there have not been any case of sars-cov- infection in the staff assigned to the autopsy activity (detected with real-time pcr on nasopharingeal swab, serological tests for specific antibodies for sars-cov- and clinical evaluation). the instrumental assessment of cardiac death in patients allowed the rapid execution of the autopsy and the collection of samples for histopathological, ultrastructural and biomolecular tests. we thus obtained visceral samples of the highest quality, comparable to those of biopsy and surgical resections ( fig. , .a-c). the comparison with the autopsy samples collected after or more hours clearly demonstrates how the latter are affected by marked post-mortem artifacts, which can distort the correct interpretation of the morphological pictures observed. in addition, immunohistochemical tests on autopsy samples taken within - hours from death are more reliable, which is particularly important when these tests are used to describe a new pathologic entity such as covid- ( fig. ) . immunohistochemistry, like biomolecular investigations performed on autopsy tissue samples, allows also to detect the presence of the sars-cov- in the body, detailing its distribution in single cell types ( fig. .d) . the rapid collection of samples for ultrastructural examinations gives the possibility to define, with a high degree of certainty, the damaging action of the pathology in progress differentiating it from post-mortem tissue and cellular changes ( fig. ). general guidance on how to perform an autopsy on a patient suspected or infected with sars-cov- have been recently published [ ] ; some autopsy studies on patients with this disease reported the observed histopathological patterns, in particular affecting the lung [ ] [ ] [ ] [ ] . in light of these indications, it seems useful to underline some practical aspects concerning the execution of j o u r n a l p r e -p r o o f autopsies in the countries where this virus is still widely spread and often undiagnosed. it is essential to consider as "potentially positive" also the patients not diagnosed with sars-cov- , because maybe asymptomatic or not included in health surveillance programmes: autopsy on these subjects must be performed with the protocol described for patients definitely infected. all autopsies should always be considered "potentially at risk of contagiousness". in each hospital a specific operating protocol that allows to quickly ascertain the patient's death must be planned out so that the autopsy can be performed rapidly without waiting for the appearance of explicit putrefactive phenomena, which is the normal procedure in italy [ ] . the study of tissues markedly modified by postmortem biological phenomena can allow us to contemplate death but not the causes and the mechanisms that produced it. in our experience the histopathological and ultrastructural frameworks that emerge from an autopsy performed within - hours after death differ significantly from those that we observed in an autopsy performed many hours or days after death and which have been presented in multiple autopsy studies on sars-cov- [ ] [ ] [ ] [ ] . the autopsy must be carried out quickly and planned before executing it, adapting its performance and methodology to the specific items of each case. these must be defined by the previous interview between the doctors and the pathologist. the maneuvers useful for macroscopically examining and sampling the organs identified as the main target in every single autopsy, will be privileged. this type of autopsy is not the occasion for elegant anatomical dissections, nor for the application of traditional teaching methods on evisceration procedures. it is essential to do it early and well, aiming for the planned goals, which may change from case to case. during the collection of material for ultrastructural, molecular or other tests, it is important to be aware that not all viscera can be sampled quickly, because the autopsy has its own procedure time and the more special samples are taken, the more time will increase. before starting the autopsy, the evisceration and sampling sequence must be planned (especially when it is necessary to take tissue for electron microscopy) and be aware that the organs sampled after one hour from the beginning of the autopsy will only be able to provide samples for histology and immunohistochemistry. in our experience the patients who undergo autopsy within few hours of death, bleed much more and the incision of very congested vessels can produce relevant blood splatters than in an autopsy performed after hours or more. in addition, patients hospitalized in intensive care for sars-cov- can be anticoagulated, a condition that increases the leakage of blood. during the autopsy the blood must absolutely not be dispersed in the environment surrounding the sector table. for this reason, the body must be positioned completely inside the sector table and the mobilization of large visceral masses outside the body should be avoided: letulle's technique (evisceration en masse) therefore does not appear adequate. blood, urine and other biological liquids must remain inside the body or aspirated by a vacuum pump in a container that can be sanitized after the autopsy. the commonly recommended use of rags or sponges to collect blood is totally inadequate and dangerous. given the multiple clinical findings of neurological symptoms in patients infected with sars-cov- [ , ] , it appears indispensable to perform the evisceration and examination of the brain and brainstem, for the completeness of the autopsy and for the very few morphological data available today on the central nervous system. our experience shows that the use of a valid circular saw with dust extraction system, combined with adequate ppe, does not put operators at risk and allows the doctor to obtain valid tissue samples. given the importance of time, it seems useless to waste it performing the swabs for the detection of sars-cov- in the usual locations for the living, when abundant material can be collected by inserting the swab directly into the lung parenchyma. to date we are conducting further investigations on the samples obtained from early performed autopsies in order to evaluate the organ damage, particularly on snc, heart and lung. at the beginning of the third millennium, it is anachronistic to engage with the challenges of our discipline with the tools available to morgagni, malpighi, vesalio or virchow, when we have new and powerful weapons at our disposal. the limited possibilities to apply modern investigation technologies to tissues severely damaged by post mortem degenerative phenomena, has led many clinicians and pathologists to believe that autopsies are obsolete although, theoretically, they may be the most complete diagnostic tool. at present the autopsy should be performed quickly after the instrumental assessment of cardiac death, to ensure that the quality of tissues examined is similar to that of biopsy or surgical samples performed on the living. the forensic autopsy is the one with the most problems because of its rapid execution is often hampered by the need to perform it in front of the consultants of the parties involved. since it is in everyone's interest to have well-preserved tissue samples available for valid histopathological, biochemical, molecular or toxicological analyses, a possible solution could be to video-transmit or video-record the stages of the autopsy that take place at the autopsy table and that, once carried out, are not repeatable. in our opinion the sars-cov- pandemic provides two major lessons. the first is that in all the areas where the virus is still circulating, even if not epidemic, autopsies must be considered at high risk of infectivity. this should prompt to increase the number of bsl autopsy rooms in order to satisfy the needs of the territories. the second lesson is that rapid autopsies for health purposes appears mandatory in every day practice, even when it is not necessary to understand the etiology and pathogenesis of a new pathology. rapid autopsies guarantee optimal tissues to apply the most sophisticated diagnostic methodologies. this supports the classic holistic autopsy examinations, which aims not only to define the cause of death but also, and perhaps above all, to reconstruct the pathological history and style of the patient's life. in conclusion even today the autopsy does not lose its etymological meaning, crucial for the correct progress of knowledge: "see with your own eyes" (from the ancient greek "autòs" -same-and "opsis" -sight-). this is the foundamental part of the scientific method (observe -make a hypothesis -verify the hypothesis) masterfully described by galileo galilei, and the first step of every medical thought process. j o u r n a l p r e -p r o o f table autopsy staff -first operator (the "dirty" one): doctor, specialist in pathology, expert in performing autopsies, even in presence of diseases with high infectious risk; performs the autopsy and, if necessary, sews the body at the end. -second operator (the "clean" one): doctor, specialist or resident in pathology, even without particular autopsy experience; takes photographs, transcribes weights, measurements, volumes and observations dictated by the first operator and assists in the collection, cataloging and storage of the samples. -technician: supports the first operator but only if he has been trained for autopsies at high infectious risk; if present, supports the first operator and sews the body at the end. external inspection of the body -body length, state of nutrition (maximum thickness of the adipose panniculus at the level of the chest and abdomen), skin (color, trophy and state of conservation), hair and nails, conjunctivae, sclerae, pupils, evaluation of any material that comes out from the mouth, nostrils or middle ear and, when possible, oral cavity and dentition. -description of any pathological or morphological abnormality detectable on external examination of the body. scalp incision and opening of the skull -bimastoid cut of the scalp, passing through vertex of the head. -the skull opening must be immediately suspended if there is a smell of bone dust in the environment: this fact indicates that the dust extraction system connected to the skull saw does not work. in this case the level of risk for the staff is unacceptable, due to biological microparticles free in the air. the operators must move away from the sector table for at least minutes (time that guarantees at least complete air exchanges in the autopsy room). evisceration of the brain -cerebrum, brainstem and cerebellum must be maintained in their anatomical continuity. -weigh the visceral block. -take samples for electron microscopy and molecular investigations, preferably collecting on a single hemisphere or visceral hemibody, preserving the other for histopathological investigations. -place the residual brain in formalin, suspending it inside the container; the brain suspension is made by passing a thin string under the basilar artery and knotting its ends at the joints of the handle of the container. -remove the pituitary gland. "y" cut of the skin and subcutaneous planes for the examination of neck and trunk cavities and viscera -the "y" cut is conducted from the two acromion processes to the xiphoid process of the sternum, and from this point to the pubis along the anterior median axis of the abdomen. -observe the characteristics of the adipose tissue, the color and trophism of skeletal muscles, the state of congestion of major blood vessels and the possible presence of pathologies or injuries. opening of the rib cage -assess the size of the cardiac area. -assess the size of the visible areas of the lungs. -assess the characteristics of pleural and pleural cavities. -measure of the maximum height of the diaphragmatic dome. opening of the pericardial sac -verify and quantify the presence of effusions or blood in the pericardial cavity. -assess the characteristics of the pericardial and epicardial surfaces -assess the content of the pulmonary veins by incision (any thrombi or clots must be taken and measured). opening in situ of the right chambers of the heart -do not damage the cardiac conduction system, particularly the sinus-atrial node, the atrioventricular node and the bundle of his. -evaluate the content of the right vessels and cardiac chambers (describing and quantifying the characteristics of any observed thrombi or clots). j o u r n a l p r e -p r o o f -evaluate the morphology of the atrium, tricuspid valve, ventricular chamber, ejection cone, pulmonary valve and artery and its branches. opening in situ of the left chambers of the heart -not open the ventricle ejection cone to avoid damaging the cardiac conduction system. -evaluate the content of the left vessels and cardiac chambers (describing and quantifying the characteristics of any observed thrombi or clots). -evaluate the morphology of the atrium, mitral valve, ventricular chamber, ejection cone, pulmonary valve and artery and its branches. eviscerate the heart -weigh it and perform three biventricular sections from the tip towards the plane of the atrioventricular valves, on transverse planes, parallel to each other and about cm apart. -on the most cranial section measure the thickness of the free walls of the two ventricles and that of the interventricular septum. -take samples for electron microscopy and molecular investigations. -place the heart completely in formalin for histological and immunohistochemical studies and, where appropriate, cardiac conduction studies. removal of the lungs by cutting them from the hilum -weigh, macroscopically examine (external and internal parenchymal surfaces) and immediately sample, collecting all the lobes and areas with significant macroscopic differences (color, consistency, content at squeezing). individual evisceration, weighing, sampling and examination of: -liver and gallbladder (in a single block). virchow technique (removal of the viscera one by one) for: lungs, spleen, kidneys, adrenals and gonads. rokitansky's technique (opening and examination in situ of viscera and apparatus) for: heart (before removing it), thorax large blood vessels, stomach, epiploon's cavity and pancreas, duodenum, large bowel, ureters, prostate, bladder and the retroperitoneal large blood vessel. ghon technique (evisceration en bloc) for: brain, tongue, organs of the neck and posterior mediastinum, liver and gallbladder, small bowel. letulle's evisceration technique (evisceration en masse) not performed to minimize the leak of blood, feces and biological fluids from the body special notes: a. to be able to eviscerate the tongue, the sublingual salivary glands and part of the hypopharynx in continuity with the trachea, it is important that the incision of the skin and subcutaneous planes starts bilaterally from the most posterior part of the acromion and not from the anterior surface of the shoulder. b. the tongue is extracted by sectioning the frenulum and the floor below the mandibular arch after lifting the skin and subcutaneous planes of the neck and upper chest, and turning the tip over on the anterior laryngeal surface. the whole block of the tongue and viscera of the neck and the posterior mediastinum is mobilized from the vertebral plane by cutting the main vessels of the neck and dissecting the esophagus after performing an obstructive ligature in correspondence with its overdiaphragmatic tract. to open the thoracic cage, dissect as far as possible the ribs at the level of their cartilage tract, to avoid producing bone spicules that may sting the operator; then disarticulate bilaterally the sternum-clavicular joint and thus remove the sternum and the anterior tract of the ribs. to increase the space of maneuver inside the chest, it is advisable to dissect the muscles of the intercostal spaces. d. removal of liver-gallbladder block cutting the hilar structures, the liver ligaments, freeing the quadrate lobe and by removing the part of the phrenic muscle corresponding to the bare area of the right lobe of the liver. e. when you need to open the bladder to remove the prostate or uterus, you need to turn it; in this case, the bladder wall must be pulled upwards, making a small full-thickness hole in it from which it will be inserted a suction cannula that will empty it. the role of the autopsy in medical malpractice cases, i: a review of appeals court decisions. autopsy committee, college of american pathologists autopsy-a procedure of medical history? post-mortem examination as a quality improvement instrument features, evaluation and treatment coronavirus (covid- ) recommendations to perform autopsies in patients with sars-cov- infection. iss working group on causes of death assessment covid- human postmortem tissue: what quality markers matter effect of fixatives and tissue processing on the content and integrity of nucleic acids assessment of specimen fixation in a surgical pathology service circ. maggio , prot. n. -indicazioni emergenziali connesse ad epidemia covid- riguardanti il settore funebre emergenza covid- . circolare ministero salute n. del . . e ordinanza del capo dipartimento di protezione civile n. del marzo . attività funebre collection and submission of postmortem specimens from deceased persons with known or suspected covid- . interim guidance legge dicembre , n. , contenente: "norme per l'accertamento e la certificazione di morte world health organization. who post-outbreak biosafety guidelines for handling of sars-cov specimens and cultures biosafety level laboratory for autopsies of patients with severe acute respiratory syndrome: principles, practices, and prospects coronavirus disinfection in histopathology cutaneous manifestations of covid- : report of three cases and a review of literature sudden infant death syndrome (sids): a study of cardiac conduction system postmortem examination of patients with covid- pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid- autopsy findings and venous thromboembolism in patients with covid- : a prospective cohort study neurologic manifestations of hospitalized patients with coronavirus disease rising evidence for neurological involvement in covid- pandemic early performed autopsy (within - hour from death) provides tissue samples for diagnosis and research of quality similar to biopsy or surgical resections early samples collection reduces post-mortem artifacts, thus preventing the wrong interpretation of the morphological pictures observed precise autopsy planning prevents risks for the staff key: cord- -v u mr i authors: bonnin, paul; miszczak, fabien; kin, nathalie; resa, cecile; dina, julia; gouarin, stephanie; viron, florent; morello, remy; vabret, astrid title: study and interest of cellular load in respiratory samples for the optimization of molecular virological diagnosis in clinical practice date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: v u mr i background: respiratory viral diagnosis of upper respiratory tract infections has largely developed through multiplex molecular techniques. although the sensitivity of different types of upper respiratory tract samples seems to be correlated to the number of sampled cells, this link remains largely unexplored. methods: our study included upper respiratory tract specimens of which negative and positive for viral detection in multiplex pcr. all samples were selected and matched for age in these groups. for the positive group, samples were selected for the detected viral species. results: among the factors influencing the cellularity were the type of sample (p < . ); patient age (p < . ); viral positive or negative nature of the sample (p = . ); and, for the positive samples, the number of viral targets detected ( . < p < . ) and viral species. conclusion: the cellular load of upper respiratory samples is multifactorial and occurs for many in the sensitivity of molecular detection. however it was not possible to determine a minimum cellularity threshold allowing molecular viral detection. the differences according to the type of virus remain to be studied on a larger scale. associated with particular diseases (respiratory syncytial virus and bronchiolitis, parainfluenza virus and laryngitis, rhinovirus and common cold, influenza virus and flu syndrome), there is no evidence for a clinical specificity, and only the virological diagnosis provides an accurate identification of the ari [ , ] . detection of respiratory viruses is of little interest in general practice, in that the infection does not present a risk of severity for the patient. however, virological confirmation of ari is needed in severe clinical presentations, requiring hospitalization in intensive care units and occurring in vulnerable subjects [ , ] . the goal of early virological diagnosis would be an optimization of patient care, which could lead to reduction in length of hospital stay, a saving of antibiotics, and complementary examinations [ ] . virological tests allow for the establishment of accurate diagnosis of infection, assessment of evolving risks (bacterial infection, acute respiratory distress syndrome), and the establishment of measures to limit its spread (isolation, wearing gloves and masks). pandemics of severe acute respiratory syndrome (sars, (sars, - and influenza a-h n ( ) lead to the development of molecular biological techniques applied to virological diagnosis, mainly based on pcr (polymerase chain reaction). performances of molecular methods in respiratory virology are so significant that they have replaced conventional techniques (culture, detection of viral antigens) as a reference method [ ] [ ] [ ] [ ] . multiplex pcr techniques are particularly suited to medical diagnosis because they can detect multiple viral targets in the same time, avoiding the virologist a selection of viral targets to search. there are now many commercial kits for the detection of a range of to respiratory viruses and some intracellular bacteria [ , , , ] . molecular techniques (real-time pcr) also make it possible to achieve a semi quantification of the viral molecular material present in the sample, giving additional information about the respiratory viral load (interest in therapeutic monitoring and infection transmission risk) [ ] . a normalized viral load can be obtained by adding a cell quantification step. the primary site for replication of respiratory viruses is the ciliated airway epithelium. the sample must be taken as soon as possible after the onset of symptoms. this is usually a nasal swab or nasopharyngeal aspiration (especially realized in children under ) [ ] . these samples are easily accessible and especially adapted to upper ari. if a rich cell collection appears to be an important prerequisite for the quality of respiratory viral diagnosis, there is currently no information on a possible cellularity threshold that would validate the result of the viral molecular detection. the main objective of this work is the study of cellularity in respiratory specimens previously characterized virologically. the results should help to define the concept of "cellular richness" and determine the factors that influence it. eight hundred respiratory samples were included in this study. all were collected between september , and february , in different departments of the university hospital of caen (france), and immediately sent to the virology department for a respiratory viral diagnosis. respiratory samples were divided into nasal swabs corresponding to nasopharynx sampling (posterior nares), collected on universal viral transport medium (utm) and nasal aspirates. after receipt in the laboratory, each sample underwent a pre-analytical step including division into aliquots: one was immediately used for the viral detection and the other two were stored frozen at - °c. one of its two frozen fractions was used for this study. this complementary diagnostic study was then conducted on residual clinical specimens, in french law, the right to use the end of the samples is written in the code of public health : code de la santé publique -article l - . these aliquots were selected in the laboratory samples bank according to their results in virological diagnosis: positive and negative for molecular detection of respiratory panel using the respifinder ® smart_ _fast technique (pathofinder, maastricht, netherlands). this kit allows for the detection of rna and dna viral targets and intracellular bacterial targets in respiratory specimens (tables and ). a total of age groups reflecting the distribution observed in practice in the laboratory were indicated as follows: infants (age < ; %; n = ), children (aged from to ; %; n = ), adults (aged from to ; . %; n = ) and elderly (age ≥ ; . %; n = ). each group is composed of half positive and half negative in molecular viral detection, and so as to be matched for age. within the group of positive samples, the distribution of detected viral species was modeled on that observed in the routine activity of the laboratory (tables and ) . after thawing the samples, the extraction of nucleic acids was performed using the plc qiasymphony® (qiagen, hilden, germany). the extraction was performed with μl of sample using qiasymphony_dsp_virus/ cell quantification was achieved by amplification and detection of a human household gene in real-time pcr (hypoxanthine phosphoribosyl transferase- ) [ , ] . cell quantification was performed on a lightcycler ii® platform (roche, meylan, france). the reaction mixture included μl of amplification premix cell_control r-gene® (argene/biomerieux, lyon, france) and μl of nucleic acid extract. each manipulation included two negative controls: one undergoing all the analytical steps, called ec (extraction control) and one introduced prior to the pcr reaction, called "negative control". cell quantification standard (qs : × cells/pcr and qs : × cells/pcr) was ready to use in the kit. an external standard curve was performed using these two standards and additional dilutions, respectively containing × and cells/pcr. the reading of the results was carried out directly from the plc software, which displays the ct values (cycle threshold) obtained and the corresponding number of cells/pcr-reaction (i.e. μl of extract). this number was converted to "cells/ml" and the final results were expressed in logarithmic scale (log /ml). the quantification kit performances were verified in our laboratory by quantifying mrc cells (human embryonic fibroblasts) of known concentration (rd-biothech, besancon, france). a range of -fold serial dilutions was made from initial mrc cell suspension in viral transport medium (utm). the nucleic acids were extracted from these cell suspensions, and cell quantification reaction was carried out under the same conditions as for the respiratory samples. descriptive statistics were used to show the characteristics of the different variables. quantitative variables were described using means and standard deviation. qualitative variables were described using frequencies and percentages. the relationships between qualitative variables were studied using the chi-square test or fisher's exact tests. the anova was used to compare the means of quantitative variables in two or more independent groups with the bonferroni post hoc test. the relationship between two quantitative variables was assessed using the spearman correlation coefficient (ρ). to look for a diagnostic threshold to divide positive and negative samples, a receiver operating characteristic curve (roc curve) was used. all the tests were two-tailed and their level of significance (p) was defined as p < . . ibm®-spss® . for windows® was the statistical software used. the -fold serial dilution range from initial mrc suspension had expected cellularity values between log/ ml (concentration given by the manufacturer) and . log/ml (last dilution). the measured cellularity values were consistent with those expected for the concentrations between and . log. the last dilutions deviated more than . log of the expected value, corresponding to concentrations between . and . log ( table ). the mean deviation from expected values was . log/ml. regarding our samples population, cell quantification was negative (no hprt- dna detection) for of the positive and for of the the negative in viral detection. comparative study of sample "swabs" and "nasal aspirates" among the cell-quantified respiratory samples, were nasal aspirates and were nasal swabs. cellularity these results are presented in table . regarding nasal swabs only, average cellularity were not significantly different between the age groups except for children compared with adults (p < . ). comparison of cellularity among the positive (n = ) and negative (n = ) samples in viral detection as the subjects were matched for age, the age distribution is identical in the two groups positive and negative (p = . ). these two groups are comparable, as expected. the average cellularity was . (+/- . ) log/ml for the positive group and . (+/- . ) log/ ml for the negative group. this difference was significant (p = . ). the results of comparison between the age groups according to the result of the viral detection (positive or negative) are presented in fig. b . within a single age group (infants, children, adults, elderly), the differences between positive and negative samples were not significant (p = . , p = . , p = . and p = . respectively). based on the results of the comparison between positive and negative samples, a roc (receiver operating characteristic) curve was performed. no minimum cellularity threshold could be defined for molecular viral detection (fig. ) . aspirates swabs fig. average cellularity of respiratory specimens depending on the sample type and age group. a nasal aspirate was the sample type that provides, on average, more epithelial cells for patients under the age of ( . log/ml for aspirates versus . log/ml for swabs). b although having the same shape, the average cellularity curve of positive samples was always located above the negatives among the selected samples, viral detection was negative in , were positive for viral target, were positive for targets and were positive for targets. the average cellularity was . (+/- . ) log/ml, . (+/- . ) log/ml, . (+/- . ) log/ml, and . (+/- . ) log/ml for these groups respectively. the average cellularity in negative samples was significantly lower than in cases of mono (p = . ), bi (p = . ) or tri-detection (p = . ). a significant tendency was observed between positive samples for one viral target and those positive for or virus (p = . ), this trend was confirmed by a spearman correlation (ρ = ) indicating a strong correlation between sample cellularity and the number of viruses detected. molecular detection, including multiplex techniques, is currently the gold standard for viral respiratory diagnosis. we have very powerful molecular tools, ensuring a quality respiratory viral diagnosis, available for all clinicians supporting hospitalized patients. one factor limiting this diagnosis is represented by the collected respiratory specimens. the main objectives of this work have been to study the cellularity of these clinical respiratory specimens, to propose a possible definition of what is commonly called "cellular richness," and to measure the impact of this marker on the molecular viral diagnosis. very few published studies have been completed in this area. however, a number of facts are commonly accepted within the medical community: respiratory specimen should be "rich" to allow for "good" viral diagnosis, the "good" samples are obtained almost exclusively in infants and children. in total we identified studies published in international journals between and , whose objective was to compare the various upper respiratory samples, in terms of sampling equipment (flocked swabs versus rayon swab), in terms of sampling site (nasal, oropharyngeal, nasopharyngeal, combining sites), and sampling modality (swab, wash, aspiration) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the number of target cells and/or the number of extracellular viral particles collected could define the respiratory sample quality during sampling. it is not unreasonable to consider that the majority of the viral sequences detected by molecular techniques are mainly located in the intracellular compartment. however, several factors could modulate this distribution: the viral species, the cytopathic effect induced in vivo, the inflammatory responses, and the sampling delay from the onset of the symptoms. this study is retrospective; it was therefore not possible to collect data from the time between sampling and clinical symptoms in a standardized way. this matter is nonetheless very interesting and remains to be explored. for this study, we considered cell quantification, or cellular richness, as the main marker of diagnostic efficacy for a sample. the studies referenced above used indirect measurement of sample richness through the virus detected in it: used molecular detection methods, and one was an antigen detection using rapid diagnostic test evaluate the number of infected and uninfected cells deposited on the slide [ ] . overall, the results are consistent and show a superiority of nasopharyngeal swab versus oropharyngeal and a superiority of flocked swab versus classical ones [ , , , ] . the superiority of the "wash" versus the "swab" is not found by all authors, and comes well counteract the widespread idea that washing is always greater than swabbing [ ] . alsaleh et al. ( ) performed a molecular cell quantification using real time pcr in order to validate viral detection on nasal swabs. the cellularity of these samples was assessed using the quantification of a human endogenous retrovirus (erv ) known to be present in two copies per diploid cell [ ] . the authors report their results by comparing the ct obtained upon erv detection and not in terms of cellular load [ ] . in our study, we made the choice to use a direct detection method for assessing the number of cells in respiratory samples. to the extent that we needed a large number of samples characterized by many markers for statistical analysis (age of the sampled patient, detected viral target, etc.), a prospective study on freshly sampled respiratory specimens could not be performed. we therefore used previously characterized and stored frozen (- °c) respiratory samples. the definition of cellular richness is not affected by freezing or thawing samples since it theoretically does not change the amount of hprt nucleic acid, i.e. copy per haploid cell, whether or not lysed. similarly, no distinction between living and dead cells was made before the freezing process because such a distinction does not affect the quality of molecular detection by pcr (detection of viral genome into infected cells, living or not). the analysis of samples, divided into positive and negative in viral detection, failed to establish a minimum cellularity threshold to invalidate the negative results in viral molecular detection. the roc curve shows that the cellularity is not a quantifiable predictor of the outcome of the virus detection. however, our work has yielded interesting results concerning the factors influencing the cellularity of samples and the impact of cellularity on the result of molecular detection of respiratory viruses. we have clearly demonstrated that nasal aspirates allowed us to collect more cells than with nasal swabs and this only in the age group under years old. this result must be tempered by the fact that % ( / ) of the nasal aspirations of the study were from children under years of age, since the distribution was random at inclusion of samples in the study. this reflects a common practice of sampling methods in clinical departments: nasal aspirates are rarely performed in adults and the elderly over years old. this gesture is considered invasive and unpleasant. our results support the idea that, de facto, it would not be appropriate to perform this type of sampling in this group. for all types of samples (swabs and/or nasal aspirates), the age of the sampled patient remains an important marker influencing cellularity, with a maximum average cellularity under the age of , a minimum average cellularity in adults group, and an intermediate average cellularity in the elderly group. the reasons for this lack of cellularity in respiratory samples from adults remain obscure. regarding the influence of cellularity on the result of viral detection, it should be noted that negative samples present, all age groups combined, an average cellularity lower than that obtained for the positive samples, even though it has not been possible to establish a predictive relationship. this difference was tenuous for the children group. this observation leads to two possible explanations: either in the positive group the infection increases the sample cellularity by promoting epithelial desquamation and, to a lesser extent, the mucus capture, containing free viral particles; or, in the negative group, there are false-negatives in virus detection, induced by a lack of cells in the sample. this result was obtained from all samples (aspirates and swabs). insofar as aspirates are evenly distributed in both positive and negative groups, we think that the result of comparison is not biased given that aspirates are richer samples. considering viral detection in its entirety, without analysis of the viral species detected, it is interesting to note that the two factors, "cellularity" and "viral codetection" (detection of or viruses) are associated positively. this suggests that the detection of several viruses is facilitated in the context of a rich sample. in cases of viral co-detection, the question of whether these are the same cells that are infected or not is not resolved, even if it is conventionally accepted that an already infected cell is less permissive to a second viral infection. it should also be noted that viral co-detection can be either a co-infection, or two or more sequential infections. such phenomena have already been discussed in the works of alsaleh et al. which showed that the positive samples in viral detection had, on average, a greater amount of genetic material of human origin than negative ones. similarly, samples where the gene erv was not detected had lower viral detection. finally, they also found that the positive samples for several viruses were also those in which cellular loads were highest [ ] . the analysis of the potential impact of cellularity on the specific detection of various viruses included in the "respiratory panel" showed results that should be confirmed with larger numbers in each group. indeed, on the one hand, the highest average cellularity was obtained in the hmpv positive samples, equally detected among adults and children groups; on the other hand, the lowest average cellularity is obtained in the rsv positive samples, mostly detected in infants and children groups. these results are surprising in that the two viruses, rsv and hmpv, belong to the same virus family (paramyxoviridae) as well as to the same virus subfamily (pneumovirinae), and are genetically close. they have many similarities in the circulatory mode and in the pathophysiology of the infection they cause. yet the cellularity of hmpv positive samples is significantly greater than that of the rsv positive specimens. the quality of samples dramatically affects the quality of results provided to clinicians. it is important that a better understanding of the sample characteristics goes along with technological developments. this work is uncommon. he tries to give answers to a trivial scientific question: respiratory specimens should they be « rich in cells » to ensure optimal virological molecular diagnosis? the cellular load is multifactorial and occurs for many in the sensitivity of molecular detection. however, it was not possible to determine a minimum threshold allowing molecular viral detection. adv, adenovirus; ari, acute respiratory infection; ct, cycle threshold; dna, deoxyribonucleic acid; ec, extraction control; erv , human endogenous retrovirus ; flu, influenzae virus; hbov, human bocavirus; hcov, human coronavirus; hmpv, human metapneumovirus; pcr, polymerase chain reaction; piv, parainfluenza virus; rhv/ev, rhino/entero virus; rna, ribonucleic acid; roc, receiver operating characteristic; rsv, human respiratory syncytial virus; sars, severe acute respiratory syndrome; utm, universal transport medium department of virology epidemiology of viral respiratory infections bronchiolitis viruses the underrecognized burden of influenza in young children viral epidemiology and severity of respiratory infections in infants in : a prospective study techniques actuelles de diagnostic des infections virales respiratoires en réanimation cost analysis of multiplex pcr testing for diagnosing respiratory virus infections superiority of reverse-transcription polymerase chain reaction to conventional viral culture in the diagnosis of acute respiratory tract infections in children increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time pcr in samples from patients with respiratory symptoms quantitation of respiratory syncytial virus rna in nasal aspirates of children by real-time rt-pcr assay molecular diagnosis of respiratory viruses comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness comparative evaluation of six commercialized multiplex pcr kits for the diagnosis of respiratory infections identification of respiratory viruses in adults: nasopharyngeal versus sampling development of an efficient qrt-pcr assay for quality control and cellular quantification of respiratory samples guideline to reference gene selection for quantitative realtime pcr comparison of flocked and rayon swabs for collection of respiratory epithelial cells from uninfected volunteers and symptomatic patients nasal swab samples and real-time polymerase chain reaction assays in community-based, longitudinal studies of respiratory viruses: the importance of sample integrity and quality control swabbing for respiratory viral infections in older patients:a comparison of rayon and nylon flocked swabs comparison among nasopharyngeal swab, nasal wash, and oropharyngeal swab for respiratory virus detection in adults with acute pharyngitis improved detection of respiratory viruses in pediatric outpatients with acute respiratory illness by real-time pcr using nasopharyngeal flocked swabs comparison of nasopharyngeal and oropharyngeal swabs for the diagnosis of eight respiratory viruses by real-time reverse transcription-pcr assays evaluation of the quidel quickvue test for detection of influenza a and b viruses in the pediatric emergency medicine setting by use of three specimen collection methods a quantification of human cells using an erv- real time pcr assay not applicable. this study was supported by national reference laboratory for measles and respiratory paramyxoviridae. we have no additional data to communicate and have incorporated article all data, tables and figures necessary for the understanding of the study.authors' contributions av and rm designed the study. pb and av wrote the main manuscript text and prepared figures. rm was responsible for statistics analysis and wrote the statistics part of the methods. cr provided protocoles for the use of cell quantification kit. av, fm, nk, jd, sg and fv participated in the successful conduct of experiments, construction of the methodology and the reading of the manuscript. pb conducted all experiments. all authors have read and approve of the final version of the manuscript. the authors have declared that no competing interests exist. not applicable. this is a complementary diagnostic study conducted on residual clinical specimens, in french law, the right to use the end of the samples is written in the code of public health : code de la santé publique -article l - . this provision permits to work on the remaining clinical specimen sampled for diagnostic test (in our study, samples were upper respiratory specimen). it was possible to perform an additional analysis (in our study, it is the cell quantification of sampling) unless the patient, previously informed, does not express its refusal. the approval of an ethics committee was therefore not necessary for this kind of study. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- - xozruq authors: jayamohan, harikrishnan; lambert, christopher j.; sant, himanshu j.; jafek, alexander; patel, dhruv; feng, haidong; beeman, michael; mahmood, tawsif; nze, ugochukwu; gale, bruce k. title: sars-cov- pandemic: a review of molecular diagnostic tools including sample collection and commercial response with associated advantages and limitations date: - - journal: anal bioanal chem doi: . /s - - - sha: doc_id: cord_uid: xozruq the unprecedented global pandemic known as sars-cov- has exercised to its limits nearly all aspects of modern viral diagnostics. in doing so, it has illuminated both the advantages and limitations of current technologies. tremendous effort has been put forth to expand our capacity to diagnose this deadly virus. in this work, we put forth key observations in the functionality of current methods for sars-cov- diagnostic testing. these methods include nucleic acid amplification–, crispr-, sequencing-, antigen-, and antibody-based detection methods. additionally, we include analysis of equally critical aspects of covid- diagnostics, including sample collection and preparation, testing models, and commercial response. we emphasize the integrated nature of assays, wherein issues in sample collection and preparation could impact the overall performance in a clinical setting. in december , health officials in wuhan, prc, reported a disease outbreak involving a cluster of cases of "pneumonia of unknown cause." since then, the coronavirus disease (covid- or sars-cov- ) outbreak has been characterized as a pandemic, spread to countries worldwide, causing , , fatalities and more than million confirmed cases (as of september , ) . the pandemic has proven to be a significant challenge to our ability to reduce the global spread of the sars-cov- . given the global scale of infections due to the novel virus and the lack of approved therapeutics and vaccines, the covid- pandemic has drawn comparisons to the deadly spanish flu pandemic. a key difference is current advances in molecular diagnostic technology that has enabled us to rapidly characterize the novel virus, identify infectious (including asymptomatic) patients, and potentially isolate them to control the disease spread. however, initial delays in assay design and supply-chain bottlenecks prevented the deployment of accurate diagnostic tests at scale globally. this was found to be a critical gap in arresting the spread of this devastating disease worldwide. nevertheless, recent developments have led to broader deployment of diagnostic tests, albeit ones that require at least a dedicated sample collection setup. nucleic acid (na) amplification tests (e.g., polymerase chain reaction (pcr)) and immunoassays (e.g., enzymelinked immunosorbent assay (elisa)) are among the most utilized tools in today's covid- diagnostic testing landscape. this review paper examines current molecular diagnostic tools (fig. ) , such as amplification-based (including crispr-cas based), antibody and antigen tests, and sequencing, utilized for the detection of sars-cov- . we discuss the capabilities offered by each of these molecular diagnostic tools and challenges encountered in utilizing them in the current pandemic, and provide an assessment of future directions in research that could help remedy some of the identified shortcomings. we also report how each of these methods plays a complementary role suited for different stages of the pandemic. several review papers in the literature describe diagnostic approaches for covid- detection. this paper underscores the importance of the integrated nature of these diagnostic tools, wherein improvements in sample collection and preparation are needed to complement advances towards sensitive and accurate diagnostic methods. this knowledge is relevant to the current scenario wherein differences in analytical sensitivity and clinical sensitivity have hampered the effectiveness of covid- diagnostics. this situation reflects the challenges faced to develop and thoroughly assess the assays, reagents, and sample handling processes required for a reliable test. also, this situation generates the need to mobilize a largescale production pipeline to meet the enormous demand for assays for a new, largely unknown pathogen, sars-cov- . furthermore, this paper discusses the challenges in ramping up testing. as an example, we discuss the problem of false-negatives that has impacted covid- testing and how improvements in sample collection could help remedy the situation. we underscore the lack of research into ensuring repeatability of respiratory sample collection. we also discuss various clinical samples that are utilized for diagnostics and how viral loads (amount of viral proteins or viral nucleic acids in a sample) vary with disease onset in the sample types. we compare and contrast the utility of each sample type from the perspective of sensitivity to utilization in self-sampling formats. in addition, we also discuss sample preparation aspects that are relevant to wider utilization and point-of-care (poc) deployment of covid- diagnostic tests (pcr, isothermal amplification, and sequencing-including library preparation). finally, we identify environmental sampling, surveillance opportunities, and advanced detection technologies being developed for commercial applications. nucleic acid (na) amplification and its subsequent detection are the most widely used method for the diagnosis of viral agents. these methods have been extensively reported for the detection of past outbreaks caused by coronaviruses such as mers and sars-cov [ ] . in particular, reverse transcription polymerase chain reaction (rt-pcr) is the current standard for the detection of active sars-cov- infections [ ] . rt-pcr broadly involves four steps-lysis of sars-cov- in the sample, purification of the viral rna, reverse transcription to complementary dna (cdna), and amplification of specific regions of the cdna, and finally, optical detection of the amplified cdna. the development of rt-pcr assays for a novel virus such as sars-cov- involves sequencing the genome and design of primers and probes. a variety of rt-pcr assays utilizing different primer/probe sets have been developed. these assays utilize different primer/probe sets targeting different regions of the sars-cov- genome. the selection of the primer/probe set affects the sensitivity of the assay [ ] [ ] [ ] . for instance, a study from the university of washington reported e gene [ ] and n gene primer/probe sets (us centers for disease control and prevention) to have better sensitivity for sars-cov- detection assay [ ] . a recent report by anantharajah et al. evaluated rt-pcr assays utilizing who-recommended primer/probe sets on clinical samples. they found substantial differences in the assay sensitivity depending on the choice of primer and probe [ ] . the difference was more pronounced for samples with low viral load (median cycle threshold, ct > ). a recent study by jung et al. compared the performance of seven primer-probe sets for the n gene and three primer-probe sets for the orf gene for the detection of sars-cov- rna. the study evaluated the specificity and sensitivity of amplification at three different reaction temperatures ( , , and °c). the primer-probe sets from the japan niid (niid_ -ncov_n) and china cdc (orf ab) were reported to exhibit the best performance without any non-specific amplification or crossreactivity for other coronaviruses (hcov- e, hcov-oc , and mers-cov rna) [ ] . as an alternative to rt-pcr, other amplification-based formats such as digital pcr (dpcr) and isothermal amplification-based assays (rt-lamp) are being utilized for covid- diagnostics. digital droplet pcr involves partitioning the sample into multiple droplets, with each droplet serving as a reactor for independent pcr. dpcr utilizes endpoint amplification for direct quantification of viral load, is less susceptible to amplification inhibitors in the sample matrix, and, unlike qpcr, does not require calibration curves. dpcr has been shown to have higher sensitivity and lower false-negative results when compared with rt-pcr [ , ] . thus, dpcr could be helpful in scenarios wherein low viral load or rna degradation has resulted in false-negative results using rt-pcr [ ] . in addition, dpcr assays could be used to identify insufficient samples by quantifying a reference gene from human rna as a control [ ] . isothermal amplification-based methods such as loopmediated isothermal amplification (lamp) and recombinase polymerase amplification (rpa) are well suited for rapid poc detection. in contrast to rt-pcr, isothermal amplification is carried out at a constant temperature, does not require thermal fig. overview of covid- diagnostic workflow-samples are collected and stored in a transport medium, lysed, rna extracted, reverse transcribed to complementary dna (cdna), and then amplified (via pcr or isothermal amplification). the amplified viral sequence is detected/quantified using fluorescent dyes or colorimetric readout. crispr-cas-based detection (sars-cov- detectr) works by the activation of cas due to the presence of a target rna sequence. the activated cas subsequently cleaves reporter labels generating a fluorescent signal. the sequencing workflow converts the cdna into a form compatible with the sequencer (library preparation) and then determines the cdna sequence via digital images (sequencing-by-synthesis) or using electrical signals (nanopore sequencing). antigen-based lateral flow assays detect the sars-cov- antigen using an immunoassay format. viral antigen forms a sandwich bound by capture and detection antibodies. the presence of the labeled detection antibody indicates the presence of antigen in the sample (image created with biorender.com) cycling, and can be carried out using minimal instrumentation. in addition, due to the higher amplification efficiency, the test is rapid in comparison to rt-pcr. abbott's id now poc platform for covid- is based on isothermal amplification and has the shortest turnaround time among fda-authorized diagnostic products [ ] . in comparison to sars-cov, the increased transmissibility of sars-cov- has been attributed to asymptomatic carriers and presymptomatic patients [ ] . the viral load of asymptomatic or presymptomatic cases has been found similar to that of symptomatic covid- patients, suggesting comparable transmissibility [ ] . there is increasing research suggesting the need for large-scale population-wide testing to identify and isolate asymptomatic and presymptomatic covid- cases [ ] [ ] [ ] [ ] . centralized and high-throughput, automated rt-pcr assay platforms [ ] are well suited to test a large number of samples in a cost-effective and timely manner. recently, a shortage of commercial reagents for na extraction and amplification has hindered efforts to scale up the number of covid- tests. alternative reagents, buffers, and protocols have been explored to remedy such shortages [ ] and will need to be utilized to support mass testing strategies. these include open-source reagents and extraction-free rt-pcr protocols. it is important to validate such alternative reagents and protocols in clinical settings on automated assay platforms before use. for instance, due to the proprietary nature of reagents used in commercial rt-pcr platforms, it is critical to evaluate the effect of alternative reagents on the sensitivity and specificity of high-throughput, automated assays. the pooling of samples is an alternative method for scaling covid- tests and could be useful for populationwide screening of covid- cases. the strategy has been recently utilized to screen millions of people for active infections in wuhan, china. these involve the pooling of multiple samples into a single rt-pcr. various approaches (such as array testing [ ] ) have been proposed for the pooling of samples for covid- testing [ ] [ ] [ ] [ ] . optimum pool size and approach depend on factors such as the prevalence of the covid- in the population, and the sensitivity of the test utilized [ , ] . dpcr, which has shown to have higher sensitivity in comparison to rt-pcr, has been proposed as an alternative for testing pooled samples [ ] . despite its wide use, amplification-based covid- assays have faced numerous practical challenges [ ] . these include vulnerabilities in the preanalytical and analytical aspects of the assay [ ] . analytical issues include those attributed to variation in viral load or timing of sample collection in relation to disease progression [ ] . a study by kucirka et al. notes that the probability of false-negatives using rt-pcr (from nasopharyngeal (nps) or oropharyngeal (ops) swabs) decreases from the day of infection ( %) to symptom onset ( %). the lowest false-negatives are observed at days after symptom onset before increasing again [ ] . preanalytical issues, such as inadequate respiratory sample collection or improper sample handling, could result in falsenegative results [ ] . in comparison to the significant advances in analytical aspects such as automated operation and sensitivity of rt-pcr assays, respiratory sample collection using swabs is a manual process and prone to errors (see "sars-cov- clinical sample collection and preparation"). issues in the collection of respiratory samples can manifest as an inadequate quantity of human respiratory epithelial cells in a sample. such issues can be avoided by including a primer/ probe set to detect the presence of the human rnase p gene in the collected samples. however, quantification of human rnase p in respiratory samples (rather than a qualitative presence) might be necessary to identify inadequate biological sampling [ ] . besides, the faulty design of the primer/probe set for the human rnase p gene in the rt-pcr assay has the potential to cause false-negative results [ , ] . proper interpretation of rt-pcr assays as a proxy for infectivity is another aspect that needs attention. recently, there have been multiple reports of recurrent positive rt-pcr results in covid- patients, even after the resolution of symptoms [ , ] . a positive rt-pcr test does not necessarily indicate a patient is infectious. for instance, wölfel and team were unable to isolate sars-cov- in culture from respiratory patient samples days after symptom onset, despite positive rt-pcr results [ ] . amplification-based tests detect the viral rna (including degraded viral rna) and not necessarily the presence of an infectious virus. more work is necessary to understand the implications of positive rt-pcr results and the potential for disease transmission. this is important from the perspective of relying on rt-pcr tests as a criterion for determining return to work conditions for healthcare personnel and other essential workers [ ] . assays that utilize droplet dpcr to measure sars-cov- rna in its intact form could be valuable to detect potentially infectious covid- samples [ ] . rt-pcr is the most widely used method for the detection of viral pathogens, including the sars-cov- virus. however, given the current challenges with rt-pcr, alternative techniques involving crispr-cas and isothermal amplification are being explored. for instance, there are severe limitations associated with the availability, costs, and the need for trained personnel to run rt-pcr tests. diagnostic capabilities with lower cost, faster turnaround times, and portability are critical, given the global scope and magnitude of the pandemic. crispr, well known as a gene-editing tool, has been leveraged for rapid, poc detection of viral pathogens [ , ] . figure describes the schematic for a molecular diagnostics assay utilizing the crispr/cas system. the crispr-cas adaptive immune system consists of a guide rna (grna) and a crispr-associated nuclease (cas). the grna consists of a nucleotide sequence, called crispr rna (crrna), which is complementary to the target sequence. the cas (a type vi crispr-cas) nuclease is activated by the presence of an rna sequence (target rna) complementary to the crrna. the cas then performs a targeted cleavage and also triggers a non-specific rnase activity, leading to degradation of nearby rna (regardless of the presence of a complementary sequence). this "collateral cleavage" activity of cas is utilized to cut rna reporter labels, which then release a fluorescent signal. other crispr-cas systems such as cas or cas , which target dna, have also been utilized for the detection of viral targets [ , ] . crispr-based diagnostics (crispr-dx) have been utilized for the detection of viral targets, including dengue virus, zika virus, and lentiviruses [ ] . these assays enable multiplexing and increased sensitivity in comparison to stand-alone isothermal amplification methods. crisp-dx combines conventional na amplification with the crispr-cas system to achieve better sensitivity and specificity. the specificity is achieved through the activation of cas via the binding of crrna to target rna. in addition, sensitivity is achieved by signal amplification due to the "collateral cleavage" of reporter labels [ ] . chiu and team reported a rapid lateral flow-based assay (sars-cov- dna endonuclease-targeted crispr trans reporter (detectr)) for detection of sars-cov- [ ] . the assay can detect the virus from respiratory swab samples with sensitivity comparable to that of the us centers for disease control and prevention (cdc) sars-cov- real-time rt-pcr assay in - min. the assay combines reverse transcription loop-mediated isothermal application (rt-lamp), crispr-cas -based detection, and a readout on a lateral flow strip. the current protocol can be incorporated into a poc instrument, which would involve microfluidic automation to achieve accurate metering and mixing of reagents along with an integrated isothermal heater [ ] . however, the rna extraction from swab samples is performed using a cdc-recommended protocol that involves the use of a conventional spin column (qiagen dsp viral rna mini kit) and a magna pure instrument. fig. schematic of a crispr/ cas-based molecular diagnostic test. adapted from [ ] with permission these steps involve bulky equipment and are not suitable for poc use. other recent reports have demonstrated the utility of crispr-cas for covid- diagnostics in clinical samples [ ] [ ] [ ] . however, rna extraction is performed manually, proving to a critical limitation to its poc use. the automation of this key rna extraction step using poc sample preparation methods and integration with coupled isothermal rt-lamp+crispr-based lateral assay readouts will enable a fast, field-deployable platform that can be used in current and potential future pandemics. another advantage of crispr-based assays is the use of lyophilized reagents and the capability to run assays directly on raw samples. gootenberg et al. developed a multiplexed crispr-cas assay (sherlockv -specific high-sensitivity enzymatic reporter unlocking-version ) capable of detecting four targets in a single reaction [ ] . this multiplexing is achieved by using distinct cleavage preferences of different cas enzymes on homopolymer reporters. in addition, they utilized lyophilized cas reagents on a lateral flow strip format. the cost of a single paper-based sherlock test was estimated to be as low as usd . [ ] . the multiplexing capability reported in this work is relevant to the current pandemic due to reports of co-infection among sars-cov- -infected patients [ , ] . multiplexed assays could be useful to identify potential false-negative assay results. for instance, the multiplexed assay could be used to detect infection from alternative respiratory pathogens with clinical symptoms similar to covid- [ , ] . building on the capabilities of sherlockv , myhrvold et al. developed a protocol (hudson) that can detect multiple viral targets directly from clinical samples, including saliva with minimal sample processing, which is relevant to the current pandemic since saliva is being explored as an alternative sample for covid- testing [ ] . despite a lower observed sars-cov- viral load in comparison with nps and ops, saliva samples have several advantages for poc screening and selfadministered tests (see "covid- testing models") [ , ] . hudson uses heat to lyse viral particles and chemical reduction to inactivate rnases subsequently. this approach obviates the need for centrifuges used in columnbased extraction and can be used in poc platforms [ ] . a protocol utilizing crispr-cas sherlock assay has been reported for the detection of sars-cov- [ ] . curti et al. reported a crispr-cas -based method to detect synthetic sars-cov- rna fragments from spiked saliva [ ] . however, the limit of detection was much lower ( copies/μl) in comparison to sars-cov- rnaspiked buffer samples ( copies/μl). recently, shine (sherlock and hudson integration to navigate epidemics), a diagnostic assay combining sherlock and hudson, was utilized to detect sars-cov- from unextracted clinical specimens (np swabs and saliva) [ ] . crispr-dx for covid- testing has shown to have comparable accuracy to cdc-recommended quantitative rt-pcr tests. in addition, these tests are simpler, are less expensive, and have a faster turnaround time. the integration of these assays with sample preparation on an automated poc format utilizing lyophilized reagents is needed, which will greatly enhance the clinical utility of crisp-dx for the covid- pandemic as an alternative to rt-pcr. given their lower cost, faster turnaround time, and higher sensitivity, crispr-dx could also be used in population-wide diagnostic screening or poc settings. next-generation sequencing (ngs) plays a critical role in identifying and monitoring a viral outbreak. unbiased ngs is the key first step in identifying a novel viral strain [ ] . the sequencing data has also enabled the determination of possible origins [ ] and transmission patterns of viral pathogens, such as sars-cov- [ ] . additionally, the identification of the sequence is the first step towards the design of primers and probes for na-based rt-pcr tests. sequencing will continue to give us important information about how the virus is evolving, guiding the development of potential vaccines and therapies [ ] . recently, sequencing has been approved as a clinical diagnostic test (under fda emergency use authorization guidelines) for covid- [ ] . sequencing approaches can be broadly divided into shortread and long-read technologies [ ] . short-read technology generates sequence data from na fragments shorter than base pairs. illumina sequencing, a short-read technology, is based on a sequencing-by-synthesis approach. the workflow involves three steps: library preparation (fragmentation of the dna to be sequenced and ligation of adapters), the amplification of dna fragments, and finally, sequencing of the amplified dna strands based on the sequencing-bysynthesis approach. the collective fluorescent signal resulting from synthesizing a large number of amplified identical dna strands allows the inference of nucleotide identity. long-read technologies include single-molecule real-time sequencing (pacbio) and nanopore sequencing (oxford nanopore), which are capable of generating sequencing data from high molecular weight na (> base pairs). nanopore sequencing works by measuring the electric current across an engineered protein nanopore, as a single strand of na translocates through it. ngs is an unbiased approach to the identification of novel infectious agents since it does not require a priori knowledge of the pathogen. ngs was used to identify the novel pathogen ( -ncov, now known as sars-cov- ) in bronchoalveolar lavage fluid (blf) from three patients in wuhan, china, exhibiting symptoms of severe pneumonia [ ] . sequencing was utilized after conventional pcr using a multiplexed rt-pcr panel failed to identify the presence of known pathogens (including human coronaviruses). ngs was able to identify the genome sequence of the causative pathogen in blf from nine patients in wuhan, exhibiting similar clinical symptoms [ ] . the sequence was shown to be . % identical to each other, which confirmed the presence of a novel, single virus. when similar symptoms were detected in patients in australia [ ] , india [ ] , and cambodia [ ] , sequencing was used to confirm that the symptoms were indeed caused by the same pathogen. timely availability of viral genome sequences is a key step in the design of rt-pcr-based assays for novel viruses such as sars-cov- [ ] . early on, sars-related sequencing information allowed for the release of primers and probes for covid- [ ] . since then, improved covid- primers have been proposed [ ] , and a bait capture hybridization probe sequence has been released [ ] . ngs is also an important tool that can provide insights into the temporal or geographic traits of pathogens. for instance, sequencing was an important tool in understanding the nature of early undetected community transmission of covid- in the usa [ , ] . in the past, researchers have tracked the evolution and spread of the zika virus in the americas [ , ] , and of the ebola virus in west africa [ ] . in the case of covid- , sharing of this genomic data has already been very helpful [ ] , with existing consortiums employed. early reports showed . % sequence homology [ ] , but, more recently, higher sequence diversity has been reported [ , ] . recent research has shown that mutations in the sars-cov- spike protein led to the emergence of a more transmissible form of the virus [ , ] . understanding of sars-cov- mutations is also important to understand the effectiveness of future vaccines and therapeutics [ ] . recently, the cdc launched a nationwide genomics consortium to utilize real-time sequencing data to support such efforts [ ] . metagenomic ngs has been proposed as a diagnostic tool for detecting a broad spectrum of pathogens [ ] , including sars-cov- in clinical samples [ ] . these include direct rna sequencing of sars-cov- , avoiding inherent amplification bias associated with rt-pcr [ , ] . however, clinical samples with low viral titers will require targeted sequencing approaches [ ] , such as amplicon-based [ ] , hybridization-based capture [ , ] , and crispr-casbased enrichment [ ] . in addition to its ability to detect coinfections, ngs can offer better sensitivity compared with rt-pcr [ ] . the ability of ngs to detect small variations in the viral genome that could guide clinical decisions is also notable [ ] . in comparison to rt-pcr, ngs has not been used extensively as a diagnostic tool in the current pandemic. this is possibly due to complex sample and library preparation protocols, expensive platforms requiring expertise to operate and analyze results, and relatively long turnaround time. the greatest difficulty is overcoming other signals in the sample: typically, < % reads are non-human, and ngs is particularly prone to contamination [ ] . ngs has also traditionally been slower (a standard illumina instrument takes > h [ ] ) and more expensive than other methods. in large-scale testing, ngs is not currently competitive with pcr-based methods; however, there is ample evidence to imply that it could become a viable testing method in the future. ngs diagnostic tests can be scaled using approaches utilizing reverse transcription (rt) primers to barcode up to , patient samples in a single sequencing run [ ] . viral genomes have been recovered directly from clinical samples [ ] , including testing of bacterial, fungal, viral, and parasitic rna from multiple body fluids and tissues for multiplexed pathogen detection [ , ] . additionally, chemiluminescence enzyme immunoassay (clia)-certified laboratories are increasingly utilizing ngs for diagnosis of encephalitis, meningitis [ , ] , sepsis, and pneumonia [ ] . in one test specific to covid- , nanopore sequencing was used to identify sars-cov- from nps within h [ ] . nanopore sequencing has a lower raw-read accuracy in comparison to sequencing-by-synthesis approaches. however, nanopore sequencers such as the minion are inexpensive, have faster turnaround time, and are portable. further advances in automation and integration of sample and library preparation (see "sample preparation") will enable the widespread adoption of its capabilities. table , summarizes the various approaches we have described in this section on the utilization of sequencing for covid- pandemic management. given the increasing global need for covid- tests, rapid and inexpensive assays are required to supplement current na amplification-based assays. these include antigen-based tests and often in the form of an immunoassay. in sars-cov- antigen detection, targets could be the nucleocapsid, spike, envelope, or membrane proteins of the virus [ ] [ ] [ ] . viral antigens bind specifically to a corresponding antibody, and the event can be detected using optical, magnetic, electrochemical, and surface plasmon resonance-based techniques, among other methods (see "nanomaterials for poc covid- diagnostics") [ ] . one of the challenges in developing effective antigen tests is the lack of antibodies for specific proteins of the sars-cov- virus. as an alternative to antibodies, selex (systematic evolution of ligands by exponential enrichment)-based strategies are useful in identifying affinity ligands such as aptamers specific to sars-cov- . recently, aptamers target the receptor-binding domain (rbd) of the sars-cov- spike protein [ ] . aptamers are significantly easier and cheaper to produce in comparison to antibodies. such efforts will enable the development of reliable and more accessible antigen-based assays. the viral load is found to be variable in covid- patients [ , ] , depending on factors such as time between disease onset and sample collection, type and quality of the sample, disease severity, and patient age [ ] . zou et al. reported ct values in the range of - in upper respiratory specimens of infected patients [ ] . antigen tests are less sensitive than rt-pcr and could be less reliable in the clinical diagnosis of covid- patients with low viral load. such challenges have affected the clinical sensitivity of antigen tests for influenza and other respiratory viruses [ ] . recent studies on four different commercial antigen tests demonstrated a wide range of sensitivities from . to % (with % specificity) in covid- clinical samples [ ] . other studies have shown sensitivity as high as . % (ci %: . - . ). however, the reported sensitivities were lower and more variable ( . %, ci %: . - . ) in samples with low viral load (ct value > . ) [ ] . table lists recent work utilizing the detection of sars-cov- antigens along with the sensitivity/ limit of detection (lod). despite these limitations, rapid antigen tests can be used as a simple and inexpensive screening tool for active covid- infections. for instance, rapid antigen tests have been proposed as a screening tool for covid- at airports and border checkpoints. sample collection remains a significant challenge in antigen testing. more ideal poc sample types, such as saliva, are less invasive, and their adoption is expected to accelerate the use of antigen tests as a much-needed screening tool. a recent evaluation reported a low sensitivity of . % for self-collected covid- positive saliva samples using antigen tests [ ] . the wide variation in the sensitivities of rapid antigen tests needs to be evaluated and understood [ ] . based on the current understanding of the sensitivity challenges associated with antigen tests for influenza, we believe advances in respiratory sample collection, antigen preconcentration, and detection modalities will drive the adoption of antigen-based screening [ ] . antibody-based tests detect antibodies generated as a result of a sars-cov- infection. antibodies are proteins generated by the immune system in response to an infection. the level of antibody response can vary with age, gender, and presence of comorbidities [ , ] . immunoglobulin g, m, and a (igg, igm, and iga) are being used as potential markers for covid- . igm is shown to be an indicator of early-stage infection, while higher igg levels are observed during late stages or post-recovery [ ] [ ] [ ] [ ] [ ] . covid- antibody can be detected based on existing methods (table ) such as colloidal gold-based immunochromatographic assay (gica) [ ] [ ] [ ] [ ] , magnetic chemiluminescence enzyme amplicon-based target sequencing using nanopore sequencer to detect sars-cov- and other respiratory organisms in clinical samples [ , ] target (amplicon-based and hybridization-based capture) sequencing using bgi sequencer to detect sars-cov- and other respiratory organisms in clinical samples [ ] direct rna sequencing of sars-cov- (from clinical specimens grown in cell culture) using nanopore sequencer [ , ] proposed scaled testing protocol using rt primers to barcode up to , patient samples in a single sequencing run [ ] outbreak surveillance analysis of sequencing data from clinical samples (using illumina sequencer) unveiled undetected community transmission of covid- in the state of washington [ ] analysis of sequencing data from clinical samples (using illumina sequencer) unveiled multiple routes of introduction of covid- into the state of california [ ] amplicon-based targeted sequencing from clinical samples (using nanopore sequencer) unveiled multiple routes of introduction of covid- into the netherlands [ ] platforms and global consortiums utilizing genomic data to track real-time spread and evolution of pathogens including covid- [ , , ] immunoassay (clia) [ ] [ ] [ ] , and enzyme-linked immunosorbent assay (elisa) [ , [ ] [ ] [ ] [ ] . pan et al. [ ] and li et al. [ ] reported the detection of sars-cov- antibodies from whole blood. the sensitivity and specificity were found to be consistent with tests performed on serum samples [ ] . antibody tests are useful in epidemiological studies to assess the number of asymptomatic cases in the population [ , ] . however, cross-reactivity of antibody is observed between human coronaviruses, such as sars-cov- , sars-cov, and mers-cov. antibody-based rapid diagnostic tests are performed on serum, plasma, or whole blood (fingerstick) poc settings. however, false-positives could lead to an inflated estimate of covid- prevalence [ ] . in addition, the temporal antibody response could vary depending on the individuals tested. also, by design, antibody-based rapid diagnostic tests do not detect the presence of sars-cov- neutralizing antibodies. tests that detect antibodies that have a high affinity for sars-cov- virus are more likely to indicate the presence of neutralizing antibodies. more research is needed to understand if a positive antibody test is indicative of immunity against future sars-cov- infections. despite these limitations, given the low cost and ease of use, with improvements in clinical sensitivity and specificity, antibody tests could be used for mass-screening of covid- prevalence. it could also play a role in the evaluation of vaccines in the future [ ] . monitoring the prominence of infection throughout a pandemic is of critical importance in all stages of a pandemic. each stage of a pandemic, as defined by the world health organization, has unique testing needs [ ] . in early and peak phases, rapid turnaround times and scale-up are of great importance. in the post-peak phases of a pandemic, easily accessible, low-complexity, inexpensive, and high-throughput monitoring is needed. current virus testing technologies and methods have proven effective in various capacities. however, each method has unique strengths and limitations, which must be considered and uniquely paired with the pandemic phase, population, available resources, and virus characteristics if an optimal response is to be achieved. while testing continues inside hospitals and clinics, other unique methods are proving to be highly effective testing models for the sars-cov- pandemic. several unique testing models have been incorporated in the current pandemic response and will undoubtedly play a role in future pandemic response planning. here, we describe these methods and their relation to diagnostic testing, and provide insight into their application. the testing of infectious viruses presents several unique challenges. traditionally, a patient visits a hospital for evaluation and diagnostic testing. the limitations of this method include increased risk of exposure due to travel and interaction with healthcare workers and social concentration of infected and non-infected persons. other concerns include overloading healthcare workers and facilities, as well as the financial cost of in-person testing. several unique testing models are being utilized to reduce exposure and resource consumption. testing can be categorized by the location of the sample collection and analysis as well as if a healthcare worker aided in this process. the combination of these possibilities results in several unique testing models. drive-thru models have distinct advantages over traditional testing and have proven effective in rapid community testing [ ] . while this model is limited in the number of communities worldwide that could utilize it, many urban and rural areas of the world could incorporate such a method. this method minimizes interaction while enabling more oversight on proper sample collection. the need for reporting of selfillness has been well argued [ , ] . self-testing models involve the collection and possibly the analysis of a sample at home by the patient. recent work by tu et al. has shown the clinical utility of patient self-collected swabs from the tongue, nasal, and mid-turbinate for covid- diagnostic testing [ , ] . the fda has recently authorized at-home selfcollection of saliva and nasal swabs for covid- diagnostics [ ] . the advantages of this method include expanded testing and reduced risk of nosocomial transmission in overcrowded healthcare settings. the primary limitation is the adaption of established testing methods to a home collection model. additionally, accuracy in sample collection and processing in self-testing could be of concern. to address this concern, a home testing method where a healthcare worker visits a patient's home to collect samples has found success in treating patients [ ] . healthcare workers can aid in sample collection and analysis to reduce the risk for error at the expense of increased risk of exposure. several companies, including labcorp, letsgetchecked, and nurx, have presented home-based poc collection devices for testing. it is encouraging to note that recent studies have reported equivalent sensitivity in self-collected samples [ , ] . point-of-care diagnostic methods with an emphasis on portability and speed are seeing a large increase in development; these tests are predominantly immunoassay based [ ] . aside from the assay itself, sample collection methods are also seeing progress. spectrum solutions llc, a developer of biosample collection devices, developed the sdna- for collecting saliva biosamples, which is currently being used in sars-cov- testing and analysis. such technologies enable self-testing models to become highly feasible. yang et al. proposed a home-based testing model wherein the patient collects and processes their sample and then images the result from a paper-based assay using a cell phone for subsequent cloud upload and evaluation [ ] . the integration of poc diagnostic assay with self-collection of samples will reduce the risk associated with the shipping of infectious samples and avoid potential issues due to sampling degradation during transport. though all the test methods are fundamentally different, the quality of the sample is crucial for successful detection. this is [ ] . increasingly, inaccuracies in rt-pcr tests have been attributed to sample collection [ , ] and handling. upper respiratory samples are the primary specimen used for covid- diagnostic testing [ ] . in comparison to advances in amplification-based tests, methods for the collection of respiratory samples need improvement. given the scale of testing required, improper sampling by untrained personnel has contributed to false-negative results [ ] . there have been efforts to develop a robot for respiratory swab sampling [ ] . however, the device was not tested in vivo or clinical settings. alternative samples like saliva or nasal swabs are simpler, less invasive, and could potentially be used to avoid improper sampling associated with respiratory swab samples. sample preparation (or preprocessing) refers to the sequence of steps required to convert a clinical sample into a form compatible with downstream analysis and detection such as amplification or sequencing. sample preparation has been a major bottleneck in widespread testing during the covid- pandemic. the availability of rna extraction kits and lysis reagents and low-throughput rna extraction protocols have resulted in increased turnaround times and delays. a rapid surge in demand for tests is threatening to overwhelm the diagnostic capabilities. a review by esbin et al. describes alternative covid- diagnostic protocols involving lysis reagents and direct addition of samples (extraction-free protocols) into the amplification reaction mix [ ] . there have been recent advances in extraction-free protocols for covid- amplification [ ] [ ] [ ] [ ] [ ] [ ] and sequencing-based tests [ ] . however, such protocols involve trade-offs between lower sensitivity and simplicity [ , , ] . automated extraction platforms utilizing liquid handling robots are useful for high-throughput, centralized covid- diagnostic testing. in addition, decentralized point-of-care (poc) diagnostic tests also have a role to fulfill. integrated poc platforms with "sample-in answer-out" capability will enable covid- diagnostic capability in doctor's office and clinics. such platforms will ensure reduced turnaround time, repeatability, and potentially lower costs by reducing reagent consumption. these will involve automation of sample preparation utilizing microfluidics and their integration with the detection assay. such methods have been extensively reviewed [ , , ] and will need to be validated with covid- clinical samples. similar approaches are required to push increased utilization of sequencing to support the covid- pandemic. for instance, the complexity and labor-intensive nature of sample and library preparation workflow could limit the widespread adoption of sequencing [ ] [ ] [ ] . efforts towards the automation and integration of sample and library preparation steps will enable increased adoption of sequencing platforms for covid- pandemic management. expanding sequencing capabilities will enable us to track mutations of the sars-cov- virus in real-time with potential implications to the efficacy of future covid- vaccines and therapeutics. it is important to treat a diagnostic assay as an integrated unit, with each step of the workflow from sample collection and preprocessing to the final detection and analysis, impacting the overall sensitivity. we have summarized some of the key factors impacting each step in the diagnostic workflow (from sample collection to detection) with reference to relevant literature (fig. ). nasopharyngeal/oropharyngeal sample nasopharyngeal/oropharyngeal swabs are generally used as the collection mode for upper respiratory samples in early detection of sars-cov- by pcr. in particular, flocked swabs are especially useful when the total volume of the sample matrix is low. these special types of nylon swabs help in collecting and retaining more analyte than traditional spunfiber swabs made from cotton, polyester, or rayon [ ] . swabs are generally taken from the nasopharynx or oropharynx [ ] , with a nasopharyngeal sample being more sensitive than oropharyngeal [ , , ] . usually, a healthcare worker collects these samples, but it has been shown that the collection can be done at home by the patient [ , ] . selfsampling reduces the risks to healthcare workers (see "covid- testing models"). following swab collection, swabs are placed into a viral rna extraction medium and subsequently amplified (e.g., by rt-pcr) [ ] . depending on the disease progression and the number of days passed since the onset of symptoms, the viral load varies considerably [ ] . in such cases, swabs from different sources can be combined to increase the probability of detection. sputum is mucus produced by the act of coughing up and spitting out the material produced in the respiratory tract (the trachea and bronchi) [ ] . it has been shown that, for covid- , sputum contains more viral rna than nps, resulting in a higher chance of detection [ , , ] . sputum collection is a much simpler process than swab sampling and can easily be done by the patient. this reduces the chances of healthcare providers getting infected while collecting samples. this process involves rinsing the mouth with water, followed by expectorating of deep cough sputum directly into a sterile, leak-proof, collection cup. however, not every patient produces sputum. alternatively, sputum can be clinically induced [ ] . han et al. showed that sars-cov- could be detected from such samples [ ] . saliva is an alternative sample that can be easily be collected by the patient at home. zheng et al. showed that saliva had a higher detection rate ( . %) for sars-cov- in comparison to throat swabs ( . %) and nps ( . %) [ ] . while most studies have found saliva to be as sensitive as nps for covid- diagnostic tests [ , , ] , few have reported slightly lower sensitivity [ ] . blf is an excellent sample for covid- diagnostic with a high detection rate ( %) compared to sputum ( %) and nps ( %) [ ] . it is generally collected from patients with severe illness or undergoing mechanical ventilation. collecting blf involves the instillation of sterile normal saline into a subsegment of the lung, followed by suction and collection [ ] . due to the complexity involved and the aerosol-generating nature of the procedure, blf is more useful in determining the recovery of admitted patients. stool samples can be collected and tested concurrently with other samples to both monitor disease progression and limit the instance of false-positives. like many other coronaviruses, large viral loads of sars-cov- can be found in stool [ , , ] . however, it should be noted that stool samples tend to be among the most difficult samples to process for viral detection. stool samples are difficult to process due to the presence of many pcr inhibitors, such as bile, polysaccharides, hemoglobin, and bilirubin [ ] . processing of stool samples often requires highly trained technicians, and results are highly susceptible to user error. therefore, the viral detection rate could be variable [ , [ ] [ ] [ ] . viral loads can be found in stool samples at early onset through the convalescent stage of illness. viral loads have been found in stool samples from as early as the onset of symptoms to as late as days after onset of symptoms [ , ] . stool samples may provide a non-invasive alternative to nps and ops. the collection of stool samples is simple and can often be performed at home. sample collection involves the capture of a stool sample in a clean, dry container and transfer of the sample into a sterile specimen cup. serum and whole blood are primarily used in antibody tests for tracking disease progression, epidemiological studies, and patient immunity. these have been discussed in the section "antibody-based tests." table lists the various clinical samples used in covid- diagnostics and important factors such as peak viral load, time to peak viral load, and average detection rate. these factors should enable guidance on their utility for different testing models such as at-home or poc testing. covid- is primarily spread via human-to-human transmission, though environmental transmission has also been reported [ ] . unlike other enveloped viruses, sars-cov- are less susceptible to environmental stresses [ ] . depending on the environment, temperature, and type of surfaces, it can survive up to days [ ] . hence, environmental surveillance for sars-cov- is useful in covid- containment and preventing transmission. air sampling can be done using an air sampling pump [ ] (e.g., skc biolite+, zefon bio-pump® plus). these pumps can sample dust/particulates, vapors/gases, bioaerosols, or environmental air samples. they use cassettes and filters to collect samples for further analysis. air samplers are efficient, adaptable, and easy to use. but for preserving the viability of samples, quick and subsequent handling of sampling is needed. air samplers are used for confined spaces such as hospital rooms and apartments. researchers are now focusing on the sampling of sewage water for covid- surveillance [ ] [ ] [ ] . this can be used to detect the presence of sars-cov- in certain populations and also to quantify the scale of infection [ ] . the persistence of sars-cov- on surfaces is important to understand the risk of infection from fomites. for instance, sampling from high-touch surfaces will help uncover modes of transmission and also the effectiveness of disinfection protocols [ ] . who has provided guidelines on potential sampling sites for such studies at covid- healthcare facilities [ ] . premoistened swabs are used to collect samples from such surfaces with rt-pcr used to detect the presence of sars-cov- [ ] [ ] [ ] . viral cultures could demonstrate the viability of sars-cov- on such surfaces. surface sampling using swabs is labor-intensive, and a large number of samples are required in surveillance studies. in april this year, the national institutes of health (nih) launched the rapid acceleration of diagnostics-radical (radx-rad) initiative to accelerate the development of novel approaches to covid- diagnostics [ ] . this initiative is also aimed at the repurposing of current diagnostic techniques towards covid- and future pandemics. the initiative plans to enable rapid covid- detection platforms for homebased and poc use. in this section, we review some of the recent work on advanced techniques utilizing nanostructured materials, developed for the detection of active covid- infections at poc. these include platforms utilizing fieldeffect transistors (fet), plasmonics, and breath-based detection of sars-cov- virus, associated antigens, or rna. fet biosensors consist of an electrode (gate) functionalized with receptors or probe molecules to specifically bind with the analyte of interest. the binding of the target analyte causes a change in electrostatic surface potential (electrostatic gating effect), resulting in a change in the current flowing between the source and drain [ ] . seo et al. developed a graphene-based fet sensor for the detection of sars-cov- in clinical samples (fig. a) [ ] . the sensor consists of graphene sheets conjugated with the sars-cov- spike antibody (receptor) that specifically binds with the sars-cov- spike protein (analyte). the binding causes a real-time change in the current response, indicating the presence of sars-cov- spike protein or sars-cov- virus in the sample. the platform was shown to be highly specific (exhibiting no response to mers-cov spike proteins) and was able to detect clinical samples with sars-cov- viral load as low as copies/ml. researchers from the university of maryland, [ , ] baltimore, developed a colorimetric assay utilizing thiolmodified antisense oligonucleotides (aso) capped on the surface of gold nanoparticles (aunps) for the detection of isolated sars-cov- rna (fig. b ) [ ] . the aso-capped aunps agglomerate in the presence of sars-cov- viral rna, causing a change in surface plasmon resonance (spr) of the agglomerates in solution. further addition of rnase h causes the aso-capped aunps to precipitate, resulting in further amplification of the spr signal and enabling a visual detection of sars-cov- rna in min. qiu and coworkers developed a dual-functional plasmonic biosensor incorporating localized surface plasmon resonance (lspr) and plasmonic photothermal (ppt) effect for the detection of sars-cov- ( fig. c ) [ ] . the biosensor consists of two-dimensional gold nanoislands (aunis) functionalized with complementary dna (cdna) receptors that hybridize with the target sars-cov- rna sequence. the hybridization event leads to an lspr phase change (response) that can be detected. in addition, when the aunis are illuminated at the plasmonic resonance frequency, a localized thermoplasmonic heat is generated, promoting the selective hybridization of the target sars-cov- rna sequence to cdna receptors. biosensors based on electrochemical sensing [ , ] , quartz crystal microbalance [ ] , and magnetoresistance [ ] have been utilized in the past for viral detection [ ] . these techniques can, in principle, be applied to covid- detection at poc. haick et al. developed an interesting alternative to covid- diagnostics involving the detection of volatile organic compounds (vocs) from the exhaled breath of patients (fig. d ) [ ] . the chemi-resistive sensor array consists of aunps linked to organic ligands that swell or shrink upon exposure to various vocs, causing a change in fig. schematic of novel covid- diagnostics platforms utilizing nanostructured materials for potential poc use. a a graphene-based fet sensor for sars-cov- detection. the device consists of the sars-cov- spike antibody conjugated to graphene sheets. the specific binding of spike antibody to sars-cov- spike protein causes a change in current (response) between source and drain. (modified with permission from [ ] . copyright © american chemical society) b a colorimetric assay based on spr utilizing thiol-modified antisense oligonucleotides capped on the surface of gold nanoparticles for the detection of isolated sars-cov- rna. (modified with permission from [ ] . copyright © american chemical society) c a dual-functional lspr biosensor utilizing gold nanoislands for sensitive detection of target sars-cov- rna sequence from a multigene mixture. (modified with permission from [ ] . copyright © american chemical society) d a chemi-resistive sensor utilizing array consisting of aunps for detection of vocs from the exhaled breath of covid- patients (reproduced from [ ] with permission from the royal society of chemistry) the electric resistance. although the technique will need to be validated in a clinical study with a larger sample size, it could prove very useful as a rapid screening tool for covid- . rapid and reliable commercial screening of sars-cov- is critical to limit the covid- pandemic. research labs and medical device companies across the world made major shifts in manufacturing desperately needed test kits at a rate that matches the rapidly increasing demand during the - pandemic. to produce effective covid- tests, companies must overcome the challenges associated with scalability, accuracy, cost, testing supply-demand, and sample collection. the usa alone has performed over million covid- tests in the first months of testing [ ] . reducing storage, transportation, and testing time are of utmost importance. covid- tests generally fall into two categories, virus rna testing (using mostly rt-pcr) to diagnose current infections or serological tests for anti-sars-cov- antibodies to determine if the patient was exposed. china first released the covid- genome on jan , , and the malaysian institute for medical research successfully produced "primers and probes" for sars-cov- the same day [ ] . because of the early and rapid deployment of tests, south korea stands out for its response to the covid- pandemic. south korean company, seegene, was one of the first commercial companies to provide large-scale testing with the development of the allplex -ncov assay, which was deployed in an impressive weeks within the release of the covid- genome. allplex™ -ncov assay is a multiplex real-time rt-pcr assay that targets three specific genes for sars-cov- , which include rdrp and n genes and e gene for all sarbecovirus, including sars-cov- . in the usa, the cdc was the first to produce a one-step process that tests samples on a -well plate and completes a full test in~ min using rrt-pcr [ ] . a major issue that hindered pcr testing is the supply chain for reagents and swabs. samples for covid- pcr are collected from the nasal, nasopharyngeal, or oropharyngeal areas using specialized synthetic fiber swabs with plastic shafts. guidelines set by the us centers for disease control and prevention (cdc) state that the sample be suspended in viral transport media and stored between and °c for up to h and after h must be stored at − °c, which poses challenges for widespread sample collection [ ] . collected samples are combined with primers and polymerase and then passed through a thermocycler for amplification and optical detection. considering the scale of required tests and the highly infectious nature of covid- , sample processing is well suited for automated devices such as roche's (basel, switzerland) rapid test polymerase chain reaction detection devices. in an -h period, these devices can reportedly process and patient samples for the cobas and , respectively [ ] . point-of-care devices remove the need for storage and transportation, saving substantial testing time. bosch (waiblingen, germany) has developed a portable point-of-care rt-pcr device called vivalytic, which is fully automatic and can complete a full in situ test in . h. serological testing [ ] provides surveillance of sars-cov- antibodies identifying who has been in contact with the virus and information on immunity. the development of antibodies typically takes weeks after infection, and for this reason, serological testing is not the preferred method for diagnosing infection. common commercialized serological tests include rapid diagnostic tests (rdts) and enzymelinked immunosorbent assay (elisa). rdts are typically point-of-care qualitative lateral flow assays where samples are collected from a finger prick, saliva sample, or nasal swab [ ] . elisa tests whole blood, plasma, or serum and is typically performed in a laboratory. patient samples are incubated with an immobilized pathogen in which antibodies in the sample can form bonds. a secondary immunolabel with a fluorescent signature is used to quantifiably and qualitatively identify the pathogen [ , ] . the sars-cov- pandemic brought to light the weaknesses of many health organizations worldwide in meeting the challenges of a global pandemic. the global financial and health costs associated with this pandemic are sure to total trillions of dollars. global pandemics will likely continue to be a significant threat in the modern world, and the pandemic response strategy is worthy of a thorough evaluation. viral diagnostic testing has been observed to be one of the areas in the sars-cov- pandemic response in need of assessment and improvement. assessment of the global response with a focus on the diagnostic methods and instruments utilized will provide recognition on what should be done to better prepare for future pandemics. we reviewed aspects of viral diagnostic tools relevant in the sars-cov- pandemic and provided insight into their current state, including advantages, limitations, and scalability, and then provided direction on future work that would enable diagnostics to be better prepared for a future pandemic response. rt-pcr remains the gold standard in sars-cov- infection testing. rt-pcr has encountered limitations in sensitivity. improved primer and probe design, along with better control over preanalytical variables such as sample collection and handling, is expected to improve performance in clinical settings. other na amplification techniques, including dpcr and isothermal amplification, are being used to a lesser degree and have distinct advantages in sensitivity and turnaround time, respectively. crispr-dx shows promise in achieving higher sensitivity and specificity over na amplification alone. additionally, crispr-based technologies appear to be more conducive to multi-target reactions, raw body sample compatibility, and poc applications. in addition to being the first step in the identification of a novel virus strain, sequencing can provide insight into the evolution, transmission, and unique characteristics of a virus. while not commonly used as a diagnostic method due to higher turnaround time and costs over traditional methods, the fda has recently granted eua to a high-throughput sars-cov- clinical testing (illumina covidseq) [ ] . the advent of nanopore sequencers combined with advances in integrated sample and library preparation will enable the widespread adoption of its sequencing for covid- pandemic management. although antibody and antigen detection methods were initially limited by low sensitivity and specificity as well as a longer development cycle in comparison to rt-pcr, they promise a cost-effective approach to population-wide massscreening and are amenable to poc use. commercial testing has relied on rt-pcr and serological testing, aiming to diagnose a current infection and previous exposure, respectively. the commercial response was limited by supply chain issues rather than by technology constraints. rt-pcr-based assays were available within a few weeks of genome discovery, while serological testing methods became available within a few months. beyond the viral diagnostic technologies themselves, the methodologies involving the collection and analysis of samples have played and will continue a critical role in effectively diagnosing viral infections during a pandemic. methods, including self-and drive-thru testing, are expected to limit transmission while reducing overall per-patient testing costs. as diagnostic testing technologies develop, it will be important to incorporate the unique user needs and requirements associated with pandemic response methodologies. finally, biological sample collection and preparation remain essential regardless of the sample detection method. the type of sample, viral load, collection method, illness phase, and preprocessing are all factors that impact the sensitivity of the diagnostic method and must be appropriately matched for ideal results. improvements to preanalytical vulnerabilities (such as ensuring repeatable sample collection and processing) should move in tandem with advances in the analytical performance of an assay to ensure the optimum clinical performance of a covid- assay. table provides a summary of various techniques reviewed in this work and their potential role in disease control and pandemic management. in conclusion, given the global spread and scale of covid- infections, the diagnostic ecosystem has encountered various bottlenecks. in spite of these challenges, various diagnostic tools will continue to play a critical and complementary role in the management of various stages of the covid- pandemic. 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real-time rt-pcr diagnostic panelfor emergency use only centers for disease control and prevention preparation of viral transport medium# roche molecular solutions, roche receives fda emergency use authorization for cobas sars-cov- test to detect novel coronavirus enzyme immunoassays in diagnostic medicine. theory and practice cellex cleared to market antibody test for covid- | north carolina biotechnology center fda emergency use authorization letter publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations conflict of interest harikrishnan jayamohan is a former employee of roche sequencing solutions inc. and owns shares in roche holding ag. all other authors have no conflicts to declare. key: cord- - sabsrgy authors: quandt, sara a.; lamonto, natalie j.; mora, dana c.; talton, jennifer w.; laurienti, paul j.; arcury, thomas a. title: covid- pandemic among latinx farmworker and nonfarmworker families in north carolina: knowledge, risk perceptions, and preventive behaviors date: - - journal: int j environ res public health doi: . /ijerph sha: doc_id: cord_uid: sabsrgy ( ) background: the covid- pandemic poses substantial threats to latinx farmworkers and other immigrants in food production and processing. classified as essential, such workers cannot shelter at home. therefore, knowledge and preventive behaviors are important to reduce covid- spread in the community. ( ) methods: respondents for families with at least one farmworker (fwf) and comparable families with no farmworkers (nonfwf) in north carolina completed a telephone survey in may . the survey queried knowledge of covid- , perceptions of its severity, self-efficacy, and preventive behaviors. detailed data were collected to document household members’ social interaction and use of face coverings. ( ) results: knowledge of covid- and prevention methods was high in both groups, as was its perceived severity. nonfwf had higher self-efficacy for preventing infection. both groups claimed to practice preventive behaviors, though fwf emphasized social avoidance and nonfwf emphasized personal hygiene. detailed social interactions showed high rates of inter-personal contact at home, at work, and in the community with more mask use in nonfwf than fwf. ( ) conclusions: despite high levels of knowledge and perceived severity for covid- , these immigrant families were engaged in frequent interpersonal contact that could expose community members and themselves to covid- . the coronavirus pandemic has posed a substantial threat to immigrant farmworkers in the united states (usa) and other workers in the food production and processing system worldwide [ ] [ ] [ ] [ ] . such workers are deemed essential workers [ , ] and are unable to practice preventive measures such as sheltering at home and working from home that may be recommended to the general population. in addition, food system workers are often of low socioeconomic status, immigrant, minority, and undocumented so that they are excluded from some of the economic legal protections of workers in other industries [ ] . in regard to the pandemic, they are specifically excluded from the social safety net provided by the coronavirus aid, relief and economic security (cares) act [ ] . they also may not be reached by rapidly evolving public health messaging or provision of personal protective equipment intended to provide them with the knowledge and materials needed to protect themselves [ , ] . in the usa, many immigrant workers exist at the poverty threshold and lack health insurance and access to health care [ , ] , further diminishing their ability to protect their health in a pandemic [ , ] . such structural factors have been found to explain the uneven distribution of covid- in the usa population during the pandemic [ ] . substantial concern was expressed in the usa about latinx farmworkers' risk of covid- early in the pandemic [ , , ] . these workers often work seasonally, and the spring work season commenced within the first months of the pandemic. workers were considered to be at risk because close contact in crowded housing [ , ] and transportation used to reach the fields could increase rates of disease transmission [ , ] . within the fields, workers often work in close proximity picking row crops; and some equipment requires two or more workers to sit side by side, e.g., on mechanical setters as they plant seedlings. they also have limited access to water and other sanitation supplies [ ] . workers could then act as a vector to their larger communities by infecting other workers and family members. such patterns were observed by april , in immigrant worker populations in meat and poultry processing facilities [ ] , further increasing the concern for seasonal and migrant crop workers who would begin work in may and june in areas such as north carolina [ ] . public health directives about covid- in the usa changed rapidly over the first few months of the pandemic [ ] . early findings that coronavirus was stable on surfaces for hours or even days [ ] led to recommendations that focused on use of cleaning products to sanitize frequently touched surfaces such as doorknobs and countertops. these were subsequently downplayed as research and modeling of effects in other countries demonstrated the importance of droplet transmission of the virus, which could be reduced through physical distancing and use of face coverings such as masks [ , ] . similarly, some early claims for treatment and cures for covid- later proved false or were subject to hurried and incomplete evaluation [ ] . communication of these messages to the public, particularly to those who did not receive communications well in english, sometimes lagged behind scientific findings. taken together, the rapidly changing messages, coupled with public concern, and limited availability of up-to-date information in formats for those with limited english proficiency created a situation in the usa in which latinx workers such as farmworkers were likely to lack consistent and accurate information and, as a result, practice ineffective behaviors to protect themselves and prevent spreading disease to their social network. this study is guided by constructs from the health belief model (hbm) [ ] . the hbm tries to understand how knowledge and personal factors lead to actions to protect or promote health. in the hbm, perceptions of one's susceptibility to a disease and its perceived severity influence actions taken. individuals must perceive that they are susceptible, in this case, to covid- , and that contracting and spreading the disease would have serious consequences. in addition, self-efficacy, the belief in one's ability to take effective action in the situation of risk to health, influences whether or not one engages in health protective or promoting actions. this suggests that having a strong sense of self-efficacy in practicing protective measures to prevent contracting and spreading covid- will lead to engaging in such measures. in this study, we measure a number of these constructs, though we do not execute a full test of the hbm. interpretation of results is placed in the framework of structural vulnerability [ ] . this argues that one's health vulnerability is the product of one's place in the social hierarchy with its diverse set of power relationships, based on ethnicity and class. when applied to immigrant workers, factors such as occupation, documentation status, and access to government benefits provide context, and in fact, limit the choices, within which health behaviors understood within the hbm can occur. we report survey data collected in a narrow time window, may , from women in a sample of latinx farmworker families and a comparison group of latinx nonfarmworker families in north carolina, usa. the paper has three aims. in all cases, we will compare farmworker families and families with no worker engaged in farm work. first, we will describe the families' respondents' ( ) knowledge of coronavirus contagion and prevention, ( ) risk perceptions, and ( ) practices used for prevention and spread of covid- . second, we will describe household social interactions and protections taken, both outside of work and at work. third, we will use these data to identify specific risks for each group, as well as areas where policy changes can help mitigate the risk for covid- . the study reported here is part of a larger two-group, prospective study examining the health and cognitive effects of pesticide exposure in children in farmworker families. the larger study uses a comparative design, with a sample of families of latinx farmworkers with children and a sample of similar families but without any farmworker members. additional details of the study can be found elsewhere [ ] . the current study used a telephone survey to reach the mother of the children in these families in may , when no face-to-face contact between study staff and study participants was permitted by the institutional review board due to covid- -related health concerns for research participants. all procedures for both the original study and this covid- study were approved by the wake forest university institutional review board. the study received a certificate of confidentiality from the national institutes of health. inclusion criteria for the families were similar in both samples when recruited from march , to december ; they reflect the purpose of the larger study. each family had to have a child aged years at baseline who had completed the first grade in the usa. all children had to be from families that self-identified as latino or hispanic, and with household incomes below % of the usa federal poverty guideline. in the farmworker sample, the mother or her partner must have been employed in farm work on nonorganic farms during the past three years. in the nonfarmworker sample, adults could not have been employed in any industry that involves routine exposure to pesticides (e.g., farm work, landscaping, or pest control) in the previous three years. families in the nonfarmworker sample could not have lived adjacent to agricultural fields in the previous three years. exclusion criteria for both samples included children having life-threatening illnesses, prior history of neurological conditions, physical condition or development disorder that would not allow them to complete or would interfere with the results of neurobehavioral tests or mris (used in the larger main study), primary language other than spanish or english spoken in the home, or refusal of mother/guardian to complete the questionnaires. in the larger study, a total of children were recruited for the farmworker sample and children for the nonfarmworker sample. for the recruitment of the original sample, the community partner north carolina farmworkers project developed a list of farmworker families with an year old child and the locations where they lived. in addition, other community organizations that served farmworker families in the recruitment area were contacted. study personnel contacted the mothers. similarly, for the original nonfarmworker sample, local recruiters in winston-salem, nc, and community members developed a list. for both samples, mothers were contacted by a bilingual staff member who explained the overall study procedures, answered questions, and, if the mother agreed to participate, obtained signed informed consent from the mother and assent from the child. as recruitment progressed, community partners worked with the study team to balance the two samples on socioeconomic status. prior to the telephone survey, children in the farmworker sample and in the nonfarmworker sample withdrew, moved away from the study area, or were lost to follow-up. the remaining children represented farmworker families and nonfarmworker families, because some families had more than one child enrolled. for the telephone sample, families refused to participate and could not be reached, all in the nonfarmworker sample. a total of farmworker families and nonfarmworker families could be reached and agreed to participate. this sample of is used in this paper. data for this study were gathered from may to june , using a telephone survey. only interviews were conducted in june. interviewers were members of the larger study team who had usual interview contact with the mothers. each interviewer participated in an individualized televideo training after which the interviewer practiced completing the form and did an oral practice interview with the study manager. to recruit participants, interviewers called the last known telephone number for the mother in each family, explained the purpose and procedures for the study, and told the mother that she would receive a $ incentive for completing it at the next in-person study visit. if there was no answer, the interviewers tried at different times of day until the participant was reached or until at least unsuccessful calls had been made. if the mother agreed to participate, her informed consent was noted, and the interviewer proceeded to conduct a standardized interviewer-administered questionnaire in the language of the participant's choice using a tablet. data were entered in real time during the interviews using research electronic data capture (redcap). redcap is hosted at wake forest school of medicine through the clinical and translational science institute. the redcap system provides secure, web-based applications for a variety of types of research [ ] . data from these interviews were later merged with selected personal, family, and household variables collected in the main study questionnaires. questionnaire items relating to the coronavirus and covid- were adapted from existing studies (e.g., mcfadden et al. [ ] ), where available, or from questions recommended for covid- research by governmental and nongovernmental agencies. because of the need for rapid data collection, validation was limited to checks on face validity and interviewer reports of difficulties experienced by respondents during practice interviews. variables from the main study baseline questionnaire were used to create measures to describe the sample. these included the following measures for the mother: age, country of origin, educational attainment, and current occupation. group assignment of the family to the farm work or nonfarm work sample was also noted from the baseline questionnaire. current household size was obtained by querying the number of adults (persons years and older) and children living in the respondent's dwelling. knowledge of covid- was measured with a series of questions that asked the respondent to identify the correct answer from a series of statements for the definition of covid- , its transmission route, the definition of "close contact" for coronavirus, and availability of treatment and vaccine. a summary variable was created by summing the number ( - ) of items answered correctly. knowledge of behaviors that can prevent exposure to the coronavirus and its transmission was measured with a set of items in which the respondent was asked whether or not each could prevent exposure for self or others. the list contained items for which the correct response was positive (e.g., wear a face mask when out in public) and items for which the correct response was negative (e.g., take herbal supplements). the number of correct responses was summed to create a summary measure of questions answered correctly, with a range of to . perceptions of risk was measured with items containing statements about health risk to self and community from covid- . responses used a -point likert-type scale with values ranging from strongly agree to strongly disagree, which was collapsed to a -point scale for analysis with values (agree), (neutral), and (disagree). the two items concerning personal risk or self-efficacy were added to create a summary measure of self-efficacy with values to . this was divided into categories of low self-efficacy ( - ) and high self-efficacy ( ) ( ) . the cronbach's alpha for this scale was . . personal behaviors to protect health and prevent spread of the coronavirus in the past month were obtained by asking the respondent if they had never, sometimes, or always practiced each of behaviors. these included the positive behaviors in the knowledge items described above, as well as additional items (avoiding travel to areas infected with coronavirus; avoiding eating outside the home). these were summed with a possible range for the summary being to , with each behavior scored as (never), (sometimes), or (always). the next section of the questionnaire included questions asking about physical distancing and mask use for protection in order to overcome any social desirability [ ] that may have affected the previous self-reports of behavior. respondents were first asked how many adults had visited in the respondent's house in the past week. response options were none, or , or , and or more. those who had had visiting adults were asked how many visitors had worn masks during their visit, with the response options of all of them, some of them, and none of them. these questions were also asked about child visitors. respondents were also asked how many different houses, apartments, or trailers of others they had visited in the last week. response options were none, or , or , and or more. those who had visited other homes were asked how often they wore a mask during their visit, with the response options of all, some, or none of the time. similar questions were asked about the household children and the respondent's spouse/partner. respondents were asked how many people they worked with, defined as the number of persons with whom they worked closely enough to have a normal conversation for at least some of the work time. response options were none, or , or , and or more. mask use was queried for coworkers, with response options of all of them, some of them, and none of them wore masks at work. similar questions were asked for the spouse/partner at work. respondents were asked if their children had been cared for in the past week at a day care, pre-school, school, after school program, or at a relative or friend's house. any positive responses were followed by asking whether all, some, or no childcare workers wore masks and wore gloves. to obtain information on large social gatherings in the past week, respondents were asked if any household member had attended church, the approximate number of attendees, and if all, some, or none of the attendees wore masks. the same set of questions was asked about whether any household member had attended a party or other social event such as a cookout, baptism, quinceañera, wedding, or funeral in the past week. frequencies and percentages were calculated to examine the variables of interest by farmworker status and significant differences were examined using chi-square or fisher's exact tests as appropriate. all analyses were done using sas v . (sas institute, cary, nc, usa), and p-values < . are considered statistically significant. respondents ranged in age from to years (table ) . about % of both samples were born in mexico; spanish was the preferred language for most. years of formal education for the respondents ranged from to college graduate, with the median in both samples being ninth grade. their spouse/partners had slightly lower education; the medians for the farmworker and nonfarmworker samples were sixth and eighth grade, respectively. there were no significant differences between the two samples for these categorical variables. total household size ranged from to (median = ) and to (median = ) in the farmworker and nonfarmworker samples, respectively. for the farmworker sample, the number of adults in the household ranged from to , while the number of children ranged from (a respondent currently separated from her family) to . for the nonfarmworker samples, the ranges were to for adults and to for children. at baseline, farmworker families reported that the most common industry in which women worked was agriculture; for men, it was construction, followed by agriculture. for nonfarmworker families, most women were not in the labor force and the majority of men worked in construction. knowledge of the coronavirus was high ( table ). all individuals in both samples had heard of the virus, and none required an explanation of what it was. the farmworker sample had more correct answers than the nonfarmworker sample on three of the four remaining items. more in the farmworker sample knew that covid- was a respiratory disease caused by a viral infection ( % vs. . %; p < . ). for the item concerning treatment or vaccine for covid- , . % of the nonfarmworker sample did not know that there is currently no cure or a vaccine for covid- , compared to only . % of the farmworker sample (p < . ). overall, knowledge in the farmworker sample was significantly higher than in the nonfarmworker sample (p < . ), with . % of farmworker sample having a perfect score, compared to only . % of the nonfarmworker sample. knowledge of behaviors to prevent exposure to the coronavirus or spread of covid- was high in both samples (table ). for seven of the items, both samples had % correct responses. more in the farmworker sample knew that avoiding touching the face with unwashed hands was protective than in the nonfarmworker sample ( . % vs. . %; p < . ). the only other items for which the samples had different responses were three of the five in the list that were negative options (e.g., taking herbal supplements). for these, the nonfarmworker sample had significantly more correct responses for using herbal supplements ( . % vs. . %; p < . ). the farmworker sample had more correct responses for eating a balanced diet ( . % vs. . %; p < . ) and getting regular exercise ( . % vs. . %; p < . ). overall, the farmworker sample had somewhat better knowledge of prevention than did the nonfarmworker sample, but the difference was not significant (p = . ). the farmworker sample respondents perceived lower risk associated with covid- for themselves and their community on most items than did the nonfarmworker sample respondents (table ) . similarly, the farmworker sample perceived that they had lower ability to protect themselves from the coronavirus, with almost all responses ( . %) falling in the lower self-efficacy category, compared to . % of the nonfarmworker sample falling in the higher self-efficacy category (p < . ). for self-reported actual preventive behaviors, the farmworker sample was significantly more likely to report practicing three behaviors (avoiding travel to areas infected with coronavirus [p < . ], avoiding eating outside the home [p < . ], and avoiding close contact with people who were sick [p < . ]), while the nonfarmworker sample was significantly more likely to report practicing four behaviors (washing hands for s [p < . ], using surface disinfectants [p < . ], avoiding touching face with unwashed hands [p < . ], and covering cough with tissue [p < . ]) ( table ). the overall difference between the two samples was significant (p = . ). slightly fewer than half of farmworker families (n = ; . %) reported that they had had adult visitors at their home in the past week. of these, reported that none of the visitors had worn a mask. similarly, of these families ( . %) reported that children had visited in their home and none had worn masks. for nonfarmworker families, more had had adult visitors (n = ; . %), but some (n = ; . %) had worn masks. a lower proportion of the nonfarmworker families had had child visitors (n = ; . %), and some (n = ; . %) had worn masks. more farmworker than nonfarmworker family respondents reported visiting the homes of others in the past week (n = , . % vs. n = , . %). both categories of respondents reported visiting or other homes, except from farmworker families who reported visiting or . none of the respondents from farmworker families reported wearing masks when visiting; . % (n = ) of the nonfarmworker respondents reported ever wearing masks while visiting. twenty-seven respondents ( . %) from farmworker families reported that their children visited other homes in the past week, and none wore masks. they also reported that . % (n = ) of their spouse/partners visited other homes, and none ever wore masks. respondents from nonfarmworker families reported fewer children (n = ; . %) and spouse/partners (n = ; . %) visiting other houses, with one spouse/partner visiting five or more houses. about a third (n = ; . %) of spouses were reported to have worn masks, though several respondents did not know, and . % (n = ) reported their children had never worn masks while visiting other homes. among respondents in farmworker families, ( . %) reported working in the past week. most (n = ; . %) worked in places with five or more employees in close enough contact to have a normal conversation at least some of the time. these respondents reported that all (n = ; . %) or some (n = ; . %) wore masks in the workplace. almost all of their spouse/partners worked (n = ; . %); . % (n = ) worked in places with five or more employees in close contact, and some or all wore masks in . % (n = ) cases. about the same proportion of respondents in nonfarmworker families worked (n = ; . %), but fewer (n = ; . %) worked in places with five or more workers in close contact. most of these respondents reported that all (n = ; %) or some (n = ; . %) of coworkers wore masks. almost all (n = ; . %) spouses worked, though less than half (n = ; . %) worked in close contact with five or more workers. in about two-thirds of these worksites ( . %), some ( . %) or all ( . %) workers wore masks. during the time women were surveyed, schools were closed, and no children attended preschools or day care centers. seven ( . %) respondents in farmworker families reported that their children were cared for at a friend or relative's house and that none of the caregivers wore masks or gloves. four ( . %) respondents in nonfarmworker families reported similar childcare arrangements. however, half reported the caregiver wore masks and gloves. five ( . %) of the respondents in farmworker families reported that a household resident had attended church in the past week. total church attendance was estimated by the respondent at ( cases), ( case), and ( cases). all attendees wore masks in four of these church services, and none wore masks in the other. only one respondent among nonfarmworker families reported that a household member had attended church in the past week. attendance was about people and all reportedly wore masks. nine ( . %) respondents in farmworker families reported that a household member had attended a party or social event in the past week. estimates of total attendees ranged from to ; none wore masks. by comparison, three ( . %) respondents in nonfarmworker families reported someone had attended a party or social event. in two cases, attendance was estimated at ; the other was estimated at . no one wore masks at two of these events. this study was designed to describe the knowledge, perceived risk and susceptibility, and preventive behaviors reported by latinx immigrant farmworker and nonfarmworker families in north carolina during the first months of the covid- pandemic. these families are of particular concern because the rates of covid- nationally are elevated in minority populations. specifically in north carolina, on june , hispanics were reported to make up % of the state's population but % of the state's covid- cases [ ] . at the same time, several farmworker camps were listed as locations of covid- outbreaks by the state department of health and human services. the study found that levels of knowledge were extremely high among the latinx families surveyed, both farmworker and nonfarmworker. all respondents had heard of the pandemic and knew what covid- is and how it is transmitted. they had somewhat less accurate knowledge about the availability of a cure or vaccine; and women in farmworker families had, overall, slightly more accurate knowledge than did the women in nonfarmworker families. both samples had strong knowledge of the health behaviors that could protect against exposure to the coronavirus and contracting or transmitting covid- . in particular, they knew the primary public health messages promoted early in the pandemic. they were less accurate in differentiating these effective behaviors from ineffective behaviors that might be promoted for health risks other than covid- , such as exercising and consuming a balanced diet. although both groups perceived that covid- presents a serious risk to health, respondents in farmworker families were significantly less likely to affirm personal susceptibility (e.g., that they would avoid going to the hospital for another illness because of risk of contracting covid- there and that they were more likely than others to get . similarly, these women in farmworker families had lower self-efficacy concerning their ability to protect themselves. the two samples affirmed different patterns of health promoting behaviors. for the farmworker families, behaviors that entailed avoiding others (e.g., not traveling to areas infected with coronavirus, avoiding eating out, and avoiding close contact with sick individuals) were affirmed significantly more often than by the nonfarmworker families. the latter were more likely to affirm behaviors related to personal hygiene: hand washing, using disinfectants, avoiding touching the face, and covering coughs and sneezes. together, these findings give a sense that, while the women in farmworker families had somewhat better knowledge, they perceived less personal susceptibility to covid- . they had low confidence that they could protect themselves. this may be underlying the protective behaviors they reported. they avoided people and places that might be contaminated but did not subscribe to practicing personal hygiene behaviors. women in nonfarmworker families had greater confidence that they could protect themselves and they claimed to practice more personal hygiene behaviors. social desirability [ ] can bias the way individuals respond to lists of health behaviors. with knowledge of recommendations, they may tend to see themselves or want to portray themselves as more positive and compliant than they actually are. in order to investigate behaviors in detail and try to avoid social desirability bias, the telephone survey included a series of questions about social interactions by household members and wearing masks. complex question sequences are thought to reduce social desirability bias [ , ] . the focus on distancing and masks was considered important in light of the developing public health messages that identified the greater importance of maintaining physical distancing and protection against spreading infected droplets with masks, rather than practices such as disinfecting surfaces that had been promoted over mask use earlier in the pandemic [ ] . the responses to these questions contrasted sharply with the other reported protective behaviors. they showed a high level of social interaction beyond the immediate household for both farmworker and nonfarmworkers families, with both adults and children coming into the homes of respondents and members of the respondent's household visiting in the homes of others. there was virtually no mask wearing reported by farmworker family respondents, and only some use of masks reported by nonfarmworker respondents. household sizes reported in this study (median for farmworker and for nonfarmworker families) are considerably larger than the usa average of . people reported for [ ] , potentially creating large social networks of contacts. many of the adult household members were reported to be working outside the home and working in situations where they had close contact with other workers. these situations, plus the sheer number of adults in the household (up to six in farmworker families and four in nonfarmworker families), allows for the spread of infection through these interconnected households [ ] . mask use was reported to be common in the workplaces, though measures of the consistency or enforcement of mask use were not obtained. the respondents and their family members reported continuing to engage in social situations with large numbers in attendance. this occurred in both samples and was particularly common among the farmworker families. although masks appear to have been worn for church attendance, little mask wearing was reported for other types of social events. in total, these results indicate that, despite relatively high knowledge, strong perceptions of risk from covid- , and claims of avoiding situations where contracting or spreading infection might be likely, many of the farmworker families included here do not practice safe physical distancing measures as recommended; and their use of masks appears to be confined to work settings. the situation for the nonfarmworker families appears to be somewhat better, with greater mask wearing reported, particularly in large social gatherings. however, the social contact is still at levels that facilitate covid- spread. the inconsistency between women in farmworker families seeing themselves as avoiding situations for infection and their actual practices may be due to their living situations and to cultural values. most live in rural environments and few women drive [ ] , so they may perceive of themselves and their households as isolated from population centers. nonetheless, it is clear that interactions take place within and between households, which can exponentially raise the possibility of transmitting infection. this is in contrast to the nonfarmworker families who live in urban environments, many in multi-unit dwellings such as apartment buildings. they may correctly perceive less ability to socially isolate themselves and, so, give greater importance to personal hygiene measures to prevent infection. for these immigrant workers (from both farmworker and nonfarmworker families), living in close proximity to extended family members plus the cultural value of familismo [ ] likely affect interpretation of public health recommendations to maintain physical distance. many immigrant workers settle in the us with extended family from their home communities-siblings, cousins, parents, aunts, and uncles. this can provide considerable social and material support while living in a new environment and working in low wage jobs; family and household boundaries are likely more fluid than they are for other ethnic groups [ , ] . these relationships are supported and reinforced by familismo. this cultural construct includes strong identification with and loyalty to family, as well as respect for family members and placing family needs over one's own needs. time spent with one's immediate and extended family is valued. in such a context, wearing masks or refusing social interaction might be considered an affront. the result can be greater contacts and less physical distancing than public health recommendations intend, increasing the risk of coronavirus infection. while covid- is an emerging issue, findings from previous research with immigrant latinx populations support the findings in this study. for example, research with immigrant latinx women has produced results supporting the lower self-efficacy seen among the respondents from farmworker families. studies of hiv and cancer prevention behavior have found low self-efficacy in latinx farmworker women, which is sometimes amenable to change with intervention [ ] , though not always when cultural norms constrain health-promoting behavior [ ] . kilanowski [ ] , in a study of farmworker child nutrition, found self-efficacy for health behavior change was inversely related to acculturation, suggesting that self-efficacy may fall with greater time in the usa. none of the families in the current study are newly arrived immigrants because of the larger study eligibility criteria. other research with farmworkers has shown that they have low levels of perceived susceptibility to other health threats, most notably pesticides [ , ] . in these cases cultural values appear to promote these ideas of low susceptibility. the farmworker families included in this study are seasonal workers, meaning that they live in the area year round, and family members work seasonally in agriculture. they may not experience the extremely crowded barrack-style sleeping quarters, kitchens, and bathroom facilities of much of the grower-provided housing where migrant workers live [ ] . however, these seasonal worker families do have crowded housing [ , ] , and they face worksite hazards for infection in crowded transportation to the fields and while working in close quarters in some situations in the fields, as well as in greenhouses or packing facilities [ ] . they also often work alongside migrant workers who live in crowded conditions. although the respondents indicate mask usage, it is difficult to know how sustained that can be, considering the high levels of heat and humidity these workers endure in the fields [ ] . the contrast between what the respondents in this study know about covid- and their seemingly contradictory behavior can be viewed through the lens of structural vulnerability [ ] . the farmworker families, as well as many of the nonfarmworker families, include those who have been deemed essential workers. these include those in farm work, in construction, in building maintenance, and in food retail. as essential workers, they need to work in order to receive income. their jobs do not provide the luxury of working from home. as immigrants, most are ineligible for government benefits provided as part of the cares safety net [ ] . in the case of undocumented families, worry about the xenophobic climate [ ] may affect decisions to work, to seek medical care, and to complain about the lack of personal protective equipment. in short, these workers are not putting themselves and their communities at risk because they are uninformed about covid- . they know how dangerous it is, and, while cultural values and practices may lead to some excess exposure, they do know how to prevent covid- . one of the strengths of this study was the concentration of data collection in a short time during which changes in national information about prevention and state regulations were relatively stable. by may, reports of emerging research had started to establish the importance of physical distancing and mask use (although publications did not appear until june [ ] [ ] [ ] ), and the initial emphasis on hand hygiene and cleaning surfaces had been downplayed. within north carolina, all families in this study would have been subject to the same governmental orders. stay-at-home orders banning gatherings of > persons and closing schools, bars, gyms, playgrounds, and restaurants (except for take-out and delivery) were put in place in march. on april, school closure was extended for the rest of the academic year. although restaurant closure was loosened on may to % of capacity for indoor dining, most restaurants took longer to implement this and many still remained at take-out and delivery only well into the summer. gatherings were limited to people on march; although -person gatherings outside with social distancing were allowed on may, indoor gatherings were kept at with no special provisions for churches. this study did not collect data on information sources about covid- available to study participants. although both groups frequently get information from spanish language radio, the nonfarmworker families may have had greater access to public health signage and other local messages in an urban context than the farmworker families did in rural settings. other study limitations include the fact that behaviors were self-reported and not observed. the women interviewed also reported for others in the household. responses could not be anonymous because they were collected by interviewers that the women had known through participation in the larger study; this could have increased the social desirability in responses concerning behavior. small sample sizes prevent more detailed analyses of data. nevertheless, this study represents a unique opportunity to document the knowledge, perceptions, and behaviors of latinx immigrants in the usa during the early days of the covid- pandemic. in particular, farmworkers are often a hidden and difficult to reach population. this study demonstrates that even with a strong knowledge base, these farmworker families lack the self-efficacy to avoid the coronavirus and covid- . while they appear to believe that they are following public health recommendations on physical distancing and wearing masks, detailed data on their social interactions and use of personal protective equipment show that this is not the case. a comparison group of urban-dwelling latinx immigrants had greater self-efficacy, which might have led to the greater use of masks as personal protection reported by respondents in these nonfarmworker families. the transmission of a highly infectious virus like the coronavirus is facilitated by close contact among individuals in a population. the large household sizes, particularly large numbers of adults working in industries deemed essential, and weak adherence to personal protective equipment such as masks make the immigrant latinx population at risk for high rates of infection. it is likely that simple public health messages encouraging physical distancing and mask wearing may not protect the population in the context of structural barriers such as crowded housing and work in essential industries, coupled with strong cultural values placed on support of large extended families. specific actions beyond what is currently being taken by public health authorities may help improve the health-related behavior reported here and curb the spread of infection in this population. developing and disseminating culturally sensitive education to help families understand the extent of their social contact and the dangers it poses is essential. using adult educational approaches [ , ] that could include interactive exercises to demonstrate the potential spread of infection would likely be more effective than education based primarily on print materials in this low literacy population [ ] . the covid- pandemic has ravaged urban populations around the world, with high population density facilitating the spread of the disease. while one might, therefore, expect urban and rural conditions in the us to be markedly different, the findings here suggest that this may not be the case for latinx workers in essential rural industries. living in large households and working in close contact with large groups of workers may negate the expected isolation of rural communities. unequally vulnerable: a food justice approach to racial disparities in covid- cases food system workers are the unexpected but under protected covid heroes disproportionate impact of the covid- pandemic on immigrant communities in the united states the lancet. the plight of essential workers during the covid- pandemic what a stay-at-home order means for migrant dairy workers latinx farmworkers and farm work in the eastern united states: the context for health, safety, and justice us policies increase vulnerability of immigrant communities to the covid- pandemic sheltering in place in a xenophobic climate: covid- and children in immigrant families delivery of health services to migrant and seasonal farmworkers providing health information to latino farmworkers: the case of the affordable care act spatial disparities in coronavirus incidence and mortality in the united states: an ecological analysis as of essential farmworkers risk covid- exposure to maintain food supply as hundreds of farmworkers test positive for covid- , many remain unprotected housing characteristics of farmworker families in north carolina housing quality among north carolina farmworker families migrant farmworker housing regulation violations in north carolina perceptions of housing conditions among migrant farmworkers and their families: implications for health, safety and social policy covid- response team. update: covid- among workers in meat and poultry processing facilities-united states the news & observer. state reports more covid- cases among farmworkers-but stops releasing names of farms aerosol and surface stability of sars-cov- compared to sars-cov- covid- systematic urgent review group effort (surge) study authors. physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov- and covid- : a systematic review and meta-analysis centre for the mathematical modelling of infectious diseases covid- working group. effects of non-pharmaceutical interventions on covid- cases, deaths, and demand for hospital services in the uk: a modelling study covid- coronavirus research has overall low methodological quality thus far: case in point for chloroquine/hydroxychloroquine historical origins of the health belief model structural vulnerability and health: latino migrant laborers in the united states using life history calendars to estimate in utero and early life pesticide exposure of latinx children in farmworker families research electronic data capture (redcap)-a metadata-driven methodology and workflow process for providing translational informatics support perceptions of the adult us population regarding the novel coronavirus outbreak social desirability bias and the validity of indirect questioning addressing the disproportionate impact of covid- on communities of color. executive order no methods of coping with social desirability bias: a review determinants of social desirability bias in sensitive surveys: a literature review the number of people in the average u.s. household is going up for the first time in over years nutritional strategies of latino farmworker families with preschool children: identifying leverage points for obesity prevention an anthropology of familismo: on narratives and description of mexican/immigrants educating hispanic women about cervical cancer prevention: feasibility of a promotora-led charla intervention in a farmworker community associations between gender norms and hiv self-efficacy among latina immigrants in a farmworker community patterns and correlates of nutrition among migrant farm-worker children applying pesticides without protective equipment in southern mexico farmworker and farmer perceptions of farmworker agricultural chemical exposure in north carolina the modern practice of adult education; the adult education company educational techniques for lifelong learners. principles of adult learning developing occupational safety and health training programs for immigrant workers: translating research to practice this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors appreciate the support and participation of their community partner, the nc farmworkers project, and of student action with farmworkers. they also appreciate the valuable contributions of our community field interviewers in carrying out participant recruitment and data collection. they especially thank the mothers who participated in this study. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. key: cord- -tncjfdtp authors: hackney, raymond w.; crawford, james j.; tulis, jerry j. title: using a biological indicator to detect potential sources of cross-contamination in the dental operatory date: - - journal: the journal of the american dental association doi: . /jada.archive. . sha: doc_id: cord_uid: tncjfdtp abstract the authors conducted a study using surveillance monitoring methodology to identify operatory contamination and to evaluate the effectiveness of infection control procedures. viridans streptococci were evaluated as biological indicators of oral contamination. viridans streptococci, abundant in human saliva, were detected on operatory surfaces after dental treatments were finished and surfaces were disinfected. the findings validate current concepts of infection control as demonstrated in barrier methods. a gallup poll indicated that two-thirds of the adult u.s. population is treated in dental offices each year. protecting a major portion of the populace from infections transmitted by saliva-and blood-contaminated operatory surfaces and equipment is an unending challenge for practicing dentists. the task of protecting patients and dental workers during the past decade has prompted dramatic change in what is required of dental practice. in addition to the bloodborne pathogens standard of the occupational safety and health administration, infection control guidelines published by the centers for disease control and prevention to protect patients have been mandated in all states, according to federal law. [ ] [ ] [ ] in recent years, instrument cleaning, disinfection and sterilization have received detailed attention and definition. [ ] [ ] [ ] [ ] [ ] patients can be protected from cross-infections only if each patient's oral tissues are not handled alternately with operatory equipment and surfaces contaminated with saliva and blood during the care of previous patients. preventing cross-contamination requires identification of the sources of contamination, as well as the careful implementation of well-designed barriers and aseptic techniques. this article addresses the difficult task of infection control assessment and monitoring for oral contamination on dental operatory surfaces handled during dental treatment. the concepts and findings we describe in this article affirm the design of current infection control methodologies. , [ ] [ ] [ ] in addition, this study also supports the importance of monitoring the potential for cross-infection in practice, research and the assessment of new dental equipment and methods. without adequate control procedures, agents of both respiratory and bloodborne diseases left on dental equipment can be transmitted to successive dental patients. intact or injured oral tissues are vulnerable to agents of hepatitis b and c, hiv, and herpes simplex and viruses. infection of oral and respiratory passages can result from transfer of pathogenic bacterial strains of streptococci, staphylococci and pneumococci; influenza, measles and mumps viruses; or varicella-zoster, cytomegalovirus, respiratory syncytial virus, rhinovirus, adenovirus, coronavirus, coxsackievirus or transmission of pathogenic yeasts and bacterial respiratory pathogens from patients' mouths to the mouths of successive patients after radiographic examinations. thus, contaminated operatory surfaces can act as fomites when infection control procedures are not followed. sampling and dye studies have shown that surfaces of operatory equipment handled during oral treatments become heavily contaminated. [ ] [ ] [ ] [ ] as saliva contamination is not visible, contaminated sites are easily overlooked. in a busy practice, time allowed between patients for thorough cleaning and disinfecting is often inadequate. these factors make rendering operatory equipment and surfaces free of contamination a difficult challenge. these observations have contributed to the development of guidelines for operatory asepsis. , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] we found nothing in the literature that provided detailed documentation and evaluation of a method of assessing contamination of contact surfaces in the dental operatory, how much contamination is encountered in private operatories or an evaluation of efforts made by private office personnel in preparing operatories for safe reuse. thus, we designed this study to establish a basis for evaluating infection control procedures and equipment, and to propose an initial standard for assessing oral contamination of operatory surfaces. although pathogens can be found in bodily fluids of infected people, shedding of those pathogens is intermittent and epstein-barr virus. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] hiv from human sources dried on contaminated surfaces becomes inactivated quite rapidly ( to percent reduction within several hours). however, hepatitis b can survive at percent humidity for seven days. staphylococcus aureus can survive on dried surfaces for a mean of five days. one group of investigators found that when dried on patients' paper charts, herpes viruses survived approximately three hours when mixed with saliva and more than four hours when mixed with blood. rhinovirus survived up to hours in saliva mixed with saline; streptococcus pyogenes survived more than two days, and s. aureus survived more than five days (viable salivary bacteria could be detected for up to five days). when plasticcone x-ray machines were inoculated with bacterial cultures, s. aureus was cultivated from the dry surface after hours, and streptococcus pneumoniae and s. pyogenes after hours. mycobacterium tuberculosis can survive for six to eight months in dried sputum protected from direct sunlight. lamp handles, bracket table handles, air-water syringes, suction hose handles, handpieces, switches, drawer handles, chair controls, clinicians' chairs and charts are frequently handled by oral health care workers whose hands are contaminated with blood and saliva. , these workers may then touch their own eyes, nose, mouth or skin lesions. a significant study by autio and colleagues demonstrated the unpredictable. thus, testing for such pathogens can be counterproductive. this problem was handled in the science of water sanitation by designing tests to detect coliform bacteria, indigenous to the healthy human intestine, in an effort to protect drinking water from all fecal waste contamination. like the intestinal tract, the oral cavity hosts specialized indigenous microbes, which can serve as indicators of oral contamination in the testing of dental office equipment and surfaces. for an oral microbe to be an indicator organism, it must meet the following criteria: dit must be common to the human mouth; dit must survive for a useful period of time outside the mouth on surfaces and equipment; dit must be present in low numbers in nondental environments in which there is low potential for oral contamination; dit must be relatively easy to recover and distinguish from other bacteria recovered from dental operatory surfaces; dit must be recoverable from operatory surfaces and equipment known to be contaminated. viridans streptococci are common to the human mouth, are easy to detect when cultured on blood agar, and would be logical indicators of oral contamination if they meet the aforementioned criteria. , [ ] [ ] we found no reports in which such oral streptococci were evaluated as indicators of oral contamination of dental equipment surfaces. we also found few data on the survival of oral streptococci with regard to environmental temperature and humidity. investigators have documented heavy contamina-tion of clinicians' smocks, cuffs and equipment used during treatments, but they did not differentiate oral bacteria from ordinary skin bacteria. seven common oral streptococcus species compose a group called viridans streptococci. they produce α-hemolysis, a zone of partial hemolysis-a greenish discoloration around each colony grown on blood agar. this characteristic makes these microorganisms easy to distinguish from other bacteria found in dust and on skin that might also contaminate clinical surfaces, suggesting the usefulness of α-hemolytic streptococci, or ahs, as standard indicators for detecting oral contamination and for evaluating operatory asepsis. in this study, we assessed the validity of oral ahs as an indicator of oral contamination in the following manner: dassessing the consistency and abundance of ahs in mouths of a sample of patients; ddetermining the distribution of ahs in nondental environments, both clinical and nonclinical; devaluating environmental survival of ahs on operatory materials; dusing ahs as an indicator of contamination after cleaning and disinfection in private dental offices. survey of dental patients' saliva for ahs. the number of ahs commonly found in saliva was determined from saliva samples of randomly selected general dentistry patients at dental school clinics at the university of north carolina at chapel hill. the mean age of the patients in the survey was . years; ages ranged from to years. of the patients, were male and were female. each saliva sample was diluted - and . milliliter was plated on sheep blood agar. the number of both ahs and nonhemolytic colony-forming units, or cfus, on each plate was counted. overall surface sampling methodology. we chose the swab-rinse method for all sampling because most of the surfaces encountered either were irregular in shape and unsuitable for replicate organism detection and counting sampling or were too large for the rinse method. [ ] [ ] [ ] the swab-rinse method consisted of using a sterile cotton swab moistened in a sterile recovery medium to sample equipment and other surfaces potentially touched by contaminated hands. the entire digital contact area of a surface was sampled. the sampled surface was rubbed several times with back-and-forth strokes (about to centimeters long); then the swab was rotated, and the surface was rubbed with strokes perpendicular to the original strokes. the swab was broken off in a tube containing . ml of the recovery medium and transported to the laboratory for inoculation of culture plates. to prevent growth of the bacteria in the recovery medium, the samples were kept on ice until they were processed. within one hour of their collection, the samples were taken to the laboratory and processed. spread plates were prepared for each sample. each tube was vortexed for . minute to release the bacteria from the cotton swabs. the spread plates were prepared by placing . ml of the sample in -millimeter petri dishes containing columbia colistin naladixic acid, or cna, sheep blood agar, an enriched medium selective for gram-positive organisms. the sample was spread evenly over the agar surface with a sterile glass spreader. the plates were incubated in candle jars at c for hours. after incubation, ahs colonies were counted. isolates were identified according to the framework described by facklam and facklam and carey. three recovery media were used: trypticase soy broth, or tsb; letheen broth; and dey/engley, or d/e, neutralizing broth. after disinfectants dry on a surface, the residual disinfectant can be reactivated when moistened by the recovery media from the cotton swab. letheen broth and d/e neutralizing broth contain ingredients that neutralize disinfectants. however, it is known that these neutralizing ingredients also can have bacteriostatic effects on, and some degree of toxicity for, the recovered bacteria. before the sampling in the private offices and in the general environment, we performed tests to determine which of the recovery media was most sensitive for recovery of ahs. letheen broth effectively neutralized residual phenolic disinfectants and was less toxic to the recovered ahs than was the d/e neutralizing broth. d/e broth was approximately six times less sensitive than letheen broth. iodophors, chlorine and hypochlorites are sufficiently neutralized by the organic material in letheen broth, or other nutrient media, such as tsb. tsb was even more sensitive than letheen broth when there were no residual disinfectants recovered in the sample. we chose tsb for sampling surfaces where disinfectants were not used, or where use was limited, because it is not toxic to the bacteria. recovery of ahs from surfaces in the general environment. we evaluated the occurrence of ahs in the general-nondental-environment by sampling surfaces commonly handled or touched in a general medical clinic, an ophthalmology clinic and a barber shop. we sampled surfaces and the entire digital contact area of each equipment handle using swabs moistened with tsb. samples were taken in the afternoon after the last of the patients or customers had been seen. the staff at the medical clinic used a phenolic disinfectant to clean the examination table between patients. other surfaces were cleaned daily with detergent and water. surfaces sam-pled in the general medical clinic included faucet handles, light handles, countertops and examination tables. in the ophthalmology clinic, a percent solution of household bleach ( . percent sodium hypochlorite) was sprayed and wiped on all surfaces in the examination room after patients with eye infections were seen. the frequency of visits by patients with eye infections varied from daily to weekly. otherwise, surfaces were cleaned with detergent and water. the ophthalmology clinic surfaces we sampled included the head adjustment handle, countertops, the scope adjustment handle, the lens adjustment handle, patient chair armrests and the patient chair headrest. disinfectants were not used on a routine basis in the barber shop. surfaces sampled in the barber shop included armrests, countertops, clippers, drawer handles, faucet handles, the vacuum/air blower handle and scissors. microbial sampling in the dental operatory. surfaces in the dental operatory were sampled before and after dental procedures were performed in a dental school clinic at the school of dentistry at the university of north carolina at chapel hill, where licensed general dentists treated patients. surfaces of equipment handles were sampled, including the entire digital contact area of each piece. swabs used to sample surfaces that had been disinfected were moistened with d/e neutralizing broth. surfaces sampled before the procedure were the handpiece base and holder, the air-water syringe handle and holder, the syringe water, suction handles and holder, the bracket tray handle and eyeglasses worn by the dentist. a total of samples were taken, before dental treatments began, from surfaces that had been cleaned. we observed the entire dental procedure, noting and counting the number of times each surface was touched by the potentially contaminated hands of the dentist, the dental assistant or both. we also noted handwashing and glove changes. after the dental treatment and before cleanup, the same sur- jada, vol. , november the tubes of recovery medium also were incubated and growth subcultured on columbia colistin naladixic acid sheep blood agar. † no α-hemolytic streptococci, or ahs, detected. ‡ counts of to cfus were estimated when no growth occurred on the plates and growth was detected in the recovery medium alone. faces were resampled, as were dental instruments and other surfaces that were touched with potentially contaminated hands. a total of samples were taken after dental treatments from surfaces that were touched or were potentially contaminated with saliva. after sampling, spread plates were prepared in the laboratory, as previously described. microbial survival in private dental offices after cleaning and disinfection. environmental surfaces in private dental practices were sampled for ahs in the morning before dental procedures began and at the end of the day after cleanup. both sets of samples were from operatories that were "clean" and ready for the next patient. surfaces sampled included items such as handpieces, air-water syringe handles and tips, suction handles, lamp handles, door handles, telephone receivers, bracket tray handles, patient seat buttons, dentist's seat controls, x-ray units and water from the air-water syringe. the surfaces were sampled with a sterile cotton swab moistened with letheen broth (broth containing lecithin and tween detergent (difco laboratories) to neutralize resid-ual disinfectants still remaining on the surfaces). we scrubbed each item or surface vigorously with the swab, using back-andforth and perpendicular strokes and rotating the swab several times. the swab was remoistened in the recovery medium two or three times for each sample; each time, the swab was pressed against the side of the tube to remove excess moisture. all items of a given type in an operatory (handpieces, for example) were sampled with a single swab. as we sampled each item, we wore gloves and used aseptic techniques. the samples were kept on ice and processed in the laboratory as previously described. the tubes with recovery medium were also incubated at c for hours. growth from the tubes was streaked on columbia cna blood agar plates. after incubation, the plates were examined for the growth of ahs colonies. survey of dental patients' saliva for ahs. the average number of ahs cfus counted in the survey was × per ml of saliva, ranging from × to × . there was an average of × nonhemolytic cfus per ml of saliva, ranging from × to × . recovery of ahs from surfaces in the general environment. ahs were detected in two of the three areas sampled; these results are summarized in ( ) / ( ) / ( ) / ( ) * samples were taken after cleanup/disinfection of operatory surfaces. samples yielded low counts of ahs colonies. ten cfus were detected in three positive samples, and cfus were detected in a fourth. dfrom the ophthalmology clinic, fewer than nine cfus were detected in one of samples. the streptococci were detected only in the tube of sampling broth, which was incubated to detect streptococci in the sample that did not grow on the . ml of plated sample. dfrom the general medical clinic, none of the samples yielded growth of ahs. didentification of the ahs detected in nondental environments showed five to be streptococcus mitis and one to be s. sanguis i. microbial sampling in the dental operatory. of samples taken before dental treatments from surfaces that had been cleaned (in clinic a), three ( percent) were positive for ahs. two of these samples were from bracket tray handles and one was from the air-water syringe handle and holder. a total of samples were taken after dental treatments from surfaces that were touched or were potentially contaminated with saliva. forty-nine ( percent) of these were positive for ahs. the four operatory surfaces most frequently touched during the dental procedures observed were the air-water syringe handle (touched times per treatment), the handpiece (touched nine times per treatment), the suction handles (touched eight times per treatment) and the lamp handle (touched seven times per treatment). the average time per treatment was . hours. ahs were detected on the handpiece and air-water syringe handle on percent of the samples. the suction handles were positive for ahs in percent of the samples. the lamp handle was not sampled because it had been covered with a plastic barrier that is removed and discarded after dental treatment. other items touched by dental personnel with potentially contaminated hands included dental instruments such as pliers, syringes, explorers, scalpels, tweezers, probes, mirror, amalgamator, camera, rubber cement container, spatula, drawer handles, lamp switch, refrigerant spray, floss holder, pencil, ruler, scissors, x-ray units, bur wrenches and cavity varnish containers. items that were positive for ahs were dental instruments, hand mirror, amalgamator, ultrasonic scaler and eyeglasses. microbial survival in private dental offices. ahs were detected on percent ( of ) of the surfaces sampled in the morning and percent ( of ) of the surfaces sampled in the afternoon, for a combined total of percent ( of ) of the surfaces sampled. the fisher exact test was used to investigate the significance of the morning sampling with the afternoon sampling results. the difference was significant (p = . ). the fisher exact test was also used to compare the afternoon sampling results in the private dental offices with the sampling results in the nondental areas ( , as the samples in the nondental areas were also taken in the afternoon. this difference was significant (p = . ). ahs were detected in all of the dental offices. in one of the offices, no ahs were detected in the morning samples, but four of samples were positive in the evening. the operatory with the highest number of contaminated surfaces had a combined total of positive samples of a total of ( percent). sampling results of the private offices are summarized in table , which also lists the type of disinfectant used in each office. the most frequently contaminated surface was the x-ray unit (eight of , percent), followed by the handpiece ( of , percent) and the patient chair buttons ( of , percent). these results are presented in table . six of the samples had high numbers of ahs: , to > , cfus. the numbers of cfus recovered in samples from the private dental operatories are summarized in table . a major goal of this investigation was to determine whether certain oral bacteria found in human saliva could serve as biological indicators of the contamination of operatory equipment. a bacterial indicator of oral contamination would have to be easy to cultivate and recognize, abundant in the mouth, present in low numbers in general environmental areas where there is a low potential for oral contamination, able to survive on environmental surfaces, and detectable on dental operatory surfaces where there is known contamination. in accordance with these criteria, literature data and the results obtained in this study, the best indicator of oral contamination appears to be ahs. the following observations support this conclusion. physiological appearance. ahs have an unusual physiological appearance that makes them easy to recognize on blood agar plates. their α-hemolysis is a result of the bacterial production of hemolysin, which causes a breakdown of red blood cells around a colony on blood agar. the zone of hemolysis is a mixture of lysed and incompletely lysed cells that results in a green or brownish color. , the term "viridans" comes from the latin term "viridis," meaning "green." ß-hemolysis, exhibited by other streptococci, appears as a clear zone of completely lysed red blood cells. all seven of the common species found in saliva have α-hemolytic strains, although the strains of streptococcus salivarius are predominantly nonhemolytic ( percent). α-hemolysis gives oral streptococci a distinguishing characteristic among the other flora growing on the cul-ture plate. ease of culturing and identification. ahs were relatively easy to culture and identify; they grew well on sheep blood agar at c. because growth conditions that provide increased carbon dioxide are favorable for streptococci, studies were conducted using candle jars in which colonies were visible after to hours. typical colonies were transparent to opaque, to mm in diameter, with an α-hemolytic zone of to mm after hours' incubation. positive samples, those with colonies exhibiting α-hemolysis, should be confirmed for the presence of streptococci with gram's stain and catalase test. presence in saliva. ahs were found in high numbers in saliva. the survey of saliva from patients who visited the dental school clinics showed that ahs averaged about × organisms per ml of saliva, ranging from × to × . the nonhemolytic colonies of the various species of the viridans streptococci averaged about half the number of the ahs colonies, although there were some patients with more nonhemolytic jada, vol. , november * numbers of colony-forming units, or cfus, are based on colony counts grown from . milliliters of the -ml recovery medium used to suspend each sample. the tubes of recovery medium also were incubated and growth subcultured on columbia cna sheep blood agar. † counts of to cfus were estimated when no growth occurred on the plates and growth was detected in the recovery medium alone. than ahs colonies. presence in general environment. ahs were detected in low numbers and frequency in the general environment. although the ahs were detected in samples from nondental environments, they were present in low numbers. one or two α-hemolytic colonies were observed on spread plates of four of the samples taken in the barber shop. none of the other spread plates for the nondental samples grew ahs colonies, although one sample from the ophthalmology clinic grew the indicator organisms in the recovery medium. the fisher exact test was used to compare the afternoon sampling results in the private dental offices with the sampling results in the nondental areas ( percent, five of ), since the samples in the nondental areas were also taken in the afternoon. this difference was significant (p = . ). the difference is attributed to the activity of saliva-contaminated hands' touching surfaces in a dental operatory despite the efforts of dental personnel to clean and disinfect those surfaces. although this activity does not take place in barber shops or medical clinics, ahs were detectable there nevertheless. ahs also are dispersed into the environment through sneezing, coughing and talking. detection of low numbers of ahs in the general environment is acceptable; however, a higher standard should be applied to an environment in which instruments and fingers of clinic personnel touch or penetrate the mucous membranes of patients. survival on dental operatory surfaces. ahs survived on environmental surfaces for several days. however, relative humidity has a pronounced ef-fect on the survival of ahs in saliva dried on surfaces. there is accelerated die-off at high relative humidities, or rh. at percent rh, the die, or d, value-the time for percent to die, or one logarithm reduction-was demonstrated to be only two hours, whereas at lower rh of percent and percent, the d value was hours and hours, respectively. we used the fisher exact test to compare the private dental office sampling results from the morning ( percent [ of ] of the samples were positive for ahs) with those from the afternoon ( percent [ of ] positive for ahs). the difference was significant (p = . ). this difference is attributed to the die-off of the indicator organisms during the approximately hours after the last patients were seen the day before. the rh in the private dental offices ranged from to percent when the samples were taken. presence on operatory surfaces. ahs were detectable on contaminated operatory surfaces. the indicator organisms were isolated from surfaces immediately after dental procedures and before cleanup. in private office operatories, the indicator organisms were found on surfaces that had been cleaned and were ready for the next patient. thirty-nine percent ( of ) of samples taken from "clean" operatories in private practices were positive for the indicator organisms, clearly showing the potential for cross-contamination between patients. we compared the findings of positive samples among total samples with the goal of zero positive samples among total sam-ples. the probability that a proportion that large would happen by chance is far less than one in , . although the primary criterion for interpretation of the monitoring results is whether or not ahs are detected, actual colony counts recovered might assist in evaluating the potential for cross-contamination. surfaces with higher counts of ahs would indicate a higher risk of cross-contamination between patients. ten ( percent) of the samples had high counts (> ), estimated to be > , cfus recovered in sampling. six of these had very high counts, ranging from to more than , a total considered too numerous to count but estimated to be from , to more than , cfus recovered. it should be understood, however, that the swab-rinse sampling methodology is not a precise measurement of the amount of contamination on a surface. colony counts would depend on variables such as the swabbing technique used and the condition of the surface sampled. colony counts also vary depending on the amount of saliva contamination on a dental care worker's gloves before he or she uses an item and the amount of digital contact he or she makes with the item. for these reasons, any indication of residual contamination should be considered significant in efforts to provide a safe treatment environment. implications of the findings. the environment presented to patients should be free of oral bacteria from previous patients; thus, the goal is that ahs should not be detected on any of the operatory surfaces. since each of the offices was sam-pled twice, a total of sets of samples were taken. as shown on table , only one of the sets was negative for all samples. this finding indicates that the time necessary for thorough cleaning and disinfection is not available in a busy dental practice. disinfection practices should include initial surface cleaning to physically remove debris and much of the contamination. well-cleaned surfaces then should be thoroughly wetted again with fresh disinfectant, allowing as much contact time as possible, according to the manufacturer's instructions. , all of the offices surveyed stated that operatory surfaces were disinfected between patients. the types of disinfectants used in each office are listed in table . however, a number of surfaces were left contaminated despite the use of various disinfectants. our finding indicates the difficulty of completely disinfecting all irregular operatory equipment surfaces with consistency. this observation supports the concept that cleaning and disinfection of equipment surfaces is not the most effective or reliable approach to infection control in the busy dental practice. asepsis implications and recommendations. alternatives to complete reliance on disinfection procedures can and should be implemented to control cross-contamination. , , , , - , , a more effective control method is the use of inexpensive, single-use, disposable plastic bags over surfaces that must be touched during treatments, such as the airwater syringe, the lamp handle, the suction handle, the dental control unit and even the chair. covers can be replaced rapidly between patients, eliminating the need for disinfection unless the bag comes off or its integrity is broken. , another effective approach is to prevent direct contact of contaminated gloved hands with occasionally contacted surfaces. this can be achieved in several ways. foot controls rather than chair buttons should be used to adjust seats and to operate water faucets for handwashing. dentists and dental assistants should use a paper towel or remove gloves to hold phones or to touch other surfaces that must not be contaminated during treatments. handpieces and other intraoral dental equipment should be designed to be removed and sterilized between appointments. sampling methodology. the swab-rinse method is preferred for microbial surface sampling in the dental operatory, because it is a simple method suitable for the irregular surfaces encountered in the operatory. the recovery medium should have disinfectant neutralizers if the surface has been treated with a disinfectant that leaves a residual that is reactivated when the surface is moistened. letheen broth effectively neutralizes phenolic disinfectants, quaternary ammonium compounds and iodophor disinfectants, and is more sensitive (not as toxic) for recovering the indicator organisms than d/e neutralizing broth. , incubating the recovery broth and then streaking the resultant culture on blood agar increases the sensitivity of the sampling method. sampling consistency is critical. it is recommended that the moistened swab be pressed firmly against the surface, using vigorous scrubbing, reversing directions, with perpendicular strokes, while rotating the swab frequently. all areas of a given surface should be sampled (unless it is too large to be practical), with the swab being remoistened two or three times during the sampling. during moistening and remoistening, the swab should be pressed and rotated against the side of the tube to remove excess moisture. more than one instrument can be sampled with a single swab. the goal in dental asepsis is to break the chain of transfer of blood and blood-contaminated saliva from each patient's mouth to surfaces in the dental operatory and to other patients via contaminated equipment or the hands of dental personnel. in this study (performed before the use of disposable plastic covers became widely recommended), the extensive detection of ahs on unprotected, inadequately disinfected surfaces should be interpreted as a potential for cross-contamination. our detection of ahs in the operatory on unprotected disinfected surfaces indicated the inadequacy of surface disinfection practices. these findings validate and reinforce current concepts of infection control advocated and used widely in dentistry , : duse of single-use plastic covers over surfaces handled with contaminated gloved hands during treatment, as barriers to contamination; davoidance of unnecessary touching of unprotected items and surfaces directly with contaminated gloves without using an additional clean barrier such as a paper towel or forceps; dsterilization of all other items or equipment that must be handled in the treatment field and cannot be protected in another fashion. this study indicates the usefulness-possibly for a number of applications-of an infection control surveillance monitoring methodology in dental practice environments using biological indicators. these surveillance methods can aid in evaluating equipment and techniques developed for infection control. sampling for indicator organisms also can be used epidemiologically to help determine the routes of infection transmission when investigating outbreaks in a dental clinic or practice. outside consultants or public health organizations required to evaluate asepsis in dental practices can use this technique for indicator organisms as part of an overall monitoring program. dental schools can use the technique as a teaching tool to show students the potential for crosscontamination and to teach or evaluate aseptic techniques and infection control practices. more imminently, sampling for indicator organisms can serve as a process control by dental practitioners. this can help identify hazards in dental practice before the public is harmed, and can be used to raise dental personnel's level of awareness of the potential for disease transmission. heightened awareness can encourage continued adherence to infection control procedures. such self-evaluation by the dental profession could eliminate any potential sources of crosscontamination that might have thus far escaped scrutiny by the profession or the public, ideally preventing any eventual need for greater outside controls of dental care asepsis. i d o you have comments or questions about this article? jada now offers an online resource called ask the author, which can put you in touch with the author of one featured article per issue. check out ask the author in the ada publishing co. portion of ada online at "http://www.ada.org". : . . occupational safety and health administration. standard, occupational exposure to bloodborne pathogens. federal register recommendations for preventing transmission of human immunodeficiency virus and hepatitis b virus to patients during exposureprone invasive procedures the public health and welfare act centers for disease control. recommended infection-control practices for dentistry infection control and management of hazardous materials for the dental team how to choose and use environmental surface disinfectants oral bacteria as biological indicators for dental asepsis (dissertation) university of north carolina at chapel hill the art and science of operative dentistry wilford hall usaf medical center and office of the assistant secretary of defense; . . crawford jj. new light on the transmissibility of viral hepatitis in dental practice and its control transmission of rhinovirus colds by self-inoculation low occupational risk of human immunodeficiency virus infection among dental professionals herpetic whitlow: report of a case with multiple recurrences getchell-white si, donowitz lg, groschel dhm. the inanimate environment of an intensive care unit as a potential source of nosocomial bacteria: evidence for long survival of acinetobacter calcoaceticus survival of herpes simplex virus and other selected microorganisms on patient charts: potential source of infection interpatient microbiological cross-contamination after dental radiographic examination miller ch, palenik cj. infection control and management of hazardous materials for the dental team barriers can minimize occupational exposure risks to contagions clinical asepsis in dentistry evaluation of spatter and aerosol contamination during dental procedures council on dental materials and devices and council on dental therapeutics. infection control in the dental office baltimore: williams & wilkins; . . crawford jj. sterilization, disinfection, and asepsis in dentistry cross infection control in dentistry: a practical illustrated guide oral microbiology a bacteriologic census of human saliva indications of the sanitation level in a dental clinic physiological differentiation of viridans streptococci biosafety committee. biosafety reference manual a comparative evaluation of methods for determining the bacterial contamination of surfaces comparative evaluation of the cotton swab and rodac methods for the recovery of bacillus subtilis spore contamination from stainless steel surfaces manual of clinical microbiology the use of inactivators in the evaluation of disinfectants ecologic studies of rheumatic fever and rheumatic heart disease taranta a, moody md. diagnosis of streptococcal pharyngitis and rheumatic fever oral streptococci with emphasis on streptococcus mutans council on dental materials and devices and council on dental therapeutics. current status of sterilization instruments, devices, and methods for dental office dental asepsis key: cord- - bwsm authors: izquierdo lara, r. w.; elsinga, g.; heijnen, l.; oude munnink, b. b.; schapendonk, c. m. e.; nieuwenhuijse, d.; kon, m.; lu, l.; aarestrup, f. m.; lycett, s.; medema, g.; koopmans, m. p. g.; de graaf, m. title: monitoring sars-cov- circulation and diversity through community wastewater sequencing date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: bwsm the current sars-cov- pandemic has rapidly become a major global health problem for which public health surveillance is crucial to monitor virus spread. given the presence of viral rna in feces in around % of infected persons, wastewater-based epidemiology has been proposed as an addition to disease-based surveillance to assess the spread of the virus at the community level. here we have explored the possibility of using next-generation sequencing (ngs) of sewage samples to evaluate the diversity of sars-cov- at the community level from routine wastewater testing, and compared these results with the virus diversity in patients from the netherlands and belgium. phylogenetic analysis revealed the presence of viruses belonging to the most prevalent clades ( a, a and b) in both countries. clades b and c were not identified, while they were present in clinical samples during the same period. low frequency variant (lfv) analysis showed that some known lfvs can be associated with particular clusters within a clade, different to those of their consensus sequences, suggesting the presence of at least clades within a single sewage sample. additionally, combining genome consensus and lfv analyses we found a total of unique mutations in the sars-cov- genome which have not been described before. in conclusion, this work illustrates how ngs analysis of wastewater can be used to approximate the diversity of sars-cov- viruses circulating in a community. introduction particular city or county, where the titers in sewage seem to correlate with the number of reported cases in the population, suggesting a potential role for sewage surveillance as an early warning tool , [ ] [ ] [ ] . therefore, sewage testing is currently considered globally as an adjunct to patient-based surveillance, and has promise as an early warning indicator of increasing virus circulation. enhanced surveillance is a key pillar of the current containment strategy aiming to control the spread of sars-cov- and includes frequent testing of people with mild symptoms, investigation of clusters of infection to identify possible common exposures, and monitoring of hospital and icu admissions. whole genome sequencing of sars-cov- directly from clinical samples has been developed as an additional tool, to provide information on diversity of circulating strains as a basis for cluster identification. particularly in areas with minimal circulation, sequencing of viruses from patients can help to identify a possible source, provided that sufficient background sequencing has been done. so far, little work is done trying to correlate the sars-cov- diversity in sewage and patients , . here we aimed to evaluate the potential of next generation sequencing (ngs) of sars-cov- , from rt-pcr positive wastewater samples, to assess if they reflect the diversity of sars-cov- circulating within the population of the netherlands and belgium. previously, sewage samples were collected from different locations in the netherlands and belgium to investigate the levels of sars-cov- in sewage using rt-qpcr . to further investigate the genetic diversity of sars-cov- a total of wastewater samples obtained from different locations in the netherlands ( samples) and different locations in belgium ( samples) with ct values of < were selected for whole genome sequencing using nanopore sequencing. the samples covered a time span of days (from march th to june rd ). two samples (franeker- and amsterdamwest- ) were sequenced by nanopore twice, while samples were also sequenced by illumina (table ) . four primers/probe sets targeting the n (n -n ) and the e genes were used to evaluate the presence and concentration of sars-cov- in sewage samples as described previously . all samples and their ct values are shown in table . the percentage of the genome covered by the assembly of nanopore reads (> x coverage per site) ranged from to . %. we observed an inverse sigmoidal correlation between the percentage of the genome assembled from nanopore sequencing reads and the ct values of both the n and the e primers/probe sets (fig. ) . the inflection point (ct value at which half of the genome can be obtained) for n , n , n and e primers/probe sets were ct values of . , . , . . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint and . , respectively. no correlation was observed between ct values and the percentage of the genome assembled from illumina sequencing data ( supplementary fig. s ). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint in order to associate specific mutations to particular a clade or cluster, all consensus sequences, including partial sequences, were compared to the wuhan-hu- reference sequence. a total of single nucleotide polymorphisms (snps) compared to the wuhan-hu- reference sequence were detected in our dataset (supplementary table s ). from these, snps were present in more than one sequence. the maximum number of mutations in individual samples compared to the wuhan-hu- reference genome were for hcov- /env/netherlands/amersfoort- -n/ , hcov- /env/netherlands/delft- -n/ and hcov- /env/netherlands/schiphol- -n/ (supplementary table fig is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint s ). the presence of clade-defining mutations in the consensus sequence suggests the dominance of a certain clade within a sample, but assessing their presence can also be used to check for virus mixtures in a sample. nextstrain has defined each clade by the presence of at least two linked mutations (https://nextstrain.org/). a is the root clade and contains the wuhan-hu- reference sequence. both b and a emerged from a, where two and three linked mutations define these major clades, respectively: t c and c t for b; and c t, c t and a g for a. clades b and c emerged from a, where the trinucleotide substitution ggg > aac at positions - defines b, and the linked mutations c t and g t define c. nucleotide substitution a g, a signature mutation of clades a, b and c, and that generates the d g amino acid substitution in the s glycoprotein, was present in . % ( / ) of the samples that were sequenced at this region (supplementary table s ). the table s ). one of the two mutations defining clades c and b (c t and t c) were found in and consensus sequences, respectively. the regions containing the other clade-defining mutations (c t for c; and c t for b) were not sequenced at high enough coverage to determine a consensus sequence in these samples. the hcov- /env/netherlands/amersfoort- -n/ sequence contained a mix of the clade-defining mutations c t, t c and ggg - aac, that define clades c, b and b, respectively. this indicates that the obtained consensus sequence is a combination of several different viruses and does not represent a single strain. in addition to the clade-defining mutations, we detected and snps in sewage samples that were not present in either the dutch ( sequences) or belgian ( sequences) gisaid datasets, respectively, but were present in the global dataset ( sequences, as th of july ), although with < % prevalence (supplementary table s ). moreover, we detected a total of novel mutations present in sewage consensus sequences that were not previously reported (supplementary table s ), of which were supported by coverage above the set thresholds to be considered as high quality (coverage > x for nanopore; and coverage > x and phred score > for illumina). additionally, it is noteworthy to mention that some samples presented discrepancies between the consensus sequences obtained by nanopore and/or illumina sequencing. for example, sample amsterdamwest- was sequenced times (twice with nanopore and once with illumina), in which positions with discrepancies were found between the consensus sequences (supplementary table s ). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint these differences were not due to an assembly error, since the alignment of the reads were manually checked and corrected for each discrepancy in every sequencing run. these differences were explained by the presence of two different nucleotides in the reads covering a particular position with varying percentages between sequencing runs, resulting in consensus sequences that could differ between each sequencing run. these results were likely caused by both or either the presence of multiple strains and low viral rna titers in the samples, leading to an amplification bias during library preparation. given that sewage samples are likely to contain a mixture of sars-cov- strains, we decided to perform a variant analysis with illumina data to determine whether lfvs were confidently present in a sample. using a coverage > x, phred score > and a frequency threshold of > % as settings, we found a total of positions with at least one sample containing major and minor variants (table ) table ).the other variants were present at similar frequencies in the dutch, belgian and global datasets ( table ) . in addition to consensus sequence, lfv analysis is of importance to be able to identify potential local outbreaks from sewage. to try to associate the presence of a minor variant to . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint sequences belonging to unique clusters, we mapped the most highly prevalent lfvs onto both dutch-belgian and global subsample phylogenetic trees (fig ) . this analysis indicated that for variants ( a, t and g) there were clear associations between the presence of the mutation and their clustering on the phylogenies (fig ) . however, when one of these variants was present as an lfv in a sewage sample the consensus sequence (blue arrows in fig. ) of this sample did not group with the cluster of clinical samples that contains the variant (magenta lines in fig. ). for example, g variant in sample tilburg- was present in two unique clusters within clade a, while its consensus sequence (hcov- /env/netherlands/tilburg- -i/ ) was clustered within clade b (fig and supplementary figs. s and s ) , suggesting the presence of both clades in this sample. although mutation t was most prevalent in clade a, it was also present scattered along the trees, indicating poor association with a particular clade. given the high chemical and biological complexity of wastewater samples, virus concentration and rna extraction methods are crucial parts of the process to reach enough is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint viral rna yield for sequencing . in this study, we showed that our method was capable of obtaining complete or near complete genomes from wastewater samples with ct values of at least or cts below the limit of detection (lod) (ct < ) and partial genomes for samples with higher ct values. therefore, only samples with enough viral rna can be used to effectively analyze sars-cov- diversity in sewage samples. in order to increase the percentage of genome covered of the samples, we used a threshold of x coverage per position to generate the consensus sequences from nanopore reads. based on previous analysis of viral sequencing data, the error rate with this threshold is less than . % table s have a coverage of at least x, which produces an error rate of in , nucleotides sequenced . the use of sewage as a tool to understand the epidemiology and diversity of sars-cov- at a community level offers many advantages over human sampling. sewage samples are relatively easy to collect, because no invasive sampling is required, there is no sampling bias towards sequences from moderate and severe cases, there are limited ethical issues, and potentially few samples are required to give a picture of the temporal changes of viral infections in the community , . nevertheless, comprehensive comparisons with clinical surveillance and other epidemiological approaches are required to determine the extent and limits of using sewage as a surveillance/early warning tool. furthermore, before the broad use of sewage samples to characterize viral diversity within a population, some obstacles need to be overcome, such as: low viral titers that complicate the retrieval of complete genomes and the distinction of multiple strains within a sample. here we have used two of the most common ngs technologies (nanopore and illumina) to study the diversity of sars-cov- found in sewage samples, from the netherlands and belgium, and compared these results with the virus diversity found in sequenced clinical samples. in order to evaluate this diversity in a comprehensive fashion, we have used nextstrain clade classification system because it is based on signature mutations to assign a sequence to a clade (https://nextstrain.org/) , facilitating the association of snps or lfv to a particular clade, especially for genome sequences with < % coverage. sewage samples can contain a mixture of sars-cov- viruses reflecting the multiple viruses circulating within a community. they may also partially reflect the presence of sars-cov- from animal origin, as sars-cov- has now been detected in domestic and livestock animals such rabbits, minks, cats and ferrets [ ] [ ] [ ] [ ] [ ] . the analysis of a consensus sequence genome from a wastewater sample may identify the predominant virus strain present in a . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint population, which might be suitable for locations where only or few introductions of closely related viruses have occurred, as it seems to be the case for previous studies in italy and usa , . nonetheless, the consensus genome approach cannot reflect the diversity of the viruses circulating in a population with a high degree of viral diversity. moreover, in some cases samples containing several diverging strains at significant levels might lead to retrieve artificial consensus genomes that do not represent an existing virus, which seems to be the case for the hcov- /env/netherlands/amersfoort- -n/ sequence, where signature mutations of different clades are present at the consensus level. in this study, we could not detect a genome belonging to the least prevalent clades ( c and b) , despite the circulation of these viruses in the human population in both countries during the same period of time (figs a and b) . although, it is necessary to mention that mutations associated with clades b and c were found in and samples, respectively (supplementary table s ). however, these consensus sequences were either too short or had a mixture of signature mutations that did not allow to confirm whether they belong to clades c and b by the nextclade tool. another reason to explain why we did not find consensus sequences belonging to these clades is the limited number of locations represented on the phylogeny by our sewage sample dataset compared with that of the clinical samples, especially for belgium (only sequences from sewage). in depth ngs analysis could help to unravel the diversity of viruses within a complex sample such as wastewater, particularly unbiased sequencing of the sewage virome can give a good picture of the general viral diversity contained in a sample . nevertheless, the detection of variants of a particular virus in a single sample can be difficult due to the relative low number of reads obtained for each virus type. targeted amplification of a genome region of the virus taxa of interest is potentially more sensitive and cheaper to perform. recently, examples of this have been described for enteroviruses, human mastadenoviruses and norovirus , , . in general, for each virus a specific small fragment (< bp) of the genome is amplified and deep-sequenced, then sequencing reads can be aligned and assigned to a particular genotype or serotype, identifying and determining the prevalence of several virus variants within a single wastewater sample , , . as the diversity of sars-cov- is still limited , this approach would not be as useful for this virus because no single small piece of the genome can reliably differentiate between clades or lineages. however, we tried to overcome this issue by using a variant analysis of sewage samples. we showed that some lfvs can be linked to particular clusters or clades within the trees (fig ) , without the need of a complete genome. although, in order to confidently determine the presence of a particular clade/cluster within a sample, at least or lfvs associated with such clade/cluster should is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint be present at significant levels. furthermore, variant analysis can also be used to monitor the prevalence of biological interesting mutations in a population. one of the most interesting is the d g (or a g) mutation in the s glycoprotein, that has been shown to increase infectivity in vitro by stabilizing the s /s interaction , and has been associated with higher transmission and mortality rates, although the latter is under debate , . unfortunately, the region containing the d g mutation was not sequenced at high enough coverage to perform a variant analysis in most of the tested samples, and we were not able to find any sample with a mixture of both variants ( d and g) . the combination of whole-genome sequencing of clinical samples with epidemiological data has shown to be important for public health decision-making wastewater can also be used to monitor novel mutations. our consensus and lfv analyses revealed a total of mutations that were not present in the global database. from these, were found at the consensus level, as lfvs and were common to both analyses. it is possible that these novel mutations were not previously detected due to several reasons: ) genetic drift eliminated these variants before they could be further spread and detected; ) these viruses cause only asymptomatic or mild disease that made them to be less likely to be detected through clinical sampling; ) they are associated with reduced transmission/replication and did not became fixed in the population; ) they originate from an unknown animal hosts; ) they are associated with enhanced enteric shedding/replication; ) they are associated with intra-host defective genomes. the latter has previously been suggested for the detection of lfvs that generate stop codons in orf ab and s genes is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint polymerase mistake during the initial pcr cycles of the library preparation can be amplified and identified as a variant. phenotypical studies could help to determine the likelihood and biological relevance of some of these novel mutations. in conclusion, this work illustrates how ngs analysis of wastewater can be used as a tool to approximate the diversity of sars-cov- viruses circulating in a community. sequencing of wastewater samples could be a powerful tool to complement clinical surveillance or be used as a standalone procedure in settings where wide clinical sequencing is not feasible. additionally, in-depth ngs analysis of wastewater samples can help in assessing changes in virus diversity to determine emergence of epidemiologically or clinically relevant mutations, aiding public health decision making. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint a total of wastewater (ww) specimens were included in this study. rna from of these ww samples (all from march th ) were collected, processed and extracted previously by kwr . the other ww specimens were collected as h flow-dependent composite samples and processed as previously described illumina sars-cov- specific multiplex pcr for nanopore sequencing was performed as described by oude munnink, et al. ( ) . in short, primers for overlapping amplicons spanning the entire genome were used in pcr pools. the amplicon length was set to bp with bp overlap between the different amplicons. the used concentrations and primer sequences have been described previously . libraries were generated using the oxford nanopore's native barcode kits (catalog numbers: exp-nbd , exp-nbd and sqk-lsk ) and sequenced on a r . flow cell multiplexing up to samples per sequence run. illumina sequencing was performed as described by richard, et al. ( ) . amplicons were generated by the sars-cov- specific multiplex pcr described above for the whole . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the resulting raw sequence data were processed as previously described by oude munnink et al. . briefly, an automated snakemake script was used to demultiplex fastq raw reads using porechop (https://github.com/rrwick/porechop), trim primers using cutadapt and perform a reference-based alignment using minimap to gisaid sequence epi_isl_ . the run was monitored using rampart (https://articnetwork.github.io/rampart/). the consensus genome was extracted and positions with a coverage < x or < x were replaced with an "n". mutations in the genome were confirmed by manually checking the alignment in ugene and homopolymeric regions were manually resolved consulting reference genomes. based on previous validation studies , mutations with a cut-off of x coverage were considered as high quality, whereas mutations with less than x coverage were marked as low quality (supplementary table s ). all the processing, reference-based alignment and variant analysis of the illumina generated data was performed using a customized workflow on the galaxy eu server (https://usegalaxy.eu/) . first, raw sequencing reads were filtered using fastp to remove adaptor contamination, ambiguous bases (n), low quality reads (phred score < ) and fragments below the length of nt. for mapping purposes, reads were aligned against the gisaid sequence epi_isl_ using the default penalty settings of the bwa-mem . reads were re-aligned using the leftalign utility from freebayes package . all reads with mapping scores of less than were discarded. both consensus sequences and variants were generated using ivar . final consensus sequences (frequency > %) were . cc-by-nc-nd . international license it is made available under a perpetuity. is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint constructed using all mapped sequence reads that covered each site at least times and had a minimum quality phred score of . for detection of low-frequency variants (lfv), parameters were set as follows: a minimum coverage of x, phred score > and a minimum frequency threshold of %. manual inspection of the aligned reads was also performed to confirm or dismiss the variant calling in ugene . variant positions are given with respect to the wuhan-hu- strain (genbank accession number: mn ) . two reference datasets were used to perform the phylogenetic analysis. the first dataset included all dutch and belgian full-length sars-cov- genomes ( and sequences, respectively) from gisaid database (https://www.gisaid.org/) publicly available up to the th of july . the second dataset is a subsample of all sars-cov- sequences available in gisaid (https://www.gisaid.org) covering the global diversity of sars-cov- genomes up to the st of june . this global 'backbone' dataset contains subsampled high-quality sequences (full length, with ns < %) to include one unique genome per country/state per week. for the maximum-likelihood (ml) trees, only sequences in this study with > % genome coverage were included in the analysis. our sequences were aligned with both datasets using mafft (https://mafft.cbrc.jp/alignment/server/). the alignment was manually checked for discrepancies and the ends were trimmed, after which iq-tree was used to perform a ml phylogenetic analysis under the gtr + f + r model for the global subsample and the gtr + f + r model for the dutch-belgian dataset as the best predicted models using the ultrafast bootstrap option with , replicates. the phylogenetic trees were visualized using figtree v . . (http://tree.bio.ed.ac.uk/software/figtree/). clades were assigned by using the nextclade tool within nextstrain (https://clades.nextstrain.org/). is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . is the author/funder, who has granted medrxiv a license to display the preprint in (which was not certified by peer review) preprint the copyright holder for this this version posted september , . . https://doi.org/ . / . . . doi: medrxiv preprint a new coronavirus associated with human respiratory disease in china global initiative on sharing all influenza data -from vision to reality a dynamic nomenclature proposal for sars-cov- lineages to assist genomic epidemiology nextstrain: real-time tracking of pathogen evolution sars-cov- productively infects human gut enterocytes evidence for gastrointestinal infection of sars-cov- infectious sars-cov- in feces of patient with severe covid- structure of the sars-cov- spike receptor-binding domain bound to the ace receptor structure, function, and antigenicity of the sars-cov- spike glycoprotein angiotensin-converting enzyme (ace ) is a key modulator of the renin angiotensin system in health and disease monitoring human enteric viruses in wastewater and relevance to infections encountered in the clinical setting: a one-year experiment in central france detection of sars-cov- in different types of clinical specimens presence of sars-coronavirus- rna in sewage and correlation with reported covid- prevalence in the early stage of the epidemic in the netherlands surveillance of influenza a and the pandemic influenza a (h n ) in sewage and surface water in the netherlands sars-cov- titers in wastewater foreshadow dynamics and clinical presentation of new covid- cases sars-cov- rna in wastewater anticipated covid- occurrence in a low prevalence area time course quantitative detection of sars-cov- in parisian wastewaters correlates with covid- confirmed cases presence and infectivity of sars-cov- virus in wastewaters and rivers temporal detection and phylogenetic assessment of sars-cov- in detection of novel coronavirus ( -ncov) by real-time rt-pcr architecture and self-assembly of the sars-cov- nucleocapsid protein wastewater and public health: the potential of wastewater surveillance for monitoring covid- sewage analysis as a tool for the covid- pandemic response and management: the urgent need for optimised protocols for sars-cov- detection and quantification validating whole genome nanopore sequencing, using usutu virus as an example susceptibility of rabbits to sars-cov- sars-cov- infection in farmed minks, the netherlands transmission of sars-cov- in domestic cats sars-cov- in fruit bats, ferrets, pigs, and chickens: an experimental transmission study susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus setting a baseline for global urban virome surveillance in genetic diversity and quantification of human mastadenoviruses in wastewater from sydney and melbourne detection of norovirus epidemic genotypes in raw sewage using next generation sequencing geographic and genomic distribution of sars-cov- mutations the d g mutation in the sars-cov- spike protein reduces s shedding and increases infectivity sars-cov- amino acid substitutions widely spread in the human population are mainly located in highly conserved segments of the structural proteins sars-cov- genomic variations associated with mortality rate of covid- rapid sars-cov- whole-genome sequencing and analysis for informed public health decision-making in the netherlands sars-cov- exhibits intra-host genomic plasticity and lowfrequency polymorphic quasispecies sars-cov- is transmitted via contact and via the air between ferrets the galaxy platform for accessible, reproducible and collaborative biomedical analyses: update fastp: an ultra-fast all-in-one fastq preprocessor aligning sequence reads, clone sequences and assembly contigs with bwa-mem haplotype-based variant detection from short-read sequencing an amplicon-based sequencing framework for accurately measuring intrahost virus diversity using primalseq and ivar iq-tree: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies key: cord- -tbah ro authors: cannovo, nunzia; cingolani, mariano; guarino, rosa; fedeli, piergiorgio title: regulation of biobanks in italy date: - - journal: front pediatr doi: . /fped. . sha: doc_id: cord_uid: tbah ro in italy, a biobank is “a non-profit organization that must be officially recognized by the appropriate healthcare authority in the member states and must guarantee the treatment, distribution and conservation of biological material according to standards of quality and professionalism,” but must not conserve material already regulated by specific laws, as is the case for organs for transplants, blood for transfusions, as well as embryos and gametes for medically assisted reproduction. the concept of biobank includes not only biological samples, but also the related database of clinical and personal information, from which the subject's lifestyle can be deduced. unfortunately, at the moment, italian law does not offer specific itineraries for achieving this legal status. the phenomenon of biobanks is relatively recent ( ) , and there has been a very sharp increase in the establishment of national and international collections of biosamples in public hospitals and private structures. as noted by fedeli et al. ( ), different definitions of "biobank" have been proposed in the national and international literature for this multifaceted phenomenon. it can be said that a biobank is "a structured collection of human biological material accessible on the basis of certain criteria" ( , ) , "in accordance with a code of good practice and correct behavior and with further indications provided by ethics committees and universities" ( ) and "in which the information contained in the biological material can be traced to a specific person" ( ), "for diagnostic, treatment and research purposes" ( ) . beginning in , the european union has provided regulations for biobanks ( ) , defining the parameters of quality and safety for donation, procurement, analysis, processing, storage and distribution of human tissues and cells, stipulating the adoption of necessary measures of protection of data, including genetic data, and other measures for safeguarding information collected in the context of activities of donation, procurement, monitoring, processing, conservation, storage and distribution of human tissues and cells to be used for applications in humans, as well as products derived from human tissues and cells, to be used in applications in humans (art. ) ( ) . in addition, the eu has provided technical prescriptions ( ) for the donation, procurement and monitoring of human tissues and cells, and has clarified the distinction between a disease biobank and research biobanks ( ) . in italy, given the delay in the passage of a national law, the regions have taken the initiative to complete local legislative itineraries to re-organize the sector. the authors provide an overview of italian legislation on research biobanks. the concept of biobank includes not only biological samples, but also the related database of clinical and personal information, from which the subject's lifestyle can be deduced. thus, the particular nature of a biobank is due to the fact that it contains genetic information that can be traced to the subjects who provided the biological material ( ). the problem of the juridical conception of biological samples for medical research lies in their two-sided nature as collections of cells and as sources of health and genetic data ( ). with emphasis on the exquisitely material nature of these samples, intense debate has revolved around the ownership of biological samples ( ) ( ) ( ) ( ) , and the legal relationship that binds the subjects to the samples, when the latter have been separated from their bodies. for some authors ( ) , when diagnostic or treatment procedures call for the ablation of material or parts from the body, even though the procedure does not cause a permanent reduction of physical integrity, these materials or parts are in effect separated from the body, and as such acquire the nature of disposable personal property (art. c.c.), within the limits of legal provisions already in vigor (art. c.c.) and are the object of ownership just as any other good ( ) ( ) ( ) ( ) ( ) ( ) . the most recent thought, however, has confuted the existence of a ius in se ipsum, arguing that after the separation, the body parts become external things, and thus can be owned. thus, there is a foundation for the claim that these tissues have been abandoned and that they can be taken by those who are interested in using them, because "for the most part, they offer the persons neither an interest of use nor an interest of exchange, " and consequently, with the separation, these goods become res nullius ( ) as derelict or abandoned, and thus can be acquired ( , ) . a certainly original theory draws a parallel between the law on separated parts of the body and that on works of creativity, based on the concept that just as a subject owns works of his/her creativity, so the individual should be deemed the owner of his/her own biological material (art. cc). according to this juridical construction, the part removed is a res that was created by the subject, albeit with the help of the surgeon, and therefore the subject should be the sole owner ( ) . the oviedo convention (art. ) ( ) re-affirmed "the prohibition of financial gain" ( - ), for biological samples. there are two possible interpretations of this principle, a more radical one and a more permissive one. an initial reading ( ) excludes a priori any possibility of constituting patrimonial rights over the body and human tissues even after their separation from the body. the more permissive theory ( ) refers to the impossibility of using individual parts of the body for gain, ensuring the freedom and spontaneity of donations on the basis of the rule of the extra-patrimonial nature of the circulation of rights over the human body. however, the solution is not easy, because the relationship of ownership entails a series of rights and faculties that must subsist in order to claim full ownership of something; clearly, this cannot be the case with biological samples, which are considered goods extra commercium, and cannot be disposed of for gain, because this would be an affront to human dignity, the protection of which is grounded in the constitution of the italian republic (art. ). this theme inevitably intertwines with the profile of the allocation of ownership of biological samples when the patient has given consent for their conservation and use ( ) . allocation of ownership to donors would legitimize a sort of commodification of the body, which could undermine scientific research and limit the range of experimentation, given the possibility that subjects may request the destruction of the samples at any time ( ) . instead, researchers are able to gain useful information from the study of these materials, as they have the technical skills needed to exploit the biological characteristics of the samples and above all to obtain information from them. however, the attribution of exclusive ownership to researchers would inevitably exclude the "donors" from participating in the biotechnological research and its profits, with the risk of creating an irreparable breach in the alliance between medical science and the community, which was established when the biological material was freely granted. also, if ownership was granted to research institutions, there could ensue a fratricidal race to "grab up" biological materials and use them for profit. thus, a new understanding of the biological sample is emerging. what was once considered special waste produced during surgery is now viewed as an irreplaceable source of medical and genetic information, an inexhaustible source for the development of biomedical science. samples are instruments of biological identity ( , ) inasmuch as they identify the body from which they came through the genetic patrimony of that person, "pieces of each subject, conserved in the very numerous databanks where the identity of the subject is sectioned and taken apart" ( ) , since samples can symbolically be considered a "crystal ball" ( ), a vehicle that provides genetic data, through which future health conditions can be predicted ( ) . in fact, from the point of view of information, "the separation of the biological sample does not mean that it has complete autonomy from the body-subject, but only that it may have autonomous circulation" ( ) . thus, the very particular nature of these samples appears evident: they have a material dimension, shaped by ownership law, and a dimension of information, expression of the personhood and identity of the subject ( , , ) . the perplexities arise from the entanglement of the uncontestable benefits that access to such data can bring to scientific evolution, and the incontrovertible need to protect the individual ( ) . on the one hand, subjects risk damage to their right to privacy ( ); if others can access their individual results, this may lead to new forms discrimination ( ) and social injustice ( ) . in such situations, genetic information can be viewed as having "multiple meanings" and being "dangerous" and "ambivalent" ( ) . on the other hand, these results are of enormous importance for scientific research, as articulated in the declaration on the human genome ( ) ( ) according to which, "benefits from advances in biology, genetics and medicine, concerning the human genome, shall be made available to all, with due regard for the dignity and human rights of each individual. freedom of research, which is necessary for the progress of knowledge, is part of freedom of thought. the applications of research, including applications in biology, genetics and medicine, concerning the human genome, shall seek to offer relief from suffering and improve the health of individuals and humankind as a whole" (art. ). in italy, biobanks are defined as "service units, without direct profit-making purposes, for the collection and conservation of human biological material used for diagnostic purposes, for studies of biodiversity and for research" ( ) . thus, a biobank is "a non-profit organization that must be officially recognized by the appropriate healthcare authority in the member states and must guarantee the treatment, distribution and conservation of biological material according to standards of quality and professionalism" ( ), but must not conserve material already regulated by specific laws, as is the case for organs for transplants, ( ) blood for transfusions ( ) , as well as embryos and gametes for medically assisted reproduction ( ) . in italy, most of the biobanks and centers of biological resources are found at structures or institutions that are part of or connected to the national healthcare service. consequently, the biological samples (sputum, serum, plasma, tissue, etc.) should be collected and stored according to international standard operating procedures and those developed ad hoc ( ) ( ) ( ) . a biobank that intends to leave behind its nature as a volunteer, not-for-profit entity, must be accredited or at least certified. unfortunately, at the moment, italian law ( ) does not offer specific itineraries for achieving this legal status, and it is necessary to refer to the forms of self-regulation adopted by biomedical laboratories, non-binding acts, and european and international law, where regulations for the phenomenon have already been established. though shared standards are lacking ( ) , it is important that the following criteria must be respected for the establishment of a biobank: systematic organization; accessibility (indicate whether or not it is open to third parties); primary purpose and directions of the research/use to be undertaken with the samples; what information will be kept; types of samples/data (living organisms and/or dna and/or sources of dna or information based on dna), with a clear separation between biological materials to be used for treatments and those to be used for research ( ) . regarding accreditation, in line with the indications of the conference of the regions and the national government, the individual regions will take responsibility, following the unified lines of conduct indicated by the national government, especially the standards of safety and quality control. european directive / of march ( ) and the subsequent / ( ) introduced and clarified the need to ensure traceability of donated tissues and cells, through the assignment of a unique code to each donation and each of the products associated with it. regarding traceability, the ministerial decree for productive activities of june , introduced the procedures for the certification of biobanks as centers of biological resources, while d.lgs / , which introduced tissue institutions for the conservation of cells and tissues for treatment purposes, transposed the provisions on quality and safety for donation, procurement, monitoring, processing, conservation, storage and distribution of human tissues and cells, which could have an interesting implementation also in the management of biobanks, in an initial phase of lack of ad hoc legislation. this legislative decree stipulates that tissue institutes must conserve the data necessary to ensure traceability in all phases, for at least years after clinical use, also in digital format. a recent italian law (law / ) ( ) allowed the collection of biological samples for research purposes. specifically, it made possible the use of biological material from previous diagnostic or treatment activities, or kept for any other purposes, on the condition of obtaining the patient's informed consent beforehand. a subsequent law, (legislative decree n. / ) ( ) assigned the task of defining the criteria for collecting biological samples to the higher institute of healthcare. as noted by cannovo et al. ( ) , the guarantor for the protection of personal data allows genetic data and biological samples ( ) collected for scientific and statistical research to be communicated or transferred to research entities and institutes, associations and other research-oriented public and private organizations, exclusively in the context of joint projects. however, these data and samples, with all personal data removed, can also be made available to third parties not participating in joint projects, for scientific purposes directly connected to those for which they were originally collected, and clearly determined in the written request for the data/samples. in this case, the subject making the request must commit to not using the data and/or samples for purposes different from those indicated in the request, and to not communicating them or transferring them to other parties. a provision established by the guarantor for the protection of personal data regulates the acquisition of informed consent. when every reasonable effort has been made to contact the subject for consent, but for particular reasons they have not met with success, the guarantor gives an active role to ethics committees ( ) . the importance that the complex phenomenon of biobanks has gained in recent years is intrinsically connected to the incessant advances in knowledge about genetics and the importance of the human genome, a special and irreplaceable source of genetic and medical data useful for the development of medical science ( ). the treatment of genetic data requires and justifies a particular legal safeguarding, but there is a need for juridical regulations that, far from limiting the freedom of research, direct the concrete developments to foster and protect the well-being and absolute value of the human person ( ) with thoughtful balancing of principles, in order to ensure respect for the rights of subjects who choose to donate their samples, and also those of the researchers and institutions that intend to use them for their scientific projects. given the fragmented and inconsistent juridical situation in italy, we believe it would be wise to consider biological samples as commons ( ) , that is, community property, or in other words, supra-individual goods at the service of the community. this should become a cardinal value in order to ensure the functionalization of these biological materials for scientific research purposes and their distribution among researchers in a democratic and transparent way. this would make it possible to disentangle the difficult knot of the dual nature of biological material, and to exploit in the best way possible the rights of intellectual property that can be obtained from it, using the model of biobanks as biotrusts, that is, third subjects, in addition to the patients and researchers. in this sense, the biobank would serve as a repository for the conservation and safekeeping of biological materials and as a guarantor of the principles underlying them. the original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s. material preparation, data collection, and analysis were performed by nc, rg, and pf. the first draft of the manuscript was written by nc. mc has contributed in revising the manuscript. all authors commented on previous versions of the manuscript, read and approved the final manuscript, and contributed to the study conception and design. cancer risk and oxidative dna damage in man informed consent for genetics research in italy working group for the protection of personal data. working document on genetic data regulations european biobank maastricht recommendation n. r( ) of the committee of ministers to member states on human tissue banks on the definition of quality and safety regulations for the donation, procurement, monitoring, processing, conservation, storage and distribution of human tissues and 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experimentation of medicines for human use, according to article , sections and of the law of ethical and deontological aspects of pediatric biobanks: the situation in italy autorizzazione generale al trattamento dei dati genetici, dicembre modificated by -provvedimento che the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © cannovo, cingolani, guarino and fedeli. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- - c mb k authors: winter, taylor; riordan, benjamin c.; pakpour, amir h.; griffiths, mark d.; mason, andre; poulgrain, john w.; scarf, damian title: evaluation of the english version of the fear of covid- scale and its relationship with behavior change and political beliefs date: - - journal: int j ment health addict doi: . /s - - - sha: doc_id: cord_uid: c mb k the covid- pandemic has many individuals around the world fearing for their lives. the constant news coverage, rapid transmission, and relatively high mortality rate, make fearfulness a natural response. to assess the fear of covid- , the fear of covid- scale (fcv- s) was developed. the primary aim of the present study was to conduct the first psychometric assessment and validation of the english version of the fcv- s. two samples were collected in new zealand. sample comprised participants of which completed all questions and were used in the analyses. sample comprised participants of which completed all questions and were used in the analyses. several psychometric tests were conducted to ascertain the scale’s reliability and validity. across both samples, the fcv- s had high internal consistency. consistent with the earlier validation studies, the fcv- s displayed a moderately strong relationship with the perceived infectability and germ aversion subscales of the perceived vulnerability to disease scale (pvds). furthermore, fcv- s scores were negatively correlated with the warwick-edinburgh mental wellbeing scale (wemwbs) scores. with respect to the motivating role of fear, there was a significant relationship between fcv- s scores and adherence to the lockdown rules that were implemented in new zealand. finally, consistent with recent reports on the politicization of the covid- pandemic, an exploratory question found that participants who rated themselves as more conservative tended to report lower fcv- s scores. the english version of the covid- s is a sound unidimensional scale with robust psychometric properties and can be used with confidence among english-speaking populations. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. behavior below for more detail on alert level ). sample comprised more males ( . %) than female participants, who were aged between and years old (mean [m] = . years; standard deviation [sd] = . years), and were largely new zealand european ( . %; . % maori or pasifika, . % asian, and . % other). sample comprised participants of which completed all questions and were used in the analyses. sample was collected during alert level . level immediately followed alert level , allowing people to expand their social circle to close family that they do not currently live with (see lockdown behavior below for more detail on alert level ). sample comprised more female ( . %) than male participants, who were aged between and years (m = . years; sd = . years), and were largely new zealand european ( . %; . % maori or pasifika, . % asian, and . % other). with respect to recruitment, both samples were recruited via posts on popular social networking sites and articles via several new zealand news websites. participants who agreed to take part in the study clicked on a hyperlink and were taken to the study webpage. the first page included a description of the study and an electronic consent form. participants who provided informed consent were asked to complete a -min survey. the study was reviewed and approved by the university of otago human ethics committee. demographic information age, gender (male, female, other), and ethnicity were collected. fear of the fcv- s is a seven-item measure assessing the extent to which a person fears covid- (ahorsu et al. ) . the scale asks participants to indicate the extent to which they agree with each item (e.g., "when i watch news and stories about coronavirus on social media, i become nervous or anxious") from (strongly disagree) to (strongly agree). both sample (α = . ) and sample (α = . ) completed the fcv- s. perceived vulnerability to disease scale the perceived vulnerability to disease scale (pvds) is a -item measure of an individual's perceived vulnerability to disease and asks them to rate from (strongly disagree) to (strongly agree) the extent to which they agree with each item (duncan et al. ). the scale has two subscales. seven items assess perceived infectability (e.g., "i have a history of susceptibility to infectious diseases") and eight items assess germ aversion (e.g., "it really bothers me when people sneeze without covering their mouths"). both sample (perceived infectability α = . , germ aversion α = . ) and sample (perceived infectability α = . , germ aversion α = . ) completed the pvds. see supplementary table for confirmatory factor analysis. scale (wemwbs) is a -item measure of mental wellbeing. participants are asked to indicate from (none of the time) to (all of the time) how often they experienced each statement (e.g., "i've been feeling optimistic about the future", "i've been feeling close to other people") (tennant et al. ). only sample (α = . ) completed the wemwbs. see supplementary table for confirmatory factor analysis. lockdown behavior a state of national emergency was declared in new zealand on march , , providing the new zealand government with access to "…extra-ordinary powers that will support delivery of an effective and timely response to covid- " (ardern ) . in brief, this allowed the implementation of a four-level alert system. alert level was implemented between march and april , with all businesses (expect essential services) and educational facilities closed. moreover, a set of rules regarding personal movement were implemented, with people instructed to stay home, only associate with individuals they live with, and limit travel to their local area. alert level immediately followed alert level , implemented between april and may . alert level allowed some businesses and educational facilities to open. however, social contact was still severely limited, with individuals only allowed to extend their social circle to close family they do not currently live with. to assess whether participants abided by new zealand's lockdown rules, participants were asked to indicate from (strongly disagree) to (strongly agree) whether they abided by the five rules (e.g., "i ensure i maintain the -meter rule when out in public") ( table ) . political beliefs given recent work suggesting covid- has become highly politicized in the usa (conway et al. ; rothgerber et al. ) , the survey also included a single exploratory item to assess participants' political beliefs (jost ; sibley and wilson ) . more specifically, following sibley and wilson ( ) , participants were given the instructions "often, people use the terms 'liberal' or 'conservative' to describe their political beliefs. how would you rate yourself in these terms?" (p. ). participants responded on a scale ranging from (very liberal), through (moderate), to (very conservative). descriptive statistics were used to describe the study participants' characteristics. the analysis also tested the skewness, kurtosis, and distributions of each scale item in the fcv- s, the internal consistency (cronbach's alpha), and inter-and item-total correlations. a confirmatory factor analysis (cfa) was conducted using maximum likelihood estimation to measure the factor structure of the fcv- s and report the factor loadings and the goodness of fit using root mean square error of approximation (rmsea where good fit is typically less than . ) and comparative fit index (cfi where good fit is typically more than . ). to date, most published psychometric reports have focused on classical test theory. the use of classical test theory, in which raw scores, linear combinations of these scores, and responses that are ordinal in scale, is considered as data on an interval scale. rasch analysis is a statistical technique traditionally for binary data, but some polytomous generalizations can also be used for interval data . standard rasch analysis is based on unidimensional models. in unidimensional models, it is assumed that only one hidden feature of the individual determines the individual's performance in the scale. if the data do not fit well with the rasch model, the unidimensional assumption is rejected. this means that more than one hidden feature has affected an individual's performance, so the feature cannot be assessed well using the scale in question . here, rasch analysis using the partial credit model was used to assess the unidimensionality and item fits of the fcv- s (masters ) . item validity was assessed using information-weighted fit statistic (infit) mean square (mnsq), and outlier-sensitive fit statistic (outfit) mnsq with values between . and . considered acceptable. the presence of disordering threshold in the fcv- s was assessed using average and step measures of the descriptors. a monotonic increase in difficulties between . and . suggests no disordering. the unidimensionality of the fcv- s was examined by conducting principal component analysis of the residuals (pcar) on the items. explaining at least % of the variance in the rasch dimension, and an eigenvalue of less than . on first contrast, provides evidence of unidimensionality (linacre ) . the response pattern across subgroups of the population (age and gender groups) was assessed by differential item functioning (dif). all analyses were conducted in r (version . . ; r core team, ) using the lavaan (rosseel ) and winsteps . software (linacre ) . to test the concurrent validity, pearson's correlations were used to assess the relationship between the fcv- s and the pvds (samples and ), the wemwbs (sample ), and adherence to lockdown rules (samples and ). finally, spearman rank order correlations were used to test the relationship between fcv- s and political beliefs. for samples and respectively, the mean fcv- s score was . and . (sd = . and . ), pvd was . and . (sd = . and . ), and wemwbs was . (sd = . ). table shows the summary statistics and item analysis for each scale item in the fcv- s (mean item scores, standard deviation, skew, kurtosis, item difficulty, item discrimination, and internal consistency if item was deleted). as seen in table , items , , and were not normally distributed (indicated by a skew and kurtosis falling outside of the − to range), due to the fact that most participants "strongly disagreed" with these items. finally, the internal consistency if an item was deleted ranged from . to . , suggesting that dropping any item in the scale would not improve overall internal consistency. cfa of the fcvs- scale showed good fit with cfi of . for sample and . for sample ( table ). rmsea was slightly higher than levels indicative of good fit, with rmsea of . for sample and . for sample , where a traditional cutoff of good fit is normally less than . . both samples, however, independently support a similar level of fit and all significantly loaded onto the same latent factor with standardized loadings between . and . . all items fitted well with their latent construct as the infit and outfit mnsq were within acceptable range ( . - . ) in both samples (table ). the most difficult item for sample and to assess concurrent validity, the fcv- s was correlated with perceived vulnerability to disease (sample and ) and wellbeing (sample ). the fcv- s demonstrated good concurrent validity. more specifically, there was a moderately strong relationship between the fcv- s and the two subscales of perceived vulnerability to disease scale: perceived infectability (sample : r = . , p < . ; sample : r = . , p < . ) and germ aversion (sample : r = . , p < . ; sample : r = . , p < . ). in addition, there was a negative relationship between fcv- s and the wemwbs (r = − . , p < . ), such that those who had higher fear scores reported lower overall wellbeing scores. finally, when assessing participants' adherence to new zealand's lockdown rules, fcv- s was significantly associated with adherence with all five rules during alert level (sample ) and three of the five rules during alert level (sample ) (table ) . finally, consistent with the view that covid- has become a politicized topic, there were modest negative correlations between the fcv- s scores and political beliefs (sample : m = . , sd = . , rho = −. , p < . ; sample : m = . , sd = . , rho = −. , p = . ). that is, participants that rated themselves as more toward the conservative end of the political spectrum tended to report lower fcv- s scores. the primary aim of the present study was to evaluate the english version of the fcv- s. across two large samples, the fcv- s had high internal consistency. mirroring the bangla (sakib et al. ) , italian (soraci et al. ) , arabic (alyami et al. ) , and hebrew (bitan et al. ) validation studies, participants tended to "strongly disagree" with items , , and . as alyami ( ) note, items , , and refer to somatic responses to covid- fear (e.g., "my heart races or palpitates when i think about getting coronavirus- "), rather than the more general level of fear tapped by the remaining items (e.g., "when watching news and stories about coronavirus- on social media, i become nervous or anxious"). consistent with the earlier validation studies, the fcv- s displayed a moderately strong relationship with the perceived infectability and germ aversion subscales of the pvd scale (duncan et al. ). moreover, the fcv- s scores were negatively correlated with the wemwbs, reflecting the fact that as fear of covid- increased wellbeing decreased. this finding is consistent with previous studies reporting positive correlations between the fcv- s and the hospital anxiety and depression scale (ahorsu et al. ; alyami et al. ) , patient health questionnaire (sakib et al. ) , and the depression and anxiety stress scale (bitan et al. ) . with respect to the motivating role of fear, there was a significant relationship between fcv- s scores and adherence to the lockdown rules that were implemented in new zealand (harper et al. ; pakpour and griffiths ; tannenbaum et al. ) . for example, fear of covid- was associated with participants maintaining the -m rule (i.e., maintain -m distance from other people when out in public) and keeping physical activity to outdoor places that could be readily accessed by foot. this finding is consistent with harper et al. ( ) , who reported a positive correlation between fcv- s scores and participants judgment of the degree to which several behaviors and practices had changed due to the pandemic (e.g., hand washing, care of children, and the elderly). consistent with the view that the politicization of covid- is not limited to the usa, participants that rated themselves as more conservative displayed lower fcv- s scores than participants that rated themselves more toward the liberal end of the political spectrum. although conservative politicians in new zealand have not gone as far as usa president donald trump, who early on referred to the virus as a hoax (franck ) , they have argued that the liberal government should reduce alert levels more quickly, effectively communicating a lower level of risk (burrows ; coughlan ) . indeed, in the usa, rothgerber et al. ( ) demonstrated that perceptions of health risk (e.g., the likelihood of being infected with covid- ) mediated the relationship between conservatism and abiding by social distancing rules. although the study comprised over participants in total, there are some limitations to consider when interpreting the results. data were not collected from a nationally representative sample (i.e., they comprised a self-selecting sample) and all the data were self-report which is subject to established method biases. given the cross-sectional nature of the data, the relationships between all the variables examined are associational, and the only way to establish the directional nature of the variables would be to carry out a longitudinal study. however, for a scale validation study, the design was robust. the current study demonstrates that the english version of the covid- s is a sound unidimensional scale with robust psychometric properties that can be used with confidence also shown is the association between fcv- s and adherence to each rule among english-speaking populations. critically, the covid- s displays sound concurrent validity and is associated with adherence to public health messages that aim to reduce the spread of covid- (e.g., maintaining the -m rule). conflict of interest the authors declare that they do not have any interests that could constitute a real, potential or apparent conflict of interest with respect to their involvement in the publication. the authors also declare that they do not have any financial or other relations (e.g. directorship, consultancy or speaker fee) with companies, trade associations, unions or groups (including civic associations and public interest groups) that may gain or lose financially from the results or conclusions in the study. sources of funding are acknowledged. ethical approval all procedures performed in this study involving human participants were in accordance with the ethical standards of university's research ethics board and with the helsinki declaration. informed consent informed consent was obtained from all participants. open access this article is distributed under the terms of the creative commons attribution . international license (http://creativecommons.org/licenses/by/ . /), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made. open access this article is licensed under a creative commons attribution . international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creativecommons.org/licenses/by/ . /. the fear of covid- scale: development and initial validation psychometric evaluation of the arabic version of the fear of covid- scale coughing while asian': living in fear as racism feeds off coronavirus panic. the guardian state of national emergency declared to fight covid- asian parents remove child from school as covid- racism spikes. stuff.co.nz fear of covid- scale: psychometric characteristics, reliability and validity in the israeli population new zealand should consider quitting lockdown early, david seymour says why are conservatives less concerned about the coronavirus (covid- ) than liberals? testing 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a meta-analysis of fear appeal effectiveness and theories the warwick-edinburgh mental well-being scale (wemwbs): development and uk validation covid- coronavirus pandemic publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations key: cord- -wbkfjz r authors: devaney, ryan; trudgett, james; trudgett, alan; meharg, caroline; smyth, victoria title: a metagenomic comparison of endemic viruses from broiler chickens with runting-stunting syndrome and from normal birds date: - - journal: avian pathol doi: . / . . sha: doc_id: cord_uid: wbkfjz r runting-stunting syndrome (rss) in broiler chickens is an enteric disease that causes significant economic losses to poultry producers worldwide due to elevated feed conversion ratios, decreased body weight during growth, and excessive culling. of specific interest are the viral agents associated with rss which have been difficult to fully characterize to date. past research into the aetiology of rss has implicated a wide variety of rna and dna viruses however, to date, no individual virus has been identified as the main agent of rss and the current opinion is that it may be caused by a community of viruses, collectively known as the virome. this paper attempts to characterize the viral pathogens associated with – -week-old rss-affected and unaffected broiler chickens using next-generation sequencing and comparative metagenomics. analysis of the viromes identified a total of dna and rna viral families, along with unidentified categories, comprised of distinct viral genera and unclassified genera. the most abundant viral families identified in this study were the astroviridae, caliciviridae, picornaviridae, parvoviridae, coronaviridae, siphoviridae, and myoviridae. this study has identified historically significant viruses associated with the disease such as chicken astrovirus, avian nephritis virus, chicken parvovirus, and chicken calicivirus along with relatively novel viruses such as chicken megrivirus and sicinivirus and will help expand the knowledge related to enteric disease in broiler chickens, provide insights into the viral constituents of a healthy avian gut, and identify a variety of enteric viruses and viral communities appropriate for further study. the overall performance of a poultry flock is dependent on various factors such as feed quality, poultry house management, and the presence of pathogenic microorganisms. one of the largest contributing factors to thriving flocks is the development and proper functioning of the gastrointestinal (gi) tract. the avian gi tract represents a site of nutrient absorption and contains many different types of microorganisms (collectively known as the microbiome) and plays host to a variety of commensal and pathogenic microbes including viruses, bacteria, and fungi. suboptimal functioning of the gi tract can result in poor performance and can lead to production problems such as a poorer feed conversion ratio (fcr; the efficiency of converting feed to body mass), uneven flock growth, and can be caused by enteric diseases such as runting-stunting syndrome (rss) in broiler chickens. rss, also known as malabsorption syndrome, was reported in broiler flocks as early as the s by kouwenhoven et al. ( a, b) who described signs such as proventriculitis, poor fcr, and runting, which is defined as undersized at hatch. further research described a wide variety of clinical signs including growth depression, irregular feathering (helicopter feathering, abnormal colouring), the presence of watery diarrhoea, other enteric problems such as intestinal lesions and pale intestines, and in severe cases mortality (smart et al., ; shapiro et al., ; otto et al., ; de wit et al., ) . globally, the incidence of rss and uneven flock growth can cause substantial economic losses to poultry producers due to culls of stunted, undersized birds that are too small to pass through the processing plant. of specific interest is the enteric viral population associated with rss of which many rna and dna viruses have been implicated and co-infections of multiple viruses such as rotavirus, chicken astrovirus (castv), avian nephritis virus (anv), and reoviruses have been detected in birds affected with rss or with poor performance (reynolds et al., ; guy, ; kang et al., ) . metagenomic research into a similar disease in turkeys, known as poult enterits complex, has helped shed some light into the range of enteric pathogens associated with avian malabsorption diseases with day et al. ( ) describing at least different rna viral genera from a pool of affected birds. other enteric pathogens associated with avian malabsorption diseases include reoviruses, parvoviruses, and members of the family caliciviridae; many of which have also been observed in broiler chickens smyth et al., ; pantin-jackwood et al., , wolf et al., . pantin-jackwood et al. ( ) demonstrated the presence of anv in chicken flocks but also in turkey flocks for the first time indicating that some level of cross-species interaction can occur. additionally, day et al. ( ) demonstrated that both turkeys and chickens exhibit species-specific viruses such as turkey astrovirus and castv. although previous studies detected many of these enteric viruses in affected flocks, and in different combinations of co-infections, some of these same viruses have been found in healthy flocks making it difficult to determine which viruses are implicated in the disease. for example, reynolds et al. ( ) demonstrated that avian astrovirus and rotavirus were detected in "normal" turkey poults as well as affected poults and pantin- reported the presence of castv and anv in healthy flocks as well as affected. some of the viruses associated with rss are difficult to cultivate in the laboratory which makes them difficult to study using conventional techniques (todd et al., ) . furthermore, a conventional approach such as viral cultivation and sanger sequencing would make it difficult and time consuming to study the community of viruses in an affected flock especially novel viruses. in order to study the complete enteric virome, and provide an unbiased comparison of affected and unaffected birds, a more comprehensive approach should be applied and high-throughput sequencing is an ideal method to examine the community of viruses inhabiting the enteric tracts of broiler flocks . high-throughput, next-generation sequencing (ngs) is a tool that has been successfully used to characterize microbial communities from a variety of complex environmental samples (mardis, ; patterson et al., ; li et al., ) . using current ngs technologies it is possible to achieve deep sequencing coverage from samples that permits comparative metagenomic analysis between samples to identify key pathogens related to avian enteric disease. bacterial metagenomics via amplicon sequencing is a wellresearched area due to the availability of the highly conserved s ribosomal rna (rrna) sequence found in bacteria (riesenfeld et al., ; gill et al., ; wang & qian, ) . as there is no viral equivalent of the s rrna gene it is more appropriate to use a shotgun sequencing approach to discover novel viruses from environmental samples. one of the major advantages of certain, newer ngs platforms is the ability to obtain longer reads from a single run; for example up to base pairs (bp) for a single read using roche's flx+ system or bp from a bp, paired end read by the miseq system from illumina, which are comparable with read lengths obtained from conventional sanger sequencing. this increases accuracy of sequence assembly in regards to rna viruses due to their high mutation rates leading to a high degree of variability and their ability to form quasispecies (todd et al., ; smyth et al., ) . this paper describes the application of ngs to characterize the dna and rna viral communities present within samples from broiler birds affected and unaffected by rss. broiler chickens of between and days of age were received from uk farms. the gut contents of between two and seven of these birds were pooled from rss-affected flocks displaying growth depression (vf - e, vf - a , vf - a , vf - a , vf - a ) and from two unaffected flocks of healthy birds (vf - a , vf - b ). from each pool g of gut content was removed from the intestinal tract of the chicken and added to ml phosphate buffered saline (pbs), ph . , in a ml centrifuge tube and was homogenized with sterile glass beads and vortexed thoroughly for min followed by centrifugation for min at × g, °c. the supernatant was transferred to a fresh ml centrifuge tube and centrifuged at × g, °c for min. the supernatant was removed and filtered through sterile . µm syringe filters (merck-millipore, billerica, ma, usa). the filtered supernatant was then ultracentrifuged (sorvall wx ultra series, thermo scientific, waltham, ma, usa) for h at , × g, °c using the sw -ti rotor (beckman-coulter, brea, ca, usa). the supernatant was removed and the pellet formed was resuspended in ml sterile pbs buffer, ph . . rnase a ( µg, thermo scientific) and u dnase (thermo scientific) were added as per the manufacturer's protocol to the enriched viral suspension and mixed by gently tapping the aliquot. the suspension was incubated in a water bath at °c for min followed by inactivation by adding ethylenediaminetetraacetic acid (thermo scientific) supplied with the dnase kit, as per the manufacturer's instructions, and incubating at °c for min. samples were divided into two fractions for dna and rna extraction and placed on ice. total rna was extracted from samples as prepared above using the ribopure rna extraction kit (life technologies, carlsbad, ca, usa), according to manufacturer's instructions and collected in a . ml tube. total dna was extracted using the viral rna mini kit (qiagen, manchester, uk) to manufacturer's instructions and collected in a . ml tube. the viral rna mini kit was shown to extract dna just as efficiently as it extracted rna and more efficiently than other dna extraction kits (unpublished results). whole transcriptome and whole genome amplification (wga) extracted dna and rna were subjected to wga and whole transcriptome amplification (wta) respectively to produce enough genomic material for sequencing. wga and wta reactions were carried out in parallel using the repli-g cell wga and wta kit (qiagen) according to manufacturer's guidelines using random primers. the wta reaction converted rna to cdna. genomic material was fragmented via sonication to achieve a - bp range suitable for sequencing by the flx+ system (roche, penzberg, upper bavaria, germany). total wga and wta material ( µl) from each sample was diluted in µl sterile pbs, ph . , and sonicated at full power using the soniprep (mse, london, uk) using an exponential probe. samples were sonicated for cycles each alternating between s sonication followed by s cooling on ice. for the gs junior system (roche) genomic material was fragmented via sonication to achieve a - bp range suitable for sequencing. this was achieved via the same method as the flx+ using nine sonication cycles instead of seven. sonicated samples were purified using the qiaquick polymerase chain reaction (pcr) purification kit (qiagen) according to the manufacturer's guidelines. purified samples were subject to end repair, followed by sequencing adaptor ligation, and removal of small fragments using the rapid library preparation reagents and adaptors kit (roche) according to manufacturer's guidelines. when preparing a single sample the rl adaptor from the kit was used. when multiplexing samples each sample utilized a different mid adaptor from the mid adaptor kit (roche). samples vf - a , vf - a , vf - a , vf - a , vf - a , and vf - b were multiplexed in a single run. this was followed by a quality check on the bioanalyser (agilent, santa clara, ca, usa) using the high sensitivity dna kit (agilent) to ensure optimum bp range was achieved. quantification for sequencing was performed on the quantifluor fluorometer (promega, madison, wi, usa). due to the µl minimum volume required by the quantifluor fluorometer (promega) a modified protocol was used. a dilution series was prepared using the rl standard from the rapid library preparation reagents and adaptors kit (roche). each standard was vortexed and centrifuged briefly at each stage of the dilution. fifty microlitres of each sample was made up to µl with te buffer in order to obtain an accurate measurement using the fluorometer. the fluorometer was set to raw readings and utilized the blue channel. relative fluorescence unit values were input into the rapid library calculator (roche, www.my .com) which calculated the dilutions required to bring each sample to a working stock of × molecules/µl suitable for emulsion pcr (empcr). once quantified, libraries were pooled together and µl was used for empcr, which was carried out to manufacturer's guidelines as found in the empcr amplification method manual -lib-l (roche). for miseq (illumina, san diego, ca, usa) library preparation the nextera xt library preparation kit (illumina) was used as per manufacturer's instructions. sequencing on the gs junior system was performed to standard protocol as found in the gs junior sequencing method manual (roche). sequencing on the flx+ system was performed in mannheim, germany by roche. sequencing on the miseq was performed to standard protocol as found in the miseq system user guide (illumina) using bp paired end reads. the pyrosequencing reads were demultiplexed and assembled separately into contigs using the newbler version . assembly software (roche). miseq (illumina) sequencing reads were assembled via basespace (illumina) using the velvet assembly tool v . . (zerbino & birney, ). contigs were input into the basic local alignment search tool (blast, geer et al., ) using the non-redundant nucleotide database searching against highly similar sequences (megablast). resulting xml files were input into metagenome analyser v . . (megan, huson et al., ) for taxonomic analysis using default settings. multiple alignments were performed using geneious v . . (http:// www.geneious.com, kearse et al., ) against reference viral genomes indicated by megan analysis. sequence data was uploaded to mg-rast (http:// metagenomics.anl.gov/, meyer et al., ) and made publically available at the following link: http:// metagenomics.anl.gov/linkin.cgi?project= . sequencing results produced , , high quality reads which were assembled into , contigs ranging between and bp with the majority of contigs ranging between and bp. short contigs were visually inspected for repetitive and low complexity sequences and removed where appropriate ( figure ). a total of contigs were assigned to dna and rna viral families (table ) along with two unclassified categories and comprised distinct viral genera and seven unclassified categories ( figure ). the remaining contigs were assigned to cellular organisms (bacteria, fungi, and avian species) or produced no hits against the basic local alignment search tool nucleotide database. across all samples the most abundant viral families identified were the picornaviridae, astroviridae, caliciviridae, parvoviridae, herpesviridae, siphoviridae, and myoviridae (table ) . within the affected samples the most abundant groups included the picornaviridae, figure . megan taxonomic analysis displaying a viral family comparison between all seven samples. vf - a and b (*) represent unaffected samples. the "viruses" and "dsdna viruses, no rna stage" categories contained viral contigs from the families siphoviridae, myoviridae, podoviridae, herpesviridae, reoviridae, retroviridae, polyomaviridae, inoviridae, baculoviridae, and poxviridae. these unassigned contigs were accounted for in table . bars located next to each taxon are proportional to the total number of contigs assigned to each category from sequencing runs. astroviridae, siphoviridae, and myoviridae. the most abundant family identified in sample vf - e was the picornaviridae with over half of the total amount of contigs ( . %) being assigned to this family and the second most abundant family being the astroviridae with . % of contigs being assigned (table ) . this sample differed from all others due to the absence of any viral contigs assigned to the bacteriophage families siphoviridae, myoviridae, and podoviridae. sample vf - a displayed the myoviridae as the most abundant family ( . %) followed by the astroviridae ( . %) and the siphoviridae ( . %). the most abundant family associated with sample vf - a was the astroviridae ( . %) followed by an even spread across the families picornaviridae ( . %), caliciviridae ( . %), parvoviridae ( . %), siphoviridae ( . %), and myoviridae ( . %). sample vf - a differed compared to all other samples with no viral contigs assigned to the astroviridae and caliciviridae and only a small number of viral contigs associated with the picornaviridae ( . %). within this sample the most abundant families were the myoviridae ( . %) and the siphoviridae ( . %). additionally, this sample had the largest amount of retroviridae contigs associated with it ( . %) compared to all other samples. sample vf - a showed a similar viral profile to that of sample vf - e as the most abundant viral families were the astroviridae ( . %) and the picornaviridae ( . %) closely followed by the herpesviridae ( . %). within the unaffected sample set, sample vf - a displayed the paroviridae as the most abundant family with . % of the viral contigs being thus assigned. this was followed by the caliciviridae ( . %) which was most abundant in this sample compared to all others (table ) . this sample also displayed a relatively even spread across the families picornaviridae ( . %), astroviridae ( . %), coronaviridae ( . %), and reoviridae ( . %). the most abundant family identified in sample vf - b was the siphoviridae with over half the viral contigs being assigned to this family ( . %). this sample was also very similar to the other unaffected sample, vf - a , in regards to the parvoviridae family with . % of the viral contigs being associated with this family (compared to . % associated with sample vf - a ). the unaffected samples displayed a larger abundance of parvoviridae contigs compared to the affected samples while the affected samples displayed a larger abundance of picornaviridae and astroviridae compared to the unaffected samples (table ) . a higher abundance of the bacteriophage families was observed in the affected samples compared with the unaffected samples. noticeably, there was an absence or low abundance of reoviridae contigs across all samples even though reoviruses have been strongly associated with rss. otto et al. ( ) described the presence of reoviruses via reverse transcriptase pcr from / ( %) of rss-affected broiler chicks tested and also from / ( %) of "healthy" control birds tested. the birds tested in the study ranged from to days of age compared to the present study which tested - week-old birds. as reoviruses are quite commonly detected in young broiler chicks it may be possible that any reovirus infection has largely cleared from these older birds and is present in relatively low abundance accounting for the absence of reoviridae contigs in the majority of samples (table ) . picornaviridae members of the family picornaviridae (order: picornavirales) were detected across all seven ( %) samples with a total of viral contigs assigned to this family ( table ). the majority of viral contigs ( ) in this metagenomic analysis were assigned to the unclassified picornaviridae category with the remaining contigs ( ) assigned to recognized genera -gallivirus, kobuvirus, and megrivirus (figure ). within these genera, viral contigs showed similarity to multiple species such as chicken gallivirus (gallivirus), aichivirus c (kobuvirus), and chicken megrivirus (megrivirus). a total of contigs characterized as unclassified picornaviridae showed similarity to the species sicinivirus , a novel picornavirus isolated from commercial broiler chickens in cork, ireland (bullman et al., ) and recently reported in mainland china (zhou et al., ) . sicinivirus contigs were almost exclusively associated with rss-affected samples with only one sicinivirus contig associated with unaffected sample vf - a . sicinivirus was commonly found alongside chicken megrivirus, chicken picornavirus , , and , and aichivirus c however the pathogenic potential of sicinivirus is yet to be determined. the remaining viral contigs ( ) associated with the unclassified picornaviridae displayed similarity to chicken picornavirus , , and , picornavirus chicken/chk /usa/ , pigeon picornavirus b, and aichivirus c. the species chicken picornavirus , , , chicken megrivirus, and aichivirus c were only associated with the affected samples compared to picornavirus chicken/chk /usa/ , pigeon picornavirus b, and chicken gallivirus which were found in both affected and unaffected samples. members of the picornaviridae are characterized by nonenveloped icosahedral virions, around nm in diameter, containing a single-stranded positive-sense rna genome, - kb in length (stanway, ; legall et al., ) . picornaviruses have been linked to disease across multiple species (such as sheep, cattle, humans, felines, and birds) and have been implicated in enteric disease (knox et al., ; tapparel et al., ) . previous metagenomic studies into the avian gut microbiome have identified multiple picornavirus species in disease-affected and healthy birds, findings which have been echoed in the present study. however, the pathogenic potential of these viruses in avian species remains relatively unknown due to the large amount of genetic variation observed among these viruses farkas et al., ; bullman et al., ; zhou et al., ) . in the present study picornaviruses were commonly found alongside the astroviridae, caliciviridae, parvoviridae, and bacteriophage families (table ) and seem to be prevalent in growth-stunted birds. due to the large number of picornavirus strains in circulation it is perhaps unsurprising that picornavirus contigs were also detected in the unaffected samples and it is possible that non-pathogenic picornavirus strains may be constituents of a healthy avian gut virome. members of the family coronaviridae (order: nidovirales) were detected in / samples and were found in both affected ( / ) and unaffected ( / ) samples although in relatively low numbers with a greater number of contigs found in samples vf - a and vf - a (table ) . all viral contigs ( ) assigned to the coronaviridae family belonged to the genus gammacoronavirus ( figure ) with all of the viral contigs displaying high similarity ( - % nucleotide identity) to infectious bronchitis virus (ibv). although both unaffected samples were observed to also have ibv contigs, this may be due to the small sample set tested. members of this family are characterized by enveloped, positive-stranded rna genomes, - kb in length (brian & baric, ; gorbalenya et al., ) . coronaviruses have been detected in a variety of wild birds and are responsible for mild to severe respiratory signs, central nervous system diseases, and gi diseases (gallagher & buchmeier, ; weiss & navas-martin, ; weiss & leibowitz, ) . the main coronavirus affecting chickens is ibv which can cause respiratory disease and can also replicate in non-respiratory areas such as enteric tissues. although ibv is not typically associated with enteric disease it may contribute to enteritis in combination with other microbial factors (cavanagh & gelb jr., ; cavanagh, ; jackwood et al., ) . a total of viral contigs belonging to the caliciviridae family were detected in / of the tested samples and were found in / affected samples and / unaffected samples. a total of viral contigs displayed similarity to calicivirus isolates calicivirus chicken/ v /bayern/ ( - % nucleotide identity), calicivirus chicken/v /bayern/ ( - % nucleotide identity), and calicivirus chicken/v /bayern/ ( % nucleotide identity) with the remaining contigs ( ) showing similarity to chicken calicivirus isolates chicken/l polyprotein gene ( - % nucleotide identity), chicken/l polyprotein gene ( - % nucleotide identity), and caliciq / polyprotein gene ( - % nucleotide identity). the caliciviridae family is comprised of five recognized genera (lagovirus, nebovirus, norovirus, sapovirus, and vesivirus) and an unclassified caliciviridae genus with the caliciviridae contigs in this study belonging to the unclassified caliciviridae genus. virions belonging to the caliciviridae family are typically non-enveloped, around - nm in diameter, and with an rna genome of around . kb in length (thiel & könig, ) . the caliciviridae family can infect a variety of host organisms including humans, birds, pigs, cattle, and avian species and have been associated with gastroenteritis in humans as early as the s (kapikian et al., ; smith et al., ; chen et al., ; wolf et al., ). calicivirus has been detected and characterized from both healthy chickens and chickens displaying growth retardation via electron microscopy, reverse transcriptase pcr and ngs wolf et al., ; . a previous metagenomic analysis of specific pathogen free (spf) and spf sentinel birds performed by described the majority of viral contigs ( , ) associated with the spf flock being assigned to the caliciviridae family ( . %) with the virus appearing to clear from sentinel birds placed on commercial farms with enteric and respiratory problems although testing of a backyard sentinel flock, with a history of enteric disease, identified contigs ( . %) assigned to the caliciviridae family. conversely, in the present study, members of the caliciviridae were more commonly associated with rss-affected samples with the most common isolate being calicivirus chicken/v / bayern/ , the same isolate described by in the spf and backyard flocks, this isolate was also detected in unaffected sample vf - a . contigs displaying similarity to the isolates l , l , q , and calicivirus chicken/v /bayern/ were only found in rss-affected samples possibly suggesting an association with enteric disease while isolate calicivirus chicken/v /bayern/ was only associated with unaffected sample vf - a and not in affected samples. interestingly, there were no caliciviridae contigs associated with affected sample vf - a (table ) . the present study has identified viral contigs displaying similarity ( - % nucleotide identity) to the avastrovirus genus (table , figure ), specifically the species castv and anv sero-or genotypes (anv ), (anv ), and (anv ). members of the avastrovirus genus, especially anv and castv, are strongly associated with rss and the current study reports a much higher viral profile associated with this genus in rss-affected samples with much lower profiles being observed in unaffected samples ( figure ). contigs associated with the astroviridae were commonly found in combination with the caliciviridae, picornaviridae, and parvoviridae families (table ) which has been described in previous metagenomic studies in chickens and turkeys . viral contigs assigned to the castv and anv species were only associated with the affected sample set. viral contigs associated with the anv and anv species were associated with both affected and unaffected sample sets with a notably larger anv profile associated with the affected samples. this may suggest greater anv strain diversity within the affected samples with certain strains exerting a greater pathogenic effect leading to stunted growth. described a very low astroviridae profile associated with a spf flock ( contigs, . %). by contrast, day reported increased levels of astrovirus contigs ( . - . %) associated with sentinel spf birds placed on commercial farms with enteric problems indicating that members of the astroviridae family are more commonly found in flocks with growth problems and enteric disease. the astroviridae family contains a group of enteric viruses which can infect multiple mammalian hosts (genus: mamastrovirus) and avian hosts (genus: avastrovirus) causing a range of enteric disease signs such as diarrhoea, enteritis, interstitial nephritis and has been linked with visceral gout and rss (yamaguchi et al., ; mcnulty et al., ; herrmann et al., ; greenberg & matsui, ; moser & schultz-cherry, ; spackman et al., ; benedictis et al., ; lee et al., ) . first identified in (madeley & cosgrove, ) astroviridae are characterized as small, non-enveloped virions with a positive-sensed rna genome around kb in length (jiang et al., ; carter, ; willcocks et al., ) . the avastrovirus genus contains at least six recognized species -anv, castv, duck astrovirus types and , and turkey astrovirus types and . previous studies have identified astroviruses in both growth-stunted and healthy avian hosts (reynolds et al., ; baxendale & mebatsion, ; kang et al., ) . avian orthoreoviruses (arv) can be present without disease in broiler chickens however reovirus infection can lead to several disease signs such as tenosynovitis, growth suppression, and enteritis and can also cause immunosuppression leaving the host susceptible to other infections (hieronymus et al., ; jones & kibenge, ; sharma et al., ; jones, ) . although reoviruses are widespread among avian hosts, the present study reports detection of reovirus in only one sample (vf- a ) which represents a healthy flock. the low detection rate of arv may be a consequence of the small sample set tested or may suggest their relative abundance is low compared to other viruses. reovirus contigs obtained from sample vf - a were assigned to the orthoreovirus genus (figure , species: avian orthoreovirus) and displayed homology to the l , l , l , s , and s genome segments and the lambdab core protein gene. the family reoviridae, consisting of two subfamilies comprising of genera, contains a group of viruses with a wide host range including invertebrates, vertebrates, plants, and fungi and has been described in both rss-affected broiler chickens and healthy chickens (robertson et al., ) and has been detected in previous avian enteric metagenomic studies . reoviruses are non-enveloped viruses that contain a segmented ( - segments) double-stranded rna genome (joklik, ; gouet et al., ; forrest & dermody, ) . the retroviridae family is comprised of two subfamilies (orthoretrovirinae and spumaretrovirinae) and contains seven viral genera and has a large host range. this study has detected the presence of one viral contig from one sample, vf - a (table ) , displaying similarity ( % nucleotide identity) to avian erythroblastosis virus (genus: alpharetrovirus) erba and erbb genes. additionally, the same sample had one viral contig displaying % nucleotide similarity to the avian endogenous retrovirus gag gene. endogenous viral elements represent either entire viral genomes or fragments of viral genomes which have become integrated into the host germ line. the remaining viral contigs ( ) from the megan analysis displayed short sequence similarity, - bp (from contigs around - bp in length), to human endogenous retroviruses and the lentivirus genus. upon analysis the full contigs displayed homologies to various bacterial genera such as salmonella, escherichia, and bacteroides. retroviruses reverse transcribe their rna genome into dna using their own reverse transcriptase followed by integration into the host genome where they replicate using the host polymerase genes (dahlberg, ; coffin, ; luciw & leung, ; gifford & tristem, ) . previous studies have described the presence of endogenous avian retroviruses within domesticated chickens which are vertically transmitted through the host germ line following genomic integration (frisby & weiss, ) . although not normally linked to disease endogenous avian retroviruses have been related to subgroup j avian leukosis virus, an exogenous avian retrovirus, which has been reported to induce myeloid leukosis, can cause mortality through the development of tumours, and can cause immunosuppression within the host (purchase et al., ; friedman & ceglowski, ; payne, ; smith et al., ; fadly & smith, ) . it is possible that the presence of avian retroviruses in rss-affected samples is not directly contributing to enteric disease however the immunosuppression caused by these viruses may leave affected birds susceptible to infection from other viral species. parvoviridae parvoviruses were detected in / samples tested with the all of the viral contigs displaying similarities to either the aveparvovirus genus ( - % nucleotide identity), specifically chicken parvovirus strains, and the protoparvovirus genus ( - % nucleotide identity), specifically to the ns , np , vp , and vp genes of the protoparvovirus genus, isolates par-vod / and parvod / , and decaesstecker et al. ( ) in which chicken parvovirus abu-p in combination with reoviruses failed to cause growth retardation in day old chicks following oral inoculation. conversely, an earlier study isolated chicken parvovirus abu-p from chickens with stunted growth and re-inoculation of embryos and day old spf chicks with this isolate caused enteritis, a decrease in egg hatchability, and severe growth retardation (kisary, ) . all affected samples contained parvovirus contigs although a substantially larger parvoviridae profile was observed in the unaffected samples compared to the affected samples. both affected and unaffected samples contained a combination of sequences from members of the protoparvovirus and aveparvovirus genera however the affected samples contained more viral contigs assigned to the protoparvovirus genus while the unaffected samples contained more viral contigs assigned to the aveparvovirus genus (species: chicken parvovirus abu-p ). the paroviridae family is divided in to two sub families (densovirinae and parvovirinae) that contain genera between them and are characterized as having a nonenveloped capsid containing a single-stranded dna genome around kb in length (berns, ; cotmore & tattersall, . the circoviridae family contains two distinct genera -gyrovirus and circovirus. they are characterized by a circular non-segmented single-stranded dna genome, around . - . kb in length (finsterbusch & mankertz, ). the present study detected two viral contigs exhibiting similarity ( - % nucleotide identity) to the gyrovirus genus in only one rss-affected sample, vf - a , which specifically showed similarity to chicken anaemia virus (cav, genus: gyrovirus) while the unaffected samples displayed no circoviridae contigs. the circoviruses can infect a range of birds and mammals such as chickens, pigs, dogs, geese, and pigeons (yuasa et al., ; tischer et al., ; todd et al., ; kapoor et al., ) . mcnulty et al. ( ) described the effects of subclinical cav infection in broiler chickens resulting in a decrease in profitability and productionspecifically a decrease in the average weight per bird and an adverse effect on fcr theorized to be caused by the immunosuppressive capabilities of cav. cav was later detected in fourweek-old growth-stunted birds in combination with cryptosporidium baileyi (dobos-kovács et al., ) and has been detected in broiler chickens displaying lymphocyte depletion (van santen et al., ) further indicating a role in immunosuppression. rosenberger and cloud ( ) also reported that the immunosuppressive aspects of cav frequently lead to secondary infections with clostridium perfringens and staphylococcus aureus. this immunosuppression, in combination with other viral pathogens, may contribute to the development of rss. although cav was only detected in one sample it is possible that the infection had largely cleared from these - -week-old birds and could have been detected if tested at an earlier age. additionally, it is possible that cav was present in samples in relatively low abundance. throughout the virus enrichment process during sample preparation there were losses associated with each of the processing stages. it is possible that the less abundant cav has been lost in these processing stages therefore not detected in the final sequencing results. the caudovirales order contains tailed bacteriophages and comprises the families siphoviridae, myoviridae, and podoviridae and were detected in all samples tested (table ) . additionally, the families leviviridae, inoviridae, and the unassigned phage category (table ) consisted of phage sequences. within the affected samples there was a large caudovirales profile associated with samples vf - a and vf - a (over % of the viral contigs for each sample) and relatively little caudovirales contigs associated with vf - a and vf - a (table ) . interestingly, affected sample vf - e contained no caudovirales contigs. unaffected sample vf - a had only three contigs associated with the caudovirales while unaffected sample vf - b had a large caudovirales profile (over % viral contigs were assigned to the caudovirales). in the present study the samples had an overall total of viral contigs assigned to phage species with the majority of contigs being assigned to the families siphoviridae ( contigs) and myoviridae ( contigs) (table ) making the bacteriophages the most abundant species identified in the current study ( . % of total viral contigs). sample vf - a had viral contigs assigned to the podoviridae family. sample vf - e displayed no similarity to any of the bacteriophage families but had one viral contig identified as an unclassified phage (table ) . this was similar to samples vf - a and vf - a which displayed a very low amount of phage contigs ( . % and . % respectively) compared to the large abundance of phage contigs identified in samples vf - a ( . %), vf - a ( . %), vf - a ( . %), and vf - b ( . %). an increase in viral contigs associated with the caudovirales may coincide with an overgrowth of specific groups of intestinal bacteria which are then infected by species-specific bacteriophages helping regulate the intestinal microbiota. the majority of the viral contigs assigned to the caudovirales were identified as enterobacteria phages, enterococcus phages, and bacteroides phages which relate to normal bacterial constituents of the chicken gi tract such as escherichia coli, enterococcus species, and bacteroides species (devriese et al., ; lu et al., ; amit-romach et al., ) . these viruses are widespread and coexist with bacterial populations across a large range of hosts and environments and aid in regulating the diversity, population, and function of microbial communities (riesenfeld et al., ) . the role of bacteriophages in relation to rss is unclear and represents an interesting group for further study since previous metagenomic studies have also detected members of the caudovirales order as part of the broiler chicken gut microbiota in growth retarded flocks (kim & mundt, ; . of the remaining viral families contigs were assigned to the herpesviridae family. samples vf - e and vf - a displayed a higher herpesviridae abundance ( . % and . % respectively) compared to all other samples which displayed a more even spread ( . - . %). contigs assigned to this family displayed similarity to the cytomegalovirus genus ( figure ) , with contigs displaying similarity to the ceropithicine herpesvirus species. within this species all contigs assigned displayed greatest similarity ( - % nucleotide identity) to stealth virus . additionally, the small number of contigs in the "unclassified virus" category (table , sample vf - e) contained three contigs displaying high similarity to the species stealth virus and ( - % nucleotide identity). stealth virus has previously been isolated from human hosts displaying signs such as chronic fatigue (martin et al., ) and encephalopathy (martin, ) however no studies have been conducted in relation to avian hosts but it has been shown to be transmissible to dogs and cats (martin & anderson, ) . this study appears to be the first study to report the presence of stealth virus in rssaffected broiler chickens however further studies must be undertaken to understand the role this virus plays in disease. most previous investigations into the enteric viruses associated with poultry have been limited to culturedependant methods and molecular assays targeting previously known viruses. the use of high-throughput ngs in this study, and a small number of other studies, presents a viable technique for the characterization of the complex viral communities present in the gi tract of disease-affected and unaffected broiler chickens. this study presents preliminary data on the viral communities present in the poultry gut from a small number of samples and has identified multiple viral families historically associated with rss co-infecting broiler guts, along with the broad characterization of other viral families such as the siphoviridae, myoviridae, and podoviridae in relation to the disease. additionally, this study attempts to characterize the virome associated with unaffected broiler chickens with the results showing a difference in the viral contigs assigned in the unaffected samplestypically many of the same families are observed in unaffected samples but with a lower number of viral contigs assigned to these families when compared to affected samples. the main viral families related to rss-affected samples from this study included astroviridae, caliciviridae, picornaviridae, parvoviridae, coronaviridae, siphoviridae, and myoviridae although many of these viral families were also found in unaffected samples indicating that certain strains within these families may be constituents of a normal broiler gut virome. one of the advantages of the sequencing platforms used is their ability 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conservative fragments in bacterial s rrna genes and primer design for s ribosomal dna amplicons in metagenomic studies coronavirus pathogenesis coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus the complete sequence of a human astrovirus genetic characterization of a novel calicivirus from a chicken molecular detection of norovirus in sheep and pigs in new zealand farms characterization of a picornavirus isolated from broiler chicks isolation and some characteristics of an agent inducing anaemia in chicks velvet: algorithms for de novo short read assembly using de bruijn graphs identification and genome characterization of the first sicinivirus isolate from chickens in mainland china by using viral metagenomics chicken parvovirusinduced runting-stunting syndrome in young broilers the authors would like to acknowledge the department of agriculture, environment, and rural affairs (daera) for funding this study. key: cord- - esrg jw authors: tam, clarence c.; offeddu, vittoria; anderson, kathryn b.; weg, alden l.; macareo, louis r.; ellison, damon w.; rangsin, ram; fernandez, stefan; gibbons, robert v.; yoon, in-kyu; simasathien, sriluck title: association between semi-quantitative microbial load and respiratory symptoms among thai military recruits: a prospective cohort study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: esrg jw background: multiplex real-time polymerase chain reaction assays have improved diagnostic sensitivity for a wide range of pathogens. however, co-detection of multiple agents and bacterial colonization make it difficult to distinguish between asymptomatic infection or illness aetiology. we assessed whether semi-quantitative microbial load data can differentiate between symptomatic and asymptomatic states for common respiratory pathogens. methods: we obtained throat and nasal swab samples from military trainees at two thai army barracks. specimens were collected at the start and end of -week training periods (non-acute samples), and from individuals who developed upper respiratory tract infection during training (acute samples). we analysed the samples using a commercial multiplex respiratory panel comprising bacterial, viral and fungal targets. we used random effects tobit models to compare cycle threshold (ct) value distributions from non-acute and acute samples. results: we analysed non-acute and acute swab samples from participants. haemophilus influenzae type b was the most commonly detected microbe ( . % of non-acute and . % of acute samples). in acute samples, nine specific microbe pairs were detected more frequently than expected by chance. regression models indicated significantly lower microbial load in non-acute relative to acute samples for h. influenzae non-type b, streptococcus pneumoniae and rhinovirus, although it was not possible to identify a ct-value threshold indicating causal etiology for any of these organisms. conclusions: semi-quantitative measures of microbial concentration did not reliably differentiate between illness and asymptomatic colonization, suggesting that clinical symptoms may not always be directly related to microbial load for common respiratory infections. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. multiplex polymerase chain reaction (pcr)-based diagnostic techniques allow rapid, simultaneous identification of a broad range of respiratory pathogens [ ] . compared to classical microbiological diagnostic methods, pcr-based assays offer higher sensitivity, specificity, and reproducibility [ ] . however, the high sensitivity of multiplex pcr diagnostics does not directly translate into clinical utility, because such assays do not distinguish between viable and dead organisms, or acute infection and asymptomatic colonisation [ ] . in the clinical setting, the etiological agent is seldom identified and unspecific respiratory symptoms are often treated empirically [ ] . although the quantification of microbial load may vary depending on the presence of co-infections, specimen type, sampling technique, or timing of sampling, quantitative or semi-quantitative microbial load data from real-time pcr assays may help define organism densities that are consistent with colonization or infection and distinguish between symptomatic and asymptomatic states [ ] . in this study, we assessed whether semi-quantitative microbial load availab from real-time pcr assays can differentiate between symptomatic and asymptomatic states for common respiratory agents in a cohort of basic military trainees at two royal thai army barracks. details of the study setting and procedures have been described previously [ ] . briefly, participants were recruited from six consecutive cohorts of basic military trainees at two royal thai army barracks between may and july . trainees entered the camps for a -week training period at the start of may and november each year. individuals aged ≥ years entering one of the two army barracks involved in the study were eligible for enrolment. suspected tuberculosis cases or individuals with immune deficiencies, such as acquired immune deficiency syndrome, leukemia or lymphoma, were excluded. throat and anterior nasal swab samples were collected using stiff synthetic swabs by trained study staff at the start and end of each training period (non-acute samples) and were placed in viral transport media (universal transport medium c ; copan diagnostics) and stored at − °c until time of transfer to the armed forces research institute of medical sciences for further testing. in addition, enrolled participants were asked to consult the camp's medical unit if they experienced respiratory symptoms during the training period. medical staff took a history, conducted a medical exam, and recorded symptoms of upper respiratory illness (uri) or influenza-like illness (ili). uri was defined as an illness with at least two of the following: (i) runny nose or sneezing; (ii) nasal congestion; (iii) sore throat, hoarseness or difficulty swallowing; (iv) cough; (v) swollen or tender glands in the neck; and (vi) fever (oral temperature > °c). ili was defined as a respiratory illness with acute onset presenting with fever and cough or sore throat. throat and nasal swab samples were collected on average . days after symptom onset from individuals who developed uri or ili during the -week follow-up (acute samples). specimens from two of the six cohorts (total number of individuals = ) were tested using a commercial multiplex real-time pcr assay comprising bacterial, viral and fungal targets according to the manufacturer's instructions (ftd kit, fast track diagnostics, esch-sur-alzette, luxembourg). these two cohorts were selected because they underwent concurrent routine environmental sampling of air and surfaces within the barracks, which were then similarly tested using the ftd kit (data not shown). multiplex testing of specimens from the remaining cohorts was not done due to resource constraints. a cycle threshold (ct) value below the detection limit of the assay (< ) was considered a positive result. non-acute samples collected at the end of the training period from participants who experienced an acute episode during follow-up were excluded from the analysis, as the ct-value might reflect post-infectious shedding. we used the mcnemar test to determine whether target-specific frequencies were significantly different in non-acute baseline samples and acute samples. in addition, we computed the chi-square (χ ) or fisher's exact test (for expected values < ) to assess whether co-detection of specific microbe pairs occurred more frequently than expected by chance in non-acute baseline or acute samples. to account for data censoring at ct-value = , random effects tobit regression models were used to compare ct-value distributions from non-acute and acute samples, or ct-value distributions from samples containing a single or multiple organisms. in addition, we used the kruskal-wallis test to compare the median delay between illness onset and sample collection between samples containing one or multiple organisms. all analyses were conducted using stata software (stata corporation). the study was approved by the institutional review boards of the royal thai army in bangkok, thailand, the walter reed army institute of research and the london school of hygiene & tropical medicine. all participants provided written informed consent. the investigators have adhered to the policies for protection of human subjects as prescribed in army regulation - . we analyzed a total of non-acute swab samples collected from recruits at the start (n = ) or end (n = ) of the training period, and acute specimens from individuals who developed one or more uri episodes during follow-up. of targets contained in the respiratory panel, were detected in at least one specimen (table ) . viruses were detected in . % ( / ) and bacteria in . % ( / ) of non-acute samples. among acute samples, viruses were detected in . % ( / ) and bacteria in . % ( / ) of specimens. haemophilus influenzae type b (hi-b) was the most commonly detected microbe ( . % of non-acute and . % of acute samples). other frequently detected bacteria included non-type b haemophilus influenzae (hi-nonb), streptococcus pneumoniae, and klebsiella pneumoniae (table ) . rhinovirus was the most prevalent virus, detected in . % of non-acute and . % of acute samples. all other viruses were detected in < % of collected specimens (table ) . hi-nonb, rhinovirus, and coronavirus were detected significantly less frequently in non-acute samples collected at the start of the training period than acute samples (p-values < . ) ( table ) . influenza b was identified in none of the non-acute, but . % of acute specimens. multiple microbes were detected in . % ( / ) of non-acute samples collected at the start of the training period. co-detection of multiple organisms was significantly higher in both non-acute samples taken at the end of the training period ( . %) and acute specimens ( . %) (p-values < . ; table ). among acute samples, specific organism pairs were co-detected more frequently than expected by chance (p-values < . ) ( were found in < % of acute specimens (table ) . no microbe pair occurred more frequently than expected by chance among non-acute baseline samples. overall, there was a substantial overlap in ct-value distributions from non-acute samples collected at the start or end of the training period and acute samples collected from symptomatic individuals during follow-up (fig. ) . this was the case even when considering only samples where a single organism was identified (fig. ) . for hi-nonb and s. pneumoniae, our tobit regression models indicated significantly lower microbial load in non-acute baseline compared to acute samples (p-values < . ) ( table ). for hi-nonb, a coefficient of . represents a . higher average ct-value in non-acute baseline samples compared to acute specimens, which corresponds to an approximately -fold lower microbial load in non-acute compared to acute samples. for s. pneumoniae, the average microbial load was . -fold lower in non-acute baseline samples compared to acute specimens. our analysis also indicated a significantly lower average rhinovirus load in non-acute samples collected either at the start or at the end of the training period compared to acute samples (p-values < . ) ( table ). this was in contrast with hi-b, for which regression analysis indicated a . -fold higher average microbial load in non-acute baseline samples compared to acute samples (p-value < . ) ( table ). for hi-non b and s. pneumoniae, there was a . -fold or . -fold increase in average microbial load in non-acute samples collected at the end of follow-up compared to acute samples collected during an uri episode, respectively (p-values ≤ . ). there was no significant difference in delay between symptom onset and specimen collection in acute samples containing one (median delay: days; interquartile range (iqr): - ) or more (median delay: days; iqr: - ) organisms (p-value = . ). six acute specimens were negative for all agents tested (median delay: . days; iqr: - ). thus, sampling delay is unlikely to account for any observed differences in ct-value distributions. we analyzed the patterns of infection with common respiratory agents in a well-defined population of military recruits. the use of highly sensitive multiplex pcr diagnostics allowed an accurate characterization of the spectrum of organisms contained in non-acute and acute samples. the data indicate co-circulation of several different viral agents, and high frequency of bacterial colonization in both non-acute and acute samples. up to one third of respiratory illness cases among army personnel are reportedly caused by viral or bacterial infections [ ] . the gathering of individuals from diverse geographic locations and the crowded living conditions increase the risk of microbe transmission in these settings [ ] . illnesses are usually self-limiting, although the emergence of highly virulent strains can lead to high morbidity and mortality [ ] . streptococcus bacteria, adenoviruses, coronaviruses and influenza are among the most widely distributed microbes in the military environment, and are implicated in > % of febrile illness cases reported at military medical facilities [ ] . we identified each of these organisms in one or more samples. for most of these microbes, overall detection frequencies were comparable in non-acute and acute samples, although influenza b and coronavirus were more commonly identified among acute specimens. other infectious agents commonly circulating among military personnel include h. influenzae, rhinovirus, and, to a lesser extent, parainfluenza, rsv, and l. pneumophila, although their presence does not necessarily imply the occurrence of clinical symptoms [ ] [ ] [ ] . h. influenzae and rhinoviruses were the most frequently detected organisms in our population in both non-acute and acute samples. we detected parainfluenza and l. pneumophila, but we did not find rsv in any of our samples. for individuals developing uri during follow-up, illness etiology could not be unequivocally determined. among acute samples, hi-b was the most frequently detected organism. it was the sole agent identified in % of acute specimens, while it was co-detected with other microbes in > % of acute samples. however, colonisation with hi-b was also common among non-acute baseline samples, where it was detected alone or in combination with other microbes in . % and . % of specimens, respectively. for organisms rarely detected among asymptomatic individuals but frequently found in acute samples, a causal association may be more likely. for instance, influenza b was detected in none of the non-acute, but . % of acute samples. similarly, the proportion of both hi-nonb-and rhinovirus-positive samples was significantly lower among non-acute specimens collected at baseline compared to acute samples. however, > % of acute samples positive for hi-non b, rhinovirus or influenza b were also positive for one or more additional microbe, so that a causal table ) association could not be determined. some agents, such as hi-non b or adenovirus, were most frequently detected in non-acute samples collected at the end of follow-up, possibly indicating post-infectious shedding or persistent infection at sub-clinical levels. in the clinical setting, overlapping clinical presentations and poor capabilities to determine the etiology of respiratory illnesses often lead to inappropriate treatment with broad-spectrum antibiotics [ ] . this might occur even more frequently in the military setting, where molecular diagnostic tools are usually inaccessible [ ] . since a considerable fraction of respiratory illnesses is caused by viruses, the unsubstantiated use of antibiotics is particularly problematic, because it can lead to negative health outcomes and promote the development of antimicrobial resistance [ ] . studies evaluating the impact of multiplex diagnostic procedures on patient management report inconsistent results. in the outpatient setting, access to rapid molecular diagnostic tools for respiratory pathogens significantly reduced antibiotic a b fig. cycle threshold value distribution in non-acute and acute samples. ct-value distribution for selected a bacteria and b viruses detected in non-acute samples collected at the start or end of the training period (orange bars) or acute samples from individuals experiencing an upper respiratory tract infection during follow-up (blue bars). a ct-value of < was considered a positive result prescription rates for patients presenting with respiratory illness [ ] . however, these findings were not confirmed in the hospital setting. pcr-based testing failed to reduce hospital admissions and duration of hospital stay in patients with acute respiratory infection [ , ] . although molecular diagnostic tools may help to differentiate bacterial and viral respiratory agents, it is unlikely that antibacterial treatment would be terminated based on the mere presence of viral agents in an acute respiratory sample, especially considering the high rates of bacterial co-infection [ ] . quantitative or semi-quantitative diagnostic tools can potentially help define clinically significant pathogen densities, and have proven highly valuable to understand the dynamics of diarrheal disease [ ] and to improve the management of gastrointestinal illnesses [ ] . among acute diarrhea patients, quantitative amplification of norovirus rna from fecal samples can help determine pathogen load thresholds that effectively distinguish between causal association and sub-pathogenic carriage [ ] . similarly, rotavirus load correlates with disease severity among children with gastroenteritis [ ] . because of the crucial role of microbial replication in viral pathogenesis, the value of pathogen load quantitation could be most clearly established for gastrointestinal illnesses of viral etiology, although some evidence is available for bacterial infections as well. for instance, microbial load of enteropathogenic e. coli is significantly higher among children with diarrhea compared to control subjects, especially when enteropathogenic e. coli is the sole agent identified [ ] . in this study, tobit regression indicated significantly lower microbial load in non-acute relative to acute samples for rhinovirus, hi-nonb, and s. pneumoniae. however, due to a substantial overlap in ct-value distributions, it was not possible to identify a ct-value threshold indicating causality for any of these organisms. previous studies assessing the association of viral load with clinical symptoms of respiratory infections reported similar findings. mean viral load for rhinovirus and six additional viruses was significantly higher in upper respiratory tract aspirates from children with pneumonia compared to healthy controls, but the overlap in viral load distribution was substantial [ ] . in pediatric patients, high rhinovirus load was associated with the presence of lower respiratory tract symptoms [ , ] , but a threshold for clinical relevance could only be determined if rhinovirus was the sole agent identified [ ] . additional studies reported a correlation between microbial load and occurrence or severity of respiratory symptoms for rsv [ ] , bocavirus [ ] , and human metapneumovirus (hmpv) [ , ] , although these findings were inconsistent [ , ] or conditional on the presence of the virus as a single microbe [ ] . we did not detect any significant association between microbial load and clinical manifestations for viruses other than rhinovirus. for both h. influenzae and streptococcus species, previous studies reported a significant correlation of bacterial densities with clinical manifestations of disease [ ] . in young patients with acute respiratory tract infection, s. pneumoniae load fluctuated with symptom incidence and resolution [ ] . among children hospitalized with pneumonia, median nasopharyngeal s. pneumoniae load was substantially higher compared to healthy controls [ ] . pneumococcal density was also associated with severity of symptoms [ ] and increased duration of children's hospital stay [ ] . similar associations were observed in pneumonic adults, although the correlation was not significant in this population [ ] . the association between microbial load and clinical manifestations may depend on specific pathogen-host interactions. if pathogenesis is primarily related to microbial replication, a stronger correlation between microbial load and illness magnitude may be observed [ ] . if clinical manifestations are largely attributable to host immune defences or bacterial toxins, the correlation with microbial load may not be obvious [ ] . temporal variations in microbial load may also play an important role if the quantity of nucleic acid is significantly more abundant at the time and location of pathology [ , ] . in acute respiratory illness patients, high bacterial colonization densities are often associated with the presence of viral co-infections [ ] , and clinical manifestations may vary depending on specific co-infection patterns [ ] . the ecology of respiratory pathogens is also likely to be influenced by the living conditions in military settings. mixing of individuals from diverse backgrounds living in close-quarters with high levels of inter-personal contact increases the potential for introduction and spread of multiple microbes in this population, which could account for the broad range of organisms and co-detections in this study. we analysed both non-acute and acute samples from a closely monitored population in a semi-closed, longitudinal setting. the study population was well-defined and relatively homogeneous with regards to demographics and living conditions. however, our findings may not be applicable to populations with different socio-demographic characteristics and populations outside the military environment, such as cohorts of children among whom the impact of respiratory infections may be greater. the frequent co-detection of multiple respiratory agents and the failure to distinguish between viable and dead organisms, or microbes that colonize the host at sub-pathogenic levels, may prevent the unambiguous interpretation of test results [ ] . a positive result may indicate illness aetiology, asymptomatic colonisation, post-infectious shedding, or an incipient infection. therefore, ct-values may not always be a reliable surrogate for infectious load. samples from only two out of six cohorts were tested by real-time pcr. although there might be bias from seasonal effects, these are usually less pronounced in the tropics. given the relatively low frequencies of viral detection, a larger sample size and a longer follow-up may have captured a more precise picture of infection patterns in this population. this study was also limited to the detection of organisms contained in the respiratory panel. we cannot exclude the presence of additional organisms in our specimens. in addition, the data were obtained from throat and nasal swab samples, but our findings may not apply to nasopharyngeal or sputum specimens. finally, the quality and quantity of material obtained through nose and throat swabs may differ significantly among subjects, and the success of pcr-based methods also depends on the availability of intact genome sequences and the absence of random mutations. overall, the multiplex respiratory panel provided a comprehensive characterization of the microbe spectrum contained in non-acute and acute respiratory samples collected among recruits. however, semi-quantitative assessment of microbial load could not reliably distinguish between symptomatic and asymptomatic samples. more research is warranted to compare new multiplex diagnostic techniques with traditional methods and evaluate their potential with regards to diagnostic accuracy [ ] and clinical utility [ , ] in the context of respiratory infections. additional file : dataset. title of data: semi-quantitative microbial load in throat and nasal swab samples from thai army recruits. description of data: semi-quantitative microbial load in non-acute and acute throat and nasal swab samples from thai army recruits, determined using a commercial multiplex real-time pcr assay comprising bacterial, viral and fungal targets; includes names, labels, and coding for individual variables. (xls kb) abbreviations pcr: polymerase chain reactionuriupper respiratory illnessiliinfluenza-like illnessftdfast track diagnosticsctcycle thresholdhi-bhaemophilus influenzae type bhi-nonbnon-type b haemophilus influenzaeiqrinterquartile rangehmpvhuman metapneumovirus we are grateful to the participants of this study, the royal thai army, and the clinical, laboratory and administrative personnel at afrims. material has been reviewed by the walter reed army institute of research. there is no objection to its presentation and/or publication. the opinions or assertions contained herein are the private views of the authors, and are not to be construed as official, or as reflecting true views of the department of the army or the department of defense. this work was supported by the united states department of defense -global emerging infectious disease surveillance (dod -geis), protocol a. the datasets analysed for the current study are available as additional file in this publication. author's contributions cct conceived the idea for this paper, vo conducted the analysis and wrote the manuscript. ka, aw, lm, de, rr, and ss participated in project oversight. sf, rg, rr, ss, and iy participated in the design of the study. all authors contributed to drafting the manuscript and approved the final submission. the study was approved by the institutional review boards of the royal thai army in bangkok, thailand, the walter reed army institute of research and the london school of hygiene & tropical medicine. all participants provided written informed consent. the investigators have adhered to the policies for protection of human subjects as prescribed in army regulation - . not applicable. cct is associate editor for bmc infectious diseases, research area viral diseases. all other authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. author details saw 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community-acquired pneumonia dna bacterial load in children and adolescents with pneumococcal pneumonia and empyema comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia new concepts in diagnostics for infectious diarrhea high nasopharyngeal pneumococcal density, increased by viral coinfection, is associated with invasive pneumococcal pneumonia clinical characteristics of children with lower respiratory tract infections are dependent on the carriage of specific pathogens in the nasopharynx development of two real-time multiplex pcr assays for the detection and quantification of eight key bacterial pathogens in lower respiratory tract infections key: cord- -vjiecd k authors: ghosh, sabyasachi; rajwade, ajit; krishna, srikar; gopalkrishnan, nikhil; schaus, thomas e.; chakravarthy, anirudh; varahan, sriram; appu, vidhya; ramakrishnan, raunak; ch, shashank; jindal, mohit; bhupathi, vadhir; gupta, aditya; jain, abhinav; agarwal, rishi; pathak, shreya; rehan, mohammed ali; consul, sarthak; gupta, yash; gupta, nimay; agarwal, pratyush; goyal, ritika; sagar, vinay; ramakrishnan, uma; krishna, sandeep; yin, peng; palakodeti, dasaradhi; gopalkrishnan, manoj title: tapestry: a single-round smart pooling technique for covid- testing date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: vjiecd k the covid- pandemic has strained testing capabilities worldwide. there is an urgent need to find economical and scalable ways to test more people. we present tapestry, a novel quantitative nonadaptive pooling scheme to test many samples using only a few tests. the underlying molecular diagnostic test is any real-time rt-pcr diagnostic panel approved for the detection of the sars-cov- virus. in cases where most samples are negative for the virus, tapestry accurately identifies the status of each individual sample with a single round of testing in fewer tests than simple two-round pooling. we also present a companion android application byom smart testing which guides users through the pipetting steps required to perform the combinatorial pooling. the results of the pooled tests can be fed into the application to recover the status and estimated viral load for each individual sample. currently, the primary method for covid- testing uses viral rna extraction followed by rt-qpcr amplification of a conserved region of the genome of the virus. the throughput and capacity for such testing is severely limited. since covid- can be transmitted from asymptomatic carriers, these testing bottlenecks have left states with the dilemma of either risking a free spread of the virus, or imposing severe lockdown and physical distancing measures with heavy economic costs. one strategy many countries have adopted to increase testing capacity is to combine samples into pools that are tested together [ , , , , ] . if such a pool is tested negative, all individual samples within the pool are declared free of sars-cov- , whereas if it is tested positive then all individuals in that pool must be retested individually in a second round. while these strategies can augment testing capacities, they do not substantially improve throughput, since two rounds of testing are still needed to identify positive samples. a challenge that such simple pooling strategies have to confront is whether to first pool samples and then perform rna extraction on the pools, or whether to individually extract rna from each sample and subsequently pool the rna. if rna extraction is done individually for each sample, then the requirement to run a positive pcr control on the rp gene (confirming rna extraction worked) for every sample would nullify any gains from pooling. if rna extraction is performed after pooling samples, then the second round of testing would require a new round of rna extraction for all individual samples from positive pools, slowing down the process. nonadaptive testing allows a way past this dilemma, by pooling samples, extracting rna from pooled samples, and returning confirmed sample-level results in a single round of testing. here we introduce tapestry pooling, a novel pooling strategy that increases both capacity and throughput beyond simple pooling. tapestry pooling gives confirmed results in a single round of testing by testing each sample thrice as part of three different pools. the pooling design is chosen in a combinatorial manner to make decoding possible. the technique is quantitative, based on ideas from compressed sensing. the decoding algorithm takes as input cycle threshold values from qpcr tests on the pools, and returns a result for each sample, along with an estimate of viral load if positive. the number of tests required with tapestry pooling compares favorably with the two-round poolings that are currently deployed in the field with comparable levels of sensitivity and specificity (see table ). the solved viral load for each sample has to satisfy a quantitative consistency check. the sum of viral loads of samples in a pool must tally with the measured cycle threshold of the pool, with some allowance for noise. this quantitative reconstruction allows more information to be extracted from positive tests than from binary group testing approaches which take into account only whether a test is positive or negative. specifically, in the binary group testing situation a positive test for a pool with one known positive sample does not convey any information about other samples in that pool. this is not the case with tapestry pooling where the quantitative values of the test and the sample can be compared to reveal if there might be other samples in the pool that are positive. as a result, tapestry pooling continues to be viable not just with low (< %) prevalence rates, but even with moderate prevalence rates ( % - %), in which regime simple pooling does very poorly. our algorithm can estimate the number of positive samples in the population from the number of tests that came out positive. based on this sparsity estimator, the algorithm has a graceful failure mode in case the prevalence rate is far above the assumed prevalence rate for which the test was designed. in such a case, our algorithm will still maintain very high sensitivity, returning almost no false negatives. it will return a list of sure positives, and a list of suspected positives with an advice on how many of the suspected positives are in fact positive. this list-decoding approach makes our algorithm viable to be deployed with prevalence rates even as high as %. we have designed an android app byom smart testing to facilitate implementation of tapestry pooling in clinical laboratory settings. the app encapsulates all the complexity of our scheme, and presents a simple and easy to execute protocol to the testing lab, vastly increasing the deployability of this scheme. the app provides a sequence of instructions in the form of visual cues to aid and direct the pipetting of samples into appropriate pools. once pooled tests are run, the cycle threshold values can be entered into the app which solves for and displays the list of positive and negative samples along with an estimate of viral loads. in this work, we focus on pcr tests since it is currently the most widely used testing methodology for covid- . in principle tapestry pooling may also be applicable to other testing assays like serological antibody tests, both for covid- testing and other purposes. we first refined and validated tapestry pooling through computer simulations. we then validated tapestry pooling analytically in a lab setting with rna and dna fragments in blinded experiments. in cases where our simulations predicted that we would, with high likelihood, recover individual sample status, our experiments indeed successfully did so. we are currently testing tapestry with clinical samples. theoretical background: let the samples be numbered , , … , and indexed by . let the viral load of the i th sample be given by . suppose the tests are numbered , , … , and indexed by . let = ( ) × be a matrix with entries either or . then entry = means that sample is not present in test , and = means that sample is present in test (see fig a) . let be the quantitative measure of viral load in the j th test where = , , … , . then ∑ = where is a mean-zero gaussian random variable denoting noise in measurement of cycle threshold for the j th sample. since most samples tested will not have the virus, we want to reconstruct a sparse (most entries ) nonnegative vector x that satisfies this noisy linear equation. this problem has been studied in signal processing literature under the name of compressed sensing [ , , ] , though the combination of a being a sparse matrix with - entries, x being nonnegative, and the multiplicative noise model is unique to this particular situation and has prompted algorithmic innovations on our part. we have explored various ways to design the matrix a including sparse expanders, explicit optimization of a loss function in a gradient descent and simulated annealing fashion, and finally using steiner triples. we find steiner triples particularly powerful and convenient for this application. in matrices constructed from steiner triples, each sample goes into pools. this makes a a very sparse matrix which has advantages for pipetting and keeps pool sizes manageable. further any two columns of a have dot product no more than ---in other words each pair of samples occurs together in no more than one test. this property ensures good reconstruction guarantees for x. further details of our decoding algorithms will be described in an upcoming theory paper. experimental methods rna and dna amplicons: a single stranded rna fragment of length kb (stock concentration ng/µl) was used as a proxy for viral rna for preliminary testing. rna fragments were diluted to clinically relevant concentrations of . pg/µl (~ copies/µl), fg/µl (~ copies/µl) and . fg/µl (~ copies/ µl) by serial dilution from the stock. we also tested our scheme with a circular dna plasmid containing the complete nucleocapsid gene from the sars-cov- virus (integrated dna technologies, -ncov_n_positive control, catalog # ). dna plasmid was diluted to clinically relevant concentrations of copies/µl and copies/µl. pooling: two pooling matrices, a × (tests × samples) and a × (tests × samples), were tested in blinded experiments where the identity and viral load of the individual positive samples were not a priori revealed to the decoding team. only the ct values corresponding to the various pooled qpcrs were communicated. the × matrix (see fig b) directs that each sample be distributed, and hence tested, two or three times while the number of samples per pool vary from six to nine, with a median pool size of seven. the × matrix was tested for five different situations, corresponding to starting with zero positive samples, one positive sample ( copies), two positive samples ( copies each), three positive samples (two with and one with copies) or four positive samples (two with , one with and one with copies). sample pools containing one or more positive samples were emulated by spiking with appropriate amounts of rna fragments to simulate the viral load after pooling. mouse rna (~ pg per reaction) was added as background to simulate the expected clinical sample matrix. a total of such sample pools were generated for downstream amplification. the many sample pools containing no positive samples were emulated by assuming that their ct values were normally distributed with a mean of . and a standard deviation of . , a distribution indicated by running qpcr experiments with no amplifiable templates, only background mouse rna. the × matrix (see fig c) directs that each sample be distributed, and hence tested, two or three times while the number of samples per pool is either six or seven with a median pool size of seven. we tested a situation corresponding to two positive samples ( copies and copies of -ncov_n_positive control) and negative samples ( aliquots of nuclease free water in separate tubes). the mock samples were variously distributed to generate sample pools for downstream amplification. qpcr amplification from rna templates: cdna was synthesized from rna templates using first strand invitrogen superscript ii system (invitrogen, catalog # - ). a mix of rna and a genespecific reverse transcription primer were denatured at °c for minutes and annealed at °c for minutes prior to the addition of the other reaction components. after the other components were added, the reverse transcription reaction was carried out for hour at °c and was followed by a heat inactivation step for minutes at °c. for cdna templates, qpcr was performed using the thermofisher sybr green mastermix system (thermo scientific, catalog #k ). a µl qpcr reaction was set up using cdna template ( . µl) and forward and reverse primers ( . µm each). each µl reaction was distributed equally as technical replicates of µl in an optical well plate as technical replicates. the thermocycling conditions were as follows: °c denaturation step for minutes followed by cycles of °c for seconds, °c for seconds and °c for seconds. the specificity of the initial test reactions were verified by primer melt curve analysis and analysis of the amplicons using agarose gel electrophoresis. qpcr amplification from dna templates: dna templates were amplified with taqpath -step rt-qpcr master mix, cg (thermofisher; catalog #a ) using the u.s. cdc designed n primer and taqman probe set (idt; -ncov ruo kit, catalog # ). the rt step was skipped during thermocycling, as we started with a dna template. the thermocycling conditions were as follows: °c denaturation step for minutes followed by cycles of °c for seconds, °c for seconds and °c for seconds. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . × pooling matrix: table shows the emulated cycle threshold (ct) values for the rt-qpcr tests corresponding to five different trials ( , , , or positive samples out of a total of samples). fig a shows the corresponding amplification curves. table shows the ground truth rna amounts for each of the samples and compares them to the values estimated using the tapestry decoding algorithm. we find that for , and positives (for which simulations suggested the matrix would perform well with high likelihood) the algorithm correctly identified the status of all samples with zero false negatives and zero false positives. for or positives, as expected, our algorithm returned zero false negatives, but some false positives. however, all the false positives are correctly identified as having a low likelihood of being a true positive, and are placed in the set of unsure positives. all sure positive and sure negative sets returned by the algorithm were correct. × pooling matrix: table shows the cycle threshold (ct) values obtained for the rt-qpcr tests corresponding to a trial with positive samples out of a total of samples. fig b shows the corresponding amplification curves. table shows the ground truth dna amounts for each of the samples and compares them to the values estimated using the tapestry decoding algorithm. the algorithm correctly identified the status of all samples with zero false negatives and zero false positives. group testing and its application to medical diagnosis by pooling goes back to a paper by dorfman [ ] . the application of compressed sensing to group testing in the presence of noise was introduced by atia and saligrama [ ] . two exciting preprints proposing applications of compressed sensing ideas to covid- testing with real-time rt-qpcr have appeared online in the last few days [ , ] . similar to our work, both these approaches argue that nonadaptive pooling has substantial advantages over adaptive pooling in the regime of low prevalence rates by enabling single-round testing of many samples with fewer tests. while [ ] is a theoretical proposal, [ ] has put out proof-of-concept studies with covid samples. we now compare our scheme with the schemes proposed in [ , ] , and point out some possible advantages our scheme may have. . sparse pooling matrix: the pooling matrix a in [ ] is based on reed-solomon error correcting codes. the pooling matrix in [ ] is based on random matrices. our pooling matrix is based on combinatorial designs known as steiner triples. each sample in our scheme goes to pools, as opposed to pools in [ ] which cuts down our pipetting time by half. in simulation experiments, we also find our matrices performing better than the other two in terms of sensitivity and specificity. . noise model: we propose an explicit noise model where cycle thresholds are measured with additive gaussian noise, leading to a multiplicative noise on the reconstructed vector y. such explicit noise models are not considered in [ , ] . . bespoke reconstruction algorithm: because of the unique combination of sparse - pooling matrix, multiplicative noise model, nonnegative x vector, and one-sided error seen in this problem, we have modified existing reconstruction algorithms and obtained a corresponding improvement in performance. . sparsity estimator and graceful failure: any pooled testing scheme that takes prevalence rate as input runs the risk that the input prevalence rate may grossly underestimate the true prevalence rate, especially as the disease progresses in a population. it is important to design the scheme so that in such settings, it can (a) recognize that it is operating in a regime where the true prevalence rate exceeds the prevalence rate it was designed for, (b) maintain very high sensitivity of the test, (c) return a list of "suspected positives" and an estimate of how many of these suspected positives are truly positives. our algorithm is able to achieve all three objectives. we employ a sparsity estimator that achieves (a). we design our recovery algorithm to guarantee (b) and achieve (c). . deployment challenges: it is clear that nonadaptive strategies, for all their advantages, are in general more complex than simple pooling strategies. this raises real questions about whether they can be deployed effectively in the real world with so many moving parts. a big part of answering such a question is coming up with a way to encapsulate the complexity so that at the end of the testing laboratory the protocol to perform is made as simple as possible. we have achieved this with our android app byom which gives clear instructions for pippetting into pools. it receives cycle threshold values, and solves for the viral loads in the cloud, and returns a list of the positive, suspected positive, and negative samples. we have demonstrated a method for creating multiple pools from a given set of samples, in such a way that by testing the pools in a single round of rt-qpcr we can reconstruct the individual viral loads of each sample with high sensitivity and specificity. the method is designed to produce zero false negatives, and with a false positive rate that can be traded off with the overall prevalence rate that the method can handle. in general, the method works best for low prevalence rates ( - % depending on the number of samples). for and samples with up to positives, we have shown using mock samples (a kb single stranded rna fragment and a circular dna plasmid containing the complete nucleocapsid gene from the sars-cov- virus) that the method works with zero false negatives and zero false positives. if the prevalence rate happens to be higher than expected, for example with samples and or positives, the method fails gracefully -that is, it still provides sure positives, sure negatives and unsure positives. moreover, the method can suggest the minimum number of new tests that need to be done to resolve the unsure casesthus the information from the first tests is not wasted. our experimental and theoretical validation of the method suggest several use cases for tapestry pooling: currently a single -well pcr run can only test individuals. tapestry pooling thus provides more than % saving, with zero false negatives and vanishingly small false positives for upto % prevalence rate. safe enough for treatment to be based on the test. this works for prevalence rates upto % with a single pcr run, providing zero false negatives and slightly higher false positives. this would therefore be appropriate for screening of asymptomatic subjects, such as hospital staff, cisf guards at airports, police personnel, delivery persons, entire apartment blocks where one person is found positive -other high-risk populations who are potential super-spreaders can be tested everyday. this is appropriate for larger groups of asymptomatic subjects, such as whole neighborhoods or large hospitals or passengers arriving at an airport. it requires two technicians for pipetting. false . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . negatives remain zero, and false positives are small for prevalence rates upto . %. if the prevalence rate is higher the algorithm has a graceful failure mode. this provides massive savings in kits, rna and pcr effort and is appropriate for populations with low prevalence rate of around %, for example passengers arriving at an airport, or testing in high population density neighborhoods. the larger amount of pipetting would require liquid handling robots. the pool size is also bigger, and experiments with real samples are needed to quantify the false negatives that might arise from such large dilution. as mentioned in the last use case, experiments are required with real samples to quantify the extent to which dilution in large pools introduces false negatives, which is necessary for calibration of our quantitative method for real-world cases, and to validate the method for clinical samples using a double-blind protocol. these experiments are ongoing. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . table . b. amplification curves corresponding to pooled tests for the × matrix. the corresponding ct values are in table . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . table . comparison of one-round tapestry pooling to two-round dorfman pooling. tapestry not only increases throughput by providing results in one round but can also reduce the required number of tests. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . fig. a . the many sample pools containing no positive samples were emulated by assuming that their ct values were normally distributed with a mean of . and a standard deviation of . , a distribution indicated by running qpcr experiments with no amplifiable templates. the algorithm was blinded to the status of the samples and only received ct values for each trial, and did not have any knowledge of which ct values were from an actual an rt-qpcr run. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . threshold not reached t threshold not reached t threshold not reached t threshold not reached t threshold not reached t threshold not reached t threshold not reached t threshold not reached t . t threshold not reached t threshold not reached t . t threshold not reached t threshold not reached t . t threshold not reached t threshold not reached t threshold not reached t threshold not reached t threshold not reached t threshold not reached t threshold not reached fig. b . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint . place an empty . ml well pcr reaction plate (or individual . ml tubes) on a -well cold block/ ice and prepare to pool samples into its wells. serially number the samples as sample , sample , etc. . starting from sample , the app will instruct the user to pipette the rna extracted from the patient samples into specified wells (indicated by flashing orange dots) of the pcr reaction plate. the grey wells indicate all the wells that will be used for the pools you may pipette any volume (we recommend between ul and ul) as long as you are consistent across all samples and have enough pooled volume in each well to perform the downstream real time rt-pcr test. clicking the next button at the bottom of the screen or swiping to the left brings up instructions for the next sample to be pipetted, while clicking previous or swiping to the right brings up instructions for the previous sample that was pipetted. alternatively, pipetting instructions are also available on the app as downloadable pdfs. . once all samples have been pipetted, the app will return to the home screen and show a success message (e.g. samples collected). the pcr reaction plate must then be tightly sealed to prevent sample loss and spun down for seconds in a plate centrifuge to collect all liquid at the bottom. the pcr reaction plate must then be gently vortexed for seconds to ensure the liquid in each well is uniformly mixed. finally, the pcr reaction plate is again spun down for seconds in a plate centrifuge to collect all liquid at the bottom. . the pooled rna samples in the pcr reaction plate are now ready for real-time rt-pcr tests. perform all steps (rna extraction, pcr setup etc.) according to the instructions of the real-time rt-pcr diagnostic panel that you will use, but using pooled samples instead of individual samples. in a typical pcr reaction, ul of the pooled samples will be used in a ul rt-pcr reaction. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . for a test that was negative, you don't need to uncheck the check box. click submit. the app will decode and display results for each individual sample. a list of positive samples, and a list of "possibly positive samples" will be displayed. all other samples are negative. . cc-by-nc-nd . international license it is made available under a is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. (which was not certified by peer review) the copyright holder for this preprint this version posted april , . . https://doi.org/ . / . . . doi: medrxiv preprint evaluation of covid- rt-qpcr test in multi-sample pools assessment of specimen pooling to conserve sars cov- testing resources low-cost and high-throughput testing of covid- viruses and antibodies via compressed sensing: system concepts and computational experiments rapid, large-scale, and effective detection of covid- via non-adaptive testing efficient high throughput sars-cov- testing to detect asymptomatic carriers the detection of defective members of large populations for most large underdetermined systems of linear equations the minimal -norm solution is also the sparsest solution stable signal recovery from incomplete and inaccurate measurements regression shrinkage and selection via the lasso boolean compressed sensing and noisy group testing • nasopharyngeal swabs stored in viral transport medium (henceforth referred to as sample.) • all patient specimens and positive controls should be considered potentially infectious and handled accordingly. • do not eat, drink, smoke, apply cosmetics or handle contact lenses in areas where reagents and human specimens are handled. • handle all specimens as if infectious using safe laboratory procedures. • specimen processing should be performed in accordance with national biological safety regulations. • use personal protective equipment such as (but not limited to) gloves, eye protection, and lab coats while performing this assay and handling materials including samples, reagents, pipettes, and other equipment and reagents. key: cord- -kji kfek authors: chakraborty, nabarun; schmitt, connie w.; honnold, cary l.; moyler, candace; butler, stephen; nachabe, hisham; gautam, aarti; hammamieh, rasha title: protocol improvement for rna extraction from compromised frozen specimens generated in austere conditions: a path forward to transcriptomics-pathology systems integration date: - - journal: front mol biosci doi: . /fmolb. . sha: doc_id: cord_uid: kji kfek at the heart of the phenome-to-genome approach is high throughput assays, which are liable to produce false results. this risk can be mitigated by minimizing the sample bias, specifically, recycling the same tissue specimen for both phenotypic and genotypic investigations. therefore, our aim is to suggest a methodology of obtaining robust results from frozen specimens of compromised quality, particularly if the sample is produced in conditions with limited resources. for example, generating samples at the international space station (iss) is challenging because the time and laboratory footprint allotted to a project can get expensive. in an effort to be economical with available resources, snap-frozen euthanized mice are the straightforward solution; however, this method increases the risk of temperature abuse during the thawing process at the beginning of the tissue collection. we found that prolonged immersion of snap frozen mouse carcass in % neutral buffered formalin at °c yielded minimal microscopic signs of ice crystallization and delivered tissues with histomorphology that is optimal for hematoxylin and eosin (h&e) staining and fixation on glass slides. we further optimized a method to sequester the tissue specimen from the h&e slides using an incubator shaker. using this method, we were able to recover an optimal amount of rna that could be used for downstream transcriptomics assays. overall, we demonstrated a protocol that enables us to maximize scientific values from tissues collected in austere condition. furthermore, our protocol can suggest an improvement in the spatial resolution of transcriptomic assays. the value added from big data is in generating information and, ultimately, knowledge by integrating a large amount of reads derived from multiple disparate assay types (marx, a; andreu-perez et al., ; gligorijević et al., ; huang et al., ) . this is due to the distinct data types that may require different and incompatible sample processing methods. it can be difficult to obtain all the desired data from a single specimen (bean et al., ; yan et al., ) . moreover, analyzing similar but not identical specimens can lead to spurious results in this type of multi-modal data acquisition (bean et al., ; gligorijević et al., ) . for example, dissecting a partially damaged whole tissue into multiple fragments for gene expression and histopathological assessment may confound the analysis, if a pathological lesion does not extend uniformly across all the fragments, or if there are subtle spatial variations in the tissue, as has been shown for tumors (yan et al., ) . this drawback attributed to the intrinsic heterogeneity of tissues that can potentially be mitigated by integrating the omics data with other "conventional" readouts (ellinger-ziegelbauer et al., ) , such as histopathology analysis. an omics-pathology integration approach could be the most effective for phenometo-genome interpretation if omics assays are conducted using the particular tissue specimen, where injury signatures are informed by histopathology image analysis (pathak and dave, ; yu et al., ) . this approach would be an ideal phenome-togenome approach, where omics readouts could be directly informed by the visualized phenotypes (yee, ; gligorijević et al., ) . in such an instance, there is an obvious and clear advantage in recycling the same tissue specimens for both gene expression and histopathology image analyses. multiple assay data generated from the same tissue specimen will improve spatial resolution of tissue's molecular landscape, enhance data integrative structure, and reduce biases in generating multidimensional information, which is the goal of making big data free from false results (marx, b; andreu-perez et al., ; gligorijević et al., ; huang et al., ) . there is a paramount need for a platform that is capable of deriving maximum information from a minimum amount of samples in space biology, as space-flown samples are scarcer. moreover, samples harvested from space-flown specimens provide uniquely valuable insights and should be exploited by multi-dimensional genome-to-phenome analysis. handling animals in microgravity is immensely challenging, which is further aggravated when the animals need to be operated on using an aseptic technique globus et al., globus et al., , . based on our own experience (dadwal et al., ) , the estimated time for handling each mouse on the international space station (iss) was five to six times longer than that required on the ground. in addition, investigators need to make every effort to minimize the physical footprint taken on a spaceflight by a given specimen and its handling equipment. to satisfy all of these constraints, freezing the animal carcass in space without any further dissection was considered the most prudent compromise. these snap-frozen carcasses had to undergo onground freeze-thaw cycles before tissue collection, which is typically expected to compromise overall sample quality and make the histopathologic analysis challenging (lyons et al., ; pikal-cleland et al., ) . increased level of interest is evident in recent years to integrate the omics data with histopathology readouts (brenna et al., ; murata et al., ) ; however these studies were typically at a risk of specimen or animal bias, since different cohorts of animals were used for omics and pathology assays, respectively. acknowledging this knowledge gap, several techniques have been developing to improve spatial, cellular, and sub-cellular resolution of transcriptomics readouts (grün and van oudenaarden, ; mignardi et al., ; dewez et al., ) . working on a similar objective, present manuscript demonstrates a protocol optimized for handling frozen samples. notably, there are several tissue preservation protocols and agents available for long term storage of biomolecules, such as rna, dna, and proteins (florell et al., ; vincek et al., ) ; however, we plan to snap freeze the tissue without using any preservatives to best simulate those austere situations (dadwal et al., ) , where various preservatives are often unacceptable owing to their levels of toxicity or viscosity or difficulty in general handling. in this context, we proposed three strategies to thaw pre-frozen specimens. we present all three strategies, as well as our recommendations after considering the advantages and disadvantages associated with each individual method. finally, we utilized recently available commercial kits to extract rna from fixed, processed tissue slices mounted on standard glass histology slides. altogether, this work is a demonstration of an optimized protocol for working with frozen samples of limited quantity collected in austere conditions. all animal experiments were approved by the institutional animal care and use committee (iacuc) at the us army center for environmental health research (usacehr), ft. detrick, md, and were performed in a facility accredited by the association for the assessment and accreditation of laboratory animal care international (aaalac). c bl/ j mice, aged weeks, were purchased from jackson labs, me. three animals were euthanized with an injection of ketamine-xylazine ( / mg/kg of mouse bodyweight). immediately after euthanasia, carcasses were transferred to a − • c freezer to simulate freezing of tissues during a space flight. all the mice carcasses were stored in the same box. after days, the carcasses were removed from the freezer, and one of the three thawing strategies was applied. thawing strategy one: incremental dry warming method following a -days holding period at − • c, three mouse carcasses were transferred to a − • c freezer and held overnight to incrementally thaw the tissues. direct transfer from one figure | flow diagram overview of the three thawing strategies, namely, strategy one, incremental dry warming method; strategy two, cold formalin immersion method; and strategy three, warm saline immersion method. nbf refers to neutral buffered formalin. freezer to the other was crucial to prevent excessive thawing of the carcasses at room temperature. the following morning, the carcasses were transferred from the − • c freezer to a • c ice bucket. they were then closely monitored for - min until sufficiently thawed to perform necropsy. for necropsy, the carcass should ideally have a firm consistency to allow proper handling of internal organs, but not have thawed to the extent that the tissues are too soft to maintain integrity during handling. following thawing, a comprehensive necropsy was performed. following a -days holding period at − • c, three mouse carcasses were placed in % neutral buffered formalin (nbf) solution at • c. after - min in % nbf, the carcasses were partially thawed. the carcasses were briefly removed from the % nbf to a sterilized plate, and a ventral midline incision was made through the skin and abdominal wall to facilitate penetration of the fixative into the abdominal cavity and internal organs. after making the midline incision, the carcass was returned to the • c % nbf solution for an additional h. after the h elapsed, the carcass was removed from the fixative, and a comprehensive necropsy was performed. a ml container was filled with ml of . % saline solution (sodium chloride, fisher scientific, pittsburgh, pa, stock solution) at • c. following a -days holding period in − • c, three mouse carcasses were submerged in the warm (i.e., • c) saline solution for min to facilitate thawing. upon removal from saline solution, a comprehensive necropsy was performed. post-thawing, the following tissues were collected: lacrimal gland, eye, brain, tongue, salivary gland, mandibular lymph node, nasal cavity, trachea, esophagus, heart, aorta, thymus, axillary lymph node, lungs, liver, gallbladder, pancreas, spleen, adrenal gland, kidney, mesenteric lymph node, inguinal lymph node, urinary bladder, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, skeletal muscle, sternum, peripheral nerve, cervical, thoracic, and lumbar spinal cord. these tissues were preserved in % nbf (fisher scientific, pittsburgh, pa). a subset of these tissues were utilized for subsequent downstream analysis ( table ) . the necropsy procedure used for tissue collection was consistent across all three thawing strategies. following necropsy and fixation, tissues received a gross inspection, placed in tissue cassettes (fisher scientific, pittsburgh, pa), and routinely processed. in detail, tissues with micron thickness were paraffin-embedded, sectioned, mounted, stained with hematoxylin and eosin (h&e), followed by the application of a coverslip, and then allowed to dry at room temperature prior to microscopic evaluation (dey, ) . all tissues were evaluated microscopically by a board-certified veterinary pathologist using representative h&e stained tissue sections. the tissues were subjected to two techniques of rna extraction from the histopathology slides. these techniques are described in the sections that follow. tissue sequestration strategy one: xylene at • c with liquid n tissue was isolated from the processed slides by incubating them in xylene (sigma-aldrich, saint louis mo) and using a wheaton slide preparation system glass staining dish in a biological safety cabinet at room temperature. different lengths of incubations of slides in xylene at • c were tried (starting at min, then min more and finally an overnight incubation) separated by incubations at room temperature for min without xylene. this was combined with removal strategies using (i) a razor or scalpel blade and (ii) encasing slides in styrofoam and repeatedly dipping them in liquid nitrogen to coax the tissue specimen off via cold snapping. tissue sequestration strategy two: xylene at • c all histology slides were deparaffinized using xylene (sigma-aldrich, saint louis mo). the slides were submerged in xylene in a wheaton slide preparation system glass staining dish and frontiers in molecular biosciences | www.frontiersin.org covered. the samples in the staining dish were placed into a biological safety cabinet for subsequent steps. the slides were shaken in a • c incubator shaker (boekel industries inc.) for min. to remove the samples from the glass slides, we used a single-edged industrial razor blade made from surgical carbon (international west chester, pa). after the incubation period, we used the sterile single-edged razor to remove the sample from each slide. rna was isolated using the allprep dna/rna ffpe kit (qiagen, carlsbad ca) as per the vendor's recommendations. the rna concentration was measured using a thermo scientific nanodrop spectrophotometer (waltham, ma), and rna quality was assessed using the agilent tapestation (santa clara, ca). using a qubit fluorometer (invitrogen) a high sensitivity assay was conducted to further validate the concentration. after evaluation, the rna was stored at − • c. freezing artifacts is characterized by the disruption of cellular membranes or tissues due to the formation of ice crystals within intra-or extracellular spaces. one of the most common causes of freezing artifacts is temperature abuse, which occurs when fixed wet tissue is allowed to freeze, thaw, and refreeze, fluctuating repeatedly above and below the freezing point of the fixative (xiong, ) . for example this may result when tissue is held in a cooler that fails to maintain a constant temperature above the freezing point of the fixative adams, . moreover, distortion or damage to cellular constituents caused by the ice crystal formation and moisture migration can confound the omics evaluation of tissues. for the present protocol, mice were flash frozen; thus, the initial risk of temperature abuse was potentially averted. minimization of temperature abuse during the thawing process remained our primary challenge. in this study, we compared the efficacy of three different methods of thawing frozen mouse carcasses. criteria of success included: (i) tissue quality-the overall quality of the tissues, any sign of damages or lesions were initially inspected by a board-certified veterinary pathologist through a microscope. figures - displays the representative pictures of the tissues with two different magnifications, respectively. furthermore, we evaluated the qualities of rna extracted from these individual tissues. rna integrity numbers (rin) and dv (i.e., the percentage measure of rna fragments > nucleotides) were used as the benchmarks to determine rna quality and in retrospect, the tissue quality. (ii) time expended with particular focus on the hands-on time during the thawing process, and (iii) implementation ease, which was primarily dependent on subjective evaluation by our technical staff. all three thawing strategies yielded similar results in terms of final tissue quality (see figures a-c , a-c, a-c). no damages or lesions were found. table describes the qualities and quantities of rna samples extracted from the tissues, which was discussed in detail in the subsequent section evaluation of the sequestration processes. in terms of time expended for each strategy, strategy three, warm saline immersion, had the quickest overall turnaround time, requiring only min per carcass for the thawing process with a minimal handling time of < min prior to the start of the necropsy procedure. strategies one and two, required significantly more overall time for the thawing process, . and h, respectively, but only about min of actual hands-on time per carcass during the process. once thawing was complete, the total necropsy time (i.e., ∼ min), was similar for all three strategies. in terms of implementation ease, strategy one, incremental dry warming, resulted in a dry, thawed, unfixed carcass which was relatively fragile when compared to a routine fresh carcass. tissues were susceptible to artifactual damage, such as tearing of skin and tubular organs as well as whole-organ breakage, during examination and handling using normal necropsy instruments. extra care during tissue handling was needed to avoid tissue damage, and to retain whole-organ tissue architecture for handling, collection, and processing prior to fixation. in contrast, strategy two, cold formalin immersion, resulted in a wet, thawed, fixed carcass that retained solid tissue architecture and was resistant to introduction of damage during examination and handling using normal necropsy instruments. starting the necropsy with a thawed, fixed carcass allowed for relatively uncomplicated tissue handling and collection prior to further processing. strategy three, warm saline immersion, resulted in a wet, thawed, unfixed carcass which most closely resembled that of a routine fresh carcass compared to the other two strategies. however, wet, warm, unfixed tissues became soft, which allowed for increased water content, making them susceptible to damage such as shearing or tearing of solid organs, and ripping of skin and tubular organs during examination and handling using normal necropsy instruments. extra care during tissue handling and collection was required to avoid tissue damage and loss of tissue architecture prior to fixation. subsequently, all the tissues of interest were fixed in % neutral buffered formalin, grossed, placed in cassettes, and then routinely processed, sectioned to a thickness of microns, and stained using hematoxylin and eosin (ellinger-ziegelbauer et al., ) . next, we selected those tissues, such as bone and muscle, which are typically challenging tissues to extract rna from. the primary challenge in this tissue sequestration protocol was optimizing an effective method to recover tissue sections from a prepared and stained glass histopathology slide. two methods for removing tissue from the slide using xylene were tested. for the first method, the slides were incubated with xylene at • c over multiple incubation periods; despite these incubations as well as cold snapping, we were unable to separate the tissue from the slide. hence, this tissue sequestration strategy was abandoned. the second method was successful; it involved incubating the slides with xylene at a higher temperature, specifically at • c. rna was extracted from the tissue specimens sequestered by the second method, and table shows the qualities and quantities of these rna samples. the rna qualities and quantities were comparable across three thawing strategies. the rin values were consistently around and dv values were lower than ; and these suboptimal scores could be attributed to the compound effects of several factors, including the compromised tissue preservation method and the effect on rna quality due to nbf (cross-linking and direct reaction with nucleotides; cox et al., ) . by certain account, the dv score is the fittest metric to decide upon the appropriateness of the samples to run for gene expression analysis (matsubara et al., ) ; while the prevalent thought is to consider both rin and dv to finalize this decision. using rna samples with rin score > . , ravo et al. claimed a complete success in microarraybased cdna-mediated annealing, selection, extension and ligation (dasl) assay; furthermore, these authors achieved similar success in dasl assays using samples with rin < . but the majority of rna size was > nt (ravo et al., ) . this concept was adapted to achieve an uniform gene sequencing coverage using rna samples with rin scores between . and . (cieslik et al., ) . other study reported nearly % mapping rate to human genome using rna sample with rin and dv scores equaled to . and , respectively (lin et al., ) . in this context, a number of commercial kits have been evaluated to score their merits in sequencing low quality rna samples (adiconis et al., ; kresse et al., ; masters et al., ; lin et al., ) and these feedbacks could be used as the benchmark for effectively analyzing the compromised rna specimens. selected from this pool of kits, we have successfully used nugen kit (nugen technologies, san carlos, ca), and systems biosciences kits (systems biosciences, mountain view, ca) to enrich and amend severely compromised rna specimens collected from the space-flown cells to produce cdna microarray data (chakraborty et al., ) . taking together, we believe that the rna specimens collected from the present methods are eligible to be treated by these commercial kits for successful gene expression analysis. present approach has certain drawbacks. the design of this study was to simulate austere conditions in a space environment by snap freezing the carcasses without accessing any preservation agents and subjecting the samples to freeze-thaw cycles that would imitate sample processing during and after space flight. actual space flight might display conditions not completely simulated by our model. nevertheless, this model provides a reliable and reproducible analysis of plausible methods to best represent how the scientific data can be accurately captured. our team has had previous exposures with space flight-related experiments (dadwal et al., ) that supplied them with a breadth of virtues and pitfalls that could be realistically associated with these methods in an actual space flight experiment. the sample size of the present study (n = ) was small but statistically viable. the histopathology slides were inspected by single boardcertified pathologist, which is an accepted practice to assess quality of the histomorphology of a slide in both diagnostic and toxicological pathology settings. furthermore, nbf is the sole source of fixative agents approved by our institute, hence present protocol precluded from testing the efficacy of any other alternatives (kiernan, ) . in conclusion, this protocol could be a paradigm shift from the typical transcriptomics assay that use homogenized tissue specimens and thereby are often criticized to miss the localized signals of stress (mignardi et al., ) . here we presented a scope to inspect the "localized signal" of partially damaged tissue through histopathology image analysis, and let the h&e information guide in selecting the most suitable tissue specimen for omics study. our technology will be greatly beneficial when tissues are collected from austere or unique conditions such as spaceflight, war theater and pandemic centers (such as the present covid- situation), where we cannot afford to obtain multiple samples, but multiple assays are necessary to comprehend the holistic picture. herein our method will inform how a single tissue can be sequentially used for multiple assays. certainly, the motivation for this study was to develop a protocol best suited for processing frozen carcasses originating from the iss; however, we can foresee that our final deliverables have potential applicability to additional research interests. all datasets generated for this study are included in the article/supplementary material. the animal study was reviewed and approved by iacuc committee of usacehr. material has been reviewed by the walter reed army institute of research. there is no objection to its presentation and/or publication. the opinions or assertions contained herein are the private views of the author, and are not to be construed as official, or as reflecting true views of the department of the army or the department of defense. research was conducted under an approved animal use protocol in an aaalac accredited facility in compliance with the animal welfare act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the guide for the care and use of laboratory animals, nrc publication, edition. nc and rh conceived the idea. cs and ch conducted histopathology study. ag, sb, hn, and cm conducted molecular assays. ag, hn, and nc carried out analysis. nc and hn drafted the text. everyone read, edited, and approved the report. all authors contributed to the article and approved the submitted version. this work was also supported by the military operational medicine research program (proposal # ). any citations of commercial organizations and trade names in this report do not constitute an official department of the army endorsement of approval of the products or services of these organizations. cs and ch wish to acknowledge the technical staff of the comparative pathology department, us army medical research institute of chemical 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reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -ufvq ngl authors: sharma, r.; goyal, s.; bist, p. title: optimal sample pooling: an efficient tool against sars-cov- date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: ufvq ngl the sars-cov- pandemic situation has presented multiple imminent challenges to the nations around the globe. while health agencies around the world are exploring various options to contain the spread of this fatal viral infection, multiple strategies and guidelines are being issued to boost the fight against the disease. identifying and isolating infected individuals at an early phase of the disease has been a very successful approach to stop the chain of transmission. but this approach faces a practical challenge of limited resources. sample pooling solves this enigma by significantly improving the testing capacity and result turn around time while using no extra resources. however, the general sample pooling method also has the scope of significant improvements. this article describes a process to further optimize the resources with optimal sample pooling. this is a user-friendly technique, scalable on a national or international scale. a mathematical model has been built and validated for its performance using clinical data. the rapid rise in sars-cov- positive cases poses a significant health threat to the world. in india, the case count is surging as government has announced significant relaxations in curbs for unlock . identifying and isolating the positive cases in the very early phase has been a major successful approach to stop the spread of the disease. , to identify such cases, the world health organization has stressed on multiple occasions the significant role of sample testing. , yet unfortunately, in the testing strategies of majority of the nations, either only symptomatic cases are included or are heavily favoured. with such approach, the patient count tracker will always find itself trailing the steps of the viral spread. multiple scientific studies state that the disease can lay asymptomatic for the first to weeks, immediately followed by the period of heavy viral shedding just as symptoms onset. [ ] [ ] [ ] [ ] [ ] [ ] [ ] thus, testing all of the suspected individuals will provide an ideal solution. however, a major challenge in implementing such approach is resource limitation. many strategies have been identified to release the constraint of resource limitation. one of such tried and tested approach is sample pooling. [ ] [ ] [ ] [ ] [ ] [ ] indian council of medical research (icmr) and other world health agencies have also suggested sample pooling as a major strategy to increase the scope of the test. , this strategy helps in multiple ways, it reduces both cost and result time. sample pooling can be achieved primarily in two ways: repeated pooling and one-time pooling. in repeated sample pooling, a sample pool which has tested positive is further broken down into sub-pools and the testing process is repeated till each of the individual samples with positive sars-cov- infection is identified. this method turns out to be more time and cost efficient as it can be designed by using algorithms like binary search tree. however, this method has serious practical limitations as -a) it is a complex procedure and requires training to implement and b) it shows significantly higher efficiency when the initial pool size is high. in contrast, during one-time pooling, only one level of pooling is done. if a pool tests positive, in the next step, each individual sample of the pool is tested for the infection. hence, one-time pooling is a more practical approach than the repeated pooling. while the current guidelines from icmr state that up to samples can be pooled, multiple studies have confirmed that the pooling size of up to does not harm the specificity and the sensitivity of the test. the latest guideline has left it up to the lab to decide the optimal pool size. multiple tools have been reported in the scientific community to find the optimal pool size. while most of such tools consider multiple parameters to arrive at the optimal pool size, complicated procedures avert the practical use. the current paper proposes a practical approach to find the optimal pool size. this approach is simple, easy to implement on a national scale and is adaptable to the stage of the viral spread. testing facilities are either already designated to test samples from a specific geography or can be easily mapped. this correlation ensures that the prevalence rate of the lab remains a continuous function. sample data verifies this hypothesis. the temporal variance in the prevalence rate is significantly lower for a testing facility level than at a country or even at a state level where the variance is extremely high. hence, in order to determine the current prevalence rate and to forecast the same, the metrics should be calculated for individual testing facilities. the determination of sample pool size for each lab using its prevalence rate would yield desired efficiency and be easy to implement. following mathematical model describes the gain factor of the sample pooling against individual testing of each sample. involved parameters are, current prevalence rate, the probability of a sample being positive is forecasted using the actual prevalence rates of the last one week, p samples to be tested, n sample pool size, x (min - , max - ) no of tests without sample pooling, n normal = n, no. of tests after sample pooling, gain factor, using equation ( ), gain factor values for different sample pool size and varying prevalence rate are simulated (table ) . was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted july , . . in the situation of prevalence rate of % or lower, though higher sample pool size yields better gain factors, yet the risk of sample dilution outweighs the miniscule gains. thus, the sample pool size of is suitable during lower prevalence rates, less than %, while for higher prevalence rates, more than %, pool size of provides the best efficiency. however, for prevalence rates above %, sample pooling should not be used. strategizing to have a common sample pool size across the nation would not yield optimum results as the variance of prevalence rates is extremely high. hence, sample pool size should be decided individually for a testing facility using the prevalence rates recorded by the same lab. our results show that a period of days can be used to forecast the prevalence rate of the next day. this prevalence rate can then be looked up on the decision matrix table to arrive at the optimal sample pool size. however, sample from the following categories should be excluded from the process: . a pool or an individual sample which has already been tested positive . special testing scenarios, such as, retesting or testing of a specific group which is bound to be having much higher risk than the normal conditions. though the mathematical model has been tested with the clinical data, a clinical study to test the performance is being planned. prevalence rate variation of a lab in clinical setting. table . decision matrix for optimal pool size selection all rights reserved. no reuse allowed without permission. was not certified by peer review) is the author/funder, who has granted medrxiv a license to display the preprint in perpetuity. the copyright holder for this preprint (which this version posted july , . . https://doi.org/ . / . . . doi: medrxiv preprint the government of india, order no. - / -dm-i (a) dated impact assessment of non-pharmaceutical interventions against coronavirus disease and influenza in hong kong: an observational study epidemiology and transmission of covid- in cases and of their close contacts in shenzhen, china: a retrospective cohort study world health organization. laboratory testing for coronavirus disease (covid- ) in suspected human cases: interim guidance world health organization. laboratory testing strategy recommendations for covid- : interim guidance ministry of health and family welfare, government of india. strategy for covid testing in india (version , dated th temporal dynamics in viral shedding and transmissibility of covid- sars-cov- viral load in upper respiratory specimens of infected patients transmission potential of sars-cov- in viral shedding observed at the university of nebraska medical center temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study asymptomatic cases in a family cluster with sars-cov- infection asymptomatic and presymptomatic sars-cov- infections in residents of a long-term care skilled nursing facility asymptomatic coronavirus infection: mers-cov and sars-cov- (covid- ) sample pooling as a strategy to detect community transmission of sars-cov- assessment of specimen pooling to conserve sars cov- testing resources pooled rna sample reverse transcriptase real time pcr assay for sars cov- infection: a reliable, faster and economical method pooled sample testing for sars-cov- evaluation of covid- rt-qpcr test in multi-sample pools efficient and practical sample pooling high-throughput pcr diagnosis of covid- advisory on feasibility of using pooled samples for molecular testing of covid- guideline for rt-pcr based pooled sampling for migrants/returnees from abroad/green zones covid- testing bulletin no uniformity in positivity rate of covid samples at laboratories key: cord- -hly ne authors: danko, david; bezdan, daniela; afshinnekoo, ebrahim; ahsanuddin, sofia; bhattacharya, chandrima; butler, daniel j; chng, kern rei; donnellan, daisy; hecht, jochen; kuchin, katerina; karasikov, mikhail; lyons, abigail; mak, lauren; meleshko, dmitry; mustafa, harun; mutai, beth; neches, russell y; ng, amanda; nikolayeva, olga; nikolayeva, tatyana; png, eileen; ryon, krista; sanchez, jorge l; shaaban, heba; sierra, maria a; thomas, dominique; young, ben; abudayyeh, omar o.; alicea, josue; bhattacharyya, malay; blekhman, ran; castro-nallar, eduardo; cañas, ana m; chatziefthimiou, aspassia d; crawford, robert w; de filippis, francesca; deng, youping; desnues, christelle; dias-neto, emmanuel; dybwad, marius; elhaik, eran; ercolini, danilo; frolova, alina; gankin, dennis; gootenberg, jonathan s.; graf, alexandra b; green, david c; hajirasouliha, iman; hernandez, mark; iraola, gregorio; jang, soojin; kahles, andre; kelly, frank j; knights, kaymisha; kyrpides, nikos c; Łabaj, paweł p; lee, patrick k h; leung, marcus h y; ljungdahl, per; mason-buck, gabriella; mcgrath, ken; meydan, cem; mongodin, emmanuel f; moraes, milton ozorio; nagarajan, niranjan; nieto-caballero, marina; noushmehr, houtan; oliveira, manuela; ossowski, stephan; osuolale, olayinka o; Özcan, orhan; paez-espino, david; rascovan, nicolas; richard, hugues; rätsch, gunnar; schriml, lynn m; semmler, torsten; sezerman, osman u; shi, leming; shi, tieliu; song, le huu; suzuki, haruo; tighe, scott w; tong, xinzhao; udekwu, klas i; ugalde, juan a; valentine, brandon; vassilev, dimitar i; vayndorf, elena; velavan, thirumalaisamy p; wu, jun; zambrano, maría m; zhu, jifeng; zhu, sibo; mason, christopher e title: global genetic cartography of urban metagenomes and anti-microbial resistance date: - - journal: biorxiv doi: . / sha: doc_id: cord_uid: hly ne although studies have shown that urban environments and mass-transit systems have distinct genetic profiles, there are no systematic worldwide studies of these dense, human microbial ecosystems. to address this gap in knowledge, we created a global metagenomic and antimicrobial resistance (amr) atlas of urban mass transit systems from cities, spanning , samples and , taxonomically-defined microorganisms collected for three years. this atlas provides an annotated, geospatial profile of microbial strains, functional characteristics, antimicrobial resistance markers, and novel genetic elements, including , novel predicted viral species, novel bacteria, and novel archaea. urban microbiomes often resemble human commensal microbiomes from the skin and airways, but also contain a consistent “core” of species which are predominantly not human commensal species. samples show distinct microbial signatures which may be used to accurately predict properties of their city of origin including population, proximity to the coast, and taxonomic profile. these data also show that amr density across cities varies by several orders of magnitude, including many amrs present on plasmids with cosmopolitan distributions. together, these results constitute a high-resolution, global metagenomic atlas, which enables the discovery of new genetic components of the built human environment, highlights potential forensic applications, and provides an essential first draft of the global amr burden of the world’s cities. introduction limitations in sequencing depth, or by missing annotations in the reference databases used for taxonomic classification, which is principally problematic with phages. it is worth noting that potentially prevalent presence and absence of species (which is robust to noise from relative abundance) and performed a dimensionality reduction of the data using umap (uniform manifold approximation and projection, mcinnes et al. ( )) for visualization (figure a) . jaccard distance was correlated with distance based on jensen-shannon divergence (which accounts for relative abundance) and k-mer distance calculated by mash (which is based on the k-mer distribution in a sample, so cannot be biased by a database) (supp. figure s a , b, c). in principle, jaccard distance could be influenced by read depth as low abundance species drop below detection thresholds. however we expect this issue to be minor as the total number of species identified stabilized at , reads (supp. figure s b ) compared to an average of . m reads per sample. samples collected from north america and europe were distinct from those collected in east asia, but the separation between other regions was less clear. a similar trend was found in an analogous analysis based on functional pathways rather than taxonomy (supp. fig s d) , which indicates geographic stratification of the metagenomes at both the functional and taxonomic levels. subclusters identified by umap roughly corresponded to city and climate but not surface type (supp. figure we quantified the degree to which metadata covariates influence the taxonomic composition of our samples using mavric, a statistical tool to estimate the sources of variation in a count-based dataset (moskowitz and greenleaf, ). we identified covariates which influenced the taxonomic composition of our samples: city, population density, average temperature in june, region, elevation above sea-level, surface type, surface material, elevation above or below ground and proximity to the coast. the most important factor, which could explain % of the variation in isolation, was the city from which a sample was taken followed by region which explained %. the other four factors ranged from explaining % to % of the possible variation in taxonomy in isolation (supp . table s ). we note that many of the factors were confounded with one another, so they can explain less diversity than their sum. one metadata factor tested, the population density of the sampled city, had no significant effect on taxonomic variation overall. to quantify how the principle covariates, climate, continent, and surface material impacted the taxo- nomic composition of samples, we performed a principal component analysis (pca) on our taxonomic data normalized by proportion and identified principal components (pcs) which were strongly associated with a metadata covariate in a positive or negative direction (pcs were centered so an average direction indicates an association). we found that the first two pcs (representing . % and . % of the variance of the original data, respectively) associated strongly with the city climate while continent and surface material associate less strongly (figure b) . next, we tested whether geographic proximity (in km) of samples to one another had any effect on the variation, since samples taken from nearby locations could be expected to more closely resemble one another. indeed, for samples taken in the same city, the average jsd (jensen-shannon distance) was weakly predictive of the taxonomic distance between samples, with every increase of km in distance between two samples representing an increase of . % in divergence (p < e , r = . , supp. figure s b ). this suggests a "neighborhood effect" for sample similarity analogous to the effect described by meyer et al. ( ) , albeit a very minor one. to reduce bias that could be introduced by samples colored by the region of origin for each sample. axes are arbitrary and without meaningful scale. the color key is shared with panel b. b) association of the first principal components of sample taxonomy with climate, continent, and surface material. c) distribution of major phyla, sorted by hierarchical clustering of all samples and grouped by continent. d) distribution of high-level groups of functional pathways, using the same order as taxa (c). e) distribution of amr genes by drug class, using the same order as taxa (c) . note that mls is macrolide-lincosamide-streptogramin. taken from precisely the same object we excluded all pairs of samples within km of one another. at a global level, we examined the prevalence and abundance of taxa and their functional profiles between cities and continents. these data showed a fairly stable phyla distribution across samples, but the relative abundance of these taxa is unstable (figure c ) with some continental trends. in contrast to taxonomic variation, functional pathways were much more stable across continents, showing relatively little variation in the abundance of high level categories (figure d ). this pattern may also be due to the more limited range of pathway classes and their essential role in cellular function, in contrast to the much more wide-ranging taxonomic distributions examined across metagenomes. classes of antimicrobial resistance were observed to vary by continent as well. clusters of amr classes were observed to occur in groups of taxonomically similar samples ( figure e ). we quantified the relative variation of taxonomic and functional profiles by comparing the distribution of pairwise distances in taxonomic and functional profiles. both profiles were equivalently normalized to give the probability of encountering a particular taxon or pathway. taxonomic to facilitate characterization of novel sequences we created geodna, a high-level web interface (figure a) to search raw sequences against our dataset. users can submit sequences to be processed against a k-mer graph-based representation of our data. query sequences are mapped to samples and a set of likely sample hits is returned to the user. this interface will allow researchers to probe the diversity in this dataset and rapidly identify the range of various genetic sequences. we sought to determine whether a samples taxonomy reflected the environment in which it was collected. to this end we trained a random forest classifier (rfc) to predict a sample's city of origin from its taxonomic profile. we trained an rfc with components on % of the samples in our dataset and evaluated its classification accuracy on the remaining %. we repeated this procedure with multiple subsamples of our data at various sizes and with replicates per size to achieve a distribution (fig. b ). the rfc achieved % on held out data which compares favorably to the . % that would be achieved by a randomized classifier. these results from our rfc demonstrate that city specific taxonomic signatures exist and can be predictive. we expanded our analysis of environmental signatures in taxonomy to the prediction of features in cities not present in our training set. to do this we collated a set of features for each city: population, surface material, elevation, proximity to the coast, population density, region, ave june temperature, and koppen climate classification. we trained a rfcs to predict each feature based on all samples that were not taken from a given city then used the relevant rfc to predict the feature for samples from the held out city and recorded the classification accuracy ( figure d ). while not all features and cities were equally predictable (in particular features for a number of british cities were roughly similar and could be predicted effectively) in general the predictions exceeded random chance by a significant margin (supp. figure s a ). this suggests that certain features of cities generate microbial signatures that are present globally and distinct from city specific signatures. the successful geographic classification of samples demonstrates distinct city-specific trends in the detected taxa, that may enable future forensic biogeographical capacities. however, unique, city-specific taxa are not uniformly distributed ( figure b ). to quantify this, we developed a score to reflect how endemic a given taxon is within a city, which reflects upon the forensic usefulness of a taxon. we define the endemicity score (es) of a taxa as term-frequency inverse document frequency where the document consists of samples from some metadata defined group such as a city or region. this score is designed to simultaneously reflect the chance that a taxon could identify a given city and that that taxon could be found within the given city. a high es for a taxon in a given city could be evidence of the evolutionary advantage that the taxon has in a particular cities environment. however, neutral evolution of microbes within a particular niche is also possible and the es alone does not distinguish between these two hypotheses. note that while the es only considers taxa which are found in a city, a forensic classifier could also take advantage of the absence of taxa for a similar metric. es show a roughly bimodal distribution for regions (fig. c) . each region possesses a number of taxa with es scores close to and a slightly larger samples for all cities are transformed into lists of unique k-mers (left). after filtration, the k-mers are assembled into a graph index database. each k-mer is then associated with its respective city label and other informative metadata, such as geo-location and sampling information (top middle). arbitrary input sequences (top right) can then be efficiently queried against the index, returning a ranked list of matching paths in the graph together with metadata and a score indicating the percentage of k-mer identity (bottom right). the geo-information of each sample is used to highlight the locations of samples that contain sequences identical or close to the queried sequence (middle right). b) classification accuracy of a random forest model for assigning city labels to samples as a function of the size of training set. c) distribution of endemicity scores (term frequency inverse document frequency) for taxa in each region. d) prediction accuracy of a random forest model for a given feature (rows) in samples from a city (columns) that was not present in the training set. rows and columns sorted by average accuracy. continuous features (e.g. population) were discretized. number close to (note that es is not bounded in [ , ]). some cities, like offa (nigeria), host many unique taxa while others, like zurich (switzerland), host fewer endemic species (supp. figure s b ). large numbers of endemic species in a city may reflect geographic bias in sampling. however, some cities from well sampled continents (e.g., lisbon, hong kong) also host many endemic species which would suggest that es may indicate interchangeability and local pockets of microbiome variation for some locations. quantification of antimicrobial diversity and amrs are key components of global antibiotic stewardship. yet, predicting antibiotic resistance from genetic sequences alone is challenging, and detection accuracy depends on the class of antibiotics (i.e., some amr genes are associated to main metabolic pathways while others are uniquely used to metabolize antibiotics). as a first step towards a global survey of antibiotic resistance in urban environments, we mapped reads to known antibiotic resistance genes, using the megares ontology and alignment software. we quantified their relative abundance using reads/kilobase/million mapped reads (rpkm) for classes of antibiotic resistance genes detected in our samples (figure a b) . , samples had some sequence which were identified as belonging to an amr gene, but no consistent core set of genes was identified. the most common classes of antibiotic resistance genes were for macrolides, lincosamides, streptogamines (mls), and betalactams, yet the most common class of antibiotic resistance genes, mls was found in only % of the samples where amr sequence was identified. despite being relatively common, antibiotic resistance genes were universally in low abundance com- pared to functional genes, with rpkm values for resistance classes typically ranging from . - com- pared to values of - for typical housekeeping genes (amr classes contain many genes so rpkm values may be lower than they would be for individual genes). in spite of the low abundance of the genes themselves, some samples contained sequences from hundreds of distinct amr genes. clusters of high amr diversity were not evenly distributed across cities ( figure c ). some cities had more resistance genes identified on average ( - x) than others (e.g. bogota) while other cities had bimodal distribu- tions (e.g. san francisco) where some samples had hundreds of genes while others very few. we note that % of the cases where we detected an amr genes had an average depth of . x, indicating that our global distribution would not dramatically change with altered read depth (supp. figure s e ). as with taxa, amr genes can be used to classify samples to cities -albeit with much less accuracy. a random forest model analogous to the one trained to predict city classification from taxonomic profiles was trained to predict from profiles of antimicrobial resistance genes. this model achieved . % accuracy on held out test data (supp. figure s a ). while poor for actual classification this accuracy far exceeds the . % that would be achieved by randomly assigning labels and indicates that there are possibly weak, city specific signatures for antimicrobial resistance genes. multiple amr genes can be carried on a single plasmid and ecological competition may cause mul- tiple taxa in the same sample to develop antimicrobial resistance. as a preliminary analysis into these phenomenons we identified clusters of amr genes that co-occurred in the same samples ( figure d ). we measured the jaccard distance between all pairs of amr genes found in at least % of samples and performed agglomerative clustering on the resulting distance matrix. we identified three large clusters of genes and numerous smaller clusters. of note, these clusters often consist of genes from multiple classes of resistance. at this point we do not posit a specific ecological mechanism for this co-occurrence, but we note that the large clusters contain far more genes than are typically found on plasmids. we performed a rarefaction analysis on the set of all resistance genes in the dataset, which we call the "panresistome" (figure (supp. figure s b ). similar to the rate of detected species, the panresistome also shows an open slope with an expected rate of discovery of previously unobserved amr gene per samples. given that amr gene databases are rapidly expanding and that no amr genes were found in some samples, it is likely that future analyses will identify many more resistance genes in this data. additionally, amr genes show a "neighbourhood" effect within samples that are geographically prox- imal analogous to the effect seen for taxonomic composition (supp. figure s c ). excluding samples where no amr genes were detected, the jaccard distance between sets of amr genes increases with distance for pairs of samples in the same city. as with taxonomic composition. the overall effect is weak and noisy, but significant. to examine these samples for novel genetic elements, we assembled and identified metagenome assembled genomes (mags) for viruses, bacteria, and archaea and analyzed them with several algorithms. this includes thousands of novel crispr arrays that reflect the microbial biology of the cities and , genomes from our data, of which did not match any known reference genome within % average nucleotide identity (ani). of the genomes were classified as bacteria, and as archaea. bacterial genomes came predominantly from four phyla: the proteobacteria, actinobacteria, firmicutes, and bacteroidota. novel bacteria were evenly spread across phyla ( figure a ). assembled bacterial genomes were often identified in multiple samples. several of the most prevalent bacterial genomes were novel species ( figure b ). some assembled genomes, both novel and not, showed regional specificity while others were globally distributed. the taxonomic composition of identifiable genomes roughly matched the composition of the core urban microbiome (section ). the number of identified bacterial mags was somewhat based on read depth and the sample count per city (supp. figure s a ). the number of bacterial mags discovered in a city which did not match a known species was closely correlated to the total number of bacterial mags discovered in that city (supp. figure s b ). bacterial mags were roughly evenly distributed geographically with the notable exception of offa, which had dramatically more novel bacterial species than other cities. we investigated assembled contigs from our samples to identify , predicted uncultivated viral genomes (uvigs). taxonomic analysis of predicted uvigs to identify viral species yielded , clusters containing a total of , uvigs and , singleton uvigs for a total of , predicted viral species. we compared the predicted species to known viral sequences in the jgi img/vr system, which contains viral genomes from isolates, a curated set of prophages and k viral mags from other studies. of the , species discovered in our data . % did not match any viral sequence in img/vr (paez- espino et al., ) at the species level for a total of , novel viruses. we note that this number is surprisingly high but was obtained using a conservative pipeline ( . % precision) and corresponded well with our identified crispr arrays (below). this suggests that urban microbiomes contain significant diversity not observed in other environments. next, we attempted to identify possible bacterial and eukaryotic hosts for our predicted viral mags. for the species with similar sequences in img/vr, we projected known host information onto , metasub viral mags. additionally, we used crispr-cas spacer matches in the img/m system to assign possible hosts to a further , predicted viral species. finally, we used a database of million metagenome-derived crispr spacers to provide further rough taxonomic assignments. our predicted viral hosts aligned with our taxonomic profiles, % of species in the core microbiome (section ) had predicted viral-host interactions. many of our viral mags were found in multiple locations ( figure d ). many viruses were found in south america, north america and africa. viral mags in japan often corresponded to those in europe and north america. we identified , crispr arrays in our data of which , could be annotated for specific systems. the annotated crispr arrays were principally type -e and -f btu a number of type two and three systems were identified as well ( figure e , f). a number of arrays had unclear or ambiguous type assignment. critically the spacers in our identified crispr arrays closely matched our predicted viral mags. we aligned spacers to both our viral mags and all viral sequences in refseq. the total fraction of spacers which could be mapped to our viral mags and refseq was similar (supp. figure s c) but the mapping rate to our viral mags dramatically exceeded the mapping rate to refseq (figure c). we present this as additional evidence supporting these novel viral mags. (tables and s ) , constituting the first large scale metagenomic study of the urban microbiome. we also identified species that are geographically constrained and showed that these can be used to determine a samples city of origin (section . ). many of these species are associated with commensal microbiomes from human skin and airways, but we observed that urban microbiomes are nevertheless distinct from both human and soil microbiomes. notably, no species from the bacteroidetes, a prominent group of human commensal organisms (eckburg et al., ; qin et al., ) , was identified in the core urban microbiome. we conclude that there is a consistent urban microbiome core ( figure , ), which is supplemented by geographic variation (figure ) and microbial signatures based on the specific attributes of a city ( figure ). our data also indicates that significant diversity remains to be characterized and that novel taxa may be discovered in the data (figure ), that environmental factors affect variation, and that sequences associated with amr are globally widespread but not necessarily abundant ( figure ). in addition to these results, we present several ways to access and analyze our data including interactive web based visualizations, search tools over raw sequence data, and high level interfaces to computationally access results. unique taxonomic composition and association with covariates specific to the urban environment suggest that urban microbiomes should be treated as ecologically distinct from both surrounding soil microbiomes and human commensal microbiomes. though these microbiomes undoubtedly interact with the urban environment, they nonetheless represent distinct ecological niches with different genetic profiles. while our metadata covariates were associated with the principal variation in our samples, they do not explain a large proportion of the observed variance. it remains to be determined whether variation is essentially a stochastic process or if a deeper analysis of our covariates proves more fruitful. we have observed that less important principal components (roughly pcs - ) are generally less associated with metadata covariates but that pcs - do not adequately describe the data alone. this is a pattern that was observed in the human microbiome project as well, where minor pcs (such as our figure b ) were required to separate samples from closely related body sites. much of the urban microbiome likely represents novel diversity as our samples contain a significant in addition to general purpose data analysis tools essentially all analysis in this paper is available as a series of jupyter notebooks. our hope is that these notebooks allow researchers to reproduce our results, build upon our results in different contexts, and better understand precisely how we arrived at our conclusions. by providing the exact source used to generate our analyses and figures, we also hope to be able to quickly incorporate new data or correct any mistakes that might be identified. for less technical purposes, we also provide web-based interactive visualizations of our dataset (typ- ically broken into city-specific groups). these visualizations are intended to provide a quick reference for major results as well as an exploratory platform for generating novel hypotheses and serendipitous discovery. the web platform used, metagenscope, is open source, permissively licensed, and can be run on a moderately powerful machine (though its output relies on results from the metasub cap). our hope is that by making our dataset open and easily accessible to other researchers the scientific community can more rapidly generate and test hypotheses. one of the core goals of the metasub consortium is to build a dataset that benefits public health. as the project develops we want to make our data easy to use and access for clinicians and public health officials who may not have computational or microbiological expertise. we intend to continue to build tooling that supports these goals. fields collected were the location of sampling, the material being sampled, the type of object being sampled, the elevation above or below ground, and the station or line where the sample was collected. however, several cities were unable to use the provided apps for various reasons and submitted their metadata as separate spreadsheets. additionally, certain metadata features, such as those related to sequencing and quality control, were added after initial sample collection. to collate various metadata sources, we built a publicly available program which assembled a large master spreadsheet with consistent sample uuids. after assembling the originally collected data at- ), which we have added to an empty sterile urine cup followed by swabbing for min (fig.s ). furthermore, the working space has been swabbed for . min / min before and after treatment with % bleach (fig. s ) figure s ). distributions of dna concentration and the number of reads were as expected. gc content was broadly distributed for negative controls while positive controls were tightly clustered, expected since positive controls have a consistent taxonomic profile. comparing the number of reads before and after quality control did not reveal any major outliers. . . batch effect appears minimal a major concern for this low-biomass studies and large-scale studies are batch effects. the median flowcell used in our study contained samples from cities and continents. however, two flowcells covered cities from or continents respectively. when samples from these flowcells were plotted using umap (see section . for details) the major global trends we described were recapitulated (supp. we used blastn to align nucelotide assemblies from case samples to control samples. we used a threshold of , base pairs and . % identity as a minimum to consider two sequences homologous. this threshold was chosen to be sensitive without solely capturing conserved regions. we identified all connected groups of homologous sequences and found approximate taxonomic identifications by aligning contigs to ncbi-nt using blastn searching for % nucleotide identity over half the length of the longest contig in each group. section ). our dilemma was that a microbial species that is common in the urban environment might also reasonably be expected to be common in the lab environment. in general, negative controls had lower k-mer complexity, fewer reads, and lower post pcr qubit scores than case samples and no major previous studies have reported that microbial species whose relative abundance is negatively cor- related with dna concentration may be contaminants. we observed a number of species that were negatively correlated with dna concentration (supp. figure s a ) but this distribution followed the same shape (but had a greater magnitude) as a null distribution of uniformly randomly generated rela- tive abundances (supp. figure s b ) leading us to conclude that negative correlation may simply be a statistical artifact. we also plotted correlation with dna concentration against each species mean rela- tive abundance across the entire data-set (supp. figure s c ). species that were negatively correlated with dna concentration were clearly more abundant than uncorrelated species, this suggests that there may be a jackpot effect for prominent species in samples with lower concentrations of dna but is not generally consistent with contamination. we analyzed the total complexity of case samples in comparison to control samples. case samples had a significantly higher taxonomic diversity (supp. figure s a ) than any type of negative control sample. we also compared the confidence of taxonomic assignments to control assignments for prominent taxa (supp. figure s b ) using the number of unique marker k-mers to compare assignments. we found that case samples had more and higher quality assignments than could be found in controls. one species, bradyrhizobium sp. btai , was not clearly better in case samples than controls but in this case we were able to assemble genomes for this species in several unique samples so we feel it is ambiguous. finally, we compared assemblies from negative controls to assemblies from our case samples searching for regions of high similarity that could be from the same microbial strain. we reasoned that uncontam- inated samples may contain the same species as negative controls but were less likely to contain identical strains. only case samples were observed to have any sequence with high similarity to an assem- bled sequence from a negative control ( , base pairs minimum of . % identity). the identified sequences were principally from bradyrhizobium and cutibacterium. since these genera are core taxa (see section ) observed in nearly every sample but high similarity was only identified in a few samples, we elected not to remove species from these genera from case samples. we generated -mer profiles for raw reads using jellyfish. all k-mers that occurred at least twice in a given sample were retained. we also generated mash sketches from the non-human reads of each sample with million unique minimizers per sketch. we calculated the shannon's entropy of k-mers by sampling -mers from a uniform , reads per sample. shannon's entropy of taxonomic profiles was calculated using the capalyzer package (section ). . . k-mer based metrics correlate with taxonomic metrics we found clear correlations between three pairwise distance metrics (supp. figure s a , b, c): k-mer based jaccard distance (mash), taxonomic jaccard distance, and taxonomic jensen-shannon diver- gence. this suggests that taxonomic variation reflects meaningful variation in the underlying sequence in a sample. we also compared alpha diversity metrics (supp. figure s d ): shannon entropy of k-mers, and shannon entropy of taxonomic profiles. as with pairwise distances these metrics were correlated though noise was present. this noise may reflect sub-species taxonomic variation in our samples. and mapped these to a set of large database using blastn (see for details). this resulted in . % reads which could not be mapped to any external database compared to . % of reads mapped using our approach with krakenuniq. we note that our approach to estimate the fraction of reads that could be classified using blastn does not account for hits to low quality taxa which would ultimately be discarded in our pipeline, and so represents a worst-case comparison. explanation ( ) as it has been demonstrated to be comparable or having higher sensitivity than the best tools identified in a recent benchmarking study (mcintyre et al. ( ) ) on the same comparative dataset. in addition, krakenuniq allows for tunable specificity and identifies k-mers that are unique to particular taxa in a database. reads are broken into k-mers and searched against this database. finally, the taxonomic makeup of a sample is given by identifying the taxa with the greatest leaf to ancestor weight. krakenuniq reports the number of unique marker k-mers assigned to each taxon, as well as the total number of reads, the fraction of available marker k-mers found, and the mean copy number of those k-mers. we found that requiring more k-mers to identify a species resulted in a roughly linear decrease at a minimum we required three reads assigned to a taxa with unique marker k-mers. this setting captures a group of taxa with low abundance but reasonable (∼ - %) coverage of the k-mers in their marker set (supp. figure s c ). however, this also allows for a number of taxa with very high ( ) duplication of the identified marker k-mers and very few k-mers per read which we believe is biologically implausible (supp. figure s d ). we filtered these taxa by applying a further filter which required that the number of reads not exceed times the number of unique k-mers, unless the set of unique k-mers was saturated (> % completeness). we include a full list of all taxonomic calls from all samples including diagnostic values for each call. we do not attempt to classify reads below the species level in this study. we further evaluated prominent taxonomic classifications for sequence complexity and genome cov- figure s b ). we chose sub-sampling (sometimes referred to as rarefaction in the literature) based on the study by weiss et al. ( ) , showing that sub-sampling effectively estimates relative abundance. note that we use the term prevalence to describe the fraction of samples where a given taxon is found at any abundance and we use the term relative abundance to describe the fraction of dna in a sample from a given taxon. we compared our samples to metagenomic samples from the human microbiome project and a metagenomic study of european soil samples using mash (ondov et al., ) , a fast k-mer based comparison tool. we built mash sketches from all samples with million unique k-mers to ensure a sensitive and accurate comparison. we used mash's built-in jaccard distance function to generate distances between our samples and hmp samples. we then took the distribution of distances to each particular human commensal community as a proxy for the similarity of our samples to a given human body site. we also compared our samples to hmp and soil samples using taxonomic profiles generated by metaphlan v . (segata et al., ) . we generated taxonomic profiles from non-human reads using metaphlan v . and found the cosine similarity between all pairs of samples. we used the microbe directory (shaaban et al., ) we analyzed the metabolic functions in each of our samples by processing non-human reads with hu- mann (franzosa et al., ) . we aligned all reads to uniref using diamond (v . . , (buchfink et al., ) ) and used humann to produce estimate of pathway abundance and completeness. we filtered all pathways that were less than % covered in a given sample but otherwise took the reported pathway abundance as is after relative abundance normalization (using humann 's attached script). high level categories of functional pathways were found by grouping postively correlated pathways and manually annotating resulting clusters. figure s : ecological relationships with taxa. a) correlation between species richness and latitude. richness decreases significantly with latitude b) neighbourhood effect. taxonomic distance weakly correlates with geographic distance within cities. c) fraction of reads assigned to different databases by blast for each region, at different levels of average nucleotide identity figure s : antimicrobial resistance genes, supplemental. a) classification accuracy of a random forest model predicting city labels for held out samples from antimicrobial resistance genes. b) rarefaction analysis of antimicrobial resistance genes. curve does not flatten suggesting we would identify more amr genes with more samples. c) neighbourhood effect. jaccard distance of amr genes weakly correlates with geographic distance within cities. d) number of amr genes detected for samples in each region. e) distribution of reads per gene (normalized by kilobases of gene length) for amr gene calls. the vertical red line indicates that % of amr genes have more than . reads per kilobase and would still be called at a lower read depth. gaussian distributions to sampling locations where the taxa was found with standard deviations based on the geographic distance between observations. top row) sampling sites in three major cities rows - ) estimated distribution of different example species in major cities row ) estimated distribution of three species together in major cities structure, function and diversity of the healthy human microbiome metaspades: a new versatile metagenomic assembler nala an, núria andreu so- mavilla a) number of species detected as k-mer threshold increases for randomly selected samples b) number of species detected as number of sub-sampled reads increase c) k-mer counts compared to number of reads for species level annotations in randomly selected samples, colored by coverage of marker k-mer set d) k-mer counts compared to number of reads for species level annotations in randomly selected samples, colored by average duplication of k-mers e) comparison of mean sequence entropy and coverage equality for core and sub-core taxa a) jensen-shannon divergence of taxonomic profiles vs mash jaccard distance of k-mers b) divergence of taxonomic profiles vs jaccard distance of taxonomic profiles. c) jaccard distance of taxonomic profilesvs mash jaccard distance of k-mers d) shannon's entropy of taxonomic profiles vs shannon's entropy of k-mers e) taxonomic richness (number of species) vs shannon's entropy of taxonomic profiles figure s : a) mash k-mer jaccard similarity to representative hmp samples metaphlan v . cosine similarity to representative hmp samples, colored by continent c) fraction unclassified dna by surface material d) cosine similarity to metaphlan v . skin microbiome profile by surface e) jensen-shannon distance between pairs of taxonomic profiles vs geographic distance f) mash k-mer jaccard similarity to representative soil samples key: cord- - obomty authors: pardon, bart; buczinski, sébastien title: bovine respiratory disease diagnosis: what progress has been made in infectious diagnosis? date: - - journal: vet clin north am food anim pract doi: . /j.cvfa. . . sha: doc_id: cord_uid: obomty when it is desired to identify infectious agents involved in an outbreak of bovine respiratory disease, a variety of possible sampling methods may be used. for field use, the deep nasopharyngeal swab, transtracheal wash, and nonendoscopic bronchoalveolar lavage are most feasible. at present, bacterial culture and polymerase chain reaction testing are most commonly used to identify infectious agents. interpretation of test results can be challenging, particularly for opportunistic pathogens. evidence-based guidelines for precise interpretation of microbiologic tests results are lacking; however, approaches that have been practically useful for the management of bovine respiratory disease outbreaks are presented. increasing pressure on antimicrobial use and the need for veterinarians and farmers to use antimicrobials more rationally. the use of diagnostic support by laboratory analysis is one of the frequently mentioned cornerstones of antimicrobial stewardship programs. however, scientifically reasoned, the evidence that systematic use of laboratory diagnostics, especially the antibiogram, would result in selection of a different first-choice therapy compared with an empiric decision preferably following (evidence-based) guidelines, is limited, especially in cattle. antimicrobial resistance in respiratory tract bacteria from cattle is present and varies highly between systems. resistance levels are generally lower in closed dairy and beef herds, substantially higher in feedlots, and most worrisome in veal calf operations, where oral mass medication is frequently used. , although there is no doubt of the presence of resistance, and multiresistance, in respiratory bacteria from cattle, to what extent this results in therapy failure when following guidelines for antimicrobial therapy is poorly documented. in recent years, guidelines specifying first-line, second-line, and third-line antimicrobial choices for the different cattle diseases have been initiated in several european union countries, including the netherlands, belgium, denmark, sweden, and germany. , , however, the amount of literature reporting the clinical benefit of every antimicrobial-bacteria combination in highly variable field settings is currently very limited. therefore, these guidelines mainly include the spectrum of the antimicrobial, pharmaceutical leaflet recommendations and follow classification of the importance of antimicrobials for human medicine of the world health organization. in contrast with human medicine, to the authors' knowledge there are no extensive, sufficiently detailed, and large-scale studies available on therapy failure caused by antimicrobial resistance in cattle. regardless of the limitations mentioned earlier, diagnostics are more and more frequently used when addressing bovine respiratory disease (brd). this increased use is understandable, because antimicrobial decision making for brd still often involves a decision to use group therapy, and, in the current climate, mass medicating without any evidence of the need for this therapy will increasingly be criticized. the authors fully acknowledge the complexity of advising on the implementation and interpretation of diagnostic tests for brd, given the huge gaps in the current knowledge. however, the need is urgent, and therefore this article provides a framework to assist practitioners and clinicians in their everyday decision-making process. this reasoning may not withstand time; this article does not represent a consensus of all leading experts, nor is the objective to provide a complete literature overview. this discussion reflects on the body site sampled, the test used, and the pathogen detected. the selected sampling site of the respiratory tract is of great importance for interpretation of the test result. and endoscopic bronchoalveolar lavage [bal] ; fig. ). nasal swabs, predominantly sampling the cutaneous part of the nose, are generally considered of limited value for infectious diagnostics. in contrast, dnss sample the respiratory and associated lymphoid epithelium of the nasopharynx and return more meaningful samples. however, the biggest issue with nasopharyngeal swabs is the large number of polymicrobial samples recovered (> %), which heavily compromises clinical interpretation when only opportunistic pathogens are retrieved. contamination can be reduced by rinsing the nares (with a single-use paper towel or a gauze with alcohol) or by using a guarded dns. however, studies specifically focusing on the effect of guarded swabs to reduce nasal contamination are, to the authors' knowledge, not available in cattle. recent reports on the respiratory microbiome in cattle also put the idea of contamination at that sampling site into another perspective, given the large variation in bacterial species normally present. , the largest disadvantage of dnss is that they do not directly sample the lower respiratory tract. despite some conflicting results, previous studies overall showed that, for most pathogens, an association between dns results and ttw or bal is present. , [ ] [ ] [ ] in addition to cotton swabs, brush swabs also exist, which cause more intensive swabbing of the mucosa (although possibly also blood staining of the sample), presumably with higher detection rates. no evidence on their benefit for use in cattle is currently available. complications of dns are rare and included nasal hemorrhage and fracture of the shaft of the swab. the latter is without any harmful consequence because the animal evacuates the remaining part of the swab either by sneezing or swallowing. to overcome the issue of nasal contamination, transtracheal sampling techniques relying on perforation of the trachea with a needle or catheter after surgical preparation of the skin have been developed. historically, transtracheal swabs have been used, but the transtracheal aspirate and wash are now common. although an aspirate (tta) only involves aspiration of mucus present in the respiratory tract, a wash (ttw) requires fluid instilment and immediate aspiration. despite the terminology tta being frequently used in the field, the technique usually used is a ttw. most frequently, for ttw in cattle, ttw kits (large animal trans-tracheal wash kit, mila international, inc, florence, ky), or human central venous catheters (eg. centracath , vygon, ecouen, france) are used, which are commercially available and sterile packed for single use. alternatively, a male dog urinary catheter can be used in combination with a -g catheter/needle to perforate the trachea in between tracheal rings. in veterinary medicine, the common thinking is that the ttw is preferred for bacteriology and bal to study inflammation (cytology). however, this recommendation generally comes from horse medicine, and seems to be expert opinion rather than supported by substantial peer-reviewed studies. in humans, ttw is generally not used for ethical reasons. the general idea is that the bronchial bifurcation is the site where the efflux of the mucociliary system of the whole of the lung comes together. hence sampling there would be representative for the whole of the lung. however, there are some counterarguments for this reasoning. first, the mucociliary system can be heavily impaired by pneumonia. second, microbial aspiration from the nasopharynx into the upper trachea is likely frequent. third, normal pathogenesis involves gradual descent of bacteria down the respiratory tree toward the lung. taking the second and third arguments into account, a positive ttw culture might equally represent a bacterial tracheitis or even an insignificant colonization or upper airway contamination, resulting in false positive diagnosis of infectious bronchopneumonia. advantages are that a new disposable catheter can easily be used for each animal, and sampling is theoretically achievable within a predictable time frame given that no active cooperation of the animal is required, in contrast with bal. however, sedation of the animal and local anesthesia of the puncture site can be done to improve animal comfort during the procedure. in a bal procedure, a bal catheter or flexible endoscope is introduced through the nose and trachea into the lower airways until it wedges into a larger (or smaller depending on catheter diameter) bronchus. next, while holding in this wedged position, a volume (usually ml in calves, if necessary followed by a second or third injection) of sterile saline is injected and immediately aspirated. classically, as in human medicine, a bal is performed by endoscopy. the major advantage is that a specific lung lobe, previously shown to be affected on radiology or ultrasonography, can be sampled. also, protective sheets or agar plugs can be used to reduce the risk of nasal contamination. the major disadvantage of the endoscope is the high operating costs and risk for equipment damage in the farm setting. also, sampling multiple animals becomes difficult because time to resterilize the endoscope between animals is needed ( - minutes minimum). to overcome the cost and risks of endoscopic bal, nbal techniques have been developed. in nbal, a bal catheter is blindly introduced through the nose, larynx, and trachea until the wedged position in a large bronchus is reached. next, a volume of saline is injected and gently aspirated. the volume used varies substantially between studies ( - ml , ), but a trend to reduce the volume for welfare/comfort reasons is present. on average, . % of the volume ( . %- . %) can be recovered in nbal, which is substantially larger than in a ttw procedure. it is important to realize that sedation not only suppresses the required responses (coughing, curving of the nose, and extroversion of the tongue) to ensure an intratracheal position but also causes systematic sampling of the diaphragmatic lung lobes, which are less likely to be affected. good restraint of the calf with the head fixed with the nose pointing upward as much as possible is advisable to ease blind introduction of the tube into the trachea. alternatively, the calf might be surprised into allowing the tube to be advanced into the airways by placing the head in a horizontal position, and introducing the catheter on inspiration visible by the opening of the nostrils. overall, in % of animals, nbal sampling can be completed within minutes. for the remaining %, the practical advice is to select another animal to sample when undertaking group diagnosis, rather than spending excessive time and causing prolonged irritation to a reluctant animal. alternatively, a technique where a double-guarded bal catheter is orally introduced into the larynx through a pvc (polyvinyl chloride) speculum has been described for calves that are at least to months old, where the guarded catheter is inserted through the larynx under visual control. the use of bal samples (especially nbal) for bacteriology is still highly controversial, mainly because of the risk of nasal contamination. although contamination is far less than with dns, . % of nbal samples were still polymicrobial. a large influence of the sampler seems to be present, likely depending not only on differences in hygienic sample handling but also on skills to swiftly introduce the catheter without touching too much of the nasopharynx. however, it is important to realize that hard evidence on substantial nasal contamination by using nbal catheters is currently not available in any species. the only available study on this matter showed pure culture and negative results in . % and . % of the nbal samples, even though dns samples of the same animals were polymicrobial. further, the currently most extensive study on sample method comparison showed very good agreement for bacteriology between dns, ttw, and nbal. interestingly, in human medicine, there are growing efforts toward the use of a mini-bal procedure for bacterial diagnosis in ventilator-assisted pneumonia. overall, sample contamination should be avoided and, in the case of nbal, this can be done by adequate training or visualization of the larynx by a video speculum (ivetscope, dairymac limited, hampshire, united kingdom) or endoscopic cameras intended for plumbers or auto mechanics. these devices are available at much lower prices than traditional endoscopes. next to the site of the respiratory tract sampled (upper or lower airway), the cultural perception of the effect of the sampling technique on animal welfare also plays an important role in what technique is currently preferred in a given country/region. no studies on the effect of respiratory tract sampling on stress or pain have yet been conducted in calves. a master of science thesis showed that both animals sampled by dns or nbal spent less time walking compared with the unsampled control group, whereas lying or eating were unaffected. for ttw as well as nbal, the required volume of saline to be instilled is unclear; it is also unclear whether the volume instilled influences bacteriology results, as it does for cytology. in summary, sampling techniques for the field need to be economically feasible both in terms of equipment/disposables cost and also invested time. the dns, ttw, and nbal best suit this profile and are currently most frequently used in the field. differences in use exist between countries, which mainly originate from historical or cultural preference. an overview of available diagnostic tests for pathogen identification in respiratory diseases in cattle is shown in table . it is beyond the scope of this article to provide a complete overview of all tests possible. the focus is on the most frequently used tests and the most promising future tests likely to become widely available for practice within the next years. in the current international context, the pressure to reduce antimicrobial use has become the main driver of diagnostic test performance for causal diagnosis of brd. a crucial aspect for field efficacy is a short turnaround time (tat), the time between sampling and availability of the test result. in order to be able to use the diagnostic test result to target therapy or initiate control measures tat needs to be as short as possible, ideally less than a day. however, having test results the next morning might also be workable for most outbreaks. also, the use of cow-side testing for a causal diagnosis of brd has great potential to reduce tat. however, to the authors' knowledge, no such tests are currently commercially available. hence, attention should be given to ensuring proper and timely transport to the laboratory. at refrigerator temperature ( c- c), the isolation rate of mannheimia haemolytica and pasteurella multocida was not reduced for hours, whereas a transport temperature of more than c resulted in reduced isolation as soon as hours later. serology serologic tests are useful to target vaccination programs, to determine protective status, and to evaluate infection dynamics at larger scale. however, they are not suitable to direct immediate therapy because they have a tat of weeks (required time for seroconversion) and only provide indirect evidence of infection. also, for the opportunistic pasteurellaceae family, maternal immunity smoothly shifts to acquired immunity, without any signs of disease or seroconversion. another important issue is that sensitivity and specificity can be highly variable between different antibody enzymelinked immunosorbent assays (elisas), hampering clinical interpretation and their use for individual animal decisions (eg, culling or purchase). for targeting therapy, direct identification of the pathogen is needed, and this can be achieved by microbial culture, matrix-assisted laser desorption/ionization time of flight (maldi-tof) mass spectrometry (ms), pcr, or ngs/third-generation sequencing. microbial culture is most frequently used for identification of bacteria. next to low operating costs, the possibility of antimicrobial susceptibility testing is an important advantage of culture. for mycoplasmata, specific media are required, and fastidious growers, especially histophilus somni, are easily overgrown, resulting in false-negative results. , sensitivity and specificity of microbial culture have not been determined for most of the bacteria involved in brd. a recent study using bayesian latent class analysis showed that mycoplasma bovis culture on solid medium containing tween is . % ( % bayesian credible intervals [bci], . to . ) sensitive and . % ( % bci, . - . ) specific. in the last decade, maldi-tof ms, which identifies bacteria by their unique protein profiles, has revolutionized routine diagnostics. it is primarily used for identification of bacteria after culturing, including mycoplasma species. however, maldi-tof ms can also be applied directly on the sample after a very short period of enhanced growth in a liquid medium. relative to classic microbial culture, these culture-enriched direct maldi-tof ms techniques allow correct bacterial identification in % of the samples (sensitivity . %; % confidence interval [ci], . - . ; specificity % [ - ]) within hours. the technique performed less well in polymicrobial samples and in samples with mixed infection. also for m bovis, a culture-enriched direct maldi-tof ms technique was developed, which was . % ( % bci, - ) sensitive and . % ( % bci, - ) specific in a bayesian latent class model including pcr and microbial culture on solid agar. tat was reduced from more than days to less than days. in addition, different maldi-tof ms methods are available for antimicrobial susceptibility testing. by means of mbt-astra (maldi biotyper antibiotic susceptibility test rapid assay), oxytetracycline resistance in p multocida could be identified with high accuracy (se . %; % ci, . - . ; sp %; [ % ci, - ]) in as little as hours, outperforming the disc diffusion antibiogram. the mbt-astra technique can be designed for every bacterium-antibiotic combination, but logistical changes are needed to create a good intralaboratory workflow. the costs of maldi-tof procedures are generally low, in line with microbial culture. pcr for the causal diagnosis of brd is now very popular. the main reasons are that multiplex pcr or multiple single pcrs allow detection of multiple bacteria and viruses, providing practitioners with a more extended view of the pathogens involved and hence more options to better target therapy, control, and prevention. fastidious and metabolically active viable but unculturable viruses and bacteria can be detected, in contrast with standard microbial culture. however, in contrast with sequencing techniques, specific primers are needed, and the pathogen of interest needs to be determined beforehand. in this way, the diagnostics are potentially biased and possibly lead to false-negative results. another problem is that viral genomes evolve rapidly and primers might become outdated, limiting the efficient detection of the pathogens of interest. pcr is generally not cheap, but, by pooling samples (dns, ttw, or bals), a group diagnosis can be reached and costs are decreased. in available studies, pools of samples from animals were shown to improve diagnostic accuracy at the group level. , the largest disadvantage of pcr is interpretative difficulty, because pcr can identify dead pathogens, opportunists currently not involved in infection, and contaminants, none of which signify a clinically meaningful test result. this disadvantage was shown, for example, for h somni. the use of quantitative pcr is more informative because the pathogen load, especially in the respiratory disease complex, is important to consider. for this reason, quantitative pcr is increasingly used in veterinary laboratories, although interpretative questions remain. especially, when multiple pathogens are detected, determining the attributable fraction of each pathogen to the clinical presentation remains very difficult. next-generation and third-generation sequencing ngs technologies are now becoming more widely available because of the democratization of the technologies, and because platforms such as minion (oxford nanopore technologies, oxford, uk) allow decentralized sequencing experiments. the first studies using ngs (metagenomics) to detect viruses involved in brd in feedlots are already reported; these detected known pathogenic viruses as well as previously unknown or incompletely understood viruses (eg, influenza d virus). , hence, the advantage of ngs is that all pathogens can be simultaneously detected, without prior selection of which pathogens to test for. also, semiquantitative results can be reported because, for most viruses, the number of reads corresponds to the initial load of the pathogen present. not only viruses but also whole genomes can be recovered at a scale that is constantly increasing. eventually, direct sequencing of bacteria will allow detection of virulence genes, phylogenetic clustering of strains during outbreaks, and ultimately prediction of antimicrobial resistance based on single nucleotide polymorphisms or resistance genes. a high total bacterial burden and low bacterial community diversity were associated with positive culture results in classic microbial culture. ngs is the basis for microbiome studies, which are discussed elsewhere in this issue. disadvantages are that ngs is costly and requires a long tat under the current conditions using the most accurate devices (eg, illumina, san diego, ca). however, with nanopore sequencing platforms (eg, minion) a higher throughput and shorter tat can be achieved. this long-read technology has been commercially available since and has made tremendous improvements in output and accuracy. in humans and pigs, minion has been used to characterize pathogens in different types of samples, even at the site of disease outbreaks, because data analysis can be done in the field on portable hardware. , on human lower respiratory tract samples, within hours of sampling, a result was given at a sensitivity of . %. however, in order to achieve wide implementation in veterinary practice, the cost, the ability to correctly interpret, and setup of an actionable logistic chain will be essential. therefore, this technology is another case in which analysis of pooled samples from multiple animals to obtain a group diagnosis of primary pathogens will be the most likely application. clinical interpretation of a diagnostic test result to determine the infectious cause of a respiratory tract disease requires information on the pathogen identified, the site of the respiratory tract sampled, the diagnostic test used, the clinical condition of the animal, and whether the sample originates from a single animal or is pooled. there is no current consensus on the way to sample the respiratory tract or to interpret diagnostic test results in humans and many other species. based on the available research, it is unlikely that an evidence-based consensus on respiratory tract sampling method, diagnostic testing, and interpretation of results in cattle can be reached. hence, this article assists readers to properly interpret results of testing by providing information not only on current recommendations but especially on the drawbacks and research gaps. the first point to consider is the nature of the pathogen retrieved, whether it is a primary or secondary pathogen. a primary or obligate pathogen (when present), per definition, induces damage to the respiratory tract, mostly followed by an inflammatory response. however, depending on the infectious dose and host immunity, infection might result in clinical disease or not. also, certain primary pathogens can chronically and even asymptomatically infect animals, resulting in carriers: for example, salmonella spp or m bovis. most primary pathogens weaken innate immunity of the airways, facilitating superinfection by opportunistic bacteria. some, such as bovine respiratory syncytial virus, bovine herpesvirus type (bhv- ), and potentially also others ( table ) , are able to induce life-threatening disease without bacterial superinfection. despite still being controversial in some scientific communities or countries, m bovis is generally considered a primary pathogen. detection of a primary pathogen can, with some caution, be interpreted straightforwardly. the primary pathogen should normally not be present, and, depending on its virulence, it can, either as a sole agent or in combination with other agents, be held responsible for the clinical picture. also, detection from any site of the respiratory tract is meaningful. for animal welfare reasons and following the pathogenesis, which starts with nasal infection, dns samples might be sufficient and even most appropriate to detect primary pathogens. however, detection rates at the different sites of the respiratory tract differ between pathogens. for bovine coronavirus, dns was more frequently positive than samples from the lower respiratory tract, whereas the inverse was true for bovine respiratory syncytial virus. for bhv- , dns is recommended given that the infection most frequently remains limited to the upper airways. the true interest of any diagnostic effort lies in extrapolation of test results from the sampled animals to the whole group. detection of primary pathogens in some animals makes involvement of the same pathogen in the part of the resident flora, differences in strain virulence described possibly resulting in some primary pathogenic strains. other studies show cattle to become ill from their own resident strain on exposure to other pathogens and/or risk factors , , chlamydia psittaci controversial, likely primary natural infections result in mild or subclinical disease (continued on next page) moraxella bovis/ovis secondary primary eye pathogen, occasionally isolated in pure culture from animals with bronchopneumonia pasteurella multocida secondary part of the resident flora. strain virulence differences exist, and some disease presentations (eg, septicemia or peritonitis) have been linked to certain strains , salmonella spp primary primary site of infection of most salmonella spp is the gastrointestinal tract. localization in the respiratory tract is possible, most likely after septicemic spread trueperella pyogenes secondary involved in purulent processes. often regarded as characteristic for chronicity. however, naturally resistant to fluoroquinolones escherichia coli, gallibacterium anatis, enterobacter hormaechei, staphylococci, streptococci, fungi secondary single reports on cattle-specific strains isolated in pure culture in an outbreak of pneumonia in calves , [ ] [ ] [ ] multiple other bacterial species can be detected in the bovine respiratory tract. this table is limited to either known primary pathogens or frequently isolated pathogens, currently assumed to have a pathogenic significance. data from refs. , , , , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] pardon & buczinski cohoused animals very likely. hence, to improve sensitivity of the group diagnosis, the use of pcr on a pooled sample (up to animals) can be considered. , a pitfall when working with pcr to detect primary pathogens is that vaccine antigen can be detected up to days after intranasal vaccination with a live vaccine, resulting in false-positives. a secondary or opportunistic pathogen can be part of the normal respiratory microbiome, without inducing inflammation. in general, breaching of innate immunity, either by another pathogen or a noninfectious cause, is needed before the opportunistic pathogen invades tissues and induces inflammation. interpretation of detection of an opportunistic pathogen is more difficult, given that they can be present in healthy animals. , , therefore, simply detecting the pathogen cannot be seen as evidence of its involvement. the pasteurellaceae family and a range of other bacteria (eg, streptococcus spp and trueperella pyogenes) are generally considered secondary pathogens. although there seems to be little discussion of p multocida, scientific opinions on the potential primary role and differences in strain virulence of m haemolytica and h somni vary greatly. , it is outside the scope of this article to review or take a position on this matter. similarly, in other species, including humans, this issue of opportunistic pathogens exists. when interpreting a positive culture result, a differentiation between contamination, colonization, and infection needs to be made. contamination is defined as the presence, usually in low numbers, of bacteria in a sample that are not expected to be present in the sampled site. colonization can be defined as the presence of a micro-organism in a host, with growth and multiplication of the organism, but without interaction between host and organisms, hence no inflammatory reaction, immune response, or clinical expression occurs. similarly, infection is isolation of a high number of bacteria from a site of the respiratory tract, but in the presence of inflammation of the mucosa, presenting either clinically or subclinically. hence, simply picking a suspected colony from an agar plate, to confirm the cause of the respiratory disease, and subsequently using an antibiogram based on this single colony may be misleading. more information can be derived from culture results if quantitative descriptions and at least the degree of contamination are described. a possible way to better describe culture results, previously used for research purposes, , is presented in table . it is also important to realize that using selective media for pasteurellaceae, as, for example, by adding bacitracin, ensures better growth and detection of these opportunistic pathogens, but information on the amount of pathogens and degree of contamination of the sample will be lost. , pasteurellaceae are part of the normal respiratory flora, and can even be abundantly present in the nasal cavity of healthy animals. an association between the presence of a pasteurella species in the nose and its presence in the lower respiratory tract is described. , , however, interpretation of dns results for opportunistic pathogens remains very difficult, especially because the composition of the nasopharyngeal microbiota seems to be heavily influenced by bioaerosols from the agricultural environment. loss of biodiversity and overgrowth of opportunistic pathogens occurs in the pathogenesis of brd, resulting in higher odds that pasteurellaceae can be cultured from nasal swabs in larger quantities in ill animals. , , however, with current knowledge on the interpretation of dns results at the individual or group level, samples of the lower respiratory tract are likely a better option to evaluate potential involvement of opportunistic pathogens. interpretation of detection of opportunists in lower respiratory tract samples remains difficult, even in ill animals, because, even with very strict clinical case definitions, pasteurellaceae can also be cultured from the lower airways in healthy animals. , previously explored ways to overcome the issue of interpreting detection of opportunistic pathogens in humans and other species are the use of quantitative cultures or cytologic evidence of inflammation. quantitative culture is derived from the assumption that, in case of a severe infection, the opportunistic pathogen will be present in larger numbers. cutoffs such as greater than colony-forming units per milliliter of bal fluid have been suggested in dogs and horses. , however, pathogen burden builds up, and sampling early in the disease process could mean that much lower numbers are detected despite the pathogen being involved in the inflammatory process. another option is derived from the assumption that a bacterial infection will result in a massive airway neutrophilia. however, clear cutoff percentages for neutrophilia differentiating a bacterial infection from a viral one or a strictly noninfectious airway inflammation have not been determined in calves. given that some bal techniques result in a larger contribution of the bronchial component in the total bal fluid, the neutrophil percentage is increased compared with a larger volume of mainly alveolar lavage fluid. primary insights in calf bal fluid analysis show that cytologic parameters coincide poorly with clinical or ultrasonographical findings or culture results for opportunistic pathogens, at least when using nbal. the presence of phagocytosis by bacteria in neutrophils or macrophages may be helpful to differentiate active infection versus simple presence of the bacteria. although interpreting culture results for opportunistic pathogens is already difficult, interpretation of pcr results is even more so. not only can insignificant quantities or even dead bacteria result in a positive pcr, the nasal passages also pick up bacterial dna, making the lower respiratory tract sample positive. quantitative pcr might overcome this issue, but, to the authors' knowledge, no guidelines on how to interpret these results are currently available in the bovine species. although laboratory costs are fixed, the return on investment of an analysis greatly depends on selection of appropriate animals to sample and on the technical sampling skills of the veterinarian. an animal in the first days of the disease, not previously treated with antimicrobials and not displaying severe respiratory signs, is first choice. by sampling in the acute phase of the disease, the odds of detecting the viral component are higher. by avoiding previous antimicrobial treatment and by sampling early in the disease course, the probability that the antibiogram derived is useful for empiric therapy increases. by avoiding sampling animals in heavy respiratory distress, the odds of aggravation of disease or even mortality can be decreased. in addition, in spite of all reasoning made earlier, veterinarians still make decisions at an individual animal level. when sampling an individual, the main interest is usually to make a decision representing the group, and to judge the utility of the diagnostic test to support this group decision. different epidemiologic approaches are possible to determine an appropriate sample size. the goal is more a detection of disease approach (being % confident that the pathogen was detected when present) rather than determining the prevalence of the pathogen in a group of animals. in the field, sample size is currently more driven by practical reasons such as available time to sample or maximum samples allowed to pool for economic reasons. for example, performing pcr on pools of animals increases sensitivity without diluting the sample too much. fig. provides an overview on the risk of not finding a positive animal in scenarios, related to % prevalence of the pathogen in the diseased population and scenario with a pretest probability that % of sick calves are affected by the pathogen (ie, where multiple pathogens are involved and can cause the same clinical disease). results of a test with % sensitivity (ie, detects the pathogen in of infected calves) and % specificity (no false-positive calves) are presented. using this test in a scenario with % of the affected animals being positive for the pathogen, after calves not finding a positive, will misclassify only . % of the herds (the scenarios assume that the pooled tests' accuracy is the same as the individual test). in the case of opportunistic pathogens, given that they can be found in healthy or subclinical animals, at this time it might be most prudent to only sample animals with evidence of clinical bronchopneumonia by using a combination of clinical scoring and thoracic ultrasonography. focusing on bacterial isolation to direct intervention strategies, without taking the clinical status into account, holds great danger for overtreatment with antimicrobials. knowledge of respiratory health is rapidly evolving in animals, following new developments in humans. in particular, better insights into the role of the respiratory microbiome and the interaction of the airway inflammatory response with different organisms and air pollutants are likely to change how the diagnostic tests discussed in this article are interpreted. the authors hope that the information, tools, and provisional advice provided can aid the large group of cattle veterinarians, already having to make rational treatment decisions today. b. pardon has received honoraria for acting as speaker or consultant for pharmaceutical (zoetis, msd, vetoquinol, dopharma, boehringer ingelheim, dechra, hipra, ceva, merial, and elanco), agricultural (algoet nutrition), and chemical (proviron) companies and nonprofit organizations (boerenbond, amcra, dgz-vlaanderen). s. buczinski has received honoraria for acting as speaker or consultant as well as research grants for pharmaceutical companies (zoetis, msd, hipra, and ceva) and companies involved in commercialization of ancillary tests used in respiratory diseases (ei medical imaging, geissler corp.). fig. . risk (probability ranging between and ) of not finding a positive animal for a given pathogen according to sample size (x axis) in a scenario where % (solid line) and % (dashed line) of affected animals are positive for a given pathogen. the graph represents a test with % sensitivity and % specificity. it assumes that there are no falsepositive results (ie, when the test indicates that the pathogen is present, this is a truepositive result). in the example where the pathogen is causing the disease in % of affected calves, the risk of not finding an infected animal after sampling n cases is ( -se)n , where se is the test 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sebastian; von stetten, felix; zengerle, roland title: diagnostic tools for tackling febrile illness and enhancing patient management date: - - journal: microelectron eng doi: . /j.mee. . . sha: doc_id: cord_uid: ixh ykrc most patients with acute infectious diseases develop fever, which is frequently a reason to visit health facilities in resource-limited settings. the symptomatic overlap between febrile diseases impedes their diagnosis on clinical grounds. therefore, the world health organization promotes an integrated management of febrile illness. along this line, we present an overview of endemic and epidemic etiologies of fever and state-of-the-art diagnostic tools used in the field. it becomes evident that there is an urgent need for the development of novel technologies to fulfill end-users' requirements. this need can be met with point-of-care and near-patient diagnostic platforms, as well as e-health clinical algorithms, which co-assess test results with key clinical elements and biosensors, assisting clinicians in patient triage and management, thus enhancing disease surveillance and outbreak alerts. this review gives an overview of diagnostic technologies featuring a platform based approach: (i) assay (nucleic acid amplification technologies are examined); (ii) cartridge (microfluidic technologies are presented); (iii) instrument (various detection technologies are discussed); and at the end proposes a way that such technologies can be interfaced with electronic clinical decision-making algorithms towards a broad and complete diagnostic ecosystem. people in low-and middle-income countries (lmics), primarily in sub-saharan africa, south-east asia, and latin america, suffer dramatically from infectious diseases, some of which are endemic, while others occur in the form of epidemics. a combination of factors leads to the perpetuation of this situation, including the tropical climate, the simultaneous presentation of multiple diseases, innate or acquired host factors (micronutrient deficiency, immunodeficiency, co-morbidities), lack of resources (low income) to procure medicines and vaccines or other preventive tools, lack/misuse (due to lack of training or information) of (suitable) diagnostic tools, increase of pathogen resistance to antimicrobial medicines, as well as social and community factors. as a result, in , % of deaths worldwide were still due to infectious diseases; pneumonia, malaria, and sepsis were the leading causes of death, particularly in africa [ ] . moreover, while the borders between tropical diseases -thought to be confined around the equator (such as ebola) -and infectious diseases classically related to northern countries (such as influenza) were discrete, climate change and the increase in population migration contributed to a blurring of these borders, thus making the spread of febrile illnesses a global reality and concern. many people with acute infectious diseases develop fever, which is a frequent complaint, observed in approximately % of pediatric and adult patients presenting with an acute medical condition to health facilities. the estimated incidence of febrile episodes in malaria endemic countries varies according to studies but is generally between and episodes per person per year in children < years [ ] . despite the fact that all these infectious diseases cause fever, they can result from very different types of pathogens -parasites, viruses, bacteriaeach of which requires a different type of treatment and management. malaria, which was a major cause of fever a few decades ago, has now https://doi.org/ . /j.mee. . . given the still high (and often increasing) numbers of febrile (and related) illness cases and accompanied mortality globally, the (re-) emergence of global threats (e.g. ebola in ) and the increasing resistance to antimicrobial medicines, there have been several conventions at the international level with the aim to define a roadmap to tackle these challenges. the most significant conclusions and decisions deriving from summit meetings are summarized below. the world health organization (who) has changed its policy regarding the case management of malaria, to shift from presumptive treatment to laboratory-based confirmation of malaria before treatment [ ] . thus, wide access to malaria diagnosis is advocated to protect available antimalarial medicines against the development of resistance. according to the who global report on surveillance and antimicrobial resistance, the emergence of drug resistance due to misuse of antimicrobials is one of the major current public health threats worldwide. new tools that could mitigate this threat are urgently required [ ] . in a summary of the lessons learned from the ebola epidemic, bill gates mentioned that: "we need to invest in better disease-surveillance and laboratory-testing capacity, for normal situations and for epidemics. routine surveillance systems should…detect early signs of an outbreak beyond their sentinel sites and be quickly scaled up during epidemics. they should be linked with national public health laboratories to enable robust monitoring and response." [ ] . in an ebola-focusing summit that was convened by the paul allen foundation in april , one of the conclusions stated that "the greatest unmet need…is a field-test for use in community or village settings… to help determine whether isolation and/or referral is indicated." [ ] . during an expert meeting at who in june focusing on interoperability standards, it was concluded that there is a clear need for interface systems between diagnostic tools and e-health technologies, aiming at both patient management and epidemics control [ ] . these conclusions and directives coming from multiple sites strongly demonstrate the need and the motivation that exists to support the development of new, innovative diagnostic tools that can efficiently resolve the aforementioned challenges related to febrile illnesses. the first step to improve the management of patients with febrile illness is to estimate the distribution of the various syndromes and pathogens that cause fever. even though the distribution of fevercausing diseases varies by geography, season, age and immunity of patients and the level of care, some findings were similar among studies [ ] . these were related to cosmopolitan diseases found in most places worldwide, such as pneumonia or urinary tract infection. for other diseases, with various levels of endemicity, such as typhoid or rickettsiosis, or that occur rather in an epidemic way, such as arbovirus infections, the prevalence varied significantly according to the geographic location and, even more, according to the season or the calendar year. in studies that have recorded the clinical presentation of patients (and not only their laboratory results), the causes of fever in outpatients could be classified into four main syndromes: ) acute respiratory infections (ari, of any type); ) diarrhea (gastroenteritis); ) fever with another clear focus (e.g. meningitis or skin infection); and ) non-specific fevers [ ] (each diagnostic platform described in section focuses on at least one of the aforementioned cases). even though important diagnostic challenges remain regarding the first three syndromes, clear clinical guidelines have been developed by who, such as the integrated management of childhood illness -imci [ ] , to help clinicians to assess their patients and to decide on their treatment. because they correspond to localized infections, these syndromes are generally diagnosed using the best available clinical predictors, while diagnostic tools are indeed not included. in contrast, regarding the th syndrome (non-specific fevers), very few guidelines are available. the main challenge faced by clinicians is the management of patients with fever without specific symptoms or signs to guide them to the source of infection, combined, in endemic areas, with a negative malaria test result (the "negative syndrome" as labeled by health workers in tanzania). these non-specific fevers, also called "fever without focus", are caused by a wide range of pathogens ( fig. and table ) that cannot be identified without accurate laboratory tests. recent studies that aimed at determining the etiologies of fever [ , [ ] [ ] [ ] [ ] have identified the following important diseases as causes of non-specific fever: (i) malaria continues to be a major global health problem. according to the world malaria report, countries were still malaria endemic [ ] . there were an estimated million cases of malaria worldwide in and an estimated , deaths, while % of all malaria deaths occurred in africa. malaria prevalence varies widely, from to % of all fevers. indeed, the decrease in malaria transmission in many places in africa [ , ] has led to significant heterogeneity in transmission. the prevalence of malaria among patients with febrile illness has, therefore, become unpredictable and has triggered the switch of the who malaria case management policy from presumptive treatment, to treatment based on laboratory confirmation [ ] . (ii) urinary tract infection was an important cause of fever in young children (generally younger than two years old) that cannot be suspected on the basis of a history of urinary symptoms at that age (and therefore, it is included in the category of non-specific fevers). the prevalence among all children under five with febrile illness was - %. (iii) enteric fever was a significant cause of fever in children older than two years old and in adults, accounting for - % of all fevers, with salmonella typhi being the most frequent pathogen identified, followed by salmonella non-typhi. (iv) other bloodstream infections, mainly due to streptococcus pneumoniae and escherichia coli, accounted for about % of all fevers. other systemic bacterial diseases, such as leptospirosis ( . - %), rickettsiosis ( - %), q fever ( - %), and brucellosis ( %) were also important causes of fever, especially in adults who were more exposed to zoonoses and other environmental-related diseases than children. (v) dengue was also an important cause and seemed to emerge in several african countries. its prevalence is extremely variable, not so much geographically but temporally (seasonal and year to year variation). (vi) cosmopolitan viruses, such as human herpesvirus , parvovirus b , epstein-barr virus, and cytomegalovirus, which are transmitted mainly through close contact within family members, were mostly prevalent in children, accounting for % of all fevers and % of non-specific fevers in children [ ] , while adults were more susceptible to vector-borne diseases such as chikungunya or west nile virus. acute hiv was found in a few patients. (vii) co-infection with two or even more pathogens was frequent (in one study, % of pediatric patients had more than one disease simultaneously, fig. ), especially when an outbreak overlapped with an endemic situation, as it happened in a fever study in tanzania in . suddenly, in january , a significant outbreak of dengue started, and at the peak of the epidemic, almost % of patients presenting with fever were infected with dengue virus of serotype [ ] . because of the absence of diagnostic tools outside the study sites, most of these patients were considered as malaria cases and treated with antimalarial medicines or, in case that malaria tests where available, they were considered as possibly infected by bacteria and treated with antibiotics. (viii) regarding hiv, chronic hiv is an important co-morbidity, especially for adults ( % were positive in two tanzanian studies, while hiv prevalence in the corresponding community was to %), and should, thus, be systematically screened for, which is unfortunately not the case presently in africa. whatever the etiology, the first step in assessing febrile patients is to triage them as having a severe rather than a mild illness [ ] . objective point-of-care (poc) biosensors (such as oximeters), in addition to the subjective assessment of clinical danger signs (such as lethargy or severe dehydration), are helpful to correctly triage patients and should be integrated in the evaluation process [ ] . when patients present with a mild disease, ambulatory management with appropriate home-based treatment is sufficient [ , ] . the main challenge is then to identify the few patients who will benefit from antimicrobial treatment among all those with self-limited infection. presumptive treatment with antimicrobials should be avoided as most of these infections are of viral origin (fig. ) , especially in children (except for malaria that can be ruled out by a rapid diagnostic test). follow-up of patients to detect persistent or new symptoms and signs will then allow for the detection of rare clinical failures. at the primary care level, the decision to administer an antimicrobial should be based on an initial classification of the condition in the four categories mentioned in section . , combined with an accurate test specific for each syndrome that can distinguish between bacterial and viral etiology. as the existing host biomarkers lack accuracy (for example, c-reactive protein to identify the few bacterial pneumonia cases among the many viral respiratory infections), they should be combined with clinical predictors (e.g. fast breathing [ ] ) to improve the overall specificity [ ] . patients with a rather severe febrile illness (most of them satisfy the criteria for sepsis) should be admitted and assessed for the different etiologies of fever, irrespective of the syndrome they present with. in sepsis, the symptoms and signs are indeed often unspecific to allow for initial classification into the four syndromes mentioned in section . and all possibilities should be considered [ ] . under these conditions, accurate laboratory diagnosis is important, and the infections listed in section . (taking into account specific exposures mentioned by the patients and the local epidemiology) should be tested for, whenever possible. the results of the laboratory tests should then still be interpreted in the clinical context, i.e. the pre-test probability of the disease and the likelihood ratios of clinical predictors. indeed, for laboratory tests that have imperfect accuracy, these elements should be considered to calculate the post-test probability and be able to safely include or exclude certain diseases [ ] . if this probability is above the "treatment threshold" (above which one treats the patients instead of continuing investigating them using tests), the appropriate antimicrobial should be administered [ ] . if the probability is below the diagnostic threshold, no specific treatment is required, besides adjunctive treatment (e.g. i.e. acute respiratory infections, gastroenteritis, skin infections, meningitis and naso-pharyngeal infections (infections due to enterovirus, adenovirus or influenza, however presenting without any respiratory symptom). source: figure provided by v. d'acremont, adapted from [ ] . classification of fever-causing pathogens in africa (parasitic, bacterial, viral, fungal systemic infections). "na": not applicable; "?": unknown; "naats": nucleic acid amplification technologies; "lft": lateral flow test. diseases not included because generally considered after more than one week of fever: sinusitis, visceral leishmaniasis, amoebic liver abscess. diseases not included because, although they are fairly prevalent, they rarely present with fever: pertussis, cryptosporidium. diseases not included because clinical diagnosis is generally sensitive enough and specific enough: measles. a medium sensitivity in hiv patients with low cd cell count (urinary test). k. mitsakakis et al. microelectronic engineering ( ) intravenous rehydration in patients with severe diarrhea) and treatment for chronic conditions. if the probability falls between these two thresholds, further testing (when available) may be required and a second-line test should then be used. in practice, antimicrobials are often administered presumptively while waiting for the test results (except for malaria that can be excluded in < min based on the results of a rapid test). in summary, evidence-based integrated management of fever is a complex process, both at hospital and primary care levels, that would clearly benefit from electronic clinical decision algorithms (ecda) based on mobile technology [ , , ] to guide clinicians through the necessary steps, reach an accurate evaluation of the disease probability, and decide on the most appropriate treatment and management. electronic clinical decision algorithms are e-health tools pertaining to the broader category of "computerized decision support systems" (cdss). contrary to differential diagnosis generators (ddx) that provide a long list of possible diagnoses and have shown poor accuracy [ ] , ecda provide structured decision pathways that align with the clinician's workflow and guide the clinician throughout the consultation, including on final treatments and advice (section ). the diagnostic tools target in-and outpatients that visit urban and rural hospitals, peripheral health facilities and laboratories, and community level health posts. in particular, the following broad categories of target groups are identified: (i) patients living in resource-limited countries are clearly in need of new diagnostic tests for infectious diseases. these patients are very diverse in their immune status, the chronic conditions they may suffer from, and their ability and willingness to pay for a diagnostic test. (ii) people traveling from endemic to non-endemic areas. although it is difficult to determine the number of returning travelers and migrants to northern countries that are infected with an endemic disease, because of variation in reporting methods for different pathogens, an estimated , - , cases of travelers with malaria are, for example, reported in europe each year [ ] . the number of people crossing borders between the southern countries is much higher and represents also an important target group. they can be tested at airports, harbors, cross-border controls, immigration control checkpoints, where reliability and short time-to-result tests are required. (iii) pre-and post-vaccination, elimination, and screening programs run by local governments, international organizations, and pharmaceutical industries. disease prevention and elimination programs should be supported by comprehensive evidence to demonstrate the underlying prevalence of the pathogen in question and the efficacy of the strategy proposed. using a multiplexingcapable diagnostic platform allows for effective modeling of the: (a) true prevalence and variation of multiple species, enabling the monitoring of impact of the intervention on disease dynamics; (b) local and regional differences in pathogen distribution. (iv) epidemics surveillance, particularly of interest in: (a) sentinel sites, where the diagnostic tools can be installed at strategic geographic locations, in order to cover several regional, cultural, environmental, and epidemiological settings; (b) ministries of health and regional alarm centers that should regularly receive reports from the sentinel sites for assessment of the frequency of patients and the diversity of diseases. the diagnostic systems should operate independently of patients' age, although there are some inherent difficulties to acquire proper quality sample matrix from an infant (e.g. sputum for alleged respiratory infection). furthermore, for some tests, such as blood culture or polymerase chain reaction (pcr) for bloodstream bacterial infections, the same diagnostic tool may have different performance in different age groups, due to different pathogen load [ ] . not all diagnostic tools are, thus, suitable for all cases. in addition, the training level of the users, the facilities available at each level of care, and the target patient population are parameters that developers of novel diagnostic tools must take into consideration. there is an extensive debate regarding whether diagnostic tests with short time-to-result, but applicable only in laboratories, are true poc technologies or if the term "poc" should be used for tests that can be performed "under the tree". considering that diagnosis should be closely associated with treatment and that, at the peripheral level, there is often shortage of health staff and medicines, the "point-of-care" becomes by default the "point" where "proper care" can be provided, and this may well be a clinic, a peripheral lab, or a hospital, depending on how well-equipped these settings are, especially in terms of human resources [ ] . the diversity of available diagnostic systems, but more importantly of target groups and local requirements, has posed the need to define generic requirements. to this scope, the "assured" criteria were developed [ ] to summarize the characteristics of the ideal diagnostic test for the developing world, which should be affordable by the target groups; sensitive and specific; user-friendly by limited trained personnel; rapid and robust; equipment-free; and delivered to those in need. these criteria were initially developed for sexually transmitted infections but are generic for developing countries-targeted diagnostics. however, it has been evident that the users' requirements call for elaboration (or "fine-tuning") of these criteria. for example, the "equipment-free" criterion may be risky, because saving the test results in the device decreases the risk of mis-interpretation by inexperienced personnel. therefore, there is an increasing trend to use mobile technology to read results provided by lateral flow tests (lfts). moreover, there is need for guidance on how to select patients who should be tested, interpret test results in the clinical context, and triage patients who should be referred and admitted due to severe illness. in case of epidemics, where several patients arrive simultaneously at the checkpoint, the capacity of the tool to process several samples at the same time is an advantage. the assured criteria, together with additional specific requirements, are summarized in target product profiles (tpps), which technology developers should meet during their development stages. examples of tpps exist for tuberculosis, ebola, and differentiation between viral and bacterial infections, and are driven by the who, the médecins sans frontières (msf), the foundation for innovative new diagnostics (find), and other organizations [ , ] . microscopy for screening of malaria parasites by examining a drop of blood smeared on a slide after proper staining remains a widely used diagnostic tool. giemsa-stained thick and thin blood films are used to detect the presence of plasmodium parasites, to quantify the parasite density, and to identify the species [ ] . considering the progress in light technology and the use of light emitting diodes (leds), microscopy has become more easy to use. however, it still requires well-trained personnel and a degree of experience to prepare the samples and to interpret the results. poor slide/ blood film preparation may lead to artifacts which are mistaken for parasites, while in reality they are bacteria, fungi, dirt, or cell debris. as a consequence, poor specificity leads to poor diagnostic quality [ ] . two studies in tanzania have shown that the low quality of malaria microscopy and the lack of trust by clinicians in microscopy outcomes resulted in higher mortality among patients with non-malaria febrile illnesses than patients with malaria [ , ] . an additional major disadvantage of this method is the fact that microscopy cannot identify sources of fever other than malaria (except for borreliosis, which is a relatively rare cause of acute fever). thus, in the case of malaria-negative diagnosis, or in the case of co-infection of malaria with some viral/bacterial agent, the microscopy-only diagnosis is not sufficient for clinicians to decide on the suitable treatment. for bloodstream bacterial infections, including typhoid fever, the main test used in equipped laboratories is conventional bacterial culture. for pneumonia, culture is also based on respiratory secretions, including sputum (but it is then impossible to distinguish between chronic carriage and infection), bronchoalveolar lavage, or pleural fluid. detection of pneumococcal bacteremia can orient the diagnosis; however, this test results in low yields, due to the relative fragility of these bacteria. indeed, the main general drawback of blood culture is the inability, except for a few cases, to diagnose localized infections such as pneumonia or urinary tract infection. for typhoid, in addition to blood culture, stool culture and/or bone marrow culture are used but are rarely available. other limitations are that cultures take several days to provide results and that collection of high quality samples from pediatric patients is rather difficult and often leads to false results [ , ] . in adults, sensitivity of blood culture is limited by the low bacterial load. specificity of blood culture is also hampered by contamination by cutaneous bacteria during blood withdrawal, because sample acquisition from two different sites to overcome this problem is rather challenging in practice, especially in children. lateral flow tests [ ] consist of a plastic device containing a composite assay strip flanked by a reagent/sample pad at one end and an absorption pad at the other. the strip contains two or more lines striped into the nitrocellulose membrane: one or more test line(s) consist of a disease-specific antigen or antibody and one line is used as a control. thus, both antigen-or antibody-specific tests can be performed. the reagent pad holds the dried conjugate consisting of colloidal goldlabeled antibodies or antigens specific for each test. the reaction is performed by adding the sample (typically finger-prick whole blood, or urine) to the sample pad, followed by the addition of a few drops of buffer. the sample is mixed with the conjugate, forming antibody-gold complexes that flow through the nitrocellulose membrane. when specific antibodies/antigens are present in the sample, they bind the respective conjugate at the test zone, indicating a positive result through the captured red-colored gold nanoparticles carrying the antibody/antigen of interest (fig. ) . multiplexing is possible with the lfts in two ways: either via multiple analytes on the same strip (one sample inlet for all), or via several strips on the same cassette (as many inlets as the number of strips). the former option is more challenging and less generic because it depends on the assays and the (compatibility of) reagents with all target proteins to be detected. the results are read visually after - min, depending on the test. a valid test result is obtained if staining of the control line is observed. the equipment-free readout, however, is not sufficiently subjective, while it becomes even more complicated when (semi)quantitative readout is desired, e.g. to assess the intensity of the strip line. in addition to the mis-interpretation and ambiguity of the test results, there is also the risk of data loss, which is not the case when a readout equipment (with data storage and transmission capacity) is used. especially in the case of multi-strip cassettes, the use of a readout unit may help to diminish the risk of confusion between results from different lines. thus, a current trend in lft diagnostics is towards an automated readout, such as the deki-reader that has been well validated in the field for malaria testing [ ] . several technologies have been developed based on colorimetric (e.g. colloidal gold or colored latex) or fluorescence detection, by commercial suppliers: esequant™ lft strip reader by qiagen lake constance (germany); an imaging system by axxin inc. (australia); the handheld image capture lft reader by detekt biomedical, lcc (usa); the cpoc™ reader from lre medical (germany); the handheld dcn fluorescent test visualizer (usa) [ ] . although these systems are well suited for developed countries, they are often not affordable for lmics. a more promising approach is the use of smartphones, which has several advantages over other systems to: (i) become increasingly accessible in africa; (ii) be used as an image processing device with the built-in camera and suitable software; (iii) be able to support an application (app) for processing the acquired data; and (iv) enable global positioning system (gps) tracking of patients during an epidemic (following the appropriate ethics rules and after related permissions). furthermore, data storage and transmission to external databases offers a high added value to lfts. the camera can also typically scan -or -d barcodes on the lft cassette in order to identify the type of test. to this direction, the non-profit global health organization "global solutions for infectious diseases (gsid)" [ ] is developing a mobile tool to read, digitize, and transmit poc diagnostic results for important infectious diseases. in cooperation with the university of washington (seattle, usa) and dimagi (boston, usa), gsid developed an open-source software to capture the results of many different poc tests and report them immediately via wireless technology. a proof-of-concept study was conducted in zimbabwe in [ ] . other examples of this approach are the mreader of mobile assay (usa), the assayquest™ from clearbridge bioloc (singapore), and the skansmart and skaneasy products from skannex (norway). extensive work on this field has been performed regarding software development, for example by novarum dx™ (uk), or the commcare mobile platform by dimagi (usa). due to the relatively low complexity in manufacturing lfts, there are > manufacturers worldwide for clinical applications (which is why we are not able to provide a list of these manufacturers). the average price of an lft is approximately us$ . several studies assessed the performance of lfts [ ] [ ] [ ] [ ] and in a non-exhaustive list of infections, lfts currently exist for malaria, dengue, typhoid, hiv, hepatitis b virus (hbv), hepatitis c virus (hcv), and recently ebola and lassa [ ] . however, the average quality of these tests is inversely proportional to the multitude of suppliers. it is often the case that their accuracy is inadequate [ ] . the main drawback of lfts that detect antibodies is that, at the acute phase, antibodies are often not yet present and the tests lack sensitivity. also, the dynamics of igm/igg can be complex and hard to interpret, especially when re-infection is possible (e.g. dengue). another problem is that antibodies -even igm -persist for several weeks after acute infection, which decreases the specificity of the test (e.g. chikungunya). for this reason, the who has a standard operating procedure for pre-qualification, and publishes and regularly reviews recommendations for lfts for particular diseases, especially malaria and hiv [ , ] . nucleic acid amplification technologies (naats) are based on sequence-specific recognition and amplification of unique target regions in the genome of pathogens to be detected. thus, naats are highly specific and sensitive, as single microorganisms can be detected. compared to microscopy (the reference in malaria diagnosis), naats are much more sensitive, while in contrast to culture methods (the reference in bacterial disease diagnosis), naat process times can be reduced from days to hours. the following sections present the most frequently used naats. polymerase chain reaction (pcr) represents the most commonly used naat-based diagnostic technology [ ] . in this reaction, a thermostable polymerase (taq, from thermus aquaticus) is employed. two primers are used that flank the target region of the template dna to be amplified. a three-step process is then repeated~ - times: (i) a denaturation step at~ °c, where the double-stranded dna (dsdna) template is denatured resulting in two single-stranded dna (ssdna) molecules; (ii) a primer annealing step at~ °c, where the primers are hybridized to the ssdna target region to be amplified; and (iii) an elongation step at~ °c, where the polymerase generates a copy (amplicon) of the target region starting from the primer in ′-to ′-end direction. in an ideal reaction, the targeted region of the template dna is doubled after each cycle. the (endpoint) amplicons are detected via gel-electrophoresis (non-specific method) or by using different kinds of specific molecular probes that generate an amplicon-specific signal. here, the signal also depends on the template concentration. the need for cyclic heating and cooling to perform the previously described steps for pcr renders this method relatively time-consuming (several hours depending on the configuration and the thermocycler) and energy-consuming (a power supply is required). recently advanced methods in dna amplification use isothermal naats (inaats) [ , ] , which overcome the need for thermal cycling. these methods are less energy consuming than pcr and can be potentially executed in battery or solar panel operated devices, which is an important advantage for their application at the point of care. furthermore, as a consequence of continuous amplification rather than cycling, the reaction will often generate detectable products faster than pcr. a disadvantage of inaats is in general (not referring to specific methods) that quantitation is difficult as they do not follow a strictly controllable reaction scheme. in all methods mentioned above and in the next sections, rna can also be amplified by the addition of a reverse transcriptase (rt), which transcribes rna into complementary dna (cdna) for further amplification. in the following sections some of the main isothermal amplification technologies are briefly described. nucleic acid sequence based amplification (nasba) [ ] is used to detect rna and is performed at °c. due to the fact that it targets rna, nasba was first used for the rapid diagnosis and quantification of hiv- [ ] . nasba reaction is facilitated by three enzymes (avian myeloblastosis virus reverse transcriptase, rnase h, and t rna polymerase) leading to the amplification product. in brief, during the amplification process, cdna is initially produced from rna templates through primer (primer ) annealing to the template rna and reverse transcription forming a cdna from the latter. during cdna production, rnase h hydrolyzes the rna template. the resulting cdna with the promoter sequence is annealed to a second primer (primer ) and double-stranded dna (dsdna) is produced with reverse transcription. following this step, t rna polymerase transcribes an rna molecule from the previously generated dsdna. the above procedure is cyclic and in each round the number of produced rna is increased. a unique advantage of this method is that it can selectively amplify rna in the presence of high dna background, while the process can also start with a dna template. in this case, the rnase h step in the non-cyclic phase is not necessary. with nasba, amplification of a nucleic acid sequence to > copies can be accomplished in~ min. loop-mediated isothermal amplification (lamp) was developed by eiken chemical [ ] and is based on a bst polymerase with strand displacement activity. thermal denaturation of dsdna is not required, as the polymerase itself can denature dsdna prior to elongation. due to the reaction temperatures employed ( - °c), dsdna is in a metastable condition and the structure may hybridize/dehybridize spontaneously. this enables primers to bind to the template dna in melted regions. six primers are used resulting in high specificity. the forward (f ) and backward (b ) primers, together with the forward inner primer (fip) and backward inner primer (bip), generate an artificial starting structure with single-stranded loop regions in each end. based on this "dumb-bell" structure, a cauliflower-like structure is generated with the fip and bip primers binding at the loop region. loop primers (lf, lb) may help to decrease the reaction time [ ] . this process results in very fast generation of dsdna products that can be detected either with intercalating dyes or with specific probes [ ] in potentially < min. in short, lamp is advantageous due to: (i) high specificity, through the use of a minimum of four primers that define six binding sites on the target; (ii) sensitivity equal to, or higher than pcr; (iii) robustness against inhibiting compounds. recombinase polymerase amplification (rpa) [ ] is typically performed at - °c. recombinase/primer filaments scan the template dna for homologous sequences. if a match is found, recombinase mediates the hybridization of the primer to the complementary sequence. the denatured part of the template dna is stabilized by singlestrand binding (ssb) proteins and the polymerase can extend the primer complementary to the template sequence. due to the low reaction temperature, the reaction may start at will, with the addition of mg + . this may be necessary, especially in applications where ambient temperature is the same as the reaction temperature. a recent international quality assessment of molecular detection of rift valley fever virus via rpa performed equally well as the best rt-pcr tests [ ] . helicase-dependent amplification (hda) is typically performed at °c. two primers are used to initiate dna replication by a polymerase. in a first step, the dsdna unwinding activity of a helicase is exploited to achieve denaturation [ ] . subsequently, the primers anneal to the single-stranded dna and then are extended by the dna polymerase. each dna duplex is doubled in one cycle. the produced dsdna molecules are separated by helicase and the chain reaction is repeated. similar to rpa, single-strand binding proteins are used to stabilize the denatured ssdna templates. the simplicity of the method, the uncomplicated cycle reaction, and its high speed (especially for circular hda (chda), speed can reach bp/s) are the major advantages of hda [ ] . hda has been demonstrated to be efficient with several different types of sample matrices, including urine, stool, blood, and plasma [ ] . further promising dna isothermal amplification techniques, such as strand displacement amplification (sda) [ ] , nicking enzyme amplification reaction (near) [ ] , strand invasion based amplification (siba) [ ] , or rolling circle amplification (rca) [ ] are available and advantageous in various applications. despite the advantages of naats regarding sensitivity, a drawback is the relatively high cost of the reagents, compared to the cost of lfts. the main cost driver in naats is typically the amplification enzyme (the polymerase). cost further increases when viral rna should be amplified and a reverse transcriptase should be incorporated in the reaction mix. in the case of some inaats, the assay design can be very complicated; for example, primer design in lamp or the simultaneous use of three enzymes, as required in nasba. costs increase further, when enzymes are not pre-stored in liquid, but in dry format (e.g. lyophilized). such approaches may be required to help enzyme stability at the challenging environmental conditions of tropical regions. however, enzyme activity loss may be observed in lyophilized compared to liquid reagents. further problems may arise as naats inherently measure both living and dead pathogens (as both can provide template dna). this may lead to false positive diagnosis, for example during monitoring the progress of a patient over a treatment period. from the infrastructure point of view, specialized laboratory facilities are needed. in this case, well-trained personnel must also be employed to achieve proper assay set-up. in case of multiplexed assays, the risk of human error (e.g. pipetting error) increases, even with experienced personnel. to reach satisfying sensitivity, it is generally still necessary to purify nucleic acids from raw sample materials, such as blood, sputum, or urine. such purification processes may represent > % of the manual workload. the labor costs for manual workflows add substantially to the overall costs. this section summarizes poc and near-patient platforms for nucleic acid extraction and amplification, representing a big category of emerging and promising technologies that address the end-users' needs expressed in sections . and . , and overcome some challenges outlined in section . related to lack of automation. in this context, naats mentioned in section should be integrated into automated systems in order to overcome the need for specialized personnel as well as laboratory infrastructure. this includes reagent pre-storage in the system for full hands-off operation. the automated platforms must withstand the challenging environmental conditions (temperature, humidity) of the tropical regions. there are dozens of technologies for diagnostics, in various stages of development, used in various settings and detecting various targets. the list is long, and it would be impossible to include all in the present review, which does not aim to provide a list of all existing technologies, but to present those that apply in the context defined in the previous sections. within this framework, we applied a specific methodology and followed certain (inclusion/exclusion) criteria to select which technologies to present. in particular, we included those that: (i) focus on febrile illness and other syndromic infectious diseases with the potential to be expanded to febrile illness (thus, we excluded technologies for blood chemistry/clinical chemistry analyzers); (ii) include at least nucleic acid amplification testing, which can also be combined with immunoassays (thus, we excluded technologies for cd cell counting); (iii) are at least at late stage development, pre-commercial, or commercial stage (thus, we excluded academic, research, or laboratorylevel solutions). the description of each platform is divided in two sections: the performance as well as the technology and operating principle behind the platform. the former refers to issues such as portability, modularity, time-to-result, etc. the latter describes the core technical component(s) and main innovative features, e.g. the detection technology, the fluidics handling, and the manufacturing technology. in short: (i) in terms of detection technology, most platforms use optical methods (fluorescence in real-time signal monitoring or imaging mode; or light scattering as in verigene®), thus, it appears that progress on the label-free technologies is still required to reach commercial level. exceptions are the q-poc™ that uses silicon nanowires for detection and easynat™ that uses an integrated lft. (ii) regarding fluidics, it is noteworthy that not all described technologies implement microfluidics but also "macro"fluidics. in particular, enigma® ml and cobas® liat use cartridges that incorporate macrofluidic handling (e.g. syringes, plunges). fluid handling is mostly pressure-driven, although centrifugal microfluidics are increasingly used (e.g. labdisk, genepoc™, liaison® mdx). (iii) manufacturing-wise, the majority of the cartridges are plastic disposables, with some exceptions that are based on cmos (complementary metal-oxide-semiconductor) technologies such as the vereplex™ chip and the q-poc™ silicon nanowire array. (iv) in terms of amplification, all platforms allow for both dna and rna analysis. most of them use rt-pcr although isothermal amplification is also increasingly used (e.g. in alere™ i, easynat™, labdisk). notably, the assembly "assay-cartridge-instrument" is common in all cases, constituting a platform-based approach. regarding performance, some of the platforms aim at single or few pathogens (e.g. genexpert®, alere™ i, easynat™, cobas® liat, q-poc™, genepoc™, liaison® mdx), while others follow a syndromic approach, offering panels of several pathogens being simultaneously tested (e.g. filmarray®, vereplex™ biosystem, verigene®, diagcore®, labdisk). furthermore, it is important to emphasize that many of the technologies have a sample-to-answer time > h (except alere™ i, cobas® liat, and q-poc™), while the cost is in the range of tens of us$ for the cartridge and several thousands of us$ for the instrument. therefore, naats seem to be outcompeted by lfts. however, the added value that multiplex naat platforms offer in comparison to the lfts should be taken into account. moreover, the cost should be considered in a broad perspective, taking into account "hidden" materials and labor costs when comparing with the standards of care. a comprehensive overview of the technologies and some of their technical features are shown in table . performance features (e.g. time-to-result, portability) are mentioned in the platform description but not in the table, as the latter intends to focus mostly on technical aspects. furthermore, the information provided regarding the performance characteristics is based on the most up-to-date search on publicly available resources. the authors cannot exclude the possibility that these data may be updated in the future by the developers/manufacturers during the progress of the platform, or that manufacturers terminate or delay the development/manufacturing/market entry due to corporate or financial reasons. furthermore, table summarizes some "customer" related information such as prices of instrument and test, pathogens/syndromes detected, and regulatory status. this information is provided for those platforms that public information is available, and it may vary between the european and us market. the genexpert® system ( fig. (a) ) from cepheid [ ] is a widely used system in developing countries implementing molecular diagnostics to identify tuberculosis (tb) [ ] [ ] [ ] . despite the complexity of its cartridge (assembly of more than five parts), the system was adopted by resource-limited countries due to the endorsement of the xpert® mtb/rif test for tb by the who in and the financial support by the global fund for aids, malaria, and tuberculosis. the system contains several additional versions to detect drug-resistant tb, hiv, mrsa, chlamydia trachomatis, neisseria gonorrhea [ ] , while it included ebola testing during the outbreak. the time-to-result is estimated to be min. the system of the xpert® series is developed in single-cartridge or multi-cartridge configurations for the end-users to decide which option is more suitable to their needs: low/medium throughput ( -, -, -, -cartridge system) for the point-of-care, or high throughput ( -cartridge station) for centralized laboratories. the most portable system of the xpert® series aims to truly decentralize tb diagnosis. the genexpert® omni* ( fig. (b) ) is a single-cartridge version, battery operated and wirelessly connected to the cloud (operable also via smartphone), which allows highly decentralized utility and real-time data transmission. the price of a test is us$ - in the us market, however there are special arrangements with ngos that subsidize this test for tb in developing countries, thus eventually costing slightly below us$ (excluding shipping and distribution; this is the sales price ex works). the genexpert® iv device cost is approximately us$ , , while the genexpert® omni* costs less than us$ , . the genexpert® is a sample-to-answer system, where sample preparation and extraction of nucleic acids take place in the cartridge in an automated way as the necessary reagents are pre-stored. only a few minutes of hands-on steps are required for liquefaction of crude samples such as sputum. regarding fluidics, the cartridge contains a total of chambers and reservoirs ( fig. (a) ). at its core, there is a cylindrical rotary valve, which is handled by a plunger rod that moves vertically, as soon as the cartridge is inserted in the processing device. the interface between the valve and the plunger activates the liquids (sample and pre-stored liquid reagents) movement between chambers, by positive or negative pressure through small openings at the base of the reservoirs. an important component of the cartridge is an integrated filter, which is located at the base of the valve body and captures the microorganisms. subsequently, a sonic horn approaches the filter area and mediates the mechanical lysis of the microorganisms and the release of nucleic acids. the latter are pumped through a chamber where beads with freezedried amplification reagents are stored. these are rehydrated upon sufficient filling of the corresponding chamber (the filling level acts also as internal fluidic quality control). the entire mixture is then directed to the pcr reaction tube, located at the back of the cartridge [ ] . regarding detection, the pcr tube is surrounded by heating/cooling plates that enable fast thermal response, and by optical blocks that enable amplification and real-time fluorescence-based pcr product detection. in contrast to traditional thermal cycling systems, in which all samples are subjected to the same time/temperature/optical protocol, each sample in a genexpert® system can be subjected to a different protocol. one more innovative feature of the processing module is the intelligent cooling/heating optical reaction (i-core) module [ ] , a sophisticated, solid-state, miniaturized microelectronic and micro-optical system capable of performing pcr reactions with multichannel fluorescence detection in < min. each i-core includes a four-channel optics system capable of exciting and detecting multiple fluorescent dyes in the same reaction tube, thereby being able to accurately measure four dna targets simultaneously. the filmarray® (fig. ) from biomérieux [ ] is a benchtop diagnostic instrument that has three fda-cleared panels: the respiratory panel [ ] , the blood culture identification panel [ ] , and the gastrointestinal panel [ , ] . filmarray® responded to the ebola outbreak and received emergency use authorization (eua) from the us fda, based on the filmarray® biothreat panel that specifically detects ebola zaire. a field study was conducted in sierra leone in patients and healthcare workers [ ] . only a few minutes hands-on preparation time is required before the cartridge is inserted in the processing device, including the loading of the pouch into the loading block, the insertion of the hydration solution into the pouch, and the loading of the sample. the simplicity of use, the lack of refrigeration storage requirements, and the multiplexing capacity are positive features for use by limited- trained personnel. up to different targets can be analyzed per test, yet the throughput is still low, as only one patient can be screened per test run [ ] . in addition, the manufacturer's list price for the reader (~us$ , ) and reagents (~us$ /cartridge) may not be affordable in resource-limited settings. the filmarray® is a sample-to-answer system, whose core technology is based on the freeze-dried vacuum reagent pouch, and on the nested multiplexed pcr that allows for the detection of multiple organisms. the microfluidic pouch ( fig. (b) ) consists of two areas, namely the fitment, where the reagents are pre-loaded and freeze-dried, and the main body. these two are bonded via heat welding. the fitment is fabricated with injection molding of polypropylene. the main body is fabricated with two sheets of a polyester/polypropylene film that contain a copolymer adhesive layer. these layers are welded together using heated plates. the fitment contains reservoirs (a in fig. (b) ) that contain the biochemical reagents. during the entire manufacturing process, vacuum is maintained in each well through a set of plunges (b). it is very important to maintain the vacuum in the fitment and the blisters because: (i) it helps to maintain the freeze-dried nature of the components and, thus, their long-term storage; (ii) it allows for the injection of the sample and the hydration solutions in the pouch in the correct volume; (iii) it minimizes the formation of bubbles in the microfluidic system. the sample matrix is a naso-pharyngeal swab for the respiratory panel (different matrices are used for other panels). a total of μl liquefied sample ( μl sample and μl lysis buffer) are used for the test. the user injects the hydration buffer (used for rehydrating the freeze-dried pre-stored reagents) and the sample in two differently designated inlets. the vacuum in the pouch automatically draws the correct volume, thus eliminating the need for precise measuring and pipetting by the user. liquid handling is applied through pneumatic pressure and "bladder" assembly. this contains a bladder plate on which several inflatable bladders are located, each corresponding to one chamber of the pouch. the assembly is subjected to different gas pressures and as it (and the bladders) is externally in contact with the pouch chambers, any change in the volume of the bladders causes liquid to move between the chambers of the pouch. the bladders are pressed through pneumatically-driven pistons (figs. , in [ ] ). the sample moves to the lysis chamber where the filmarray® performs mechanical lysis (bead beating) using ceramic (zirconium silicate) beads. the procedure follows the "boom chemistry" [ ] . the released nucleic acids are bound on silica-coated magnetic beads, which are transported from the lysis chamber into the purification chamber, using a magnet integrated in the device. in this chamber, any remaining cellular or viral debris are removed through washing. an elution step releases the nucleic acids from the magnetic beads. the st stage pcr follows ( - cycles, including a reverse transcription step to convert rna into cdna), where dozens of primer pairs of all target molecules are mixed for multiplex pcr. a dilution step follows with the addition of a mixture of a fresh mastermix containing an intercalating fluorescent dna dye. subsequently, the mixture is aliquoted in a high-density array of microfluidic wells where the nd stage (nested) pcr [ ] takes place. the -step pcr is used to eliminate limitations associated with single-step multiplex pcr. at the end of the nd stage pcr, the temperature is slowly increased and the fluorescence in each well is measured to create a melting curve, the shape and position of which can be used for differentiation of amplification products with differences of < °c in melting temperature [ ] . for the two stages of pcr, the instrument has two peltier elements. a blue led is used to illuminate the array at the nd stage pcr and the fluorescence emitted by the dyes is imaged by a ccd camera. alere inc. markets the molecular diagnostics point-of-care system alere™ i (fig. (a), [ ] ) for the detection of influenza a and b [ ] , human respiratory syncytial virus (rsv), as well as group a streptococcus (clia-waived, different cartridges for virus and bacterium detection). recent studies have shown that the alere™ i system exhibited at least equal or superior sensitivity over conventional rapid tests [ , ] . the rapid isothermal amplification technology implemented by alere™ i (near, [ ] ) enables the sample-to-answer analysis within min. the instrument itself is portable and stand-alone (no need for a laptop), has a user-friendly interface, data storage capacity, and interface ability. the test price, as of , is approximately us$ - while the device costs approximately us$ , . the sample matrix is a naso-pharyngeal swab eluted in transport medium. the analysis procedure is from sample to answer with prestored reagents. the single use disposable test cartridge consists of three parts (fig. ) . the test base is inserted into the first available device slot. the test base has two reaction tubes, each of which contains a lyophilized pellet with a pair of templates (similar to primers) for the specific amplification of the target nucleic acid, and a fluorescentlylabeled molecular beacon designed to specifically identify the amplified targets. the sample receiver is inserted in the second available device slot. this cartridge contains the elution buffer to elute the pathogens from the swab ( s stirring of the swab in the cartridge). the transfer cartridge is pressed against the sample receiver to collect the necessary amount of material and then transferred and locked over the test base in order to initiate amplification. the amplified products are detected via dual-channel fluorescence, deriving from the fluorescent probes. indepth technical design or information regarding the cartridge are not publicly available in detail for this system. another system from the same manufacturer is the alere™ q hiv- / detect (fig. , [ ] ). it is a fully automated platform for hiv- and hiv- detection within min based on real-time pcr with multiplexing capability [ , ] . it is suitable for point-of-care use due to its portable nature, battery operation, no laptop requirement, easy to use touch screen, while the cartridge can be stored at room temperature without need for cold chain. the alere™ q platform has been launched in multiple developing countries, initially for qualitative hiv, and allows for finger-prick detection of hiv virus in whole blood collected directly into the cartridge ( μl are sufficient) that is then inserted into an instrument for automated processing, extraction, amplification, and detection. technology and operating principle of the alere™ q alere™ q hiv- / detect is a sample-to-answer system. lysis is based on chaotropic salts acting upon the sample and releasing the nucleic acids. biotinylated oligonucleotides that are specific to hiv- and hiv- genome hybridize with the post-lysis released viral rna, and are subsequently bound on streptavidin-sepharose particles. after washing, reverse transcription and pcr follow. detection of the pcr product is the core technological innovation of the system. it is based on competitive reporter monitored amplification (cma) technology utilizing an array of immobilized oligonucleotide probes and complementary fluorescently labeled reporter oligonucleotides in solution [ ] . these reporters compete with the amplicons for hybridization on the surface probes. as in the beginning there are only few amplicons, labeled reporters hybridize with the surface-probes, thus, the initial signal is high. as the pcr reaction progresses, the labeled reporters preferably bind to the amplicons, therefore, less reporters are bound on the microarray. consequently, the progress of pcr is monitored via the decrease of fluorescence from the microarray spots until a plateau in the amplification reaction is reached. fluorescence imaging follows the progress of the reaction, as one image is collected during each annealing cycle. regarding fluidics (fig. (b) ), the sample is inserted in a capillary (position ), the inlet being specially designed to receive finger/heel prick. a control window ( ) helps the user to assess the filling level. the cap ( ) ensures hermetic sealing and minimizes the risk of sample spilling or aerosols exiting the cartridge, thereby avoiding any contamination risk. dry reagents are pre-stored in internal compartments, along with a buffer reservoir ( ) . a fluidic network of valves in the cartridge regulates the liquid/air movement. a septum, which is pricked by a needle connected to the pneumatic module of the cartridge, is used to apply air pressure and move the liquids into the different compartments. the compartment ( ) is the reaction chamber where pcr takes place and the microarray is located. the enigma® ml (minilab™) (fig. ) is a point-of-care platform commercialized by enigma® diagnostics, which, since mid- , is in the process of selling their intellectual property underlying its point-of-care molecular diagnostics platform [ ] (nevertheless the platform is described here as one of the most representative ones). although the main application of the system has been on respiratory tract infections [ ] , its functional and performance characteristics would enable potential use in febrile illness diagnosis. indicatively, the pipeline of infectious diseases includes influenza a and b, as well as rsv. the enigma® ml requires no laptop due to its internal controller and user-friendly touch screen. the instrument is available in a modular configuration, i.e. in a single-module up to a six-module option (for one to six cartridges, respectively). each module can operate independently via the central controller, therefore, different assays (requiring different protocols) from the same or different specimens can be performed at the same time. the overall analysis time for each module is min. the analytical and diagnostic verification process of the fluab-rsv assay on the enigma® ml that led to the subsequent ce marking was completed in july , at guy's and st. thomas' nhs foundation trust, london, uk [ ] . the molecular analysis is based on real-time pcr. all required reagents are pre-stored in the cartridge allowing for fully automated analysis with a very short hands-on time of min, while there is no need for cold chain as the reagents can be stored at ambient temperature. the biochemical analysis is initiated with a combination of thermal and chemical lysis to release nucleic acids, which are then captured by magnetic beads. magnetic beads are robotically transferred in situ to two washing buffers for purification and removal of any inhibitory agents. the last step before amplification is the elution of the nucleic acids from the beads, followed by mixing with the amplification reagents (freeze-dried). each test contains bacteriophage ms as an internal process control. the cartridge consists of three main blocks (fig. ) : (i) the sample inlet block, where the swab is introduced in the collection tube that is then clamped onto the cartridge; (ii) the block with the pre-stored (liquid) reagents (indicated with yellow in fig. ) ; and (iii) the block with specific tools (movable components such as pipettors, magnetic wand, and foil cutter). the core of the cartridge operation is based on robotic actuation of fluids between the vessels in order to facilitate the biochemical reactions, handling of beads, etc. in particular, a robotic arm picks any movable items and inserts them in the corresponding liquid tubes [ ] . a highly innovative feature of the system is the pcr reaction vessel [ ] . it consists of an electrically conducting polymer (ecp), which acts as a resistive heating element. at the same time it is arranged in such a way that the electrically conducting profile differs in different areas of the vessel (along its axis), thereby controlling thermal gradients that are necessary for thermocycling. the different electrical conducting profile is regulated during the manufacturing of the vessel by varying its radial thickness (key parameter), thus varying unevenly its resistance profile. the vessel consists of a combination of a highly conductive material (aluminum) and an ecp, the two being separated by an insulating layer. the aforementioned induced temperature gradients also assist in convection-based liquid mixing in the reaction vessel. the ecp fabrication technology is typically injection molding. detection is achieved via hybridization probes and fluorescent resonant energy transfer (fret) [ ] . taking advantage of this effect, the enigma® ml system uses two probes to hybridize with the amplified dna/rna product, acting as energy donors and acceptors. when amplification occurs, the two probes hybridize and are located in close proximity, thus, the energy of the donor (induced by the excitation source) is transferred to the acceptor, which, in turn, emits this energy as fluorescence. the latter is then monitored in real time during the amplification process, as the amount of the acceptor fluorescence is proportional to the amount of the generated pcr product. roche diagnostics offers the cobas® liat (lab-in-a-tube) system [ ] as a compact solution for point-of-care molecular diagnostics (fig. ) . the system is currently used for detecting: (i) influenza a and b and rsv viral rna in naso-pharyngeal swab specimens from patients with signs and symptoms of respiratory infection in conjunction with clinical and epidemiological risk factors; (ii) streptococcus pyogenes (group a β-hemolytic streptococcus, strep a) in throat swab specimens from patients with signs and symptoms of pharyngitis. however, it is not a multiplexed system, as the aforementioned infections are detected in different cartridges. the instrument is compact and stand-alone (laptop-free) with small footprint operating in such way that minimizes possible interference by, and contamination of the user. with minimum training needed, the system can provide results in as fast as min. the cost of the device and cartridge is approximately € , and € , respectively. although the system has not yet been used for febrile illness diagnosis in developing countries, its performance features make it a good candidate. technology and operating principle cobas® liat is a sample-to-answer platform thanks to the full integration of all necessary reagents in its cartridge. the in situ sample preparation starts with mixing the sample ( μl) with chaotropic agents inducing pathogen lysis, followed by the "bind-wash-elute" protocol (boom-chemistry [ ] ), which involves binding of nucleic acids in silica-shelled magnetic beads, subsequent washes and a final elution step. nucleic acids are then ready for real-time pcr with internal controls. the core technology of the system is the cartridge, which consists of a rigid frame and a transparent flexible tubule. the latter consists of several pouch segments, which are separated via breakable seals and are flexible enough to be compressed. the system uses peristaltic fluidic actuation for liquid handling. after the cartridge is loaded, the analyzer, which contains clamps, plunges, and actuators, controls the selective release of reagents from tube segments, leading to mixing and propulsion of liquids by precisely applying pressure to the pouches. the pressure needed to compress the tubule to unload the pouch is approximately atm. the analyzer also provides control of reaction conditions at different temperatures, and alternating movement of the reaction mix between two different temperature zones for rapid pcr (fig. ) . the outcome of the reaction appears as a real-time fluorescence signal, detected by the six-channel photometer module in the analyzer [ ] . from the fabrication perspective, the tubule is based on polyolefins and is fabricated via injection molding or blow molding. in order to serve specific processing needs, some tubules are surface-processed via plasma treatment or introduction of additives. the wall material is multi-layered, with the inner layer being biocompatible, while the outer is resistant to gas permeability. the flexible tubule is supported by a rigid frame, which is made of polypropylene with lower melting temperature than the tubule to ensure uniform melting during sealing. according to the relevant patent [ ] (although the patent does not ensure that these exact methods are used in the final production) sealing between these components is achieved via welding of the flexible tubule on the outer frame using thermal and/or ultrasonic sources. an alternative approach is to seal the two components with a hot-melt adhesive joint with ethylene vinyl acetate, or with uv cure epoxy. the dimensions of the frame, surrounding the tubule cartridge, are mm (height) × mm (width). the agency for science, technology and research (a*star), singapore, stmicroelectronics, italy, and veredus laboratories, singapore [ ] have partnered to develop a lab-on-a-chip for diagnosis of infectious diseases (fig. ) . the system is already in the market and was used by the brazilian authorities as the tool of choice during the world cup. a number of application-specific cartridges have been developed, some of which are: vereflu™ (for respiratory infections); veremtb™ (for tuberculosis); verethreat™ (for biothreat agents such as anthrax, small pox, plague and tularemia); verevet™ (for multiple avian pathogens); veremers™ (for the middle east respiratory syndrome coronavirus). in the partnership announced the launch of veretrop™ for multiple tropical diseases, providing the necessary multiplexity that febrile illness diagnosis requires. the cost per veretrop™ test is approximately us$ . sample preparation is performed ex situ, either via spin columns or an automated extraction kit, and the overall time-to-result (including extraction, amplification, optical readout) adds up to approximately . h. vereflu™ [ ] and veretrop™ [ ] assay chips and multi drug resistant tuberculosis [ ] have been clinically validated. the core of the system is based on a lab-on-a-chip cartridge (the "in-check" platform, fig. ) in the size of a standard microscope slide. it contains a silicon chip covered with a glass coverslip and bonded to a plastic support. an on-chip printed circuit board (pcb) provides the necessary electric contacts as an interface to the processing instrument (e.g. for temperature control). the silicon chip has distinct processing areas (fig. ) : (i) the sample inlet microfluidic ports with buried microchannels to facilitate easy chip filling and fluid displacement. (ii) the pcr area ( × . mm ), which contains four pcr chambers, with μl liquid volume capacity, each. four pairs of independently controlled aluminum resistors are used to heat the silicon substrates and fluidic reactors, thus providing the thermocycling environment. the temperature is regulated by the temperature control system (tcs) in the chip processing device. (iii) the outlet holes, which provide microfluidic contact to (iv) the post-pcr hybridization and detection area, which consists of a . × mm microarray with spots, where oligonucleotide probes are spotted using a piezo-array system [ ] . fabrication is performed with a semiconductor-based mems process technology, named simich at ″ wafer diameter, which allows for the fabrication of micro-mechanical, sensor, and microfluidic components. more details on the process steps are available in [ ] . fig. shows some key microfabricated components of the chip and a cross-sectional view of the entire lab-on-a-chip device. post-fabrication processing includes surface chemical treatment in order to immobilize dna probes in the microarray area, and to achieve biocompatibility of the pcr area for dna amplification [ ] . post-amplification detection is performed in a separate, dedicated instrument, through fluorescent microarray detection of chip-hybridized amplification products using an image acquisition camera. quantumdx [ ] is a uk-based nanomedtech company that develops and commercializes affordable handheld sample-to-result diagnostic and dna sequencing technologies using nanowire biosensors and innovative microfluidic technologies. their collaboration with the institute of microelectronics (ime) of the agency for science, technology and research (a*star), singapore, is strategic for the fabrication of the key microelectronic components. the q-poc™ platform (fig. ) of quantumdx is a handheld, battery-powered molecular diagnostic device that promises a sample-to-answer dna-based result in as few as min. it contains integrated reagents with current stability targets of up to °c for up to months. the system can test one patient per run and is a pioneering approach to the handheld sequencing market. in , quantumdx partnered with find in order to develop a solution for the combined detection of tb and drug susceptibility determination for the causative agent of tb. the q-poc™ system seems to be especially promising for antimicrobial resistance profiling given that many different genes can be screened through a nanowire array. there are several other assays in the pipeline, for diagnosis of e.g. hiv, sexually transmitted infections, neglected diseases, as well as for tumor profiling [ ] . the core of q-poc™ lies in its biosensor chip, which is equipped with a silicon nanowire (sinw) array based field-effect biosensor. sinws have large surface-to-volume ratio, tunable electric properties, and exhibit high potential as biosensors, thus enabling next-generation point-of-care diagnostics. the binding events are detected in a labelfree way through changes in the nanowires' resistance, effectively converting the genetic code into a binary code and providing the output in a digitized format. in particular, upon hybridization of the target dna on the sinw, the charge density (and subsequently the electric field) changes. this is reflected as a change in the resistance of the nanowire, which produces the biosensing signal (fig. ) . the more negative charges present during hybridization, the higher the resistance of the n-type sinw sensor. this principle was shown by zhang et al., by varying the hybridization sites (thus varying the distance of the charge layer) but keeping the total number of charges unchanged [ ] . functionalization of sinws is performed with peptide nucleic acid (pna), instead of dna oligonucleotides. the neutral nature of pna minimizes the formation of dense charge layer near the nanowire surface, subsequently minimizing the signal to noise ratio. the n-type sinws feature typical dimensions of (w × h × l) nm × nm × μm and are fabricated using standard cmoscompatible top-down approaches, through the oxidation of polysilicon beams patterning using conventional photolithography on p-type test wafers (more details available in gao et al. [ ] ). this method is compatible with mass-manufacturing, is reproducible and convenient, and allows for scaling the sensing area and multiplexing as required by the application. with a capacity of nanowires in each silicon biosensor chip, the multiplexing potential becomes huge [ ] . the sample-to-answer operation is achieved by housing the biosensor chip in a microfluidic cartridge (fig. ) . ustar biotechnologies (hangzhou) ltd. [ ] commercializes its proprietary isothermal amplification technology into an equipment-free diagnostic system, the easynat™ (fig. ) . therefore, the system is especially attractive for resource-limited settings and is intended for batch testing in microscopy centers. however, the system lacks multiplexity ( target/cartridge) and requires hands-on extraction steps (centrifuge, vortex, syringe). the extraction is performed ex situ using an equipment-free extraction kit. after boiling lysis, a syringe is used to extract and elute the dna using appropriate buffers. initial tests with easynat™ kit have been performed in clinical studies in tanzania [ ] and china [ ] for tuberculosis. the manufacturers note that the reagents require refrigeration ("storage in − °c"). the system is based on an isothermal amplification method, called cross priming amplification (cpa), which uses multiple ( - ) crosslinked primers to amplify dna or rna at a constant temperature of reprinted with permission from [ ] . copyright © american chemical society. °c [ ] . due to its isothermal nature, amplification does not require any complex temperature control system: a water bath or a heating block are used in order to maintain constant temperature and incubate the cpa assays. following the min amplification step, the reaction tube is transferred to the cartridge, where a lateral flow immunochromatographic strip provides the visual readout after - min. the patented cross-contamination-proof (xcp) nucleic acid detection device (fig. ) offers the advantage of minimizing the risk of amplicon cross-contamination [ ] , through: (i) maintaining closed reaction tubes after amplification (thus avoiding release of amplicon aerosols); (ii) using an enclosed unit (the amplicon tube) to insert the reaction tube; (iii) placing the amplicon tube into an additional enclosed unit (the detection device). running buffer and reaction tubes are inserted in the amplicon cartridge. the amplicon cartridge is subsequently inserted into the detection chamber. as the sealing of the detection chamber is closed, it activates a blade that punctures simultaneously the two tubes. the running buffer drives the amplicon to the adjacent lateral flow strip and amplification results are visually readable (fig. (c) ). the detection principle in the lateral flow nucleic acid testing strip is based on a combination of the nucleic acid hybridization and immunoassay principles. a probe is used, which carries a colored particle, and can hybridize with the amplicon. the complex is then captured by the antibody on the nitrocellulose membrane of the test strip. in the absence of an amplification product, there is no interaction/hybridization with the probe, which then flows uncaptured through the membrane to the absorbent pad ( fig. (b) ). (i) meander-shaped microfluidic channel filled with reagents that are released at will to area (g)/(h). in particular, reagents may include various nucleotide mixtures and wash solutions, all separated by air bubble(s), in order to flow sequentially and in a well-controlled way. when nucleotides flow sequentially over a nanowire-immobilized single stranded oligonucleotide, then according to the hybridization sequence, the local charge changes, causing a resistance change in the sinw. image source: used from [ ] . luminex corp. commercializes the verigene® rp flex system, (fig. ) (developed and previously commercialized by nanosphere), which received an fda approval in [ ] . the target application is respiratory tract infections and the system covers a broad panel including viruses and bacteria [ ] . analysis is performed from naso-pharyngeal swabs. verigene® also offers other panels such as gastrointestinal, while it also identifies eight gram-negative organisms and resistance markers from blood culture [ ] . the full-automation mode of the system (pre-stored reagents) requires minimum hands-on time (< min), while the overall time-to-result is h. the system is benchtop and consists of two components, the reader (as a controller) and the processor sp, performing the automated test processing steps. it is a modular system since one reader, which acts as the user interface and central control unit, can handle several processors sp. each verigene® test uses four single-use consumables: an extraction tray, a tip holder assembly, an amplification tray, and a test cartridge. these are all loaded in the processor sp for all automated processing the glass support of the test cartridge, which carries the microarray, is inserted in the reader of (a) for detection. image sources: (a) used from [ ] ; (b,c) reprinted from [ ] , copyright © , with permission from elsevier. steps, specimen extraction, target amplification, and hybridization. fluidic handling is performed via an in-system servo actuator and positive displacement pump, e.g. syringe pump, in combination with pneumatic valves. a comprehensive sequence of steps is provided in one of the company's patents [ ] and the system's technical bulletin [ ] . after sample processing in the extraction and amplification trays, nucleic acids are transported to the test cartridge. at the end of the procedure, the substrate (which supports a glass slide with the microarray for capturing the target nucleic acids) must be removed from the cartridge and placed in the reader (readout takes a few seconds). all necessary reagents are contained in the cartridge. the amplification technology is based on multiplex rt-pcr between heating and cooling zones followed by hybridization to target specific capture oligonucleotides in a microarray format. end-point detection is achieved with gold nanoparticles (aunps), which constitute the core technology of the system (proprietary nanogrid technology, fig. ). with dimensions of - nm, the advantage of aunps over conventional fluorophores is the increased sensitivity by several orders of magnitude (e.g. the light scattered from one nanoparticle is equivalent to the light emitted from , fluorophores [ ] ). after amplification is completed, the workflow for detection is as follows: inside the test cartridge, the amplified targets are hybridized to capture oligonucleotides that are pre-immobilized on the glass slide microarray. after removal of the unbound dna, the hybridization products are labelled with aunps. subsequently, intermediate oligonucleotides are flushed into the chamber and bind specifically to the immobilized complex, leaving a poly-a tail sticking out and being available for subsequent attachment of poly-t-bound aunps, which act as probes. after removal of unbound aunps, captured aunps are covered by silver nps (during the analysis procedure) to enhance the scattered light. thus, the overall size of the nps becomes larger and the subsequent optical signal is enhanced. illumination begins with an led (λ max nm) shining through the edge of the glass slide (acting as a waveguide to the led illumination). the evanescent wave that is generated through the glass slide and towards the np coated surface causes light scattering from the illuminated au/silvernps towards the camera, which captures images of the array multiple times and at various exposure times [ ] . notably, the aunp-based detection technology may be adapted for protein analysis as well. the spanish molecular diagnostics company stat-dx [ ], acquired by qiagen in january , has been developing the diagcore® (fig. ) platform operating as a near-patient testing solution for low test volumes or as a platform to cover mid-throughput requirements. the company announced in early the approval of ce-ivd clearance for the system and the respiratory panel. stat-dx expects full commercial launch of the diagcore® in the nd quarter of . the the cartridges of diagcore® are pre-filled with dry and liquid reagents with a shelf-life stability of months at room temperature. the cartridges can be inoculated either using swab directly (with the appropriate cartridge housing for sample elution [ ] ), or using liquid samples of volumes from μl to . ml. automated cell lysis is used for releasing nucleic acids from pathogens. nucleic acid purification follows using high quality extraction columns. the sample is then subjected to rapid thermocycling for amplification; eight real-time pcr reaction chambers with six-plex capability allow for the simultaneous detection of a total of targets. regarding fluidics, the cartridge includes a transfer module that moves laterally along a guide that is located within the cartridge in order to align various fluid ports and to control the movement of fluids through the cartridge channels and chambers [ ] . fig. (a) shows a typical cartridge from the back side, where the areas designated with the numbers # , # , and # represent the testing unit, the dilution/dosing unit, and the fluid guiding unit between various chambers, respectively. the testing unit is shown in more detail in fig. (b) . the fluidic testing arrangement is aligned with the gravity field in order to avoid formation of bubbles in the various chambers. the inlet port of the structure is indicated as # , while the top chamber (# ) is unvented and acts as an air reservoir for the air trapped within the microchannels. by avoiding venting, the elimination of aerosol leakage out of the cartridge is achieved. channel openings (# a, # b) are used as fluidic controls, where optic sensing defines whether there is liquid or not. a mixing chamber is included (# ). its large crosssection causes turbulent liquid flow, which enhances mixing (passive mixing). the chamber # is the actual reaction chamber (with a volume of up to μl) serving also for detection. chamber # contains dry stored reagents. by applying variable pressure in the inlet port # , the liquid may be pushed to chamber # , rehydrate the reagents, which are then pulled back to chamber # in order to participate in the reaction. the structure indicated in fig. (b) may be repeated several times, with a common inlet port and different stored reagents, in order to serve in geometrically multiplexed reactions. the concept of centrifugal microfluidics (in contrast to pressuredriven microfluidics that all the aforementioned platforms use) has evolved to a mature and powerful technology for automation of diagnostic workflows in the fields of clinical chemistry, immunodiagnostics, nucleic acid analysis and others. this microfluidic approach benefits in particular from the straightforward mechanism for liquid actuation based on centrifugal forces that are a result of simple rotation of the cartridge with defined spinning frequencies [ ] . no external pressure sources or complicated (micro)pumping systems are required, which reduces the risk of cross-contamination (due to openings in the cartridge) and allows for the building of simple, portable processing devices with small footprint and weight. these facts qualify centrifugal microfluidics as a technology for automation of diagnostics at the point of care. a main advantage of centrifugal microfluidics is the employment of microfluidic modules ("unit operations"), each one serving a particular fluidic functionality for: liquid handling (e.g. metering and aliquoting [ , ] , valving and switching [ ] [ ] [ ] , pumping [ , ] or mixing [ , ] ); assay integration (reagent pre-storage and supply [ , ] ); separation [ , ] ; detection [ , ] ; blood plasma separation; nucleic acid purification and amplification, or washing and blocking. a comprehensive overview on unit operations and process chains is provided by strohmeier et al. [ ] . combination of these unit operations and process chains allows for the integration of complex laboratory processes into one system for fully automated sample-to-answer analysis. challenges are mainly in the field of cartridge-integrated sample preparation required for completely integrated sample-to-answer solutions [ ] [ ] [ ] . apart from the world-to-disk interface that defines how the sample can be added by the user to the cartridge, the preparation of complex samples such as sputum, a classical specimen in diagnostics e.g. for detection of tuberculosis [ ] , is an unsolved issue yet, as samples can be diverse in their physical constitution, and harsh chemicals or strong forces are required for pretreatment. further challenges are the handling of increased sample volumes (> ml) of blood or urine that are required when the pathogen load is low or when the required clinical sensitivity for diagnosis is high. the maximum sample volume is typically limited by the available footprint of the disk and it has to be considered that e.g. for nucleic acid extraction by classical bind-wash-elute chemistries, equal volumes of lysis and binding buffer are required for sample treatment, hence posing further challenges to cartridge integrated reagent pre-storage. from the research perspective, increased application of disk-based systems has been recently observed in resource-limited settings, especially in the field of immunodiagnostics. a disk-based test for detection of a four-parameter liver assay panel for defining liver function has been demonstrated to be completed within min by nwankire et al. [ ] using a portable, battery powered processing device and including field testing in a laboratory in nigeria. furthermore, hosseini et al. [ ] have implemented a novel mixing method using microspheres on disk in order to detect dengue virus antigen ns . the following sections provide a short overview of some centrifugal microfluidic platforms that are at late development or market stage. the inclusion criteria in this review were that the technologies focus on infectious diseases and have at least a few publications in the field. one representative platform of centrifugal microfluidics at late stage development is the labdisk (fig. ) from hahn-schickard [ ] and imtek, university of freiburg, germany. the technology has been applied in a variety of applications ranging from clinical chemistry [ , ] , to immunoassay [ ] and nucleic acid analysis, indicating universal use. in the latter field, two significant advances in fully automated sample-to-answer (nucleic acid extraction and amplification) analysis were announced, particularly for neonatal sepsis, detecting s. warneri, h. influenza, e. coli, and s. agalactiae [ ] , as well as for h n influenza virus [ ] . the amplification was based on pcr and the time-to-result was - . h. yet, when the labdisk was used to integrate rpa isothermal amplification for the detection of biological warfare pathogens (b. anthracis, f. tularensis, and y. pestis) the total time-toresult was < min [ ] . in addition, the processing device for isothermal amplification operates with a power consumption of < w, which facilitates its application in remote environments [ ] and its potential use with battery or solar panels. the application of the labdisk at the point of care in resourcelimited settings has been demonstrated with the detection of yellow fever virus in laboratory and field settings in senegal using rpa and min amplification time [ ] . recently, a labdisk was developed for the detection of malaria, dengue, chikungunya, pneumonia, and typhoid fever that used lamp providing the necessary means for nucleic acid based multiplex amplification with a time-to-result between and min [ ] . currently, one disadvantage of labdisk is that, while several parameters can be analyzed per disk, only one disk, corresponding to one patient, can be processed per test run. the concept behind the labdisk is to translate laboratory processes and steps into microfluidic unit operations and interface them towards fully functional sample-to-answer solutions. sample volumes can range from (sub)μl to several hundreds µl. one of the labdisk innovations is the pre-storage of liquid reagents in stick-packs [ ] . these pouches tightly seal the buffers and release them on demand upon centrifugation. the manufacturing technology is microthermoforming of polymer foils [ ] , a technology that is routinely used in the macroscale for blister packages, but was adapted to the microscale for micro/nanostructures in the labdisk (fig. ) . typical foil thicknesses are between and μm. a mold and a thin polymer foil placed above the mold are heated at a temperature near the glass transition temperature of the polymer foil. when the conditions are favorable, air is blown from the top so that the foil takes the shape of the mold. main process parameters are the applied air pressure and the temperatures of mold and foil. various materials can be used, with most preferable candidates being the cyclic olefin polymer/copolymer (cop, coc) and polycarbonate (pc), depending on the application requirements. sealing is performed either via a pressure sensitive adhesive foil (containing nanocapsules, which break and release adhesive upon application of pressure) or via thermal sealing using one more foil material (e.g. coc on coc). the elastomeric molds are suitable for prototyping, where fast iterations are required. however, each elastomeric mold may be worn out after repeated use for some tens of cartridges. thus, for large manufacturing series it is more efficient to use a metal mold. it may be more challenging to deform the foil (and methods such as coating on metal are used), however it is economically more sustainable and the durability of one metal mold can be sufficient for tens of thousands of disks. from the commercial point of view, the genepoc™ system (fig. ) from genepoc inc. [ ] is a simple and integrated portable instrument for the prevention and early detection of infectious diseases. the system received in fda clearance for its clostridium difficile assay and a ce mark for its group b streptococcus assay. in the future, genepoc inc. plans to develop cartridges for detection of respiratory, gastrointestinal, neonatal, and hospital-acquired infections, as well as sexually transmitted diseases. the system operates in a sample-to-answer mode, with minimum hands-on steps (a few minutes). the manufacturers declare a time-to-result of up to h. importantly, the system operates in a stand-alone mode, without the need for laptop, which is replaced with a touch screen instead. its universal sample-to-cartridge interface (accepts liquefied matrix) allows compatibility with several types of swabs, urine, etc. [ ] . the cartridge consists of only three pieces of plastic and has a wedge-shaped form (unlike an entire disk), which allows the device to process up to eight cartridges simultaneously, thus offering an increased throughput that is often necessary in cases of "extreme" poc testing [ ] . the genepoc™ disposable is composed of three distinct operational sectors: (i) world-to-chip interface: to μl of clinical sample conditioned in genepoc™ sample buffer is inserted in the disposable with a transfer pipette. the developers mention that the use of blood is not feasible yet, as it would require off-chip microbial preconcentration. subsequently, the inlet chamber is sealed with a lid that prevents aerosols from leakage. a series of siphons and passive valves prime the liquid to the next chambers. (ii) universal sample preparation, starting with pathogen mechanical lysis: a metallic disk is magnetically actuated in the chamber where glass beads are present leading to efficient lysis and release of nucleic acids. the lysate is centrifugally driven to a dilution chamber, where pcr inhibitors are inactivated by heating up to °c and with proper ionic condition adjustment of the extracted material in order to optimize subsequent amplification. (iii) the amplification and detection sector, which is the only assay-specific area of the cartridge where rt-pcr is performed (reagents are drystored). this sector has three chambers available for amplification (thermocycling is achieved via air heating of the overall process chamber). the device includes four non-overlapping excitation and emission wavelengths for fluorescence detection of chemical dyes, therefore, up to targets ( in each chamber) can be detected simultaneously, and under rotation. further details regarding cartridge material and fabrication methods are not available by the manufacturer. the liaison® mdx (fig. , formerly m cycler with simplexa™ integrated assays) is a centrifugal-based automated platform commercialized by diasorin inc. (formerly by focus diagnostics, which is currently owned by, and is part of diasorin group [ ]). the system has acquired fda/ce-ivd approval and its target applications currently aim at respiratory tract infections for influenza a and b as well as rsv viruses, hsv and , group a streptococcus, and c. difficile [ ] . the system is classified as benchtop and is operated via a laptop. the cost per cartridge is approximately € , while the instrument costs approximately € , . the device is compatible with two types of disks, targeting different types of end-user specifications (both are made of polypropylene with a nucleic acid compatible adhesive): (i) the direct amplification disk has eight individual slots/cartridges (all are connected into one disk, but one or more can be used simultaneously). the sample volume is μl, which is mixed with buffer ( μl) and the sample-to-answer time is min based on high-speed thermocycling and rapid rt-pcr using high heating/cooling rates (> °c/s and > °c/s, respectively). samples require no pre-processing (dna/rna extraction) and direct amplification is applicable. (ii) the universal disk is a -well singleuse cartridge able to perform multiple assays per disk. the reaction volume is μl. the device has four excitation and emission wavelengths for optical multiplexing in addition to the geometric (multichamber) multiplexing. when the universal disk is used, extraction should be performed ex situ and the eluate is inserted into the disk. a rather different approach from the typical benchtop-based or fully-automated systems has been suggested by the "diagnostics-in-a-suitcase (dias)", initially developed for the emerging avian influenza a (h n ) virus [ ] . in contrast to the aforementioned systems, where the core was a (micro)fluidic cartridge handled in a fully automated way, the dias system does not proclaim miniaturization or fully-integrated components and requires a few hands-on steps. yet, it smartly includes all the necessary biochemical components (extraction and amplification reagents), tools (gloves, pipettes, tube tracks, pipette boxes, waste container), and equipment (a tube scanner, vortex, microcentrifuge, even a mask in case of a highly infectious disease/epidemic), within a suitcase for a complete on-site analysis. it is an entire portable lab, which functions with solar panels or power packs (fig. ) . the workflow includes a typical sample preparation starting from the lysis of the pathogens, extraction of nucleic acids by binding to magnetic beads, two washing steps, and elution. this process requires min. the subsequent amplification step is conducted via rt-rpa and the time-to-positive is min. in total, nine pipetting steps are required, which can be reduced by replacing some liquid components with lyophilized reagents. during the ebola outbreak, the system was applied in field trial in guinea in collaboration with the institut pasteur in dakar, the public health institute of guinea, the university of stirling, the robert koch institute, and twistdx ltd. [ ] . this innovative approach tackled several problems including lack of (constant) electricity; lack of cold chain for material storage; risk and time loss from transferring infectious material from the field to central laboratories for analysis; and the need for fast time-to-result for healthcare providers who screened patients for ebola virus. a natural drawback of the dias system is the lack of connectivity with all the accompanied consequences (manual registration of data, risk of loss, ambiguous storage of data, etc.). furthermore, it requires a certain degree of user training for the handling of manual steps. along a similar concept, msf is developing a small-scale, autonomous diagnostic bacteriology laboratory based on appropriate and feasible, existing or adapted techniques, at affordable cost, with high accessibility and ease of use, which may respond to clinical needs in rt-pcr/isothermal . the feasibility of using robust blood culture bottles with visual readout, with a new type of media that bypasses the need for blood and, thus making it easy to use by non-experts, is being currently evaluated. a simple pathogen identification system, restricted to genus, as well as an antibiotic sensitivity testing system, restricted to the six antibiotics used frequently at the peripheral level, have also been developed. the target patient populations are children presenting with severe illness, those with risk factors for bloodstream infections, such as neonates, malnourished or hiv positive children, those who fail to respond to first-line antibiotic treatment, hospitalized war patients, and patients with trauma or prior surgery by providing organism-directed antibiotic therapy. with the developed autonomous diagnostic laboratory, it will be possible to acquire information regarding more general questions such as proportions and antibiotic resistance profiles of key bacterial pathogens, thereby providing evidence-based information for antibiotic policy (appropriate prescribing) and allowing for the improvement of epidemiological investigations as well as supporting healthcare infection control in emergencies and in neglected populations in resource-limited settings. novel diagnostic platforms, such as those mentioned in the previous sections, require validation in the target patient population, as well as in users. this should be a requirement for all new diagnostic tests, but it is even more important for point-of-care tests that are often used in nonselected patients (for example, any patient with fever attending any level of the health system) and by users with no technical background. before large-scale implementation in resource-limited countries is decided, the test should also preferably be endorsed by who and by international donors and governments that provide funding. unfortunately, many point-of-care tests have been launched in the market with only limited laboratory validation using convenient patient samples (for example that have a high pathogen load), or even cultured pathogens in relatively high concentration. field evaluation of the performance of the test in real patients, who often harbor low pathogen loads, is indeed often undertaken only after registration of the product. this is due to the lack of national and international regulatory mechanisms applicable to diagnostic tests, which, in contrast, exist for medicines [ ] . the net result is that health workers, especially those working in private clinics, and even patients going to pharmacies, have access to poc tests that have in fact very low sensitivity and/or specificity. for example, lft for chikungunya is commercialized, although it has a sensitivity of maximum % in the field [ ] [ ] [ ] . the gap between the performance measured in the laboratory and that in the field is due to several factors, depending on the type of pathogen and technology used. the sensitivity of naats to detect bacteria in the blood compared to blood cultures is often disappointing [ ] . even though naats are able to detect additional cases compared to blood-culture, they are not positive in all blood culture-positive patients [ ] because bacterial load can be very low, especially in adult patients with robust immunity. this explains the failure of numerous molecular-based multiplex platforms licensed in northern countries, aiming at diagnosing rapidly the cause of sepsis in admitted patients. fig. . the whole diagnostic test development process, from innovation, to validation and application. this development process is in reality a reiterative process that is more circular than linear. source: v. d'acremont. these tests work well for blood cultured for a few days but not for native blood, which implies that the potential gain of time, essential in these patients, is lost [ ] . tests based on the detection of antibodies, even of igm type, often lack sensitivity during the very first days of an acute febrile episode, but also specificity because of antibody persistence for several weeks. these challenges show how important it is to undertake clinical trials in real conditions to assess first the real performance of the test in the field (accuracy against the best available standard), and then the impact of using such tests on the management of patients (efficacy, measured through the impact on the health outcome of patients, cure rate after a certain number of days, secondary hospitalizations, or deaths and sequelae), done by routine health staff in their real working environment (effectiveness, measured generally through the level of compliance to test results and guidelines, and the appropriateness of antimicrobial prescriptions) (fig. ). outcomes measured in these trials should be related to the health outcome of patients and the public health benefits, and not only to the diagnosis based on laboratory reference standards (which for some diseases are anyway suboptimal or even non-existent). "we have to treat the patient, not a test result". to follow this adage, it is even more important nowadays that a continually increasing number of diagnostic tools is becoming available at the point of care. that should, however, not be interpreted as it has been for years with malaria microscopy, as an invitation to disregard test results and treat diseases presumptively based on clinical elements with low sensitivity and/or specificity [ ] . this adage rather suggests that, because no diagnostic test is % perfect, their results do not provide a black-orwhite answer, but rather an indication on the disease probability. this probability depends on two factors: (i) the prevalence of the disease in the corresponding patient population (pre-test probability), and (ii) the performance (positive and negative likelihood ratios) of the test used. when a disease is rare in the population (low pre-test probability) even if the test has a reasonable accuracy, the post-test probability after a positive result remains low. for example, if a typhoid rapid test with % sensitivity and % specificity (and thus, a likelihood ratio (lr) of . /( - . ) = ) is used in an area of asia where prevalence of typhoid fever among patients with febrile illness is %, the post-test probability will be % (it can be calculated using fagan's nomogram [ ] ), which suggests that it is reasonable to implement an antibiotic treatment to that patient. if the same test is used in an area of africa where the prevalence is %, the post-test probability will only be . %, which hardly justifies treating the patient for typhoid but rather to consider alternative diagnoses. based on these principles, decision charts can be built to precisely guide clinicians on how to use diagnostic tests and prescribe medicines in the most rational way. paper versions of such guidelines have been developed by who and unicef, and details on their strengths and weaknesses can be found in the literature [ , , ] . these guidelines, however, were deemed difficult to be adopted and properly implemented, due to several reasons including the long duration of the training required and the conflicting recommendations provided in different guidelines due to the time required for harmonization and then update of all paper material at large scale. mobile electronic technologies are promising tools to overcome these challenges. an electronic algorithm is, indeed, able to guide clinicians through the specific branches that apply to their patients, to accelerate training by teaching health workers directly on the mobile support (and even integrating training modules into the device), and to help to disseminate quickly to all health workers updated versions of e-algorithms through g connection. fig. provides a schematic representation of an integrated tool that aims at managing children with febrile disease at primary care level in resource-limited countries (derived from the existing e-poct tool [ ] ). the tool integrates a few key clinical data, biosensors measuring respiratory rate, oxygen saturation, cardiac rate, glycemia, and hemoglobinemia, as well as rapid diagnostic tests detecting malaria antigen (other pathogens can be added according to local endemicities) and host biomarkers (to differentiate bacterial from viral infections). it then provides guidance to health workers on when to refer a sick child, and whether to prescribe or not an antimicrobial (and other type of medicines), while it also calculates drug dosage based on weight or age. it is beyond the scope of this review to elaborate on the content and performance of existing e-algorithms and platforms for the integrated management of children with febrile disease, however, the reader can refer to a recent review published by keitel et al. [ ] . the consultation process data collected through the computerized decision-support tools used by clinicians and sent with g remote connection to a userfriendly data management system can be used for surveillance and outbreak detection. to document the cause of patient's fever, and at the same time the cause of the potential epidemic, a decision-support system should integrate the results of a pathogen poc test, through a reader directly connected to the tablet supporting the clinical e-algorithm, and if possible also remotely to the central server. the source of information would be patients seen at routine health facilities tested by poc tools that have a direct impact on case management (e.g. malaria or typhoid), but also from a restricted number of sentinel sites where additional poc test, aimed at detecting pathogens of particular interest (that cause large epidemics, even if non-treatable such as dengue, or rare such as ebola virus disease), would be used. the data accumulated on a daily or weekly basis (depending on the level of g connection availability) at the central level would allow health authorities to conduct real-time disease surveillance, through weekly/monthly reports, and would also enable immediate outbreak alarms when the preset incidence threshold for the surveyed disease has been reached. from the central server, automated feedback reports can also be created, sent to clinicians' tablets and discussed during supervision visits at the health facility. training e-tools, aimed at improving management of patients in general or in the particular situation of an outbreak, could also be sent to the tablet to guide clinicians. finally, a patient file supported by an electronic card owned by the patient, could be synchronized at each new consultation with the data collected by clinicians in the tablet (fig. ) . such a fully integrated system seems to be overoptimistic; however, each of its components has already proven to be feasible and the challenge now is to find appropriate and sustainable diagnostic and information and communication technology (ict) platforms that can be deployed at large scale and in the long term. to address the clinically complex differential diagnosis of an episode of fever, that is a frequent presenting syndrome, this review outlined some point-of-care and near-patient diagnostic technologies that can assist to this scope. in the long, but non-exhaustive, list of described technologies, none seems to satisfy simultaneously all criteria of end-users' needs, target product profiles, and the assured criteria. regarding performance, some technologies may be fully automated and multiplexed, but they are prohibitively expensive; some are cost-effective, but compromise sensitivity or multiplexity. in contrast, some solutions offer multiplexity, yet they are benchtop, require laptop, electricity, and are barely portable (due to size and weight). regarding technology, most of the described platforms implement nucleic acid amplification (pcr-or isothermal-based) and almost none of them includes a parallel immunoassay analysis to measure the host response to an infection. a few of the suggested approaches are equipment-free but therefore lack connectivity and interfacing ability. however, one can observe a common rationale in the structure of the described technologies. the vast majority are based on the combination of three components: the assay, the cartridge, and the readout device. several variations may exist in the format of the assay, the cartridge, or the detection principle; however, this three-element model seems to be the backbone of all described diagnostic platforms. this modular nature should motivate the developers and manufacturers to consider more (semi)open and adaptable platforms to address end-users' needs in a dynamic way, being able to quickly modify their panels and adapt to endemic or epidemic needs. the review concludes with the need of electronic decision trees to support diagnostic tools, as interactive assistance to clinicians. such algorithms are derived from paper-based guidelines to computerized versions (which enforce the provision of output for any possible combination of clinical elements, which is not the case for traditional guidelines), operating on g mobile tools such as tablets. they receive, as input, data from several (poc and non-poc) diagnostic tests, which are processed via clinical algorithms, to provide clinicians with guidance regarding the handling of the patient (admission, treatment, etc.). such interoperability between ict components and interfaced physical systems (diagnostic tests) for decision-support can provide solutions of clinical and public health utility, therefore, this trend is expected to increase rapidly, offering high impact diagnosis in developing countries, and transforming the global healthcare landscape. this process, being rather complex and requiring know-how from a variety of disciplines and partners, requires multiple interactions between universities, industries, governmental and non-governmental organizations, as well as philanthropic institutions, that are not only based in wealthy countries but also in resource-limited countries. to increase the chances to make progress, most successful technologies should be supported and developers be motivated to partner with each other. it is only with such a spirit that the development pathway can be coherent and harmonized, and the new tools eventually may be implemented at large scale. looking into future perspectives, it seems that a combination of technological and non-technological factors will be critical to decide whether the diagnostic poc technologies can be used in the management of patients with febrile illness. from the technological perspective, it would be interesting to observe the landscape in the next - years and whether label-free detection technologies can be transferred from the laboratory to the clinics, and then to the market. magnetic field, electrochemical, gravimetric (e.g. surface acoustic waves), and micromechanical (e.g. cantilever based) detection technologies are some laboratory-mature fig. . schematic representation of the potential integration of a mobile patient management system (efever) and a centralized electronic disease surveillance system (emergence). source: valérie d'acremont. candidates that could potentially substitute fluorescence detection and offer the advantage of cheaper read-out devices. regarding label-free technologies, it is important to consider how they can potentially be integrated into sample-to-answer systems, in view of the requirements outlined in sections . and . . in addition, it is important to keep track of the progress on the lft-based nucleic acid analysis and detection, a concept that is rapidly catching up and combines the easiness of lfts with the molecular analysis. the transition from the laboratory to the market is crucial and depends on multiple parameters. a fundamental one is the sustainable and scalable manufacturability of the disposable cartridges (in terms of materials and processes). ideally, developers should have access to fabrication facilities that could perform the scale-up manufacturing in two phases: first, from prototyping to medium scale manufacturing (a few thousands or tens of thousands cartridges/chips/components per year) in order to serve clinical validation studies; and in a next level to aim for large scale manufacturing (tens of thousands to hundreds of thousands, that are fairly realistic quantities for infectious diseases of global impact). special attention should be given to the heterogeneous integration of components, i.e. when a system consists of not only plastic/silicon/glass core components, but integrates biological and biochemical agents, which are necessary for the sample-to-answer analysis. issues of stability and compatibility of (bio)chemical reagents with cartridge materials and manufacturing processes, adsorption (and loss) of reagents may prove critical for reaching the desired limit of detection. a future perspective is also that the diagnostic tools are no longer operating in a stand-alone mode, but in a broader "ecosystem". to this direction, integration of ict is of primary importance, as it is the means to connect a variety of different tools and generate networks (existing "traditional" approaches, reference methods, and new innovative tools), create databases, and process data (since we are in the era of big data). along these lines, the combination of nucleic acid with protein biomarker testing seems to be a path for more reliable diagnosis, especially in case of syndromes caused by several different pathogens of bacterial, viral, parasitic, or fungal nature. further elaborating on the ecosystem approach, and especially for diagnostics for resource-limited countries, special business model developments are required that would render marketization in the target areas attractive. large industries or small and medium enterprises (smes) may choose their own way to market, but a new path may be to try marketing in partnership with the pharmaceutical industry. it is obvious that diagnostics and therapeutics are the two sides of the same coin, and only through their combination and interplay can healthcare truly progress. especially in an era that antimicrobial resistance rises dramatically, this exact issue could and should become the bridge to unite the worlds of diagnostics and therapeutics. one parameter that should also be considered during the transition of the poc technologies from the laboratory to the market is the healthimpact modeling, including health economics and cost-impact analysis. one technology may be (comparatively) cheap but may not offer added clinical value; another may be (seemingly) expensive but it may prove overall cheaper than the existing test because the clinical impact is higher. the developers/manufacturers should compare the manufacturing cost of their technology to the real cost of the same test within their healthcare systems. a substitution of an existing standard of care by an innovative technology would be welcome if: it can lead to the same clinical impact at an overall lower price; or it can offer higher clinical impact at the same price. when assessing novel technologies, one should consider what they actually aim to substitute (material savings, time and labor savings, etc.) in order to have a complete view of the cost savings. last but not least is the perspective of adaptability and usability by the potential end-users. whether in resource-limited countries or in the more wealthy part of the world, a technology may be blocked from being used, even if it performs in the best way, due to reasons related to constraints and reluctances that end-users have. a behavioral change of the end-users (through several means, e.g. electronic decision algorithms, 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caring for newborns and children in the community the authors k.m., s.h., f.v.s., and r.z. acknowledge the european commission for funding under the fp project "discognosis" (ga- ), the h project "diagoras" (ga- ), and the h project "dmc-malvec" (ga- ).the author v.d.a. thanks blaise genton for critical reading of the manuscript. conflicts of interest: none. key: cord- -vhprfb o authors: tram, dai thien nhan; wang, hao; sugiarto, sigit; li, tao; ang, wee han; lee, chengkuo; pastorin, giorgia title: advances in nanomaterials and their applications in point of care (poc) devices for the diagnosis of infectious diseases date: - - journal: biotechnol adv doi: . /j.biotechadv. . . sha: doc_id: cord_uid: vhprfb o nanotechnology has gained much attention over the last decades, as it offers unique opportunities for the advancement of the next generation of sensing tools. point-of-care (poc) devices for the selective detection of biomolecules using engineered nanoparticles have become a main research thrust in the diagnostic field. this review presents an overview on how the poc-associated nanotechnology, currently applied for the identification of nucleic acids, proteins and antibodies, might be further exploited for the detection of infectious pathogens: although still premature, future integrations of nanoparticles with biological markers that target specific microorganisms will enable timely therapeutic intervention against life-threatening infectious diseases. according to the world health organization (who), in infectious diseases claimed million lives worldwide (world health, ) . among them, human immunodeficiency virus (hiv) and tuberculosis were the leading causes of death at all age groups. in , hiv claimed . million lives in sub-saharan africa alone (tarantola et al., ) . the extent of damage exerted by a particular infectious disease could reach well beyond the people directly plagued by the germs. a recent ebola outbreak caused so much trouble for the healthcare system in west africa that there were insufficient resources available for measles vaccination programs, thereby further adding to the death toll (takahashi et al., ) . an even more recent outbreak is represented by the zika virus, currently spreading in the americas and the pacific region. this has resulted in increased infections during pregnancy and microcephaly, as well as guillain-barré syndrome in adults. the transmission of pathogens is not limited to just humans. a number of transmissible microbes originated from animal vectors (e.g. birds, bats, ticks, etc.) could subsequently switch host to humans. severe acute respiratory syndrome (sars) virus, hantavirus, nipah virus and human immunodeficiency virus (hiv) are just a few of such examples (morse et al., ) . in the past few decades, the spread of once dreaded maladies such as smallpox and poliomyelitis have generally been kept under control, but these rigorous vaccination programs are far from being equally practiced across the globe (fonkwo, ) . in developing countries, a lack of proper sanitation, technologies, equipment, and human resources has been hampering efforts to provide timely treatments (batt, ) . identification of microorganisms by observing characteristic features of cultures has been in practice for decades. however, several limitations render this classical technique impractical for on-site diagnosis of infectious diseases, especially in resource-poor regions (kaittanis et al., ) . being time-consuming is one of the principal flaws of current diagnostic approaches. for preliminary results, each analysis takes - days. for more definite results, it might take up to - days. detection of salmonella typhimurium consumes - days before yielding results (he et al., ) , whereas diagnosis of tuberculosis via microbiological means may take weeks (dinnes et al., ) . an additional complication derives from the fact that, in order to procure meaningful observations, the initial serum samples must contain pathogen loads above a certain threshold level. this prerequisite might not be met if the patients are in early stages of infection. to worsen the situation, the life cycle of some bacterial strains includes a dormancy state, whereby organisms do not grow significantly in number when cultured. this could culminate in false negative results that critically undermine diagnoses. interferon gamma (inf-γ) release assay detects inf-γ produced by t-cells when the patient is exposed to mycobacterium tuberculosis antigen. however, a tuberculosis patient is usually affected by hiv at the same time. concurrent presence of hiv could readily impair the patient's immune systems. the resulting low t-cell count could mask a clinically relevant quantity of mycobacterium tuberculosis, hence leaving tuberculosis undetected (diel et al., ) . in the case of microbes more diminutive than bacteria (e.g. viruses, with average size of only about one-hundredth that of the average bacterium), an electron microscope is required for detailed visualization of the viral particles (i.e. virions). the growth of viral particles also necessitates a more sophisticated protocol than the one adopted for bacterial cultures (shinde et al., ) . technological advances have empowered medical professionals with a wide range of diagnostic tools. however, even state-of-the-art techniques are still far from being suitable for application in resourcepoor contexts, wherein infectious diseases have proven to be the most widespread. as of , the gold standard for hiv diagnosis is an enzyme immunoassay which detects igm antibodies in the patient's serum, followed by western blot (branson, ) . two popular methods are enzymelinked immunosorbent assay (elisa) and nucleic acid test (nat) . in order to credibly detect a few virions in μl of plasma sample, most commercially available methods require nucleic acid amplification (calmy et al., , fiscus et al., , rouet and rouzioux, . fourth-generation elisa, a combination assay capable of detecting both hiv igg/igm and the capsid protein p , has a limit of detection (lod) of pg/ml (speers et al., ) , thereby removing the need for nucleic acid amplification. the main downside is its high cost. amidst the outbreak of severe acute respiratory syndrome coronavirus (sars-cov) in , real-time polymerase chain reaction (rt-pcr) (chan et al., ) was widely employed. however, sensitivity of the assay represented the main limitation. in specifics, it would appear below clinically established standards, were the patients infected fewer than six days before the sample extraction date (vasoo et al., ) . while a refinement of specimen extraction process does improve the sensitivity level, it leaves the cost issue unaddressed. another pathogen whose diagnosis utilizes rt-pcr as the standard test is the avian flu h n . commercially available immunochromatography-based strip for the diagnosis of h n (welch and ginocchio, ) is not as costly, but low sensitivity and specificity limit its clinical utility (lee-lewandrowski and lewandrowski, , posthuma-trumpie et al., ) . other than diagnosis, nat sees extensive use in screening of blood supply for common pathogens such as hiv, hepatitis b virus (hbv), and hepatitis c virus (hcv) (fiscus, cheng, ) . it is also employed to monitor patient progress throughout treatment courses. genexpert is the first fully integrated nat system. it could produce test outcomes in h. despite the relatively shorter assay time, the problems of cost and energy consumption remain (meyer-rath et al., ) . according to the college of american pathologists, poc testing could be considered as on-site diagnostic tests carried out using mobile devices readily accessible to the patients and the in-charge physicians (lamb et al., ) . another more concise definition is 'testing done in the proximity of patient care' (kiechle et al., ) . the portable devices employed can be either hand-held or transported on a cart (urdea et al., ) . the acronym "assured" was coined by who to denote the fundamental criteria of poc testing: affordable, sensitive, specific, userfriendly, rapid and robust, equipment-free, and deliverable to end user (sista et al., ) . as mentioned above, there is an increasing demand for diagnosis of infectious diseases in resource-poor regions. a paucity of laboratory technicians with necessary know-hows is, among others, a major concern under such circumstances. hence, poc devices need to be userfriendly with easy-to-follow instructions. as such, routine tests may be performed by family members, or even the patients themselves. apart from mobility, short assaying time is crucial to poc testing. in cases where such time reduction does not hold much clinical significance, cutting down on the waiting time helps alleviate patients' discomfort (holland and kiechle, ) . conventionally, poc tests are classified into main categories : ) those whose speediness is the most valuable attribute, to reinforce decision on treatment regimen of lethal conditions (e.g. meningitis); ) those whose short assaying time is also a crucial element for prompt measures to restrain an outbreak (e.g. mrsa in hospitals); ) those simply for verification of the disease-causing microbes; ) those for self-monitoring by patients who do not attend follow-ups (e.g. in the case of patients with sexually transmitted diseases). despite this classification, poc devices are still at their infant stage. by , several poc systems had been investigated in clinical trials. however, none had been released for commercial uses (liao and huang, ) . in general terms, a poc device is designed to detect, either qualitatively or quantitatively, the presence of a specific biomarker characteristic of the malady at stake. at the moment, the analytes of interest range from nucleic acids of the microbes or proteins released by them during their time residing in the host, to antigens located on the surface of the microbes themselves. therefore, the review of the current pocs has been organized into ) nucleic acids, ) whole pathogens and ) proteins & antibody detection systems. many diagnostic techniques have been revamped and adopted for detection of microorganisms' genetic materials: electrophoresis, spectrophotometry, rt-pcr, etc. (cagnin et al., ). however, adopting them not only for laboratory experiments, but also clinical applications at patient sites has proven to be a challenge. electrochemical (ecm) sensing was originally contrived for applications in laboratories, but recent advancements in technology have refined its suitability for the development of poc devices (liepold et al., ) . on its own, ecm already has desirable properties (lucarelli et al., , pohlmann et al., , wakai et al., , wei et al., . firstly, ecm sensors do not require much expertise to maneuver. the steps entailed in sample manipulation process are straightforward as well. in terms of resources, these systems can work with sample size smaller than usual, which ranges from a couple of microliters all the way to nanoliters, and do not consume much energy. all these fortes render ecm sensors suitable for poc assays. a typical ecm sensor consists of an electrode, a capture probe, and a reporter probe (fig. s (siangproh et al., ) and s ). capture probe is essentially an oligonucleotide whose sequence is complementary to that of the target nucleic acid. in most cases, the probe is conjugated to a surface, such as an electrode. after sample introduction, binding of target dna/rna to the capture probe generates a series of changes that eventually trigger the release of ecm signals by the reporter probe. even though a number of disposable electrodes such as glassy carbon (rivas et al., ) and pyrolytic carbon (stoner et al., ) have been in use for quite some time, non-disposable alternatives (e.g. indium tin oxide, pencil graphite, screen-printed carbon) are slowly but steadily taking over. after all, the latter are more economical and easier to produce (yeung et al., ) . for instance, indium tin oxide electrodes were exploited in a silicon-and glass-based microchamber for simultaneous diagnosis of escherichia coli and bacillus subtillis (xu et al., ) . capture probes were attached to the electrode surface by electrochemical copolymerization. nanoparticles (nps) have lately been used to complement electrodes in immobilizing probe (fig. ). sun et al. employed gold nanoparticles (aunps) and multi-walled carbon nanotubes (mwcnts) to conjugate single-stranded dna probes for the detection of staphylococcus aureus dna . gold electrodes were also included in the set-up. rather than the common purpose of immobilizing capture probes, the electrode was instead used to concentrate the nano-sized anchors. in another example, each aunp served as the core for coconjugating a hairpin sequence of dna and a reporter dna. interestingly, the reporter dna only carried a sequence complementary to half of the target helicobacter pylori sequence. the other half was recognized by a capture probe anchored to a gold electrode (cui et al., ) . with a great surface-to-volume ratio, nps could potentially bind a greater density of capture probes. this indirectly magnifies the ecm signals ultimately generated (cui et al., ; sun et al., ) . at the same time, signal-to-noise (snr) ratio is improved. out of various kinds of nps, aunps are arguably the most extensively investigated as far as ecm sensing is concerned (table ) . like most other nano-sized materials, aunps have inherently large surface area and surface free energy. these properties facilitate the adsorption of nucleic acid strands. nonetheless, there exist some challenges in terms of reliability, reproducibility, scalability in manufacturing aunp-based biosensing assays and long-term stability; these aspects have hampered the successful translation into clinical trial research. indeed, nanoparticles with different size ranges might show variable surface area, reactivity and orientation towards biosensing molecules. quite recently, aunps have been produced with very narrow size distribution (e.g. . ± nm (lu et al. ) or even smaller, . ± . nm ) and further research efforts have resulted in samples with desirable polydispersity indices (b . ). this process could be further enhanced via functional groups such as thiols and disulfides (galow et al., , niemeyer and ceyhan, ) . since aunps are capable of forming table examples of diagnostic poc systems whose target analytes are nucleic acids. key features ecm (yeung et al., ) analyte: escherichia coli dna or bacillus subtillis dna sample size: μl sample pretreatment: • genome isolation using avidin-coated magnetic particles • amplification using pcr detection range: - cells clinical/real-world samples tested? no performance: • capture probes were thermally stable throughout thermal cycling process • insignificant interference with quantification due to non-specific adsorption onto the electrodes • use of magnetic particles for dna isolation was compatible with pcr process (baeumner et al., ) analyte: dengue virus rna sample pretreatment: • amplification using isothermal nucleic acid sequence-based technique • mixing with liposomes assay time: min (excluding rna amplification process) clinical/real-world samples tested? yes (human serum) performance: • sensitivity and specificity comparable to that of lab-based techniques • accurately detect dengue serotypes , and in clinical samples • minimal cross-reactivity with dengue serotype (authier et al., ) analyte: -base pair hcmv dna sample pretreatment: • dna extraction from cell culture • amplification using pcr • denaturation in alkaline media at room temperature • -fold dilution with coating solution reproducibility: • ensured by slicing off a small segment at the end of the electrodes in between trials • enhanced by maintaining the screen-printed microband electrodes in a solution of (ferrocenylmethyl)trimethylammonium hexafluorophosphate • undermined with the use of manual screen-printer detection range: - pm clinical/real-world samples tested? no performance: • aunps label employed for hybridization assay proved to be more stable than radioisotopic or enzymatic labels • non-specific binding present in low level • selectivity demonstrated by testing against non-complementary human ets gene • lod better than that reported in an electrochemiluminescent hcmv dna method tested on -base pair hcmv dna (boom et al., ) analyte: staphylococcus aureus nuc gene sample size: μl (after purification) sample pretreatment: • filtration of tap water samples through μl membrane • inoculation with different amount of staphylococcus aureus • centrifugation at , rpm for min • dna extraction using rapid boiling method • amplification using pcr • dilution with te buffer solution, followed by denaturation in boiling water bath detection range: • fm- nm of nuc gene • - cfu ml − for real-world samples clinical/real-world samples tested? yes (tap water) performance: • achieved a lower lod (for tap water samples) than other reported methods such as aunp-based immunosensors (hejazi et al., ) or fluorescence-based assay using cdse quantum dots(yang and lai, ) optical (chen et al., ) analyte: detection of hev rna sample pretreatment: • reverse transcription of hev rna, followed by denaturation at °c • amplification using rt-lamp assay time: b min (excluding amplification step) detection range: n hev rna copies clinical/real-world samples tested? yes (human serum) performance: • selectivity demonstrated by testing against three other hepatitis strains hav, hbv, and hcv • results validated using agarose gel electrophoresis (phillips et al., ) analyte: escherichia coli dna sample size: μl reproducibility: • reproducible fluorescence signals • extent varied depending on bacterial strains and species assay time: within min performance: • selectivity demonstrated by testing against twelve other species of bacteria (griffin et al., ) analyte: hcv rna detection range: - pm clinical/real-world samples tested? no performance: • no tagging is required • about two orders of magnitude more sensitive than some common colorimetric techniques • selectivity down to the level of single-base mismatch • quantitative signal intensity varied with the length of target rna sequence (nam et al., ) analyte: nucleotide sequence indicative of anthrax lethal factor sample size: μl assay time: - h detection range: zm- fm performance: • sensitivity on par with that of pcr-based methods, but did not require enzymatic amplification process • selectivity down to the level of single-base mismatch analyte: hiv subtypes (a, b, c, d, e, g, and panel) sample pretreatment: • system capable of effectively sequestrating separating viruses without the need for pretreatment reproducibility: strong covalent bonds with sulfhydryl groups, thiolation reaction could be readily achieved (daniel and astruc, ) . in contrast, nucleic acid strands with adenosyl phosphothiolate tails could be conjugated in a more direct manner (patolsky et al., ) . ease of functionalization and excellent biocompatibility (liu and ju, ) render aunps a great asset in both optical (cao et al., ) and electronic (park et al., ) dna detection methods. to incorporate aunps into ecm biosensors for poc devices, researchers have devised a handful of approaches for fully exploiting their potential . aunps, while being immobilized on genosensors, could be directly detected. in one experiment, target nucleic acid strands were first anchored onto au quantum dots (qds) (pumera et al., ) . binding between target sequence and the capture probe, by then already conjugated to paramagnetic beads, led to the formation of a complex that enabled voltammetric detection of the gold qds. alternatively, it is possible to quantify au + ions generated after aunps are exposed to a mixture of hydrogen bromide and bromine (i.e. acid dissolving step). such a strategy was employed in the detection of human cytomegalovirus (hcmv) dna sequence (authier et al., ) . since many au + ions are released as each aunp is suspended in a medium, the ecm signal is enhanced. as a result, a limit of detection (lod) of pm could be achieved. however, the mixture used for dissolving aunps is extremely harmful (hydrogen bromide is highly corrosive and irritating by inhalation). as such, this could limit the practicality of the technique (lucarelli et al., ) . for signal amplification, silver enhancement could be employed. cai et al. conjugated dna capture probe to aunps, and targeted dna to a glassy electrode (cai et al., ) . a silver enhancer solution was introduced to allow metallic silver to coat itself onto aunps. such coating process helped boost the voltammetric signals by more than times. alternatively, signal amplification may be achieved by letting aunps act as carriers of electroactive labels. the incorporation of ferrocenylhexanethiol decreased the lod level to pm for a sample size of μl . aunps could be combined with other nano-size materials for application in ecm sensors. watanabe et al. recently combined the use of aunps and magnetic nps (mnps) in the detection of meca gene, a popular biomarker for mrsa (watanabe et al., ) . two dna probes were employed. one was anchored to mnps, whereas the other to aunps alongside ferrocene. the sequences of these probes were designed to be complementary to nearby regions located on meca gene. the co-binding of mnps permitted the isolation and enrichment of analyte complex prior to ecm measurement. even without the help of nucleic acid amplification via pcr, this system managed to detect as low as pm of target dna. disposable biosensors are slowly but steadily assuming a larger role in poc technology, given the troubles commonly associated with nondisposable counterparts. a poc device that could be reused requires thorough cleaning after every assay to ensure no cross-assay contamination occurs, hence preserving the reliability of the assay. with that prerequisite, there is still the issue of how to do the washing without inadvertently damaging the integrity of the test reagents. this renders non-disposable poc devices unpractical in resource-poor settings. however, calibration and sterility problems have been reported for example in disposable clinical sensors, where the sensor-imbedded device required sensor calibration and/or validation by the clinician immediately prior to each use. some recent advances have enabled the production of pre-calibrated and pre-validated sensors (e.g. us b patent), but they still require optimization in lowering the costs and increase performance before becoming suitable for a single-use sensor application. aunps have also been integrated into the design of disposable biosensors. they were used together with screen-printed electrodes for the diagnosis of respiratory pathogens such as mycoplasma pneumonia, streptococcus pneumonia, and chlamydophila pneumonia (bessede et al., ) . in another example of disposable biosensors, liposomes (another type of nps) were employed as carriers of dyes (ho et al., ) . this strategy was examined for the detection of serotype-specific rna fragments of dengue virus (baeumner et al., ) . a portable reflectometer was utilized for quantification of the nucleic acid materials. taking into account its many fortes (i.e. portable, inexpensive, user-friendly with easy-to-follow instructions, etc.), this system seems very promising. following isothermal nucleic acid sequence-based amplification, which requires only basic tools such as water baths, it only took another min to produce results. the same research group has adopted a similar system for detection of viable escherichia coli in drinking water (baeumner et al., ) . thus far, we have discussed systems in which ecm labels play a major role in the quantification process. however, label-free sensors have also been investigated by numerous researchers. there are two main methods via which ecm sensing works without having to rely on electroactive labels (siangproh et al., ) . the first one is rather straightforward. nucleotide bases do have intrinsic redox properties. thus, they can generate ecm signals, which are indicative of the amount bound to the electrode. in the second approach, molecules which stably orient themselves into the groove of target dna duplex (e.g. methylene blue, daunomycin, aromatic amines, co( , '-bipyridyl) + ) are employed. after the two dna strands get detached, these duplexintercalating entities are freed, hence generating ecm signals. the latter strategy has been adopted in the investigation of infectious pathogens such as escherichia coli, mycobacterium tuberculosis, hiv (haddache et al., ) , and hbv (meric et al., ) . however, a grave downside of this technique is the interaction between the chemicals and dna duplex, which makes them potentially mutagenic (watanabe et al., ) . depending on the nature of the pathogens (e.g. dna or rna viruses, according to the baltimore classification (baltimore, ) ), rna detection is another viable strategy that could benefit from the advent of nanotechnology into the development of poc systems. for instance, aunps were incorporated into a lateral flow test strip for the diagnosis of hiv through the quantification of viral rna in plasma samples (rohrman et al., ) . lateral flow nucleic acid test strips derived from the well-established immunochromatographic strips (mao et al., ) . at the moment, this kind of device has several limitations (carter and cary, , corstjens et al., ) . before letting the sample flow through the devices, nucleic acid hybridization process is normally carried out in advance. this results in the addition of - min to the total assaying time. in general, quantification done with lateral flow assays necessitates the use of expensive equipment. the high cost, however, is not necessarily translated into great sensitivity of the assays (he et al., ) . there have been several attempts to augment the sensitivity of lateral flow assays. most of these ended up further complicating the protocol without really addressing the other issue (i.e. high cost) (rohrman et al., ). an example of such attempts to improve sensitivity exploited antigens and antibodies to detect hbv, hcv, and hiv viruses (dineva et al., ) . • surface chemistry of nps demonstrated to be reproducible to a considerable extent • analysis results reproducible for several hiv subtypes assay time: h of capturing and min of detection and analysis detection range: • varied between different subtypes • ranging from ± copies/ml (subtype d) to , ± , copies/ml (subtype e) clinical/real-world samples tested? yes (unprocessed whole blood) indeed, the specific detection hiv- rna is particularly challenging (rohrman et al., ) . it is known that the level of hiv genetic material present in the patients' bloodstream is naturally not very high. to be more precise, it is only a few copies per milliliter of blood. therefore, duplication of the nucleic acids prior to the assay is a vital prerequisite for an adequately sensitive test. to this end, rohrman et al. opted for isothermal nucleic acid sequence-based amplification, a popular technique mentioned above (baeumner et al., ) . this particular system has many laudable qualities (rohrman et al., ) , including a low cost (each strip costs no more than one us dollar) and simple production steps that involve commercially available reagents. from the very beginning to the completion of the assay, the user only needs to perform three steps. it takes in total only about min, which is much shorter than the duration of any assay discussed thus far. in addition, simpleto-operate and relatively inexpensive instruments (e.g. heat block, scanner, camera, and pipette) are sufficient to perform the assay. in addition, when tested under different temperature conditions, the results produced by the assay remained essentially consistent. such a commendable level of robustness testifies to its suitability for use in africa and other places where a high temperature is a normal occurrence. consistent performance was also observed when the system was tested against varying storage periods. however, this technology is still far from ideal, as the use of heat blocks does consume a considerable amount of energy (labarre et al., ; liu et al., ) . it could be replaced by heater equipment that runs on battery, hence cutting down on costs. instead of imaging instruments, a color scale could be exploited to qualitatively simplify data interpretation process. up to this point, it should be fairly apparent that ecm sensing is one of the most extensively studied methods for the detection of pathogenic nucleic acid. another method that has also attracted much attention in the field of poc technology is optical sensing. in essence, presence of the biomarker of interest in the sample will trigger a chain of biochemical reactions. the end result is a change in optical properties of the system. such variation is designed to be proportional to the amount of the nucleic acid sequence to be analyzed (fig. ) . one key advantage of optical sensing is that electroactive labels are not indispensable (shafiee et al., ) . once again, nanotechnology plays a huge role. metal nps especially show tremendous promises. among them, gold and silver nps are the better options, since they are less susceptible to oxidation than their copper counterparts (jain et al., ) . reportedly, nanorods (bi et al., ) and aunps were employed in a colorimetric assay for the diagnosis of hepatitis e virus (hev) (chen et al., ) . to ensure that the concentration of the biomarker fell within detectable range, real-time loopmediated isothermal amplification (rt-lamp) was employed as part of sample preparation process. streptavidin molecules were conjugated to aunps, which were then added to the sample that already underwent rt-lamp. if the sample was hev-positive, aunps would clump together as a response. this would in turn trigger a color change from red to purplish blue, hence permitting visual readout by naked eyes without any need for sophisticated instruments. on the contrary, had there been no hev genetic material in the test sample, the biotin added during rt-lamp process would help stabilize the aunps. as a result, the solution would remain red. a notable advantage demonstrated by this system was a remarkably short assay duration. not including rna amplification process, the test consumed only min in total. surface plasmon resonance (spr)-based sensors have rapidly emerged as a popular type of optical biosensors (homola, , tokel et al., . they work by measuring changes in refractive index of metal-dielectric interface, which could occur following binding events between the analyte molecules and capture probes. surface plasmon bands absorbed by metal nps are closely related to the size of their aggregates, which come about as a consequence of nucleic acid hybridization (hazarika et al., ) . the more np aggregates there are, the greater the resulting red-shift becomes. an example of spr-based biosensor was used for the detection of hbv (chuang et al., ) . aunps were also employed in the said assay, which was economical and exhibited a lod of fg/ml with just min of assay time. nat has always been a valuable diagnostic tool of hbv infection. its utility is even more conspicuous during the 'window period' where other common techniques (e.g. immunoassays) are not practically reliable. the reason for this setback of immunoassays is the lack of antibodies against hbv in the body throughout the 'window period' (yildiz et al., ) . nat is also particularly useful in the diagnosis of occult hbv infection (ozsoz et al., ) . while dna of occult hbv are present in the bloodstream, there is no trace of their surface antigens. given its unique capability, nat is the preferred technique when it comes to scanning of blood transfusion sources (stramer et al., ) . fluorescence-based assays are categorized under optical sensing as well. storhoff investigated the use of this type of assay for the detection of meca gene of mrsa (storhoff et al., ) . the integration of nanosized materials (i.e. aunps) helped augment the sensitivity of the assay relative to that of similar methods. conveniently, nucleic acid amplification prior to quantification process was not necessary. a separate study adopted fluorescence-based sensors for the detection of escherichia coli dna (esteban-fernandez de avila et al., ) . in details, anionic molecules of poly(para-phenylenethynylene) (ppe) were immobilized onto aunps, which were already functionalized with ammonium groups. the resulting complex efficiently subdued fluorescent property of ppe. after sample introduction, if bacteria were present, there would be electrostatic interaction between their surface and the various positive charges lining along the surface of aunps. this triggered the release of ppe from the complex. once freed, ppe molecules regained their fluorescence, hence allowing quantitative measurements of the bacteria. as additional advantage, the assay is capable of identifying three distinct bacterial strains within min. fluorescence-based assays can also offer a final readout by naked eyes. zhang et al. reported a poc device that made use of microcapillaries for detection of two rna biomarkers that belong to different hiv strains . a simple uv-flashlight was sufficient to generate visible readouts of fluorescence signals from the indicator, calcein. in addition, the system is self-sufficient in the sense that it did not utilize any external source of electricity. more precisely, a pocket warmer was all it needed. the use of capillaries to introduce and hold samples permitted concurrent analysis of several samples. in brief, this system did manage to tackle some of the most troubling issues associated with diagnostic tools in resource-poor settings, namely time and energy consumption. these assays are generally based on immunoreactions between antibodies and antigens, which are characteristics of individual strains. relative to nat, immunological tests are generally capable of producing more robust results within a shorter span of time (shinde et al., ) . however, their level of specificity and sensitivity often pales in comparison. over the time, different kinds of antibodies (e.g. conventional, heavy chain, monoclonal, polyclonal, and recombinant antibodies) have been investigated in immunological tests. none of them have proven to be perfectly suitable for the role (o'kennedy et al., , shinde et al., . polyclonal antibodies could be produced in a more rapid and economical manner than monoclonal antibodies, but their intrinsically poor specificity represents a valid concern. the latter have their fair share of shortcomings though. the production of monoclonal antibodies requires more well-trained personnel, and more sophisticated machineries, whose cost is a setback. moreover, recombinant antibodies do not promise an acceptable level of sensitivity and affinity. to worsen the matter, they are rather vulnerable to interference from contaminants. monoclonal antibodies were utilized in the commercially available architect qualitative assay by abbott (lou et al., ) . this system was designed to detect hepatitis b surface antigens (hbsag) by conjugating anti-hbsag monoclonal antibodies to paramagnetic nps. recognition of the hbsag in plasma samples, now bound to paramagnetic nps, was handled by another set of acridinium-functionalized antibodies. upon the introduction of hydrogen peroxide and sodium hydroxide into the system, chemiluminescence signals indicative of the amount of hbsag were emitted. they were exploited by architect system optics for quantification purposes. in immunological tests, ecm sensing plays a significant role ( table ). aunps served as the carriers for five different antibodies in an ecm immunosensor array that concurrently detects five separate strains of hbv (tang et al., ) . while its performance was comparable to that of conventional elisa, it was demonstrated to be more energy-efficient and able to produce results within min (ye et al., ) . if aunps attract all the limelight in nat, a wide range of nps have found utility in the detection of pathogenic antigens. one such nanosized material is graphene. by virtue of its distinguished electron transfer quality, graphene film was used to construct electrodes for detecting rotavirus (liu et al., ) (fig. s (a) ). antibodies specific to the viral particles were anchored onto the surface of the graphene-based electrodes. a wide range of graphene-based nanomaterials have been investigated for application in the field of nanomedicine. that alone is proof of the utility of graphene nps. however, development of graphenebased biosensors has been to some extent impeded by a lack of reproducibility and scalability of the manufacturing processes. graphene nps have also been exploited for other functions. for instance, graphene oxide nps were employed by chen et al. as fluorescence quenchers in an assay capable of simultaneously detecting both human enterovirus (lods: . ng/ml) and coxsackievirus b (lod: . ng/ml) . this assay also made use of qds, another type of nps. for the detection of two unrelated species of viruses, two kinds of qds, which possessed distinct optical behaviors for selective quantification, were required to conjugate the two kinds of antibodies. both kinds of qds were in turn functionalized with graphene oxide, which efficiently suppressed fluorescent signals from qds. when either human enterovirus or coxsackievirus b was present in the samples, the corresponding qds detached themselves from graphene oxide nps and emitted fluorescence. the signals could be picked up and quantified. in another study, graphene oxide nps were used together with silver nps (agnps) for simultaneous diagnosis of hbv, hiv and treponema pallidum (liu et al., ) . one major defect of graphene-based nanomaterials is their high hydrophobicity, which is responsible for formation of clumps in solution. these bulky aggregates indiscriminately bind biomolecules other than the desired targets, and could cause denaturation of the sample (yildiz et al., ) . mnps were employed in a sandwich-type immunoassay for the detection of salmonella typhimurium (gehring et al., ) . anti-s. typhimurium antibodies were conjugated onto superparamagnetic beads while being functionalized with alkaline phosphatase. phosphatase was the key element of ecm sensing mechanism in that investigation. after sample introduction, the inherent magnetism of mnps allowed to attract the complex towards disposable graphite ink electrodes. this step was included in the protocol to enhance the efficiency of ecm detection. a drawback of this particular assay was that it took in total min, which made it much more time-consuming than many other poc systems. given the fact that gehring et al. reported this immunoassay more than a decade ago, the lack of efficiency in assay time was understandable. in an enzyme-free ecm immunosensor, silicon nanowires were functionalized with antibodies for the detection of influenza a virus (patolsky et al., ) (figure s (b) ). a change in conductance was recorded once the complex was exposed to viral particles. such quantifiable change was observed when the sample contained paramyxovirus and adenovirus, but not influenza a virus. this helped establish that the assay possessed a clinically relevant level of selectivity. remarkably, samples containing single viruses could be detected without compromising the selectivity. this level of performance would compare favorably against the mainstream pcr-based techniques. moreover, the system was demonstrated to be capable of multiplexing. • excellent specificity ( . %) when tested on specimens • performed better than other hbsag assays in terms of accurate detection • capable of detecting more substitution mutants than an earlier versions (tang et al., ) analyte: multiple types of hepatitis virus antigens (hav, hbv, hcv, hdv, hev) reproducibility: inter-assay imprecision level at . % assay time: min detection range: • lod slightly varied between antigen types, ranging from . ng/ml for hbv to . ng/ml for hcv and hev • same upper limit of linear range ( ng/ml) clinical/real-world samples tested? yes (human serum) performance: • quality of results comparable with that of conventional elisa • some cross-reactivity between adjacent sites (≤ . %) purpose: enterovirus and coxsackievirus b assay time: shorter than equivalent methods (e.g. rt-pcr) detection range: • (perez et al., ) analyte: herpes simplex virus or adenovirus sample size: μl sample pretreatment: minimal detection range: • viral particles in μl samples • viral particles in μl samples clinical/real-world samples tested? no performance: • superior than common pcr-based techniques • capable of analyzing complex turbid samples • more sensitive than elisa assays (lien et al., ) analyte: dengue virus serotype sample size: μl sample pretreatment: • the concentration of anchored antibody does influence performance of the assay (reducing the concentration from mg/ml to mg/ml lengthens the detection range) • selectivity demonstrated when tested against escherichia coli o :h and liseria genus (zhao et al., ) analyte: escherichia coli o :h sample pretreatment: • no amplification or enrichment required assay time: min clinical/real-world samples tested? yes (spiked ground beef) performance: • could even detect a single bacterium in the samples (verified using two distinct quantitative techniques) as mentioned in the previous section, mnps have been integrated into ecm immunosensors before. nonetheless, they are more extensively applied in poc devices, which utilize magnetic resonance detection method ( fig. (a) ). a phage-based magnetoelastic biosensor was adopted to analyze salmonella typhimurium on fresh tomato surfaces (li et al., ) . use of filamentous e phages facilitated the binding of the analytes. the resonance frequency generated by the wireless biosensors could be quantified via magnetic fields. traditional magnetic beads typically used in biological separation have a diameter of about − μm. in contrast, mnps are much smaller (b nm in diameter). as such, they have a substantially larger surfaceto-volume ratio (josephson et al., ) . superparamagnetic iron oxide nps were used to anchor antibodies for the detection of herpesvirus or adenovirus in μl of sample volume (perez et al., ) . the iron oxide nps were coated with dextran. this layer could be further functionalized with amino groups, thereby facilitating antibody conjugation (josephson et al., ) . existence of viral particles in the samples triggered self-aggregation of the mnps to form a complex with augmented magnetic properties. this change in structure then allowed for quantitative detection. it was observed that the percentage of serum of the samples did have an impact on the sensitivity of the assay. in % serum samples, the lod was virions, but it dropped to as low as virions when % serum samples were investigated. an immunosensor with that impressive lod is undoubtedly promising. while it can efficiently scan serum samples for viral infections, its application in the detection of bacteria seems far from being ideal (kaittanis et al., ) . upon being exposed to a low bacteria count, the mnps would clutter together on the surface of the pathogens. however, if the count was above a certain threshold value, the mnps would revert back to a dispersed state just like how they would behave under pathogen-free circumstances. therefore, the assay could potentially produce false negatives. with this serious flaw left unaddressed, applications of this system are confined to analysis of infectious diseases generally known to display a low serum pathogen count (e.g. mycobacterium avium spp. paratuberculosis (map)) (kaittanis et al., ) . a separate study investigated the use of dextran-coated iron oxide nps, this time in the form of nano-sized rods, also for the diagnosis of map (liao et al., ). an lod of cfu could be achieved after just min. one principal shortcoming of magnetic resonance detection method is the requirement of machineries such as magnetic relaxometers or other instruments specialized to perform nuclear magnetic resonance (nmr). aside from being too costly, their operation demands a certain level of technical skill of the personnel. this ultimately undermines the suitability of this detection method for application in poc devices. magnetic properties of mnps have not only been exploited for magnetic resonance sensing, but also for deliberate isolation of the pathogens of interest from the sample (fig. (b)(c)(d)). such maneuver allows for sample enrichment. lien et al. immobilized antibodies onto mnps to trap dengue virus from the sample (lien et al., ) . using a planar micro-sized coil, a magnetic field gradient was set up to attract the viral particles captured on mnps. the separation efficiency achieved was fairly high at %. as a result, pathogenic quantification could then be executed with a greater level of sensitivity. to be specific, the lod was brought down to fu/ml. in another study, xia et al. employed mnps to bind and extract escherichia coli virions from the flow of a solution that simulated human blood samples (xia et al., ) . in place of a planar microcoil, a high-gradient magnetic field concentrator was used to generate the necessary magnetic field gradient. it was noticed that the separation efficiency of the system did not deteriorate with time. at flow rates from to μl/h, separation efficiency of mnp-bound bacterial cells ranged from % to over %. raising the cell density of input flow brought about a great increase in throughput rate. one common setback faced by both systems was a poor capacity for concentrating samples. nevertheless, it is not universally observed among experiments that perform magnetic separation using mnps. in a microfluidic chamber device, which employed a close-packed columns of polydispersed iron nps and a ndfeb permanent magnet, . ml of hiv-inflicted plasma samples was concentrated by times. this remarkable concentrating power would significantly raise the sensitivity level of any quantification method subsequently employed for the diagnosis of hiv (chen et al., a) . ecm sensing was one of the methods which have been coupled with magnetic separation to enhance sensitivity. escherichia coli o :h , an enterohemorrhagic serotype of escherichia coli, was subjected to diagnosis using this combination of techniques (gu et al., ) . setterington et al. took the modification a step further and bioconjugated the bacterial cells with polyaniline after they were magnetically separated (setterington and alocilja, ) . the electroactive label allowed quantification using cyclic voltammetry. this played a huge role in strengthening the ecm signals emitted. consequently, a lod of cfu/ml was attained. notwithstanding the relatively lengthy sampling time of min, this experimental poc system can be carried out using compact and mobile devices, hence confirming its suitability for a broad range of relevant applications. it has been demonstrated that magnetic separation of the pathogenic particles also allows for microscopic identification of the microorganisms. in one study, vancomycin was conjugated to mnps in order to trap vancomycin-resistant enterococci (gu et al., ) . with the help of an external magnetic field, the mnps-bound bacteria were accumulated into an area around mm large. biological separation was followed by observation using optical microscope, and finally verification with the help of electron micrograph. a lod as low as cfu/ml was achieved. this kind of assays has several advantages (ghindilis et al., , lin and ju, , warsinke et al., . it has inherently excellent sensitivity relative to other kinds of diagnostic tests, could easily be adopted as poc technology, and is, above all else, cost-effective. nano-sized materials have been widely studied as add-ons to accompany transducers so as to facilitate electron transfer process, magnify the snr of the system, or to heighten the efficiency of antibody conjugation . in some cases, nps complex have also been explored as ecm labels, and anchor points for antibodies. it has been demonstrated that antibodies do retain their biological binding activity after being conjugated to nps (e.g. aunps) (liao et al., ) (table ) . a range of nps have been investigated for application in proteinsensing ecm immunosensors ( fig. s (a) ) (hansen et al., , liu et al., , yuan et al., . among them, qds are fairly popular in multiplexed assays for parallel detection of several biomarkers at the same time (fig. s (b) ) (liu et al., ) . to this end, one set of antibodies is immobilized onto magnetic beadseach type of antibody belonging to the other set is conjugated to a distinct type of metal sulfide semiconductor. the examined qds included cds, zns, pbs, and cus, which all have comparable levels of sensitivity. the hydroxyl terminals of these nano-sized colloidal tracers enabled the antibody functionalization process via carbamate bonds. the use of sets of antibodies served as the central elements of an ecm sandwich immunoassay. the antigens of interest, be it proteins or a human antibodies, were captured between magnetic beads and qds with the corresponding antibodies. each individual antibody-antigen binding event would produce a characteristic voltammetric peak. the position and magnitude of the peaks provide detailed information on how much of each table examples of diagnostic poc systems whose target analytes are proteins and antibodies. author key features electrochemical (ambrosi et al., ) analyte: human igg sample size: μl sample pretreatment: • mixing with antibody-coated magnetic beads • labeling with double codified aunps detection range: varied between the two quantification methods employed • spectrophotometry: lod of pg/ml • ecm: lod of pg/ml clinical/real-world samples tested? no performance: • more sensitive than conventional elisa techniques • selectivity demonstrated by testing against goat igg (yuan et al., ) analyte: c-reactive protein (hscrp) and soluble cd ligand (scd l) sample size: μl sample pretreatment: minimal reproducibility: • acceptable level of reproducibility • inter-assay standard derivations were . % and . % for hscrp and scd l respectively detection range: . - ng/ml • lod of hscrp: . pg/ml • lod of scd l: . pg/ml clinical/real-world samples tested? yes (human serum) performance: • stability of system demonstrated by storing the immunosensor at °c for days in between assays ( . % and . % of initial response achieved for hscrp and scd l respectively) optical (zhu and yang, ) analyte: anti-rabbit human igg sample size: μl sample pretreatment: minimal assay time: min detection range: - μg/ml clinical/real-world samples tested? no performance: • reducing sample size to μl helps reduce assay time to around min, but at the same time compromises the sensitivity (higher lod of μg/ml) • selectivity demonstrated by testing against bovine serum albumin (at much higher concentration than the target analyte) biomarker (e.g. β -microglobulin, igg, bovine serum albumin, and creactive protein) was present in the sample. qds were accompanied by aunps and thiolated aptamers in an ecm immunesensor system, which reportedly yielded an lod of ng/l (fig. s (c) ) (hansen et al., ) . aptamers are synthetic nucleic acid ligands which have been studied as substitutes to antibodies. they possess several desirable qualities, out of which resistance to denaturation is perhaps the most laudable. in general, aptamer-based biosensors could achieve excellent sensitivity. this forte was apparent in this particular investigation, whereby trace amounts of proteins could be detected. being energy-efficient, easily miniaturized, and economical, the assay satisfied a handful of assured criteria. in another study, cadmium tellurite qds were conjugated to silica nps for magnification of ecm signals. the system was designed for detecting epstein-barr virus-derived latent membrane protein (lmp- ) (chen et al., b) . the especially large surface area of nano-sized carriers allowed for the immobilization of numerous qds. this served as the basis for the augmentation of ecm signals detected by square wave voltammetry. as a result, an lod of pg/ml was achieved. the invariable efficiency of qds immobilization process also worked in our favor by ensuring excellent reproducibility of the assay. the flexibility of qds is apparent when we take into account the properties that make them excellent fluorescence emitters. by adjusting the size of these nano-sized semiconductors, it is possible to manipulate their emission wavelength range (pinaud et al., ) . ergo, a single absorption wavelength could be used to trigger a range of emission wavelength, given that qds of varying size are employed. this can be tapped on for potential development of fluorescence-based multiplexed assays (hare et al., ) . multiplexed assays can also be achieved with mnps. in one study, hybrid nps, which consisted of a nife o core enclosed within a sio shell, were used to anchor different kinds of antibodies for concurrent detection of four distinct biomarkers (tang et al., ) . the extent of ecm signal interference between adjacent electrodes (each electrode designed to detect one biomarker) was minimal. ambrosi et al. reported an immunoassay compatible with two separate detection methods (ecm and optical) for quantifying human igg (ambrosi et al., ) . aunps were conjugated with antibodies specific to human igg. these antibodies were then bonded to horseradish peroxidase. detection step could be done spectrophotometrically by measuring the intensity of the solution's color emanated from aunps. alternatively, innate ecm behaviors of the aunps could be quantified with stripping voltammetry. the use of paramagnetic beads enabled magnetic separation of the labeled antibody complex. as a consequence, the sensitivity of the assay outperformed conventional elisa tests. the lod of optical and ecm detection methods was pg/ml and pg/ ml respectively. in addition, the use of mnps helped curtail incubation and washing time, which then contributed to a more desirable total assay time. just considering optical detection method alone, europium (iii) nps (eunps) have been contemplated as an excellent substitute for aunps. they were meant to help reduce the sophistication of the assays without compromising their sensitivity (tang et al., ). the optical properties of these nano-sized fluorophores render them suitable for immunosensors. after all, eunps are capable of producing robust and lasting fluorescence (hemmila et al., ) . in one investigation, they were encapsulated inside polystyrene nps for the detection of anthrax protective antigen (tang et al., ) . the system was considerably reliable, since no false negatives were observed. meanwhile, the assay attained a level of sensitivity times greater than that of conventional elisa, whose lod was known to be around ng/ml (moayeri et al., ) . poc devices which permit visible readouts are generally associated with a more affordable cost, since the need for advanced instruments for quantitative detection is eliminated. it is therefore a welcomed addition to poc technology, considering how it helps realize one key element of the assured criteria. one recent example microfluidic immunoassay with naked-eye readouts leveraged on the changing appearance of liquid crystals (zhu and yang, ) (fig. s (a) ). binding events between antigens and immobilized antibodies triggered a shift of the lqc appearance from dark to bright. this phenomenon could be visualized without the need for sophisticated devices. the technique exhibited good robustness, a lod of μg/ml. in addition, good specificity was demonstrated using bovine serum albumin as the non-target interference ( -fold concentration compared to the target analyte). another example is the volumetric bar-chart chip reported by song et al. for the detection of disease-specific proteins (song et al., ) (fig. s (b) ). in this study, catalase and antibody molecules were both conjugated onto the surface of silica nps. if the target analyte was present in the sample, the enzyme would catalyze the decomposition of hydrogen peroxide in the solution to give out oxygen. the extent of pressure build-up inside the enclosed columns was proportional to the amount of oxygen gas produced. the rise in column pressure elevated the ink columns upwards. in a nutshell, the extent of elevation was designed to be indicative of the amount of the protein biomarkers in the sample. the presentation of quantitative results in the form of bar charts, as suggested by the name of the device, could be readily interpreted with just naked eyes. moreover, the oxygen-producing reaction facilitated by catalase occurred very rapidly, within seconds (george, ) . this was arguably the key feature of the system, which helped shorten the total assay time. duration was further decreased by virtue of this poc system's impressive multiplexing power, which permitted up to concurrent tests. apart from the aforementioned strengths of this system, it did exhibit certain limitations (zhu et al., ) . the biocatalytic capability of the enzyme could possibly be impaired during the conjugation step. the fact that catalase enzyme itself is highly susceptible to hydrolysis further cast doubt on the reliability of the assay. more recently, zhu et al. developed an immunoassay based on similar concepts, but with certain alterations to expunge the existing drawbacks (zhu et al., ) . to tackle the problem at its root, the easily degraded catalase was replaced by hybrid nps comprised of a gold shell and a platinum core (au@ptnps). these au@ptnps were encapsulated inside aptamer-modified hydrogels. when the biomarker of interest was added, its interaction with the aptamers found on the exterior of the hydrogels would trigger their disintegration. au@ptnps would then come into contact with hydrogen peroxide molecules already present in the test solution. in this manner, the hybrid nps assumed the role of catalase. this led to the formation of visible bar-chart displays, as explained above in the study of zhu et al. (zhu and yang, ) . this novel system could be conveniently adopted for detecting numerous protein and antibody biomarkers. after all, a diverse selection of aptamers could be procured through different means (ellington and szostak, , tuerk and gold, ) . subramaniam et al. demonstrated another system with visible readouts, referred to as metal-amplified density assay (subramaniam et al., ) . levitation of diamagnetic polystyrene beads was employed as the parameter for interpretation by naked eyes. in essence, antibodies specific to the biomarkers were conjugated onto these nano-sized beads. after sample introduction, successful immunoreactions would bring about a change in the density of the polystyrene beads. at first glance, such physical alteration proved to be too minute to be reliably detected. to address this issue, the authors revised the protocol by incorporating aunps into the poc system to amplify the change in density. visible readouts using floating height of small particles are much more attractive than those dependent on colorimetric interpretation (martinez, ) . the former have shown promises in parallel testing capacity by virtue of several kinds of colored beads. for instance, it was explored for simultaneous diagnosis of syphilis and hepatitis c (subramaniam et al., ) . however, its protocol required a handful of steps which could appear confusing to on-site test performers. by producing diagnostic test results within a short period of time, at low cost, and without the need for advanced instruments or welltrained technicians, poc devices are undoubtedly dream companions for medical professionals in resource-poor regions. sometimes, the availability of poc devices could very well make the difference between life and death, given their ability to produce timely test outcomes. for those mobile devices whose operational instructions have been sufficiently simplified, the task of carrying out the tests could be entrusted to the caretaker, or even the patients themselves if the need arises. notwithstanding its far-reaching medical applications, poc technology in general, and nano-sized materials in particular, are still in early phases of development. throughout this review, we have discussed several poc devices currently in experimental stage. they all excel in certain aspects, but at the same time fail to satisfy every single assured criterion. this makes a case for further improvements. one direction for improvement is to develop a platform which is able to deal with samples without the requirement of preprocessing (e.g. hiv in whole blood or pathogen on fresh tomato surface (li et al., ) ). this can be truly helpful to users for a one-step diagnosis. two feasible methods may be adopted for this purpose. one is further enhancement of sensitivity and another is having integration with microfluidic components for sorting and purification functions (bi et al. ) . advancements in the field have rendered the synthesis of inorganic nps (e.g. aunps (craig et al., ) , sio nps (li and zhao, ) ) largely reproducible, with respect to physical parameters such as size distribution. attaining uniformity of organic nps used to be a real challenge, but progresses have been made in that aspect. recently reported syntheses achieved acceptable levels of reproducibility and desirable polydispersity indices (b . ). since nps are primarily employed as carriers onto which biomolecules are conjugated, their size distribution is one of the key factors predisposing the performance of the poc systems. therefore, future researches into fine-tuning reproducibility of nps will further enhance the reliability of poc systems. it also helps that the developments in poc technology are easily adopted horizontally, as long as the biomarkers belong to the same class (e.g. nucleic acids). with the current amount of time and effort invested into researches on poc technologies, it is not much of an exaggeration to state that breakthroughs are bound 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gold electrode a dna biochip for on-the-spot multiplexed pathogen identification recent advances in micro/nanotechnologies for global control of hepatitis b infection a simultaneous electrochemical multianalyte immunoassay of high sensitivity c-reactive protein and soluble cd ligand based on reduced graphene oxide-tetraethylene pentamine that directly adsorb metal ions as labels point-of-care multiplexed assays of nucleic acids using microcapillary-based loop-mediated isothermal amplification a rapid bioassay for single bacterial cell quantitation using bioconjugated nanoparticles microfluidic immunoassay with plug-in liquid crystal for optical detection of antibody au@pt nanoparticle encapsulated target-responsive hydrogel with volumetric bar-chart chip readout for quantitative point-of-care testing this research has been supported by the national university of singapore, department of pharmacy ((acrf) tier -frc grant r- - - - , r- - - - ; nusage grant n- - - - ), by moe of singapore (grant moe -t - - , r- - - - ) and by a-star-serc (r- - - - ). leung kai fook grant (r- - - - ). supplementary data to this article can be found online at http://dx. doi.org/ . /j.biotechadv. . . . key: cord- -d wbfrx authors: nakayasu, ernesto s.; nicora, carrie d.; sims, amy c.; burnum-johnson, kristin e.; kim, young-mo; kyle, jennifer e.; matzke, melissa m.; shukla, anil k.; chu, rosalie k.; schepmoes, athena a.; jacobs, jon m.; baric, ralph s.; webb-robertson, bobbie-jo; smith, richard d.; metz, thomas o. title: mplex: a robust and universal protocol for single-sample integrative proteomic, metabolomic, and lipidomic analyses date: - - journal: msystems doi: . /msystems. - sha: doc_id: cord_uid: d wbfrx integrative multi-omics analyses can empower more effective investigation and complete understanding of complex biological systems. despite recent advances in a range of omics analyses, multi-omic measurements of the same sample are still challenging and current methods have not been well evaluated in terms of reproducibility and broad applicability. here we adapted a solvent-based method, widely applied for extracting lipids and metabolites, to add proteomics to mass spectrometry-based multi-omics measurements. the metabolite, protein, and lipid extraction (mplex) protocol proved to be robust and applicable to a diverse set of sample types, including cell cultures, microbial communities, and tissues. to illustrate the utility of this protocol, an integrative multi-omics analysis was performed using a lung epithelial cell line infected with middle east respiratory syndrome coronavirus, which showed the impact of this virus on the host glycolytic pathway and also suggested a role for lipids during infection. the mplex method is a simple, fast, and robust protocol that can be applied for integrative multi-omic measurements from diverse sample types (e.g., environmental, in vitro, and clinical). importance in systems biology studies, the integration of multiple omics measurements (i.e., genomics, transcriptomics, proteomics, metabolomics, and lipidomics) has been shown to provide a more complete and informative view of biological pathways. thus, the prospect of extracting different types of molecules (e.g., dnas, rnas, proteins, and metabolites) and performing multiple omics measurements on single samples is very attractive, but such studies are challenging due to the fact that the extraction conditions differ according to the molecule type. here, we adapted an organic solvent-based extraction method that demonstrated broad applicability and robustness, which enabled comprehensive proteomics, metabolomics, and lipidomics analyses from the same sample. author video: an author video summary of this article is available. given metabolite can be measured by metabolomics, which can result from the regulation of either its biosynthetic or degradation pathways. however, also measuring the levels of enzymes of each pathway using proteomics can reveal which mechanism is being regulated. further, measurements of the enzyme rna levels can also provide key information on whether the regulation occurs at the transcriptional or posttranscriptional level. for example, bordbar et al. built a metabolic network model based on available genomic sequences to study macrophage activation and subsequently used transcriptomics, proteomics, and metabolomics information to further refine the model, which led to a better understanding of the impact of metabolism during an inflammatory response ( ) . in the context of multi-omics analyses, being able to perform multiple measurements on the same sample can also decrease experimental variation. additionally, this approach can be very useful when samples are difficult to obtain, i.e., for some environmental and patient samples (e.g., biopsy specimens) and for samples from high-biosafety-level laboratories, where working conditions are not optimal and are otherwise rigorously controlled. in addition, limited volumes or amounts of samples may preclude splitting them prior to performing parallel extractions and sample processing. recent studies have evaluated the use of variations of chloroform/methanol extraction methods to isolate proteins, metabolites, and lipids or to sequentially extract dna, rna, proteins, metabolites, and lipids, sometimes with the use of different commercial kits, and all from the same sample ( ) ( ) ( ) ( ) ( ) . while the use of chloroform/ methanol mixtures is well established for metabolomics and lipidomics sample preparation (we routinely use such a protocol in our laboratory), the reproducibility of proteomics, transcriptomics, and genomics measurements and their applicability for a diverse range of samples require further investigation. indeed, we have found only a single report of an evaluation of the reproducibility of extraction of rna and protein and of the reproducibility of the resulting proteomics data from a single sample type; weckwerth et al. found that rna and protein that were extracted from arabidopsis thaliana had coefficients of variation (cvs) of % and %, respectively, when using a multi-omic extraction protocol based on the use of chloroform/methanol ( ) . targeted quantification of peptides mapping to proteins showed cvs of % on average. recent analysis of the material obtained using different kits for multiple extractions showed reduced yields and/or quality of the end products ( ) . this could have been due to the fact that optimum buffers and solutions differ for extracting dna, rna, proteins, or metabolites and that longer extraction protocols may lead to material degradation. methods employing organic solvent extractions, such as the combination of chloroform, methanol, and water, have been widely used for extracting lipids and other metabolites ( , ) . in this procedure, a chloroform and methanol solution is added to samples resuspended in water or aqueous buffer, or directly to samples that have sufficient water content, so as to induce the formation of two solvent layers-an upper aqueous phase, containing hydrophilic metabolites, and a lower organic phase, containing lipids and other hydrophobic metabolites-while proteins precipitate in the interphase. since organic solvent extraction is a simple and quick procedure, we reasoned, as others have ( ) ( ) ( ) ( ) , that it would prevent protein loss by degradation and make possible the simultaneous extraction of lipids, metabolites, and proteins for subsequent omics analyses. furthermore, organic solvents can be easily removed by evaporation, minimizing the introduction of artifacts during sample preparation. in this work, we sought to develop a robust protocol for simultaneous metabolite, protein, and lipid extraction (mplex) from the same samples for integrative multi-omic analyses. we based the protocol on a chloroform-methanol-water extraction method routinely used in our laboratory to simultaneously prepare metabolite and lipid extracts from the same sample. others have demonstrated the reproducibility of the resulting metabolomics and lipidomics data in using variations of this protocol for select sample types ( , , , ) . to evaluate the broad applicability of expansion of this method for proteomics, we performed comprehensive proteomics analyses of the protein material extracted with the mplex procedure from a variety of samples, including a gramnegative bacterium, an archaeon, an environmental microbial community, a plant leaf, a murine tissue, a human body fluid, and a cell line. we found that the proteome coverage for this diverse set of samples was very similar to that seen with matched control samples prepared in parallel using a standard proteomics sample preparation method, suggesting the broad applicability of the protocol. we then applied this methodology and integrated proteomic, lipidomic, and metabolomic analyses in the study of middle east respiratory syndrome coronavirus (mers-cov) infections in a lung epithelial cell line, which showed the impact of viral infection on different host metabolic pathways. impact of different metabolite extraction methods on proteomic analysis. integrative multi-omics analysis is a powerful approach to study complex biological responses and has gained popularity in recent years ( ) ( ) ( ) . in this context, the prospect of being able to perform multiple omics measurements on the same sample is very attractive but the method is still difficult to implement, likely due to the distinct optimal conditions for extracting different types of molecules. aiming to develop a protocol for global multi-omic analyses of the same sample, we modified an extraction approach based on a chloroform-methanol-water solution to simultaneously obtain metabolite, protein, and lipid fractions. since the protocol is well established and since we have applied it successfully for the analysis of lipids and other metabolites in several studies ( ) ( ) ( ) ( ) ( ) ( ) , we focused our efforts on determining if the method is applicable for global proteomic analysis and the associated quantification of relative amounts of proteins (i.e., the determination of fold increase or decrease in protein expression). we tested the mplex method with the gram-negative bacterium shewanella oneidensis by extracting its proteins, lipids, and metabolites (n ϭ ). as a comparison, we also performed extractions using % methanol (meoh) or % acetonitrile (acn) (n ϭ [each]), which are commonly used solvents for metabolomics extractions. we found that significantly reduced total protein fractions were recovered after extraction of metabolites and lipids by all three methods compared to control samples prepared using a standard protocol (control) (fig. a) . these results are in agreement with previous data from the literature showing that some protein mass is lost during precipitation procedures ( ) . we then evaluated if these protein losses affected the ability to obtain useful proteomic data, since a method that can simultaneously extract multiple omics sources from the same sample would be extremely useful for systems biology experiments and subsequent integrated data analysis, as well as in cases where limited sample amounts are available (e.g., a survey of data from the national cancer institute showed that obtaining an adequate number of samples to conduct a study is a major difficulty facing researchers [ ] ). thus, we investigated whether extraction with organic solvents would have a major impact on the coverage and the quantitative aspect of the associated proteomic analysis. to explore this issue, proteins extracted with mplex, acn, and meoh methods were digested in parallel with control samples, normalized by bicinchoninic acid (bca) assay, and analyzed by liquid chromatography mass spectrometry (lc-ms) using the accurate mass & time (amt) tag approach ( ) . the results of the proteomic analysis of samples extracted with different methods showed that the numbers of peptides detected in the mplex samples were very similar (no significant difference) to the numbers seen with controls (fig. b) . a significant increase in the levels of peptides was identified in samples extracted with acn, but no significant differences between the control and meoh extractions in the numbers of peptides were observed (fig. b) . the overlap of the numbers of peptides identified in samples extracted with all protocols was very high, as shown by a similarity matrix (fig. c) . the similarities between samples were even higher at the protein level ( fig. d and e). the similarity of the proteome coverage results obtained by the different extraction methods is remarkable, considering that much larger (up to -fold to -fold) differences are observed just by digesting proteins using different buffers, surfactants, or denaturing agents, even without any previous extraction ( , ) . our results show that despite some protein mass losses, the choice of extraction protocol did not significantly affect the proteome coverage. the selective loss of a few proteins during the extraction procedure is expected and has been shown in a study carried out with human plasma samples only ( ) . another important feature for multi-omic analysis is that of being able to accurately identify differentially expressed or abundant molecules. in this context, if the extraction procedure affects the quality of the proteins, then it might increase the variance across different samples. thus, we examined the correlation of the proteomics data between samples extracted with different organic solvents, and the results showed remarkable similarity at both peptide and protein levels ( fig. f and g). we then calculated the variance of protein measurements by comparing different extraction protocols. indeed, no significant differences in the distributions of coefficients of variance (cv) were observed comparing mplex with controls, with the cvs of the great majority of the proteins smaller than %, with peaks of Ͻ % (fig. h ). meoh extraction led to cvs that were similar to but slightly smaller than those seen with the mplex and control samples (fig. h ). on the other hand, acn extraction had a bimodal distribution, with very low and very high cvs (fig. h) , suggesting that some proteins are not reproducibly precipitated with this solvent. this phenomenon might be due to the fact that acetonitrile does not fully precipitate small proteins ( ) . taken together, these results showed that mplex did not affect the proteome coverage or the results of quantitative analysis of the s. oneidensis samples. to investigate whether the mplex protocol is robust and broadly applicable, we performed proteomic analyses of a very diverse set of samples that included the archaeon sulfolobus acidocaldarius, a unicyanobacterial consortium ( ) , mouse brain cortex tissue, human urine, cells of the calu- human lung epithelial cell line, and leaves from arabidopsis thaliana. whereas we compared mplex results to control results for most of these samples, the a. thaliana sample results were compared to results of extractions performed with saturated phenol or trichloroacetic acid (tca), because plant leaves are rich in phenolic compounds that need to be removed and that otherwise would interfere with mass spectrometric analysis, and these alternative protocols have been shown to perform well in preparations of plant samples ( ) . as observed for s. oneidensis, the proteome coverage was very high at both the peptide (see fig. s in the supplemental material) and protein ( fig. ) levels across the diverse set of samples when using mplex and comparable to that obtained using the standard protein extraction protocol, although minor differences were detected for the unicyanobacterial consortium and human urine samples. in the case of a. thaliana, similar proteome coverage results were observed in samples extracted using either tca or mplex ( fig. e ; see also fig. s e). however, despite repeating the experiment twice, we had very limited success in extracting leaf proteins using the phenol protocol. in terms of quantitative measurements, similar correlations were observed across different samples by comparing mplex results to control or tca extraction results at both the peptide and protein levels, although minor differences were observed in the results from the human urine samples ( fig. ; see also fig. s ). overall, comparing mplex to control or tca extraction, the levels of proteome coverage and correlation between samples were very similar (see fig. s ), suggesting no qualitative losses. the fact that the proteome coverage, correlation, and variability results of comparisons of samples using mplex are not different from those seen with the standard protocol indicates that the relative quantification of proteins, which is the type of quantification employed in the vast majority of proteomics studies, is not compromised. nonetheless, we investigated any losses of specific proteins that could affect studies focusing on absolute quantification of protein copy numbers. only . % and . % of the proteins in shewanella oneidensis were shown to be significantly enriched and depleted by more than -fold, respectively ( table ). the acn extraction showed a smaller number of significantly enriched or depleted proteins, which was likely a consequence of the higher variability in the replicates observed using this solvent (table ). in contrast, the meoh extraction showed much higher losses than mplex (table ) . with the exception of the human urine sample, all samples had losses corresponding to less than % of the proteins (table ) . to investigate possible causes of protein enrichment or depletion using mplex, several physical-chemical properties of the significantly enriched or depleted proteins were examined, including the number of proteins with transmembrane domains, molecular weight, length, hydrophobicity calculated by grand average of hydropathy (gravy), and isolectric point (pi). no pattern was consistently observed across the different samples for any of the tested physical-chemical properties, indicating that the small amount of enrichment or depletion of proteins induced by mplex is not based on such properties. although these small differences in protein extraction results seen using mplex should be considered in proteomics studies employing absolute quantification, they likely do not introduce artifacts in the results, as these studies typically have very small (up to %) errors when stable isotope-labeled peptides are used as internal standards ( ) and up to -fold to -fold variations in label-free analyses ( , ) . although protein oxidation is an important physiological posttranslational modification, it is also an artifact introduced during sample processing for proteomic analysis. considering that there is more o dissolved in organic solvents than in water ( ), it is reasonable to suspect that extraction performed with such solvents could increase the oxidation of peptides. thus, the number of peptides containing oxidized methionine residues was counted in each sample, and an increase in methionine oxidation was observed only in the s. acidocaldarius sample extracted with the mplex protocol (see fig. s in the supplemental material). however, the opposite trend was observed in s. oneidensis, mouse brain cortex, and unicyanobacterial consortium samples, and no difference was observed in the other samples (see fig. s ). these results suggest that the oxidation of peptides is sample dependent and that it is not induced by mplex. taken together, our data show that mplex is a robust protocol and can be applied for a variety of sample types without compromising the proteome coverage or quantitative measurements or inducing oxidation artifacts. to illustrate an application for mplex and the value of multiple omics measurements obtained from the same sample, we applied the method to study mers-cov infection. we specifically chose mers-cov because it is a deadly emerging infectious agent with subsequent disease mortality rates of approximately % and because there are currently no effective drugs available for treatment ( ) . since mers-cov is a newly emergent virus, information about the mechanism of virulence of the infection is very scarce in the literature and any new data would immensely contribute to a better understanding of the disease. in addition, experiments investigating mers-cov need to be performed in biosafety level (bsl ) facilities, which require extensive safety and decontamination procedures. thus, being able to analyze multiple omics from the same sample would significantly reduce the time of exposure risk of the researcher inside the biosafety facility. for this experiment, we used human lung epithelial calu- cells, which we initially tested as described above and which showed good proteome coverage (fig. d) . nine replicates of cell cultures were infected for h with mers-cov, while replicates were left uninfected as mock controls. samples were subjected to mplex and submitted for global proteomic, metabolomic, and lipidomic analyses. in total, , proteins, metabolites, and lipid species were identified and quantified (see tables s to s in the supplemental material). data from all three global measurements were then integrated using the metscape plugin of cytoscape (fig. a) ( , ) . we also performed a function-enrichment analysis based on the kegg database using the lrpath tool ( ) and combined this information into metscape. the lrpath analysis showed that pathways were significantly enriched in differentially abundant proteins (see table s ) and that of the pathways were from the central metabolism of the cell (fig. a) . from these pathways, we chose the glycolysis and gluconeogenesis pathways due to their complexity and the fact that these two pathways share most of the metabolites and enzymes therein. being able to determine which of these pathways is affected more during infection would result in valuable information for better understanding the disease. in fig. a , the nodes highlighted in yellow represent the glycolysis/gluconeogenesis pathway, which was separated into a subnetwork in fig. b for a better visualization. this pathway showed several proteins that were downregulated during mers-cov infection, which are represented in metscape by the small nodes (fig. b ). this pathway was then manually curated and visualized using the vanted tool ( ) (fig. c) , showing quantitatively that almost all proteins in the glycolysis/gluconeogenesis pathway were reduced in abundance during the infection with mers-cov (fig. c) . although limited numbers of metabolites from the glycolysis/gluconeogenesis pathways were detected, the reduced levels of glucose -phosphate (g p), dihydroxyacetone phosphate (dhap), and -phospho-d-glycerate ( pg) further support the idea of a decrease in activity of this central pathway (fig. c) . since glycolysis and gluconeogenesis share the same enzymes, proteomics alone is insufficient to determine exactly which process is affected. however, results from the addition of metabolomics, specifically, the observation that the initial substrate, glucose (glc), had accumulated, indicated that glycolysis was more likely than gluconeogenesis to have been affected by the viral infection (fig. c) . to conclude, the proteomics analysis by itself would show differences only in the abundances of the enzymes from the glycolysis/ gluconeogenesis pathway, but the addition of metabolite measurements helps confirm that the pathway activity is reduced and which direction is the more affected, clearly illustrating the advantage of integrating multi-omic measurements for studying specific metabolic pathways. to further demonstrate the utility of multi-omic analyses facilitated by the mplex protocol, we investigated mers-cov-stimulated changes in the calu- lipidome by integrating the measurements of sphingolipids and glycerophospholipids from the lipidomics analysis, free fatty acids from the metabolomics analysis, and enzymes from the proteomic analysis using the vanted tool (fig. ) . increases in the levels of all detected fatty acid species were observed in mers-cov-infected cells compared to mock controls (fig. ) . the increases in fatty acid levels appear unrelated to lipid synthesis itself, since almost all the enzymes of the synthesis pathway are downregulated with infection (fig. ) . conversely, the decrease in levels of enzymes in the fatty acid degradation pathway might be contributing to the accumulation of fatty acids (fig. ) . in addition, degradation of phosphatidylcholines (pc), lyso-pc, phosphatidylserines (ps), and lyso-ps by phospholipases might also have been contributing to the accumulation of fatty acids during infection (fig. ) . although the responsible phospholipase was not detected in the proteomic analysis, it seems to be specific to pc and ps, since other classes of glycerophospholipids and glycerolipids remained mostly unchanged during infection (fig. ) . more-extensive changes in abundance were observed in members of sphingolipid classes than in phospholipids. the abundance of hexosylceramide increased during mers-cov infection, seemingly due to a decrease in the levels of its degradation enzyme glucosylceramidase (gba) (fig. ). an increase of ceramide levels was also detected during infection which did not appear to be related to synthesis, since the abundance of serine palmitoyltransferase (sptlc ), the enzyme that catalyzes the first step of ceramide synthesis by condensing serine and palmitate into -ketosphinganine, was decreased (fig. ) . the accumulation of ceramides was most likely due to the degradation of sphingomyelin in combination with a decrease in levels of the ceramidase (asah ) (fig. ) . sphingolipids have been reported to play an integral role in viral uptake, replication, maturation, and budding during viral infection. membrane domains enriched with ceramides have been proposed to facilitate the entry of enveloped viruses into host cells by changing the membrane fluidity and enhancing vesicular fusion ( ) . ceramides are also known to trigger apoptosis and death of the host cells ( , ) . indeed, apoptosis has already been reported in bronchial epithelial cells infected with mers-cov ( ), but its relationship with the increased levels of ceramides still needs to be further investigated. overall, the lipid metabolic network built by integrating multi-omics measurements shows a much more complete and likely more accurate view of the lipid landscape compared to lipidomics alone and provides more insights concerning the mechanism of lipid regulation. integration of multi-omics measurements has been consolidated as a technique for studying complex biological systems ( - ) . thus, methods that enable multiple omics measurements on the same sample are not only attractive but the only choice in cases of samples with limited availability. in this context, the mplex method can be an excellent alternative since it has been shown to be robust and applicable for a variety of samples ranging from bacterial cells to environmental samples to animal tissue. it is worth noting that, in addition to metabolomics, proteomics, and lipidomics, it is very likely that mplex can be used for the analysis of posttranslational modifications. indeed, a preliminary unpublished phosphoproteomic analysis using mplex led to the identification of several thousand phosphopeptides, although more careful analysis is required to determine if there are losses in this process. to conclude, we demonstrate the utility of multi-omics integration using mplex to study a lung epithelial cell line infected with mers-cov, which showed major differences in central carbon and lipid metabolism during infection. samples. for this study, we chose a variety of sample types: plant leaves from arapdopsis thaliana, human urine as an example body fluid, the gram-negative bacterium shewanella oneidensis, the cultured tissue cell line calu- , a unicyanobacterial consortium isolated from hot lake, wa, usa ( ) , mouse brain cortex tissue, and the archaeon sulfolobus acidocaldarius strain dsm . calu- cell infection with mers-cov was performed as described in text s in the supplemental material. s. oneidensis, the unicyanobacterial consortium, and s. acidocaldarius cells were lysed by bead beating in a bullet blender (next advance, averill park, ny) with . -mm-diameter zirconia beads at speed for min at °c, and the lysate was spun into a falcon tube at , ϫ g for min at °c. additional lysis was done via pressure cycling technology (pct) using a barocycler (pressure biosciences inc., south easton, ma). the suspended cells were subjected to s of high pressure at , lb/in followed by s of ambient pressure for cycles. a. thaliana leaves were frozen with liquid nitrogen and mechanically disrupted on a mortar with a pestle. mouse brain cortex tissue was homogenized in ice-cold nanopure h o at full speed with a hand-held omni tool and a disposable probe (omni, kennesaw, ga) for s, allowed to cool, and homogenized again. extraction methods. each sample was processed in replicates using the following protocols. (i) metabolite, protein, and lipid extraction (mplex). the extraction procedure was adapted from the method of folch et al. ( ) by keeping the same final solvent proportions; however, the monophasic extraction step was not performed, as water was initially added to the sample along with the chloroform and methanol to simultaneously extract and partition molecules into the three different phases. cell pellets or lysates were resuspended in water, and volumes of cold (Ϫ °c) chloroform-methanol ( : [vol/vol]) solution was added to the samples. samples were incubated for min on ice, subjected to vortex mixing for min, and centrifuged at , rpm for min at °c. for the samples for which metabolomics and lipidomics analyses were performed, the upper aqueous phase and bottom organic phase, containing hydrophilic metabolites and lipids, respectively, were collected in glass autosampler vials. the interphases, containing proteins, were washed by adding ml of cold (Ϫ °c) methanol, vortex mixed for min, and centrifuged at , rpm for min at °c. the supernatants were discarded, and the resulting pellets were dried in a vacuum centrifuge for min. (ii) phenol extraction. powdered a. thaliana leaves were resuspended in ml of phenol extraction buffer ( . m tris-hcl [ph . ], containing . m sucrose, . m kcl, mm edta, % [vol/vol] ␤-mercaptoethanol, and mm phenylmethanesulfonylfluoride), and then ml of phenol solution saturated with mm tris-hcl (ph . ) was added to each tube. samples were shaken for min at °c and centrifuged at , ϫ g for min at °c. the upper phenolic phase was collected into a fresh tube and washed twice by adding ml of phenol extraction buffer, followed by centrifugation at , ϫ g for min at °c, and discarding of the lower phase. the upper phenolic phase was collected in a fresh tube, and volumes of . m ammonium acetate in methanol was added. samples were incubated overnight at Ϫ °c and centrifuged at , ϫ g for min at °c. protein pellets were then washed twice with ml ice-cold methanol and twice with ml ice-cold acetone by adding the solvent, centrifuging at , ϫ g for min at °c, and discarding of the supernatant. the resulting protein pellet was dried under a stream of n . (iii) tca extraction. ml of freshly prepared ice-cold tca-acetone extraction buffer ( . m trichloroacetic acid- % acetone) was added to powdered a. thaliana leaves, and the mixture was incubated overnight at Ϫ °c. proteins were then precipitated by centrifuging for min at , ϫ g at °c, and the supernatant was discarded. the protein pellet was washed three times by adding ml of ice-cold acetone, followed by centrifugation for min at , ϫ g at °c, and discarding of the supernatant. the resulting protein pellet was dried under a stream of n . (iv) acetonitrile extraction. lysates were resuspended in volumes of ice-cold (Ϫ °c) pure acetonitrile and incubated for min at °c to precipitate the proteins. the samples were centrifuged for min at °c at , ϫ g to pellet the protein. the supernatant was removed, and the protein pellets were dried by evaporation before digesting with trypsin. (v) methanol extraction. the methanol extraction was performed with the exact same procedure as the acetonitrile extraction, with the difference that the organic solvent was replaced by methanol. proteomic, lipidomic, and metabolomic analyses. the detailed methodology of proteomic, lipidomic, and metabolomic analyses are provided in text s in the supplemental material. for proteomic analysis, proteins were digested with trypsin into peptides and analyzed using the accurate mass & time (amt) tag approach ( ) . peptides were separated by nano-capillary liquid chromatography (nano-lc), and eluting peptides were directly analyzed using ltq-orbitrap velos or exactive mass spectrometers (thermo fisher scientific). peptides were identified by matching to the appropriate mass tag database, and the peak areas were extracted using viper ( ) . matching results were filtered with statistical tools for amt tag confidence and uniqueness probability scores ( ) . lipids extracted from calu- cells infected with mers-coronavirus were analyzed by lc-tandem ms (lc-ms/ms) using an ltq-orbitrap velos mass spectrometer as previously described ( ) . then, raw data files were analyzed using liquid (lipid informed quantitation and identification) software developed in-house for semiautomated identification of lipid molecular species followed by manual validation of identified species. polar metabolites extracted from calu- cells infected with mers-coronavirus were derivatized with n-methyl-n-(trimethylsilyl)trifluoroacetamide (mstfa) and analyzed by gas chromatography-mass spectrometry (gc-ms) as described previously ( ) . the raw data files were processed using metabolitedetector ( ) and manually validated. comparative analysis of different extractions. the proteomic analyses comparing the different extraction methods were performed by rolling up the intensity values of peptides into values corresponding to proteins using the r rollup function of inferno rdn (formerly dante) ( ) . only proteins with two or more peptides that were unique were considered for further analysis. the intensity values were transformed to log values and submitted to standard paired t tests and g tests ( ) (considering only proteins present in or of replicates). for analyses of proteomics, lipidomics, and metabolomics data from the calu- cells infected with mers-cov, the quantitative data profiles were evaluated for extreme outlier behavior ( ) . no outlier samples were observed in the metabolomics and lipidomics data; however, one proteomics replicate from the infected group showed extremely poor coverage and correlation, indicating an issue with the protein extraction. that one sample was removed from subsequent analyses. further quality assessment of the proteomics data included evaluation of individual peptides to identify those with inadequate coverage for either statistical analyses or protein quantification ( ) . metabolomic and lipidomic data were normalized via standard median centering, and proteomics data were normalized via median centering against a rank-invariant peptide subset identified to reduce bias ( ) . to allow evaluation of the proteomic data at the protein level, a signature-based protein quantitation methodology was employed ( ) . finally, the protein, metabolite, and lipid data were evaluated for quantitative differences between the results of mock infection and mers-cov infection via a standard two-sample t test. multi-omics data integration. accession numbers from proteomics data of the mers-cov-infected cells were converted into entrez gene identifiers (id) and uploaded to lrpath for function-enrichment analysis ( ) . then, expression values of metabolomics, lipidomics (both converted to kegg compound ids), and proteomics data were integrated using metscape v. supplemental material for this article may be found at http://dx.doi.org/ . / msystems. - . text s , docx file, . mb. figure s , tif file, . mb. figure s , tif file, . mb. figure s , 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no-mediated endothelial cell apoptosis via ceramideregulated glycogen synthase kinase- beta and nf-kappab activation bilateral entry and release of middle east respiratory syndrome coronavirus induces profound apoptosis of human bronchial epithelial cells a simple method for the isolation and purification of total lipides from animal tissues viper: an advanced software package to support high-throughput lc-ms peptide identification a statistical method for assessing peptide identification confidence in accurate mass and time tag proteomics metabolitedetector: comprehensive analysis tool for targeted and nontargeted gc/ms based metabolome analysis dante: a statistical tool for quantitative analysis of -omics data combined statistical analyses of peptide intensities and peptide occurrences improves identification of significant peptides from ms-based proteomics data improved quality control processing of peptide-centric lc-ms proteomics data a statistical selection strategy for normalization procedures in lc-ms proteomics experiments through dataset-dependent ranking of normalization scaling factors bayesian proteoform modeling improves protein quantification of global proteomic measurements key: cord- - w qcv c authors: ayginin, andrey a.; pimkina, ekaterina v.; matsvay, alina d.; speranskaya, anna s.; safonova, marina v.; blinova, ekaterina a.; artyushin, ilya v.; dedkov, vladimir g.; shipulin, german a.; khafizov, kamil title: the study of viral rna diversity in bird samples using de novo designed multiplex genus-specific primer panels date: - - journal: adv virol doi: . / / sha: doc_id: cord_uid: w qcv c advances in the next generation sequencing (ngs) technologies have significantly increased our ability to detect new viral pathogens and systematically determine the spectrum of viruses prevalent in various biological samples. in addition, this approach has also helped in establishing the associations of viromes with many diseases. however, unlike the metagenomic studies using s rrna for the detection of bacteria, it is impossible to create universal oligonucleotides to target all known and novel viruses, owing to their genomic diversity and variability. on the other hand, sequencing the entire genome is still expensive and has relatively low sensitivity for such applications. the existing approaches for the design of oligonucleotides for targeted enrichment are usually involved in the development of primers for the pcr-based detection of particular viral species or genera, but not for families or higher taxonomic orders. in this study, we have developed a computational pipeline for designing the oligonucleotides capable of covering a significant number of known viruses within various taxonomic orders, as well as their novel variants. we have subsequently designed a genus-specific oligonucleotide panel for targeted enrichment of viral nucleic acids in biological material and demonstrated the possibility of its application for virus detection in bird samples. we have tested our panel using a number of collected samples and have observed superior efficiency in the detection and identification of viral pathogens. since a reliable, bioinformatics-based analytical method for the rapid identification of the sequences was crucial, an ngs-based data analysis module was developed in this study, and its functionality in the detection of novel viruses and analysis of virome diversity was demonstrated. an increase in globalization, climate change, and interaction with livestock animals has resulted in the emergence of novel viral pathogens or zoonoses [ ] , which pose a serious health problem for birds and animals and ultimately for humans. the natural reservoirs of pathogens, such as birds, bats, rodents, and bloodsucking arthropods play a significant role in the sustenance and transmission of zoonotic infections. migratory birds warrant special attention, as their rich diversity and migratory behavior contribute to the spread of infections to considerable distances. such migrations are strongly associated with the emergence of the epidemic and enzootic outbreaks as well as the formation and activation of natural sources of viral infections. wild birds are widely acknowledged to be reservoirs and transmitters of pathogens responsible for emerging infectious diseases such as severe acute respiratory syndrome virus (sarsv), avian influenza virus a (h n ), and west nile virus (wnv), to domestic animals and humans [ , ] . the rich biodiversity of the advances in virology wild bird population may increase the risk of spread of pathogens to domesticated poultry. understanding the viral diversity is critical for predicting future risks of transmission or possible outbreaks of viral diseases. however, identifying and monitoring the transmission of novel viruses are one of the vital requisites for responding to outbreaks. the asymptomatic carriage of viruses, which could be attributed to certain characteristics of bird metabolism and the adaptive capabilities of their immune system, provides ideal conditions for coevolution, leading to the emergence of mutant and recombinant strains of viruses. hence, the majority of widely used molecular diagnostic methods, such as those employing polymerase chain reaction (pcr), are not sufficiently suitable for the identification of a wide variety of viruses, as the techniques are usually designed for the detection of highly conserved regions in the genomes, which limits the search to a restricted group of viral agents and prevents the identification of new viruses or viral variants. in addition, the sanger sequencing technique, which has been a standard diagnostic tool for the detection and identification of various pathogens, provides limited sequence information at a higher cost per nucleotide base and can only be used to identify pathogens with a high titer. moreover, a preliminary evidence of the presence of the certain viruses is required for performing the pcr, in the absence of which the process of pathogen identification could take a significant amount of time, which can be a major obstacle in the prevention and control of the infection. dna barcoding is a method which uses a short part of organism's genome (so-called barcode) to identify whether it belongs to a particular family, genera, or even species, by using extensive parallel sequencing technologies (or more commonly ngs [next generation sequencing]). this method was initially developed for studying bacterial communities (e.g., studies on gut microbiota), but today it is widely employed for various tasks, including detection of food adulteration [ ] , the study of the diets of marine communities [ ] , and biofuel analysis [ ] . unlike other taxa, viruses lack a universally shared phylogenetic marker (such as s for bacteria, cytochrome c oxidase for birds and mammals, rbcl and matk for plants, and internal transcribed spacer (its) for fungi or plants), which makes it impossible to design a universal primer pair to amplify and differentiate diverse viral sequences. furthermore, the viral taxonomy gives a better indication of the signs of diseases caused, rather than genetic similarity. this fact complicates the barcode (a short, standardized nucleotide sequence of an organism's dna) selection, even for one genus (for example, mammarenavirus can be serologically, phylogenetically, and geographically divided into two major complexes: the old world complex prevalent in africa, europe, and asia, and the new world complex found in north and south america [ ] ), let alone higher taxa. however, metagenomics still allows the detection of different viral pathogens using shotgun sequencing [ ] . despite the constant reduction in the cost of dna sequencing, this approach is still considerably expensive and is not feasible for screening a large number of samples. also, metagenomic ngs data-sets are usually predominantly composed of host-derived sequences with only a minor fraction of pathogen sequences. often, even an approximate fraction of pathogen content is initially unknown, making it practically impossible to estimate how many sequencing reads are needed per sample, in order to detect the pathogen in the final sequencing data file. another common problem for most ngs-based tests is that complex multistep workflows may pose challenges in the reproducibility of results. recently, briese et al. [ ] had developed a virome capture sequencing platform for vertebrate viruses (vircapseq-vert), which consisted of ∼ million biotinylated dna-probes for target enrichment of viral nucleic acids to increase the sensitivity of sequence-based detection and characterization of viruses. the described method allowed the identification (and possibly even sequencing the whole genome assembly of detected viruses) of a large number of viruses including the novel ones. however, the overall cost of sequencing per sample remained considerably high. other methods of enrichment are based on the targeted amplification of cdna region using genus-specific primer pairs in pcr. this approach has been known for a long time [ ] and has been used successfully by various researchers, both in the studies of the representatives of individual viral genera and in the analyses of the diversity of viruses in different types of biological material [ ] [ ] [ ] . till recently, a large number of such primers have been described [ ] [ ] [ ] . however, most of them were designed for the detection of certain species of viruses. moreover, it was impossible to use them in multiplex reactions due to primer-specific annealing temperatures, nonspecific amplification, and potential selfcomplementarity. thus, in order to analyze one sample, it is necessary to carry out a number of pcr experiments corresponding to the number of primer pairs. the effectiveness of the study could be significantly increased by combining genus-specific pcr and ngs. in this approach, the products of different pcr assays for each sample would be pooled in one tube, purified, eluted in a minimal volume, and prepared for ngs [ ] . however, this approach does not exempt the requirement of many pcrs per sample, which is a problem when a large number of samples are studied simultaneously. in addition, there are restrictions on the use of different protocols for the library preparation, particularly when the protocol includes the emulsion pcr stage, which strictly requires pcr products of the same length. in this study, we have introduced a method for designing oligonucleotide panels for targeted enrichment of viral nucleic acids, where the main objective is to use a minimum number of primer oligonucleotides to cover the maximum number of diverse viral taxa within a single pcr reaction. we have applied this approach to design genus-specific primer pairs for targeted enrichment of cdna from zoonotic rna viruses and have evaluated it using several samples from birds. we have also demonstrated a considerable increase in the viral genome coverage. panel. this section contains the technical details of the algorithm for the design of genus-specific primer panels. to enable us to process the enrichment pcr reaction in a single tube, a number of restriction parameters were applied to the positions and structures of oligonucleotides. the availability of validated reference viral nucleic acid sequences is crucial for efficient usage of the algorithm. although several viral reference sequence databases are publicly available [ ] , the medically relevant and model organisms are largely overrepresented in most of them, and the genomic diversity of circulating strains is often underrepresented. one such source is the open-source database "the virus pathogen database and analysis resource (vipr)" [ ] , which was developed back in . the most important advantage of this database is the authenticity of nucleic acid sequences, as the data are curated and managed by experts in virology and bioinformatics. this source was used to retrieve the sequences of the polymerase genes of target viral genera (or other genes in cases where the sequences of polymerase genes were not available). the sequences were filtered by length (≥ bp), quality, and intrageneric similarity and were combined to the corresponding fasta files. in order to create consensus sequences, nucleotide sequences within each fasta file specific to a genus were aligned using clustalw [ ] . if a minimum of two consensus subsequences with lengths of bp and a maximum of four ambiguous positions with minor nucleotide frequency ≥ % were not identified, the original fasta file was iteratively clustered using cd-hit [ ] , with decreasing threshold. the clusters obtained at each step were aligned independently using clustalw to identify consensus subsequence(s) for all the subsets, which must collectively represent at least % of different species within the genus. finally, at least one aligned fasta file was obtained for every genus. for extracting the common subsequences from multiple sequence alignments, a "sliding window" was used within a specified range of lengths. this window "slides" from the 'to '-end of the alignment with a step of one nucleotide and identifies all subsequences fulfilling the following criteria: (i) proportion of ambiguous positions (p amb ) ≤ %; (ii) proportion of unique species, which share the subsequence and do not contain gaps (p sh ) ≥ %; (iii) gc content of the consensus sequence (p gc ) within - % interval; (iv) absence of self-complementary regions; (v) absence of formation of homodimers; (vi) absence of formation of heterodimers with previously selected oligonucleotides. an amplicon length between two subsequences (primers) was then adjusted between and bp to make the panel compatible with the most popular sequencing platforms (for example, illumina miseq or ion s from thermo fisher scientific). two subsequences were considered to be a pair if they together covered at least % of species related to a target genus or cluster and shared over % of the species. the pairs were then filtered according to their annealing temperature ( ∘ c ≤ t a ≤ ∘ c). the selected primer pairs were then aligned with the nr database using blast to check for their specificity. nonspecific candidates were eliminated. the possibility of formation of heterodimers between sequences in the pair, and between the sequences and previously selected primers, was calculated using the software primer [ ] . then the parameters described above were calculated and the pairs were sorted accordingly. the "best" primer pair was selected as a "genus-specific pair." the parameters described above were then calculated, and the pairs were sorted accordingly. the primer pair with the best fit was selected as a "genus-specific pair." the sequences of primer pairs for the reference viral genera, designed using the developed algorithm, are presented in tables and . the ability of the developed panel to enrich the target cdna from zoonotic rna viruses was assessed using high titer solutions of viral rna (concentration ranging from to copies per ml) from species of viruses, belonging to viral genera within viral families (table ). viral rnas were sourced from the collection stored at the central research institute for epidemiology, moscow, russia. all viral rnas were stored at − ∘ c till further use for the study. the h o sterile (amplisens, russia) was used as a negative control in all experiments. the control samples cdna was obtained by reverse transcription reaction performed on l of the extracted rna using the reverta-l rt kit (amplisens; total volume of the reaction mixture is l); after that l of the reaction mixture containing cdnas was further used for evaluation of the ability of the primer pair to amplify the targeted region of viruses, both in single and in multiplex pcr format. mode. the pcr reaction mix ( l) was prepared using l of the cdna template, l of h o (milliq, amplisens), l of pcr mix fep/frt, l of . m of each primer (in the single-plex format) or . m of each primer (total concentration of . m in the multiplex format), . l of dntps ( . mm; amplisens), and . l of taqf polymerase (amplisens). the thermal cycling parameters were initial denaturation at ∘ c for min, followed by cycles of ∘ c for s, ∘ c for s, ∘ c for s, and final extension at ∘ c for min. pcr products of appropriate lengths were resolved by electrophoresis on . % agarose gel containing ethidium bromide. the amplicons were purified using a qiaquick pcr purification kit (qiagen), following the manufacturer's instructions. the purified pcr products were then sequenced using abi prism sequencer (applied biosystems) to confirm the specificity of the reactions in a single-plex format. bird samples (cloacal swabs and/or feces) were collected from the enisei ecological station, mirnoe (russian federation). the samples were collected from birds captured using mist-net for routine ornithological examination or from droppings left on the ground by geese at their stop-over sites. to prevent contamination, separate . . rna extraction and reverse transcription. total rna was extracted from l of the resuspended sample with rneasy lipid tissue mini kit (qiagen) using robotic workstation qiacube (qiagen), following the manufacturer's protocol. the cdna was obtained by reverse transcription reaction on l of the extracted rna using a reverta-l rt kit (amplisens), according to the manufacturer's instructions. the primer panel was tested with a reference set of samples ( table ). the results are presented in figure . in all cases, the products of the specified range of lengths were obtained. the multiplex system was then tested with the same set of viruses and the pcr products were reamplified using the genus-specific primer pairs. in all cases, unspecific amplification was observed as well. however, the reamplification reactions with the genus-specific primers showed the presence of the target products. the multiplex system was first tested with three bat samples infected by several known viruses (sample n : betacoronavirus, sample n : betacoronavirus, sample n : orthoreovirus). the reamplification of the pcr products was carried out with the genus-specific primers ( figure ) . the obtained results clearly demonstrated the presence of the products with target length in all three samples. advances in virology the targeted sequences were enriched by multiplex pcr with the designed primer pool described above (table ) . pcr products were cleaned using carboxyl-coated magnetic particles, commercially available as sera-mag speed beads (ge healthcare). the concentrations of the fragments were measured using qubit dsdna hs assay kit with a qubit . fluorimeter (invitrogen). the preparation of the amplicon libraries involved phosphorylation of the '-end and incorporation of barcoded adapters, followed by amplification of the final library. for this purpose, t polynucleotide kinase and t dna ligase (both from new england biolabs, neb) were used according to the manufacturer's protocol with slight modification. amplification was performed using pcr-mix fep/frt (amplisens). the two total rna libraries for ion s high-throughput sequencing were prepared from total rna of two bird samples (b and b ). the first strand of cdna was synthesized using random primers and reverta-l rt kit (amplisens). the second strand of cdna was prepared with nebnext ultra second strand synthesis module of kit #e (neb). the double stranded dna was fragmented by ion shear plus reagent kit (thermo fisher scientific) and advances in virology amplicon libraries were separated by . % agarose gel electrophoresis stained with ethidium bromide, and the fragments of target lengths were cut out and purified using the minelute gel extraction kit (qiagen). size selection of the final total rna libraries was done using % e-gel sizeselect ii agarose gels (thermo fisher scientific) on the e-gel electrophoresis system (thermo fisher scientific). sequencing was carried out on the ion s platform using ion / kit-chef reagent sample preparation kit and employing ion chips on the ion chef instrument (thermo fisher scientific). raw sequencing reads obtained from the platform were first filtered using the prinseq-lite tool [ ] to eliminate too short (< bp) and low-quality (min mean < ) fragments. the mean, median, and th and th percentiles of read lengths distributions for selected unfiltered fastq files are shown in the supplementary table s . the bwa software [ ] was used then to align the filtered reads to the reference birds' genomes database. the ideally aligned reads (full read length and no mismatches) were marked as nonviral reads and were eliminated. the software cd-hit [ ] was used to reduce the redundancy, i.e., the number of reads per sample, by clustering (with a similarity threshold %) and selecting the representative sequences for each cluster. briefly, the cd-hit algorithm sorts the sequences from long to short and processes them sequentially. the first sequence is automatically classified as the first cluster representative sequence. then each query sequence from the remaining sequences is compared to the representative sequences identified before it and is classified as redundant or representative based on whether it is similar to one of the existing representative sequences. the described filtering and clustering steps significantly reduced (by ∼ times) the computational time of the further steps. the software blast was then employed to first compare the representative sequences (rs) against virus-only nucleotide and protein databases that were collected by selection of virus sequences from genbank nt and genbank nr databases, respectively, to identify viral nucleotide candidate rs (nrs) and protein candidate rs (prs) with e-value cutoffs of - and - , respectively. since virus-only databases are much smaller than ncbi nt and nr databases, pairwise sequence comparisons are much faster, which maximizes the speed and allows for the more efficient use of computational resources. we subsequently aligned these candidate nrs and prs to the entire genbank nt and nr databases, respectively, to eliminate potential false positives (sequences with higher similarity to nonviral reference sequences than to viral ones in the database) among the candidates and to select the true positives. the additional step was applied to select true positives from viral prs: the prs aligned to nr database were compared with nrs aligned to nt database to eliminate viral prs, which correspond to nrs aligning to nonviral nucleotide sequences with a high e-value. to get an idea of the total number of true viral reads belonging to the same virus, the numbers of reads for each rs were eventually summed up. virus hits e< e- figure : a schematic picture of the bioinformatics pipeline developed for the analysis of the ngs data in this study. specific third-party tools that were employed are shown in parentheses. a schematic picture of the bioinformatics analysis scheme is presented in figure . a panel of primers including forward and reverse primers was designed using the developed algorithm and was synthesized accordingly (tables and ) . a preliminary analysis using the available control samples (table ) demonstrated the ability of the designed primers to perform targeted cdna enrichment in both single-plex and multiplex formats. this was confirmed by the presence of specific bands of expected lengths in electrophoresis, followed by capillary sequencing (figures and ) . subsequently, we deployed the primer panel to study bird samples (as described in the methods) and the sequencing results are presented in table and figure . only those samples, for which at least viral reads (filtered by quality and length) were identified in the final fastq files, are presented in the table. the total number of sequencing reads in such samples is also given to exemplify the percentage of pathogen reads. the alignment of sequencing reads with viral reference sequences was visually inspected to eliminate most of the potential artifacts (such as primer-dimers). as evident from table , the genera of viruses (samples b , b , and b , highlighted in bold) having a specific primer pair in the panel showed significant amplification, and percentage of the corresponding viral reads ranged between . % and . %. this confirms that our primer panel can efficiently enrich the target genera. other sequencing reads belonged to bacteriophages, bacteria, and host species. however, we also observed a significant enrichment in the samples b and b , for which a significant number of reads corresponding to sanxia water strider virus and duck adenovirus were, respectively, detected. we scrutinized these results in further detail by carrying out a blast analysis of all possible versions of the primers (since they are very degenerated) against the sequencing reads and observed that these findings were very likely due to nonspecific amplification from the primers designed for other genera (gammacorona f and flavi r), though we have not checked this by a direct experiment with a panel that lacks these primers. although nonspecific amplification allows an even more exhaustive search, it also introduces potentially undesirable enrichment. this could be a problem, especially when the lengths of obtained amplicons are outside the standard intervals suitable for sequencing on most popular platforms. we also observed a considerable number of viral reads corresponding to various genera in some samples (b , b , b , b , b , and b ), despite the absence of specific primers in the panel for their enrichment. the number of reads varied from . % (tunis virus) to . % (watercress white vein virus). the latter is a plant virus and its presence is expected as the feces of the host birds mainly contained grass. these results are possibly due to a weak, nonspecific pcr amplification. we then checked whether the obtained viral reads for samples b , b , b , b , and b were due to the amplification with the designed primers in the panel. these five samples were chosen as they indicated the most significant amplification, with . - . % of total sequencing reads in the fastq files being viral. this was done by repeating a blast analysis of all possible versions of the primer sequences against the obtained viral reads for the aforementioned samples and calculating the percentage of those containing at least one primer. we observed that over % of the sequences contained the primers validating the effectiveness of the panel. finally, in order to confirm the effectiveness of the panel's enrichment, we selected two samples (b and b ) for which the presence of viral rna was shown using amplicon sequencing data (see table ). notably, the b possessed a significant number of reads that correspond to coronaviruses ( . %) and our panel had specific primers for these genera. as for the b sample, it had a small number of reads that correspond to adenoviruses ( . %) but we did not design oligonucleotides for them. we then prepared total rna-seq libraries for these samples, and subsequently sequenced them, yielding ∼ . million of reads per sample. the percentage of the coronavirus reads in the b sample was found to be significantly lower ( . %) than that in the same sample that was previously enriched by the panel ( table ). as for the b sample, we identified that . % of reads belong to the adenoviridae family, and most of them were identified as being similar to the duck and psittacine adenoviruses. this is similar to the percentage of reads found in the b figure : a graphical representation of the percentage of the detected viral reads (grey) with respect to the total number of sequencing reads obtained for the samples listed in table . fastq file ( . %) when the primer panel was applied, i.e., no noticeable enrichment was observed as expected. at the same time, we lost ∼ . % of adenoviruses in the b , but since the amount of data was very different in the two experiments, there was a significant amplification of coronaviruses with almost a half of the reads belonging to these genera; this outcome is not totally surprising. thus, our approach allows a significant reduction in the cost of sequencing. besides, for a successful identification of viruses in a preenriched sample, , - , reads per sample are generally enough. in this study, we have presented a method of oligonucleotide design for the enrichment of viral nucleic acids. we used this method to design a primer panel, which showed a high efficiency in the detection and identification of several viruses using ngs sequencing. this is especially important for the detection of viruses known to persist in natural zoonotic reservoirs including birds, bats, and rodents, those with a high genetic diversity, and those which can be potentially dangerous to humans and animals. these factors make the development of rapid test-systems essential for the effective management of potential epidemics. the described method can be recommended to the researchers investigating diverse viromes across different types of biological samples. we deployed our panel for the screening of a number of bird samples and demonstrated a great efficiency. in future studies, the panel will be expanded to target more diverse viruses and tested with bird samples from other regions. all the fastq files used to support the findings of this study are available from the corresponding author upon request (kamil khafizov; kkhafizov@gmail.com). all applicable international, national, and/or institutional guidelines for the care and use of animals were followed. an earlier version of this work was presented as an abstract at the rd febs congress prague - july . zoonosis emergence linked to agricultural intensification and environmental change reservoirs and vectors of emerging viruses relationship between domestic and wild birds in live poultry market and a novel human h n virus in china detection of plant-based adulterants in turmeric powder using dna barcoding dna metabarcoding for diet analysis and biodiversity: a case study using the endangered australian sea lion (neophoca cinerea) comparative and joint analysis of two metagenomic datasets from a biogas fermenter obtained by -pyrosequencing lassa virus validation of metagenomic next-generation sequencing tests for universal pathogen detection virome capture sequencing enables sensitive viral diagnosis and comprehensive virome analysis detection and analysis of diverse herpes-viral species by consensus primer pcr a strategy to estimate unknown viral diversity in mammals hantavirus in bat, sierra leone identification of a novel coronavirus in patients with severe acute respiratory syndrome novel genus-specific broad range primers for the detection of furoviruses, hordeiviruses and rymoviruses and their application in field surveys in south-east australia rt-pcr detection and identification of three species of cucumoviruses with a genus-specific single pair of primers genus-specific detection of alphaviruses by a semi-nested reverse transcription-polymerase chain reaction retrospective diagnosis of two rabies cases in humans by high throughput sequencing the th annual nucleic acids research database issue: a look back and upcoming changes vipr: an open bioinformatics database and analysis resource for virology research clustal w and clustal x version . cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences primer -new capabilities and interfaces quality control and preprocessing of metagenomic datasets fast and accurate short read alignment with burrows-wheeler transform the authors declare that there are no conflicts of interest regarding the publication of this paper. key: cord- -nro aa authors: valitutto, marc t.; aung, ohnmar; tun, kyaw yan naing; vodzak, megan e.; zimmerman, dawn; yu, jennifer h.; win, ye tun; maw, min thein; thein, wai zin; win, htay htay; dhanota, jasjeet; ontiveros, victoria; smith, brett; tremeau-brevard, alexandre; goldstein, tracey; johnson, christine k.; murray, suzan; mazet, jonna title: detection of novel coronaviruses in bats in myanmar date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: nro aa the recent emergence of bat-borne zoonotic viruses warrants vigilant surveillance in their natural hosts. of particular concern is the family of coronaviruses, which includes the causative agents of severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and most recently, coronavirus disease (covid- ), an epidemic of acute respiratory illness originating from wuhan, china in december . viral detection, discovery, and surveillance activities were undertaken in myanmar to identify viruses in animals at high risk contact interfaces with people. free-ranging bats were captured, and rectal and oral swabs and guano samples collected for coronaviral screening using broadly reactive consensus conventional polymerase chain reaction. sequences from positives were compared to known coronaviruses. three novel alphacoronaviruses, three novel betacoronaviruses, and one known alphacoronavirus previously identified in other southeast asian countries were detected for the first time in bats in myanmar. ongoing land use change remains a prominent driver of zoonotic disease emergence in myanmar, bringing humans into ever closer contact with wildlife, and justifying continued surveillance and vigilance at broad scales. infectious diseases are considered to be "emerging" if they appear in a new population or geographic region or are occurring with greater frequency than the expected background rate [ ] [ ] [ ] . emerging infectious diseases (eids) are capable of causing debilitating health effects and financial instability, especially in less developed countries with insufficient capacity to mount health interventions, and thus pose a significant global public health challenge in the st [ ] . an estimated - % of eids are comprised of zoonotic diseases; of these, more than % have purportedly originated in wildlife species [ ] [ ] [ ] . spillover has been largely attributed to changes in anthropogenic activity subsequent to exponential human population growth since the latter half of the th century. large-scale land use change, such as deforestation and land conversion for agriculture, can alter host-pathogen relationships and increase human encounter rates with wildlife and their pathogens, making cross-species transmission events more likely [ , ] . for established pathogens, human-mediated biodiversity loss often leads to reduced populations of suboptimal host species and increased numbers of competent or amplifying hosts, potentially precipitating higher infection rates in people [ ] . in addition, intensification of livestock and poultry production systems results in artificially dense populations of domestic animals, which can lead to pathogen amplification and spillover to humans [ ] . approximately two-thirds of human pathogens occupy complex, multi-host systems, and pathogens with multiple animal hosts, including some wildlife species, are more likely to become emergent [ ] . bats are increasingly recognized as the natural reservoirs of viruses of public health concern [ ] [ ] [ ] [ ] . the capacity of bats to carry and transmit zoonotic pathogens has been hypothesized to be due to their unique life history traits, including their ability for sustained flight, potential for long-distance dispersal, aggregation into densely populous colonies, and adaptation to peri-urban habitats [ , ] . historically, bats have been linked to highly pathogenic viruses that pose a serious threat to human health, including the coronaviruses responsible for severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), the hemorrhagic ebola and marburg filoviruses, and paramyxoviruses such as nipah virus [ , , [ ] [ ] [ ] [ ] [ ] [ ] . more recently, a pandemic of an acute respiratory syndrome originating in wuhan, china in december was linked to a coronavirus (designated "sars-cov- ") that shared % identity with a bat-borne coronavirus at the whole-genome level [ ] . in some cases, these viruses can subsequently spread through person-to-person contact following spillover from animals, increasing their epidemic potential [ , , ] . the - sars epidemic, the emergence of mers in people in , and the ongoing covid- pandemic have prompted substantial interest in detecting coronaviruses of bat origin due to public health concern and their pandemic potential [ , [ ] [ ] [ ] [ ] [ ] [ ] . coronaviruses (cov) are a family of enveloped, single-stranded rna viruses that commonly infect the respiratory and gastrointestinal tracts of their mammalian and avian hosts [ ] . the alphacoronaviruses and betacoronaviruses are of particular importance to human health, with sars-cov, sars-cov- , and mers-cov-which have caused the most severe disease in humans to datebelonging to the latter group [ , , ] . mounting evidence indicates that bats are the evolutionary hosts and origin for these cov lineages [ , [ ] [ ] [ ] [ ] . in addition to human-associated covs, bats are also hosts of coronaviruses that infect production animals, and have been implicated in the emergence and origin of swine acute diarrhea syndrome (sads), transmissible gastroenteritis virus (tgev) in pigs, and porcine epidemic diarrhea (ped), which can cause considerable losses [ ] [ ] [ ] [ ] . thus, bat-borne covs can pose a significant threat to human health and food production. in spite of these infectious disease threats, bats are an indisputably essential component of ecosystems. they provide critical services such as seed dispersal, pollination, control of insect populations (including crop pests and disease vectors), and fertilization via guano, making them invaluable assets to agricultural industries and small-holder farming [ ] . the importance of bats to ecosystems and human communities while being the natural reservoirs of many zoonotic pathogens presents a challenge for disease control. the potential threats posed by bat-borne coronaviruses to human and livestock health necessitate the identification and characterization of these viruses at high-risk interfaces among humans, domestic animals, and wildlife. particular attention is needed in developing regions of high biodiversity, where eids are most likely to arise, and where substantial losses in agricultural production may be a source of financial insecurity [ ] [ ] [ ] [ ] [ ] . myanmar is a particularly vulnerable country due to the interplay of ecological and human factors, which increase opportunities for viral spillover. the nation is situated in the heart of the southeast asia region, a hotspot for eids, including some neglected tropical diseases and some of pandemic potential like sars and h n influenza [ , ] . a combination of biological, ecological, socioeconomic, and anthropogenic factors renders the region particularly susceptible to emerging zoonoses that could impart a considerable public health and economic burden [ , ] . our study aimed to detect coronaviruses in free-ranging bats living in close proximity to human communities. (fig ) . these sites were targeted as potential high-risk human-animal interfaces due to land use change increasing human proximity to wildlife and potential human exposures through livelihood, recreational, commercial, and religious or cultural activities. two of these sites also featured popular cave systems where people were routinely exposed to bats through guano harvesting, religious practices, and ecotourism. sites and consisted of several smaller sub-sites where bat capture and sampling events occurred. all surveillance activities were conducted in collaboration with three of myanmar's government ministries: ( ) the ministry of livestock, agriculture, and irrigation; ( ) the ministry of health and sports; and ( ) the ministry of natural resources and environmental conservation. all work conducted was approved through a letter of agreement, ethical review committee, and memorandum of understanding, respectively. bat sampling was performed by trained field personnel in collaboration with myanmar's ministry of agriculture, livestock and irrigation (moali) and ministry of natural resources and environmental conservation (monrec). all bats were captured using mist nets, with each individual manually restrained for species identification, morphometric evaluation, and sample collection. no anesthetic or immobilization agents were used during capture or handling. oral and rectal swabs were collected when possible using sterile polyester-tipped applicators (animal size often precluded rectal swab collection). naturally voided guano samples consisting of combined urine and feces were also collected from the environment using plastic tarps. at site , the tarps were placed on the floor of the caves and left overnight, with sample collection occurring the following morning. at sites and , the tarps were placed at cave entrances and under roosting areas in the evening as the bats emerged to forage, and samples were collected immediately. guano pellets were collected randomly from the tarps and pooled. tarps were disinfected between each use and gloves were changed in between each sampling event. pooled guano samples were attributed to a presumptive host species based on field identification of species in caves when possible, otherwise were designated as "unidentified chiropterans" when multiple species were present. all sample types were collected into μl viral transport medium (thermoscientific microtest tubes, fisher scientific, pittsburgh, pa, usa) or μl trizol reagent (invitrogen trizol reagent, fisher scientific, pittsburgh, pa, usa), transported from the field in liquid nitrogen, and transferred to a - ˚c freezer within five days and stored until time of testing. bats were humanely trapped, handled, and sampled from according to protocols approved by the institutional animal care and use committee of the university of california at davis (protocol ) and smithsonian institution (protocol - ) and with approvals from moali and monrec. bats were released within km of the sample testing was performed at the uc davis one health institute laboratory and the veterinary diagnostic laboratory, livestock, breeding, and veterinary department (lbvd) in myanmar. μl was used from each sample for rna extraction per kit instructions, and to ensure availability of an additional aliquot should a second extraction or other downstream analyses be needed. rna was extracted using direct-zol rna columns (zymo research corp), and μl rna was used for cdna transcription using superscript iii (invitrogen). samples were screened for coronaviruses using two broadly reactive consensus conventional polymerase chain reaction (pcr) assays targeting two non-overlapping fragments ( bp and bp) of the rna-dependent rna polymerase (rdrp) of orf ab of covs [ , ] . bands of the expected size were cloned (pcr -topo vector; invitrogen corp.) and sanger sequenced (abi capillary electrophoresis genetic analyzer; applied biosystems, inc., foster city, ca). sequences were analyzed and edited using geneious prime (version . . ), uploaded to genbank (s table) , and compared with known sequences in the database. coronavirus sequences were classified as belonging to viral taxa according to established cut-offs and methods [ ] . virus sequences that shared less than % identity to a known sequence were labelled sequentially as predict_cov- , - , - etc; while groups sharing � % identity to a sequence already in genbank were given the same name as the matching sequence. based on these criteria, the cov sequences detected were assigned to discrete viral taxa. viral culture and isolation were not attempted for any positive samples. bat samples positive for a cov-including positive pooled guano samples-were barcoded to confirm the host species using pcr assays targeting fragments of the cytochrome b gene (cytb) and the cytochrome oxidase subunit genes (co ) [ ] . one pcr amplicon was selected for sequencing and compared to reference sequences in genbank using blast tools. a threshold of % sequence identity was used to confirm the species. sequences with < % sequence identity were classified to the genus. dna barcoding was also performed on a subset of the cov-negative pooled guano samples. pooled guano samples were assigned a presumptive origin species based on host barcoding. a total of bats representing at least species across eight genera from six families were captured and sampled (table ). both insectivorous microbats and fruit bats were represented in our study population. a total of samples were collected and tested ( oral swabs, rectal swabs, guano samples). a total of samples were collected in the dry-season sampling ( oral swabs, rectal swabs, and guano samples) and samples ( oral swabs, rectal swabs, and guano samples) in the wet season. covs were detected in samples: one oral swab and seven rectal swabs from seven individual bats and pooled guano samples (table ). viral fragments were detected from one unidentified tomb bat (taphozous sp.), three horsfield's leaf-nosed bats (hipposideros larvatus), and three greater asiatic yellow house bats (scotophilus heathii). thirty-six of the positives detected in guano were attributed to h. larvatus, while the host species for the remaining four positive pooled guano samples was identified as wrinkle-lipped free-tailed bats (chaerephon plicatus). overall viral prevalence across all bat taxa and all coronaviral genotypes was approximately . %. the vast majority of positive detections ( . %) were made from pooled guano samples, while oral swabs had the lowest yields. positive detections were made from samples collected during the dry season ( . %), while wet-season sampling resulted in positive detections from eight samples ( . %). both sites and accounted for positive detections, while no coronaviral sequences were detected at site . fifty-four total sequences were recovered, clustering within seven distinct coronaviral genotypes. using established cut-offs and methods [ ] , we detected four alpha coronaviruses (predict_cov- , , , and ) and three betacoronavirues (predict cov- , , and ). of these, the alphacoronavirus predict_cov- was previously known, having been found in scotophilus kuhlii, unidentified myotis, and other unspeciated host bats in the neighboring countries of cambodia and vietnam from to [ ] . the remaining six coronaviruses were novel (three alphacoronaviruses and three betacoronaviruses). predict_cov- was the most commonly detected coronavirus, found in pooled guano samples attributed to h. larvatus (table ) . interestingly, three coronaviruses were only found as co-infections: predict_cov- was detected with predict_cov- , predict_cov- with - , and predict_cov- also with - . indicates at least one instance of co-infection. did not meet the % nt identity threshold for identification to the taxonomic level of species. https://doi.org/ . /journal.pone. .t three new alphacoronaviruses, three new betacoronaviruses, and one previously described alphacoronavirus were detected in bats in myanmar. none of the viruses appeared to be closely related to sars-cov, mers-cov, or sars-cov- . guano samples accounted for the majority of positives, suggestive of an important transmission route for cov shedding from bats [ , , ] and a possible risk to people during the act of guano harvesting [ , ] . viral detection in guano also has implications for future surveillance, as our study demonstrates the value of non-invasive collection of guano for viral surveillance, potentially obviating the need for handling individual bats for coronaviral detection. our findings supplement those of he et al., who profiled the virome of insectivorous bats from northern myanmar but did not detect coronaviruses in that study [ ] . a difference was found in positives for cov by species, as samples from h. larvatus represented % of positives. a wide diversity of covs has been found in hipposiderid bats [ , , , ] , and our study is consistent with those findings. four covs detected in our surveillance study were found in a single host species each: predict_cov- was found only in s. heathii; and predict_cov- , - , and - were found only in h. larvatus (table ) . these findings may possibly suggest limited host-switching and viral sharing for certain viruses within our study populations, a pattern consistent with prior observations that viral groups are likely significantly associated with host taxa at the family level [ ] . however, further evidence is needed to elucidate host-viral relationships and ecology in the region. our findings also likely reflect a bias in our sampling effort. although h. larvatus samples accounted for the most positives, these were largely detected in guano samples collected from the environment, as individuals were not frequently caught by mist net. overall in our study, the numbers of individual bats handled and sampled per species were relatively low, ranging from one to (table ). viral prevalence may vary widely with the species of host and pathogen. anthony et al. suggested a sample size of at least individuals per species in order to maximize our ability to detect covs. targeting more host species, specific taxa (hipposideridae), and larger sample sizes might have improved our detection rate in the species where no covs were found [ , ] . currently, active pathogen surveillance at human-wildlife interfaces in myanmar is limited. despite relatively small sample sizes, our study detected several coronaviruses in insectivorous bats, suggesting that more may remain to be uncovered. given the potential consequences for public health in light of expanding human activity, continued surveillance for coronaviruses is warranted, especially in other species and human-wildlife interfaces. anthony et al. estimated that over , covs occur in bats, most of which remain undiscovered [ ] . enhancing our sampling effort to incorporate more diverse bat families and larger sample sizes may enable us to identify more covs in bats in myanmar. additionally, because only short fragments of the conserved rdrp gene ( bp and bp) were amplified in this study, protein sequence and phylogenetic analyses were not pursued, and identification of recombination events was not possible. while this is an inherent shortcoming of our methodology, the purpose of this study was not to fully characterize specific viruses, but to broadly screen for viruses in bats living in proximity to human communities to better understand potential sources of zoonotic transmission in the context of these human-wildlife interfaces. further studies may consider complete genomic sequencing for more comprehensive profiling of the bat viromes in this ecosystem. in particular, evaluation of the spike gene sequences may provide insights into host range, including potential viral host-sharing or host-switching events [ ] . land use change will likely continue bringing people into closer proximity with bats, raising encounter rates and opportunities for spillover, facilitating the emergence of zoonotic viruses, and supporting the need for surveillance [ , ] . historically, human activities have arguably played a significant role in interspecies transmission events. following the sars outbreak, coronaviruses have since been detected in numerous bat species globally, including in asia, africa, europe, the americas, and the australasian region [ , [ ] [ ] [ ] [ ] [ ] [ ] . mounting evidence supports the role of bats in the transmission of viruses of public health concern-including sars--cov and mers-cov-and the zoonotic potential of unknown bat-borne coronaviruses warrants vigilant, continued surveillance [ ] . understanding their ecology and prevalence in their natural hosts can improve our ability to detect, prevent, and respond to potential public health threats. finally, given the essential ecosystem services provided by bats, public health efforts should advocate for preventative 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circulation of alphacoronavirus, betacoronavirus and paramyxovirus in hipposideros bat species in zimbabwe recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission bats, coronaviruses, and deforestation: toward the emergence of novel infectious diseases? front microbiol detection of group coronaviruses in bats in north america detection of novel sars-like and other coronaviruses in bats from kenya detection and prevalence patterns of group i coronaviruses in bats coronavirus infection and diversity in bats in the australasian region coronaviruses in bats from mexico detection of coronaviruses in bats of various species in italy we also thank the livestock breeding and veterinary department (lbvd) within the ministry of agriculture, livestock, and irrigation (moali); ministry of natural resources and environmental conservation (monrec); and the department of medical research (dmr) within the ministry of health and sports (mohs), myanmar, with whom we collaborated closely on surveillance activities. thanks also to the invaluable field and laboratory staff who provided technical skill and expertise and were critical in the research process. key: cord- - buawes authors: liebing, j.; völker, i.; curland, n.; wohlsein, p.; baumgärtner, w.; braune, s.; runge, m.; moss, a.; rautenschlein, s.; jung, a.; ryll, m.; raue, k.; strube, c.; schulz, j.; heffels-redmann, u.; fischer, l.; gethöffer, f.; voigt, u.; lierz, m.; siebert, u. title: health status of free-ranging ring-necked pheasant chicks (phasianus colchicus) in north-western germany date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: buawes being a typical ground-breeding bird of the agricultural landscape in germany, the pheasant has experienced a strong and persistent population decline with a hitherto unexplained cause. contributing factors to the ongoing negative trend, such as the effects of pesticides, diseases, predation, increase in traffic and reduced fallow periods, are currently being controversially discussed. in the present study, free-ranging pheasant chicks were caught within a two-year period in three federal states of germany; lower saxony, north rhine-westphalia and schleswig-holstein. the pheasant chicks were divided into three age groups to detect differences in their development and physical constitution. in addition, pathomorphological, parasitological, virological, bacteriological and toxicological investigations were performed. the younger chicks were emaciated, while the older chicks were of moderate to good nutritional status. however, the latter age group was limited to a maximum of three chicks per hen, while the youngest age class comprised up to ten chicks. the majority of chicks suffered from dermatitis of the periocular and caudal region of the head ( – %) of unknown origin. in addition, intestinal enteritis ( %), pneumonia ( %), hepatitis ( %), perineuritis ( %), tracheitis ( %), muscle degeneration ( %) and myositis ( %) were found. in % of the cases, various mycoplasma spp. were isolated. mycoplasma gallisepticum (mg) was not detected using an mg-specific pcr. parasitic infections included philopteridae ( %), coccidia ( %), heterakis/ascaridia spp. ( %) and syngamus trachea ( %). a total of % of the chicks were avian metapneumovirus (ampv) positive using rt-pcr, % positive for infectious bronchitis virus (ibv) using rt-pcr, and % positive for haemorrhagic enteritis virus (hev) using pcr. all samples tested for avian encephalomyelitis virus (aev), infectious bursal disease virus (ibdv) or infectious laryngotracheitis virus (iltv) were negative. the pool samples of the ten chicks were negative for all acid, alkaline-free and derivative substances, while two out of three samples tested were positive for the herbicide glyphosate. pheasant chick deaths may often have been triggered by poor nutritional status, probably in association with inflammatory changes in various tissues and organs as well as bacterial and parasitic pathogens. theses impacts may have played a major role in the decline in pheasant populations. a a a a a the original distribution area of the ring-necked pheasant (phasianus colchicus) ranged from the black sea over the dry areas of central asia to the east of asia to south korea and siberia [ ] . the romans introduced the pheasant to europe around ad, from where it spread through regular release throughout central and western europe [ ] . according to current published data, the pheasant mainly prefers structurally semi-open land, using trees and hedges as cover, and also occupies adjacent sparse forests and reedy areas [ ] . most pheasants seek shelter under trees to be protected from natural predators. however, some subspecies spend the night on the ground or among dense reeds. their resting places during the day are usually well-hidden hedges, where sand-baths are taken in carved hollows [ ] . adult pheasants mainly feed on plants, consuming different parts of the plant such as seeds, berries, tubers, root shoots and leaves, as well as green sprouts. however, on occasions, their diet is supplemented by animal protein, preferably in the form of insects [ ] . for chicks, smaller ground-level insects are especially important during the first weeks of life. they feed on a variety of species of insects such as spur cicadas (delphacidae), bugs (heteroptera), sawfly wasps (tenthredinidae) and butterfly caterpillars (lepidoptera larvae) [ , , ] . this diversity is particularly important for a healthy growth [ , ] . for example, a diet based only on aphids can lead to delayed plumage development due to inadequate amino acid supply [ ] . in germany, the pheasant is a typical soil-breeding bird of the agricultural landscape. the main part of the german population is found in southwest lower saxony, north rhine-westphalia and schleswig-holstein. the population level reached its plateau between and in lower saxony. during this period, the hunting bag statistics (state registered numbers of hunting animals, in this case pheasants), i.e. the absolute number of pheasants killed, amounted to approximately , pheasants in germany [ ] . in the severe winter of and the following wet spring of , the population of pheasants and many other wild living animals declined [ , ] . the hunting bag was reduced to an average of about , pheasants and declined further. not only was the pheasant population subjected to this decline, but also that of many other farmland birds [ , , ] . around / , the population showed another severe decline of unknown cause. in germany, the renewable energy sources act (erneuerbare-energie-gesetz: renewable energy sources act describes the implementation of ecological energy generation in germany) amendment of with an advancement in biogas, triggered the doubling of corn cultivation. consequently, huge areas of fallow land disappeared in lower saxony [ ] . the contributory factors to the ongoing decline in the pheasant population, such as the effects of pesticides, infectious agents, predation, increasing traffic and human populations as well as reduced fallow periods, are currently the subject of controversial discussion among different stakeholders [ , , , ] . some authors see a correlation between the changes in agriculture and the decline in the populations of many farmland birds [ , ] . in the third week of life, % of the chicks' diet consists of insects. gradually, the insect percentage is reduced. from the sixth week of life, the diet is similar to that of adult birds. previous studies [ , ] associated the decline in the number of many farmland birds with the use of insecticides. if chicks are unable to find a sufficient number of insects during the first weeks of life, they have to search a larger range of their habitat, which can lead to malnutrition and weakening. thus, harmless ubiquitous pathogens may have negative effects on chicks [ , , , , , ] . investigations carried out led to the assumption that there is no specific epidemic infectious agent currently circulating in the adult pheasant population [ ] . many hunters report that especially the number of chicks has declined, with more older birds making up the hunting bag. however, the authors found serological evidence of certain viruses (infectious bronchitis virus (ibv), avian encephalomyelitis virus (aev) and infectious bursal disease virus (ibdv)) which typically cause chick mortality. these pathogens infected adult and young pheasants, but the pathogenicity in chick and subadult populations is considerably more serious than in adult birds [ , ] . in addition, other factors may weaken the population and pathogens become more important. based on these findings, our study focused on pheasant chicks up to eleven weeks of age. previous studies on pheasants indicated that the most sensitive age class for infectious diseases was pheasant chicks, possibly due to a higher susceptibility [ , ] . the aim of our research was to assess the health state of free-living pheasant chicks in order to check the animals for lesions indicative of infections or toxic substances. the findings should contribute to understanding the causes for the decline in the pheasant population in north-western germany. in and , the institute for terrestrial and aquatic wildlife research (itaw), university of veterinary medicine hannover, foundation, hannover and the wildlife research institute, state office for nature, environment and consumer protection of north rhine-westphalia caught free-living ring-necked pheasant chicks from lower saxony (cuxhaven, grafschaft bentheim, emsland, osnabrück, vechta), north rhine-westphalia (coesfeld, warendorf) and schleswig-holstein (dithmarschen) to assess the health state by means of pathological, microbiological, virological, parasitological and toxicological investigations. the caught chicks were grouped into three age classes (ac) based on the feather markings of the hand-wings. age class one (ac ) included chicks up to three weeks of age, ac , chicks from four to six weeks of age and ac , chicks older than six weeks and up to weeks. an animal experiment permit was obtained from the responsible veterinary office of the lower saxony state office for consumer protection and food safety (laves) (permit number: . - - - / ). the study areas comprised hunting regions with traps in lower saxony (hemmoor, meppen, neuenkirchen, osten, strücklingen, vechta, wilsum,) regions with traps in north rhine-westphalia (ahlen, dülmen, lippstadt, welte) and districts with traps in schleswig-holstein (warwerort) (fig ) . the catching period lasted from may until august. in , the investigated chicks were three to weeks old. in , the age of the chicks varied from one-day-old to eleven-week-old chicks. at the age of weeks, the young pheasants were considered as sexually mature. after the catch, the mother hen was released and at maximum, half of the chicks in the trap were taken for analysis (mostly onethree chicks at random). in , the traps had a size of . m and were covered with iron bars with a mesh-size of cm . in , the traps were slightly adapted based on experience from , using a cover made of loose polyethylene netting with a mesh-size of cm . a piece of string was used as a trigger so that both trap doors closed when the chicks moved forward. corn was used to attract the hen and her chicks. afterwards, the chicks were transported alive to the university of veterinary medicine hannover for examination. the time span from catch to examination took on average about five hours. in , the chicks were stunned by a head blow and killed by exsanguination. in , the chicks were euthanised with an intravenous injection of pentobarbital-sodium (boehringer-ingelheim, ag & co. kg, ingelheim, germany). the nutritional condition score was evaluated macroscopically by the thickness of the pectoral muscles and the body fat percentages as good, moderate, poor or cachectic. as described by curland et al. [ ] , animals in a good body condition revealed a vast amount of fatty tissue within the thoracic and abdominal regions, whereas animals with a moderate body condition demonstrated reduced amounts of body fat tissue. animals in a poor body condition possessed only low amounts of fat reserves, these frequently associated with pectoral muscle atrophy. in contrast, cachectic animals lacked fat reserves and exhibited serous atrophy of the coronal myocardial fatty tissue. the necropsy was carried out in accordance with the standard protocol [ ] . representative samples of the following tissues and organs were collected, fixed in % neutral-buffered formalin and routinely embedded in paraffin wax: the skin of the head and abdomen, skeletal muscle (musculus pectoralis, musculus quadriceps), ischiadic nerve, brachial plexus, nose with infraorbital sinus, eye with lacrimal gland, bone with bone marrow, trachea, thymus, thyroidal gland, lung, heart, liver, pancreas, spleen, kidney, crop, proventriculus, gizzard, intestine, adrenal gland, gonads, bursa of fabricius and brain. paraffin sections of - μm were stained with haematoxylin and eosin (he) for histological examination. in selected cases, periodic acid schiff (pas) reaction, ziehl-neelsen stain, brown-brenn stain and turnbull's blue stain were performed [ ] . for parasitological examinations, samples of the small intestine were collected from all chicks at necropsy. at the same time, skin and plumage of the chicks were macroscopically examined for ectoparasites. for coproscopical examination, the combined sedimentation-flotation method was performed: the faecal sample was filled into a tea strainer (mesh size mm) and rinsed in a beaker with a jet of water. the filtrate containing helminth eggs and protozoan oocysts was allowed to sediment for min. afterwards, the supernatant was decanted and the sediment transferred to a -ml centrifuge tube filled with saturated zinc sulphate solution (znso , specific gravity . ) and centrifuged at x g for min. the liquid surface was transferred onto a slide with a wire eyelet and examined microscopically. if at least one egg or oocyst was detected, the sample was classified as positive. a semiquantitative classification was applied using the following key: one-two eggs or oocysts were categorised as mild, six-ten eggs or oocysts as moderate, - eggs or oocysts as severe; if more than eggs or oocysts were detected, the shedding intensity was classified as by mass [ ] . the fresh samples, consisting of brain, trachea and caecal tonsils, as well as the bursa of fabricius were placed in rnalater (sigma-aldrich chemie gmbh, münchen, germany). samples were analysed by rt-pcr for avian metapneumovirus (ampv), infectious bronchitis virus (ibv), avian encephalomyelitis virus (aev) and by pcr for infectious bursal disease (ibdv) and infectious laryngotracheitis as described in [ , ] . serum was taken from all birds to check for antibodies against avian influenza virus (aiv) subtypes h , h and h . eight additional liver samples from chicks with hepatitis were analysed for the presence of haemorrhagic enteritis virus (hev) by pcr [ ] . for microbiological investigations for mycoplasma, tracheal swabs, six tracheal tissue samples and three periorbital skin tissue samples were taken [ ] . the samples were directly transferred to mycoplasma cultivation medium (sp ). detection of mycoplasma by pcr. for dna extraction, swabs were soaked and rubbed in μl phosphate buffered saline (pbs). using the dneasy blood & tissue kit (qiagen gmbh, hilden, germany) in accordance with the manufacturer's instructions, μl of the liquid was taken for dna extraction. for dna extraction of tissue samples and the single colony subcultures, the fluid medium from culturing ( ml) was centrifuged at x g for minutes. the remaining pellet was incubated with μl lysis buffer (atl buffer, qiagen, gmbh) and μl proteinase k (qiagen gmbh) for two hours at ˚c. all samples and single colony subcultures were screened via mycoplasma-genus-specific pcr (target: s rrna gene sequence) for dna of mycoplasma spp. as described by [ ] and modified [ ] . from all single colony subcultures, an additional pcr (target: s- s rrna sequence (intergenetic transcribed spacer region)) was performed [ ] . furthermore, all samples were examined via mycoplasma gallisepticum-specific pcr [ ] . the pcr products were sequenced by a commercial dna sequencing service (lgc genomics gmbh, berlin, germany). the sequences of the pcr products were aligned with the s rrna gene and s- s rrna isr sequences of mycoplasma spp. in the ncbi database using blast (ncbi, bethesda, md, usa) algorithm [ ] . mycoplasma culture. the samples were cultured using sp liquid and agar media produced in house as described previously [ ] . each sample was immersed in the sp broth and afterwards removed and stored for further investigations. the broth was diluted (ten-fold dilution up to − ) and an aliquot of μl each was transferred onto agar media. both, liquid and solid media were incubated at ˚c with % co in a humidified environment for up to ten days. broth was examined for colour change and agar plates for colony growth daily. in case of colour change, or after five days, an additional "subculture" on agar media was performed. in case of mycoplasma growth, several single colony subcultures were performed at least twice in order to ensure pure species cultures. each third single colony subculture was stored at - ˚c until further investigation by molecular biological methods [ , ] . liver samples of nine pheasants were screened for herbicide glyphosate and other pollutants (for details see s table) . of these nine samples, one sample was taken from a ten-chick ratchet in ac , while the remaining eight samples were single-samples from ac with liver or kidney inflammation. the samples were stored directly after autopsy at - ˚c. toxicological samples (n = ), g liver pool samples, were used to detect substances by means of the gas chromatography-mass spectrometry ( during the two-year-period, a total of chicks were caught: birds in lower saxony, in north rhine-westfalia and six in schleswig-holstein. fourteen chicks were allocated to ac , to ac and to ac . of the investigated animals, were female, male and for chicks, macroscopic gender estimation was unknown; histological samples were not taken. the nutritional status in ac was predominantly poor (n = ; . %) or cachectic (n = , . %). in ac , approximately nine ( . %) of the chicks were well fed and seven ( . %) were moderately fed. the majority of birds in ac were well fed (n = , . %), some were moderately fed (n = , . %) and one chick ( . %) was in a poor body condition (table ) . to an excessive amount, mild to severe cutaneous abrasions with feather loss, lacerations and/ or subcutaneous haemorrhages of the head were noticed in one out of two chicks ( %) in ac , nine out of chicks ( %) in ac and out of chicks ( %) in ac trapped with the tt . one out of animals ( %) in ac and nine out of individuals ( %) in ac that had been trapped with tt were more mildly affected by those lesions. histological examination of the skin from the head revealed various types of inflammatory alterations which occurred solely or concurrently in one individual (table ). in all age classes and independent of trap type, mainly perivascular predominantly lympho-histiocytic dermatitis admixed with occasional heterophils and plasma cells of varying degrees was present (fig ) . this type of inflammation was in some cases accompanied by follicular aggregations of lymphocytes sometimes with secondary follicle formation (ac , tt ). in addition, ulcerative (fig ) , occasionally necrotising, suppurative and pustular inflammatory changes were found more often in chicks trapped with tt than with tt , these in many cases being associated with dermal and/or subcutaneous haemorrhages of varying degrees. only a few animals showed no cutaneous alteration in this localisation. the abdominal skin of the chicks was rarely affected by perivascular dermatitis; single individuals were mainly affected by lymphohistiocytic or pustular dermatitis. crops, glandular stomachs and gizzards were variably filled (table ). however, it should be noted that the chicks had spent up to five hours in the traps. the mentioned parts of the digestive tract contained grains, green food, and, inside the gizzard, grit stones. in one chick ( %) in ac , in seven chicks ( %) in ac and in ten chicks ( %) in ac , the quality of the intestinal content was associated with hyperaemic intestinal mucosa and perianal attachment of faeces suggestive of catarrhal enteritis. histologically, one animal ( %) in ac showed focally severe ulcerative stomatitis at the gums and one chick in ac focally moderate lympho-histiocytic ingluvitis. in single chicks in ac , nematodes without reactive inflammatory changes were found in the crop. furthermore, single individuals showed erosive and heterophilic inflammation of the gizzard, occasionally associated with intralesional nematodes which were not differentiated here. the intestinal mucosa in all animals showed a mild to moderate infiltration with eosinophils, lymphocytes and a few plasma cells. in eight out of chicks ( %) in ac and in four out of chicks ( %) in ac , reproduction stages of protozoal organisms, most likely coccidia sp., were found histologically within the epithelium (fig ) . predominantly mild focal lymphohistiocytic hepatitis was observed in one out of chicks ( %) in ac , in four out of chicks ( %) in ac , and in ten out of chicks ( %) in ac . single individuals in ac showed multifocal granulomatous hepatitis with severe acute coagulation necrosis (fig ) . in eight out of chicks ( %) in ac , a mild to severe diffuse fatty change in hepatocytes was present. nematodes with the morphology consistent with syngamus trachea were found in the trachea of none of the chicks in ac , in five out of chicks ( %) in ac and in ten out of ( %) chicks in ac . histologically, tracheal parasitism in most animals was associated with multifocal lympho-histiocytic, occasionally granulomatous or ulcerative tracheitis of variable extent. in single animals, subepithelial lymphoid follicles were found. in the lung, focal or multifocal interstitial, mild to moderate lymphohistiocytic pneumonia was observed in one out of chicks ( %) in ac , in three out of chicks ( %) in ac , and in six out of chicks ( %) in ac . focal or multifocal, mild to moderate granulomatous, occasionally necrotising pneumonia was present in three individuals ( %) in ac and in two individuals ( %) in ac . one animal in ac suffered from severe suppurative to necrotising pneumonia. there was no evidence of viral, bacterial, fungal or parasitic agents in these lungs, even in the histological special stains. hyperplasia of bronchus-associated lymphoid tissue was noticed in two chicks ( %) in ac and in three chicks ( %) in ac . numerous lungs showed acute haemorrhages. the kidneys displayed focally mild interstitial infiltrations consisting mainly of lymphocytes and macrophages in two chicks ( %) in ac and five chicks ( %) in ac . independent of the age class, a mostly moderate diffuse infiltration with plasma cells was observed in almost all examined lacrimal glands. miscellaneous findings included focally mild lymphocytic myocarditis in one chick ( %) in ac (fig ) , focally moderate lymphohistiocytic perineuritis (n. ischiadicus) in one chick in both ac ( %) and ac ( %), severe subacute hyaline degeneration of skeletal muscles with histiocytic infiltration in one chick ( %) in ac , focal chronic suppurative myositis in another chick ( %) in ac , and single protozoal cysts, most likely sarcosporidia sp., in the skeletal musculature of two individuals ( %) in ac without inflammatory changes. in many brains, perivascular and parenchymatous haemorrhages were observed without reactive changes. of all chicks tested, % of the chicks were positive for avian metapneumovirus (ampv) using rt-pcr, % positive for infectious bronchitis virus (ibv) using rt-pcr, and % for haemorrhagic enteritis virus (hev) using pcr. none of the chicks tested for hev were positive using pcr. tracheae of chicks and caecal tonsils of ten birds were tested by the coronavirus-rt-pcr and were negative for the respective virus. all samples tested for avian encephalomyelitis virus (aev), infectious bursal disease virus (ibdv), or infectious laryngotracheitis virus (iltv) were negative. the pool samples of the ten chicks were completely negative for all acid, alkaline-free and derivative substances as listed in s table which summarises the substances tested and the detection limits. two out of three samples tested for the herbicide glyphosate were positive ( . mg/kg, . mg/kg). since the s, a population decrease in ring-necked pheasants has been observed. especially in / , the population decline intensified [ ] . the present investigation revealed that the randomly trapped pheasant chicks displayed inflammatory lesions in different organs. in association with environmental stressors and a depleted nutritional status, these health changes may increase pheasant chick mortality, thus contributing to the population decrease. the dermatitis detected was often of a non-purulent character, mostly perivascularly accentuated with different cellular compositions of gradual variable infiltrations by lymphocytes, plasma cells and macrophages. especially on the head, this alteration additionally displayed pustular and lymphocytic inflammation. it was possibly itch-or parasite-induced. avian pox was excluded due to lack of pathognomonic and histological changes [ ] . these alterations occurred in chicks as young as one or two days of age. six out of chicks ( %) in ac already showed these alterations, with different types and degrees of inflammation. m. gallisepticum (mg) is an important etiological differential diagnosis, especially inducing periocular dermal swelling with lymphocytic inflammation [ ] . however, this pathogen was ruled out by the investigations. nevertheless, various mycoplasma spp. were isolated in out of ( . %) of the investigated chicks. however, the role of these mycoplasma spp. as a potential cause of periorbital skin alterations in pheasants is still unclear, but should be considered in following investigations in pheasants. as some birds were rt-pcr positive for ibv or ampv, it has to be elucidated further whether these viruses may have contributed to these lesions, as they are known to be respiratory disease associated. the inflammations might be itch induced following insect or tick bites. furthermore, the head injuries with lacerations and haemorrhages resulted from catching caused by the chicks jumping against the iron bars of the traps. these injuries did not appear anymore after exchanging these bars for loose nylon mesh. a total of % of the pneumonia cases were of an eosinophilic character and were most likely caused by syngamus trachea in ten cases. all cases of granulomatous inflammation were free of acid-fast bacteria as shown by the ziehl-neelsen stain. therefore, the cause of this granulomatous inflammation remains unknown. other possible agents able to induce pneumonia, bronchopneumonia, tracheitis and bronchitis were not detected. a prevalent eosinophilia tracheitis ( %) occurred in almost all cases in connection with detected parasites at different stages including coccidia, heterakis/ascaridia spp. and syngamus trachea. the degrees of inflammation were mainly mild up to moderate so that the clinical relevance is rather subordinate. the proventriculitis can have many origins. a histologically similar disease, that of gizzard erosion in broilers is often caused by an interaction between vitamin deficiency, fungal infections and stress situations after consuming mycotoxins. with periodic acid schiff reaction (pas) and brown-brenn stain, fungi, gram-positive and gram-negative bacteria were excluded [ ] . as ampv, ibdv, coronavirus and siadenovirus were excluded by pcr, a viral cause is relatively unlikely. marek's disease is doubtful as well due to the lack of other typical organ changes [ ] . it is possible that the birds may have been exposed to mycotoxins or pesticides that caused proventriculitis. based on localisation, size and shape of the eggs found in the proventriculus, a nematode-infection with dyspharynx nasuta probably resulted [ ] . the inflammation of the livers showed lymphocytic and lymphohistiocytic characters. the causes for these inflammatory changes are manifold and may include infectious as well as noninfectious agents. in three cases, the inflammation was granulomatous and necrotising. using ziehl-neelsen stain, acid-resistant bacteria were excluded. differential diagnoses for granulomatous and necrotising hepatitis include toxic, ischemic or infectious causes [ , ] . in the presented investigations, only a limited number of samples could be investigated for pesticides. therefore, it is difficult to directly link pathological findings to any of the investigated chemicals. further investigations are needed to elucidate the role played by pesticides in the declining pheasant populations as their habitat is regularly exposed to different chemicals used in agriculture. the main findings in the study were the poor nutritional status in the younger age groups and the increasing occurrence of various inflammation when the birds were ageing. as no direct cause for the inflammation was found and the inflammation affected various organs, it might be more a sign of various pathogens affecting the chicks. this seems to be more a sign of a weakened immune system, unable to defeat facultative pathogenic organisms. this is in line with the poor nutrition status, which triggers the development of diseases. no suspected virus infection was detected though. virus infections cannot be ruled out completely as a cause as viruses obviously circulate in the adult pheasant population and infected chicks die quickly. therefore, such cases were not among the sampled animals as the study focused on live chicks which still followed the hen. due to predation, decomposition and vegetation in the field, diseased pheasants are difficult to retrieve for health examinations and therefore were not included here. concerning parasites, low coccidian infections can be regarded as desirable to build up a protective immunity against reinfections. however, intestinal changes of the chicks show that coccidia sometimes considerably damage the intestinal mucosa due to severe infections, which may lead to a reduction in nutrient uptake. furthermore, a severe syngamus sp. infection can occlude the tracheal lumen, resulting in suffocation of the chicks, or their general condition deteriorates to such an extent that they become easy prey for predators. all these findings point to an effective complexity that either chicks die of starvation or their immune system becomes weakened. it seems that when the effects of maternal antibodies slowly diminish and the chicks have to mobilise their own immune system, the chicks become weakened as their immune system is not sufficiently developed. also, it is known from poultry that the development of the immune system is influenced by nourishment and that malnutrition can negatively influence the immune system [ , , ] . this hypothesis has not been confirmed yet in pheasants. not only pheasants, but also many other farmland birds have to cope with the intensive agricultural landscape. this change in habitat and the use of pesticides make it increasingly difficult to find insects that are vital for the chicks during their first weeks of life. a whole concatenation of circumstances could be explained by this connection; namely, the rather poor nutritional status, a possibly weakened immune system and the increased susceptibility to diseases. additionally, it is possible that the chicks are easier prey for predators due to various inflammations or poor physical condition, too. also, the weather can have a greater influence. supporting information s table. toxicological investigation of substances and limits of detection. highlighted in bold are the substances found in the pheasant samples. 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(unpublished) jägerstiftung natur+mensch. institute for terrestrial and aquatic wildlife research retrospektive zum rückgang des fasans agricultural intensification and the collapse of europe's farmland bird populations the second silent spring? farming and birds in europe: the common agricultural policy and its implications for bird conservation eeg stellt kulturlandschaft auf den kopf effects of cropping practices on declining farmland birds during the breeding season responses of plants and invertebrate trophic groups to contrasting herbicide regimes in the farm scale evaluations of genetically modified herbicide-tolerant crops weeds in fields with contrasting conventional and genetically modified herbicide-tolerant crops. i. effects on abundance and diversity influence of autumn applied herbicides on summer and autumn food available to birds in winter wheat fields in southern england the swiss agri-environment scheme promotes farmland birds: but only moderately birds as useful indicators of high nature value farmlands: using species distribution models as a tool for monitoring the health of agro-ecosystems declines in insectivorous birds are associated with high neonicotinoid concentrations host mortality, predation and the evolution of parasite virulence parasites and the dynamics of wild mammal populations differential body condition and vulnerability to predation in snowshoe hares prevalence, intensity and aggregation of intestinal parasites in mountain hares and their potential impact on population dynamics. international host manipulation by parasites in the world of dead-end predators: adaptation to enhance transmission? interactions between sources of mortality and the evolution of parasite virulence investigation into diseases in free-ranging ring-necked pheasants (phasianus colchicus) in northwestern germany during population decline with special reference to infectious pathogens effects of early feeding and dietary interventions on development of lymphoid organs and immune competence in neonatal chickens: a review romeis mikroskopische technik kompendium der geflü gelkrankheiten modified live infectious bursal disease virus (ibdv) vaccine delays infection of neonatal broiler chickens with variant ibdv compared to turkey herpesvirus (hvt)-ibdv vectored vaccine experimental haematobiochemical alterations in broiler chickens fed with t- toxin and co-infected with ibv prevalence of mycoplasmas in eggs from birds of prey using culture and a genus-specific mycoplasma polymerase chain reaction genus-and species-specific identification of mycoplasmas by s rrna amplification high inter-species and low intra-species variation in s- s rdna spacer sequences of pathogenic avian mycoplasmas offers potential use as a diagnostic tool das vorkommen von mykoplasmen bei storchnestlingen in brandenburg und sachsen-anhalt basic local alignment search tool recovery of mycoplasmas from birds wild und jagd-landesjagdbericht / . ni ministerium für ernä hrung, landwirtschaft, verbraucherschutz und landesentwicklung a retrospective studie of skin lesions in wild turkeys (meleagris gallopavo) in the eastern usa, - mycoplasma gallisepticum in pheasants and the efficacy of tylvalosin to treat the disease hard af segerstad c. mycotic proventriculitis in gray partridges (perdix perdix) on two game bird farms causes of gizzard erosion and proventriculitis in broilers first report of five nematode species in phasianus colchicus linnaeus (aves, galliformes, phasianidae) in brazil / primeiro registro de cinco espécies de nematóides em phasianus colchicus linnaeus (aves, galliformes, phasianidae) no brasil necrotizing hepatitis in a domestic pigeon (columba livia) hepato nephropathology associated with inclusion body hepatitis complicated with citrinin mycotoxicosis in a broiler farm early nutrition programming (in ovo and posthatch feeding) as a strategy to modulate gut health of poultry nutrition-mechansims of immunosuppression we wish to thank the hunting associations of lower saxony, north rhine-westphalia and schleswig-holstein for supporting the study. furthermore, special thanks go to the laboratory personnel for their excellent technical assistance in the laboratory investigations. key: cord- - dannjjm authors: nan title: research abstract program of the acvim forum denver, colorado, june – , date: - - journal: j vet intern med doi: . /j. - . . .x sha: doc_id: cord_uid: dannjjm nan clinics Ãà also see infectious disease abstracts id- -id- (thursday, june , : pm - : pm) Ãà also see pharmacology abstracts p- -p- (thursday, june , : pm - : pm) Ãà also see gasteroenterology abstracts gi- - june , hypertrophic cardiomyopathy (hcm) is the most commonly observed myocardial disease in cats. beta-blockers and calcium channel inhibitors are frequently administered drugs to cats with preclinical hcm despite the fact that neither drug category has been proven to slow disease progression or improve survival. ivabradine (procorolan s , servier, france) is a novel negative chronotropic agent used in the treatment of ischemic heart disease in people. little is known about its efficacy and safety in cats. the purpose of this study was to determine the short-term effects of ivabradine on heart rate (hr), blood pressure, left ventricular (lv) systolic and diastolic function, left atrial (la) performance, and clinical tolerance in healthy cats after repeated oral doses. ten healthy laboratory cats were involved in the present study. physical examination, systolic blood pressure measurement, and transthoracic echocardiography were performed in all cats at baseline and after oral administration ( weeks each) of ivabradine ( . mg/kg, q h) and atenolol ( . mg/cat, q h; . - . mg/kg) in a prospective, double-blind, randomized, active-control, fully crossed study. a-priori non-inferiority margins for the effects of ivabradine compared to atenolol were set at % (f . ) based on predicted clinical relevance, observer measurement variability, and in agreement with fda guidelines. variables were compared by use of -way repeated measures anova. ivabradine was clinically well tolerated with no adverse events observed. hr (ivabradine, po . ; atenolol, po . ; ivabradine vs. atenolol, p . ) and rate-pressure product (ivabradine, p o . ; atenolol, p . ; ivabradine vs. atenolol, p . ) were not different between treatments. at the dosages used, ivabradine demonstrated more favorable effects than atenolol on echocardiographic indices of left ventricular (lv) systolic and diastolic function and left atrial performance. ivabradine is non-inferior to atenolol with regard to effects on hr, rate-pressure product, lv function, la performance, and clinical tolerance. clinical studies in cats with hcm are needed to validate these findings and further assess safety. the aim of this study was to compare outcome from cpa in dogs following initial administration of either epinephrine or vasopressin during cardiopulmonary resuscitation (cpr). dogs having cpa in the er or icu of a university hospital were randomized to receive either iv epinephrine ( . - . mg/kg) or vasopressin ( . - . u/kg) in a blinded fashion immediately following establishment of iv access and again three minutes later. a standardized cpr protocol was followed. other vasopressors were not permitted during the six minute study period, at the end of the study period additional cpr interventions were at the discretion of the managing clinician. the primary end point was return of spontaneous circulation (rosc) within the study period; secondary end points included rosc at any point, survival to minutes, and survival to one hour. sixty dogs completed the study, received epinephrine and received vasopressin. rosc within six minutes was % ( vasopressin, epinephrine p . ), rosc at any time was % ( vasopressin, epinephrine p . ). survival to minutes was % ( vasopressin, epinephrine p . ), survival to one hour was % ( vasopressin, epinephrine p . ). five dogs survived to hours, one survived to hospital discharge. of animals dying after rosc, / were euthanized and / rearrested. no advantage of routine substitution of vasopressin for epinephrine was seen for rosc, a small survival advantage at one hour was seen in the group receiving epinephrine. the study also demon-strated that prospective clinical cpr research in animals is both possible and practical. three dogs were evaluated in phases. phase- : single-dose diltxr at approximately mg/kg po. phase- : same dose q h for . days. phase- : after a day wash-out the single-dose protocol was repeated using cut tablets to assess affect on extended release properties. blood pressure (bp), -lead ecg, echocardiogram, and h-ambulatory ecg were performed at baseline, and conclusion of phase- . blood samples and bp was obtained , , , , , , and h after final dosing. peak median plasma diltiazem concentrations (mcg/ml) measured by hplc for each phase were . , . , and . respectively. diltiazem concentrations were below the limit of detection in the majority of samples in phase- . median diltiazem concentration reached purported therapeutic concentrations ( . - . mcg/ml) by h post-pill in phase- and h post-pill in phase- . therapeutic concentrations were maintained for h in phase- , but only h in phase- . median bp (mmhg) was . at baseline and . at peak concentration in phase- . median heart rate (bpm) was . at baseline. h-ambulatory ecg analysis revealed the median hourly heart rate was . at baseline and during phase- . median heart rate at peak concentration in phase- was . . lack of detectable plasma diltiazem during phase- may be due to up-regulation of drug metabolism via p-glycoprotein (abcb - ) mutations. ongoing data collection and analysis will include mutation testing. adiponectin (adpn) is a cytokine produced by fat cells which has been shown to be correlated with adverse cardiac conditions in humans. in the heart, adiponectin activates several pro-survival reactions, including the ampk pathway and cox receptors, which protect the heart following ischemic injury. recent studies have shown that higher levels of adpn influence cardiac remodeling signaling, inhibiting protein synthesis and suppressing pathological cardiac growth. in humans, adpn plasma levels rise with decreased activity of the sympathetic nervous system and b-adrenergic agonists inhibit adpn at the level of gene expression. in contrast c-reactive protein (crp), a marker of systemic inflammation is elevated in humans with congestive heart failure (chf) and correlates to the severity of disease. first, we hypothesized that dogs with chf would have reduced adpn and elevated crp compared to normal dogs and that cytokine concentrations would predict severity of chf. second, we hypothesized that adpn receptor- (r ) and adpn protein would be elevated in the myocardium of chf dogs reflecting a compensatory process. we collected serum from dogs ( healthy and chf). circulating adiponectin and crp levels were quantified using a mouse/rat adiponectin elisa and a canine crp elisa. we found lower mean crp concentrations in normal dogs ( . ae . mg/ ml) than dogs with chf ( . ae . mg/ml), however, the results were not statistically significant due to the large variability seen among the chf dogs (p . ). we found greater mean adpn concentrations in normal dogs ( . ae . mg/ml) than chf dogs ( . ae . mg/ml) (p . ). in general, the greater the severity of the heart failure, the lower the level of serum adpn. when the tests the purpose of this study was to determine if there are any clinically important differences between the approaches (including devices) used in non invasive transvascular (interventional) closure of patent ductus arteriosus (pda) in dogs in our institution. initial and follow up records from all dogs (n ) that underwent attempted transvascular pda occlusion from january -december were examined. dogs were placed into groups depending on the device and route of vascular access (transvenous or transarterial). group : amplatz s canine ductal occluder (acdo) (transarterial) - dogs; group : gianturco s or mreye flipper s detachable embolization (flipper) coil (transarterial) - dogs; group : amplatzer s vascular plug (avp) (transarterial) - dogs; group : flipper coil (transvenous) - dogs. statistical comparisons were made using the kruskal-wallis test with mann-whitney tests to compare pairs of groups when significance was detected. p o . was considered significant. there was no significant difference in ages between the groups. there was a significant difference in body weight between groups with dogs receiving a coil either transarterially or transvenously (groups and ) being significantly smaller than dogs receiving an acdo or avp. this was by design since the acdo and avp cannot be used in small dogs. overall, the success rate of the total procedure (including vascular access and satisfactory pda occlusion) was high ( %) with success rates being comparable between groups ( - %). there was a significant difference in complication rate between groups (p o . ) with the acdo group having a markedly lower complication rate than the remaining groups ( % for acdo versus - % for the other groups). total fluoroscopy time ranged from - minutes (median minutes). fluoroscopy time for the transvenous method was significantly longer (median minutes; range - minutes) than in the remaining groups (median minutes; range - minutes) (p o . ). number of dogs with residual flow immediately following the procedure and hrs later was significantly less in the acdo group than in the remaining groups ( dogs from group and from group had moderate persistent flow while dog from group and from group had severe persistent flow hours after the procedure). the acdo appears superior in ease of use, complication rate, completeness of occlusion and fluoroscopy time than other devices. the remaining limiting factor with this device is patient size. until a smaller acdo device is marketed, coils remain the only choice for interventional closure in very small dogs ( o . kg). previously presented at the university of california davis, house officers seminar day. subvalvular aortic stenosis (sas) is one of the most commonly reported canine congenital heart defects and is inherited in newfoundland dogs and human beings. the golden retriever and rottweiler are breeds over-represented in dogs with subvalvular aortic stenosis; however, a genetic cause of this disease in these breeds has not been described. we performed genome wide association analysis in both normal and sas affected rottweilers and golden retrievers to identify chromosomal regions of interest that could implicate a causative mutation by high density single nucleotide polymorphism (snp) array. ( unaffected/ affected) adult golden retrievers and ( unaffected/ affected) adult rottweilers were included in this study. criteria for affected included a subcostal continuous-wave doppler aortic velocity ! . m/s and presence of a left basilar systolic ejection murmur; criteria for unaffected included a doppler aortic velocity . m/s. dna samples were obtained from anticoagulated blood. genotypes were obtained using high density ( , ) snp arrays, and genome wide association with sas was evaluated for each breed. significance cut-off was set at p  À , and all snps meeting this criterion were plotted within each breed and compared across breeds using plink. affected golden retriever data implicate the most significant region of genetic variation on chromosome at location (p .  À ; odds ratio . ) with other significant surrounding snps . affected rottweiler data also implicate the most significant region of genetic variation on chromosome at location (p .  À ; odds ratio . ) with other significant surrounding snps . other regions of statistical significance were on chromosomes and in the golden retriever and and in the rottweiler. genome wide association with subvalvular aortic stenosis in the golden retriever and rottweiler implicate overlapping chromosomal regions of interest for causative mutations on chromosome . the different secondary chromosomal regions of interest (chr , in golden retrievers and , in rottweilers) supports the known familial nature of this disease within different breeds and may suggest the presence of multiple mutations or breed specific disease modifiers. these data highlight the need for candidate gene evaluation on chromosome in golden retrievers and rottweilers with sas. heart valves share developmental signaling pathways with bone and cartilage. degenerative aortic valve disease in humans is characterized by valve stenosis and calcification. recent evidence suggests that degenerative aortic valves are undergoing pathologic processes that mimic osteogenesis. degenerative mitral valves in dogs and humans are characterized by valve regurgitation, and rarely undergo calcification. we tested the hypothesis that canine and human degenerative mitral valves might be undergoing pathologic processes that mimic chondrogenesis. to test this hypothesis, expression of bone morphogenic protein (bmp ), a chondrogenic growth factor; sox , a chondrogenic transcription factor; aggrecan, a proteoglycan abundant in cartilage; and type ii collagen were evaluated utilizing immunohistochemistry. normal canine mitral valves, different stages of canine degenerative mitral valves (early, intermediate, and late), and late-stage human degenerative mitral valves were studied. canine and human degenerative mitral valves showed focal areas that co-expressed all four markers of chondrogenic signaling and phenotype. valve interstitial cells and surrounding extracellular matrix in these focal areas adopted a morphologic appearance reminiscent of cartilage. focal chondrogenesis was present in all stages of canine degenerative mitral valves, but not normal canine mitral valves. focal areas of chondrogenesis did not coincide with nodular areas of glycosaminoglycan accumulation on the leaflet edge, but rather seemed to occur at points of chordae attachment to leaflets. in conclusion, canine and human degenerative mitral valves undergo pathologic processes that mimic chondrogenesis. this finding suggests that mitral valve degeneration may be recapitulating developmental signaling pathways shared by heart valves and cartilage. the triggering events for chondrogenesis in mitral valves remain unknown; as does the reason why aortic and mitral valves appear to be undergoing different pathologic processes. the fact that humans exhibit degeneration of both the aortic and mitral valve, and that dogs commonly exhibit only the latter could eventually provide insight into both processes. arrhythmogenic right ventricular cardiomyopathy (arvc) is a familial cardiomyopathy characterized by right ventricular fibrofatty infiltration and ventricular ectopy of left bundle branch block morphology (vpc) . a deletion in the striatin gene has been associated with arvc in at least some boxer families. syncope and sudden death (sd) occur in some affected dogs, although many affected dogs survive for years. the objective of this study was to define clinical characteristics of arvc in boxers that experienced sd, and compare them to those of a contemporaneous group of arvc boxers that had not died suddenly (nsd). data for both groups were collected from adult boxers enrolled in a long term prospective study of arvc in which echocardiograms and hour ambulatory ecg (aecg) are evaluated annually. aecg are quantitated for vpc numbers and arrhythmia grade ( - ). arvc diagnosis requires at least vpcs/ hours in the absence of other disease. forty three adult boxers that entered the study had died suddenly at the time of analysis (sd defined as the absence of observed clinical signs within hours prior to an unexpected and unexplained death). striatin genotype was available for of the sd dogs ( heterozygotes, homozygotes); were female ( intact) and were male ( intact). sd occurred at a mean age of years (range, - ); sd dogs ( %) had no prior history of syncope. twelve sd dogs ( %) were on antiarrhythmics at the time of death (metop-p oooooooooooprolol ( ), sotalol ( ), amiodarone ( ), procainamide ( ), mexiletine & atenolol ( ), atenolol ( )). eleven sd dogs ( %) had decreased myocardial systolic function defined by a shortening fraction (%fs) o % (range - , mean ) on the most recent echocardiogram prior to sd. median vpcs/ hours on annual aecg was , (range - , ) with a median arrhythmia grade of (range - ). twenty one contemporaneously entered arvc boxers that had survived to at least the median age of the sd group with nsd were available for comparison; / were genotyped ( heterozygous, homozygous, negative), were female ( intact) and male ( intact). twelve nsd dogs ( %) had no prior history of syncope. median nsd group age was years (range, - ); / ( %) were on an antiarrhythmics (sotalol ( ), mexiletine & sotalol ( ), mexiletine & atenolol ( )). one nsd dog had decreased %fs (nsd group %fs range - , mean ). the nsd median number of vpcs was , (range - , ), median arrhythmia grade was (range - ). striatin genotype was significantly associated with sd. no significant differences were found between groups with respect to vpc numbers or arrhythmia grade. shortening fraction was significantly lower in the sd group (p o . ). sd in arvc appears to be associated with the presence of the striatin mutation and reduced % fs, it does not appear to be associated with number of vpcs or arrhythmia grade. coughing in the small breed dog may be related to cardiac causes associated with myxomatous mitral valve degeneration (mmvd) including pulmonary edema and compression of the mainstem bronchus by a severely enlarged left atrium, or due to respiratory causes such as tracheal and/or bronchial collapse or chronic bronchitis. the purpose of this study was to evaluate the association between left atrial enlargement and large airway collapse in dogs with mmvd and chronic cough. we hypothesized that airway collapse was independent of degree of left atrial enlargement. twelve dogs with mmvd and a chronic cough in the absence of congestive heart failure were prospectively evaluated with thoracic and cervical radiography, echocardiography, fluoroscopy, bronchoscopy and bronchoalveolar lavage (bal). group dogs (n ) had moderate to severe left atrial enlargement based on an echocardiographically calculated left atrial:aortic surface area [la:ao(a)] . group dogs (n ) had no to mild left atrial enlargement [la:ao(a) ]. the site and severity of airway collapse was graded on bronchoscopy and bal cytology was assessed for evidence of inflammation or infection. the occurrence of bronchoscopic abnormalities was compared between groups using fisher's exact test. p o . was considered significant. age and body weight did not differ between groups. left atrial size was interpreted radiographically as moderately to severely enlarged in of dogs in group and as moderately enlarged in of dogs in group . fluoroscopy revealed variable degrees of airway collapse during normal respiration and induced cough in both groups. radiography and fluoroscopy were not accurate in identifying site and degree of collapse in either group when compared to bronchoscopy. cervical tracheal collapse was identified during bronchoscopy in both group ( of ) and group ( of ) dogs but was subjectively less severe in group dogs. bronchial collapse % was evident at multiple sites in both groups of dogs with no difference between groups. all dogs had suppurative and/or lymphocytic inflammation on airway cytology. infection was not present in either group of dogs, although non-specific light bacterial growth was detected in of group dogs and of group dogs (p . ). preliminary results failed to identify an association between left atrial enlargement and airway collapse in dogs with mmvd but did suggest that airway inflammation is common in affected dogs. further studies are needed to identify factors contributing to airway collapse in dogs with and without mmvd. atenolol is often used empirically in cats with asymptomatic hcm, even though clinical and experimental evidence of efficacy is lacking. cardiac biomarkers play a critical role in the early detection of subclinical cardiac disease, in the prediction of long-term prognosis, and in monitoring the response to therapy in humans. we hypothesized that serum concentrations of the biomarkers, nterminal pro-brain natriuretic peptide (nt-probnp) and cardiac troponin i (ctni), would improve following the chronic administration of atenolol po to asymptomatic cats with hcm. six maine coon or maine coon cross cats with severe hcm from the research colony at ucdavis were administered atenolol ( . mg po twice a day) for days. no cat had severe left ventricular dynamic outflow tract obstruction due to systolic anterior motion of the mitral valve. the concentrations of nt-probnp and ctni were assayed prior to drug administration and on the last day of drug administration. there was no statistically significant difference identified in nt-probnp [median before: pmol/l (range: - pmol/l), median after: pmol/l (range: - pmol/l); p . ] or ctni [median before: . ng/ml (range: . - . ng/ml), median after: . ng/ml (range: . - . ng/ml); p . ] concentrations before and after drug administration using the wilcoxon matched pairs test. the ctni finding suggests that atenolol does not reduce chronic myocyte death in cats with hcm. the lack of improvement in nt-probnp suggests that atenolol does not improve myocardial wall stress in cats with hcm. a clinical trial is warranted to confirm or refute the findings from this study. therefore, leptin-gene expression was investigated in blood samples of dogs with congestive heart failure (chf; n ) in comparison to dogs presented for cardiac screening (n ) without abnormalities. additionally myocardial samples (interventricular septum, right and left atrium and ventricle) of dogs with no cardiac abnormalities (controls), seven dogs with acquired and three with congenital cardiac diseases were investigated using quantitative rt-pcr. leptin blood levels were significantly higher in dogs with chf in comparison to dogs without diseases (p . ). there was an association with gender with higher myocardial leptin levels in female dogs with cardiac diseases (p . ). differences between cardiac regions were present (p o . ) and cardiac diseases resulted in an increase in atrial leptin levels in both sexes (p . ). interestingly, a significant reduction of myocardial leptin was present in dogs with congenital cardiac diseases (p . ), whereas acquired cardiac diseases resulted in an increase in leptin (p . ) in comparison to controls. these results suggest that the heart might be a target of leptin action in the dog and myocardial leptin production might play a role in regulating cardiac function in an auto-and paracrine manner. predicting risk of chf in asymptomatic dogs with mitral valve disease (mvd) is challenging. we examined ability of nt-probnp to identify asymptomatic dogs at high risk for chf. dogs with isachc- b (la:ao . ) were prospectively recruited; dogs with current or previous chf or diuretic therapy were excluded. physical examination, radiography, echocardiography, and nt-probnp were performed at - mo intervals for dogs (median follow-up d, range, - d). thirty-one patients reached a study endpoint of radiographic pulmonary edema; remained asymptomatic. parameters from the visit immediately previous to onset of chf (future-chf) or prior to the most recent visit without chf (remain-asympt) were analyzed. median nt-probnp of future-chf ( pmol/lpmol/l, iqr - ) was significantly different from remain-asympt ( pmol/l, - ; p . ). median time to chf of future-chf was d (iqr, . groups also differed in median la:ao (future-chf . [ . - . ]; remain-asympt . [ . - . ], p . ); vhs (future- ]; remain-asympt . [ . - . ], p . ); and lvidd:ao ]; remain-asympt . [ . - . ], p . ). roc analysis to predict if chf would occur prior to the next visit yielded auc . ( %ci, . - . ). sensitivity was . % or . % and specificity . % or . % for nt-probnp pmol/l or pmol/l, respectively. mean increase in nt-probnp between penultimate visit and two visits prior to endpoint: future-chf . pmol/l vs. remain-asympt . pmol/l. within mo, . %, . %, . %, and . % of dogs with nt-probnp o , , and pmol/l developed chf. nt-probnp and heart size helped assess risk of chf in asymptomatic mvd. increasing the assay's upper limit of detection would likely improve utility of nt-probnp. piiinp is a serum biomarker of collagen biosynthesis and is described as a marker of myocardial fibrosis in human patients. we hypothesised that piiinp concentrations would vary according to the degree of remodelling demonstrable in dogs with naturallyoccurring myxomatous mitral valve disease (mmvd). serum piiinp concentrations (mg/ml) were measured in dogs with mmvd and healthy controls using a validated commerciallyavailable radioimmunoassay. results are reported as (mean ae sd). non-normally distributed variables were logarithmically transformed. comparisons of continuous variables were made between groups using t-tests and one-way anovas with tukey's post-hoc comparisons. univariable analyses were used to evaluate associations between piiinp and clinical characteristics (age, breed [cavalier king charles spaniel (ckcs) yes/ no], sex, weight, heart rate [measured from ecg], treatment with acei [yes/ no], treatment with diuretics [yes/ no] and echocardiographic measurements [la/ao, lvedd/ lvfwd, lveddn, lvesdn]). multivariable analysis was initially performed with all dogs included and then repeated excluding all dogs receiving therapy. dogs with mmvd were divided into those with no cardiomegaly (nc) (la/ao o . and lveddn o . ), those with cardiomegaly (la/ao ! . and/ or lveddn ! . ) but no clinical signs (c) and those dogs with cardiomegaly requiring treatment for congestive heart failure (chf). one hundred and fifty-four dogs with mmvd and control dogs were studied. there was no difference in age (p . ) or weight (p . ) between the mmvd and control groups. there was a significant difference in serum piiinp (p . ) between normal ( . ae . ), nc ( . ae . ), c ( . ae . ) and chf ( . ae . ) groups. post-hoc comparisons demonstrated a difference between nc and chf groups (p . ). there was no difference in serum piiinp between genders (p . ). in the univariable analysis ckcs (yes/ no) (p . ) was positively associated with serum piiinp. age (p o . ), log (la/ao) (p . ) and lveddn (p . ) were negatively associated with serum piiinp. in the multivariable model including all dogs, lveddn (p o . , b À . ( %ci À . to À . )), age (p . , b À . ( %ci À . to À . )) and ckcs (yes/ no) (p . , b . ( %ci . to . )) were independently associated with serum piiinp. in the multivariable model including only dogs not receiving therapy (n ), lveddn (p . , b À . ( %ci À . to À . )), age (p . , b À . ( %ci À . to À . )) and ckcs (yes/ no) (p . , b . ( %ci . to . )) were independently associated with serum piiinp. in conclusion, serum piiinp decreases with age and with increasing lveddn. ckcs have higher serum piiinp measurements independent of age and lveddn, which may reflect a difference in collagen turnover in this breed. left atrial (la) chamber dilation and congestive heart failure (chf) are common consequences of cardiac conditions in cats. in some cases chf is manifest as right-sided chf (r-chf) or pleural effusion, in other cases chf manifests as left-sided chf (l-chf) or pulmonary edema. it is not always readily apparent as to which cats will develop what form of chf. a general impression has been that la enlargement is associated with the average burden of elevated filling pressures, but little attention has been paid to the function of the la chamber itself. since chf is classically preceded by abnormal atrial chamber dilation and alterations in atrial chamber function, we want to understand how these changes may help us manage or predict chf in the cat. we hypothesized that cats manifesting r-chf have la failure with the la acting primarily as a conduit, resulting in greater pulmonary hypertension, whereas l-chf cats maintain some booster pump and reservoir function. we measured la maximum and minimum areas from right parasternal long axis four-chamber views on d echo, and la m-mode dimensions at maximum, minimum, and beginning of atrial contraction. la area change, fractional shortening, active emptying fraction, and expansion index were calculated from these measurements. right ventricular internal diastolic diameter was also measured on m-mode views. preliminary data revealed that maximum left atrial size is not significantly different between r-chf and l-chf cats on d or m-mode measurements due to high variability. however, total left atrial fractional shortening is significantly reduced in r-chf cats ( . % ae . ) compared to l-chf cats ( . % ae . )(p . ), and r-chf cats have reduced left atrial active emptying fraction ( . % ae . ) as compared to l-chf cats ( . % ae )(p o . ). left atrial expansion ability is poorer in r-chf cats ( . % ae . ) than in l-chf cats ( . % ae )(p . ). these findings may suggest that atrial stiffness and poorer atrial function is associated with a greater degree of pulmonary venous and thus secondary pulmonary arterial hypertension resulting in pleural effusion (r-chf). right ventricular diameter on m-mode was increased in r-chf cats ( . mm ae . ) when compared to l-chf cats ( . mm ae . )(p . ) and normal cats ( . mm ae . )(p . ), which may also be evidence for a greater degree of pulmonary arterial hypertension in these cats. episodic weakness and syncope are common in boxer dogs. reported causes include rapid ventricular tachycardia (vt) and exertion-excitement triggered neurally-mediated bradycardia (nmb) .the purpose of this retrospective study is to describe the features of presumed nmb in boxers. to be included in the study, each dog must have been overtly healthy with a history of exertion-excitement triggered syncope or presyncope; had a normal echocardiogram (ec); had absence of vt and fewer than ventricular premature complexes (vpc) on an initial and subsequent hour holter recordings; and been alive and overtly healthy for at least six months following the initial evaluation. a total of boxers were identified. sixteen were male and were female. most ( %) dogs were either less than or more than years of age. most dogs had multiple, but infrequent, episodes and heart rhythm was documented at the time of an episode in only ( %) and only once (bradycardia) on the first holter recording. owners were instructed to attempt to precipitate episodes. bradycardia related episodes were subsequently recorded in : during the nd ( ), rd ( ) or th ( ) day of hour holter recordings and during the th day of an event recording ( ). collapse and bradycardia were documented during auscultation in additional dogs. the heart rate during syncope was never documented in ( %) dogs. a presumptive diagnosis of nmb was based on the absence of initial and follow-up of ec abnormalities and the presence of no or few vpc during extended ecg monitoring. multiple holter recordings ( - hours) were performed in of ( %) dogs and event monitoring of days ( ) and days ( ) was performed in additional dogs. in conclusion, documentation of the heart rhythm during episodes of collapse was difficult, accomplished in only % and was unlikely during the first holter recording. in boxers with suspected nmb, extended ecg monitoring and implantable loop recorders may be best for hr documentation. arrhythmogenic right ventricular cardiomyopathy (arvc) is an inherited myocardial disease with high prevalence in the boxer dog population, and is associated with sustained monomorphic ventricular tachycardia, sudden cardiac death, and replacement of myocardium with fatty or fibro-fatty tissue. though several genes have been linked to the disease both in humans and in boxers, the etiology of arvc is still unclear. several mechanisms for the development of arvc have been suggested, including dysfunction of the canonical wnt pathway, which results in an arvc phenotype in the mouse. the canonical wnt pathway has been linked to many cellular functions, including growth and differentiation of adipocytes. with the recent discovery that the gene encoding striatin, a protein involved in wnt signaling, may be involved in the development of boxer arvc, we hypothesized that changes in the wnt pathway may also play a role in the etiology. here, we show changes in the localization and decreased amount of proteins affiliated with the canonical wnt pathway. afflicted boxers were identified by -hour holter monitoring and histopathological examination of the heart. samples from the right ventricle rv) of arvc afflicted boxers, and unafflicted dogs ( beagle, mongrels, and german shepherds) were collected, fixed in % formalin, processed, treated with antibodies recognizingcatenin, striatin, and calnexin, and examined using confocal microscopy. western blots were performed on unafflicted rv samples, and arvc afflicted rv samples. frozen tissue samples were homogenized in laemmli buffer, and mg of protein was loaded into each well of a - % gradient gel. -catenin, an integral modulator of the wnt pathway, and striatin were colocalized with the endoplasmic reticulum (er) marker, calnexin. in the unafflicted animals, -catenin localized at sites of cell-to-cell apposition, and striatin localized in a diffuse intracellular pattern, with no detectable localization in the er. in contrast, in the arvc boxers, bothcatenin and striatin were colocalized with calnexin in an er pattern. in the afflicted samples, -catenin and striatin were not visualized to the intercalated disc and intracellular space, respectively. western blots of striatin and -catenin revealed no changes in the amount of protein. interestingly, a western blot for the wnt protein revealed a decrease in the amount of protein in arvc samples, compared to unafflicted samples. our preliminary data suggest that disturbances of the canonical wnt pathway may play an etiological role in the development of arvc in the boxer dog. there are numerous benefits of omega- fatty acid supplementation in human heart disease, including reduction in arrhythmias, decreased incidence of sudden death, and improved survival in heart failure. antithrombotic effects of omega- fatty acids have been demonstrated in people, which may have particular benefit in cats given their risk of thromboembolic complications with cardiac disease. benefits also have been found in canine heart disease, and reduced serum concentrations of the omega- fatty acids, eicosapentaenoic acid (epa) and docosahexaenoic acid (dha) have been found in dogs with congestive heart failure (chf) secondary to dcm. to the authors' knowledge, no studies to date have investigated fatty acid concentrations in cats with cardiomyopathy. the purpose of this study was to measure serum fatty acid concentrations in normal cats and cats with cardiomyopathy. serum fatty acid concentrations were measured in normal cats and cats with cardiomyopathy using gas chromatography. cats with cardiomyopathy and at least mild left atrial (la) enlargement (la to aortic ratio . on two-dimensional echocardiography from a right parasternal short axis view) were candidates for study. normal cats had a normal history, physical examination, echocardiogram, packed cell volume, total solids and platelet count. cats with evidence of other systemic disease or those receiving anticoagulants were excluded from the study. normally distributed and skewed data were compared between the cardiomyopathy and control groups with independent t tests or mann whitney u tests, respectively. statistical significance was set at p o . . thirty cats with cardiomyopathy ( neutered males and neutered females) and healthy controls ( neutered males and neutered females) were enrolled. median age was yr (range, - yr) in the cardiomyopathy group and yr (range, - yr) in the control group (p . ). cats in the cardiomyopathy group were classified in the international small animal cardiac health council stage b (n ), (n ), a (n ) and b (n ). compared to control cats, cardiomyopathic cats had higher concentrations of palmitic acid (p . ) and dha (p o . ), and lower concentrations of linoleic acid (p . ). among cats with cardiomyopathy, there was no significant correlation between any serum fatty acid concentration and left atrial size or age. these findings warrant further investigation into the role of fatty acids in cats with cardiac disease. platelet mapping is an application of thromboelastography that relies on the generation of at least three tracings: ma thrombin (maximum platelet activity),ma fibrin (fibrin activity only), an-dma aa or adp (platelet activity not inhibited by arachidonic acid or adp receptor antagonists, respectively). using these three tracings, the % inhibition of platelets can be calculated. the purpose of this study was to evaluate the platelet mapping assay in normal cats and assess platelet inhibition in cats receiving clopidogrel. employee-owned cats with normal history, physical exam, echocardiogram, thromboelastography, packed cell volume, total solids and platelet count were eligible. clopidogrel ( . mg po q h) was administered for days with platelet mapping performed on days and . platelet mapping values were compared using a paired t test, with significance set at p o . . seven cats ( fs, cm, aged - years) were enrolled. compared to day , ma adp (p o . ) and ma fibrin (p o . ) were lower on day . the latter unexpected result prompted measurement of fibrinogen concentrations at day and in the last of these cats. fibrinogen was not different from day to in these cats. these results suggest that platelet mapping may be a simple, outpatient clinical tool to measure antiplatelet activity in cats receiving clopidogrel. this clopidogrel dose resulted in significant platelet inhibition as measured by ma adp in all cats. further studies correlating antiplatelet effects measured by platelet mapping with clinical outcomes in cats with cardiomyopathy are warranted. this study investigated the hemodynamic effects of application of an itd in a canine model of cardiopulmonary arrest. laboratory beagles which were part of a separate terminal study were anesthetized and instrumented for continuous measurement and recording of right atrial pressure, arterial pressure and carotid blood flow. following euthanasia, cpr was performed for one minute, then a pause of one minute followed by a second one minute period at a compression rate of - /minute, ventilation with % oxygen was delivered at eight breaths/min and ml/kg tidal volume. cpr was performed with the itd in place (itd-cpr) and without the itd (s-cpr) for one period each in a randomized fashion with the rescuer blinded to its application. baseline, s-cpr and itd-cpr data were assessed for normality, a kruskal-wallis one-way anova on ranks was used (baseline v cpr). when appropriate a pairwise multiple comparison procedure (dunn's method) was used. percentage of baseline s-cpr v itd-cpr was assessed using the student t-test. the right atrial diastolic pressure was significantly more negative with the itd attached than without (p . ), the coronary perfusion pressure and carotid blood flow were significantly higher during cpr with the itd than standard cpr (p . , p . ). no significant differences in diastolic, mean or systolic arterial pressure or end tidal co were seen. application of the itd resulted in significantly improved hemodynamics during cpr in dogs. clinical evaluation of the device is warranted to examine whether this translates into improved success in resuscitation and survival. left ventricular (lv) systolic dysfunction is a common problem in dogs and can be due to a variety of etiologies. one potential etiology for systolic dysfunction is persistent or paroxysmal tachyarrhythmias, leading to tachycardia-induced cardiomyopathy (ticm). in humans, ticm carries a relatively good prognosis in that remodeling may be reversible with normalization of heart rate. differentiating between primary and secondary tachyarrhythmias in dogs with systolic dysfunction is critical for prognostic purposes as primary tachyarrhythmias may be associated with a better outcome. the goal of our study was to describe a population of dogs with ticm and to determine if treatment of arrhythmias was associated with reversible cardiac remodeling as indicated by standard echocardiographic parameters. medical records of dogs referred to the cardiology service of ksu vmth from to were reviewed. ticm was defined as the presence of severe tachyarrhythmias that were reversible with treatment, systolic dysfunction or ventricular enlargement that improved with treatment of the arrhythmia, or dogs with severe tachyarrhythmias and systolic dysfunction of breeds with that are atypical for idiopathic dilated cardiomyopathy. exclusion criteria were dogs with congenital heart disease, severe mitral regurgitation, and endocarditis. transthoracic echocardiography, thoracic radiographs and electrocardiography (ecg) were performed in all dogs. ventricular enlargement and systolic dysfunction were defined according to standard echocardiographic parameters. arrhythmias were confirmed with a holter monitor in dogs. a total of dogs were included in the study. mean age was years (range - years) with males ( intact, castrated) and females ( spayed, intact). four dogs had pulmonary venous congestion or pulmonary edema and two dogs had ascites. at initial presentation, the meanaesd values were as follows: heart rate ae bpm, m-mode fractional shortening (fs) . ae . %, ejection fraction (ef) using the area-length method . ae . %, and left atrial to aortic root ratio (la/ao) . ae . . initial meanaesd m-mode derived lv internal dimensions corrected for body weight were as follows: diastolic . ae . and systolic . ae . . at least one of the following tachyarrhythmias were identified in each dog: atrial fibrillation ( ), supraventricular tachycardia ( ), junctional tachycardia ( ), and ventricular arrhythmias ( ). ten dogs were available for follow up. seven dogs improved in at least one of the following parameters: resolution of tachyarrhythmia ( ), improved systolic function ( ) antidiuretic hormone (adh) has been shown to be elevated in humans with congestive heart failure (chf). recently, antidiuretic hormone antagonists were successful during investigational treatment of refractory congestive heart failure in humans. adh levels have been only modestly investigated in dogs with cardiac disease, primarily due to the technical difficulty in measuring adh levels via radioimmunoassay. based on the homologous structure of canine and human adh, we aimed first to determine the feasibility of measuring adh in dog plasma using a human elisa kit, and secondly to investigate the level of adh in dogs with congestive heart failure due to acquired cardiac disease. elisa assay kit validation was performed using six healthy dogs with normal clinical and echocardiographic examinations. pooled canine plasma was spiked with synthetic adh and intra-assay precision, dilutional parallelism, and linearity were assessed. to address the second aim of the study, samples were collected from normal dogs and dogs with heart failure due to one of two types of acquired cardiac disease: chronic degenerative valve disease (cdvd) or dilated cardiomyopathy (dcm). patients underwent clinical, radiographic, and echocardiographic examination to confirm diagnosis, assess severity of disease, and determine presence of pulmonary edema. whole blood was collected into edta tubes containing protease inhibitors, cold centrifuged, and plasma was stored at À until analysis. following ether extraction, plasma adh in each sample was measured in duplicate using a human elisa kit. statistical analysis included a d-agostino and pearson test for normality; group results were compared using a nonparametric mann-whitney test. adh was measured in canine plasma using the human elisa kit with acceptable intra-assay precision, linearity, and dilutional parallelism. intra-assay coefficient of variation was %. twenty-four dogs were recruited for the second phase of the study. six normal dogs and twelve dogs with radiographic evidence of pulmonary edema due to either cdvd or dcm were selected for participation. the remaining six dogs were excluded due to lack of definitive radiographic evidence of congestive heart failure. median adh values were . ae . pg/ml for the normal group (n ) and . ae . pg/ml for the heart failure group (n ). median adh values for the two groups were statistically different (p . ). our preliminary results indicate that measuring canine adh using a human elisa kit is feasible and provides results with an acceptable coefficient of variation. we also showed that dogs with congestive heart failure due to cdvd and dcm have elevated adh levels in comparison to normal dogs. our findings motivate further investigation to assess the degree of plasma adh level elevation and the possible use of adh antagonists as an adjunct treatment for refractory congestive heart failure in dogs. aortic thromboembolism (ate) occurs in both cats and dogs. whereas ate in cats is strongly associated with structural heart disease and typically has an acute catastrophic presentation; the pathogenesis and presentation of ate in dogs is less well known or understood. further, an effective antithrombotic strategy for ate in dogs has not been reported. medical records of dogs diagnosed with ate between and were examined retrospectively. diagnosis of ate was based on ultrasonography, doppler flow studies, and diminished or absent femoral pulses. dogs were treated with various acute and chronic antithrombotic therapies. the severity of ambulatory dysfunction was graded as none, mild, moderate, severe, or non-ambulatory at presentation and after therapy. a cohort of dogs in this study received a standardized protocol of chronic warfarin therapy with or without antiplatelet drugs. target international normalized ratio for warfarin therapy was to . twenty-six dogs were diagnosed with ate. all had an apparent mural aortic thrombus caudal to the renal arteries with most having evidence of embolization to the iliac and femoral arteries. none had structural heart disease at diagnosis. twenty dogs ( %) were still ambulatory at diagnosis. the median duration of ambulatory dysfunction prior to presentation was . weeks (range day - weeks). a majority of dogs ( %) had no concurrent conditions at diagnosis. nine dogs ( %) had protein-losing nephropathy. four dogs ( %) were hypothyroid. fourteen dogs were treated with a standard warfarin protocol for a median period of . months (range . - months). eight dogs were treated concurrently with aspirin, dogs were treated concurrently with clopidogrel, and dogs were treated with warfarin only. ambulatory function improved between and grades in dogs treated with chronic warfarin. the median period until clinical improvement was . days (range - days). two dogs treated with chronic warfarin therapy had documented resolution of ate, and dogs had complete resolution of ambulatory dysfunction. none of the dogs treated with chronic warfarin became nonambulatory, died, or underwent euthanasia because of ate. the median period of freedom from an adverse event was . months. no serious hemorrhagic events were reported. four dogs were treated with tpa. three of these had an unfavorable outcome. two dogs were ambulatory before tpa and become non-ambulatory after treatment. two dogs underwent surgical thrombectomy. one had a favorable outcome until ate recurred months after surgery. in conclusion, the pathogenesis of ate in dogs is not associated with structural heart disease or left atrial thrombus formation. the presentation tends to be for chronic ambulatory dysfunction. most dogs are still ambulatory at presentation. warfarin, with or without concurrent antiplatelet therapy, is an effective antithrombotic treatment strategy for dogs with ate. information is known. through previous work, investigators have encountered norfolk terriers (nt) with echocardiographically apparent dmvd in the absence of a heart murmur. in order to more fully understand dmvd in this breed of dog, we sought to characterize findings from the physical and echocardiographic exam, biochemical, biomarker, and nutritional profile, and select environmental variables from a cohort of apparently healthy nt. overtly healthy nt ! yrs old were recruited by different veterinary hospitals and underwent historical, physical, ecg, and d/color-flow doppler echocardiographic exam. anterior mitral valve length, maximal thickness, area, and prolapse were measured from d images. presence of dmvd was defined as thickened, prolapsing, or flail mitral valve leaflets in the presence of color flow doppler evidence of mitral regurgitation. blood samples were obtained for serum biochemistry and serotonin, plasma nt-probnp, amino acid profile, c-reactive protein, and cardiac troponin-i. forty-eight dogs were entered into the study (median age, yrs iqr [ - ]; gender, f, m; median bcs, ). of the dogs, ( %) had murmurs, ( %) had mid-systolic clicks, ( %) had ecg p-pulmonale, and ( %) were deemed to have echocardiographic evidence of dmvd, including nt without a murmur. seven ( %), ( %), and ( %) dogs were classified as isachc , a, and b, respectively. mean indexed echocardiographic mitral leaflet length (p o . ), thickness (p . ), prolapse (p . ), and la:aod (p . ) were significantly different between isachc a/b vs . between isachc a/ b and , there were no differences in serum amino acids, c-reactive protein, troponin, diet, or environmental factors; however different amino acids (ala, gly, phe, pro, trp, tyr) were significantly higher in isachc b vs. a. median serum serotonin was increased in dogs with a/b vs. (p . ). dogs whose diets contained some canned food (p . ) and dogs residing in suburban environments (p . ) had higher serotonin concentrations. nt-probnp tended (p . ) to be higher in isachc a/ b vs. . dmvd appears to be relatively common in nt and echocardiographic changes consistent with mild dmvd can be seen in dogs without a heart murmur. the results of this study establish a foundation of useful information upon which additional prospective studies can be developed. left ventricle (lv) evaluation is one of the most important contributions of echocardiography in the assessment of cardiac function. however, lv analysis can be made from images obtained by different modes and views of the heart. the aim of this study was to compare lv measurements, shortening fraction (sf) and ejection fraction (ef) obtained from four methods: m mode in short-axis and in long-axis, bidimensional mode in short-axis and in long-axis views of the heart. forty normal adult german shepherds were selected. echocardiographic study of lv of each animal was performed by the four methods described above. ancova test was used to examine the effects of axis, mode, weight and gender over lv measurements. isolated effect of the axis was observed for lv end-diastolic diameter (lvedd), with greater values obtained from short-axis views. there was isolated effect of mode over ef and sf, with greater measurements derived from bidimensional mode methods. weight correlated with all linear lv measures at least in one method, but not with ef and sf. weight had positive effect over lv endsystolic diameter and lv end-diastole posterior wall thickness in all methods, except from m mode in short axis in the last one. gender had isolated effect over lvedd, males showing greater values than females in bidimensional mode in short and long axis. the combined effect of axis, gender and weight was identified in interventricular septal end-diastolic thickness. we concluded that normal reference values obtained by different echocardiographic modes and planes should not be used interchangeably. abstract c- assessment of left ventricular diastolic func-tion by color tissue doppler imaging echo-cardiography in maine coon cats tested for mypbc-ap mutation hypertrophic cardiomyopathy (hcm), characterized by increased cardiac mass and diastolic dysfunction, is the most common feline heart disease. myocardial analysis by color tissue doppler imaging (tdi) is more sensitive than conventional echocardiography. this study evaluated diastolic dysfunction in various stages of feline hcm. maine coon cats (n ) were screened for the mybpc-a p mutation and examined with both echocardiography and tdi. then, were phenotypically classified in: normal (n ), suspects for hcm (n ) and hcm group (n ); and genotypically classified in: negative (n ), heterozygous (n ) and homozygous group (n ). myocardial velocities, measured in the basal and mild ventricular segment of the interventricular septal wall (ivs), left ventricular free wall (lvw) and in radial segment of left ventricular wall, was compared among different groups. a significant decreased (p , ) longitudinal em/am at the basal segment of ivs was observed in hcm cats compared with suspects and normal cats. a significant increased (p , ) longitudinal e/em at the basal segment of ivs was observed in hcm cats compared with suspects and normal cats. and a significant decreased (p , ) longitudinal sm at the basal segment of the lvw was observed in heterozygous cats compared with negative cats, both without hypertrophy. there was a significant positive correlation between summated early and late diastolic velocities (emam) and heart rate (p o , ); and a positive correlation between sm and em velocities and heart rate (p o , ). the mybpc-a p mutation is not consistently associated with ventricular hypertrophy and negatives cats can also develop hcm. the tdi alone is not able to identify cats with mutation before myocardial hypertrophy. diastolic dysfunction occurs in many cats with hypertrophic cardiomyopathy (hcm) but less is known about systolic function in various stages of hcm. myocardial strain analysis by tissue doppler imaging is a noninvasive echocardiographic method to assess systolic function. this study evaluated systolic function in various stages of feline hcm. maine coon cats (n ) were screened for the mybpc-a p mutation an examined with both echocardiography and strain. then, were phenotypically classified in: normal (n ), suspects for hcm (n ) and hcm group (n ); and genotypically classified in: negative (n ), heterozygous (n ) and homozygous group (n ). peak myocardial strain, measured in the basal and mildventricular segment of the interventricular septal wall (ivs), left ventricular free wall (lvw), left ventricular anterior wall (lvaw), left ventricular posterior wall (lvpw) and radial segment of left ventricular wall, was compared among different groups. whereas conventional echocardiography demonstrated an apparently normal contractile state based on fractional shortening, myocardial strain (at mildventricular segment of ivs) in hcm cats was significantly decreased compared with normal group (p , ). myocardial strain (at basal segment of lvaw) also was significantly decreased in heterozygous cats compared with negative group (p , ); and was significantly decreased in heterozygous cats compared with negative group, both without ventricular hypertrophy (p , ). there was a significant negative correlation between strain values and wall thickness (p o , ). this method allows detection of abnormal systolic deformation in maine coons cats with hcm mutation despite apparently normal systolic function. the abnormal systolic deformation already can be present in heterozygous cats without hypertrophy and increased with progressive ventricular hypertrophy. recently, multiple advanced resting electrocardiographic (ecg) techniques have been applied in humans for detection of cardiac autonomic and repolarisation function. this has improved the diagnostic and/or prognostic value of short-time ecg in detection of common human cardiac diseases even before onset of symptoms or changes in the standard ecg. therefore, this study investigates, if advanced ecg can predict the severity of mitral regurgitation (mr) in dogs with myxomatous mitral valve disease (mmvd) and thereby improve the diagnostic value of ecg. the study included privately owned cavalier king charles spaniels (ckcss) (age . ae . years; males and females). all dogs were examined by echocardiography and a short-time ( - min) high-fidelity -lead ecg, with the dog in a resting position and in sinus rhythm. dogs were divided into groups according to the degree of mr estimated as the percentage of the left atrium area using color doppler mapping ( %; % o jet %; % o jet %; jet %; jet % and with clinical signs of congestive heart failure). ecg recordings were evaluated via custom software programs to calculate different parameters, including heart rate variability (hrv), qt variability (qtv), t-wave complexity, wave morphology and -d ecg. one-way anova determined ecg parameters, which were significantly different (p o . ) between the dog groups. principal component factor analysis identified a factor model with . % explained variability. qrs dipolar voltage and two repolarization indices of qtv increased significantly with mr severity, whereas total power of the frequency spectrum of rr interval and the standard deviation of qtv decreased significantly with mr severity. for the selected parameters the prediction of mr jet value was tested by multiple linear regression. a correlation coefficient (r) of . indicated that the prediction value was significant (p o . ). if age was included in the multiple linear regression the prediction value was further increased (r . ). our results indicate that for a cut-off criteria of mr ! % jet the five selected ecg parameters could predict the severity of mr caused by mmvd in ckcss with sinus rhythm with sensitivity % ( % with age inclusion) and specificity % ( % with age inclusion) (p o . ). nt-probnp concentration is increased in canine patients with heart disease. relatively little is known about the stimuli for release of nt-probnp in dogs. physical activity independent of cardiac disease and the stress of being in the hospital could influence nt-probnp release and affect diagnosis and management of patients. we hypothesized that nt-probnp concentration in healthy dogs would not exceed the normal reference value ( pmol/l) following a period of exercise. the goal of this study was to examine whether physical activity could elevate plasma nt-probnp and cause false positive results in healthy dogs. the study population included healthy dogs yr of age without heart murmur or known systemic disease, and normal d/color flow echocardiographic exam. plasma samples for nt-probnp were obtained before, immediately after, and hour after a standardized -minute submaximal exercise regimen. the study included dogs with a median age of . yrs and included females and males. there was no statistical difference in median plasma nt-probnp concentration across the three time points (baseline median, [iqr, immediately post, ; p . ). the average coefficient of variation of nt-probnp concentration across the exercise regimen was . ae . %. in of dogs ( . %), nt-probnp increased from to pmol/l immediately after exercise. the results of this study demonstrate that submaximal exercise does not significantly change median nt-probnp concentration and the incidence of false positive results is low. further studies should investigate effects of exercise on nt-probnp concentrations in dogs with heart disease. obesity is an increasing problem in veterinary medicine. obese human patients are shown to present lower levels of natriuretic peptides, regardless of an increased volume and pressure load, what raises the possibility that the natriuretic response is impaired in these individuals. considering the controversial findings in obese humans, and the lack of studies reported in dogs, this study proposed the evaluation of nt-proanp concentration in obese dogs. nt-proanp concentration was determined prospectively in obese dogs ( females; males; - months) and in non-obese dogs (controls; females; males; - months) from a veterinary hospital population. obesity was determined by body condition score [ ( / ); ( / ); ( / ); ( / ); ( / ); ( / )]. dogs were excluded if they had any primary cardiac disease, renal insufficiency, endocrine disease, or if they were receiving diuretics, vasodilators, antiepileptic drugs or corticosteroids. commercial kits were used (vetsign s canine cardio screen nt-pro-anp vc -guildhay/biomedica). mann whitney test was used for group comparison. results are presented as median; interval; p and p ). nt-proanp was significantly lower in obese dogs [ . fmol/ml ( . - . ); p . ; p . ] than in controls [ . fmol/ml ( . - . ); p . ; p . ]; (p . ). results were similar to what has been found in obese humans. lower levels of natriuretic peptides are also seen in obese heart failure patients. this study provides important information regarding nt-proanp concentration in obese dogs, which should be better explored characterizing the behavior of natriuretic peptides after weight loss, and also in obese dogs with primary heart disease. left-to-right shunting patent ductus arteriosus (pda) is one of the most common canine congenital cardiovascular defects. human studies have shown that bnp and nt-probnp concentrations are elevated in patients with pda, and can be used to detect hemodynamically significant pda. the purpose of this study was to measure nt-probnp concentrations in dogs with pda, and to assess whether additional indicators of hemodynamics correlate with ntprobnp. we hypothesized that nt-probnp will serve as a simple non-invasive marker of hemodynamic significance in dogs with pda prior to and following transcatheter ductal occlusion. nt-probnp was measured in client-owned dogs with echocardiographically normal hearts. ten dogs with pda were initially evaluated with thoracic radiographs, transthoracic and transesophageal echocardiography, pulmonary capillary wedge pressure (pcwp) and nt-probnp. nt-probnp and echocardiography were repeated at day and months following ductal occlusion. pcwp was repeated at months. baseline nt-probnp was significantly higher in pda dogs compared to control ( ae pmol/l (mean ae sd), ae ; p o . ). at day and months following ductal occlusion, nt-probnp was ae and ae , respectively. the following decreased significantly from baseline: pcwp ( . ae . to . ae . mmhg; p . ), and indexed left ventricular internal dimensions in diastole ( . ae . to . ae . ; p . ) but not significantly in systole ( . ae . to . ae . ; p . ). nt-probnp is elevated in dogs with pda and transductal closure is associated with a reduction in nt-probnp, pcwp and left ventricular size. cardiac biomarkers, particularly nt-probnp, are becoming more commonly used in dogs and cats as part of a diagnostic work up. multiple studies already have documented the correlation of this peptide with cardiac disease status and potential clinical implications. in a portion of these reports the manner in which samples were handled was placement of whole blood into an edta tube, followed by centrifugation and decanting of the supernatant that was ultimately stored at À c or À c prior to shipment, either with or without protease inhibition. our objective was to compare the nt-probnp concentrations in feline plasma collected using the previously reported methods to the california animal hospital (cah) collection method using tubes containing a protease inhibitor. this study compared nt-probnp concentrations using the protease inhibitor tubes vs. edta tubes from privately owned feline patients, with confirmed cardiac disease, and control feline patients. for all study participants, we performed a full history and physical examination, a hematology and chemistry panel, thoracic radiographs, ecg, and echocardiogram. in each study participant, at least ml's of whole blood was drawn from a peripheral vein, and transferred to a plastic edta tube. the sample was centrifuged within hour after collection. ml of plasma was then transferred to a tube containing a protease inhibitor, which was stored at c until being shipped within hours of collection. the remaining plasma was placed into separate microtubes, which did not contain a protease inhibitor. one microtube was then stored and shipped as previous studies have reported (À c, styrofoam container, shipment within hours), and the second microtube was frozen at À c. all samples were shipped, received and analyzed within hours of collection. results of this study showed that no difference was found between the frozen sample methods ( pmol/l and pmol/l p . ). it was determined that both frozen methods had lower nt-probnp levels ( and pmol/l) when compared to plasma samples shipped in protease inhibitor tubes ( and pmol/l). the findings of this trial demonstrate that the nt-probnp levels are significantly different between samples placed in edta tubes vs. contain protease inhibitor (p . and p . ). utilizing protease inhibitor tubes allows more accurate measurement of plasma nt-probnp. as for its relevance for future research and publications, authors should take care to investigate the manner in which blood samples were handled and the conclusion/results of these studies should be taken in light of the methodologies used in collecting, storing, shipping and analyzing the samples. degenerative mitral valve disease (dmvd) is one of the most common heart disease and is present approximately % of the canine heart disease. although the high prevalence exists in small dogs, the underlying molecular mechanism of its pathophysiology is rarely known. dmvd is functionally and pathologically similar in humans and dogs, thus, there will be a common pathogenesis in human and dogs with naturally occurring dmvd. serotonin and serotonin-related mechanisms have been implicated as a cause of valvular disease in human and animals, including spontaneous dmvd in dogs. increased circulating ht concentration as a potential source of heightened ht signaling is demonstrated in small dogs with dmvd. the aim of this study was to investigate whether serum ht concentrations were associated with severity of naturally occurring dmvd in small dogs and to investigate potential associations of dog characteristics on serum ht concentrations in our study population. forty-eight dogs were included in this study and were classified into control and dmvd groups according to the results of physical and echocardiographic examinations. based on the la:ao ratio, dogs with dmvd were classified as follow: control (la:ao ratio . and no mr), mild (la:ao ratio . and mr), moderate ( . o la:ao ratio . and mr), severe (la:ao ratio . and mr). serum serotonin concentrations were measured by elisa. an overall significant difference (p o . ) was found among groups and ht concentrations (control, . ng/ml [ . - . dmvd, ). significantly higher ht concentrations were observed in dogs with moderate (p o . ) and severe (p o . ) dmvd, compared with concentration in control group. additionally, ht concentration in dogs with moderate dmvd were significantly higher (p o . ) than concentration in dogs with mild dmvd. also, dogs with severe dmvd had significantly higher ht concentration than dogs with mild (p o . ) and moderate (p o . ) dmvd. there was no significant association of age, platelet, and lvidd, on serum ht concentration, however, weak correlation between serum ht increased significantly and la:ao ratio (r . , p o . ) was observed. the results of this study indicate that serum ht concentrations were higher with increasing severity of spontaneous dmvd, which may be the potential cause to advance the progression of dmvd. further studies should be performed to reveal the role of ht in inducing and accelerating spontaneous dmvd and to investigate if lowering serum ht concentration could alter the progression of dmvd. the objective of this prospective study was to evaluate the utility of cardiac troponin i (ctni) in differentiating between underlying etiologies of pericardial effusion in the canine patient. patients were prospectively recruited at time of diagnosis of novel pericardial effusion. serum samples were collected prior to pericardiocentesis. patients were evaluated by echocardiography and classified with the diagnosis of hemangiosarcoma (hsa), heart base tumor (hbt), or unknown etiology at the initial evaluation based on established characteristic echocardiographic findings. idiopathic pericardial effusion (ipe) was defined by histopathology, echonegative for a mass lesion with no recurrence of pericardial effusion months, or symptom free months from time of enrollment. patients were excluded from analysis if a diagnosis could not be established based on above criteria or concurrent moderate azotemia (creatinine . mg/dl) was present at time of sample collection. serum samples were frozen and analyzed in batches within days of collection by a ctni assay with a . ng/ml lower limit sensitivity. sixty-three patients were recruited over a one year period with patients excluded due to lack of diagnosis ( ) or azotemia ( ). median ctni levels of dogs with hsa (n ), hbt (n ), and ipe (n ) were . ng/dl (interquartile range (iqr) o . - . ), . ng/dl (iqr o . - . ), and o . ng/dl (iqr o . -o . ) respectively. concentrations of ctni differed significantly between dogs with hsa and hbt (p . ) and ipe (p . ). there was no difference between ctni concentrations between hbt and ipe dogs (p . ). receiver operating curve analysis to determine the optimal cutoff for differentiation of dogs affected with hsa and both hbt and ipe revealed a significant (p o . ) area under the curve ( . ). a cut-off point of ctni of . yielded a sensitivity of % ( % ci, - %) and specificity of % ( % ci, - %). utilizing a higher cut-off point of . yielded a lower sensitivity of % ( % ci, - %), but a higher specificity of % ( % ci, - %) which may have more clinical utility given the disparity in prognoses of the etiologies compared. in conclusion, this study supports the diagnostic utility of ctni concentrations to delineate between patients with hsa and other etiologies of pericardial effusion, but does not reliably differentiate between dogs with ipe and other neoplastic etiologies. the pathogenesis of degenerative mitral valve disease (dmvd) in dogs remains to be fully elucidated. the high sheer stress caused by mitral regurgitation damages the endothelial surface of the valve, and a previous study demonstrated increased transcription of intercellular adhesion molecule- (icam- ) and e-selectin in affected mitral leaflet tissue. we hypothesized that this may be responsible for platelet recruitment and adhesion, and initiation of a proliferative cascade, resulting in further myxomatous changes. the goal of this study was to compare plasma levels of icam- and e-selectin in healthy dogs and those with dmvd. the study population included dogs with echocardiographic evidence of dmvd and healthy control dogs year old with no heart murmur or known systemic diseases. dmvd dogs underwent d/color-flow doppler echocardiographic exam. blood samples were obtained for plasma icam- and e-selectin analysis using commercially available elisa kits. the study included dogs, of which had dmvd and were normal. the dmvd group had a median age of . yrs ) and included females and males. two ( %), ( %), ( %) and ( %) dogs were classified as isachc a, b, and a, respectively. of the control dogs, median age was . yrs [ - . ], with females and males. there was no statistical difference in plasma e-selectin between control dogs (median . [ . - . ]) and those with dmvd ( . [ . - . ]); p . . plasma icam- concentrations were higher in dmvd dogs ( . [ . - . ]) than controls (median . [ . - . ], but this difference did not reach statistical significance (p . ). linear regression analysis showed no significant correlation between icam- or e-selectin and serum serotonin level, nt-probnp or echocardiographic measures of dmvd severity (la:ao, lvidd:ao, lvids:ao). the results of this study demonstrate no significant difference in circulating adhesion molecules icam- and e-selectin in dogs with dmvd as compared with healthy controls. further studies investigating adhesion molecules within the mitral valve tissue itself are likely needed if icam- and e-selectin play a role in the pathophysiology of dmvd. the rate of glucose utilization in the heart is greater than in other tissues, and impaired glucose uptake may play a major role in the pathogenesis of heart failure (hf). glucose uptake across the sarcolemma is regulated by a family of membrane proteins called glucose transporters (gluts), which includes glut- , the major cardiac isoform, and glut- , a recently discovered isoform, the role of which is unknown in the heart. in addition, despite the wellknown regional differences in myocardial structure and function, potential regional patterns in glucose transport have not been investigated. thus, we hypothesized that glut- and - protein and gene expression would be chamber specific in healthy dogs and during chronic hf. using a canine model of tachypacing induced chronic hf, glut protein and messenger rna in both ventricles and atria were investigated by immunoblotting and real time pcr. in control dogs, glut- , but not glut- , protein expression were significantly higher in the atria compared to the ventricles, with the highest content in the right atrium (ra, p o . ). glut- and - mrna were homogeneously expressed in all the cardiac chambers. during chronic hf, glut - and - expression was highest in the left ventricle (lv, by . and . fold, respectively, p o . ), with a concomitant increase in glut- and - mrna (p o . ). glut - , but not glut- , was decreased in ra during chronic hf (p . ). our data suggest that glut- protein was differentially expressed across the cardiac chambers in the healthy heart. during chronic hf, lv was the primary site dependent on both glut and glut -mediated glucose transport, which was transcriptionally regulated. in addition, the paradoxical decrease in glut content in ra may induce perturbations in atrial energy production during chronic hf. some obese dogs are suspected to have cardiac disease because they have enlargement of the heart on thoracic radiograph. it has been reported in cats that the fat increases the cardiac silhouette, while echocardiograms revealed normal cardiac dimensions. the purpose of this study was to determine whether obesity overestimates cardiac dimension in radiographs compared to echocardiographic findings in dogs. twenty three obese dogs and controls were included based on a - body condition scoring (bcs). computerized radiography was obtained and vhs measurement was performed as previously described. echocardiographic measurements were interpreted based on reference values according to lean body weight regression equations. results for echo and vhs measurements were classified in scores as normal, mild, moderate or severe increase. student's t test was used for comparison of vhs between groups. mann-whitney rank sum test was used to assess echocardiographic scores between groups. spearman rank order correlation was used to assess relationships between any pairs of variables between echo and vhs scores, echo vs bcs and vhs vs bcs. groups were similar regarding age [obese ( ae ); control ( ae ); p . ], breeds and gender distribution. obese dogs had significantly higher vhs and echo scores compared with controls [vhs: ( . ae . ) vs ( . ae . ); p o . ; echo score: range ( - ) vs ( - ); p . ]. there were no relationships between any pair of variables analyzed. these results show that there are changes both in echo and radiographic appearance of the heart in obese dogs, but vhs overestimates cardiac silhouette compared to echo, probably related to pericardial fat accumulation. heart rate variability (hrv) is an indirect measurement of the autonomic modulation of heart rate (hr). reduced hrv measured from short-time electrocardiography is seen in dogs with heart failure (hf) secondary to myxomatous mitral valve disease (mmvd). however, hrv is suggested to increase with disease severity at early stages of mmvd. the aims of this study were ) to associate hr and hrv with severity of mmvd in cavalier king charles spaniels (ckcs) and ) to compare hr and hrv between ckcs and other dog breeds in a group of dogs in hf secondary to mmvd. one-hundred dogs were examined by echocardiography and hour electrocardiography. the dogs were divided into five groups: ) ckcs with no/minimal mitral regurgitation (mr) (mr jet % of the left atrial area using color doppler mapping) and no murmur, ) ckcs with mild mr ( % o jet %), ) ckcs with moderate/ severe mr (jet %) and no clinical signs of hf, ) ckcs in hf (hf defined as left atrium to aortic root ratio (la/ao) . , clinical signs of hf and furosemide responsiveness) and ) non-ckcs in hf. dogs in hf were allowed hf therapy. both hr and hrv were analyzed over a -hour period, while hrv were also analysed over a -hour nightly period. analyses of variance were performed with hr or hrv as response variables and the explanatory variables dog group and echocardiographic indices of mmvd were included separately. all p-values were bonferroni corrected. minimum-and mean hr were significantly higher in ckcs with moderate/severe mr and in hf compared to ckcs with no/ minimal and mild mr (all p o . ). seven out of hrv variables were significantly decreased in ckcs with moderate/ severe mr and in hf compared to ckcs with no/minimal and mild mr (all p o . ). another hrv variables showed the same groupwise differences (all p o . ), except that the difference between ckcs with mild mr and ckcs with moderate/severe mr did not reach statistical significance. mminimum hr, mean hr and the hrv variables ( and ) differing between dog groups, also consistently decreased with increasing mr, la/ao and the proximal isovelocity surface area in ckcs. non-ckcs in hf had a lower minimum hr compared to ckcs in hf (p . ) and a higher triangular index measured in both periods (all p o . ). in conclusion, hr increased and most hrv variables decreased with increasing severity of mmvd in ckcs, even prior to the development of hf. other breeds in hf secondary to mmvd had lower minimum hr, but higher triangular index compared to ckcs in hf. although the cells in the specialized conduction system in the heart are capable of initiating their own impulse, the rate in which those impulses are generated can be influenced by autonomic nervous system. different types of respiratory patterns can stimulate autonomic nervous system in different manners. thus, non-sedated rabbits were studied during forced respiration aiming to evaluate the influence of this breathing pattern on heart rate. twenty male, one-year-old healthy new zealand rabbits were enrolled in the study. animals were set in right lateral recumbency and maintained that way by physical contention. chemical sedation was not used. partial nasal obstruction by digital compression was applied to those rabbits for five seconds, eliciting a forced inhaling and exhaling against semi closed nostrils. heart rate was obtained by measurement of two consecutives rr intervals in the computerized electrocardiography, recorded continuously prior and during the maneuver. heart rate before the intervention was ae bpm (mean ae standard deviation). all rabbits submitted to the maneuver showed a dramatic reduction in this parameter. after nasal partial obstruction, heart rate was ae bpm. data was submitted to statistical analysis by paired student's t test and a significant difference between the heart rate before and after the maneuver was observed (p o . ). although the exactly mechanism involved in this response was not elucidated, the presented data support the applicability of this maneuver as an efficient method for non-pharmacological heart rate reduction in rabbits. obesity can affect cardiac function due to effects on cardiac rhythm, ventricular volume and blood pressure. the purpose of this study was to determine the effects of obesity and overweight on noninvasive systemic blood pressure and doppler echocardiographic parameters in cats without others causes of cardiac hypertrophy. the study groups comprised fifteen obese cats with mean body score index (bsi) of , , seven overweight cats (bsi , ) and seven cats with ideal bsi ( , ). the blood pressure was measured by doppler method and the doppler echocardiography was performed in conscious animals. the statistical analysis was performed by analysis of variance followed by tukey's test and pearson's correlation. the blood pressure values of the obese cats were superior ( , ae , mmhg, p o , ) than in overweight ( , ae , mmhg) and normal cats ( , ae , mmhg) and % of the obese cats had blood pressure higher than mmhg. there were observed differences on the ratio of early (e) and late (a) left ventricular filling velocity (p , ) of obese animals (e/a , ae , ) compared to overweight ( , ae , ) and normal cats ( , ae , ). seven obese cats ( %) had inversion of e/a compatible with diastolic dysfunction and there were negative correlation (r À , , p , ) between the e/a ratio and blood pressure values. other differences observed were increases in left ventricular septum in diastole (p , ) and in free wall in diastole (p , ) and systole (p , ) of the obese animals compared to overweight and normal cats. these results demonstrate the possibility of cardiovascular effects related to obesity in cats, such as systemic arterial hypertension and secondary diastolic dysfunction. diuretic therapy reduces preload, and relieves congestion secondary to cardiac dysfunction. torsemide (torasemide) is a loop diuretic with longer duration of action, less diuretic resistance, and adjunctive aldosterone antagonism as compared to furosemide. we hypothesized that torsemide was no less effective than furosemide at diuresis, control of clinical signs, and maintenance of quality of life in dogs with congestive heart failure. a double-blinded, randomized, crossover clinical trial was performed in dogs with stable heart failure receiving bid furosemide and adjunctive medications. dogs were randomized to their current furosemide dose or torsemide (calculated as / of the daily furosemide dose divided into bid dosing). crossover occurred at day and the study ended on day . clinical, laboratory, radiographic, and owner-perceived quality of life variables were evaluated on days , and . no dog developed recurrent heart failure during the study. average furosemide dose on day was . mg/kg/day (range, . - . ). following torsemide treatment, blood urea nitrogen (p . ), albumin (p . ), and albumin:globulin ratio (p . ) were significantly increased, and urine specific gravity (p . ) and chloride (p . ) were significantly decreased as compared to baseline and/or furosemide dosing (one-way anova with bonferroni correction). no differences in qol were found. results indicate that torsemide is equivalent to furosemide at controlling clinical heart failure in dogs, and might in fact, achieve greater diuresis vs. furosemide. larger clinical trials evaluating furosemide resistance and/or torsemide as a first-line loop diuretic for congestive heart failure in dogs with heart failure are warranted. the purpose of this study was to investigate the feasibility of speckle tracking echocardiography (ste) in healthy cats and to determine whether or not it can detect myocardial dysfunction in cats with diseased heart. radial and circumferential strain and strain rate were measured by ste using left ventricle short-axis view in clinically healthy cats. eighteen cats with hcm whose lv thickness at end-diastole with mm or more were evaluated with ste analysis, and compared with healthy cats. index of left ventricular synchrony (trs-sd) was also assessed in cats with hcm, and compared to healthy subjects. ste resulted in technically adequate images in % of the cats. fusions of early and late diastolic (e and a) wave in strain rate were seen in of cats. percent errors in analysis with or without simultaneous ecg monitoring were . - . % in all parameters. inter-and intraobserver variability of ste parameters in healthy cats was minimal ( . - . %) except for the systolic circumferential strain rate. sedation using buprenorphine and acepromazine did not affect any ste parameter. e wave in radial and circumferential strain rate of hcm cats was significantly decreased compared with healthy cats. no significant difference was seen in trs-sd. ste analysis was considered clinically feasible to assess cardiac function in cats, and could detect myocardial dysfunction in cats with hcm. further study is warranted to investigate to assess whether or not ste can differentiate the etiology of left ventricular concentric hypertrophy since it is clinically important. carvedilol, a rd generation non-selective beta-blocker with ancillary alpha -blocking and antioxidant properties may have therapeutic implications for multiple diseases in cats; however, pharmacokinetics and bioavailability of commercially prepared oral carvedilol has not been determined. hplc for carvedilol measurement in feline plasma was validated and standardization curves created. the pharmacokinetics (pk) of carvedilol was evaluated in apparently healthy male neutered adult cats (average weight of kg) following single dose intravenous (iv) of . mg/kg and single dose oral administration of . to . mg/kg. concentrations of the active parent compound, carvedilol, were detected in plasma using hplc analysis. lower limit of quantification was ng/ml. the mean peak concentration after iv administration of carvedilol was ng/ml (range, to ), elimination half-life was . hours (range, . to . ), and clearance was . l/hr/kg. the volume of distribution was . l/hr. after a single oral administration of carvedilol, the time to peak plasma concentration was minutes (range, to minutes) and the mean residual time was . hours. the half life was . hours. maximal concentration ng/ ml and the mean bioavailability was . % with a median of . % (range, . % to %). these data demonstrate a low bioavailability of oral carvedilol and a wide variation in cats. all cats tolerated the oral dose of carvedilol with no major adverse effects. also, a mean residual time of . hours would suggest that a more frequent dosing schedule may be required to maintain therapeutic plasma levels. pharmacodynamic studies investigating beta-adrenergic blockade duration may provide a more accurate dosing interval of carvedilol. abstract c- effects of sildenafil citrate on dogs with ei-senmenger's syndrome. k nakamura, m yamasaki, h ohta, m takiguchi. department of veterinary clinical sciences, graduate school of veterinary medicine, hokkaido university, sapporo, hokkaido, japan. sildenafil has shown to be effective for dogs with pulmonary hypertension; however, its efficacy for dogs with eisenmenger's syndrome (es) and secondary erythrocytosis has not yet been determined. the objective of this study is to determine the effect of sildenafil for dogs with eisenmeger's syndrome and secondary erythrocytosis. this was a prospective, single arm, open-label study. five clinical dogs with es and secondary erythrocytosis were included. new york heart association (nyha) functional class, pcv, and pulmonary artery acceleration time to ejection time ratio (pa at : et) were evaluated before and after sildenafil therapy ( . mg/kg, bid). nyha functional class was significantly improved after (median ; range - , p . ) and months (median ; range - , p . ) of sildenafil therapy, compared with the baseline (median , range - ). pcv was significantly decreased after month ( . ae . %, p . ) and months ( . ae . %, p . ) of therapy, compared with the baseline ( . ae . %). at : et was significantly increased after month of therapy ( . ae . , p . ) from the baseline ( . ae . ). sildenafil resolved the clinical signs and secondary erythrocytosis in dogs with es. sildenafil therapy could be the treatment of choice for dogs with es. sepsis is the number one cause of mortality in neonatal foals. the role of the raas and hpaa in systemic inflammation and response to stress is well documented in critically ill human neonates, but limited information exists in foals. we hypothesized that in septic foals the raas and hpaa will be activated by systemic inflammation and hypoperfusion and the degree of activation will be associated with severity of sepsis and mortality. blood samples were collected on admission from septic (sepsis score ), sick non-septic (sns), and healthy foals of o days of age. blood concentrations of corticotropin-releasing hormone (crh), adrenocorticotropin (acth), cortisol, aldosterone, angiotensin-ii (ang-ii), arginine vasopressin (avp) and plasma renin activity were determined by radioimmunoassays. acth, cortisol, aldosterone, ang-ii and avp concentrations were higher while crh was lower in septic and sns foals compared to healthy foals (p o . ). septic non-survivor foals had higher concentrations of aldosterone, cortisol, acth and avp and lower concentrations of ang-ii and crh than survivors. avp was associated with ang-ii in septic, and with acth in septic and sns foals (p o . ). there was no difference in renin activity and ang-ii concentrations among foal groups. septic foals had a higher acth:aldosterone ratio than healthy foals (p o . ). this study shows that in response to sepsis there is raas and hpaa activation in critically ill foals. we propose that in sick foals avp is more important than crh in regulating acth secretion. the increased acth:aldosterone ratio further supports relative adrenal insufficiency in septic foals. this prospective, cohort study aimed to characterize alterations in coagulation and blood-derived inflammatory biomarkers in adult horses that developed diarrhea during hospitalization. physical and hematological parameters were evaluated at times (onset of diarrhea), , , and h, then every h until resolution of diarrhea or death. each hematological analysis included a complete blood count (cbc), thromboelastography (teg), partial-thromboplastin-time (ptt), prothrombin-time (pt), plasma concentrations of lactate, tumor necrosis factor alpha (tnf-a), interleukin (il)- , il- , il- and nt-proc-type-natriuretic peptide (pcnp). horses were categorized into three groups based on the duration of diarrhea and evidence of systemic inflammation. group : diarrhea o h without systemic inflammation (si); group -diarrhea ! h without si; group -diarrhea with si. assessment of vital parameters and cbc established a diagnosis of si as previously described (levy, ) . descriptive and univariate outcome analyses were based on data normality. horses were enrolled, of which ( . %) survived to discharge. the mean age was . /- . years. eight horses ( . %) were categorized as group- , ( . %) as group- and ( . %) as group- . two horses developed thrombophlebitis. based on the results of teg, / ( . %) were normocoagulable, / ( . %) were hypocoagulable and / ( . %) were hypercoagulable, at one or more time points. of these, / ( . %) group- horses were coagulopathic. additionally, group- horses had a significantly lower ma than group- horses at baseline ( . ae . vs. . ae . ) and h ( . ae . vs. . ae . ). biomarker analyses are pending. in conclusion, si was associated with coagulation disorders in horses with hospital acquired diarrhea. clostridium difficile and clostridium perfringens are commonly associated with colitis and diarrhea in equines but asymptomatic carriers exist. reported carrier rates of toxigenic c. difficile and c. perfringens strains in feces range between - % and - % respectively. toxigenic c. difficile has also been isolated from the small intestine of diseased foals and is implicated as etiologic agent of duodenitis/proximal jejunitis in adult horses however scarce information is available on prevalence in gastrointestinal compartments other than feces in healthy horses, and it is unclear whether fecal samples are good predictors of the status of proximal intestinal sites. the objectives of this study were to investigate the presence of c. difficile and c. perfringens in various intestinal compartments of healthy adult horses and to molecularly characterize isolates. intestinal contents were collected from the stomach, duodenum jejunum, ileum, cecum, right dorsal and left ventral colon, small colon and rectum of euthanized horses free of apparent gastrointestinal disease. enrichment culture was performed for c. difficile and c. perfringens and c. difficile isolates were further characterized via toxin gene detection and ribotyping. c. difficile was isolated from / ( %) samples from / ( %) horses. between zero and three sites were positive per horse, and multiple sites were positive in three horses. isolates were recovered from duodenum (n ), right dorsal colon (n ), small colon (n ) and rectum (n ). in one horse, the rectal sample was negative but c. difficile was isolated from a proximal site, all other horses were positive on the rectal sample if a more proximal compartment was positive. in three horses multiple compartments were positive however different strains were always present within the same horse (n ). all isolates possessed genes encoding toxins a and b. five isolates were ribotype and also possessed genes encoding the binary toxin. the other isolates were ribotype and were negative for the genes encoding the binary toxin. despite using a method with a detection level as low as cfu/g of feces, no c. perfringens was recovered. rectal samples were a good predictor of overall c. difficile carrier status ( / horses), however rectal samples were not always representative for the ribotype carried in more proximal compartments. the presence of variable strains within the same horse suggests transient passage of the bacterium through the gastrointestinal system rather than actual colonization although further study testing multiple colonies per site is needed. the predominance of ribotype is consistent with recent emergence of this strain in this region, as earlier studies found other strains ( , ) to be more prevalent and a variety of ribotypes were typically recovered from horses. interestingly ribotype has recently emerged as a hypervirulent strain in humans in our area. clostridium difficile, clostridium perfringens and salmonella are important enteric pathogens in horses, however some healthy animals also harbour these pathogens. point prevalence studies have reported these carriage rates, but there are no data regarding longitudinal prevalence of these enteric bacteria, information that would be useful to better understand the epidemiology of these pathogens. additionally, antimicrobial resistance is a pressing concern. commensal e.coli is often used as an indicator organism to evaluate antimicrobial resistance of enteric bacteria, yet there are limited data from horses on farms. the objectives of this study were to longitudinally investigate the above enteric pathogens over the course of one year, molecularly characterize obtained isolates and determine the antibiotic susceptibility profile for e. coli. fecal samples were collected from adult horses from five farms on a monthly basis over the course of one year. selective cultures were performed for c. difficile, c. perfringens, salmonella and e. coli. c. difficile isolates were characterized via toxin gene pcr and ribotyping. broth microdilution was performed to assess antimicrobial susceptibility profiles of e. coli. clostridium difficile was isolated from / ( . %) samples from / ( %) horses. four horses were positive on more than one occasion, three were positive in two consecutive months. different ribotypes were found in two of the latter horses. most isolates were ribotype (n ) with ribotype (n ) and ribotype c (n ) also identified. ribotypes and c possessed genes encoding toxins a, b and binary toxin, while ribotype only possessed toxin a and b genes. despite a detection threshold of cfu/g feces, c. perfringens was not detected in any samples, nor was salmonella. e coli was isolated from / ( %) samples. resistance to ! antimicrobial was present in only / ( . %) isolates. multidrug resistance (! antibiotics) was present in / ( %). most commonly, isolates were resistant to sulfisoxazole ( / ) and trimethoprim sulfamethoxazole ( / ). the overall detection rate for toxigenic c. difficile in fecal samples of healthy horses was . % which is consistent with previous studies. the cumulative prevalence of % was striking but only one horse shed the same strain for more than one month, indicating c. difficile shedding is a transient and dynamic state. the predominant isolation of ribotype is consistent with the suspicion that this strain has emerged and become widely disseminated in this region in recent years. the low prevalence of c. perfringens and salmonella is in agreement with some other studies. the low prevalence of antibiotic resistance in commensal e. coli was encouraging and suggests that healthy horses on pleasure horse farms are not likely a major reservoir of resistance in enteric bacteria. type polysaccharide storage myopathy (pssm) in horses is associated with a dominant missense mutation (r h) in the skeletal muscle glycogen synthase gene (gys ). since disease severity varies between affected horses, we hypothesised that some clinical variability could be accounted for by the underlying genotype. belgian / percheron horses were genotyped using a validated restriction fragment length polymorphism assay enabling grouping of horses as homozygotes (hh), heterozygotes(hr) or normal (rr). subsequently, semimembranosis muscle samples were biopsied from each of six matched sedentary horses from each group; one sample was formalin-fixed and one fresh frozen. sections were stained using haematoxylin and eosin, periodic acid schiff /diastase. anti-dystrophin, nnos and myosin heavy chain immunohistochemistry was performed to examine sarcolemmal intregrity, there were significant differences in resting ck activity (p . ) (median hh u/l interquartile range(ir) - ; hr u/l ir - ; rr u/l ir - ) and ast activity (p o . ) (ast mean hh u/l sd ; hr u/l sd ; rr u/l sd ) and muscle pathology between the groups, with severity increasing rr o hr o hh. there were significantly more type a (p . ) and fewer type x fibres (p . ) in homozygotes ( a % sd . ; x % sd ) compared with the other groups (hr a % sd . , x % sd . ; rr: a . % sd . x % sd . ). more type a fibres contained polysaccharide inclusions in homozygotes ( % sd . ) than in heterozygotes ( . % sd . ) (p o . ). both dystrophin and nnos expression was normally localised to the sarcolemma in pathologically normal and vacuolated fibres from mutant horses. in conclusion, sedentary homozygotes have more severe skeletal muscle pathology and higher resting plasma ck and ast activities than heterozygotes, and pssm is associated with a fibre type shift towards type a. although subsarcolemmal vacuolation likely disrupts the contractile apparatus's attachment to the sarcolemma, the latter's integrity appeared intact. the recumbent horse presents a logistic, diagnostic, and therapeutic challenge to the equine practitioner. there is currently very little data available on the prognosis and outcome of horses that are recumbent. therefore, the purpose of this study was to investigate the outcome of hospitalized horses that had been recumbent in the field or in the hospital and the factors affecting their survival. records of horses admitted to the school of veterinary medicine, university of california davis from january of to december of with a history of recumbency or horses that became recumbent while hospitalized were evaluated. a horse was defined as recumbent if it was unable to stand on its own. the medical record was examined for the following criteria: history pertaining to the current illness including treatment by the rdvm, breed, age, weight, date of presentation, physical and neurological examination findings, cbc and biochemical profile results, initial drugs administered on arrival, time spent recumbent, time spent in a sling, diagnosis, and hospitalization costs. statistical analysis correlating factors associated with survival was performed using logistic regression. overall there were non survivors and survivors. factors that favored survival included early initiation of treatment in the field by the rdvm, horses that tolerated a sling and spent more time in a sling, increased duration and costs of hospitalization, horses that were recumbent post anesthesia, and those recumbent due to disease of the musculoskeletal system. factors that increased likelihood of non survival included horses that were ataxic on presentation, horses with increased bun, horses that spent more time recumbent, those that did not tolerate a sling, and horses diagnosed with botulism and spinal cord disease. in conclusion, this retrospective study demonstrated that both the cause of recumbency and the ability of horses to tolerate a sling had a direct effect on survival. abstract e- plasma peak and trough gentamicin concentra-tions in hospitalized horses receiving once daily gentamicin. jr read , pa wilkins , rd nolen-walston . university of pennsylvania, new bolton center, kennett square, pa. university of illinois, champaign-urbana, il. gentamicin is often used to provide gram negative antimicrobial coverage in horses at . mg/kg iv every hours. therapeutic drug monitoring in our hospital suggests larger doses are required in many clinical cases to achieve the desired concentration ( -  minimum inhibitory concentration) for common bacterial isolates (peak target range - mg/ml). the aim of this study was to determine the correlation between gentamicin dose and plasma concentration in hospitalized horses receiving gentamicin treatment in order to identify an optimum dose range for this population. review of records ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) identified horses ! months old receiving once-daily gentamicin with peak and trough assays performed (n sets). spearman rank correlation coefficient analysis revealed a weak (r . ) but statistically significant correlation (p . ) between gentamicin dose and peak plasma concentration. horses receiving . - . and . mg/kg gentamicin (groups and ) had higher median peaks ( mg/ml) than horses receiving . - . mg/kg (group ; . mg/ml). higher doses were more likely to result in peaks mg/ml ( and %, groups and respectively) than horses receiving . - . mg/kg (group ; %). all hour post-gentamicin administration trough values were o mg/ml. no correlation was found between dose and change in plasma creatinine during treatment, nor dose and trough level. these data suggest that gentamicin dosage in horses should be individually determined by therapeutic monitoring. additionally, these data support an initial dose of . - . mg/kg iv every hours in order to achieve desired peak concentration and an appropriately low trough concentration. heaves is a common respiratory inflammatory disease, characterized by a pulmonary neutrophilia. this disease is also characterized by an activation of circulating neutrophils after antigen challenge but their specific role in heaves is not well understood. also, there are anecdotal studies concerning heaves-affected horse to be more susceptible to infection. however, to our knowledge, the antibacterial host defense role mediated by circulating neutrophils was not investigated in heaves-affected horses. the objective of this study was to compare phagocytosis activity and bacterial killing by circulating blood neutrophils of heaves-affected and control horses. peripheral neutrophils were isolated from heaves-affected (n ) and control (n ) horses using a density gradient technique. the killing capacity was assessed by incubating neutrophils with streptococcus equi spp equi and spp zooepidemicus. after h of bacterianeutrophil coculture, total viable bacterial cells were measured by quantitative plating. the phagocytosis was evaluated by flow cytometry using fluorescent beads and gfp-transformed streptococcus suis suilysin-negative mutant strain. circulating neutrophils from heaves-affected horses showed a significant decrease in their killing capacity toward s. zooepidemicus (p . ). a reduced, although not significant (p . ), killing capacity of s. equi by these neutrophils was also observed. the phagocytosis activity was not different between groups. this impairment of blood neutrophil bactericidal activity in heaves-affected horses could contribute to an increase susceptibility to infection. obesity is a common disorder of the horse, with current prevalence estimated at %. in people, obesity is associated with dyslipidemia, insulin resistance, mitochondrial dysfunction and downregulation of lipid and glucose metabolic pathways. in the horse, obesity is similarly associated with insulin resistance and alterations in lipid profiles; however, metabolic regulatory gene expression profiles have not been fully characterized. we hypothesized that obese horses have decreased expression of metabolic regulatory genes and decreased mitochondrial content in skeletal muscle compared with non-obese horses. sixteen light breed horses, - years of age were included. body condition score (n ) and neck circumference (n ) were recorded. post-mortem biopsy samples of the semi-membranosus muscle were obtained. dna and rna were isolated. relative expression of the metabolic genes, peroxisome proliferator activated receptor g (pparg), pparg coactivator- a (pgc- a), fatty acid translocase (fat) and estrogen related receptor a (erra) was determined by quantitative polymerase chain reaction (qpcr). mitochondrial content was assessed by determining mitochondrial dna/nuclear dna ratio by qpcr, using nadh-dehydrogenase subunit and cytochrome oxidase subunit as mitochondrial genes and beta actin as the nuclear reference gene. non-normal data was log transformed for analysis and a pearson coefficient of correlation was calculated for relative gene expression, body condition score and neck circumference. a value of p o . was considered significant. body condition score was strongly correlated with neck circumference (n , r . , p . ). relative expression of erra and glut- increased with body condition score (erra: n , r . , p . ; glut- : n , r . , p . ). copy number of the mitochondrial genes (nadh-dh and cox- ) was not related to body condition score or metabolic gene expression. expression of glut- , erra, pparg, and fat were strongly correlated to each other, but not pgc- a. there was a strong trend towards correlation between pparg and pgc- a in horses with body condition score (n , r . , p . ). in this study, there was no change in mitochondrial content in obese horses. assessment of mitochondrial function in obese horses and horses with ems is under way. the strong correlation between pparg and pgc- a observed only in horses with high body condition scores suggests this pathway is activated with obesity. the role of pparg and pgc- a in equine obesity should be further investigated to determine their potential as therapeutic targets. upregulation of erra and glut- in horses with increasing body condition score is unexpected, and may indicate a compensatory response to dysfunction of a downstream pathway. further studies to better define the role of metabolic regulatory gene expression in obese horses and those with ems are ongoing. previously presented at the th annual harold hamm diabetes center research retreat, oklahoma city, ok. inflammatory bowel disease is a cause of weight loss, decreased performance, and colic in horses. this condition is difficult to diagnose and clinicians rely upon absorption tests to document malabsorption. the purpose of this study was to compare glucose and xylose absorption tests in normal horses and determine the repeatability of these procedures. eight horses received mg/kg dextrose or d-xylose powder mixed as a % solution in water or water alone via nasogastric intubation on three different occasions within the same week for three consecutive weeks ( tests/horse). a crossover design was employed and the order of treatments was randomized. blood samples were collected at time , , , , , , and min. data were analyzed by repeated measures anova and t-tests. results showed that the xylose response over time differed significantly from the glucose response over time (test  time; p o . ). mean time to maximum concentration differed (p o . ) between tests (glucose min; xylose min). within-horse area under the curve, maximum concentration, and time to maximum concentration values for dextrose and xylose did not differ significantly when tests were repeated. results indicate that glucose and xylose absorption tests are repeatable within the same horse, but plotted curves differ between tests, with peak concentrations occurring at a later time point for the glucose absorption test. we conclude that both tests provide repeatable measures of intestinal absorption, but glucose and xylose appear to differ in their rates of absorption and clearance. the purpose of this study was to examine the records of a population of thoroughbreds with cervical vertebral malformation (cvm) and to determine which factors have an effect on these horses achieving athletic function. this was a retrospective case study of thoroughbreds with cvm treated medically from to . forty-one were euthanized after diagnosis, while the remaining were discharged for treatment. racing records were reviewed to determine which horses raced after treatment. horses were separated into groups based on whether or not they raced. medical records were reviewed, and results of neurologic examination, radiographic and laboratory findings, treatments, and outcome were assessed and compared between groups. twenty-one of horses treated medically ( %) improved enough to race. median neurologic grade between groups was significantly different (p o . ), with a hind limb grade of . (range - ) for the raced group and . (range . - ) for the unraced. intravertebral sagittal ratios measured from standing lateral cervical radiographs were equivocal between groups. radiographs of all horses were examined for kyphosis, dorsal over-riding arch, caudal epiphysitis, degenerative joint disease, cystic bone lesions, and cranial stenosis of the vertebral canal. horses with kyphosis (p . ), degenerative joint disease (p . ), or cranial stenosis (p . ) at any site were less likely to return to racing. racing prognosis for horses with cvm treated conservatively is equivalent to that of those treated surgically as reported by rush moore et al (javma, ) . radiographic changes and neurologic grade may help serve as indicators for whether a horse will respond to conservative therapy. since pain assessment is vital for management of colic, a valid, reliable and feasible tool for assessing the severity of acute abdominal pain in horses is urgently needed. our aim was to construct and validate a behavior-based pain scale by methodology utilized in construction of pain scales in non-verbal humans. the project consisted of four stages. firstly, behaviors to include in a scale were empirically identified. thirty equine clinicians noted behaviors in each of random film clips of horses with colic using a checklist. nine behaviors (e.g. rolling, pawing, and flank watching) demonstrated good inter-observer agreement without bias (multi-rater kappas: . - . ). secondly, the clinical judgment of experts was utilized to identify and to weight behaviors. six expert clinicians independently expressed opinions as to which of behaviors to include and the severity of pain they indicate. two contending scales (equine acute abdominal pain scales (eaaps) & ) were constructed based on both the empirical and the judgmental approaches. each included identical behaviors with a - point score range; eaaps- with gradations to some of the behaviors and eaaps- without. in the third stage, blood cortisol and lactate levels and heart rate were shown to only approximate pain since they correlate poorly with degree of pain as assessed by visual analog scale (vas) in horses with colic and controls (spearman rho; lactate . ; cortisol . ; heart rate . ). finally, reliability and validity of the pain scales were evaluated including constructs of pain; face validity, convergent and discriminate validity and extreme groups. thirty of films of horses with colic were randomly presented to expert equine clinicians internationally who were randomly allocated into three groups to score pain; one group by both vas and numerical rating scale (nrs)), and two groups, each by one of the two eaaps scales. inter-observer reliability of both eaaps scales was excellent (intraclass correlation . ). intra-observer reliability based on scores given for identical films demonstrated; % and % agreement, kappa . and . , and spearman's rho . and . for eaaps- and , respectively. both scales varied by score between observations. face validity; each group reported their scale to be valid ( % & %). convergent validity; the scales compared favorably with vas/nrs scores (spearman's rho: . - . ). discriminate validity; correlation to heart rate, lactate and cortisol levels was predictably low (rho . - . ). extreme group validity; colic horses scored significantly higher than control horses; scores of . - . in controls versus . - . in cases. in conclusion, methodology established in human medicine but novel in veterinary medicine was used to construct and validate two clinically feasible equine abdominal pain severity scales that showed excellent reliability and validity. further refinement of the eaaps scale is advised prior to introduction into clinical practice. aortic valve prolapse (avp) is a common echocardiographic finding in horses, but when compared with mitral valve prolapse in dogs, little is known about the natural progression of this condition. previously published data has shown that echocardiographic identification of avp in horses is reliable, diagnostic criteria have been established and that development occurs with training. the aims of this study were to evaluate the different rna and protein expressions of smooth muscle actin (sma), transforming growth factor-b (tgf), nitric oxide synthase (nos) and the concentrations of elastin and collagen in normal, prolapsing and diseased cusps to evaluate what structural changes may predispose them to prolapse. valve cusps were harvested and processed from a group of horses at a commercial abattoir following disease classification using echocardiography. horses were aged . ae . years, weighing ae kg and with a median body condition score of / . cusps were collected in rnalater s and stored at À c prior to processing. cdna was produced from half a valve using a standard protocoland qrt-pcr performed to assess relative rna expression of sma, tgfb , endothelial (enos) and inducible nos (inos) and compared with the housekeeping gene s. a quarter of cusp was processed using an adapted commercial protocol to evaluate protein expression of sma, tgf b , enos and compared to vimentin. specific antibody binding was assessed with western blotting and protein expression evaluated using dot blots. the remaining quarter cusp was used to measure soluble collagen and elastin concentrations using commercial assays . statistical analyses included one way anova with post-hoc bonferoni, paired student's t-test, linear and logistic regression. there was no effect of gender or age on any of the measurements. valves from animals with avp had lower expression of sma and elastin compared to normal and diseased valves, increased expression of tgfb and enos, whereas inos expression was greater than normal valves (table ) . collagen content of valves from horses with avp was increased compared to normal but lower than horses with valve disease. prolapsing cusps appear to be a different phenotype from diseased cusps. further studies will help to elucidate the significance of these findings in vivo. a clear association between heart rate (hr) and body mass has been observed across a wide range of mammalian species. furthermore, it is well known that electrocardiographic (ecg) time intervals vary with heart rate and body mass. within the equine species, small breeds are generally thought to have higher heart rates than large breeds. however, despite the large differences in size among different equine breeds, there is little information about normal heart rates and normal ecg time intervals in horses and ponies of different body size. similarly, the relationship between hr and body mass in dogs of various breeds and sizes is still under debate. the goal of this study was to investigate the relationship between heart rate and ecg time intervals to body mass in apparently healthy horses and ponies and to calculate normal ranges for different weight groups. adult horses and ponies at an age of . ( - ) y [median (range)] and a body weight of ( - ) kg were included in the study. all animals were considered clinically healthy based on history and physical examination. a standard base-apex ecg was recorded at a speed of (n ) or mm/s (n ) using a multiparameter monitor (datascope passport). during the procedure, the horses were unsedated, standing quiet in a box stall. mean hr over sec was determined for each recording. the following ecg time intervals were measured in triplicate and averaged for further analyses: pq interval, qrs duration, qt interval, and difference between qt and qrs (qt-qrs duration). the relationship between hr, ecg time intervals, and body mass was assessed using linear regression analyses. normal ranges ( . % to . % percentile) were calculated for different weight groups. the level of significance was p . . heart rate was inversely related to body mass (p o . , r . ). the pq interval (p o . , r . ), qrs duration (p o . , r . ), qt interval (p o . , r . ), and qt-qrs duration (p o . , r . ) were directly related to body mass. normal ranges for hr, pq, qrs, and qt within the different weight groups were - bpm, - ms, - ms, - ms (o kg); - bpm, - ms, - ms, - ms ( - kg); - bpm, - ms, - ms, - ms ( - kg); - bpm, - ms, - ms, - ms ( - kg); and - bpm, - ms, - ms, - ms ( kg). we conclude that in healthy horses there is a significant but weak relationship between body mass and hr and between body mass and ecg time intervals, respectively. this study therefore supports the hypothesis that within the equine species, small breeds have faster heart rates and shorter ecg time intervals than large breeds. therefore, body mass has to be considered when comparing hr and ecg time intervals to normal ranges in horses. horses with pituitary pars intermedia dysfunction (ppid) often have elevated plasma acth concentrations. however, ppidaffected horses rarely have resting serum cortisol levels above the reference range or adrenal hyperplasia. we hypothesized that this apparent dissociation between plasma acth levels and adrenal response in horses with ppid is due to the secretion of acth that is less biologically active than that from normal horses. to test our hypothesis, a bioassay to evaluate acth activity was developed. adrenocortical explants were harvested aseptically from normal horses at euthanasia and stimulated with plasma from healthy (n ) and ppid-affected horses (n ). the assay was performed three times with explants obtained from different horses. cortisol secreted by the explants and plasma acth levels were measured with commercially available radioimmunoassays. cortisol secretion stimulated by each sample was standardized to the respective explant protein concentration. cortisol data was normalized for acth concentration in each plasma sample and expressed as a cortisol:protein:acth ratio. ratios from horses with ppid and normal horses were compared by unpaired t-test. horses with ppid had significantly lower cortisol:protein:acth ratios compared to normal horses. (assay : . ae . vs. . ae . , p o . ; assay : . ae . vs. . ae . , p o . ; assay : . ae . vs. . ae . , p o . ). these results suggest that plasma acth from ppid horses is less biologically active than plasma acth from normal horses. our findings give further insight into the pathophysiology of ppid and may aid in the development of novel diagnostic testing protocols. an online survey was conducted to determine perceived needs of potential employers of new acvim-laim diplomates. the survey was designed as the first step in determining what is needed for success in the various sectors of practice employing acvim-laim diplomates. demographic and background data were collected using questions and drop-down menus on the first page. the survey evaluated skills or concepts in areas of veterinary practice. participants answered questions about each skill or concept using drop-down ranked lists. those participants that had completed an acvim-laim training program were asked additional questions about whether they were taught the skill or concept during their own residency. data were collated and descriptive statistics calculated. the mean scores or frequencies of use for each skills or concepts were ranked to determine which of the skills or concepts were most important for an entry-level diplomate. eighty-eight individuals participated in the survey with respondents being acvim diplomates, respondent was not board-certified and respondent was an act diplomate. nineteen respondents were diplomates of acvim and an additional specialty. eighty-three respondents had completed an acvim residency. the majority of respondents were in academia ( %) with % being in private practice. equine specialists prevailed ( %) followed by mixed large animal ( %) and then food animal only specialists ( %). the distribution of years post-residency was slightly skewed toward younger diplomates, but overall there was a good distribution of diplomates across years of experience. most respondents stated that they did not make hiring decisions in their practice. competency in disciplines other than internal medicine was expected with ultrasonography and radiology being the most desirable followed by theriogenology and lameness. surgical skills, both equine abdominal ( %) and food animal general surgery ( %) were considered important by some respondents. thirty-six per cent of respondents thought that a new diplomate should expect to make o $ , per annum, while only % of respondents thought that a new diplomate should expect to make ! $ , per annum. not all respondents answered questions on all skills or concepts. the mean number of skills or concepts evaluated was (sd ) with only respondents answering all . all skills or concepts evaluated were found to be at least somewhat important, were estimated to be used at least occasionally, were recommended for inclusion in training programs as core or elective, and some level of knowledge was expected. at least some of the respondents were taught each of the skills or concepts during their residency, practiced the skill or concept at least occasionally during their residency, and some degree of competency was expected at the time of completion of their residency. these data will provide a framework for designing future laim residency programs. abstract this study evaluated pharmacokinetics and clinical safety of an oral paste formulation of a commercially available cox -sparing nsaid in clinically healthy pony foals in a randomized controlled clinical trial. values for complete blood count, serum chemistry profile, urinalysis, pharmacokinetic assay, and gastric endoscopy were evaluated in eighteen shetland pony foals treated with firocoxib ( . mg/kg, po, q h) or placebo for days. foals were divided into treatment groups. group and foals received firocoxib while a rd group was administered an oral placebo. gastric endoscopy was performed on group and foals prior to treatment and on days and to monitor for the presence of gastric ulcers. group and foals had blood and urine samples taken sequentially for pharmacokinetic analysis, cbc, serum chemistry evaluation, and urinalysis. physical examinations were performed prior to treatment and daily for days. data were analyzed using anova and paired t-tests (p o . ). none of the foals presented adverse clinical effects. there were no significant changes in cbc, biochemical profiles within groups, or differences between groups. pretreatment gastric endoscopy scores were not significantly different from evaluations at and days. firocoxib was quickly absorbed with an observed maximum concentration at hr, the first sampling interval, for the majority of animals. firocoxib plasma concentrations decreased in a log-linear manner after reaching the maximum concentration and steady state concentrations were achieved by the th dose. based on the sampling times after the final and th dose, an average half life of . days was estimated. administration of firocoxib did not cause any adverse effects on gastrointestinal, or hematological or serum biochemical variables, appears to have been well tolerated, and follows a predictable pharmacokinetic pattern in - week old foals. equine herpesvirus (ehv- ) is highly prevalent in most horse populations. horses are routinely vaccinated against ehv- , and neutralizing antibodies have helped to prevent disease. however, the usda has recently classified ehv- myeloencephalopathy (ehm) as an emerging disease, in response to the apparent increase in incidence, morbidity, and mortality of ehm that suggests a change in virulence of the virus. it has been reported that cellular immune mechanisms, in particular cytotoxic t-cells (ctls), are important in controlling ehv- viremia. interferon-alpha (ifn-a) has a key function in innate immune regulation by inducing the differentiation and maturation of ctls. here, we investigated the influence of abortogenic (racl , ny ) and neuropathogenic (ab ) ehv- virus strains on ifn-a, il- and il- secretion in equine pbmc. equine pbmc were infected with racl , ny or ab ehv- strains or kept in medium for hours. ifn-a, il- and il- secretion was detected in the supernatants by a fluorescent bead-based cytokine assay. the production of ifn-a increased with increasing viral doses and similarly for all three ehv- strains. the production of the antiinflammatory cytokine il- was significantly decreased after ab infection compared to racl and ny strains at viral infection doses of moi . - . at high doses (moi ), il- production was suppressed by all three ehv- strains. the results suggested that abortogenic and neuropathogenic ehv- strains equally induce antiviral ifn-a production in equine pbmc. they also illustrated the differences in the ability of ehv- strains to modulate anti-inflammatory il- . neuropathogenic ab strain had an increased potential to down-regulate il- production suggesting specific viral mechanisms that interfere with the control of inflammation in the host. the variations in innate il- secretion might influence the development of protective immunity and might offer an explanation why neuropathogenic ab induces more severe disease, including myeloencephalopathy, than abortogenic ehv- strains. previously presented at a conference of research workers in animal disease. rhodoccocus equi is the major cause of pneumonia in foals during the first six months and control measures are frequently ineffective. treatment protocols are long, expensive and do not always produce good results. rhodococcosis prevention through immunization of foals using a safe and efficient vaccine is still a challenge. recent studies are based on the use of the virulence associated protein a (vapa) which has been described as an important inducer of immunity against r. equi. the present study evaluated the clinical and immune response of foals vaccinated with an attenuated strain of s. enterica typhimurium expressing vapa antigen (test group) or s. enterica typhimurium without the vapa gene (control group), previous to and following experimental challenge. two experimental phases were established according to the immunization route: intranasal or oral vaccination up to hrs following birth and at days of age. the experimental and control groups were challenged on day with a virulent stain of r. equi. clinical examination, cbc and image complementary exams were used to evaluate the development of clinical signs. immune response patterns were evaluated though immunoglobulin dosage, cytokine expression, lymphocyte proliferation assays, isolation of r. equi and cytological profiles of tbw. clinical manifestation was less intense in the test group during the second experimental phase, and death occurred only in the control group ( / ) and was due to r. equi pneumonia. the test group produced a more intense iggb response when compared to controls however no statistical difference was observed. lymphoproliferation and th cytokine expression were higher in the test group. in contrast, controls produced an il- response. local iga was significantly higher in animals immunized with salmonella carrying vapa. immunization protocols produced no severe toxic effects. the vaccination of neonatal foals with s. enterica typhimurium expressing vapa was considered safe, produced efficient modulation of the immune response and is apparently able to protect against experimental r.equi infection. this study was conducted to test the hypothesis that the kd protein, myristolated alanine-rich c-kinase substrate (marcks), is involved in equine neutrophil migration and adhesion. in other species, marcks phosphorylation and dephosphorylation causes the protein to cycle between the cell membrane and cytosol, respectively. to investigate marcks phosphorylation in horses, neutrophils were isolated from whole blood and stimulated with platelet activating factor (paf), leukotriene b (ltb ) or phorbol myristate acetate (pma). western blot was performed using specific phospho-marcks and total marcks primary antibodies. these results determined marcks phosphorylation is maximal seconds following stimulation and that dephosphorylation occurs within minutes. to investigate the requirement for marcks in equine neutrophil chemotaxis, isolated neutrophils were pre-treated with mans (a cell permeant peptide identical to the n-terminal amino acids of marcks), rns (a control peptide) or vehicle control (vc) prior to a migration assay toward known neutrophil chemoattractants (ltb or paf). pre-treatment of equine neutrophils with mans significantly inhibited migration while rns pre-treatment had no effect. to investigate marcks requirement in equine neutrophil adhesion, mans, rns or vc treated cells were stimulated to adhere to immulon plates coated with % fbs. pre-treatment of equine neutrophils with mans significantly inhibited adhesion while rns pre-treatment had no effect. inhibition of marcks using a cell permeant peptide identical to the protein's n-terminus significantly inhibited equine neutrophil adhesion and migration. these results indicate that marcks is an important regulator of equine neutrophil chemotaxis and represents a potential target for anti-inflammatory therapy. amongst other tests, a thorough neurologic examination of horses may include walking with the head elevated and during blindfolding, in order to help differentiate normal from abnormal and to help with neuroanatomically localising any lesion(s) i.e. in the ataxic horse. consensus amongst equine neurologists suggests that gait abnormalities associated with these specific tests are often exacerbated in horses with underlying proprioceptive deficits however the effect of these tests on temporal gait characteristics in normal horses has not previously been assessed quantitatively. we hypothesized that head elevation or blindfolding, in comparison with walking in a straight line would result in a compensatory decrease in lateral (left front-on to left hind-on and right front-on to right hind-on) and diagonal coupling intervals (left front-on to right hind-on and right front-on to left hind-on) in normal horses. four thoroughbreds without any history or clinical signs suggestive of neurological disease (age range to years) were included in the study. retroreflective markers were applied to the withers, to the sacrum and to left and right tuber coxae; for each limb, lateral fetlock markers and dorsal and lateral hoof wall markers were used. a minimum of trials each with - walk strides for each task were analysed as horses walked across an -force-plate runway i surrounded by a -camera kinematic system. ii force-plate data were processed with semi-automated custom written matlab iii scripts. data were analysed with a mixed model using the statistical software r. there was a significant fixed effect of normal walk on a straight line and head elevation on left and right lateral coupling intervals (p o . ) and of the left and right diagonal coupling intervals (p o . ). there was no significant effect of blindfolding on neither lateral nor diagonal coupling intervals. the random effect of horse had no influence on the coupling intervals. the decrease of the lateral coupling intervals indicates a tendency towards a pacing gait during head elevation. we conclude that there is a significant change in temporal gait characteristics of non-neurologic horses when the head is elevated but not during blindfolding compared to normal walking. current results suggest that pacing and increased variation in foot-placement during head elevation should be interpreted with caution however further work is required to determine whether the change differs between horses with neurological disease and non-neurologic disease. hereditary equine regional dermal asthenia (herda) is an autosomal recessive connective tissue disorder associated with a mutation in cyclophillin b that leads to impaired collagen folding, aberrant wound repair, and corneal abnormalities. it affects young quarter horses, appaloosa, and paints. herda shows similarities to the human hereditary connective tissue syndrome ehlers danlos (eds). many eds patients suffer from joint pain and osteoarthritis (oa) as adults. the similarity between eds and herda raises the question whether horses suffering from herda develop oa. in oa, excess production of inflammatory mediators such as prostaglandin e (pge ) activate enzymes that degrade cartilage as well as impede wound healing. the present study examined articular cartilage from yearling horses afflicted with herda. we hypothesized that chondrocytes from these horses are continually activated to produce inflammatory mediators. to test this hypothesis, articular cartilage from carpal and tarsal joints of herda horses were evaluated using histology. pge production by chondrocyte cultures was measured by elisa and analyzed by one-way anova, tukey post-hoc test, p o . significance. we also determined the antiinflammatory effects of an avocado/soybean unsaponifiables (asu), glucosamine (glu), and chondroitin sulfate (cs) mixture (ingredients found in cosequin s asu) and phenylbutazone (pbz) on chondrocytes. cosequin s asu and pbz are used alone or in combination for the management of oa. chondrocyte cultures were incubated for hrs with control media alone, a clinically relevant concentration of pbz ( mg/ml), or the combination of asu (nmx s , . mg/ml) glu (fchg s , mg/ml) cs (trh s , mg/ml). articular cartilage from joints of five herda-afflicted horses showed gross and histologic evidence of osteoarthritic lesions. chondrocyte cultures from cartilage of horses suffering from herda spontaneously produced greater pge than chondrocytes from normal horses ( -fold). pbz significantly decreased pge production by $ % (p o . ). the combination of asu -glu cs also significantly reduced pge production by $ % (p o . ). the present study supports anecdotal findings that horses suffering from herda are likely to develop oa. the inhibition of pge synthesis by asu glu cs suggests that this combination may be beneficial for the management of oa in herda. research supported by nutramax laboratories, inc. equine inflammatory airway disease (iad) is a common condition often treated empirically with corticosteroids. gene expression analysis in the bronchoalveolar lavage fluid (balf) may help understand the effects of corticosteroids in iad. the first part of the study aimed at identifying reference genes in the balf of iad horses treated with corticosteroids. the second part of the study investigated the effects of dexamethasone and fluticasone propionate treatments on the mrna expression of il- b, il- , il- and il- . the expression stability of seven candidate reference genes was determined in balf taken pre-and post-treatment with dexamethasone and fluticasone propionate in horses with iad. primers' efficiencies were calculated using linregpcr. normfinder, genorm and qbaseplus softwares were used to rank the genes according to their stability. gapdh was the most stably expressed gene whereas b m was the least stable gene. in addition, genorm analysis revealed that the number of genes required for optimal normalization was four (gapdh, sdha, hprt, rpl ). in the second part of the study the mrna expression of il- b, il- , il- and il- was measured in balf samples from seven iad horses treated in a randomized cross-over design with dexamethasone ( . mg/kg sid, days) or inhaled fluticasone propionate ( mcg bid with aerohippus, days). the balf samples were taken at baseline and after each treatment period. there was no significant effect of the corticosteroids treatment on the mrna expression of il- b, il- and il- in the balf. the mrna expression of il- was suppressed by dexamethasone and fluticasone propionate treatments. pneumonia is observed in horses after long distance transportation in association with confinement of horses' head position leading to a reduction in tracheal mucociliary clearance time (tmct). we hypothesize that clenbuterol, a beta- agonist shown to increase tmct in the horse, will ameliorate the affect of a fixed head position on large airway contamination and inflammation in a long-distance shipping model. six adult horses were enrolled in a cross-over design prospective study. horses were housed with their heads in a fixed position for hours to simulate long distance transport, and treated with clenbuterol ( . ug/kg po q h) or a placebo starting hours before simulated shipping. tmct was measured using a charcoal clearance technique. data were collected at baseline and hours, and included tmct, tracheal wash cytology and quantitative culture, rectal temperature, cbc, fibrinogen, and serum tnfa, il- and il- levels. there was a -week washout between study arms, and each horse served as its own control. the data was analyzed using regression analysis and wilcoxon rank-sum tests. no statistically significant difference was seen between treatment and placebo groups for any of the variables investigated. tmct did not differ after treatment ( . ae . cm/min) versus placebo ( . ae . cm/ min; p . ), and intratracheal bacterial counts were similar for treatment (  ae  cfu; p . ) and placebo (  ae  cfu) groups. a reduction of tracheal b hemolytic streptococcus. spp. after clenbuterol versus placebo was also nonsignificant ( % versus %; p . ). in conclusion, treatment with clenbuterol does not appear to combat the deleterious effects of this long-term shipping model. breathing cold air during strenuous exercise is associated with airway inflammation. under these conditions, warming and humidification of inspired air occurs in the lower respiratory tract resulting in mucosal cooling, desiccation, and hyperosmolarity. the purpose of this research was to test the hypothesis that airway hypertonicity causes inflammatory cell migration and alterations in cytokine expression associated with exercise induced airway inflammation. horses (n ) were examined in a randomized crossover design after exposure to hypertonic aerosols ( minute nebulization with solutions of either isotonic or hypertonic mannitol). airway leukocytes were harvested and hours post aerosol challenge via bronchoaveolar lavage, and were used to determine total and differential nucleated cell count and expression of cytokinespecific mrna. hypertonic aerosol challenge resulted in an increase in total number of cells hr after challenge, characterized by increased macrophage (p . ) and neutrophil (p . ) concentrations, but there was no effect on airway leukocyte concentrations hours after nebulization. no significant changes in the relative quantity of mrna for airway cytokines were noted at either time point. these data demonstrate that transient airway hypertonicity can cause airway leukocyte influx and may be responsible for the airway inflammation commonly found in athletes that exercise in cold conditions. however, our data do not support the hypothesis that hypertonicity is the sole initiating cause of changes in cytokine expression secondary to cold weather exercise. it is likely that factors such as airway temperature, shear stress or epithelial damage also play a role in this phenomenon. we studied the importance of abdominal sonograms in neonatal foals suffering from gastrointestinal conditions. we hypothesized that there would be a subgroup of neonates with sonographically detectable pneumatosis intestinalis (pi) as a reflection of a necrotizing component of the disease. records of foals days of age hospitalized between and with signs of gastrointestinal disease were evaluated (n ). the association of sonographic, clinical, pathological and clinicopathological signs with outcome and severity of disease was determined. pneumatosis intestinalis was imaged in foals. twenty-seven foals were classified as having necrotizing gastrointestinal disease based on the presence of gastrointestinal signs (colic, diarrhea, gastric reflux or abdominal distension) and pi detected sonographically ( ), surgical ( ) or pathological ( ) evidence of gastrointestinal necrosis. there was a difference between overall survival rate ( %) and survival rate in foals with necrotizing disease ( %, p . ) or foals with pi detected sonographically ( %, p . ). pneumatosis intestinalis was the only sonographic finding associated with outcome. sonographic abnormalities in peritoneal fluid, stomach, duodenum, jejunum, cecum, umbilicus or the presence of meconium were associated (p o . ) with surrogates of severity of disease (hospitalization cost or days of hospitalization). hypoproteinemia was associated with pi (p . ). the presence of blood in the feces, reflux and abdominal distension was associated with necrotizing gastrointestinal disease (p o . ). abdominal sonograms have prognostic value in neonatal gastrointestinal disease. pneumatosis intestinalis was a common sonographic sign that worsened the prognosis. the therapeutic implications of detecting a necrotizing component of the gastrointestinal disease deserve further study. the interaction of insulin and the microvascular endothelial insulin receptor (irc) plays an important role in the normal and insulin resistant (ir) individual. while endothelial irc signaling is normally vasodilatory, this effect is well-documented to reverse in the ir individual, resulting in vasoconstriction. although vascular dysfunction has been reported in sepsis-associated equine laminitis, the role of the laminar microvasculature in endocrinopathic laminitis remains poorly characterized. the purpose of this study was to characterize the pattern of irc expression in digital laminae in ponies subjected to a dietary carbohydrate challenge that mimicked abrupt exposure to pasture rich in nonstructural carbohydrates (nsc). mixed-breed ponies (body weight . /- . kg) received a diet of hay chop (nsc $ % on a dm basis) for weeks prior to initiation of the experimental feeding protocol. following conditioning, ponies either remained on the control diet (n ) or received the same diet supplemented with sweet feed and oligofructose (total diet $ % nsc; n ) for a period of days. serum insulin concentrations were measured prior to and after completion of the feeding protocol. at the end of the feeding protocol, sections of numerous tissues, including dorsal digital laminae, were collected immediately following euthanasia. the samples were formalin-fixed for hours, transferred to % ethanol, and paraffin-embedded. laminar sections were stained immunohistochemically for irc using a commercially-available antibody (abcam); the number of irc ( ) cells was quantified in x light microscopy fields (n ) for each section. the total number of irc ( ) cells was greater in the laminae of challenged ponies than control ponies (p . ), and there was a significant correlation between the change in serum basal insulin concentration and number of laminar irc ( ) endothelial cells (r . ; p o . ). while the number of irc ( ) endothelial cells was significantly greater in the dermal laminae of challenged ponies (p . ), there was no difference in the number of interstitial irc ( ) cells (p . ). no epithelial irc ( ) cells were observed in any laminar section, and irc ( ) cells were conspicuously absent from the deep dermal tissue (including vessels). up-regulation of irc expression in the laminar vasculature occurs acutely in response to dietary carbohydrate challenge and accompanies hyperinsulinemia in ponies. the dramatic increase in endothelial irc expression in the laminar microvasculature in nutritionally challenged ponies, with no apparent epithelial irc present, suggests that hyperinsulinemia associated with exposure to increased dietary nsc may induce laminar injury by causing a similar vasoconstriction in ir equids as described in the microvasculature of ir humans. glucose transport from the blood stream into cells, the limiting step in whole-body glucose utilization, is regulated by a family of glucose transporter (glut) proteins in insulin-sensitive (i.e., muscle and adipose) tissues. we previously demonstrated that glut , the major isoform, is a key factor in the pathogenesis of equine insulin resistance (ir). while it has been recently demonstrated that glut (a newly discovered isoform) increases insulin-stimulated glucose transport in human muscle, its role in other tissues, particularly in the setting of ir, is not well characterized in any species. in addition, as has recently emerged as a key downstream signaling molecule regulating translocation of glut to the cell surface, the rate-limiting step in glucose uptake. we hypothesized that glut content would be differentially expressed across tissues and that ir would induce alteration in glucose transport by affecting active cell surface glut . biopsies of skeletal muscle, and subcutaneous and visceral adipose tissue were collected from light-breed horses, characterized as either insulin sensitive or compensated ir, based on the results of an insulin-modified frequently-sampled intravenous glucose tolerance test (n /group). we specifically quantified active cell-surface glut in these biopsies, using an innovative exofacial bismannose photolabeled assay, which has not been previously applied to glut . total glut protein expression was measured by western blots, as well as total and phosphorylated (indicating activation of) as . glut was expressed in all the depots with a significant regional effect. total glut protein content was increased (p o . ) in visceral (omental and mesenteric) compared to subcutaneous (nuchal ligament and tailhead) adipose tissue and skeletal muscle of healthy horses. ir did not induce alterations in active cell-surface and total glut content nor in total and phosphorylated as in any of the tissues evaluated. our data suggests that glut is abundant in visceral adipose tissue and is therefore likely to play a substantial role in the regulation of glucose transport. however, neither glut translocation nor as activation are impaired in insulin-sensitive tissues of ir horses. it is concluded that, in contrast with glut , glut does not appear to contribute to glucose transport alterations during naturally-occurring equine ir. insulin resistance (ir), characterized by exaggerated glycemic or insulinemic responses to glucose challenge, is a key metabolic disturbance in horses that develop obesity-associated laminitis. in addition to obesity, diet and age have been demonstrated to affect tissue sensitivity to insulin in other species but these factors have received limited investigation in horses. we hypothesized that there would be greater glycemic and insulinemic responses to a sweet feed meal in aged horses, as compared to adult horses, as well as in horses adapted to a forage-only diet. three diets, grass hay only, grass hay plus sweet feed (starch and sugar-rich, ss), and grass hay plus a fat and fiber (ff) feed, were fed to mares, adult ( - yr) and aged ( yr), for a -week adaptation period in a randomized design. glycemic and insulinemic responses to a standardized meal of sweet feed ( g/kg bw offered for hour) were determined for hours from the onset of feeding. peak glucose and insulin concentrations and areas under the glucose or insulin vs. time curves (auc-g, mg/ dl/ min, and auc-i, mu/ml/ min) were determined and data were analyzed by one-and two-factor repeated measures anova. there were no differences between age groups in glycemic responses to any of the diets. however, in aged horses peak glucose concentration (p o . ) and auc-g (p o . ) were greater after adaptation to the forage-only diet, as compared to the other two diets. in contrast, aged horses had a greater peak insulin concentration (p o . ) and auc-i (p o . ) than adult horses on all diets but no differences in peak insulin concentration or auc-i was found between diets within age groups. as hypothesized, the insulin response, but not the glycemic response, to a sweet feed meal was greater in aged horses, regardless of background diet. further, the glycemic response was greatest after adaptation to a forage-only diet, but this finding was only significant in aged horses. morbidity, mortality, and economic loss to the equine industry. in obese humans and rodent models of nutritional obesity, systemic insulin resistance and hyperinsulinemia are followed temporally in a majority of individuals by decreased glucose tolerance, pancreatic bcell failure, and type ii diabetes mellitus. in stark contrast to humans, obese horses and ponies chronically remain in what is termed a ''prediabetic'' state in human ir, characterized by hyperinsulinemic euglycemia. few data exist describing the biology of the equine endocrine pancreas in the chronically ir animal that may both: ) explain this unique equine endocrine physiology and ) characterize the animal at-risk for hyperinsulinemia-associated laminitis. the purpose of the study reported here was to characterize the morphology and physiology of the equine endocrine pancreas in response to a dietary carbohydrate challenge. twenty-two mixedbreed ponies (body weight . ae . kg) were conditioned to a diet of chopped hay (nsc $ % on dm basis) for weeks; following conditioning, ponies either remained on the control diet (n ), or received the same hay supplemented with sweet feed and oligofructose (total diet $ % nsc; n ) for days. serum insulin concentrations were measured prior to and after completion of the feeding protocol. at the end of the feeding protocol, sections of numerous tissues, including pancreas, were collected immediately following euthanasia. the samples were formalin-fixed for hours, transferred to % ethanol, and paraffin-embedded. immunohistochemistry was performed on pancreas sections using a commerciallyavailable anti-insulin antibody (abcam), and measurements of islet surface area and b-cell surface area were performed (n islets per tissue section) using a commercially-available computer software program (image j). there was a trend for greater total islet surface area in pancreatic tissue from ponies fed the high nsc diet when compared to the ponies on the hay diet (p . ); however, no difference was noted in b-cell surface area between diet treatments (p . ). the change in serum insulin concentration was significantly greater in the high nsc-fed ponies than in controls ( . /- . miu/l vs. . /À . miu/l; p . ); however, this variable was not correlated with total islet surface area (r . ; p . ) or b-cell surface area (r . ; p . ). due to the relatively modest changes in pancreatic islet surface area that accompany marked increases in serum insulin concentrations in ponies fed a high nsc diet, it is important to assess both b-cell function and insulin clearance mechanisms in future studies to delineate the mechanism(s) of hyperinsulinemia in this model. humans that suffer from obesity show exaggerated inflammatory responses and this may be relevant to the association between increased adiposity and laminitis in horses with equine metabolic syndrome (ems). this study was performed to test the hypothesis that inflammatory responses to endotoxemia differ between healthy horses and those affected by ems. six healthy adult mares and horses with ems received an intravenous infusion of lipopolysaccharide (lps; ng/kg in ml sterile saline) or saline alone. a crossover design was employed with a -day washout period. physical examinations were performed hourly for h and whole blood was collected at , , , , , and min for assessment of inflammatory cytokine gene expression. a liver biopsy was performed between and min postinfusion. data were analyzed using mixed model anova. mean rectal temperature, heart rate, and respiratory rate increased following lps infusion (treatment  time; p o . ), with higher heart (group  treatment; p . ) and respiratory rates (group; p . ) detected in ems horses. lipopolysaccharide infusion significantly increased whole blood gene expression of tumor necrosis factor a (tnfa), interleukin (il)- b (p o . ), il- (p o . ), il- (p o . ), and il- (p . ), and hepatic gene expression of il- (p o . ), il- (p o . ), and il- (p . ). inflammatory gene expression did not differ significantly between groups, so our hypothesis was not supported. heart rates tended to be higher when lps was administered to horses with ems. elevated serum concentration of cardiac troponin i (ctni) is a biomarker for myocardial damage in horses. preferred times to test blood for ctni levels following athletic performance or other events that may cause myocardial injury are not yet established and would be affected by time of release from the myocytes, location of release within the myocytes, duration of release and half-life of ctni in the horse. this information would be necessary to more accurately and reliably test horses for myocardial injury. the aim of this study was to determine the elimination half-life (t / ) of equine ctni. to establish the t / of equine ctni in horses, ctni was recombinantly expressed in e.coli. two healthy ponies received intravenous injections of recombinant equine ctni and plasma ctni concentrations were measured with a point-of-care ctni analyzer at multiple time points after injection. standard pharmacokinetic analysis was performed to establish the elimination half-life of ctni. for comparative purposes, data were subjected to pharmacokinetic models describing a single versus biphasic elimination profile. elimination of recombinant equine ctni following intravenous administration exhibits a short half-life. establishing the t / of troponin provides critical information in understanding the clinical application of this cardiac biomarker in clinical practice. this study describes a true biological ctni t / , which has not been documented in any species thus far. stall-side assessment of this cardiac biomarker in horses should enhance the ability of clinicians to detect myocardial damage and aid in the management and treatment of horses with cardiac disease. the objective of the study was to evaluate the between-pony, within-pony, between-analyser and within-analyser variation of flow-mediated vasodilation (fmd) measurement in healthy ponies, to investigate the hypothesis that fmd occurs in healthy ponies. six healthy, native breed, unrelated pony mares of varying weight ( - kg), body condition score ( / - / ) and age ( - years) were used. the median artery was occluded for minutes. twodimensional ( d) ultrasonographic images of the artery were recorded for seconds prior to and for minutes after occlusion. the peak luminal diameter was compared to baseline diameter to calculate the relative percentage increase in luminal size (fmd). images were obtained from six ponies on one occasion and from one pony on six occasions. analysis of images was performed by two independent analysers and by one analyser twice. the mean (sd) fmd in ponies was . % ( . %) and in pony ( occasions) was . % ( . %). coefficients of variation were . % and . % respectively. agreement between analysers was fair (icc . ) and within analyser was poor (icc . ). fmd is used to assess endothelial function in humans and has recently been assessed for its use in canine subjects. fmd occurs and measurement is feasible in ponies. fmd could be used to assess endothelial function, in the context of laminitis or other cardiovascular diseases. current state-of-the-art technique for measuring blood pressure (bp) in the horse is invasive and involves cannulation of the facial artery. indirect techniques, such as oscillometry, have proven useful in the anaesthetised horse, but have not become routine in the standing horse. monitoring bp can be indicated for the diagnosis and treatment of the hypotensive patient (ie. caused by endotoxemia, hypovolemia, systemic inflammatory response syndrome and cardiac failure) or the hypertensive patient (ie. due to equine metabolic syndrome or pain). the objective of this study was therefore to a) describe the methodology for application of oscillometric bp using a cuff applied to the tail in the standing horse and b) and to determine accuracy and precision of this method applied to the normotensive standing horse. the oscillometric method is simple to apply in a clinical setting. a pneumatic cuff is snugly applied to the unclipped tail-base with the cuff bladder centered over the middle coccygeal artery. the tail circumference must match the manufacturers description of the cuffs diameter range. the oscillometric apparatus inflates the cuff and obtain systolic, diastolic and mean arterial bp (sap, dap and map). at least consecutive measurements must be obtained. a correction of . mmhg/cm vertical distance between cuff and heart level is added to the measurement to correct for hydrostatic pressure difference. for determination of accuracy and precision of indirect sap, dap and map, eight healthy horses (age to years), was equipped with an intra-arterial catheter ii in the facial artery and a commercial tail-cuff oscillometric apparatus. i measurements were recorded every minutes for minutes. the data were analysed with the statistical software r using a mixed model with repeated measurements and a bland-altman analysis corrected for repeated measurements. oscillometric bp was accurate and precise for map (mean bias, lower confidence level, upper confidence level, variation in difference, all mmhg) (À . , À . , . , . , respectively) in the conscious horse but not for sap (À . , À . , . , . , respectively) and dap ( . , . , . , , respectively) . there was no significant contribution to the statistical model of either horse or measurement number. all horses tolerated the tail-cuff well and the method was simple to apply. only map could be measured with acceptable accuracy and precision in the normotensive standing horse using the described oscillometric method. reference intervals for thyroid hormone (th) concentrations have not been established for donkeys. therefore, clinicians must use reference ranges from horses, potentially leading to misdiagnosis of thyroid diseases. we hypothesized that th concentrations are different between donkeys and horses. the purposes of this study were: a) to compare th concentrations between donkeys and horses and, b) to determine whether the age may influence th concentrations. thirty-eight healthy donkeys ( . ae . years), mixed breeds, and healthy andalusian horses ( . ae . years) were used. donkeys were divided into three groups: o years (n ), - years (n ), and years (n ). serum concentrations of total triiodothyronine (tt ), free triiodothyronine (ft ), total thyroxine (tt ), free thyroxine (ft ), reverse triiodothyronine (rt ) and thyroid-stimulating hormone (tsh) were quantified by radioimmunoassay. all blood samples were collected the same day. neither horses nor donkeys had received any treatment for days before sampling and both farms had similar production conditions. total t , ft , ft and tt concentrations were higher (p o . ) in donkeys than horses. in contrast, no statistical differences were found for rt and tsh concentrations. young donkeys ( o years) had higher ft , tt and rt concentrations compared to other donkey groups (p o . ). old donkeys ( years) had lower tt and ft concentrations than both younger donkeys groups (p o . ). this study shows that there are differences in th concentrations between donkeys and horses, raising awareness on the possibility of misdiagnosis of thyroid gland dysfunction when using values from horses, being necessary to determine exclusive reference intervals for donkeys. ovariectomy is associated with alterations of responses to many hormones, not just those associated with reproductive function. in humans and rats, ovariectomy leads to insulin resistance, increased adiposity and altered fat mobilization. the effects of ovariectomy on energy metabolism have not been reported in horses. ovariectomized mares have been shown to respond normally to an acth stimulation test, but the response to suppression of the hypothalamo-pituitary-adrenal axis has not been previously described. the aim of this study was to evaluate the effect of ovariectomy on insulin response in mares and to determine if mares exhibit alterations in response to dexamethasone administration after ovariectomy. six healthy mares underwent an intravenous glucose tolerance test (ivgtt), an insulin sensitivity test (ist) and a dexamethasone suppression test (dst) before and weeks after bilateral ovariectomy. body weight, cortisol values at baseline, and hours after dexamethasone injection and acth values at baseline, and hours after dexamethasone injection, basal insulin/glucose ratio, time to reach a % decrease in blood glucose in the ist, time to reach baseline glucose concentration in the ivgtt and area under the curves plotting blood glucose and time to injection of glucose or insulin were compared before and after ovariectomy using a paired t-test or an anova for repeated measures. significance level was p o . . average body weight was decreased after surgery ( kg ). the injection of dexamethasone resulted in a serum cortisol concentration of less than mg/dl in all mares before ovariectomy, whereas after ovariectomy, dexamethasone injection resulted in a serum cortisol concentration of less than mg/dl in out of mares. in all cases, acth concentration was within the reference range ( - pg/ml) before and after ovariectomy. however, acth concentrations at t and at t were significantly higher after ovariectomy. each mare had a normal ivgtt, both before and after ovariectomy. additionally, no significant differences were observed in basal blood glucose ( ae mg/dl before and ae mg/dl after) or in the time to reach glucose baseline ( ae min before and ae min after). serum basal insulin concentration and insulin/glucose ratio was not significantly different before or after ovariectomy ( . ae . miu/ml and . ae . miu/ml and . ae . and . ae . , respectively), nor was the average time to reach a % decrease in blood glucose after insulin injection ( ae min and ae min, respectively). these findings suggest that, as reported in other species, the shortterm effect of ovariectomy may modify dexamethasone response in mares and that, contrary to other species, it may not modify insulin response. equine gastric ulcer syndrome (egus) is a common medical problem in horses. the high prevalence of gastric ulcers, vague clinical signs and negative effect on performance make it a significant clinical and economic problem within the horse industry. current pharmaceutical treatments are expensive and alter the acidic environment of the stomach. berries and pulp from the seabuckthorn plant (hippophae rhamnoides) are a rich source of vitamins, trace minerals, amino acids, antioxidants, and other bioactive substances and have been used successfully to treat stomach ulcers in man and rats. the purpose of this study was to evaluate the efficacy of a commercially sold, liquid extract of seabuckthorn berries (seabuck tm sbt gastro-plus) for treatment and prevention of gastric ulcers in horses. eight thoroughbred and thoroughbred-cross horses ( - years of age, geldings & mares, - kg) were used in a blinded two-period cross-over study. treatments consisted of control (untreated) and treatment (seabuck tm sbt gastro-plus) twice daily mixed with the grain meal. horses were treated for weeks followed by a week alternating feed-deprivation period to induce or worsen existing ulcers. gastroscopies were performed on all horses on day , week , and week (at the end of the alternating feed-deprivation period). gastric juice was aspirated and ph was measured. during gastroscopy, gastric ulcer scores were assigned to each stomach based on lesion number and severity. horses acted as their own controls, and between each treatment period the horses had a -week washout period. data was analyzed by anova for repeated measures via the glm procedure (sas inst. inc., cary, nc). when significant differences (p o . ) were observed, a post-hoc tukey's test was used to determine differences. non-glandular gastric ulcer scores significantly increased in all control and sbt-treated horses from week to week , after the feed-deprivation phase of the study. there was no significant difference in the non-glandular gastric number (p . ) and nonglandular gastric severity (p . ) scores in sbt-treated horses compared to non-treated controls. glandular ulcer number (p . ) and glandular ulcer severity (p . ) was significantly lower in the sbt-treated horses compared to the control horses. there was no significant difference in the ph (p . ) in sbt-treated horses compared to non-treated controls. thus, seabuck tm sbt gastro-plus, mixed in the feed twice daily, may be efficacious in controlling the severity of glandular ulcers in horses during stress, without increasing stomach ph. the availability of rapid and accurate quantitative fibrinogen measurements may be useful for evaluation of hospitalized equine patients. the abaxis vspro analyzer was evaluated for precision using two levels of human fibrinogen controls ( mg/dl and mg/dl), four different vspro machines, and two different lots of cartridges, assessed over subsequent days. the coefficients of variation of the assay ranged from % ( mg/dl) to % ( mg/ dl). we subsequently evaluated the abaxis vspro fibrinogen assay compared to fibrinogen concentration measured using the beckman coulter acl- in equine samples of varying fibrinogen concentrations obtained from horses with gastrointestinal disease. all samples were measured in citrated plasma. fibrinogen samples measured on the acl- ranged from to mg/dl (median mg/dl). vspro samples were run in duplicate, and the mean compared to the acl values. pearson correlation coefficient analysis generated an r value of . (p o . ). duplicate measurements on the vspro were strongly correlated to each other with an r value of . (p o . ). bland-altman analysis of these samples for the vspro compared to the acl- noted a bias of À ae mg/dl the results of this study indicate that the vspro benchtop fibrinogen analyzer provides accurate and precise fibrinogen data compared to the acl- reference analyzer. the immune response of foals to r. equi is incompletely understood and believed to be responsible for clinical disease caused by this pulmonary pathogen. in a recent study foals receiving a large inoculum exhibited th skewing with pneumonia and a small inoculum exhibited th skewing without clinical disease. we hypothesized that cytokine/chemokine production by pulmonary alveolar macrophages, in vitro, would increase with the infective dose and that the magnitude of the response would differ between foals and adults. alveolar macrophages were obtained by bronchoalevolar lavage from healthy mares and their -week-old foals. macrophage cultures were infected with r. equi ( or -) at a multiplicity of infection (moi) of or . total rna was harvested and hours post-infection, reverse transcribed and used as template for quantita-tive pcr. the ddct method was used to calculate relative gene transcripts for il- , il- p , tnfa and cxcl . cellular infections at moi resulted in significantly higher expression of il- , il- p and tnfa mrna transcripts compared to moi . however, the dose-effect was reversed for cxcl with significantly lower expression at the higher moi. there was no difference in magnitude of cytokine/chemokine responses by the alveolar macrophages between adults and foals. dose-dependent responses of alveolar macrophages may represent a novel mechanism by which r. equi could modulate immune responses and therefore disease. significant down-regulation of cxcl mrna transcripts associated with a higher dose is of particular interest as this chemokine plays a role in development of protective th responses. the intent of this study was to develop likelihood ratios (lrs) for infection attributable to corynebacterium pseudotuberculosis in horses based on synergistic hemolysis inhibition (shi) test titers. medical records for horses presented to the uc davis veterinary teaching hospital with serum submitted for shi titer determination were evaluated and cases met study inclusion criteria. these cases were grouped based on evidence of internal and/or external infection attributable to c. pseudotuberculosis and likelihood ratios with % confidence intervals determined. results showed increasing lrs indicating increasing odds for any form of active disease as titer increased with all cases considered. lrs for internal infection were for titers ! overall and for titers with external abscess cases excluded. no difference from (and therefore no significant change in pre-test to post-test odds) was seen in any lrs for internal disease when only cases with external disease were examined (external and internal disease vs. external only). overall, the shi test results showed usefulness in determining internal c. pseudotuberculosis infection in horses with no evidence of external abscessation. overall, however, higher titers were more indicative of active external or internal disease than internal disease specifically in contrast to previous reports. the shi test was unable to distinguish internal infection when external abscesses were present. salmonella enterica is a zoonotic pathogen that has tremendous impact on many different animal production and management systems. rapid detection of s. enterica in fecal samples may facilitate effective infection control practices. current detection methods require - hours (polymerase chain reaction or pcr) or - hours (enriched aerobic culture) to obtain results. alternatives have been developed, lateral flow antigen detection systems (lfads), which are currently marketed for salmonella detection related to food safety microbiology. the objective of this study was to evaluate two commercially available rapid salmonella detection systems in equine feces. fecal samples collected from repeatedly culture-negative horses were inoculated with known concentrations of salmonella enterica serotype typhimurium (five uninoculated control samples, and samples of each -fold dilution [ .  - .  cfu/gram of feces]). all samples were aerobically cultured using a standard enrichment technique. in a blinded fashion, samples were tested using two different lfads as well as plated on agar media for confirmatory testing. at hours of incubation, using bacterial culture as the reference method, test was correctly identify % of samples ( bacterial contamination of stalls with salmonella sp. is a serious problem in equine hospitals. salmonella sp. exposure to horses in the facility can result in nosocomial infections which results in temporary facility closure, until the organism is eradicated. hospital closure can result in loss of revenue, damage to reputation and interference with patient care. the purpose of this study was to evaluate three stall cleaning methods on eradication of salmonella sp. at an equine veterinary teaching hospital (vth). horses admitted to the vth were assigned to salmonella sp.negative stalls within areas of the vth during the study period (september -january . when the horses were discharged stalls were randomly assigned to one of three cleaning methods (pressure-washing only [pw] , pressure washing and hand scrubbing [pws] , or hand scrubbing only [s]) in a single period, non cross-over design. all stalls were stripped of bedding and surfaces sprayed with tap water and cleaned with a disinfectant quaternary-ammonia solution (super hdq neutral, spartan chemical co., inc, maumee, oh). the pressure-washing system (psc cleaning systems, inc., toronto, canada) used, provided a pressure of psi and a temperature range of - f. following cleaning, each stall was allowed to air dry and within hours, stall surfaces were sampled using three  sponges moistened with sterile saline. the person collecting the samples was masked to the method of cleaning. sponges were submitted to the louisiana animal disease diagnostic laboratory (laddl) for culture of salmonella sp. a chi-squared analysis was used to determine significant differences (limit p o . ) between cleaning methods and salmonella sp. isolation. during the study period, stalls (pw [n ]; pws [n ]; s [n ] were included. all stalls had negative environmental salmonella sp. cultures prior to beginning the study. for pw cleaned stalls, / ( . %) were salmonella sp.-positive, for pws cleaned stalls, / ( %) were salmonella sp.-positive, and for s cleaned stalls, / ( . %) were salmonella sp.-positive. although, there were fewer salmonella sp.-positive stalls ( . %) in the handscrubbed stalls, cleaning method did not significantly (p . ) affect the isolation of salmonella sp. from the stall environment. in conclusion, power washing alone, power washing and hand scrubbing, and hand scrubbing alone, using a quaternary-ammonia solution did not significantly affect environmental isolation of salmonella sp. from stalls surfaces in the vth during this study. the objectives of this study were to determine the plasma and pulmonary disposition of gamithromycin in foals and to investigate the in vitro activity of the drug against streptococcus equi subsp. zooepidemicus (s. zooepidemicus) and rhodococcus equi isolates. a single dose of gamithromycin ( mg/kg of body weight) was administered intramuscularly. concentrations of gamithromycin in plasma, pulmonary epithelial lining fluid (pelf), bronchoalveolar lavage (bal) cells, and blood neutrophils were determined using hplc with tandem mass spectrometry detection. the minimum inhibitory concentration of gamithromycin required to inhibit growth of % of r. equi and s. zooepidemicus isolates (mic ) was determined. additionally, the activity of gamithromycin against intracellular r. equi was measured. mean peak gamithromycin concentrations were significantly higher in blood neutrophils ( . ae . g/ml) and bal cells ( . ae . g/ml) compared to pelf ( . ae . g/ml) and plasma ( . ae . g/ml). mean terminal half-lives in neutrophils ( . h), bal cells ( . h), and pelf ( . h) were significantly longer than that of plasma ( . h). the mic of s. zooepidemicus isolates was . g/ml. the mic of gamithromycin for macrolide-resistant r. equi isolates ( g/ml) was significantly higher than that of macrolide-susceptible isolates ( . g/ ml). the activity of gamithromycin against intracellular r. equi was similar to that of azithromycin and erythromycin. intramuscular administration of gamithromycin at a dosage of mg/kg would maintain pelf concentrations above the mic for s. zooepidemicus and phagocytic cell concentrations above the mic for r. equi for approximately days. eight western stock yearling horses were infected with ehv- (ab ) by nasopharyngeal instillation. venous blood samples for collection of plasma were collected in na-citrate tubes on the day prior to infection (d - ) and on d through d . in addition, clinical data, nasal swabs and peripheral blood mononuclear cells (pbmc) for detection of viremia were collected on the day before infection (d - ) and on d through d post-infection. d-dimer concentrations were determined in citrated plasma samples using a latex agglutination test (minutex d-dimer, biopool, ireland). viral load in pbmc was determined using quantitative pcr. all horses showed bi-phasic fevers typical for ehv- infections. one horse developed acute ehm on d and was euthanized after samples were collected. in all horses d-dimers were undetectable on d - and on d , and . in contrast, all horses had increased ddimer concentrations for to consecutive days starting on day post-infection. d-dimer concentrations in horses increased to ug/ml and one of these horses was the horse with acute ehm. interestingly, mean increased d-dimer concentrations showed timely overlap with the mean fever curve and, delayed by day, with the mean viremia curve. because plasma samples for d-dimer measurements were not collected during the first days post-infection, which are typically associated with a primary fever, conclusion on the association of d-dimers with fever of viremia await analysis of a second study currently conducted in our laboratory. in conclusion our data indicates that during ehv- infection with neuropathogenic strains activation of the coagulation cascade and production of cross-linked fibrin is wide-spread; not limited to horses with clinical signs of ehm, and can be expected between days and post-infection. lawsonia intracellularis is an emerging pathogen in horses and the causative agent in equine proliferative enteropathy (epe). the goal of this study was to evaluate the exposure of pre-weanling foals and broodmares to lawsonia intracelluaris on several farms in louisiana with a history of epe and compare the results to several farms with no known clinical cases of epe in foals. an additional goal of the study was to identify whether a relationship exists between lawsonia intracelluaris and other gastrointestinal pathogens in foals. whole blood and fecal samples were collected from mares and foals from four breeding farms in louisiana. farms a and b had no known clinical cases of epe, while farms c and d had previous know cases of epe in . serum samples were examined for the presence of antibodies against lawsonia intracellularis using an immunoperoxidase monolayer assay (ipma). dna was extracted from fecal samples using a commercial dna kit and molecular detection of lawsonia intracelluaris was assayed using real-time pcr. fecal ova were determined using quantitative sucrose floatation. the presence of fecal clostridium difficile toxin was measured using a commercial enzyme linked immunosorbent assay (elisa). three of the farms examined had foals and mares with exposure to l. intracellularis as evidenced by serum antibodies against the organism. of the total population sampled, foals ( . %) and mares ( . %) had evidence of antibodies to l. intracellularis based on serology. three foals ( . %) tested positive for l. intracellularis organism by fecal pcr, and all of these foals were located on farm c. of these, one of the foals was seronegative, while the other two were seropositive. farm c also had the highest percentage of mares ( . %) serologically positive for l. intracellularis, while farm a had the highest percentage of foals ( . %) with antibody titers against l intracellaris. farm c also had the only pairs (n ) of serologically positive mares with seropositive foals. while farm a and b had seropositive mares and/or foals, none of the foals were positive for l. intracellularis fecal shedding by pcr. all serum and fecal samples were negative for evidence of l. intracellaris on farm d. ten foals ( %) had fecal egg counts greater than egg per gram and foals ( %) were positive for c. difficile toxin. this study demonstrated evidence of natural exposure to l intracellularis on farms both with and without a history of epe in louisiana. further, this study failed to establish a relationship between l intracellularis and other gastrointestinal pathogens. the objective of this study was to examine the clinical, hematological, biochemical, and outcome data from equids infected with anaplasma phagocytophilum presented to a primary care field setting in southeastern pennsylvania. computerized medical records from febrile equids with confirmed anaplasma phagocytophilum infection were reviewed. confirmation of anaplasma phagocytophilum was defined by the presence of granular inclusion bodies seen within leukocytes or eosinophils on microscopic blood smear evaluation and/or a positive polymerase chain reaction (pcr) for anaplasma phagocytophilum. horses and donkey presented with a mean fever of . f and mean fever duration of hours. the mean age at presentation was . years and the mean pack cell volume was . %. / cases were diagnosed in the months of may to december. equids ages to years had significantly lower platelet counts. / cases were positive on blood smear for inclusion bodies and / cases were positive for anaplasma phagocytophilum on pcr. treatments included intravenous oxytetracycline, oral doxycycline, or both. mean treatment duration was . days and mean treatment cost was $ . / cases were normothermic within hours. the treatment used in the two remaining cases was changed from oral doxycycline to intravenous oxtetracycline and was successful. this is the first case series of equine granulocytic anaplasmosis in the mid-atlantic states. all cases were examined and treated in the field. in order to make a definitive diagnosis, some cases required pcr. treatment failures were documented with the use of oral doxycycline alone. % of the cases survived. a high incidence of clinical and possibly genetic abnormalities has been reported amongst friesian horses including dwarfism, hydrocephalus, dissecting aortic aneurism and esophageal dysfunction. the purpose of the current study was to develop a new electromyography (emg) method to assess neurophysiological function of the esophagus especially for friesian horses. five friesian horses with esophageal dysfunction were included (ranging in age from . - years and comprising mares and a stallion) and two friesian control horses (a -and -year-old gelding). all five horses with esophageal dysfunction had a history of recurrent esophageal obstruction and were examined histopathologically post-mortem. barium contrast radiography was used as the gold standard to distinguish the diseased from the control horses. an endoscopically-guided percutaneous needle emg procedure (viking quest r ; software version . ) was performed just caudal to the larynx and just cranial to the thoracic inlet (to monitor striated and smooth muscle, respectively) to visualize esophageal motility. esophageal contractility in both control horses was predominantly reflected by interference patterns associated with longer duration and lower amplitude in smooth muscle compared to striated muscle. mean (ae sd) values were . ae . ms and . ae . mv (n readings) and . ae . ms and . ae . mv (n readings), respectively. in diseased horses, aperistalsis in smooth muscle was the most remarkable finding suggesting a loss of inhibitory neurogenic input resulting in aperistalsis and thus esophageal dysfunction. preliminary findings suggest that endoscopically-guided percutaneous needle emg might become a valuable method in elucidating the pathophysiology of dysfunction of esophageal motility especially in friesian horses. lymphoma affects horses of all ages. unlike in humans, no etiologic agent has been discovered. a year old thoroughbred/warmblood cross mare presented with signs of upper and lower respiratory disease and was subsequently diagnosed with lymphoma and equine multinodular pulmonary fibrosis (empf) and was positive for equine herpes virus (ehv- ) in both the pulmonary tissue and the lymph nodes. retrospective polymerase chain reaction (pcr) testing of six lymphoma cases found that of of the cases were positive on pcr for ehv- ( . %, p . , rr . ). electron microscopy was performed on one sample and herpes virus particles were identified. of the samples in which immunohistochemistry was performed ( of ), only t-cell rich b-cell lymphoma was identified. samples of mesenteric or submandibular lymph nodes from clinically healthy horses were submitted for ehv- pcr analysis; % were positive. gamma herpesviruses in humans have been associated with lymphoproliferative diseases such as kaposi's sarcoma and burkitt's lymphoma. equine herpesvirus , also a gamma herpesvirus, is found in association with equine lymphoma; although the exact role this virus plays in the initiation or perpetuation of lymphoproliferative neoplasia remains unknown. pathologic events reported to occur in the digital laminae in early stages of sepsis-related equine laminitis include leukocyte extravasa-tion into the laminar interstitium, pro-inflammatory cytokine expression, and epithelial stress. while these events have been documented early in the disease process at both a developmental stage and at the onset of obel grade (og ) lameness in the carbohydrate overload (cho) model of laminitis, the later events occurring at the onset of obel grade lameness(og , time point at which structural failure of the laminae usually occurs) have not been determined. we hypothesized that the inflammatory events described above are sustained through og lameness, likely playing an injurious role culminating in laminar failure. our objectives were to determine pro-inflammatory gene expression, leukocyte extravasation, and epithelial stress at og induced using the cho model. archived laminar tissue samples (snap frozen and paraffin embedded sections) were used from a previous cho study at louisiana state university (control group [n , water], cho group [n , corn starch]. calprotectin (cp) immunohistochemistry (ihc) was used to assess both laminar myeloid leukocyte numbers and epithelial stress; rt-qpcr was used to assess inflammatory gene expression. minimal inflammatory changes were present at og compared to published values at og stage in the cho lameness model including decreased mrna concentrations of cytokines (i.e. -fold increase in il- at og vs. -fold increase at og , no increase in il- b at og vs. -fold increase at og ), chemokines (no change in mcp- at og vs. fold increase at og , -fold increase in il- at og vs. fold increase at og ) and adhesion molecules (no change in e-selectin at og vs. -fold increase at og ). laminar leukocyte emigration was also decreased at the onset of og lameness compared to previously reported leukocyte infiltration at og . interestingly cox- , underwent a greater increase at og (approx. -fold) compared to that reported at og lameness ( -fold). finally, epithelial stress at og evidenced by cp ihc did not follow the uniform widespread distribution reported at og lameness, but instead was present in focal areas in which secondary epidermal laminae on either side of a common primary dermal vascular supply demonstrated increased cp signal. overall, laminar inflammation appears to be subsiding at og lameness, with epithelial stress possibly more dependent on vascular dysregulation instead of inflammatory events. the sustained increase in cox- , central to the induced production of vasoactive prostanoids in disease processes, may play a role in vascular dysregulation. this study was conducted to characterize clinical, laboratory and postmortem findings associated with oleander toxicosis in equids and to determine factors predictive of survival in these cases. retrospective analysis of medical records from our veterinary medical teaching hospital from january , to july , was completed. records of equids demonstrating detectable oleandrin in serum, plasma, urine or gastrointestinal fluid samples or detectable serum digoxin in the absence of pharmaceutical cardiac glycoside administration were included. descriptive statistics were used to evaluate the history, physical examination, and laboratory and postmortem data of affected individuals. logistic regression analysis was used to detect physical examination and laboratory factors significantly associated with survival. thirty equids met inclusion criteria of the study. three of subjects were dead on arrival or died immediately upon arrival ( %). of the remaining equids, % presented with gastrointestinal abnormalities, % were azotemic and % had cardiac arrhythmias. mortality was % for all subjects and % for those treated. the predominant cause for non-survival was cardiac dysfunction. factors significantly associated with survival included relatively decreased hematocrit and serum glucose, relatively increased serum chloride, absence of cardiac arrhythmias, and increased duration of hospitalization. equids with oleander toxicosis frequently present with gastrointestinal upset and may develop cardiac and renal disturbances. patients with cardiac arrhythmias and relatively increased hematocrit and serum glucose and decreased serum chloride are significantly less likely to survive. oleander intoxication is a differential diagnosis for colic in endemic areas, particularly with concurrent azotemia or cardiac dysrhythmia. the quantitative physicochemical approach emphasizes the importance of strong ions (na, k, cl, lactate), pco , and the plasma protein concentrations in determining plasma ph. serum concentrations of strong ions, proteins, and total co are reported on modern biochemical profiles. we hypothesized that the results of serum biochemical analysis can be used for acid-base interpretation in horses. the objective was to determine whether blood ph, anion gap, and strong ion gap could be quantitatively estimated and clinically used based on the results of serum or plasma biochemical analysis. horses ( adults and foals) presented to the isolation unit of our veterinary teaching hospital for suspected infectious diseases were prospectively enrolled. a venous serum sample was analyzed using a hitachi or copas c automated machine. measured parameters included strong ion difference (sid {na k}-{cl lac-tate}), total protein concentration (tp), and total co (tco ), with lactate being measured by blood gas analyzer. a second venous blood sample was collected into a na-heparin blood gas syringe and analyzed for ph (ph m ), pco and concentrations of na, k, cl, and lactate using a radiometer flex blood gas analyzer; sid was calculated from the measured values, and total solids (ts) were estimated using refractometry. serum/ plasma ph (ph calc ) was calculated using stewart's factor equation from the results of serum or plasma biochemical analysis, assuming pco mmhg for serum and pco accurate for plasma. anion gap (ag) was calculated as: ag (na k)-(cl tco ). strong ion gap (sig) was calculated as: sig . x[total protein, g/l]/ ( {pka-ph} )-ag. linear regression analysis was used to compare ph calc to ph m, as well as ag and sig to blood lactate concentrations. measured ph ranged from . to . ( . ae . ). measured sid from serum biochemistry (sid sb ) ranged from . to . meq/l ( . ae . meq/l) and sid from blood gas analyzer (sid bg ) from . to . meq/l ( . ae . meq/l; r . ; sid bg .  sid sb ). sid sb and sid bg showed small variability in measurements. tp ranged from to g/l ( . ae . g/l) and ts from - ( . ae . g/l; r . ; ts .  tp). using sid sb and tco values with constant pco , ph calc was poorly associated with ph m (r . ; ph calc . . ). in contrast, using sid bg with accurate pco , ph calc was closely associated with ph m (r . ; phcalc . . ) and the equation was not different from the line of identity. anion gap and sig (meq/l) calculated were significantly linearly correlated with lactate concentrations (mmol/l); ag .  [lactate] . (r . ), and sig À .  [lactate] . (r . ). we conclude that ph calc using sid sb , tco and constant pco values is not accurate. however, variability of measured biochemical parameters between machines was small, permitting use of serum biochemistry for clinical metabolic acid-base abnormalities interpretations of patients. these results reemphasize the importance of strong electrolytes and proteins in acid-base balance. metalloproteinases (mmps) are critically important in remodeling processes and in wound healing. however, excessive activation of mmps by pro-inflammatory mediators including cytokines, prostaglandin e , and nitric oxide lead to tissue breakdown. this is observed in osteoarthritis (oa) which is characterized by erosive lesions in articular cartilage. in hereditary equine regional dermal asthenia (herda), afflicted horses exhibit collagen abnormalities and can have associated chronic inflammation and aberrant wound repair. herda affects horses with quarter horse bloodlines and is similar to the human hereditary connective tissue syndrome ehlers danlos (eds). many adult eds patients suffer from joint pain and oa. we hypothesized that chondrocytes from articular cartilage of herda horses have increased activity of mmps. to test this hypothesis, chondrocytes were retrieved from articular cartilage of homozygous herda carpal and hock joints. chondrocytes from normal horses were also obtained for comparison. chondrocytes were seeded at x /ml into -well plates and incubated at c, % co for up to seven days. activity of secreted mmps was determined by zymography using equal amounts of proteins for loading. secreted mmps were analyzed by western blot. zymography showed that normal chondrocytes secreted two major bands with gelatinolytic activity observed at and kda suggestive of the latent form of mmp- and mmp- , respectively. less intense bands of gelatinolytic activity were observed at about and kda suggestive of the active form of mmp- and mmp- . another band of activity was also seen at kda which is suggestive of a dimer of mmp- that has been reported when mmps are in excess of tissue inhibitors of metalloproteinases (timps). chondrocyte cultures from homozygous herda cartilage showed a similar profile but with decreased activity by % at kda and - % increased activity at kda compared to normal chondrocytes. western blot analysis detected mmp- and mmp- immunoreactivity in chondrocyte culture media of herda-afflicted and normal horses. the present study demonstrates for the first time that horses suffering from herda have increased mmp activity which may predispose them to the development of lesions in articular cartilage. research supported by nutramax laboratories, inc. equine polysaccharide storage myopathy (pssm) type is a dominantly inherited glycogenosis caused by a mutation in the gene coding for skeletal muscle glycogen synthase type (gys- ). the disease has been reported to affect the haflinger breed but so far its prevalence is unknown. aim of this preliminary study was to estimate the occurrence of the gys- mutation in austrian haflingers and establish which of the seven haflinger sire lines appear mostly affected. gys- genotyping of randomly chosen haflingers was performed with a validated restriction fragment length polymorphism assay. resting and post-exercise muscle enzyme activities (creatine kinase (ck), aspartate aminotransferase (ast), lacate dehydrogenase (ldh)) and blood lactate concentrations were compared between horses with and without the mutation. among the horses were heterozygous (hr) carrier of the mutation. no homozygotes (hh) were identified. all horses with the gys- mutation were descendents of the a-or w-sire lines. the estimated hr prevalence was % ( % ci: . - . %). ck activity after exercise (p . ) was significantly higher in hr horses compared with horses not carrying the mutation (rr). ast activity was significantly higher in the hr group at rest and after exercise (p o . ). there was no statistically significant difference in resting ck, resting and post exercise ldh activity or blood lactate between hr and rr. results suggest that the prevalence of hr in the austrian haflinger population is higher than in the overall quarter horse population and might be as high as %, similar to some draft horse breeds. further research is needed to establish the prevalence within the different breeding lines. hereditary equine regional dermal asthenia (herda) is an autosomal recessive connective tissue disorder affecting quarter horse lineages. although a mutation in the gene encoding cyclophilin b has been genetically linked to herda, its causal association with the disease is not yet documented. previously, we demonstrated reductions in ultimate tensile strength (uts), modulus of elasticity, and energy to failure (toughness) of skin from many corporal regions of herda animals. given the presumed relationship between her-da and abnormal collagen structure, and the predominance of type i collagen in skin, we hypothesized that altered biomechanical properties would be detected in tendons which are rich in type i collagen. to evaluate this hypothesis we compared the uts, modulus of elasticity, and energy to failure of forelimb deep digital flexor tendons (dft) from six herda horses to six age-matched controls. isolated dft was secured and pulled to failure on an instron s universal testing instrument using purpose-built cryogenic clamps. analysis of variance was executed using sas . proc glimmix program (sas institute, ). p-values . were identified as significant. uts and modulus of elasticity were significantly lower in herda dft when compared with controls (p o . ); energy to failure did not differ between groups. these findings document abnormal biomechanics in herda tendon, leading us to postulate that lower uts and modulus of elasticity associated with the herda defect could convey a competitive advantage in the athletic disciplines in which this defect has segregated. (references on request). a proprietary herbal biocontamination product (bios) approved for cosmetic use in france, inhibits proliferation of medically relevant bacteria, mold, and viruses. these properties make bios potentially useful as a topical wound medication, prompting us to compare bios to silver sulfadiazine (ssd) in a distal extremity wound healing model in horses. using general anesthesia, two . cm wounds were aseptically created on the dorsomedial aspect of all limbs. for the duration of the study, two contralateral limbs were randomly chosen to be bandaged; the other two limbs were un-bandaged -with one limb of each group being treated with % bios and the other with ssd. for each limb the most proximal wound served as an untreated control. every hours wounds were evaluated, digitally photographed, and perimeter and area determined using morphometric software (imagej, nih). analysis of variance did not identify significant differences between ssd or bios treatment for wound perimeter (p . ) or area (p . ). at individual time points the effect of bandaging was significant when area was evaluated (p . ) and trended toward significance for perimeter (p . ) comparisons, substantiating published reports that bandaging modifies wound healing. difference in perimeter and area between control and treatment were highly significant (p o . ), substantiating the importance of topical treatment. over the study duration, effects of bandaging (p o . ) and topical treatment (perimeter p o . ; area p . ) continued to be highly significant. bios performance in the equine distal extremity wound model was equivalent to ssd. both bandaging and topical treatment significantly impacted wound healing. this effect was compounded when both variables were evaluated over time. radiolabeled leukocytes are the only scintigraphic method currently available for identifying sites of infection and/or inflammation in horses; however the clinical applicability of this technique is limited by expense and poor efficacy. this pilot study compares the accumulation of m tc-labeled igg, peg-liposomes and leukocytes in an equine muscle abscess model. three mixed breed adult horses had  cfu s. equi zooepidemicus inoculated into the right semitendonosis to create an abscess. peg-liposomes were prepared via the film hydration method and labeled using mci m tc-hexamethyl-propylene-amine-oxime ( m tc-hmpao). autologous leukocytes were obtained from ml whole blood and labelled using mci m tc-hmpao. commercial equine polyclonal igg was conjugated with the chelator hydrazinonicotinamide (hynic) and labelled with mci m tc. radiopharmaceutical administration was initiated hours after inoculation. horses and received mg m tc-igg, . mmol/kg m tc-liposomes and m tc-leukocytes, with a hour interval between each radiopharmaceutical. horse received only m tc-leukocytes. scintigraphic examinations were performed at and hours post injection (p.i.) with each radiopharmaceutical. after the final study, horses were euthanized and tissue samples collected. the percentage of injected dose per kilogram of tissue (%id/kg) was calculated for the region of the abscess, normal muscle and multiple organs. scintigraphic examinations demonstrated increased radiopharmaceutical in the region of the abscess with all three techniques at both time-points. at hours p.i. abscess-to-background ratio was highest using m tc-igg ( . ae . ). at hours p.i. abscess to background ratio was highest using m tc-liposomes ( . ae ). tissue biodistribution data revealed abscess to muscle ratios of ( m tc-igg), ( m tc-liposomes), and . ( m tc-leukocytes). this preliminary data demonstrates that m tc-liposomes, m tc-igg and m tc-leukocytes exhibit long circulating characteristics and accumulate at inflammatory/infectious foci after intravenous injection in horses. m tc-igg and m tc-liposomes appear to be superior to m tc-labelled leukocytes in this model. due to its low cost and ease of preparation, m tc-igg has great potential for clinical use where identification of infectious or inflammatory foci is necessary. digital hypothermia is used clinically to decrease the incidence of sepsis-related equine laminitis, a disease causing structural failure of digital laminae resulting in crippling lameness. due to the fact that hypothermia was recently reported to effectively decrease laminar expression of inflammatory molecules including pro-inflammatory cytokines, chemokines and cox- in equine laminitis, our laboratory is investigating the effect of hypothermia on central upstream signaling cascades which may induce expression of these diverse inflammatory molecules. the p mapk pathway has recently been reported to be a central component of inflammatory signaling in multiple diseases including human sepsis, and is currently being assessed as a therapeutic target. we thus hypothesized that ) p mapk is upregulated and activated in affected laminae in equine laminitis and ) digital hypothermia inhibits inflammatory mediator expression by blocking p mapk phosphorylation (indicator of p mapk activation). western hybridizations using both a total p mapk and a phospho-p mapk antibody were performed on archived pooled laminar samples from black walnut extract (bwe) model ( control, developmental (dev) groups [ . h & h post bwe administration] and the onset of obel grade lameness (og ) [n each]) and carbohydrate overload (cho) models (con [n ], dev [n ], og [n ]) of laminitis, and individual laminar samples from two groups of horses from a digital hypothermia (dh) study. in the dh study, one forelimb of each horse was kept at approximately c in ice water and the other at ambient temperature following administration of g/kg oligofructose (of). dorsal laminae were harvested for snap freezing at either hours after of administration (dev, n ) or at the onset of lameness (og , n ) using protein extracted from treated and untreated digital laminae of each horse. increased laminar concentrations of phospho-p mapk were present in the developmental periods ( . h and h) in the bwe model, and in both the dev and og periods in the cho laminitis models. however, digital hypothermia had no effect on laminar phospho-p mapk concentrations. thus, p mapk is activated in affected laminae in multiple models of laminitis, but does not appear to be the central signaling cascade through which hypothermia works to block the expression of inflammatory molecules. therefore, p mapk is not likely to be a viable therapeutic target as a sole source for blocking the multiple inflammatory signaling mechanisms inhibited by local hypothermia. abstract e- does cefquinome penetrate the blood brain barrier in the normal horse? hollis ar duggan ve and corley ktt . scott dunn's equine clinic, berkshire, uk; university college dublin, dublin, ireland; anglesey lodge equine hospital, the curragh, ireland. meningitis is a rare but serious condition that occurs in both foals and adult horses. there is currently a restricted choice of antimicrobials that are both safe to use in horses and penetrate the blood brain barrier. cefquinome is a fourth generation cephalosporin that has activity against streptococcus, the most commonly reported causative organism in adult horse meningitis. therefore, if cefquinome were to achieve therapeutic concentrations in cerebrospinal fluid following routine administration, this would be an exciting advance for the treatment of meningitis in the horse. mature, healthy horses were used on separate occasions, seven days apart, in a crossover design. each horse was administered either cefquinome ( mg/kg) or saline (equivalent volume). cerebrospinal fluid was collected via atlanto-occipital puncture under general anaesthesia and hours after administration of cefquinome or saline placebo. blood samples were collected prior to, and and hours after administration of cefquinome or placebo. all samples were analysed for the presence of cefquinome by a laboratory masked to treatments administered. cefquinome was detectable in the cerebrospinal fluid in all horses hours after intravenous administration, and in horses hour after administration. cefquinome penetrates the blood-brain barrier and it is therefore a potential treatment for equine meningitis. further investigation of the pharmacokinetics and pharmacodynamics of cefquinome in the cerebrospinal fluid is warranted to establish the optimum intravenous dose. the purpose of this study was to determine if enrofloxacin alters the pharmacokinetics of firocoxib in the horse. firocoxib is a coxibclass nonsteroidal anti-inflammatory drug (nsaid) approved for use in horses to control pain and inflammation associated with osteoarthritis. dosages of firocoxib are species dependent, with the recommended dose for horses being . mg/kg as an oral paste every h. the main elimination pathway of firocoxib is hepatic; however the effects of concurrent administration of drugs that may inhibit its metabolism have not been evaluated. enrofloxacin is a synthetic antibacterial agent from the flouroquinolone group developed for veterinary use. it is primarily used for gastrointestinal, urogenital, skin and respiratory tract infections in various animals. a well acknowledged problem associated with flouroquinolone usage is their effect on the metabolism of other drugs. co-administration of multiple drugs can result in unpredictable therapeutic outcomes. often it is either diminished therapeutic efficacy or increased toxicity of one or more of the administered drugs. various pharmacokinetic interactions between antimicrobials and nsaids have been described. six healthy, adult mares were administered . mg/kg of firocoxib orally. samples were collected by direct venipuncture of the jugular vein at (control), , , and min, , , , , , , , , and h after administration. after a day washout period the six horses were pretreated days with enrofloxacin mg/kg intravenously every h then on the fourth day given . mg/kg of firocoxib orally. samples were collected at (control), , , and min, , , , , , , , , and h after administration. all samples were stored at À c until analysis using a validated hplc method. the t / , c max , t max , auc - and auc -f after firocoxib administration were . angiotensin converting enzyme (ace) inhibitors improve survival and quality of life in humans and small animals with cardiovascular and renal disease. there is limited information regarding their effects in horses. the purpose of this study was to determine the pharmacokinetics of quinapril and its effects on ace inhibition in horses. six healthy horses were administered quinapril at mg iv, mg po or mg po in a -way crossover design. blood was collected at predetermined times for measurement of quinapril and quinaprilat concentrations using high pressure liquid chromatography, as well as ace concentrations using a radioenzymatic assay. normally distributed data were analyzed with one way repeated measures analysis of variance (rm-anova) and non-normally distributed data were analyzed using friedman rm_anova on ranks. significance was set at p o . . no adverse effects were observed during the study period. plasma quinapril concentrations were low and rapidly declined after iv administration. quinaprilat concentrations were below the limit of quantification ( . mg/ml). ace activity was significantly decreased from baseline at . and hour after iv dosing and at all timepoints after oral dosing. maximum % ace inhibition was , and % with the iv, high and low oral doses, respectively. these results suggest that, despite low plasma concentrations, quinapril has sufficient oral absorption and results in inhibition of ace in healthy horses. controlled studies in clinically affected horses are indicated. this study determined the pharmacokinetic profile of firocoxib in healthy neonatal foals. foals are more sensitive to the side effects of nsaid, primarily due to immature renal clearance mechanisms and ulcerogenic effects on gastric mucosa. firocoxib, a novel, second generation nsaid, is reported to have reduced side effects due to cox- selectivity. the pharmacokinetic profile of firocoxib in neonates has not been established. we hypothesized that firocoxib given po to neonatal foals would achieve therapeutic concentrations in plasma. seven healthy foals of mixed gender were administered . mg/kg firocoxib po q h for nine consecutive days, commencing at h old. blood was collected for firocoxib analysis at (dose # only), . , . , , , , , and h after doses # , and . for all other doses ( , , , , and ) blood was collected immediately prior to the next dose ( h trough). elimination samples were collected after dose # . plasma was stored at À c until analysis. physical examinations were performed on foals daily and body weight obtained every two days during the sampling period. analysis of plasma samples by liquid chromatography-mass spectrometry revealed firocoxib was rapidly absorbed. after the initial dose, a maximum plasma concentration was reached in min, minimal accumulation after repeat dosing occurred and steady state was obtained after approximately four doses. after the final dose, plasma drug concentration decreased in a linear manner with an estimated terminal t / of h. seventy-two hours after the final dose, firocoxib was not detectable (o ng/ml). erythrocytosis is reportedly a rare finding associated with hepatocellular carcinoma in horses. the purpose of this study was to determine the relative frequency of erythrocytosis and the clinicopathologic abnormalities and hepatic histopathology associated with erythrocytosis in horses with liver disease. ninety-seven horses aged ! year with clinicopathologic or clinical signs of liver disease, a complete blood count (cbc), and hepatic histopathology were included. information on cbc, biochemical variables, and hepatic histopathology was collected from records. data from horses with erythrocytosis (packed cell volume %) were compared to those without using the mann-whitney rank sum test with significance set at p o . . there were no differences between groups in white blood cell count, gamma-glutamyl transferase, sorbitol dehydrogenase, aspartate aminotransferase, and alkaline phosphatase activities, total protein, albumin, globulin, blood urea nitrogen, or glucose concentrations. fibrosis ( %), biliary hyperplasia ( %), inflammatory infiltrate ( %), megalocytosis ( %), degeneration ( %), necrosis ( %), cholestasis ( %), anisocytosis and anisokaryosis ( %), and lipidosis ( %) were observed in livers of horses with erythrocytosis. neoplasia ( %) was observed rarely. this study reports a high frequency of erythrocytosis in horses with liver disease. erythrocytosis is associated with higher total bilirubin and serum bile acids concentrations. common histopathologic changes include fibrosis, biliary hyperplasia, and inflammatory infiltrate. hepatic neoplasia was rare. this study was performed to determine if horses diagnosed with equine proliferative enteropathy (epe) from lawsonia intracellularis (li) infection had long term effects from disease based on their sale price as yearlings and race earnings. a retrospective review of medical records of thoroughbred horses that were treated for lawsonia intracellularis infection between january , and january , at hagyard equine medical institute in lexington, kentucky was performed. three criteria were used for inclusion in this study. first, each horse had presumptively been diagnosed with li based on physical examination findings such as ventral edema, diarrhea, lethargy, or poor body condition. second, horses had hypoalbuminemia of less than . mg/dl (normal reference range: . - . mg/dl). third, each horse had a positive fecal polymerase chain reaction (pcr) for li, a positive serum immunoperoxidase monolayer assay (ipma), or both. an ipma titer greater than or equal to was considered positive for disease. horses met the initial criteria. of the horses sold at public auction as yearlings. the sale price of these horses was compared to the average sale price of all yearlings by the same sire as the affected horse (control group). of the horses raced in the united states. their monetary earnings from racing were compared to the average monetary earnings of all progeny by the same sire as the affected horse (control group). earnings of horses that were between and years of age ( / horses) at the conclusion of the study were compared to the lifetime average earnings of the stallion's progeny. earnings from horses that were two years of age ( / ) at the end of the study were compared to the two year old average earnings of the stallion's progeny. monetary earnings from all races prior to december , were included in the study. horses both sold at public auction and raced. as well as being included in the total number of horses that sold and raced, their sale records and monetary earnings were compared to the averages from their respective sire as a separate group. this retrospective study indicated that yearling horses previously infected with li do not sell for as much at public auction as their herdmates, but their monetary earnings from racing are not significantly different from other horses. these results should assist practitioners in guiding owners in determing if treatment of horses with epe is appropriate and it may aid in reassuring owners that despite the poor condition of the horse during and shortly after the course of disease, horse may still have future athletic potential. this abstract was presented at the aaep in december . bronchopneumonia caused by streptococccus equi subsp. zooepidemicus (s. zooepidemicus) is one of the most important causes of morbidity in weanling foals. ceftiofur crystalline free acid (ccfa) is a long acting third-generation cephalosporin antimicrobial recently approved for the treatment of bronchopneumonia associated with s. zooepidemicus in adult horses. the objective of the present study was to determine the disposition of ccfa in plasma and pulmonary epithelial lining fluid (pelf) of weanling foals. six healthy -to month-old weanling foals were administered a single intramuscular injection of ccfa at a dose of . mg/kg of body weight. concentrations of desfuroylceftiofur acetamide (dca) and related metabolites were measured by use of ultra-high performance liquid chromatography and tandem mass spectrometry. following im administration, median time to maximum plasma and pelf concentrations was h ( - h) . mean (ae sd) peak dca concentration in plasma ( . ae . mg/ml) was significantly higher than that in pelf ( . ae . mg/ml). terminal half-life of dca in plasma ( . ae . h) was not significantly different from that of pelf ( . ae . h). time above the therapeutic target of . mg/ml was significantly longer in plasma ( ae h) than in pelf ( ae h). based on the results of the present study, intramuscular administration of ccfa at a dose of . mg/kg would be appropriate for the treatment of bronchopneumonia caused by s. zooepidemicus and other susceptible pathogens in weanling foals. fgf- is secreted by osteocytes and osteoblasts in response to hyperphosphatemia. fgf- enhances phosphaturia and is postulated to have a central role in the development of secondary renal hyperparathyroidism. hyperthyroid cats have elevated plasma phosphate and parathyroid hormone concentrations, which may in part be associated with underlying chronic kidney disease (ckd). the aim of this study was to determine if plasma fgf- concentrations were associated with the presence of underlying ckd in hyperthyroid cats, and to investigate the changes in plasma fgf- concentrations that occur following treatment of hth. hyperthyroid cats were recruited from two london-based first opinion practices between and . cats that were azotemic at diagnosis were excluded. hth was treated with anti-thyroid medication alone or in combination with thyroidectomy. cats were included in the study if they had a plasma total thyroxine concentration o nmol/l documented for a six month period following commencement of treatment. cats were classified as having azotemic ckd if they developed renal azotemia within six months of establishment of euthyroidism. otherwise cats were deemed to have normal renal function. stored edta plasma samples were assayed for fgf- using a recently validated elisa. the mann-whitney u test and the wilcoxon signed rank test were used to compare between the groups and assess the response to treatment respectively. results are reported as median [ th , th percentiles]. correlations were made using spearman's correlation coefficient. thirty one cats with hth ( azotemic and non-azotemic) were included in the study. plasma phosphate concentrations decreased following treatment in cats that did not develop azotemia ( . [ . , . ] mg/dl vs. . [ . , . ] mg/dl; n , p . ) whereas plasma phosphate concentrations did not change significantly following treatment in cats that did develop azotemia ( . [ . , . ] mg/ dl vs. . [ . , . ] mg/dl; n , p . ). plasma fgf- concentrations were significantly higher in cats that developed azotemia than cats that did not at both pre treatment ( . [ . , . ] pg/ml vs. . [ . , . ] pg/ml; p . ) and post treatment ( . [ . , . ] pg/ml vs. . [ . , . ] pg/ml; p . ) timepoints. plasma fgf- concentrations increased following treatment in both azotemic (p . ) and non-azotemic groups (p . ). plasma fgf- concentrations and plasma phosphate concentrations were not correlated at baseline (r s . , p . ) or following treatment (r s . , p . ). plasma fgf- concentrations were higher in pre-azotemic cats than non-azotemic cats and increased following treatment of hth. the reason that fgf- concentrations increased following treatment, particularly in the face of decreasing plasma phosphate concentrations in cats that remain non-azotemic, is unclear but may be related to the decline in glomerular filtration rate. hyperthyroidism is a disorder resulting from the excessive production and secretion of t and t by the thyroid gland. although the disorder and its pathological lesions have been well studied and described the cause remains illusive. whole blood and solid tissue samples from non-diseased, severe disease and mild disease cats based on t levels and thyroid histology were used in this study. whole blood samples from non-disease cats, severe disease cats and mild disease cats as well as solid thyroid tissue samples from non-disease cats, severe disease cats and mild disease cats were collected and processed. the resulting total rna samples were used for genechip analysis using our custom feline gene chip designed by affymetrix. data analysis was performed using the partek s gs software for gene expression data. the robust multichip average algorithm was used for background adjustment, normalization, and probe-level summarization of the raw data. anova analysis was performed to find significant differentially expressed genes with a minimal false discovery rate control of . and a fold change of . in each direction. during the mild disease state, pathways associated with dna damage and apoptosis are most prominent. at later stages when the histopathological disease is more severe in addition to the aforementioned pathways others associated with tgf-beta signaling, cell adhesion and extracellular matrix remodeling take more prominence. the analysis of this unique data set generated from the use of our proprietary genechip revealed molecular mechanisms that are associated with the transition from non-disease, to mild disease to severe disease, in the thyroid tissue as well as the blood. these mechanisms could provide insights into the causes of the disease and identify potential new therapeutic and diagnostic targets. although it is well established that concurrent chronic kidney disease (ckd) develops in about % of hyperthyroid cats, no one has reported the use of the iris staging system for ckd before and after treatment of these hyperthyroid cats. the purpose of this study was to compare the effects of treatment in hyperthyroid cats with known stage and ckd in order to determine the effects of restoring euthyroidism or inducing hypothyroidism has on the iris stage in these cats. we evaluated hyperthyroid cats (median age, years) in this study. one day prior to treatment, serum t concentration, serum chemistry analysis, complete urinalysis, and urine protein-to-creatinine ratio (upc) were measured. all cats were again evaluated with the same parameters again months after treatment with i. prior to treatment, ( %) of the cats had no evidence of azotemia (serum creatinine o . mg/dl), whereas cats ( %) had stage ckd (serum creatinine, . - . mg/dl). in the cats, iris staging revealed proteinuria in cats ( %), with borderline proteinuria (upc, . - . ) and with overt proteinuria (upc . ). hyperthyroidism was cured in all cats (median post-t , . mg/dl). all cats had a good response to treatment; there were no signs of ckd except for polyuria and polydipsia in some cats. a significant (p o . ) increase in median values for both serum urea nitrogen ( mg/dl to mg/dl)) and creatinine ( . to . mg/dl) occurred after treatment. nine of the cats ( . %) classified as nonazotemic or iris stage prior to i progressed to stage ckd after i. all cats with stage ckd before treatment remained azotemic after i, with cats remaining in stage ckd, and cats progressing to stage ckd (serum creatinine, . - . mg/dl). there was a significant inverse relationship (p . ) between pretreatment urine specific gravity (usg) and post-treatment serum creatinine in the cats. of the cats with post-treatment serum creatinine values . mg/dl (stage to ckd), ( %) had pretreatment usg of o . . in contrast, in the cats with post-treatment serum creatinine values o . mg/dl, only ( %) had pretreatment usg of o . . a significant (p o . ) decrease in median upc from . to . occurred after treatment, but there was no relationship between degree of proteinuria and iris stage in these cats. two cats developed iatrogenic hypothyroidism after i, diagnosed by finding low serum t and high ctsh concentrations. both hypothyroid cats had progressed from stage before treatment to stage and ckd, respectively, after i; after thyroxine replacement, serum creatinine decreased to near pretreatment concentrations in both cats. conclusions: ) iris stage ckd is common in untreated hyperthyroid cats. ) progression to next higher iris stage is common after treatment, but most cats with remain relatively asymptomatic for ckd. ) usg may be helpful in predicting which cat's iris stage will progress after i. ) iatrogenic hypothyroidism worsens azotemia, an effect that appears reversible with replacement therapy. home blood glucose monitoring (hbgm) of diabetic pets is likely to result in superior glycaemic control, minimizing episodes and impact of dangerous hypoglycaemia and reducing costs. nevertheless, it has proven difficult to objectively establish a clear benefit of hbgm using biological parameters (clinical signs, blood glucose, fructosamine). the current study aimed to assess the impact of hbgm on owner perceived quality of life (qol) aspects of diabetes mellitus (dm) treatment, using the recently validated psychometric tool diaqol-pet. owners of insulin treated diabetic cats were recruited to complete the -item tool, evaluating areas affecting the cat's and owner's qol, including: worry about pet's dm, hypoglycaemia, costs, owner's desire for autonomous control over the pet's dm, etc. item-weighted-impact-scores (iwis), reflecting frequency and importance ratings of each item, were calculated, as well as averageweighted-impact-scores (awis; average iwis of all items), as an overall measure of diabetes dependent qol. frequencies, iwis and awis were compared between owners practising hbgm and those who did not using mann whitney u test (significance p o . ). two hundred and eleven owners of insulin treated diabetic cats completed the diaqol-pet; owners practised hbgm, whereas the remaining did not practise any form of home monitoring (including urine glucose). iwis for 'excessive drinking' and 'owner wanting more control' were significantly different between the hbgm-group (mean /-standard deviation: À . /À . and À . /À . ) and the non-hbgm-group (À . /À . and À . /À . ). there was no significant difference between the groups with regards to the iwis for other items, including 'worry about hypoglycaemia' or 'worry about pet's dm'. polydipsia was reported significantly more frequently in the non-hbgm-group and this was the reason for the difference between groups in this item's iwis as it was considered of equal importance. frequency and iwis of reported occurrence of hypoglycaemia signs were not significantly different. awis for both groups was not significantly different (hbgm: À . /À . ; non-hbgm: À . /À . ). the current study suggests that hbgm is predominantly practised by owners who desire more autonomous control over their cat's dm. the frequency of polydipsia was lower in the hbgm-group perhaps suggesting superior control. however, hbgm did not detectably affect the impact of the majority of qol-items, nor the frequency of hypoglycaemic episodes. overall diabetes dependent qol of diabetic cat and owner, as measured per diaqol-pet, was unaffected by hbgm. these data argue for the use of hbgm in selected pet-owner combinations rather than as part of a practice's standard dm management protocol, although further studies are indicated. insulin resistance is associated with impaired activation of the insulin signaling pathway in peripheral tissues such as skeletal muscle, visceral and subcutaneous (sc) adipose tissue. high plasma glucose, fatty acid and endotoxin levels are three major causes of insulin resistance in feline and human obesity and in type diabetes mellitus. however, the mechanisms by which these factors influence insulin action are still unclear. therefore, our aim was to investigate the tissue-specific expression of crucial mediators of insulin action such as the insulin-receptor substrate (irs ), the serine/threonine protein kinase b (pkb/akt) and of the principal insulin-dependent glucose transporter protein (glut ) in feline models of hyperglycemia, hyperlipidemia and subacute endotoxemia. healthy cats were infused through the jugular vein with glucose (n ), lipids (n ) or lipopolysaccharide (lps; n ) for days to clamp their blood concentrations at the approximate level found in untreated feline diabetes (glucose: - mmol/l; triglycerides: - mmol/l) or to induce a systemic low-grade inflammation (lps; rectal temperature: . - . c), respectively. healthy control cats were infused with saline (n ). on day , specimens were collected from skeletal muscles, visceral and sc fat and processed for irs mrna expression, total and phosphorylated pkb/akt and glut protein expression. gene transcripts of irs were not different between the groups. compared to controls, skeletal muscle pkb/akt phosphorylation was % lower in cats infused with glucose (p o . ); lipid-infused cats showed a trend for a decrease in pkb/akt phosphorylation ( % lower than saline) and had decreased glut expression (p o . ) in muscle. total (p o . ) and phosphorylated (p o . ) pkb/akt protein expression were decreased in the sc adipose tissue of lps-infused cats compared to controls. in these cats, phosphorylation of pkb/akt protein was also decreased in visceral fat (p o . ). sustained hyperglycemia and, to a lesser extent, hyperlipidemia impaired insulin signaling and glucose transport pathways primarily in skeletal muscle; endotoxemia reduced insulin sensitivity mainly in adipose tissues. thus, the development of insulin resistance in response to hyperglycemia, hyperlipidemia or endotoxemia might be affected by tissue-specific mechanisms in cats. separately used, single photon emission computed tomography (spect) and computed tomography (ct) both lack sensitivity and are additionally hampered by a poor anatomical location capacity and a lack of specificity, respectively. these drawbacks suggest an interest in the fusion of images obtained by the techniques. the aim of this study is to test spect/ct fusion performance in dogs with insulinoma. inclusion criteria were: / a biological diagnosis of insulinoma; / an examination by high resolution ct scan and in-pentetreotide spect followed by spect/ct fusion; / a surgical or post mortem examination completed by histopathological analysis. spect examination showing abnormal foci and ct scan showing pancreas, lymph nodes (ln) or liver abnormalities were considered positive. in case of double positivity, presence (imp ) or absence (imp-) of superimposition of abnormal images was noted. ten dogs were included. in / dogs, superimposition of abnormalities couldn't be tested. ct scan detected abnormal images [ pancreatic nodules (pn), enlarged ln (eln)] while spect failed to show any abnormal uptake. both dogs became euglycemic after removal of pn and ln designed by ct scan. in / , all abnormal images were classified as imp [ pn, eln and diffuse hepatic infiltration (dhi)]. surgery performed on / resulted in euglycemia in ; dog remained hypoglycemic after partial removal of pn. pn localization and dhi were confirmed after necropsy in the th dog. in / dogs imp and imp-images were both recorded. in dog, a dhi was classified as imp but pn localization was imp-: localized in the left lobe by ct scan and in the corpus by spect, the latest localization being confirmed after necropsy. in the other dog pn localization was imp but a diffuse spect signal superimposing to the liver considered as normal on ct scan was noted. hepatic biopsy confirmed spect results. this study confirms an imperfect sensitivity of both ct scan and spect. it confirms that ct scan can be associated with unspecific abnormal images. subject to a confirmation on a larger cohort of dogs, it indicates that imp images provide specific detection and accurate localization of canine insulinomas' primary lesions and metastasis. the majority of dogs with primary hypoadrenocorticism (ph) reveal clinical and laboratory abnormalities of gluco-and mineralocorticoid deficiency. in some of them sodium and potassium levels are normal, a phenomenon currently called atypical addison's. it has been postulated that in those cases adrenal destruction is confined to the zona fasciculata/reticularis, resulting in isolated glucocorticoid deficiency. however, there are no histological studies confirming a normal zona glomerulosa and in most reported cases diagnosis was based solely on low post-acth cortisol levels. the aim of the study was to evaluate aldosterone (aldo) levels in dogs with ph with and without electrolyte abnormalities. seventy dogs with newly diagnosed ph were included. aldo concentrations (ria, coat-a-count s , siemens) were measured before and min after administration of mg synthetic acth (synacthen s , novartis) iv. results were compared to those of healthy dogs and dogs with diseases mimicking ph. to confirm that peak concentrations were not missed aldo was additionally measured , and min after acth in dogs ( with ph, with ph mimicking diseases). results were analysed by means of non-parametric statistical methods (p o . ). post-acth aldo was significantly lower in dogs with ph ( - pg/ml, median pg/ml) than in healthy dogs ( - pg/ml, median pg/ml) and in dogs with ph mimicking diseases ( - pg/ml, median pg/ml). low post-acth aldo was found in / dogs with ph, in / of them levels were below the detection limit of the assay. normal sodium and potassium levels were found in / dogs ( %), / dogs ( %) had hyponatremia and normal potassium, / dogs ( %) had hyponatremia and hyperkalemia. electrolyte abnormalities ranged from mild to severe. there was no correlation between post-acth aldo and sodium and a weak correlation between post-acth aldo and potassium (r À . ). aldo concentrations were not different , and min after acth. the results demonstrate that aldo levels are low in most dogs with ph independent of the degree of electrolyte abnormalities. this implicates that all three zones of the adrenal cortex are compromised and that there are mechanisms which allow maintenance of a normal electrolyte balance without aldo. definitive diagnosis of canine hypoadrenocorticism (ha) is based on inadequate cortisol secretion following adrenocorticotropic hormone (acth) administration. an abnormal serum sodium to potassium (na:k) ratio can be used to determine whether an acth stimulation test is warranted. the aim of this study was to examine the utility of combining the na:k ratio with white blood cell counts to determine whether an acth stimulation test is warranted. a retrospective review of medical records of dogs examined between and was performed. dogs diagnosed with ha and control dogs, in which a diagnosis of ha was excluded during the study period, were included. inclusion criteria for all dogs were hospitalization with intravenous fluid therapy, a complete blood count, and serum na and k measurements at the time of initial examination. dogs were included in the ha group if they also had pre and post acth stimulation serum cortisol concentrations . mg/dl. dogs were included in the control group if they had resting or post acth stimulation serum cortisol concentration . mg/dl. exclusion criteria were recent administration of glucocorticoids, prior treatment of hyperadrenocorticism, or serum cortisol concentration . mg/dl but . mg/dl. continuous variables were compared between groups using the mann-whitney u test. receiver operating characteristic (roc) curves were produced to assess the sensitivity and specificity of detecting ha with various cutoffs for each variable. data is presented with % confidence intervals (ci) and statistical significance was defined as p o . . the na:k ratio, neutrophil count and neutrophil:lymphocyte ratio were significantly lower in dogs with ha than in dogs without ha (p o . for each). lymphocyte and eosinophil counts were significantly higher in dogs with ha compared to dogs without ha (p o . for each). the areas under the curve by roc analysis were largest for na:k ratio ( . , ci: . - . ) and lymphocyte count ( . , ci: . - . ). a na:k ratio . was % sensitive (ci: - %) but only % specific (ci: - %) for detecting ha. a lymphocyte count ! . x cells/ml was % sensitive (ci: - %) and % specific (ci: - %). conversely a na:k ratio . was % sensitive (ci: - %) but % specific (ci: - %) and a lymphocyte count ! .  cells/ml was % sensitive (ci: - %) but % specific (ci: - %). a na:k ratio . was % sensitive (ci: - %) and % specific (ci: - %) for detection of ha and a lymphocyte count ! .  cells/ml was % sensitive (ci: - %) and % specific (ci: - %) for detection of ha. a combination of this na:k ratio ( . ) and lymphocyte count (! .  cells/ml) was % sensitive (ci: - %) and % specific (ci: - %) for detection of ha. these results indicate that the combination of lymphocyte count and na:k ratio results in a better screening test for ha than the use of the na:k ratio alone. pheochromocytoma is a malignant, catecholamine-producing, adrenomedullary tumor. clinical signs resulting from excessive catecholamine secretion are typically non-specific, making differentiation from other adrenal tumors a challenge. elevated plasma concentrations of the catecholamine breakdown products metanephrine (mn) and normetanephrine (nmn) are used to identify pheochromocytoma in humans. this study tested the hypothesis that plasma metanephrine concentrations are greater in dogs with pheochromocytoma than in dogs with other adrenal neoplasms, healthy dogs and dogs with non-adrenal illness. edta plasma was collected from healthy dogs and unwell, hospitalized dogs with non-adrenal illness, pheochromocytoma and cortical tumors between april and october . samples were stored at À c before measurement of free mn and nmn concentrations using high pressure liquid chromatography at the central laboratory for clinical chemistry at the university of groningen ( samples) or the mayo clinic, rochester, minnesota ( samples). kruskal-wallis tests followed by dunn's multiple comparison analysis were used to compare results between groups. significance was set at p o . . results are reported as median [range] . eight dogs with pheochromocytoma, healthy dogs, dogs with non-adrenal illness and dogs with cortical tumors were sampled. pheochromocytoma was diagnosed histologically ( dogs) or cytologically ( dog). cortical tumors were diagnosed histologically ( dogs) or by response to trilostane treatment after obtaining consistent endocrine test results ( dogs occult hyperadrenocorticism (hac) has been theorized to exist in which excess adrenal sex hormone secretion induces the clinical signs and laboratory changes associated with classic hac. however, the ability of sex hormones to cause such alterations has never been closely evaluated. if sex hormones can cause a syndrome similar to classic hac, they should be able to induce expression of classic glucocorticoid-induced genes. the purpose of the study was to determine if in vitro expression of the gene for corticosteroid-induced alp (cialp) could be induced by clinically relevant concentrations of cortisol and sex hormones believed to cause occult hac. canine hepatocytes were purchased from a commercial source (cellzdirect or invitro) in -well plates. upon arrival ( - plates per shipment), the cells were allowed to recover in general media per supplier recommendations. after hrs, media was changed to william's e media (-l-glutamine) containing concentrations of cortisol or sex hormones that have been documented in the literature in dogs with hac or with purported occult hac. each plate was treated with a different hormone (cortisol, -hydroxyprogesterone [ ohp], progesterone, estradiol or androstenedione), and each well contained a different concentration (starting with no hormone added as a negative control) to evaluate a dose response. media was changed daily. after days of hormone exposure, rna was extracted. reverse transcription was performed and the product used for quantitative pcr for cialp and beta-actin (roche lightcycler) using a gene-specific fluorescent probe for detection. standard curves were created for each gene. all samples and standards were run in duplicate. using the lightcycler software (vers . ), cialp expression was normalized to that of beta-actin. fold change in expression was determined relative to the negative control. each sex hormone was used to treat plates; one plate in each shipment was treated with cortisol as a positive control. for cortisol, a dose response was seen in expression of the cialp gene. compared to no cortisol, , , , , and nmol cortisol increased expression . , . , . . . , . and . fold, respectively. a -fold increase is considered significant (j.vandesompele et al genome biol ). expression of cialp was not significantly induced in response to any concentration of ohp ( nm maximum), progesterone ( nm maximum), estradiol (max pm maximum) or androstenedione ( nm maximum). we conclude that in vitro these sex hormones do not induce expression of the cialp gene which is classically induced by cortisol in vivo; indeed, elevated serum cialp activity is a hallmark of classic hac. thus, the ability of the sex hormones to induce the gene in vivo must be questioned and evaluated. measurement of sex hormones has been advocated as an adjunct means for diagnosing typical hyperadrenocorticism (hac), i.e. disease due to excess cortisol secretion, as well as for diagnosis of atypical hac, i.e. disease due to excess adrenal sex hormone secretion. however, measurements in either setting have not been widely studied. therefore, our objectives were: . to determine the sensitivity of -hydroxy-progesterone ( ohp) and estradiol concentrations pre-and post-acth for diagnosis of typical hac. . to determine the specificity of ohp and estradiol concentrations preand post-acth for diagnosis of occult hac. dogs that had pdh (n ), dogs that were suspected to have hac but proven not to (had non-adrenal illness [nai, n ]) or dogs that were healthy (n , used to establish reference ranges [rr]) were enrolled. acth stimulation tests were performed ( mcg/kg cosyntropin iv); blood samples were drawn pre and min post; ohp and estradiol were measured by previously validated radioimmunoassays. a kruskal-wallis rank sum test was used to compare values between the groups. significance was set at p o . . for basal and acth-stimulated ohp concentrations, the rr were determined to be . - . ng/ml (mean ae s.d.; range . - . ) and . - . ng/ml (range . - . ), respectively. in pdh dogs, and had basal and post-acth ohp concentrations above the rr, respectively; in the nai group, and dogs had concentrations above the rr, respectively. thus, the sensitivity of basal and post-acth ohp measurement for diagnosis of hac is % and %, respectively. specificity of diagnosis is % and %, respectively. post-acth ohp concentration was significantly different between groups. for basal and stimulated estradiol concentrations, the rr were determined to be - pg/ml (range - ) and - pg/ml (range - ), respectively. for both basal and stimulated estradiol, pdh dogs (n ) had concentrations above the rr; for those with nai (n ), and had concentrations above the rr, respectively. thus, the sensitivity of estradiol measurement for diagnosis of hac is % for both pre-and post-acth. specificity of estradiol for diagnosis for hac is % and % for pre-and post-acth, respectively. overall, dogs with nai had at least one elevated estradiol concentration (total specificity %). post-acth estradiol concentration was not significantly different between groups. we conclude that use of ohp and estradiol concentrations for diagnosis of hac can be problematic. sensitivity and specificity are relatively low, potentially leading to misdiagnoses. diabetes mellitus is one of the most common feline endocrinopathies and is considered to have a similar pathophysiological basis to human type diabetes. several studies have identified risk factors for development of diabetes mellitus in cats, which include age, obesity, inappropriate diet and physical inactivity. however, to date, no specific genetic risk factors have been identified. genome-wide association studies in humans have identified several genes that predispose to obesity and/or diabetes mellitus, one of which is the melanocortin receptor (mc r) gene. the aim of the current study was to identify polymorphisms (snps) in the feline mc r gene and to use these to perform a case:control study to determine whether these candidate gene snps were associated with diabetes mellitus in cats. genomic dna from cats ( domestic short hair [dsh], burmese) was initially analysed by pcr and direct sequencing using felmc r-specific primers, which identified a missense mutation (mc r:c. c t) in the region encoding the extracellular domain of the receptor protein in dsh cats only. one hundred and nineteen dsh cats were subsequently recruited into the case:control study. fifty nine cats were obese diabetic ( male, female), mean age . years (range - y); mean weight . kg (range . - kg). sixty lean cats were used as controls ( male, female), mean age . years (range - y), mean weight . kg (range . - . kg). the t to c base change alters a restriction site in the sequence recognized by the enzyme bstoi, such that dna from cats with the mutant (c) allele can be cut, whereas that from the wild-type (t) allele cannot. primers were designed that flanked the mutation to allow pcr amplification of this region of mc r from genomic dna obtained from edta blood. the pcr products were purified and subject to restriction fragment length polymorphism (rflp) analysis. bstoi digestion products were then analysed by agarose gel electrophoresis. of the diabetic cats, ( %) were homozygous for the mutation (cc), compared to ( %) of control cats. statistical analysis (two tailed fisher's square test) revealed that this difference between groups was statistically significant (p . ). in conclusion, this pilot study has identified a missense mutation in the coding sequence of mc r. this could be an important predisposing factor for development of diabetes and/or obesity in dsh cats. polymorphisms in a similar region of human mc r predispose to obesity, which in turn is a major risk factor for type diabetes. hyperadrenocorticism (hac) is one of the most common endocrine disorders of dogs. the two most effective medical treatments are trilostane (vetoryl s ) and mitotane (lysodren s ). previous studies evaluating the effect of treatment on aldosterone secretion measured the hormone at min post-acth administration. however, the optimal sampling time would be at the time of maximal secretion, which occurs minutes after the mg/kg dose commonly used for the test (carlson et al, jvim, ). thus, the true effect of either medication on aldosterone secretory capacity is unknown. our objectives were: ) to assess and compare the effect of treatment with trilostane and mitotane in dogs with pituitarydependent hac (pdh) on aldosterone secretory reserve at min post-acth stimulation and ) to determine if changes in aldosterone concentration at that time correlate with changes in serum sodium and potassium concentrations. forty-six dogs being treated for pdh with either mitotane (n ) or trilostane (n ) have been enrolled. the dogs could be treated for any length of time. all had acth stimulation tests performed ( mcg/kg cosyntropin iv); blood samples were drawn before and at and min post-acth for monitoring of cortisol and aldosterone concentration using previously validated radioimmunoassays. ten historical normal controls were also included. serum sodium and potassium concentrations were measured in the basal samples. a kruskal-wallis rank sum test was used to compare values between normal dogs and those treated with mitotane or trilostane. linear regression analysis was used to determine if a correlation existed between electrolyte and aldosterone concentrations or between cortisol and aldosterone concentrations. significance was set at the p o . level. acth-stimulated aldosterone concentrations in mitotane-treated but not trilostane-treated dogs were significantly lower than that in normal dogs at both the and min time points. no difference was detected between aldosterone concentrations at and min after acth injection in either treatment group. a positive correlation existed between the -min cortisol and -min aldosterone concentrations in the trilostane-treated group (r . ), i.e. the peak post-acth concentration for each hormone, but not in dogs treated with mitotane. basal serum sodium and potassium concentrations were not correlated with the basal aldosterone concentration in either treatment group. in conclusion, treatment with mitotane resulted in decreased aldosterone secretory reserve, but this did not correlate with hyperkalemia or hyponatremia. measurement of aldosterone concentrations is not predictive of electrolyte concentrations. previously presented at the auburn university phi zeta research emphasis day, november , . antioxidant depletion is documented in humans with hyperthyroidism, and is reversible with treatment. in addition, antioxidant depletion has been shown to increase the risk of methimazole toxicity in rats. the primary aim of this study was to determine whether deficiencies in glutathione (gsh), ascorbate (aa), or vitamin e, along with increases in urinary -isoprostanes, were present in hyperthyroid cats, and were reversible after radioiodine treatment. a secondary aim was to determine whether antioxidant abnormalities were associated with a prior history of methimazole toxicity. ongoing prospective, controlled, observational study. otherwise healthy client-owned hyperthyroid cats presenting for radioiodine therapy (n to date) and healthy age-matched controls (n to date) were recruited. all cats were screened with cbc, biochemical panel, urinalysis, and t , as well as red blood cell (rbc) gsh, plasma aa, plasma vitamin e, and urinary -isoprostanes. hyperthyroid cats were re-evaluated months after radioiodine treatment. unlike in humans, median blood antioxidants were not significantly different in hyperthyroid cats (gsh . mm; aa . mm, and vitamin e, g/ml) compared to controls (gsh . mm; aa . mm, and vitamin e, g/ml). results for urinary isoprostanes are pending, and associations with methimazole toxicity will be investigated after full recruitment. rbc gsh concentrations did increase significantly (to . mm; p . ) after radioiodine treatment. however, this modest change is unlikely to be clinically significant. preliminary data do not indicate clinically significant blood gsh, ascorbate, or vitamin e deficiencies in hyperthyroid cats. with appropriate insulin therapy and a low carbohydrate diet, up to % of newly diagnosed diabetic cats are eventually able to maintain euglycemia without insulin administration, and these cats are considered to have achieved remission. there are currently no published data reporting the glucose tolerance status of cats classified as being in remission, and it is unknown whether these cats are truly in diabetic remission, or should be classified as non-insulin dependent diabetics, or having impaired glucose tolerance, and/or impaired fasting blood glucose. the aim of this study was to determine fasting blood glucose concentrations and glucose tolerance status of cats in remission. the study was a prospective study in a feline-only clinic. for inclusion, diabetic cats had to have achieved remission through insulin therapy, and insulin withheld for a minimum of two weeks. five diabetic cats in remission and five matched non-diabetic cats were enrolled in the study. blood samples were obtained via the ear vein but where the cat's temperament precluded this, from the jugular.glucose concentration was measured using a meter calibrated for feline blood (abbott alphatrak). a simplified glucose tolerance test was performed after food was withheld for hours. a g catheter was placed in a cephalic vein three hours before the gtt was commenced, to minimize the effects of stress on blood glucose concentration. blood glucose concentration was measured at time and then a g/kg dose of glucose was administered slowly via the intravenous catheter. further blood glucose measurements were made at hours and then hourly until glucose had returned to o mg/dl (o . mmol/l). in the control group, all cats had a fasting blood glucose below mg/dl, and following glucose administration, glucose had returned to o mg/dl by hours. fasting blood glucose in the remission group was o mg/dl ( mmol/l) in all cats except one, which had fasting blood glucose of mg/dl ( . mmol/l). following glucose administration, all five cats in remission had blood glucose above mg/dl ( . mmol/l) at three hours, four were o mg/dl at four hours, and one returned to o mg/dl at five hours. the cat with impaired fasting glucose subsequently became diabetic after steroid administration. the results of this study show that these cats, while no longer diabetic, have mildly impaired glucose tolerance compared to nondiabetic cats, and a minority have impaired fasting glucose. the objective of this study was to determine the role of iodine restriction in the nutritional management of cats with naturally occurring hyperthyroidism. five domestic shorthair cats ranging in age from - years were confirmed to have hyperthyroidism based on persistently increased serum total thyroxine concentrations (tt ), palpable thyroid nodule and weight loss. serum tt concentrations ranged from - nmol/l (reference range - nmol/l). the cats were then fed a low iodine containing food ( . ppm iodine dmb, as measured by epiboron neutron atomic activation). serum tt concentrations were measured every weeks. biochemistry parameters were also evaluated at weeks , and . at weeks, serum tt concentrations had decreased in all cats with of cats ( %) being euthyroid (mean nmol/l; range - nmol/l). the remaining hyperthyroid cat had an initial serum tt of nmol/l, which decreased to nmol/l after being fed the iodine-restricted food. mean decrease in tt for all cats was nmol/l (range - nmol/l). renal parameters remained stable in all cats. these cats along with additional newly diagnosed hyperthyroid cats were transitioned to a similar food that contained less iodine ( . ppm dmb). baseline serum tt concentrations in the new cats ranged from - nmol/l. serum tt and other biochemical parameters were monitored every weeks for weeks, and then every weeks for an additional weeks. with the . ppm iodine food the four new cats became euthyroid with a mean tt concentration of nmol/ (range - nmol/l). the euthyroid cats from the earlier feeding study had a further decrease in tt concentration (mean tt nmol/l, range - nmol/l). the single non-euthyroid cat from the first study had a serum tt concentration of nmol/l, a decrease from the baseline concentration of nmol/l. the average decrease in serum tt for all cats was nmol/l (range - nmol/l). finally, of the cats were fed a third iodine-restricted food ( . ppm dmb) along with one other newly diagnosed hyperthyroid cat ( nmol/l serum tt ) and evaluated every weeks. all cats in this evaluation were euthyroid (mean tt nmol/l; range - nmol/ l). this result included the cat whose serum tt remained in the hyperthyroid range in the first two evaluations. the average decrease in tt was nmol/l (range - nmol/l). biochemical features of renal function remained stable and no other biochemical abnormalities were observed. in summary, the results of these three feeding studies demonstrate that feline hyperthyroidism can be managed effectively with dietary iodine restriction. we have shown previously that restriction of dietary iodine (i) is a safe and effective method for decreasing serum thyroxine concentrations (tt ) in cats with hyperthyroidism. the objective of this study was to determine the maximum level of iodine in a nutritionally balanced feline mature adult food required to maintain normal serum tt concentrations in hyperthyroid cats currently being controlled on a food containing . ppm i (dmb) as measured by epiboron neutron atomic activation. all cats were previously diagnosed at least months prior to the start of the study and their tt concentrations were maintained in the normal range by dietary iodine restriction for a minimum of months (range months- years). serum tt concentrations ranged from - nmol/l (reference range - nmol/l) at the beginning of the study. the cats were divided into two groups each containing cats. groups were similar in age and gender distribution (mean age . years, range - years). one group (group a) was placed on a food that was formulated for mature adult cats containing . ppm i (dmb). the other group (group b) was placed on a similar food that differed only in that it contained . ppm i (dmb). blood was collected from all cats every three weeks and analyzed for serum tt concentration. biochemistry parameters were also evaluated at weeks , and . all group a cats exhibited increases in serum tt concentration (mean increase of nmol/l above baseline, range - nmol/l). seven of the cats remained in the euthyroid range (mean serum tt nmol/l, range- - nmol/l). two cats exceeded the upper limit of the reference range ( and nmol/l respectively). the cats in group b also exhibited increases in serum tt concentration but to a greater degree than the cats in group a (mean increase nmol/l, range - nmol/l). four cats remained in the euthyroid range (mean serum tt , range - nmol/l). the five remaining cats all exceeded the upper limit of the reference range (mean serum tt nmol/l, range- - nmol/l). all cats returned to a euthyroid state within month of being returned to a diet containing . ppm i (dmb). it was determined that serum tt concentrations are not ideally controlled in the normal range in hyperthyroid cats fed a food containing ! . ppm i (dmb). hyperthyroidism is a common disease in old cats. excessive production of thyroid hormones is the hallmark of the disease. three main treatments for feline hyperthyroidism include radioactive iodine, thyroidectomy, and antithyroid drugs such as methimazole. previously we have shown that limiting dietary iodine to or below . ppm induces euthyroidism in cats with hyperthyroidism compared with a similar diet containing . ppm iodine. the objective of this study was to test whether dietary iodine at . ppm would induce euthyroidism in cats with naturally occurring hyperthyroidism. fourteen cats with hyperthyroidism confirmed by serum tt and ft measurements were stratified into two groups based on gender and age. one group (control: males and females, age ranged from to years) was given a positive control dry cat food ( . ppm iodine) while the other group (test: males and females, age ranged from to years) was fed a commercial dry cat food ( . ppm iodine) for at least weeks before the study. afterwards (week ), the control cats continued to receive the same food while cats in the test group were given a test food ( . ppm iodine) for additional weeks. all cats had free access to their food and deionized water during the study. blood samples were collected during weeks , , , and of the study. the control cats maintained euthyroidism during the study. the test food significantly reduced serum tt ( ae , ae à , ae à , ae à nmol/l in weeks , , and , respectively; à : p o . compared with week , dunnett's t test). it also significantly reduced ft at the end of the study ( ae vs. ae pmol, week vs. week ; dunnett's t test, p o . ). serum ft was within the reference range ( - pmol/l) in cats in both groups. serum tt , ft , and tsh were not affected by the test food and were within the reference ranges (tt : . - . nmol/l, ft : . - pmol/l, and tsh: - mu/l) in cats of both groups during the study. this study demonstrates that dietary iodine at or below . ppm provides an effective and inexpensive therapy for cats with naturally occurring hyperthyroidism. radioactive iodine ( i) is a widely used treatment for feline hyperthyroidism. prior to i administration, many cats receive methimazole therapy. it has been suggested that recent withdrawal of methimazole prior to i may increase the risk of hypothyroidism, inhibit the response to therapy, or have no effect. to further address this question, a retrospective medical records search was performed to identify hyperthyroid cats that received i therapy after methimazole treatment. inclusion criteria included documentation of the time interval between discontinuation of methimazole and i administration, and measurement of thyroxine (t ) at - days after i. cats were divided into groups: those receiving i within day of stopping methimazole, and those receiving i treatment or more days after stopping methimazole. sixty cats met the inclusion criteria. forty received i within day of stopping methimazole. of those, ( %) had a low t (o . mcg/dl), ( . %) had a normal t ( . - . mcg/dl), and ( . %) had an elevated t ( . mcg/dl) at - days after i therapy. fourteen cats received i or more days after stopping methimazole: ( %) had a low t , ( %) had a normal t , and ( %) had an elevated t at - days after i therapy. the results were compared with a fisher's exact test and there was no difference between the groups (p . ). these findings indicate that stopping methimazole therapy within day of i therapy does not inhibit the response to therapy. pharmacokinetic studies evaluating synthetic insulin analogs such as glargine necessitate the ability to measure the blood concentrations of glargine without cross-reactivity to endogenous insulin. although the cross-reactivity between endogenous human insulin assays and synthetic analogs is often known for commerciallyavailable assays, the degree of cross-reactivity of human insulin assays with feline insulin is not. the purpose of this study was to evaluate the cross-reactivity of feline insulin with a commerciallyavailable human insulin elisa with known cross reactivity to several synthetic analogs. pre-and post-prandial blood samples were collected from four healthy cats immediately prior to and approximately minutes following a meal, for a total of samples. dextrose was added to the meals given to two of the cats. blood samples were immediately centrifuged and the serum was collected, aliquoted, and stored at À c until analysis. serum insulin levels were determined in parallel with commercially-available feline insulin and human insulin elisas. the elisas were run in duplicate and according to the manufacturer's instructions. concentrations of serum insulin measured by the feline insulin elisa ranged from . ng/l to ng/l. despite the wide range of concentrations of feline insulin, all samples evaluated with the human insulin elisa yielded absorbance readings equal to or lower than the absorbance of the negative control, indicating no crossreactivity between the evaluated human insulin assay and feline insulin. since this assay is reported to cross-react significantly with glargine, it is a great candidate for determination of serum glargine concentrations in cats. the aim of this prospective, controlled study was to compare the efficacy of two trilostane protocols for treatment of canine pituitary-dependent hyperadrenocorticism (pdh). among the client-owned dogs diagnosed with pdh, only the dogs weighing o kg were selected (n ). group a (n ; low-dose treatment group) and group b (n ; high-dose treatment group) received . ae . mg of trilostane/kg orally every hours and mg of trilostane/ body orally every hours, respectively. all of the dogs were reassessed at , , , and weeks after the initiation of treatment. the improvement in post-acth stimulation serum cortisol concentration, as well as clinical signs in group a, required more time than group b; however, of dogs in group b had clinical signs and abnormal laboratory findings consistent with hypoadrenocorticism after treatment for weeks. twenty-four weeks later, all of the dogs of both groups improved the abnormal clinical findings. the present study suggests that twice daily, low-dose administration of trilostane is effective in the management of canine pdh and may be safe without the potential adverse effects of once daily, high-dose treatment. however, because this study involved only a small number of dogs, a population-based control study will be needed to clarify the efficacy of low-compared to high-dose trilostane treatment. cobalamin is essential for a variety of metabolic processes in many tissues and organs, and has effects on cell growth and peripheral and central nervous system function. chronic distal small intestinal disease in humans, cats, and dogs has been shown to cause cobalamin deficiency. an immunoassay for the measurement of serum cobalamin concentration in these species is being used in routine practice for the diagnosis of cobalamin deficiency. in pigs, the role of cobalamin has not yet been extensively investigated. thus, the aim of this study was to analytically validate an immunoassay, labeled for use in humans, for the measurement of cobalamin in porcine serum samples and secondly to determine serum cobalamin concentrations in weaned pigs. for the analytical validation of the assay, serum cobalamin concentrations were measured using the commercially available immulite s cobalamin immunoassay (siemens healthcare diagnostics ltd., deerfield, il, usa) in surplus porcine serum samples from a variety of studies. validation of the assay consisted of determination of dilutional parallelism, spiking recovery, and intra-and inter-assay variability. additional surplus serum samples from piglets from four litters at a texas a&m university farm were obtained. each piglet had been bled twice, the first at weaning ( days of age) and the second one days later. to investigate results in comparison between age groups, serum cobalamin concentrations were compared using a wilcoxon matched pairs test. significance was set at p o . . observed to expected ratios (o/e) for serial dilutions ranged from . to . % (mean ae sd: . ae . %) for four different serum samples at dilutions of : , : , and : , and from . to . % (mean ae sd: . ae . %) for one serum sample at dilutions of : , : , and : . o/e for spiking recovery ranged from . to . % (mean ae sd: . ae . %) for five different porcine serum samples that had been spiked with each other in a : dilution. intraassay coefficients of variation (%cv) for five different serum samples were . , . , . , . , and . %. inter-assay %cvs for five different serum samples were . , . , . , . , and . %. serum cobalamin concentration was significantly lower in piglets post weaning (median: ng/l) compared to those at the time of weaning (median: ng/l; p . ). the immulite s cobalamin immunoassay labeled for use in humans is linear, accurate, precise, and reproducible for measurement of serum cobalamin concentrations in pigs. this study also showed that piglets that differ in age by only days have significantly different serum cobalamin concentrations. further investigations of cobalamin concentrations in both sows and piglets at different stages of weaning are warranted. primigravid dairy heifers can be infected with mastitis pathogens during the periparturient period. the prevalence of intramammary infection (imi) ranges from - % of quarters pre-partum and - % at parturition. some pre-partum infections self-cure before parturition, however a number of these imis persist into early lactation. these imis may impact milk production and quality and may serve as a reservoir for contagious pathogens. no study has specifically investigated the risk of an imi persisting from the prepartum period into early lactation. the objectives of this study were to describe the prevalence of mastitis pathogens in heifers on a grazing dairy before and after parturition and calculate the relative risk (rr) and attributable fraction of population (afp) for the association between a post-partum and pre-partum imi. two-hundred-ninety-four heifers were systematically assigned to of groups: g ) pre-partum secretions from all mammary quarters (n ), g ) no pre-partum secretions collected (n ) and g ) pre-partum secretions from two diagonal quarters (n ). group assignments were designed to assess whether pre-partum sampling increased the likelihood of imi at calving. mammary quarter secretions were collected for bacterial culture approximately weeks prior to expected calving date. quarter milk samples were collected for bacterial culture once weekly during the st -weeks of lactation. bacterial isolates were classified as staphylococci, non-agalactiae streptococci and gram-negatives. mammary quarter samples yielding different bacteria were classified as mixed infections and those yielding ! bacterial types were classified as contaminated. bacterial isolates were speciated using gene sequencing methods and strain-typed using pulse-field-gel-electrophorysis to evaluate the relatedness of bacteria isolated from pre-and post-partum samples from the same mammary quarter. relative risk and afp were calculated using  tables. forty-five percent of mammary quarters had a pre-partum imi. during the st weeks of lactation the mean prevalence of imi was . % of quarters. staphylococci were most frequently isolated bacteria from pre-partum secretions and milk with s. chromogenes and s. aureus being the most common species. using data from mammary quarters, the rr and afp for the association between a post-partum and pre-partum imi were and %, and %, and and % for all staphylococci, s. aureus only and cns only imis, respectively. mammary quarters sampled pre-partum were no more likely to have a post-partum imi than those not sampled (chisquare, p ! . ). these data demonstrate that pre-partum imis persist into early lactation and that pre-partum secretion cultures may be a useful, not only in predicting imi at calving, but also in assessing risk of introducing new contagious mastitis pathogens, e.g., s. aureus, into the lactating herd. despite concerns about antimicrobial resistance and clostridium difficile in food animals, there has been little study of the prevalence or mechanisms of resistance. this study evaluated the impact of tetracycline treatment on c. difficile shedding in veal calves and the impact on resistance. calves arriving on veal farm received oral oxytetracycline for days as per farm protocols. calves were sampled at arrival and days later. selective culture for c. difficile was performed. isolates were ribotyped, and tested for tetracycline susceptibility and the presence of tetracycline resistance genes. multivariable logistic regression models were used to determine the relationship between tetracycline resistance and the presence of tetracycline resistance genes. clostridium difficile was isolated from % ( / ) and % ( / ) calves, at the first and second samples, respectively. the percentage of tetracycline resistant isolates increased from % to %. isolates from the second sample were times more likely to be tetracycline resistant (p . ) and times more likely to possess tet(m) (p . ). tet(m) was detected in % ( / ) and % ( / ), tet(o) in % ( / ) and % ( / ) and tet(w) in % ( / ) and % ( / ) of isolates from first and second samples, respectively. tet(l), tet(k) and tet(s) were not detected. resistant isolates were not carrying any of the genes investigated. routine tetracycline use may have had an impact on both the prevalence of c. difficile, as well as the strain distribution and resistance patterns. this is the first report of presence of tet ( the objectives of this study were to ) estimate the prevalence of antimicrobial resistance in the study population and ) to investigate the associations between exposures to antimicrobial drugs and antimicrobial resistance in fecal non-type specific e. coli (ntsec) recovered from individual feedlot cattle. two-stage random sampling was used to identify cattle for enrollment at western canadian feedlots. a fecal sample was collected per rectum from each individual at arrival and in the middle of the feeding period when cattle were rehandled as part of standard feedlot protocol. from samples collected at this second time point, a total of , ntsec isolates were tested for susceptibility to antimicrobial drugs by disk diffusion. parenteral and in-feed exposures to antimicrobial drugs were recorded for each individual enrolled in the study. the least square means estimates and % confidence intervals for the prevalence of resistance at each time point were modeled using poisson regression. multivariable logistic regression was used to investigate associations between antimicrobial resistance and exposure to antimicrobial drugs. regression models were adjusted for clustering of observations among individuals and pens. the most common resistances identified in arrival samples were sulfisoxazole ( . %; %ci: . - . ), streptomycin ( . %; %ci: . - . ) and tetracycline ( . %; %ci: . - . ). at the second sampling point, resistance prevalence was . % ( %ci: . - . ) for sulfisoxazole, . % ( %ci: . - . ) for streptomycin, and . % ( %ci: . - . ) for tetracycline. logistic regression modeling identified weak associations of exposures to tetracycline and macrolide classes of drugs with antimicrobial resistance at the second time point. abstract fa- premature/dysmature syndrome in cria: a ret-rospective study of cases ( ) ( ) ( ) ( ) ( ) ( ) ( ) . c. gerspach, d. anderson. the ohio state university, columbus oh. prematurity is widely acknowledged as risk factor for subsequent morbidity and mortality in llama and alpaca cria. a review of medical records for premature cria alive at the time of admission to the veterinary teaching hospital between and was performed to determine risk factors of prematurity and to report the outcome and related conditions or diseases in affected cria. medical records for premature or dysmature cria were included in this study. of these cria, were alpaca and llama, were female and were male. reasons for referral were prematurity, failure of passive immunity, dyspnoea, weakness and failure to gain weight. cria were presented at a mean age of . days and were premature by a mean estimated time of . days. overall survival rate was . %, with all llama cria surviving. a multivariate logistic regression model was used to identify risk factors associated with not surviving. cria receiving camelid colostrum had a significant better outcome than cria receiving no colostrum or colostrum from different species. dyspnea and tachypnea was associated with a poor outcome. all cria that were able to nurse, without assistance prior to referral, survived. clinical pathology parameters most commonly associated with death were hyperphosphatemia and acidosis. enrofloxacin is approved for the treatment of swine respiratory disease, however there are no published studies describing the pharmacokinetics of enrofloxacin at the approved dose and route in pigs ( . mg/kg subcutaneously). furthermore no studies have assessed the unbound concentrations of enrofloxacin at its site of action, the extracellular tissue fluid. therefore the objective of this study was to use an in-vivo ultrafiltration method to measure the active fraction of enrofloxacin, and the metabolite ciprofloxacin, at tissue sites relevant to pigs, and to compare these concentrations with plasma concentrations collected at similar time points. six healthy pigs were used in this study. pigs were recently weaned and weighed an average . kg. on the day before the experiment, pigs were anesthetized for the placement of jugular vein sampling catheters and interstitial fluid collection probes. three ultrafiltration probes were placed in each pig in a subcutaneous site near the right shoulder, an intramuscular site along the epaxial muscles, and in the pleural space of the chest cavity. each pig received an injection of enrofloxacin (baytril , bayer animal health) at a dose of . mg/ kg subcutaneously behind the left ear. plasma and interstitial fluid samples were collected at pre-determined time points, and enrofloxacin and ciprofloxacin concentrations were measured using hplc with fluorescence detection. protein binding was determined with a microcentrifugation system. pharmacokinetic data was analyzed using a one compartment model. the analysis of plasma and isf showed that only a small fraction of ciprofloxacin was produced in these pigs, therefore ciprofloxacin concentrations were not used in pharmacokinetic measurements. the plasma half-life (t / ), volume of distribution, clearance, and peak concentration (c max ) for enrofloxacin was . hr (ae . ), . l/kg (ae . ), . l/kg/hr (ae . ), and . mg/ml (ae . ), respectively. the concentrations from each of three tissues were not different in each pig. when pharmacokinetic values from all tissues were combined for the isf, the t / was . hr (ae . ) and the c max was . mg/ml (ae . ). the enrofloxacin plasma protein binding was . % (ae . ) and . % (ae . ) at a high and low concentration, respectively. this study has demonstrated that the concentration of biologically active enrofloxacin in tissues exceeds the concentration predicted by the unbound fraction of enrofloxacin in pig plasma. the half-life of enrofloxacin is longer in tissues and plasma than has been reported in previous studies. the high tissue concentrations and long half-life produce an auc/mic ratio sufficient for the pathogens that cause respiratory infections in pigs. ceftiofur crystalline free acid (ccfa), a long-acting ceftiofur formulation labeled for use in cattle, pigs, and horses for treatment of respiratory disease has been used for treatment of ovine respiratory infections in clinical practice. pharmacokinetic data, however, do not exist for ccfa administered subcutaneously in sheep. the present pharmacokinetic study evaluated the single dose subcutaneous administration of ccfa in sheep (n ) at . mg/kg body weight. concentrations of ceftiofur free acid equivalents (cfae) in plasma were measured by high performance liquid chromatography for days following drug administration. pharmacokinetics of subcutaneous ccfa in sheep were best described using a single compartment model with the following average (ae sd) parameters: area under the concentration time curve ! ( . hÃug/ml ae . ), observed maximum plasma concentration ( . ug/ ml ae . ), and observed time of maximum plasma concentration ( . h ae . ). no significant adverse drug reactions were observed. adequate cfae plasma concentrations were attained to effectively treat respiratory tract pathogens associated with pneumonia in sheep. the purpose of this study was to assess, using thoracic ultrasonography, the prevalence of lung lesions in pre-weaned dairy calves. subsequent aims were to describe ultrasonographic changes within the lung, clinical respiratory score, and treatment of respiratory disease. a longitudinal study was performed using female dairy calves from commercial dairy farms in new york state. calves were enrolled based on age. thoracic ultrasound and clinical respiratory scoring were performed on each calf at time points. a standard mhz linear ultrasound probe was utilized to evaluate intercostal spaces through of each hemi-thorax with the calf in lateral recumbency (us ) or standing (us ). lesion appearance, size, and location were recorded. respiratory score (rs) was assigned based on a previously published protocol incorporating fever, nasal discharge, cough, ocular discharge and ear droop, with a higher numerical score corresponding to more severe disease. abnormal lung on ultrasound was defined as one or more areas of ! cm width or depth of non-aerated lung. farm records were evaluated to identify treated calves. calves were treated for respiratory disease at the farm manager's discretion, not based upon ultrasound findings or rs. non-parametric methods were used to evaluate the data. ninety-one calves were enrolled into the study, with lost to follow-up. an average of minutes was spent performing the rs and ultrasound on each calf. the median ages at first (us ) and second (us ) examination were (interquartile range - ) and (interquartile range - ) days, respectively. the majority of calves had a low rs (o ) and only . % of calves had a rs high enough to warrant treatment based on previous recommendations (rs! ). the prevalence of calves that had abnormal lungs on ultrasound but a low rs (o ) was . % (us ) and . % (us ). the prevalence of calves that had abnormal lungs on ultrasound and a high rs (! ) was % (us ) and . % (us ). of the calves that had abnormal lungs on ultrasound but a low rs, % were treated with antimicrobials within days of examination. none of the calves with high rs and abnormal lungs on ultrasound were treated with antibiotics within days of examination. this study demonstrates a high prevalence of abnormal lungs, as detected by thoracic ultrasonography, without significant clinical signs in pre-weaned dairy calves. the relatively low treatment rate in these calves may suggest an area of opportunity for improvement in calf health, welfare, and herd longevity. further studies and follow up are needed to elucidate the significance of these findings and whether or not treatment is indicated. literature regarding diseases causing lameness in beef cattle is limited. this retrospective study was undertaken to examine beef cattle presented for lameness. medical records of beef cattle having a lameness examination done during the period to were reviewed and descriptive statistics generated. lameness was classified based on clinical diagnosis. the medical records of beef cattle were reviewed of which . % were male and . % were female. beef cattle presented for lameness most often during the summer months ( %) and least during autumn ( %). causes of lameness were categorized as infectious ( . %) or non-infectious ( . %) and infectious lameness subcategorized as either a primary disorder or a secondary infection. all cases of a primary infectious disorder were interdigital phlegmon. secondary infections diseases included sole abscess ( . %), septic arthritis ( . %), tenosynovitis ( . %), and pedal osteitis ( . %). non-infectious lameness included proximal limb lameness ( . %), foot trauma ( . %), hoof horn cracks ( . %), hoof defects ( . %), interdigital fibromas ( . %), overgrown hooves ( . %), sole bruise ( . %), subclinical laminitis ( . %), white line disease ( . %), osteoarthritis ( . %), heel erosion ( . %), sole ulcers ( . %), and sole hemorrhage ( . %). the most frequently affected claw was the lateral digit of the hind limb ( . %), followed by the medial digit of the front limb ( . %), lateral digit of the front limb ( . %), and the medial digit of the hind limb ( . %). the findings of this study suggest significant differences in the frequency of disease causing lameness in beef cattle compared to published reports for dairy cattle. in people, endoscopic ultrasound (eus) has become the technique of choice for assessing pancreatic disease and eus-guided fineneedle aspiration (eus fna) has proven a useful and safe modality for characterizing pancreatic lesions. reported complications include infections, bleeding and acute pancreatitis. in dogs, laparoscopic-assisted pancreatic biopsy has been suggested to be a safe procedure, however eus and eus fna have not been evaluated in dogs so far. thus the aim of the present study was to assess the practicability and safety of eus examination of the abdominal cavity as well as pancreatic eus fna in healthy dogs. this study was approved by the cantonal committee for the authorization of animal experimentation, zurich, switzerland. the study population consisted of healthy beagle dogs with a median bodyweight of . kg ( . - . ). eus was performed with an olympus gf-uc p-echoendoscope and fna were performed using g needles (cook echotipultra). after completion of the eus-examination of the abdominal cavity from the stomach (liver, gallbladder, bile ducts, kidneys, adrenals, pancreas), the scope was advanced into the duodenum and eus fna of the pancreas was performed. fna tissue acquisition was made applying negative pressure and to needle passes were made. all dogs received mg/kg metimazole im after eus fna and were re-checked ultrasonographically minutes post eus fna. postoperative activity was assessed using a standardized scoring system. a cbc, serum biochemistry, urinalysis and spec cpl s were measured before, as well as and h after eus fna. the eus examination was complete in / dogs, the pancreas could not be visualized in dog. the pancreas was hypo-( / ) to isoechoic ( / ) to the surrounding mesenterium in all cases. in / dogs parts of the pancreas presented hyperechoic. the mean measured thickness was . cm. the pancreas was aspirated in dogs using a transgastric approach ( ) or transduodenal approach ( ). duodenal transmural puncture was not accomplished in dog where a re-sterilized needle was used. a minimal amount of peripancreatic fluid was observed in / dogs after eus fna. all dogs recovered uneventfully and required no further analgesia. all laboratory results including the spec cpl s measurements were within reference ranges on all three time points. cytologically, conglomerates of exocrine pancreatic cells were seen in / cases, duodenal villous epithelial cells were seen in / cases. in dog the aspirated pancreatic material was sufficient for a histological assessment. the aspirates with exocrine pancreatic cells on cytology were obtained by transgastric ( ) and transduodenal ( ) aspirations. in conclusion, ( ) eus examination of the abdomen is feasible in medium-sized dogs, ( ) the healthy canine pancreas can be difficult to visualize completely, and ( ) eus-guided pancreatic fna using a g needle is a safe procedure in healthy dogs. studies evaluating its use in dogs with pancreatic disease are warranted to assess its clinical utility. miniature schnauzers have a high prevalence of idiopathic hyperlipidemia, which is characterized by an increased serum triglyceride (tg) concentration, with or without an increased serum cholesterol (chol) concentration. a common initial therapeutic approach for the management of hyperlipidemia is the use of a low-fat diet. also, it is believed that low-fat diets may be beneficial in the treatment of pancreatitis in dogs. however, the efficacy of this approach has not been evaluated for either condition. the aim of the present study was to evaluate the effect of a commercially available low-fat diet on serum concentrations of tg, chol, and canine pancreatic lipase immunoreactivity (cpli; measured as spec cpl s ) in apparently healthy miniature schnauzers with hypertriglyceridemia. blood samples were collected from apparently healthy miniature schnauzers with hypertriglyceridemia (serum triglyceride concentrations mg/dl). common causes of secondary hyperlipidemia were excluded based on historical information, physical examination findings, and the measurement of serum glucose, total t , and free t (by ed) concentrations. the owners of the dogs were asked to switch their dog to the study diet (royal canin gastrointestinal low fat s ; fat content: . g/ , kcal) and have a second blood sample collected weeks after their dog had been on the new diet. all blood samples were collected after food had been withheld for hours. serum tg, chol, and spec cpl concentrations were measured both before and after the diet change. results were compared between the two time-points using the wilcoxon signed rank and fisher's exact tests. serum tg concentrations were significantly higher before (median: mg/dl) than after the diet change (median: mg/dl; p . ). the proportion of dogs with hypertriglyceridemia was significantly higher before ( / ) than after the diet change ( / ; p . ). also, the proportion of dogs with serum tg mg/dl was significantly higher before ( / ) than after the diet change ( / ; p . ). serum chol concentrations were significantly higher before (median: mg/dl) than after the diet change (median: mg/dl; p . ). the proportion of dogs with hypercholesterolemia was significantly higher before ( / ) than after the diet change ( / ; p . ). finally, the difference in serum spec cpl concentrations before (median: mg/l) and after the diet change (median: mg/l) approached but did not reach significance (p . ). also, the proportion of dogs with high serum spec cpl concentrations before ( / ) and after the diet change ( / ) was different, but this difference was not significant (p . ). in summary, a commercially available low-fat diet was effective in reducing serum tg and chol concentrations in miniature schnauzers with hypertriglyceridemia. toll-like receptor (tlr ) is an extracellular pattern recognition receptor which recognizes flagellin present in motile bacteria. we have previously demonstrated a significant association between three non-synonymous single nucleotide polymorphisms (snps) in the tlr gene (g a, c t and t c) and inflammatory bowel disease (ibd) in german shepherd dogs (gsds). recently, we have confirmed that two of these tlr snps (c t and t c) are significantly associated with ibd in other canine breeds. to further substantiate the role of tlr in canine ibd functional analysis of these polymorphisms would be needed. therefore the aim of this study was to determine the functional significance of the tlr snps by transfecting wild-type and mutant receptors in to human embryonic kidney cells (hek) and carrying out nuclear factorkappa b (nf-kb) luciferase assay and il- elisa. the tlr gene containing the risk haplotype for ibd (acc) and wild-type haplotype (gtt) as determined by the case-control analysis in gsds with ibd were cloned into plasmids expressing yellow-fluorescent protein (yfp). these were then stably transfected into hek cells. nf-kb activity was measured by transiently transfecting the cells with nf-kb firefly and hsv-thymidine kinase promoter (prl-tk) renilla plasmids. the cells were then stimulated with various ligands ( . mg/ml flagellin, . mg/ml flagellin, mg/ml lps, mg/ml pam csk and media control). firefly and renilla luciferase activities were measured using the dual-glo luciferase assay system (promega, uk) according to the manufacturer's recommendations. the supernatants were harvested and used in an il- elisa (r&d systems). human tlr transfected hek cells (invivogen) served as positive controls in all experiments. independent t-test was used to determine the significance of relative luciferase activity and il- concentration between wild-type and mutated tlr cells. although there was no significant difference between the wild-type and mutated receptor when they were stimulated with . mg/ml of flagellin (p . ), there was a significant increase when the cells with mutated tlr were stimulated with . mg/ml of flagellin compared to the cells expressing wild-type tlr (p . ). similarly, there was a significant increase in il- concentration in the supernatants in the cells with the mutated tlr receptor when stimulated with . mg/ml flagellin compared to the wild-type (p . -one-tailed, . -two-tailed) but not with . mg/ml flagellin (p . ). we show for the first time that polymorphisms associated with ibd are functionally hyper-responsive to flagellin compared to the wild-type receptor. this suggests that tlr may play a role in canine ibd and that blocking the hyper-responsive receptor found in susceptible dogs with ibd may alleviate the inappropriate inflammation seen in this disease. however, further in-vivo functional analysis of tlr , especially at the intestinal mucosal level would be needed to confirm these findings and predict the usefulness of any future therapeutic interventions. tlr has been shown to play a role in the inappropriate inflammation seen in human inflammatory bowel disease (ibd). similarly, we have recently demonstrated a significant association between three non-synonymous single nucleotide polymorphisms (snps) in the canine tlr gene (g a, c t and t c) and inflammatory bowel disease (ibd) in german shepherd dogs (gsds). therefore the aim of this study was to determine the functional significance of the tlr snps in the breed of gsds. the tlr gene containing the risk haplotype for ibd (acc) and wild-type haplotype (gtt) were stably transfected into hek cells. nf-kb activity was measured by transiently transfecting the cells with nf-kb firefly and hsv-thymidine kinase promoter (prl-tk) renilla plasmids. the cells were stimulated with various tlr ligands ( . mg/ ml flagellin, . mg/ml flagellin, mg/ml lps, mg/ml pam csk and media control). firefly and renilla luciferase activities were measured using the dual-glo luciferase assay system (promega, uk). the supernatants were harvested and used in an il- elisa (r&d systems). peripheral whole blood from dogs carrying the wild type and mutant tlr genes was cultured and stimulated with tlr ligands as above. canine tnf-alpha was measured in the supernatant by commercially available elisa (r&d systems). t-test was used to determine differences of relative luciferase activity, il- concentration and tnf-alpha concentration between wild-type and mutated tlr cells. there was a significant increase in nf-kb activity when the cells with mutated tlr were stimulated with . mg/ml of flagellin compared to the cells expressing wild-type tlr (p . ), which correlated with il- expression in the supernatant (p . ). similarly, in the whole blood assay the tlr risk haplotype for ibd in gsds (acc) was significantly hyperresponsive to flagellin at a concentration of . mg/ml compared to the tlr wild-type haplotype (gtt) (p . ). we show for the first time that polymorphisms associated with canine ibd in gsds are functionally hyper-responsive to flagellin compared to the wild-type receptor. blocking the hyper-responsive receptor found in susceptible dogs with ibd may alleviate the inappropriate inflammation seen in this disease. proton pump inhibitors (ppi) are widely used in human and also veterinary medicine. side-effects of ppi treatment reported in people are atrophic gastritis, gastric and esophageal cancer, and rebound hyperacidity following cessation of treatment, which has been speculated to be due to a sustained increased in circulating gastrin concentration. moreover, long-term ppi treatment has been associated with an increased risk for osteoporosis in people. little is known about the effect of ppi treatment on serum gastrin concentration or calcium metabolism in dogs. eight healthy adult research dogs ( males and females) were enrolled into the study. the dogs received an average dose of . mg/ kg of omeprazole orally twice daily for days. blood samples were collected prior to initiating the treatment and every days during the days of treatment and during the days after discontinuation of treatment for determination of serum gastrin, ionized calcium, pth, and oh vitamin d . gastric fluid was collected via gastroscopy after an overnight fast for measurement of gastric ph prior to, during, and after the omeprazole treatment period. normally distributed data were compared with a repeated measures anova and post hoc dunnett's test. data that were not normally distributed were compared with a friedman's test and a post-hoc dunn's test. gastric fluid ph was significantly higher (p o . ) at the end of the treatment period (median: . ; range: . - . ) when compared to pretreatment values (median: . ; range: . - . ). serum gastrin concentrations increased significantly from a median baseline of . ng/l (range: . - . ) to a maximum median of . ng/l (range: . - . ) at day of treatment (p o . ). serum gastrin remained significantly increased above baseline values from day to day of the treatment, but was not different from pre-treatment values days after the end of the treatment. omeprazole treatment had no effect on ionized calcium or pth for the duration of the study. marginal, but significant changes of oh vitamin d were observed at day (end of the treatment period -increased by . %) and day ( days after the end of the treatment -decreased by . %). this study shows that treatment with omeprazole for weeks results in a profound and sustained increase in serum gastrin concentration in dogs. this effect is rapidly reversible after cessation of the treatment. no effect on calcium metabolism was observed. however, this study documents only the effect of short-term treatment and it is possible that the effects of long-term administration are different. omeprazole treatment has been associated with small intestinal bacterial overgrowth and a higher risk for infectious enteropathies in humans. using a semi-quantitative sequencing approach, we have previously shown that omeprazole treatment may lead to alterations in both duodenal and gastric bacterial populations in healthy dogs (acvim ). however, a sequencing approach can only estimate relative proportions of genomic bacterial targets. therefore, significant changes in the total number of bacteria could not be evaluated. the aim of this study was to quantify gastric and duodenal bacterial populations in dogs undergoing omeprazole treatment. eight month-old healthy research dogs ( males and females) were enrolled. the dogs received an average dose of . mg/kg of omeprazole orally twice a day for days. endoscopic gastric and duodenal biopsies were harvested and days before starting omeprazole treatment, on the last day of treatment (day ), and days after the end of treatment (day ). all biopsies were fixed in % formalin for hours, processed, and embedded in paraffin blocks. fluorescent in situ hybridization was used to quantify mucosa-associated bacteria using fluorescently-labeled probes targeting the s ribosomal rna. statistical analysis aimed to compare changes in helicobacter spp. in gastric biopsies and total bacteria in both gastric and duodenal biopsies using the glimmix and npar way procedures in sas s . . bacteria were counted in , and microscopic fields ( Â) obtained from and gastric and duodenal biopsies, respectively. in the stomach, omeprazole treatment led to a decrease in helicobacter spp. (log of average counts ae standard error: . ae . at day ) when compared to the counts ( . ae . , p . ) and ( . ae . , p . ) days before treatment. after completion of omeprazole treatment, helicobacter spp. increased and returned to baseline counts ( . ae . at day , p . vs day ). also, in the stomach, non-helicobacter spp. bacteria were observed more often during omeprazole treatment (median: , range: - ) than on days (median: , range: - ) and (median: , range: - ) before and days after (median: , range: - ) omeprazole treatment; however, statistical comparison across time points did not reach significance. in the duodenum, while the median number of bacteria for all time points was zero, non-parametric comparison of median scores (number of points above median) revealed significantly higher numbers of bacteria during omeprazole treatment (p . ). our results suggest that omeprazole treatment for weeks leads to a lower abundance of helicobacter spp. organisms in the stomach of healthy dogs. also, this transient decrease in helicobacter spp. was accompanied by a higher abundance of other bacteria in both the stomach and the proximal duodenum. the smartpill ph.p s capsule (the smartpill corporation) is a wireless motility capsule that measures ph, pressure, and temperature as it passes through the gastrointestinal (gi) tract. analysis of this data allows the calculation of gastric emptying time (get), small and large bowel transit time (slbtt), and total gi transit time (tgtt). this study evaluated the variability associated with repeated measurement of gi transit times and the effect of oral administration of ranitidine (zantac s ) on gi transit times in dogs using this system. it was hypothesized that ranitidine would reduce gi transit times. six privately owned healthy adult dogs weighing between . kg and . kg were used. on occasions each dog was fed a standard meal followed by oral administration of a capsule. data were recorded until the capsule had passed in the dog's feces. on a th occasion each dog was given mg of ranitidine po q hrs starting hrs prior to testing. the dogs were then fed the test meal and the capsule was administered as above. ranitidine was given until the capsule had passed in the dog's feces. proprietary smartpill software was used to calculate get, slbtt, tgtt, and the median gastric ph (mgph). mean intra-individual and inter-individual coefficients of variation (cv%) were calculated for get, slbtt, and ttt for the first time points. transit times and gastric ph recorded at all time points were compared using a repeated measures anova. where significant differences were identified, post-hoc testing was performed using a bonferroni's multiple comparisons test. significance was set at p o . . a sharp rise in ph indicating exit of the capsule from the stomach was identified in each experiment. mean (ae sd) get, slbtt, and tgtt without ranitidine were ae , ae , and ae min, respectively. mean get, slbtt, and tgtt during treatment with ranitidine were ae , ae , and ae min, respectively. mean intra-individual cv% before ranitidine for get, slbtt, and tgtt were . , . , and . %, respectively. mean inter-individual cv% before treatment with ranitidine for get, slbtt, and ttt were . , . , and . %, respectively. no significant differences in get, slbtt, or tgtt were found at any of the time points. the mean mgph during treatment with ranitidine (ph . ) was significantly higher than at all other time points (overall mean ph for the time points: . ; p o . ). the smartpill system is an easy to use, ambulatory, non-invasive, non-radioactive method for assessing gi transit times in medium to large breed dogs. measurements of gi transit times, especially slbtt, were subject to considerable intra-individual and interindividual variation. no significant effect of oral ranitidine on gi motility was identified in this group of dogs. however, as expected, oral ranitidine caused a significant increase in gastric ph. the intestinal microbiota has been implicated in the pathogenesis of various gastrointestinal disorders in both humans and dogs. recent metagenomic data suggest that specific bacterial groups, including bacteria within the clostridium clusters iv and xiva (i.e., faecalibacterium spp., ruminococcaceae, and lachnospiraceae) and bifidobacterium spp. are decreased, while proteobacteria are increased in dogs with clinical signs of gastrointestinal disease. the objective of this study was to establish quantitative polymerase chain reaction (qpcr) assays for these specific bacterial groups and evaluate their abundance in healthy dogs and dogs with clinical signs of gastrointestinal disease. fecal samples were collected from healthy dogs ( females and males) and dogs with clinical signs of gastrointestinal disease ( females and males). novel quantitative pcr assays were established for faecalibacterium spp., ruminococcaceae, and lachnospiraceae by aligning respective group specific sequences against canine specific sequences obtained from s rrna gene clone libraries and sequences available from the ribosomal database project. primers for bifidobacterium spp. and proteobacteria were selected from previously published studies. the specificity of the qpcr assays was confirmed by sequencing of obtained qpcr amplicons. the bacterial dna abundance in fecal samples was compared between healthy dogs and dogs with clinical signs of gastrointestinal disease using a mann-whitney u test. significance was set at p o . . a significantly lower abundance of faecalibacterium spp. (p o . ) and ruminococcaceae (p . ) was observed in dogs with clinical signs of gastrointestinal disease when compared to healthy dogs. proteobacteria were more abundant in dogs with clinical signs of gastrointestinal disease, but this difference did not reach statistical significance (p . ). there was no significant difference in the abundance of lachnospiraceae (p . ) and bifidobacterium spp. (p . ) between both groups. in conclusion, we established novel qpcr assays for faecalibacterium spp., ruminococcaceae, and lachnospiraceae. we observed significant decreases in the abundance of faecalibacterium spp. and ruminococcaceae in dogs with clinical signs of gastrointestinal disease. these bacterial groups are considered major short-chain fatty acid producers and studies are warranted to determine if a decrease in these bacterial groups is associated with decreases in short chain fatty acid production. further studies are also needed to determine if these bacterial shifts are associated with specific gastrointestinal disorders. the pathogenesis of chronic enteropathies (ce) in dogs likely involves complex interaction between the mucosal immune system and the intestinal microbiota. while the application of bacterial s rdna sequence-based analysis has shown an association between altered microbial composition and duodenal inflammation in dogs, relatively little is known about alterations in non-invasive mucosal and luminal bacteria seen with diseases involving the ileum and colon. the present study sought to evaluate the relationship of enteric bacteria to type and severity of mucosal inflammation affecting the ileum and colon of dogs with ce. eleven client-owned dogs with ce involving both the small and large intestines were prospectively enrolled. ce was diagnosed on the basis of a history of chronic gastrointestinal signs, exclusion of identifiable underlying disorders, and histopathologic evidence of intestinal inflammation. mucosal bacteria were detected in formalinfixed ileal and colonic tissue sections with fluorescence in situ hybridization (fish) using s rdna-targeted probes directed against all bacteria, enterobacteriaceae, e. coli, eubacterium rectale-clostridium coccoides group, bacteroides/prevotella, and helicobacter spp. sections were examined by epifluorescence microscopy and the number of bacteria and their spatial distribution (luminal, superficial mucus, epithelial adherent, within mucosa) was determined in ten x fields of each section. microbial composition in ce dogs was compared to the ileal/colonic microbiota of healthy control (hc) dogs using a mixed effect anova model. p values o . were considered significant. the final diagnoses for dogs with ce included ibd (n ) and lymphosarcoma (n ). when compared to hc dogs, dogs with ce showed regional (ileum versus colon) imbalances in microbiota composition characterized by selective enrichment of mucosa-associated populations. evaluation of colonic biopsies in dogs with ce showed that the total number of bacteria (p o . ), clostridium (p o . ), enterobacteriaceae (p o . ) and e. coli (p o . ) were increased in the adherent mucus regions of dogs with ibd as compared to hc dogs. total bacteria (p o . ) and e. coli (p o . ) were also more numerous in dogs with lsa versus hc and ibd dogs (p o . for e. coli). ileal biopsies from ce dogs similarly showed variable dysbiosis with increased total bacteria (p o . ) but decreased helicobacter spp (p o . ) and bacteroides (p o . ) observed within inflamed intestines as compared to hc tissues. the spatial distribution of these bacteria was also appreciably different from hc dogs, with higher numbers of bacteria generally found within the adherent mucus compartment as compared to other ileal regions. our data demonstrate that dogs with ce affecting the ileum and colon have altered microbiota composition that may be a cause or consequence of mucosal inflammation. recognition of these microbiota imbalances may provide new opportunities for therapeutic intervention. trichomonads have been rarely reported in the feces of dogs and their pathogenicity remains uncertain. although pentatrichomonas hominis (ph) is considered to be a commensal that may overgrow in dogs with other causes of diarrhea, little is known regarding the history, clinical presentation or prevalence of concurrent gi infections in dogs with trichomonosis. the aim of this study was to determine whether dogs with diarrhea and trichomonosis could be distinguished from dogs having diarrhea without trichomonosis on the basis of clinical signs or presence of concurrent enteric infections. fecal samples from dogs were submitted to ncsu from - for trichomonas spp. pcr testing. dna was extracted using a zr fecal dna mini-prep kit and absence of pcr inhibitors verified by amplification of bacterial s rdna. pcr for ph and tritrichomonas foetus (tf) was performed as well as real-time pcr assays for possible concurrent enteric infectious agents. obtainable medical records were reviewed. all submitted fecal samples were submitted from dogs with diarrhea that was variably described as soft, mucoid, hemorrhagic, or watery. mean age of the dogs was . years (median . ; range: . - months) and represented a total of breeds. ph, tf, or concurrent ph and tf were diagnosed in , , and dogs respectively (group a). the remaining dogs were negative for ph and tf by pcr no dogs were identified as infected with canine distemper virus or parvovirus. five samples from each group had insufficient quantity or quality of dna for concurrent infectious disease testing. in this large study of canine trichomonosis, no differences in age, clinical signs, or prevalence and identity of concurrent enteric infection between diarrheic dogs with or without ph were identified. thus, these findings do not appear to support a primary pathogenic role for ph as a causative agent of diarrhea in dogs. gastrointestinal motility disorders are a common clinical problem in domestic animals. many of the g.i. motility disorders have been treated previously with -ht agonists although limited availability of drugs in this classification have stimulated interest in the use of new (and old) drug therapies. the dopaminergic antagonists are a group of drugs with well-known anti-emetic effects at central dopamine d receptors, and putative gastrointestinal prokinetic effects at peripheral d receptors. domperidone has been shown, for example, to reverse gastric relaxation induced by dopamine infusion in the dog. similar studies have not been reported in the cat or rabbit, two species at risk for distal gastrointestinal motility disorders. our aim was to study the effects, mechanisms, and sites of action of domperidone in feline colonic and rabbit gastrointestinal smooth muscle contraction. portions of stomach (fundus and antrum), intestine (duodenum and ileum), cecum (rabbits only), and colon (ascending and descending) were obtained from healthy cats and rabbits from - months of age. longitudinal and circular smooth muscle strips from each site were suspended in physiologic (hepes) buffer solution, attached to isometric force transducers, and set to optimal muscle length (l o ) using acetylcholine (ach; À m). muscle strips were treated with domperidone (d; À to À m) in the presence or absence of ach ( À to À m), and maximal force output (p max ) was normalized for cross-sectional area (n  newtons/m ). domperidone (d) had a minor direct effect of inducing feline and rabbit gastric, cecal, and colonic smooth muscle contraction. direct effects were similar whether in the longitudinal or circular muscle orientation. the direct effect of domperidone was dose-dependent and maximal (feline colon p max . - . n; rabbit colon p max . - . n) at a dose of À m. domperidone had a much greater indirect effect in augmenting cholinergic (ach; À m) contractions in feline and rabbit gastric, cecal, and colonic smooth muscle. domperidone-augmented cholinergic contractions were - % (feline colon p max . ae . n ach only; feline colon p max . ae . n ach d) of baseline cholinergic contractions. domperidone contractions were of a similar magnitude to those induced by cisapride. domperidone effects were similar in mucosaintact and mucosa-dissected preparations. domperidone contractions were unaffected by prazosin (a receptor antagonist), yohimbine (a receptor antagonist), or terbutaline (b receptor agonist), but were somewhat attenuated by dopamine (d receptor agonist) and a non-specific cholinergic antagonist (atropine). in vitro studies show for the first time that domperidone has minor direct and major indirect effects in augmenting cholinergic contractions of feline and rabbit gastrointestinal (stomach, cecum and colon) smooth muscle. as recognition of acute and chronic pain in dogs has increased, so too has the use of non-steroidal anti-inflammatory drugs (nsaids) often in conjunction with tramadol. in people and rats, co-administration increases the risk of perforation and gastric injury over nsaids alone. using an ex vivo model of acid injury in canine gastric mucosa, we examined the effects of indomethacin and tramadol on gastric permeability and concentrations of gastroprotective prostaglandin e (pge ). mucosa from the gastric antrum was harvested from shelter dogs immediately after euthanasia, and mounted on ussing chambers. the tissues were equilibrated for -minutes prior to addition of acidic ringer's solution (ph, . ). after -minutes of injury, the acid was replaced with neutral ringer's and the tissues were treated with indomethacin, tramadol or both. tissues were maintained for minutes total, during which time permeability was assessed electrically. prostanoid concentrations were quantified using a commercially available elisa. western blots were performed for cox- and À . recovery of gastric barrier function after acid injury was inhibited by co-administration of tramadol and indomethacin ( figure ) but not by tramadol or indomethacin alone (data not shown). prostaglandin e increased with acid injury. the increase in pge was inhibited by co-administration of indomethacin and tramadol (in pg/ml: acid injury . ae . , indo tramadol . ae . ). there was no significant effect of treatment on cox- or À expression. co-administration of tramadol with a non-selective nsaid inhibits the return of gastric mucosal barrier function after acid injury in canine tissue, suggesting that caution is required in prescribing concurrent use of these drugs in dogs at risk for gastric ulcers. these drugs may exert this effect by decreasing levels of gastroprotective prostanoids. further study is needed to understand the mechanism of this drug interaction. an increased intestinal permeability (ip) has been suggested to be both cause and consequence of gastrointestinal (gi) disease, such as inflammatory bowel and celiac disease, in people. a novel tight junction regulator, larazotide acetate (alba therapeutics, baltimore, md) has been shown to significantly decrease ip in rats and in humans with celiac disease. the purpose of this study was to determine if larazotide acetate reduces ip in soft coated wheaten terriers (scwt) and norwegian lundehunds (nl) with chronic gi disease and ameliorates clinical signs. four nl ( females, males; median age: . yrs, range: . - . yrs) and scwt ( females, males; median age: . yrs, range: . - . yrs) were enrolled based on presence of clinical signs of gi disease and hypoalbuminemia, increased fecal alpha proteinase inhibitor (a -pi) concentrations, and/or hypocobalaminemia. scwt with protein-losing nephropathy were excluded. dogs were fed q hrs and received . mg ( nl and scwt) or . mg ( scwt) of larazotide acetate po before each meal for days. prior to start of treatment (day ) and at the end (day ), ip was evaluated by calculating the lactulose/rhamnose (l/r)-ratio in serum samples obtained at , , , and min after oral dosing. also, consecutive fecal samples each were collected prior to day and day for n-methylhistamine (nmh) measurement. pre-and post-treatment data were compared using a wilcoxon signed rank test. the . mg vs. . mg dose groups were compared using a mann-whitney u test. statistical significance was set at p o . . l/r-ratios (medians) for the min sampling time point were significantly lower on day ( . ) than on day ( . ; p . ). dogs treated with . mg q hrs had significantly lower min l/r ratios on day than dogs treated with . mg ( . vs. . ; p . ). no difference was found between breeds. fecal nmh concentrations were not different between time points, treatment groups, or breeds. fecal a -pi concentrations were available for of the dogs and were significantly higher on day compared to day (p . ). no differences were found between pre-and post-treatment serum albumin or cobalamin concentrations. weight gain was seen in all nl. resolution of diarrhea, vomiting, hyporexia, as well as an increased activity was seen in scwt. another scwt had resolution of diarrhea and a decrease in pruritus. no changes in clinical signs were reported in the remaining scwt. this study indicates that larazotide acetate might be able to reduce ip in dogs. this effect may be dose-dependent. however, not all dogs showed an improvement in clinical signs, suggesting that factors other than increased ip might have been responsible for the clinical signs in these dogs. breed-related effects cannot be ruled out, and further studies are warranted to determine the efficacy of larazotide acetate in dogs of other breeds with gi disease. to analyze different biochemical markers, calculate clinical activity scores, and assess survival in dogs with ple and compare them with those in dogs with food-responsive diarrhea (frd) without protein loss. dogs with ple and dogs with frd, referred to the university of bern, ch, were enrolled. selection criteria included a history of chronic diarrhea ( weeks), exclusion of identifiable underlying causes, and histopathologic evidence of intestinal inflammation, but not neoplasia. underlying disorders were excluded based on cbc, chemistry profile, urinalysis, fecal analysis, trypsinlike immunoreactivity, cobalamin, folate, and transabdominal ultrasound. also, canine pancreatic lipase immunoreactivity (spec cpl s ), c-reactive protein (crp), calprotectin and alpha -proteinase inhibitor (a -pi) were measured in serum from dogs and compared with dogs with frd without ple. all dogs were scored using the canine ibd (cibdai) and the canine chronic enteropathy (cce) clinical activity index (ccecai). total protein, albumin ( - . g/l), and total calcium ( . - . mmol/l) were decreased in all dogs. cobalamin was decreased in all but dogs ( o - ng/l). spec cpl was mildly increased in / dogs with ple and normal in / ple and all frd dogs. crp was normal in / dogs with ple ( / frd), mildly increased in / ( / frd), and moderately increased in / ple dogs ( / frd). calprotectin was slightly higher in dogs with ple, but all ple and frd dogs yielded values in the normal range. serum a -pi was significantly lower in dogs with ple than in those with frd (p o . ), with / ple dogs below the reference range ( / frd). cibdai ranged from to and ccecai from to . at the end of the study, / dogs were still alive with survival times between and days. / dogs died with a median survival of days (range - days). dogs with mildly increased crp died earlier than dogs with a normal or moderately increased crp (p . ), whereas albumin, calcium, spec cpl, calprotectin, cibdai, and ccecai had no significant impact on outcome and survival. in conclusion, dogs with ple have a significantly lower a -pi in the serum than dogs with frd. furthermore, most dogs with ple have an increased crp and a decreased cobalamin. a mild increase in crp appears to be a poor prognostic factor. while hypoalbuminemia is a common finding associated with chronic enteropathies, its impact on survival in this population is poorly defined. the aim of this study was to compare dogs with chronic enteropathies on the basis of their serum albumin concentration at the time of presentation. we hypothesized that dogs with a protein losing enteropathy (ple) have a significantly shorter survival time compared to dogs with chronic enteropathies which are not hypoalbuminemic (controls). information obtained from the medical records included signalment, duration and characteristics of clinical signs, physical examination findings, clinicopathologic data and survival time. one hundred seventeen cases fit the inclusion criteria; in the ple group and controls. there was no statistical significance between groups for age (p . ), weight (p . ), weight loss (p . ) and body condition score (p . ). compared to control dogs, ple dogs had decreased serum concentrations of cobalamin (p . ), total calcium (p o . ), globulin (p o . ), cholesterol (p o . ) and ionized calcium (p o . ). survival analysis revealed a significantly decreased survival time for ple dogs (p . ); median survival was days for ple dogs and , days for controls. while the ple group did not survive as long, survival was not directly associated with severity of hypoalbuminemia; patients with albumin concentration o . g/dl survived longer than those with mild hypoalbuminemia ( . - . g/dl). this study supports the observation that chronic enteropathy patients have decreased survival time when presented with hypoalbuminemia; however this study suggests the severity of hypoalbuminemia is not a reliable indicator of survival. cobalamin (vitamin b ) deficiency in the chinese shar pei (shar pei) is suspected to be hereditary. inherited causes of cobalamin deficiency have been reported in humans and may affect absorption, transport, or cellular processing of cobalamin. based on human and veterinary studies, an increased serum methylmalonic acid (mma) concentration has been suggested to reflect cobalamin deficiency at the cellular level. in this context, it has been shown in humans that mma concentrations are higher in patients with genetic disorders affecting intracellular processing than in patients with genetic defects affecting gastrointestinal processing and extracellular transport of cobalamin. therefore, the aim of this study was to evaluate serum mma concentrations in shar peis and dogs of six other breeds with cobalamin deficiency. from in conclusion, serum cobalamin deficient shar peis had a times higher median serum mma concentration compared to cobalamin deficient dogs of six other dog breeds. further studies are needed to investigate the intracellular processing of cobalamin in shar peis with cobalamin deficiency. chinese shar peis (shar peis) have a high prevalence of cobalamin deficiency. two other conditions reported frequently in this breed are shar pei fever and cutaneous mucinosis. shar pei fever is an autoimmune disorder causing periodic flare-ups and is associated with increased serum concentrations of c-reactive protein (crp), a nonspecific marker of inflammation. cutaneous mucinosis is characterized by excessive deposition of mucin in the dermis. also, hyaluronic acid (ha), the main component of mucin, was shown to be significantly higher in serum from shar peis with cutaneous mucinosis than in healthy controls. to date, a possible association between shar pei fever and/or cutaneous mucinosis on one side and cobalamin deficiency on the other has not been investigated in shar peis. thus, the aim of this study was to compare serum concentrations of ha (an indicator of cutaneous mucinosis) and inflammatory markers (crp, calprotectin, and s a ), assumed to be increased in episodes of shar pei fever, in shar peis with and without cobalamin deficiency. serum samples from shar peis, collected from to , were analyzed. serum ha and crp (reference interval (ri): . - . mg/l) were quantified by using commercial elisa kits (echelon biosciences, salt lake city, ut, usa and tridelta, maynooth, ireland; respectively). serum calgranulin concentrations were measured using an in-house elisa (calprotectin; ri: . - . mg/l) and ria (s a ; ri: . - . mg/l), respectively. mann-whitney u tests were used to compare serum ha, crp, calprotectin, and s a concentrations between shar peis with and without cobalamin deficiency. significance was set at p o . . fourteen shar peis were severely cobalamin deficient, defined by an undetectable serum cobalamin concentration ( o ng/l). in the remaining dogs, serum cobalamin concentrations were within the reference interval ( - ng/l). serum concentrations of ha, crp, calprotectin, and s a were not significantly different between cobalamin deficient shar peis (medians: . ng/ml, . . fifty percent of cobalamin deficient shar peis had serum calprotectin concentrations above the upper limit of the reference interval, and % had serum s a concentrations above the suggested upper reference limit. in this study, serum concentrations of ha, crp, and the calgranulins did not differ between cobalamin deficient shar peis and shar peis with a normal serum cobalamin concentration. this finding leads us to speculate that increased ha and/or inflammatory markers are not associated with cobalamin deficiency in shar peis. further studies are needed to investigate serum cobalamin concentrations in patients with shar pei fever or cutaneous mucinosis. cobalamin deficiency (cd) has been associated with gastrointestinal and pancreatic disease in dogs. hereditary cd has been demonstrated in giant schnauzers and single case reports have suggested congenital cd in the border collie (bc) breed. clinicopathologic findings of cd vary and can be unspecific as cobalamin acts as a co-factor for a multitude of enzymatic reactions. the two most important reactions concern the conversion of methylmalonyl-coa to succinyl-coa and the re-methylation of homocysteine (hcy). these two metabolites increase when cobalamin is lacking and act as markers for cobalamin availability on a cellular level. preliminary data from dogs suggested that measurement of methylmalonic acid (mma) may be a better diagnostic test for cd than serum cobalamin concentration. therefore the goals of the study were ( ) to establish reference values for serum cobalamin, urine mma and plasma hcy in healthy pet dogs, ( ) to screen a larger bc population from switzerland for cd, and ( ) to perform genomic analyses on bc with cd. for determination of reference values healthy pet dogs were used. serum cobalamin was measured using an automated chemiluminescence assay (immulite ), urine mma was determined using gas chromatography and expressed as a ratio to urine creatinine and plasma hcy was measured using high pressure liquid chromatography and fluorimetric detection. to calculate reference ranges the th and th percentile were used. data were analyzed using non-parametric tests. reference ranges for cobalamin, hcy, and mma were: cobalamin . - . ng/l; urine mma - . mmol/mol creatinine; and plasma hcy . - . mmol/l. the screened bc population comprised purebred dogs and bc (median . months; range - ) suffering from congenital cd could be identified. clinical signs differed and consisted of tiredness ( ), stunted growth ( ), anemia ( ), dysphagia ( ) and persistent fever ( ). median (ranges) results for healthy bc and bc with cd were: for cobalamin ( - ) and . ( - ) ng/l; for urine mma ( - ) and ( - ) mmol/mol creatinine; for hcy . ( . - . ) and . ( - . ) mmol/l. strikingly, healthy bc with cobalamin concentrations well within the reference range had significantly higher urine mma concentrations compared to control dogs. under the assumption that the four affected bc are inbred to a single founder animal, first results of genotyping on the k illumina canine_hd snp chip suggest that mutations in the cubn and amn gene can be excluded to cause the observed cd in these dogs. we conclude that cd is a rare familial disease in bc with variable clinical signs. to define the genomic region responsible for cd further genetic analysis is in progress. it remains to be determined why some bc have high urine mma concentrations despite a serum cobalamin concentration within the reference range. calprotectin is a protein complex that plays an important role in the innate immune response. preliminary data suggest that canine calprotectin (ccp) is a useful marker for the detection of inflammation in dogs. recently, a radioimmunoassay for the measurement of ccp has been developed and analytically validated, but this test requires the use of a radioactive tracer. therefore, the aim of this study was to develop and analytically validate an enzyme-linked immunosorbent assay (elisa) for the quantification of ccp in serum and fecal specimens from dogs. canine calprotectin (ccp) was purified, antiserum against purified ccp was raised in rabbits, monospecific antibodies were purified by affinity chromatography, and a sandwich-elisa was developed. purified antibodies were used for capturing and, after coupling with horseradish peroxidase (hrp), for reporting. a hrp substrate was used for color development. the assay was analytically validated by determination of analytical sensitivity and specificity, dilutional parallelism, spiking recovery, and intra-and inter-assay variability. control intervals for serum and fecal ccp were established from and healthy pet dogs, respectively, using the central th percentile. sensitivity of the assay for serum samples assayed in a : dilution and for fecal extracts assayed in a : , dilution was . mg/l and . mg/g, respectively. over a wide range of the assay, there was no cross-reactivity with cs a , the closest structural analogue of ccp available. observed to expected ratios (o/e) for serial dilutions ranged from . - . % (mean ae standard deviation [sd]: . ae . %) for four different serum samples, and from . - . % (mean ae sd: . ae . %) for five different fecal extracts. o/e for spiking recovery ranged from . - . % (mean ae sd: . ae . %) for four different serum samples and different spiking concentrations, and from . - . % (mean ae sd: . ae . %) for different fecal extracts and different spiking concentrations. intra-assay coefficients of variation (cv) for different serum samples were . , . , . , and . %, and . , . , . , and . % for different fecal extracts. inter-assay cv for different serum samples were . , . , . , and . %, and . , . , . , and . % for different fecal extracts. the control intervals for serum and fecal ccp were established as . - . mg/l and . - . mg/g, respectively. we conclude that this new elisa for the measurement of ccp is analytically sensitive, linear, accurate, precise, and reproducible, and does not cross-react with canine s a . further studies evaluating the clinical usefulness of measuring serum and/or fecal ccp are currently under way. the syndrome of hemorrhagic gastroenteritis (hge) is characterized by a peracute onset of hemorrhagic diarrhea, vomiting, depression, and anorexia, and can be associated with a high mortality if untreated. the etiology of hge is unknown, but it is speculated that an abnormal response to bacterial endotoxins, bacteria, or dietary components may play a role. hge is characterized by an increased vascular/mucosal permeability, thought to represent a type i-hypersensitivity reaction, whereas inflammation and necrosis appear to be rare. however, markers of gastrointestinal (gi) inflammation and changes in the intestinal microbiota have not been studied extensively in dogs with hge. therefore, the aim of this study was to evaluate fecal canine calprotectin (cp) and s a (a ), a -proteinase inhibitor (a -pi, a marker of gi protein loss), and bacterial groups that have previously been shown to be decreased (i.e., faecalibacterium spp., ruminococcaceae, bifidobacterium spp.) or increased (i.e., proteobacteria) in fecal samples from dogs with hge. fecal samples from consecutive days were collected from dogs with hge. fecal cp, a , and a -pi concentrations were measured by in-house immunoassays. bacterial dna was extracted from each fecal sample and was analyzed for faecalibacterium spp., proteobacteria, rumino-coccaceae, and bifidobacterium spp. using quantitative pcr assays. concentrations of fecal cp, a , and a -pi, and the abundance of bacterial dna were compared using a friedman test with dunn's post-hoc tests. significance was set at p o . . at the time of diagnosis (day ), fecal cp, a , and a -pi were above the suggested reference intervals in , , and of the dogs, respectively. until day , this number decreased to , , and , respectively. decreases in concentrations were significant between days and for a (p . ), and between days and for a -pi (p . ), but not for cp despite a trend (p . ). no differences in the abundance of faecalibacterium spp. (p . ), bifidobacterium spp. (p . ), or proteobacteria (p . ) were observed. however, the abundance of rumino-coccaceae was significantly lower on day when compared to day (p . ). in this study, fecal markers of inflammation and gi protein loss were increased in dogs with hge. although the number of patients was small, following initiation of treatment, two of the markers decreased significantly. these results suggest a loss of protein into the gi tract at the onset of hge. the lack of significant increases of faecalibacterium spp., bifidobacterium spp., and ruminococcaceae, and decreases in proteobacteria may suggest gi dysbiosis. further longitudinal studies are needed and are currently under way to evaluate gi dysbiosis in canine hge patients. the most recent antiemetic approved for use in dogs is maropitant citrate (cerenia s , pfizer animal health). maropitant is a selective nk receptor antagonist that acts by blocking the binding of substance-p within the emetic center and chemoreceptor trigger zone. label dosage recommendations for maropitant citrate are mg/ kg sc or mg/kg orally once daily for up to consecutive days (acute emesis) and mg/kg orally once daily for up to consecutive days (motion sickness). the study objective was to determine when steady-state is reached and the pharmacokinetics of maropitant administered at label oral dosages once daily for consecutive days. two groups of eight healthy beagles were administered maropitant citrate at or mg/kg orally once daily for days. concentrations of maropitant and its metabolite were measured in plasma using a lc-ms/ms assay. pharmacokinetic parameters were estimated using non-compartmental pharmacokinetic techniques and a modeling approach was used to estimate steady-state. the accumulation ratio for maropitant was . (auc - ) and . (cmax) for the mg/kg dose; and . (auc - ) and . (cmax) for the mg/kg dose after days. the model estimate for the number of doses required to reach % of steady-state was . for mg/kg and . for mg/kg. three dogs experienced a single episode of vomiting. dosing maropitant citrate beyond the label duration was well tolerated by healthy dogs. steady-state was reached after approximately doses for daily mg/kg and doses for daily mg/kg oral dosing. previously presented at the veterinary cancer society, november . cobalamin (vitamin b ) is involved in a variety of metabolic processes. altered serum cobalamin concentrations have been observed in dogs with gastrointestinal disorders, such as exocrine pancreatic insufficiency (epi) or severe and longstanding ileal disease. this study was conducted to identify breeds with a higher proportion of a decreased serum cobalamin concentration that were submitted to the gastrointestinal laboratory. the study was also aimed at investigating serum trypsin-like immunoreactivity (tli) concentrations that were diagnostic for epi in the dogs with a decreased serum cobalamin concentration. except for csp, breeds identified here, have not previously been identified to have a higher rate of a decreased serum cobalamin concentration. also, a possible association between an undetectable serum cobalamin and a decreased serum tli in ai needs to be further investigated. calprotectin (cp) is a widely used marker for the diagnosis and monitoring of gastrointestinal (gi) inflammation in humans. studies in humans usually report fecal cp concentrations based on a single stool sample although considerable day-to-day variability of fecal cp was found in patients with gi disease and in healthy controls. intra-individual variation of canine cp (ccp) was also substantial in a small number of healthy dogs but has not been determined in dogs with chronic gi disease. thus, the aim of this study was to compare the day-to-day variation of fecal ccp in dogs with chronic gi disease before and during treatment to that in healthy dogs. we hypothesized that fecal ccp would be less variable in patients with chronic gi disease than in healthy controls, and thus collection of a single fecal sample would be sufficient. fecal samples from consecutive days were prospectively collected from dogs (group a; median age: . years) referred for diagnostic work-up of chronic signs of gi disease, from dogs (group b; median age: . years) with stable gi disease while being treated, and from healthy adult dogs (group c; mean age: . years). fecal samples were extracted and ccp was measured by an in-house immunoassay. mean ccp, standard deviation, coefficient of variation (cv), and difference between maximum and minimum ccp for the -day sample collection period were calculated for each dog and were compared among groups using a kruskal-wallis test. fecal ccp ranged from . - . mg/g (median: . mg/g) in dogs with gi disease (group a), from . - . mg/g (median: . mg/g) in dogs of group b, and from . - . mg/g (median: . mg/g) in healthy controls (group c). cvs were - . % in group a (median: . %), . - . % in group b (median: . %), and - . % in group c (median: . %), respectively. patients in group a appeared to have less variable fecal ccp than dogs in group b and c, but this difference was not significant (p . ). the difference between maximum and minimum ccp for the -day sample collection ranged from - . mg/g in group a (median: . mg/g), from . - . mg/g in group b (median: . mg/g), and from - . mg/g in group c (median: . mg/g), and were not significantly different between any of the groups (p . ). in this study, considerable day-to-day variation of fecal ccp was found in dogs with chronic gi disease (regardless of treatment) and was comparable to that in healthy dogs. results of this study suggest that for evaluating fecal ccp in dogs with clinical signs of gi disease, three consecutive fecal samples rather than a single fecal sample should be analyzed. because we did not intend to evaluate the clinical usefulness of fecal ccp as a marker of gi disease in dogs, disease severity, quality, and location differed among dogs in groups a and b. the diagnostic utility of fecal ccp in dogs with gi disease is currently being investigated. it has been suggested that diagnosis of clostridium perfringens related enteropathy should be based on the detection of the c. perfringens enterotoxin gene (cpe-gene) by pcr and/or c. perfringens enterotoxin (cpe) by elisa in feces. however, the prevalence of the cpe-gene and cpe in dogs and especially cats with gastrointestinal disease has not yet been reported. also, there is limited information about the stability of cpe in fecal samples at various storage conditions. the aim of this study was to evaluate the prevalence of the cpe-gene and cpe and the stability of cpe in fecal samples from dogs and cats. to evaluate the prevalence of the cpe-gene, a total of fecal samples from dogs and cats with clinical signs of gastrointestinal disease ( dogs and cats) and fecal samples from those without such signs ( dogs and cats) were examined using pcr. to evaluate the prevalence of cpe, a total of fecal samples from dogs and cats with clinical signs of gastrointestinal disease ( dogs and cats) and dogs without such signs were evaluated using a commercially available elisa kit (techlab, blacksburg, va). the results were analyzed using a fisher's exact test. significance was set at p o . . to evaluate the stability of cpe, fecal samples from dogs and from cats with clinical signs of gastrointestinal disease that were positive for cpe were examined. also, cpe negative samples from dogs were evaluated as negative controls. each sample was subdivided into aliquots and evaluated on day ; on days , , and after being stored at room temperature (rt) or c; and on day after being stored at À c. the prevalence of the cpe-gene was not significantly different between dogs with signs of gastrointestinal disease ( / ; . %) and dogs without ( / ; . %; p . ). also, the prevalence of the cpe-gene in cats with signs of gastrointestinal disease ( / ; . %) was not significantly different compared to cats without ( / ; . %; p . ). pcr and elisa results were available for samples. of the pcr positive samples, only ( . %) were elisa positive. of the pcr negative samples, only ( . %) was elisa positive. the prevalence of cpe was not significantly different between dogs with clinical signs of gastrointestinal disease ( / ; . %) and those without ( / ; . %; p . ). the prevalence of cpe in cats with signs of gastrointestinal disease was / ( . %), but no samples from cats without such signs were available. when evaluating the stability of cpe, results for all aliquots were consistent with the initial result, except for one sample (on day , stored at rt, which was initially cpe positive). these results indicate that only a small proportion of samples that are pcr positive for the cpe-gene are also positive for cpe. studies are warranted to further compare the prevalence of cpe among animals with gastrointestinal disease and those without. furthermore, the results indicate that cpe is relatively stable in fecal samples at various storage temperatures. clostridium perfringens has been implicated as a cause of diarrhea in dogs. the main study objective was to compare two culture methods for the identification of c. perfringens. a secondary objective was to evaluate c. perfringens toxin genes a, b, b , e, ı and cpe from canine isolates using a multiplex pcr and determine their prevalence in a group of normal and diarrheic dogs. fecal samples were collected from clinically normal (nd, n ) and diarrheic dogs (dd, n ) at a primary care veterinary facility. isolation of c. perfringens was performed using direct inoculation of feces onto % sheep blood agar (sba) as well as enrichment of stool in bhi broth followed by inoculation onto sba. isolates were tested by multiplex pcr for the presence of a, b, b , e, ı and cpe genes. c. perfringens was isolated from % ( / ) of nd fecal samples using direct culture and . % ( / ) with bhi enrichment (p . ). in the dd, corresponding isolation rates were . % and . % (p . ). all isolates possessed a toxin gene. b, b , e, ı and cpe toxin genes were identified in . %, . %, . %, . % and . % of nd isolates, respectively. in the dd group, b and b were identified in %, e and ı were not identified and the cpe gene in . % of isolates. bhi enrichment did not significantly increase the yield of c. perfringens compared to sba but increased time and cost involved. c. perfringens (p . ) and c. perfringens toxin genes were present in equal proportions in nd and dd groups (p ! . ). culture of c. perfringens and pcr for toxin genes are of limited diagnostic utility due to the high prevalence of c.perfringens in normal dogs and the lack of apparent difference in toxin gene distribution between normal and diarrheic dogs. endoscopic biopsies are a relatively convenient, non-invasive test for feline infiltrative intestinal disorders. commonly, only the duodenum is examined due to cost, risks and time required to prepare the colon using lavage solutions, cathartics and/or enemas. the purpose of this study was to evaluate the consistency between endoscopic biopsies of the duodenum and ileum in cats. endoscopic biopsies from cats which had duodenal and ileal tissue specimens were evaluated retrospectively. all slides were randomized and reviewed by a single pathologist (jm) for quality, number of biopsies, and diagnosis according to wsava standards. no information regarding history, clinical signs, endoscopic findings, or previous histological diagnosis was made available to the pathologist. statistical comparison of the diagnosis of sc-lsa and ibd by intestinal location was conducted using fisher's exact test (p o . significant). of cats ( . %) were diagnosed with sc-lsa in the duodenum and/or ileum. of these cats, ( . %) were diagnosed with only duodenal sc-lsa, ( . %) were diagnosed with only ileal sc-lsa, and ( . %) had sc-lsa in both duodenum and ileum. in cats with only ileal sc-lsa, had severe ibd in duodenal biopsies, possibly consistent with early sc-lsa. of these had duodenal biopsies without evidence of sc-lsa. our results suggest there is a population of cats in which diagnosis of sc-lsa may only be found by evaluating ileal biopsies. clinicians should consider performing both upper and lower gi endoscopic biopsies in cats with suspected infiltrative small bowel disease. periodontitis is one of the most common diseases in cats and is mainly due to the presence of plaque and calculus. in this study, we investigated putative correlations between dental tartar and gingivitis and also between gingivitis and subgingival bacteria in cats. twelve cats (median age: years; range: - years; dsh and persians; females and males) were enrolled. dental tartar was obtained during scaling for a dental prophylactic procedure. all cats were negative for felv and fiv infection as assessed by a commercial elisa test (snap s fiv/felv combo test). severity of gingivitis (scores: - ; normal, mild, moderate, and severe) and dental tartar (scores: - ) were scored in each cat. endodontic paper points were applied for collecting a bacterial sample from the subgingival area and transferred to thioglycollate transporting media for bacterial culture. the relationship between gingivitis and tartar thickness scores was analyzed by spearman correlation. a student's t-test was used to compare the mean differences (gingivitis and tartar thickness scores) between upper and lower teeth. the association between severity of gingivitis and bacterial type was tested by chi square test. the spearman correlation coefficient for the average gingivitis score and the average tartar thickness score was . (p o . ). interestingly, the average tartar thickness scores from the upper jaw were significantly higher than those from the lower jaw (p o . ). the highest scores were found for the molar teeth in all cats. bacterial culture revealed . % anaerobic bacteria species (i.e., bacteroides spp., peptostreptococcus anaerobius, and eubacterium aerofaciens) and . % aerobic bacteria species (i.e., pasteurella multocida, streptococcus spp., enterococcus spp., staphylococcus spp., bacillus cereus, escherichia coli, and pseudomonas aeruginosa). anaerobic bacteria were found mostly in cats with higher gingivitis scores ( - ; chi square: p o . ), while pasteurella multocida was found mostly in cats with lower gingivitis scores ( - ; chi square: p o . ). antimicrobial sensitivity testing indicated that all of the anaerobic bacteria were sensitive to clindamycin, chloramphenicol, metronidazole, cefoxitin, or tetracycline, % were sensitive to erythromycin, and % were sensitive to penicillin. the most abundant aerobic bacterial species, pasteurella multocida, was sensitive to cefoxitin in all cases in which it had been cultured. these results suggest that anaerobic bacteria may be associated in the pathogenesis of severe gingivitis. these data warrant further studies of the prophylactic use of antibiotics in cats undergoing dental prophylactic procedures. inflammatory bowel disease is the most common cause of vomiting and diarrhea in dogs. although it can occur in any canine breed, certain breeds are more susceptible. we have previously shown that polymorphisms in the tlr and tlr gene are significantly associated with inflammatory bowel disease (ibd) in the german shepherd dog (gsd), a breed at risk of developing this disease. it would be useful to determine if these polymorphisms are significant in other canine breeds as this may allow the development of novel diagnostics and therapeutics to be applied to all canine breeds with ibd. therefore the aim of this study was to investigate whether polymorphisms in canine tlr and tlr genes are associated with ibd in other non-gsd canine breeds. four non-synonymous snps in the tlr gene; t c, g a, a t and g a and three non-synonymous snps in the tlr gene; g a, c t and t c previously identified in a mutational analysis in gsds with ibd were evaluated in a case-control study using a snapshot multiplex reaction. sequencing information from unrelated dogs with ibd consisting of different non-gsd breeds from the uk were compared to a breed-matched control group consisting of unrelated dogs from patients treated for noninflammatory disease at the royal veterinary college, london, uk. as in the gsd ibd population the two tlr snps; c t and t c were found to be significantly protective for ibd in other breeds included in this study (p . and p . respectively). this study confirms the protective effects of the two tlr snps (c t and t c) in other canine breeds with ibd. this highlights the importance of tlr in the pathogenesis of canine ibd and may represent common pathological pathways of ibd in different canine breeds due to the high degree of haplotype sharing seen among breeds. this may allow for the future expansion of novel diagnostics and therapeutics to be applied to all canine breeds with ibd. further functional studies looking at the role of tlr in the pathogenesis of canine ibd are needed to confirm these findings. toll-like receptor (tlr ) is an extracellular pattern recognition receptor belonging to the innate immune system. we have recently shown that three non-synonymous single nucleotide polymorphisms (snps) in the tlr gene (g a, c t and t c) are significantly associated with inflammatory bowel disease (ibd) in german shepherd dogs. in addition, we confirmed that two of these tlr snps (c t and t c) were significantly associated with ibd in a population consisting of different dog breeds. in order to determine if other novel snps exist in the tlr gene in addition to the ones identified in the gsd population, mutational analysis was carried out in seven boxer dogs with ibd. polymerase chain reaction was carried out to amplify the tlr coding region in the seven dogs with ibd. sequencing was carried out using sequence based typing with the abi prism sequencing kit (applied biosystems, uk) and analyzed using an abi automated sequencer (pe applied biosystems). sequencing information from seven boxer dogs with ibd from the uk were compared to the reference sequence published on the ensemble webserver (www. ensembl.org/canis_familiaris). in addition to the three snps identified previously in the tlr gene, a novel non-synonymous snp; t c was identified in the boxer dog population with ibd. this snp has never been reported before and was present as the homozygote genotype in three dogs with ibd and in one dog as the heterozygote genotype. using the simple modular architecture research tool (smart) web server (http:// smart.embl.de/) we were able to map the t c snp to the leucine rich repeat domain of the tlr protein. the leucine rich repeat domain is involved with ligand binding and therefore a change in the amino acid in this region may affect function, especially as the t c snp results in a change in the amino acid from non-polar to polar. our study further confirms the role of tlr in the pathogenesis of canine ibd. our results suggest that in addition to shared risk polymorphisms amongst breeds, individual breeds may harbor unique snps arising after breed formation which may further affect their susceptibility to this disease. however, a case-control study would be needed in the boxer dog to confirm the significance of the tlr t c snp and further functional data would be needed to elucidate the exact role of this polymorphism in canine ibd. an automated power driver device (oncontrol, vidacare) has recently become available for bone marrow aspiration (bma) and bone marrow biopsy (bmb) in humans. the purpose of our study was to compare this automated technique to the traditional manual technique for bone marrow sampling in cats. twelve healthy research cats were anesthetized using a standardized protocol on different occasions, days apart, to have bmas and bmbs performed by the same operator. on day , half of the cats were randomized to have a bma performed at both the proximal humerus and the iliac crest, and a bmb performed at the iliac wing, using the oncontrol device ( -gauge needle for bma; -gauge needle for bmb). the other half of the cats had the same procedures performed using a manual technique ( -gauge illinois needle for bma; -gauge jamshidi needle for bmb). on day , each cat had bma performed at the opposite humerus and iliac crest, and a bmb performed at the proximal humerus using the opposite technique from day . for each procedure, the operator was given a maximum of attempts to successfully collect a sample. the rate of success, as well as the number of attempts were recorded. the ''ease of use'' of the device was rated by the operator on a -point scale after each procedure. using previously determined criteria, the macroscopic and microscopic qualities of the bma and bmb samples were assessed by a board-certified pathologist, blinded to the technique used. the level of pain experienced by each cat was evaluated , , , , and hours following each set of procedures, using a previously validated pain scoring system. two sample t-tests were used to compare the automated technique to the manual technique and to compare the humerus to the iliac crest site for bmas and the humerus to the iliac wing site for bmbs. for all procedures, at all sites, the ''ease of use'' was better for the automated technique than for the manual technique (p o . ). the duration of the procedure and the number of attempts to collect a sample were significantly lower with the automated technique for bma at the proximal humerus (p o . ). there was no significant difference in the level of pain at any time point following each set of procedures with either technique. performing bma at the proximal humerus was associated with a higher rate of success (p o . ), a lower number of attempts (p o . ), a shorter duration of the procedure (p o . ), a higher-rated ''ease of use'' of the technique (p o . ), and a better quality sample (p o . ) when compared to sampling from the iliac crest, in conclusion, we found the automated bone marrow sampling technique suitable for use in adult cats. this technique was easier to use than the manual technique for both bma and bmb, and reduced the duration of the procedure and the number of attempts for successful bma at the proximal humerus. performing bma at the proximal humerus was faster, easier and allowed collection of better quality samples than at the iliac crest, independently of the technique used. the fractious nature of some feline patients sometimes makes sedation or general anesthesia necessary for routine procedures such as blood collection for hematologic analyses. it has been anecdotally reported that sedation or general anesthesia could induce variations in hematologic parameters in cats, making it important for the clinician to be able to anticipate potential changes on hematologic parameters that could result from chemical restraint. this study evaluated the effects of a standardizecd anesthetic protocol using ketamine ( mg/kg, iv), midazolam ( . mg/kg, iv) and buprenorphine ( mg/kg, im) on the hematologic parameters of healthy adult research cats. each cat had blood samples collected before and after induction of anesthesia on different occasions, days apart. in total, pairs of complete blood counts were obtained. analyses were performed at a certified veterinary laboratory. paired sample t-tests were used to determine whether there were any statistical differences between hematologic parameters before and after induction of general anesthesia, for each cat, on different occasions. compared to preanesthetic values there was a significant decrease in red blood cell count, hemoglobin concentration, hematocrit, lymphocyte count and plasma total protein concentration after induction of anesthesia. there was no significant difference in the segmented or band neutrophil, eosinophil, basophil, monocyte and platelet counts between the samples taken before and after induction of anesthesia. on average, there was a . % decrease in the red blood cell count ( .  /l to .  /l) (p o . ), a % decrease in hemoglobin concentration ( . g/l to . g/ l) (p o . ), a . % decrease in the hematocrit ( . l/l to . l/l) (p o . ), a . % decrease in the lymphocyte count ( .  /l to .  /l) (p . ), and a . % decrease in the plasma total protein concentration ( . g/l to . g/l) (p o . ) when samples taken before and after induction of anesthesia were compared. if only the hematocrit was considered as a marker of anemia, % of the samples from these healthy cats, taken while they were under general anesthesia, would have been misinterpreted as belonging to anemic patients (hematocrit o . l/l), using the reference interval established in our laboratory. none of the cats would have been considered anemic before induction of general anesthesia. in practice, the decrease in lymphocyte count following anesthesia is unlikely to be of clinical relevance, as all the samples except had a lymphocyte count that was within the reference interval for cats established by our laboratory. this study suggests that complete blood counts performed on blood taken under general anesthesia with this combination of anesthetic drugs in cats should be interpreted cautiously in order not to make a false diagnosis of anemia. the mechanism responsible for the decrease in circulating red blood cell mass following anesthesia induction in cats is unknown and requires further investigation. rivaroxaban is an oral inhibitor of activated coagulation factor x (xa). it is expected to have similar coagulation effects as low molecular weight heparin, without the need for injection, making it an attractive alternative for long-term anticoagulant therapy in cats. citrated blood obtained from five healthy adult cats was exposed in vitro to varying concentrations of rivaroxaban, followed by coagulation testing. the rivaroxaban was extracted from commercially available tablets (xarelto s ) and dissolved in dmso prior to addition to the blood. tests performed included kaolin-activated thrombelastography (teg), prothrombin time (pt), dilute pt (dpt), activated partial thromboplastin time (aptt), and anti-factor xa (axa) activity. dose-dependent prolongations were seen in all coagulation parameters. similar to human data, therapeutic axa levels (between . - . axa units) were achieved at in vitro concentrations between and mg/l. at mg/l, dpt measurements were clinically prolonged in all cats ( . ae sec vs. . ae . sec, p . ), while aptt values were only mildly prolonged from baseline ( . ae sec vs. . ae sec, p . ). significant prolongations were seen in dpt at ( . ae ec, p . ). teg r time did not prolong from baseline values until concentrations of mg/l were reached ( . ae min compared to . ae . min, p . ). rivaroxaban has similar coagulation effects in the cat as in other species and may play a role in feline thromboprophylaxis. kaolinactivated teg does not appear to be sensitive to low concentrations of rivaroxaban in the cat. anticoagulated blood is required for platelet function studies. sodium citrate, a calcium chelater, is the most commonly used anticoagulant to measure coagulation parameters including platelet aggregation but it may have a negative effect on platelet responsiveness. dogs are generally considered moderate responders to collagen on platelet aggregation and are notorious for being poor or inconsistent responders to adp-induced platelet aggregation using citrated whole blood. hirudin, a selective thrombin inhibitor, can also be used as an anticoagulant for coagulation assays and is the anticoagulant of choice for certain assays including the multiplate s platelet function analyzer. ten adult healthy dogs were used to compare whole blood platelet aggregation between citrated and hirudinated blood samples. venous blood was collected atraumatically from the external jugular vein directly into tubes containing . % trisodium citrate or hirudin. whole blood platelet aggregation was performed (whole-blood lumi-aggregometer, chrono-log corporation, havertown, pa, usa) with collagen ( mg/ml) and adp ( mm) as agonists. maximal platelet aggregation (ohms) was recorded. there was a significant increase in collagen-induced platelet aggregation from the hirudinated samples compared to the citrated samples ( . ae . vs. . ae . o, p o . ). there was also a significant increase in adp-induced platelet aggregation from the hirudinated samples compared to the citrated samples ( . ae . vs. . ae . o, p . ). the results of this study show a significant difference in platelet responsiveness between citrated and hirudinated whole blood using the chrono-log impedance aggregometer. while both collagen and adp-induced platelet aggregation was attenuated from citrated blood samples, this was most notable for adpinduced aggregation where almost all samples had no objective measurable platelet aggregation. it is suggested from this data that future whole blood platelet aggregation studies performed on the chrono-log impedance aggregometer should use hirudinated blood samples although new reference limits would need to be established. low-molecular-weight heparin (lmwh) is now used to prevent thrombotic complications in dogs. a functional assay such as the calibrated automated thrombogram (cat) may provide a new approach for monitoring lmwh therapy. we hypothesized that cat would detect decreased endogenous thrombin potential (etp) in healthy dogs receiving lmwh (fragmin s ). twenty-four healthy adult beagles were included in this study and divided equally in four groups. one dose of u/kg, u/kg or u/kg of lmwh were given subcutaneously to healthy dogs and compared to a control group. platelet poor plasma (ppp) was collected over a hour period. using a repeated-measure linear model, effect of lmwh on etp was time and dose dependent with a significant interaction (p o . ). compared to control dogs, significant differences were obtained for group u/kg at t (p . ), for group u/kg at t (p . ) and between t -t minutes (p o . ) respectively, and for group u/ at t (p . ), between t -t minutes (p o . ) respectively and at t (p . the cat assay can be employed to measure the effects of lmwh at different doses in healthy dogs, resulting in significant time and dose-dependent decreases in etp and warrants further investigation as a tool for monitoring lmwh therapy in dogs. the purpose of this study was to determine the effects of prednisone and prednisone plus ultralow-dose aspirin on coagulation parameters in healthy dogs, with an emphasis on thromboelastography (teg). this was a prospective, randomized, blinded study utilizing fourteen dogs determined to be healthy based on normal physical examination, complete blood count, biochemistry, urinalysis, and fecal floatation. dogs were evenly divided into either prednisone plus aspirin (pa) or prednisone plus placebo (pp) groups. baseline values for teg parameters (r, k, angle, ma, ly , ly , g, ci) were measured twice two days apart, and thrombin-antithrombin complexes (tat), and traditional coagulation parameters (prothrombin time, activated partial thromboplastin time, d-dimer, antithrombin (at), fibrinogen) were measured once. each dog received mg/kg/ day of prednisone, and either . mg/kg/day of aspirin (pa group) or placebo (pp group) for days. a complete blood count, biochemistry profile, teg, tat, and traditional coagulation parameters were then repeated on each dog. day to day variation was calculated for the teg parameters using the two baseline measurements. the change from baseline between and within each group were compared using t-tests, or wilcoxon sample test where appropriate, for teg, tat, traditional coagulation parameters, and hematocrit. day to day variation in teg was acceptable ( %) for ma, g, and angle, unacceptable ( %) for r, k, ly and ly , and not meaningful for ci. within both groups, ma, g, ci and fibrinogen significantly increased from baseline (p o . ). within both groups, ly and at significantly decreased from baseline (p o . ). for the pp group, ly significantly decreased from baseline (p . ), and approached significance for the pa group (p . ). all other within group changes from baseline were not statistically significant (p-values . ). for all parameters, there was no difference between groups for change from baseline (p values . ). day to day variation in some teg parameters is high and may preclude their clinical utility. prednisone causes hypercoagulability in healthy dogs based on increased g, ma, and ci. the addition of ultra-low dose aspirin to prednisone has no effect on the parameters measured in this study. 'aspirin resistance' has been identified in people and dogs that develop thrombi despite low dose aspirin therapy. variability in platelet cyclooxygenase (cox) isoform expression is one proposed mechanism for aspirin resistance in people. two isoforms (cox- and cox- ) have been identified in canine platelets. high (antiinflammatory) dose aspirin inhibits platelet function and alters expression of both cox isoforms in most dogs. this study evaluated the effects of low dose aspirin on platelet function and cox expression in normal dogs. twenty-five healthy client-owned dogs were evaluated before and at two time points (days and ) during aspirin therapy ( mg/kg po sid). platelet response to aspirin (siemens pfa- s ; collagen/ epinephrine cartridges), was stratified into one of three groups [aspirin responders ( dogs), non-responders ( dogs), or inconsistent responders ( dogs)]. flow cytometry identified platelet cox- and cox- expression. an elisa was used to measure urine -dehydro-thromboxane b ( -dtxb ). there were no significant differences between groups for cox- , cox- or -dtxb at any time point. when all dogs were considered as a single group, there was a significant increase (p o . Ã) in cox- and cox- mean fluorescent intensity (mfi) from baseline to day , . % ae . (mean ae sd) and . % ae . , respectively. there was a significant decrease in mean urine -dtxb :creatinine on day and by . % (p . à ) and % (p o . à ). as with our previous high dose studies, cox- expression was increased with aspirin exposure. however, there was a significant increase in cox- expression with low dose aspirin in contrast to the decrease seen at higher doses. our study suggests that levels of platelet cox- and cox- expression do not influence aspirin response in dogs. although thromboxane levels decreased in most ( of ) dogs on low dose aspirin, platelet function was consistently affected in only % of dogs, suggesting that differences in response to thromboxane may play a role in the variable affects of low dose aspirin on canine platelet function. delayed postoperative bleeding is common in retired racing greyhounds (rrgs), despite normal results of routine hemostasis assays. the excessive postoperative bleeding in the rrgs is not due to primary or secondary hemostatic defects, and may be due to enhanced fibrinolysis or to a clot maintenance dysfunction. providing a method to prevent or minimize the severity of postoperative bleeding in rrgs will not only have major economic impact for owners, but also will markedly decrease the associated complications of minor or major surgeries in the breed. epsilon aminocaproic acid (eaca) is a potent inhibitor of fibrinolysis that also supports clot maintenance due to unknown mechanisms. the objective of this double-blinded, prospective, randomized study was to evaluate the effects of eaca versus placebo on the prevalence of bleeding in rrgs, and to investigate its mechanism of action by using thromboelastography (teg). we compared the effects of eaca and placebo in rrgs that underwent elective ovariohysterectomy or orchiectomy at the veterinary medical center, the ohio state university during years. the main endpoint was bleeding (prevalence and severity); minor endpoints included most teg parameters. thirty percent ( / ) of the rrgs in the placebo group had delayed postoperative bleeding starting to hours after surgery, compared to % ( / ) in the eaca group (p . ). on the teg parameters, the r time (clot formation time) was significantly different between treatment groups (p . ). the postoperative administration of eaca significantly decreased the prevalence of postoperative bleeding in rrgs. thromboembolism associated with protein losing nephropathy (pln) has been long recognized as a serious and unpredictable complication in dogs, however its prevalence remains unknown. in humans, surrogate indicators are frequently used to assess thromboembolic risk. this study aimed to investigate the prevalence of hypercoagulability in pln dogs based on thromboelastography (teg), and to determine whether hypercoagulability in these patients could be predicted by clinical assessments that identify systemic hypertension (systolic blood pressure mmhg), hypoalbuminemia (serum albumin o . mg/dl), antithrombin activity (o %), and degree of proteinuria (urine protein:creatinine ratio [upc] ! ). between march -september , twenty-seven dogs were identified with pln at the animal medical center. the prevalence of hypercoagulability based on a teg g-value . was . %. there was no statistically significant relationship, either categorically or continuously, in univariate as well as multivariate analyses of all variables. univariate logistic regression (odds ratio; lower and upper confidence limit; p value) for hypertension was À . ; . , . ; . ; for albumin - . ; . , . ; . ; and for antithrombin activity - . ; . , . ; . . thus, in this patient population, in the absence of teg, prediction of hypercoagulability using abnormalities in commonly measured clinicopathologic variables was not helpful. however, given the documented high prevalence of hypercoagulability in patients with pln, early institution of prophylactic anti-platelet or anticoagulant therapies should be considered. thromboelastography (teg) is a test of global hemostasis. due to the effects of extrinsic factors on whole blood coagulation, sample collection method (scm) may influence results. the purpose was to determine if scm influenced teg using kaolin-activated citrated whole blood (wb). healthy dogs with normal platelet counts were prospectively enrolled. three wb samples were obtained from each dog at least hours apart from alternating jugular veins in a randomized order of three methods: ) vacutainer s into citrated tube (vac), ) citrated syringe with transfer into plain tube (cit), or ) plain syringe with transfer into citrated tube (plain). draw time was recorded in seconds. kaolin-activated teg was performed, with measurement of reaction time (r), clot formation time (k), maximum amplitude (ma), and alpha angle. eleven dogs were enrolled. there were no significant differences in teg indices between vac samples and either cit or plain samples. cit samples had a significantly higher k value (p . ) and a lower alpha angle (p . ) compared to plain samples. draw times ranged from - seconds. a longer draw time was significantly correlated (p . ; r À . ) with a shorter r time. higher platelet count was significantly correlated (p . ; r . ) with a higher ma. scm did not have a significant effect on teg parameters when comparing vac samples to either cit or plain samples. minimizing sample collection time and trauma during venipuncture may be important in minimizing hypercoagulable changes in teg indices. liquid plasma (lp) is defined as either plasma collected and refrigerated immediately after collection or fresh frozen plasma (ffp) that is thawed and stored refrigerated until use. stability studies in people have shown that adequate clotting factor activity is preserved for at least days. lp is transfused in human level i trauma centers to critically ill people requiring rapid infusion of clotting factors as the time required to defrost ffp is considered prohibitive. the use of lp has not been described in veterinary critical care. the purposes of this study were to ) determine the length of time required in a water bath for a unit of canine ffp to thaw and ) describe the use of lp in a busy university emergency room (er). for part : six units ( ml) of canine ffp were individually thawed in a c water bath. the duration of time (in minutes) to thaw was recorded. for part : the transfusion log was reviewed for dogs receiving lp in the last months. the indications and outcome were recorded. the mean time ae sd thaw time was . ae . minutes. ten units of lp were transfused to critically ill or injured dogs during the study time. indications for lp transfusion included hypovolemic shock due to intra-abdominal hemorrhage in dogs ( traumatic, non-traumatic) and rapid correction of hemorrhage following parenteral tissue plasminogen activator administration in dog. lp volume transfused ranged from . to . ml/kg. no transfusion reactions were identified. effect on coagulation was not consistently evaluated. time required to thaw a unit of ffp is greater than minutes which could be detrimental in a bleeding, coagulopathic dog. lp was transfused without incident to critically ill and injured dogs and represents a potential new addition to the armamentarium of treatments in a veterinary er setting. further investigation of canine lp is warranted including evaluation of in vitro factor stability and in vivo efficacy in correcting coagulopathy. immune mediated thrombocytopenia (imt) is associated with increased morbidity and mortality. large prospective research studies in dog platelet antibodies and clinical utilization of platelet immunoglobulin assays are limited. potential explanations include limited availability and low specificity due to nonspecific binding. the focus of this study is to evaluate optimized direct and indirect platelet surface associated immunoglobulin (psaig) and staining with anti-cd antibodies (cd ab) for the utilization in classifying thrombocytopenic dogs. one hundred clinically ill and apparently healthy dogs were prospectively evaluated. data collected included a history of hemorrhage, physical examination evidence of bleeding, complete blood count, and measurement of psaig and cd ab. the psaig assay utilized polyvalent antibodies with correction for non-specific binding by subtraction of background fluorescence with control antiserum. thrombocytopenia was defined as o , /ml and all dogs were clinically classified into of groups (g): g imt, n , g thrombocytopenia from non-immune mediated diseases, n , g ill with normal platelet counts, n , g healthy dogs, n . median platelet counts, by groups, were g , , ; g , , ; g , , ; and g , , /ml. for the direct and indirect psaigs in dogs with itp (g ), more dogs (n and n ) with clinical evidence of bleeding had antibodies compared to those who were not bleeding (n and n ). considering only direct psaig the sensitivity and specificity was % and %, respectively for the diagnosis of imt. for indirect psaig the sensitivity and specificity was % and %, respectively, for the diagnosis of imt. when considering both direct and indirect psaig together, the sensitivity was % with a specificity of %. in g interference from high control antiserum background staining was noted in . % of dogs and resulted in a negative direct psaig classification. minimal background interference was noted in g , g , or g . the percentage of platelets stained with cd ab was significantly less in g (median , p . ) vs. g (median , p . ) vs. g (median . , p . ) and g (median . ). these findings indicate the optimized platelet surface associated immunoglobulin assay has a high specificity, however poor sensitivity, for the diagnosis of imt. the decreased cd staining in g (imt group) may reflect decreased surface gpiiia expression, blocking by anti-gpiiia antibodies or other proteins, clearance by macrophages, or increased non-platelet debris and has potential applications in the diagnosis and treatment of imt. greyhounds have lower serum concentrations of a-globulin than other breeds, explained by negligible levels of haptoglobin (hp) measured using different methods (colorimetric, immunoturbidimetric and protein electrophoresis). the purpose of the present study was to characterize the hp gene in greyhounds. we isolated dna and rna from blood samples of akc-registered and retired racing greyhounds (akcg, rrg), and a german shepherd dog (gsd). we sequenced the hp exons and splice sites, and conducted array comparative genomic hybridization to identify associated dna structural variation (custom m agilent oligonucleotide array). additionally, we tested for the presence of one or multiple haplotypes spanning hp in greyhounds using a high density snp array ( k illumina hd). sequencing results of hp in both dna and cdna revealed three synonymous snps in the racing greyhound. we did not identify structural variation overlapping or near the hp gene. notably, we detected that the rrg and akcg do not appear to share a specific haplotype spanning hp. despite having low or undetectable serum concentrations of hp, we did find that rrg hp mrna is expressed and lacks amino acid variation. this suggests that the clinical absence of the hp is attributable to post-transcriptional hp effects or to an unknown physiological interaction. finally, given the existence of distinct rrg and akcg haplotypes spanning hp, it is important to characterize serum levels of hp in akcg in follow on studies. we reported that hemoglobin in retired racing greyhounds (rrg) has higher oxygen carrying properties and affinity than other breeds. surprisingly, very little is known about canine hemoglobin genetics. the purpose of this study was to characterize genetics of canine beta globins. using computational blast analysis of the dog genome, we identified five beta globin genes in a single locus: two human hbelike followed by three hbb-like genes. we isolated dna and rna from blood of rrgs, akc registered greyhounds (akcg), and german shepherd dog (gsd). all beta globin exons and splice sites were sequenced, and the beta globin locus was examined by array comparative genomic hybridization (custom m agilent array). additionally, we determined the number of common haplotypes that span this locus in rrgs and akcgs using high density snp array ( k illumina hd). expression and sequence analysis of cdna showed all five beta globin genes are actively expressed in adults. canhbb and were created by relatively recent segmental duplication and have identical protein sequence. canhbb / are abundantly expressed in adults; canhbb is expressed at greatly reduced levels. sequencing results revealed one rare non-synonymous single nucleotide polymorphism (snp) in hbe of rrgs, but no variation that could explain their abnormal hemoglobin. we did not detect structural variation overlapping or near the beta globin locus. notably, rrg and akcg do not share haplotypes spanning the beta globin locus. this is the first characterization of canine hemoglobin genetics, and the first report of canine embryonic hemoglobins and their expression in adults. sampling of the bone marrow in the dog from the costochondral (cc) junction can be performed with minimal to no sedation and readily available equipment but is not in widespread use in the united states. the aim of this study was to compare the number of attempts needed to successfully obtain a sample, the time needed for the procedure, and the sample quality between aspirates obtained from the cc junction and more traditional sites (humerus or femur) in healthy dogs when performed by novice and seasoned practitioners. samples were obtained from healthy anesthetized laboratory reared adult dogs after undergoing terminal endoscopic surgery. paired samples from separate dogs were obtained by each practitioner using either a gauge needle and cc syringe at the cc junction or an gauge rosenthal needle and cc syringe from either the proximal humerus or femur (clinician preference). three small animal veterinary interns, one experienced technician and one boarded internist were monitored for number of attempts to success and length of time needed for success of each procedure. slides were prepared by a single investigator and read by a blinded clinical pathologist. data were compared using the paired t-test for normally distributed data and wilcoxen signed rank test for non-gaussian distributions. five pairs of samples from three dogs were evaluated. two dogs had two pairs drawn from opposite limbs and ribs. mean number of attempts to success for traditional sampling sites ( . /À . ) and time to success ( . minutes /À . ) did not differ significantly from attempts ( . /- . , p . ) or time ( . /- . , . ) needed when aspirating from the cc junction. subjectively, samples were of similar quality with regards to cellularity and number of particles present when compared within practitioners. myeloid: erythroid ratio and percentage of lymphocytes were also not significantly different between sites (m:e ratio p . , lymphocyte % p . ) and were within normal limits. while there were no significant differences between the two sites in terms of number of attempts or time to success, it should be noted that the ''seasoned'' practitioners had never performed an aspirate at the cc site and had an increased number of attempts compared to the traditional sites. if the number of attempts needed for success decreases with experience, it is likely the time required would decrease as well. both subjectively and objectively, there were no significant differences in quality or cell populations between the two sampling sites in healthy dogs. based on this data, bone marrow aspiration from the cc junction appears to be equivalent to more traditional sampling sites in healthy dogs. larger studies in clinically ill dogs should be performed before routinely using the site in the clinical setting. recent research on iron homeostasis has elucidated the tightly controlled intestinal uptake of iron. hepcidin, the major hormone limiting iron absorption and release from macrophages, is downregulated by matriptase- , a transmembrane serine protease (tmprss ) produced by the liver. while iron deficiency is commonly caused by chronic blood loss anemia and rarely dietary deficiency or intestinal disorders in dogs and other species, we report here the clinical to molecular investigations of a dog with iron-refractory iron deficiency anemia (irida) caused by a matriptase- deficiency homologous to a recently described autosomal recessive disorder in humans. the proband, a spayed female cocker spaniel without any clinical signs except for recent occasional idiopathic seizures, exhibited a lifelong history of microcytosis and hypochromasia but not anemia. there was no evidence of any blood loss and the dog was receiving an appropriate meat-based diet. mean values of complete blood cell counts, performed from . - years of age, were: hematocrit % (normal reference range - ); rbc count .  /ml ( . - . x ); mcv fl ( - ); mchc g/dl ( - ). serum iron parameters revealed severe iron deficiency with serum iron mg/dl ( - ); total iron binding capacity mg/dl ( - ); serum iron saturation o % ( - %), and ferritin ng/dl ( - ). prolonged courses of oral ferrous sulfate supplementation and several short courses of intramuscular (dextran) injections and intravenous iron infusions did not result in improvement of any red cell or serum iron parameters. however, this dog was never anemic and the partial seizures could not be directly related to the iron deficiency status. no family members were available for further studies. genomic dna was extracted from the proband's edta blood and the exons of the tmprss gene were amplified with flanking primers and then sequenced. in comparison to the normal canine tmprss sequence and that of a sequenced control dog we found a homozygous missense muation, r h, toward the c-terminal end of the protein in the proband's gene. in conclusion, the severe microcytosis and hypochromasia, low serum iron parameters and lack of a response to oral and parenteral iron therapy led to the diagnosis of irida. the missense mutation in the matriptase- at position from an arginine, which is conserved across all species currently deposited in the genbank, to a histidine is likely the disease-causing mutation. this is the first report of an irida in the dog with features very similar to those observed in humans. dogs with naturally-occurring irida may be helpful in developing and assessing novel therapies. accidental ingestion of copper-coated zinc pennies minted after is the most common causes of zinc toxicity anemia in the dog. zinc toxicity anemia may also be seen with ingestion of zinc from other sources as ingestion of metallic foreign material other than pennies, medicines containing zinc, and zinc supplements. the purpose of this study was to determine if there is a weight below which dogs are more susceptible to zinc toxicity anemia secondary to metallic foreign body ingestion. records of dogs presented to the internal medicine service at the veterinary medical center of long island for metallic foreign body ingestion were reviewed for signalment, weight, presenting pcv, and type of metallic foreign body ingested. eighteen dogs met the inclusion criteria and were compared. of the dogs, there were cases of coin ingestion ( %), with ( %) involving ingestion of or more pennies. the other cases involved ingestion of a metallic object ( ), decorative garland ( ), and bb pellets ( ).of the dogs exposed to zinc, ( %) were less than pounds ( . kgs). of those cases ( %) had ingested one or more pennies. eleven out of the ( %) zinc exposure dogs were anemic at presentation. the average weight of the dogs was . pounds ( kg). this study showed that dogs less than pounds appear to be more susceptible to developing anemia secondary to zinc toxicosis, with the majority of cases due to ingestion of pennies minted after . zinc toxicity anemia secondary to penny ingestion is more commonly seen in small dogs. we suspect larger dogs are able to pass pennies through the pyloric sphincter and thus not develop clinical signs. although thrombocytopenia is common in hospitalized dogs, canine cryopreserved platelet concentrate (pc) is used infrequently. data suggest in vitro efficacy of pc and when administered to research dogs, but efficacy is unknown in clinical patients. study objectives were to determine clinical characteristics of dogs receiving pc as well as safety and efficacy of pc in thrombocytopenic dogs. medical records were evaluated retrospectively to identify dogs that received pc. information evaluated included patient characteristics, platelet count, acute transfusion reactions, and survival. twenty six dogs met study criteria. dogs receiving pc ranged in age from - years (mean . years) and / ( . %) were spayed or intact females. hemorrhage was reported in / dogs ( . %) prior to pc transfusion. platelet counts prior to transfusion ranged from to  /ul (mean . /À .  /ul). change in platelet count was measured in dogs and the mean change was . /À .  /ul. dose of pc administered ranged from . to ml/kg with a mean of . /À . ml/kg. no acute adverse reactions were reported. there was no correlation between transfusion dosage and platelet count change post transfusion. survival to discharge occurred in / ( . %) of dogs. the only variable correlated with survival was age with survivors being younger than non-survivors ( . years-old ae . vs. . years-old ae . .; p . ). efficacy of cryopreserved pc transfusions for improving clinical outcome in dogs with thrombocytopenia is yet to be determined; however, pc is well tolerated in clinical patients. fresh frozen plasma (ffp) is used to treat coagulopathies in dogs. current transfusion guidelines recommend that ffp be administered within hours of thawing to avoid decreasing clotting factor function and bacterial contamination. the purpose of this study was to assess clotting factor activity and bacterial contamination of ffp that had been thawed and refrigerated for days. blood was collected from client-owned healthy dogs with no known history of coagulopathy or of administration of drugs affecting coagulation. plasma was separated from whole blood and frozen (À c) within minutes of collection. thawed plasma was maintained at c ( /À c). aerobic and anaerobic bacterial culture, prothrombin time (pt), activated partial thromboplastin time (ptt), and factor ii, vii, ix, and x analyses were tested at time of whole blood collection, ffp thaw, hours post-thaw, hours post-thaw, and hours post-thaw. there were no statistically significant differences in pt and ptt at any of the measured time points. statistically significant differences occurred between initial measurements of factors ii, vii, ix, and x and subsequent time points, but there was no difference in activity levels of the factors once ffp was thawed. one bacterial colony was grown from each of two samples from post-thaw plasma. thawed plasma protocols do not significantly decrease the function of factors ii, vii, ix, and x or prolong pt and ptt. bacterial contamination of the plasma supply seems unlikely, but strict aseptic technique should be used when obtaining plasma for patient use. erythrocyte pyruvate kinase (pk) deficiency is the first and most common erythroenzymopathy described in dogs, cats, and humans. the pk enzyme plays a crucial role in the erythrocyte energy metabolism and its absence causes severe hemolytic anemia, often misdiagnosed as autoimmune hemolytic anemia. the disease is inherited as an autosomal recessive trait and affected dogs also develop osteosclerosis. in dogs, the enzymatic diagnosis is complicated by the anomalous expression of malfunctioning m -pk expression, but breed-specific r-pk mutation tests have been reported for basenjis, west highland white terriers (whwt), and beagles. we report here on a survey of canine pk deficiency studied at the penngen laboratory. a biased group of samples were received for screening from dog breeds with known mutations as well as from dogs with chronic, prednisone-and antibiotic-resistant hemolytic anemia and their relatives. edta blood samples and/or cheek swabs as well as medical record information were received and genomic dna and/ or enzyme activity testing were performed. among the whwts % and % were found to be homozygous deficient dogs or carriers, respectively, with a mutant allele frequency of . . the average age at the time of diagnosis was . years ranging from months to years of age; some samples came from europe and south america. of the beagles studied, % were affected and % were carriers (mutant allele frequency . ). the average age at the time of diagnosis was years ranging from months to years. surprisingly, very few samples from basenjis were received for screening, and none showed the mutant allele. while pk-deficient basenjis lived o years, whwt and beagles often show milder signs and can reach years of age. several dogs from other breeds were also examined because of chronic regenerative anemia and none had any of the known mutations seen in the other breeds. however, based upon pk enzyme activity studies, chihuahua, dachshund, miniature poodle, spitz, eskimo toy, and labrador retriever dogs were found to be affected; they also had osteosclerosis and at least one labrador retriever developed severe hemochromatosis (hepatic iron , ppm; normal o , ppm, analyzed on a dry weight basis). moreover, sequencing of the r-pk cdna from a pk-deficient labrador retriever revealed a new nonsense mutation in exon . in conclusion, pk deficiency appears to be a common cause for hemolytic anemia in certain breeds, and mutation testing makes screening simple. pk deficiency should also be considered in dogs of other breeds which may require the more cumbersome enzyme testing. studies to identify new mutations will confirm and simplify the diagnosis. supported in part by nih grant rr . immune-mediated hemolytic anemia (imha) is a common hematological condition observed in dogs. the diagnosis is based on clinical history, presenting signs and hematological evidence of imha such as regenerative anemia, leucocytosis and presence of spherocytes. the definitive diagnostic procedure is the coomb's test (direct antiglobulin test, dat) which is known to be highly specific but lacks diagnostic sensitivity. direct flow cytometric assay (fca) for igg, igm or c coated red blood cells (rbcs) detection might be more sensitive and thus could be introduced as an alternative diagnostic tool. to investigate the usefulness of fca for imha diagnosis, evaluation of igg, igm or c coated rbcs was performed from dogs presented at the veterinary hospital at usp that fulfilled clinical and hematological criteria for imha. thirty eight healthy dogs were included as controls. dat was performed with polyvalent and monovalent anti-dog sera with twofold serial dilutions of each one, incubated with % rbcs suspension at c and c. for fca, % rbcs from anemic and healthy dogs were incubated with fitc anti-dog igg, anti-dog igm and anti-dog c and submitted to flow cytometry evaluation. specific software and mann whitney u test were used for data analysis. five dogs showed positive results for dat with polyvalent coombs reagent at c (titer to ) and c (titer to ) but only three of them had agglutination titer for anti-igg at c ( to ) and c ( to ). no positive results were observed for anti-igm and anti-c dat. by fc, percentage of igg, igm and c coated rbcs in normal and anemic dogs were, respectively, , % and , % (p o , ); , % and , % (p o , ); , % and , % (p o , ). igg coated rbcs percentage were higher in dogs showing dat positive results (min. , %; max. , %; median , %). direct flow cytometric erythrocyte immunofluorescence assay is more sensitive than dat for detection of antibodies coated rbc in anemic dogs and may provide quantitative data useful for laboratorial diagnosis of imzha. bone marrow aspirates from cats are typically obtained from the ilium, humerus or femur, but may be difficult to obtain and/or of poor quality. in this study the feasibility, safety, and nature of sternal aspiration in cats was investigated. under general anesthesia, bone marrow aspirates were obtained in a randomized order by a single investigator from the sternum and ilium of healthy cats weighing . - . kg, with body condition scores of - (on a scale of - ). for sternal aspirates, cats were positioned in sternal recumbency and a -inch, - ga hypodermic needle attached to a cc syringe was inserted into the cranial manubrium and directed caudally along the long axis of the sternum. aspirates were also obtained from the right iliac crest using an ga illinois needle attached to a cc syringe. difficulty of site localization, needle insertion and advancement, and specimen aspiration, were scored from (easiest) to (hardest). bone marrow smears were prepared by one investigator and reviewed by a pathologist blinded to aspiration site and cat. sample quality was scored from (no marrow particles) to (excellent) based on the number of wellsmeared marrow particles on the slide. particle cellularity was scored from ( % fat) to (o % fat). post-procedure, cats were treated with tramadol ( - mg/kg, po, q h) for days, and assessed for post-biopsy pain (colorado state university feline acute pain scale, range [no pain] - [maximum]) and site swelling (range [none] - [marked]). data were analyzed by ancova accounting for effects of weight and body condition score. pneumothorax was not identified. it was significantly easier to perform sternal than iliac aspiration, but the quality of the sample was significantly better for iliac than for sternal aspirates. because of limitations due to sample quality, bone marrow morphology in sternal samples could not be compared to iliac samples in all cats. for samples that could be compared, cellularity was identical for sternal and iliac samples from cat but underestimated in the sternal sample from another cat. myeloid:erythroid ratios and lymphocyte numbers were the same for sternal and iliac samples in and cats, respectively. megakaryocyte numbers were the same in one sample, less in sternal samples compared to iliac samples from cats, and overestimated in the sternal sample from cat. bone marrow cell morphology was normal in all acceptable samples. it was concluded that sternal aspiration of bone marrow using a - ga hypodermic needle is ) easier to perform than iliac aspiration; ) safe; but ) provides samples of lower quality than iliac aspiration in cats. the diameter of - ga jamshidi-type needles makes bone marrow core biopsy difficult in cats. in this study, biopsies of the left humeral head were taken under anesthesia using a -inch, ga needle (ez-io s intraosseous infusion system, vidacare) from healthy cats weighing . - . kg with body condition scores of - (on a scale of - ). humeral biopsies were compared to biopsies taken from the left iliac crest using a -inch, ga jamshidi needle. biopsies were performed in randomized order by one investigator. biopsy was repeated to a maximum of attempts until a specimen ! mm long was obtained. difficulty of site localization, needle insertion and needle advancement were scored from (easiest) to (hardest). specimens were wrapped in tissue paper and placed in davidson's fixative for min and then transferred to formalin. biopsy sections were reviewed by a pathologist blinded to biopsy site and cat. biopsy length on the slide was measured, and biopsy quality was scored from (no hematopoietic tissue) to (! intertrabecular spaces free of artifact). post-procedure, cats were treated with tramadol ( - mg/kg, po, q h) for days, and assessed for postbiopsy pain (colorado state university feline acute pain scale, range [no pain] - [maximum]) and swelling of biopsy sites (range [none] - [marked]). data were analyzed by ancova accounting for effects of weight and body condition score. there were no significant differences between ga and ga biopsies except for post-biopsy swelling, and there were no significant effects of body weight and body condition. six ( %) of ga and ( %) of ga biopsies were considered acceptable specimens for assessment of bone marrow architecture and morphology; all intact spaces in these biopsies had normal hematopoiesis and cell morphology. comparison of acceptable ga to ga biopsy specimens from cats showed no significant differences for cell density and lymphocytes/plasma cells, while cellularity, assessed as high in of the ga biopsies, was assessed as medium in corresponding ga biopsies; and megakaryocytes, assessed as - /low-power field in one ga biopsy, were assessed as /low-power field in the ga biopsy. myeloid:erythroid ratios were greater in ga biopsies compared to ga biopsies in cats, and less in the ga biopsy in one cat. discordant results between biopsies were not related to differences in quality. in conclusion, ga bone marrow biopsy of the humerus was as likely to yield a specimen of acceptable quality as was ga biopsy of the ilium, and resulted in less post-biopsy swelling. reports on canine acute liver failure (alf) include individual or small case series of animals with a specific diagnosis. the aim of this study was to describe the clinical course, outcome and etiology of alf in dogs presenting to a referral hospital. medical records ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) were reviewed for a diagnosis of alf (elevated serum bilirubin or icterus with concurrent coagulopathy or hepatic encephalopathy (he)). fifty cases were identified representing breeds: labradors retrievers, golden retrievers, german shepherds, and cocker spaniels. median age was years ( m to humerus, ga . ae . . ae . . ae . . ae . à ( . - . ) ( - ) ( - ) ( - ) ilium, ga . ae . . ae . . ae . . ae . . ae . . ae . à ( . - . ) ( - ) ( - ) ( - ) ( - ) ( - ) yrs). presenting signs included anorexia ( / ), vomiting ( / ), polydipsia ( / ) and neurologic signs ( / granulomatous hepatitis (gh) is a histopathological diagnosis characterized by focal aggregations of activated macrophages mixed with other inflammatory cells that is usually part of a systemic disease process (wsava). published case reports describe many potential infectious causes, but only one retrospective study involving nine dogs with gh has detailed clinically relevant findings. the aims of this study were to describe the clinical and clinicopathologic findings in dogs with a histopathological diagnosis of gh, and to identify infectious agents using differential staining techniques, pcr, and fluorescence in-situ hybridization (fish) in archival paraffin-embedded tissues from dogs with gh. medical records of dogs with a histopathological diagnosis of gh (n ) were reviewed and signalment, historical toxin exposure or evidence of other systemic diseases, clinical signs, physical exam findings, clinicopathologic test results, imaging findings, concurrent diagnoses, treatments administered, and case outcome, when available, were extracted and summarized. twelve archival formalin-fixed, paraffin-embedded hepatic tissue samples were available for special staining and molecular diagnostic testing. two of these samples had sufficient tissue for only pcr. the mean age of dogs with gh was years (median . years; range to years) and included males and females representing different breeds. common presenting complaints included inappetance or anorexia (n ), weight loss (n ), lethargy (n ), fever (n ), and vomiting (n ). high mixed liver enzyme activity ( / ) was the most common clinicopathologic abnormality. leukemia was diagnosed in one dog and copper-associated hepatopathy in dogs. no infectious agents were identified using differential staining techniques. bartonella species dna was not pcr amplified from the extracted archival tissue. furthermore, no bacteria were identified by means of fish using a universal eubacterial probe. these data suggest a possible role for copper accumulation in the genesis of gh in dogs and support further evaluation of dogs with gh for evidence of copper-associated hepatopathy. future studies should include detailed environmental histories, the collection of adequate sample volumes for quantification of hepatic copper content and the examination of frozen tissues using novel molecular diagnostic platforms. hepatocyte copper and iron accumulation contribute to cell loss, inflammation, and fibrosis. the purpose of this study was to compare copper and iron accumulation in feline liver samples with different disease processes. liver biopsies (n ) submitted between july , and june , were evaluated using wsava guidelines and categorized as non-hepatic/normal, congenital, inflammatory/infectious, neoplastic, and other. copper (by rubeanic acid) and iron (by prussian blue) accumulation were graded by increasing amounts ( - ) and location (centrilobular cl, midzonal mz, periportal pp, random r). associations between metal scores and diagnosis category were assessed using the kruskal-wallis test. histologic diagnoses were non-hepatic/normal (n ), congenital (n ), neoplastic (n ), infectious/inflammatory (n ), and other (n ). ninety-two samples were negative for copper; remaining samples were graded (n ), (n ), and (n ). histologic diagnoses (pattern) for positive samples were congenital ( cl), infectious/inflammatory ( : cl, mz, pp, r), neoplastic ( pp), and other ( cl). iron staining was negative in samples; remaining samples were graded (n ), (n ), and (n ). distribution was primarily cl (n ) or r (n ), though mz (n ) and pp (n ) distribution occurred. there were no significant differences by kruskal-wallis analysis for amount or location of hepatocellular iron or copper for the different disease categories. in this study, copper accumulation was rare, had variable distribution and occurred primarily in samples with inflammatory/ infectious disease. in contrast, iron accumulation was common and did not correlate with disease category. further prospective evaluation of copper and iron accumulation in feline liver disease and association with outcome may be warranted. chronic hepatitis (ch) in dogs is a progressive condition without clearly defined treatment. glucocorticoids are commonly used to stop progressive inflammation and fibrosis but are associated with significant side effects including a steroid hepatopathy that complicates enzyme monitoring. cyclosporine is proposed as an alternative therapy, but there are no published reports of its use for canine ch. patient records at the csu veterinary teaching hospital were searched for histologically confirmed cases of ch treated with cyclosporine. data were compiled on cyclosporine dosing, concurrent medications, clinical course and biochemical parameters. patients over a -month period were identified. serum alanine aminotransferase (alt) decreased by an average of % in dogs. the alt normalized completely in of dogs treated for days. in of dogs on mg/kg/day, the alt also normalized. five of the patients that exhibited clinical signs prior to treatment showed measurable improvement (weight gain, fewer gastrointestinal signs). eight patients had hyperbilirubinemia or ascites prior to treatment; these resolved in . post-treatment histopathology, available in one patient, revealed decreased severity of ch. five patients exhibited adverse effects including gastrointestinal signs ( ), gingival hyperplasia ( ), and papillomatosis ( ). cyclosporine was discontinued in dogs with gastrointestinal signs. cyclosporine was an effective therapy for many cases of ch and should be considered for patients who are refractory to or cannot tolerate glucocorticoids. prospective clinical trials with histological documentation are needed to better define cyclosporine's effectiveness in ch. insertion of the veress needle and establishment of pneumoperitoneum is associated with to % of all laparoscopic complications in humans. the purpose of this study was to determine the accuracy of interpretation of tissue impedance measurements for veress needle location. two laparoscopists, blinded to impedance measurements, placed reusable veress needles in cadaverous dogs euthanized for reasons unrelated to the study. placement order was randomized. a third individual evaluated impedance measurements using a handheld device (sensormed, knoxville tn) to determine correct versus incorrect needle placement. veress needle locations were marked using contrasting colors of india ink; tissues were dissected to determine ink locations. impedance measurement interpretation identified / correct and / incorrect placements, respectively. sensitivity, specificity, accuracy, and precision for correct veress needle placement are listed below. agreement was moderate (kappa . , p . ) for placements by operator and very high (kappa . , p o . ) for placements by operator . results for tissue impedance measurement interpretation are superior to published data for currently available tests. impedance measurements accurately detected all incorrect needle placements. comparison of needle placement with and without tissue impedance feedback will be necessary to determine whether it increases operator detection of inappropriate veress needle placement and decreases installment phase complication rates. delayed detection of intestinal perforation during veress needle insertion is associated with high mortality. the purpose of this study was to evaluate the accuracy of tissue impedance measurement interpretation for veress needle location. two laparoscopists, blinded to impedance measurements, placed reusable veress needles in cadaverous cats. placement order was randomized. a third individual evaluated impedance measurements (sensormed, knoxville, tn) to determine placement location. needle locations were marked using india ink; tissues were dissected to determine ink locations. impedance measurement interpretation identified / correct and / incorrect placements. all undetected incorrect placements were located within the retroperitoneal fat pad. sensitivity, specificity, accuracy, and precision for correct veress needle placement are listed below. correlation was absent (kappa À . , p . ) for placements by operator and substantial (kappa . , p o . ) for operator . there was no association between correct or incorrect placement and operator on chi-squared analysis. failure of impedance measurements to identify placement in the retroperitoneal fat pad resulted in poor accuracy and discordant kappa statistics. small cat size limited the number of appropriate placement sites, perhaps resulting in excessively dorsal placement. comparison of needle placement with and without tissue impedance feedback will be necessary to determine whether impedance measurements increase detection of inappropriate veress needle placements or decrease installment phase complication rates. best clinicopathologic tests detecting portosystemic shunting (pss) in dogs remains controversial. this retrospective study examined performance of single random "fasting" and paired serum bile acids (sba; pre-and -hr post-feeding) in a large population of non-icteric dogs with confirmed pss (abdominal ultrasound, colorectal scintigraphy, radiographic or spiral-ct portography, laparotomy, or necropsy). sba were measured by enzymatic colorimetric method with normal o mmol/l. dogs meeting inclusion criteria (n ) included portosystemic vascular anomalies (psva; extrahepatic [e-psva], intrahepatic [i-psva]), and acquired pss (apss). signalment and laboratory parameters were recorded. non-parametric statistical analyses were used, two-tailed p o . applied with bonferroni corrections. median age and weight of breeds were . ( . - ) yrs and . ( . - ) kg, with equal gender distribution. random "fasting" sba detected % psva and % apss, whereas post-feeding sba detected % psva and % apss. low protein-c (o % activity) occurred in % psva and % apss. low mcv and creatinine occurred in % and % of psva dogs, respectively; other tests were less helpful. in apss, post-feeding sba was superior. compared to apss, psva had significantly (p . ) lower mcv, cholesterol, bun, creatinine, glucose, and protein-c. compared to e-psva, i-psva had significantly (p . ) lower post-feeding sba, mcv, albumin, urine specific gravity, and protein-c but higher cholesterol and glucose. post-feeding sba reflect physiologically provoked bile acid challenge and should be the preferred sba test in non-icteric dogs for pss detection. protein-c assists in identifying psva but its utility in apss may be complicated by concurrent coagulopathies and inflammation. this study compared outcomes of treatment with adjunctive nonsteroidal anti-inflammatory drugs (nsaids) or anti-inflammatory glucocorticoids in dogs with severe pulmonary blastomycosis. medical records were reviewed for dogs diagnosed with blastomycosis at the university of illinois veterinary teaching hospital between and . dogs with a presenting pao of mmhg, and clinical or radiographic signs of respiratory blastomycosis were included. all dogs were treated with either itraconazole, fluconazole, amphotericin b, or a combination of these. group (g ) dogs were treated with nsaids and group (g ) dogs were treated with glucocorticoids as anti-inflammatory adjunctive therapy. the following comparisons were made: days of oxygen supplementation, days in hospital, survival to discharge, and long term patient survival. mann-whitney u tests and chi-squared tests were performed on continuous and categorical data, respectively. p o . was considered significant. sixty-eight dogs fit the inclusion criteria. g consisted of dogs and g consisted of dogs. the two groups were found to be similar in weight, age, and sex distribution. there was no significant difference between the two groups with regard to duration of oxygen supplementation, duration of hospitalization, survival to discharge, and patient survival. there does not appear to be a difference between the clinical course or patient outcomes between groups of dogs with severe pulmonary blastomycosis treated with nsaids or anti-inflammatory glucocorticoids. further studies need to be performed to fully evaluate the impact these adjunct treatments have on prevention of ards and additional respiratory complications. diagnosis of feline histoplasma capsulatum infection traditionally relies upon identification of organisms in circulating monocytes or affected organs. in recent years, an antigen assay (aa) was developed for the diagnosis of disseminated histoplasmosis in human patients, but there is little information describing this test in cats. the goal of this study was to determine the sensitivity and specificity of h. capsulatum aa in cats with clinical disorders suggestive of histoplasmosis. urine and serum h. capsulatum aa results for feline patients from veterinary hospitals were evaluated. medical records were reviewed for confirmatory evidence of histoplasmosis (based on cytological or histopathological findings) or an appropriately supported alternate diagnosis. aa results were available for cats; initial testing was performed on urine samples, serum samples, and unspecified sample. of these cats, / had a definitive diagnosis of histoplasmosis based on organism identification, and had a definitive alternate diagnosis (e.g., neoplasia, other infection) based on necropsy findings (n ) or other clinical data (n ). an additional cats had a clinical alternate diagnosis with no cytological or histopathological evidence of histoplasmosis in the affected body system(s). the remaining cats had unverified histoplasmosis (n ) or an open diagnosis (n ). of the cats with confirmed histoplasmosis, were positive on initial urine aa. one cat (with rectal involvement) was negative, indicating a test sensitivity of %. one cat was positive on urine aa but negative on serum aa. all of the cats with definitive or clinical alternate diagnoses had negative results on the aa, suggesting an excellent specificity ( %). however, this result should be interpreted with caution, as the possibility of primary or concurrent histoplasmosis was only definitively excluded in the patients who underwent necropsy examination. these findings suggest that the aa for h. capsulatum is a reliable diagnostic tool in this species. a positive result appears to reliably support the presence of infection, but a small percentage of infected cats may be negative on aa. in addition, tests performed on urine may be more sensitive that those performed on serum. the purpose of this study was to evaluate the sensitivity and specificity of an aspergillus galactomannan antigen enzyme immunoassay (ga-eia) for the diagnosis of canine systemic aspergillosis. serum and urine samples were collected from sick dogs at hospitals (ucd and tamu). group dogs were diagnosed with systemic aspergillosis using culture (sterile site) or microscopy and culture (non-sterile site). group dogs had clinical findings suggestive of aspergillosis but an alternate diagnosis was established. group dogs were not suspected to have aspergillosis. samples were tested using the ga-eia and results expressed as a galactomannan index (gmi). gmis . were considered positive. comparisons were performed using the mann-whitney test. there were dogs in group , in group , and in group . serum was collected from all dogs, and urine from , , and dogs, respectively. serum gmis did not differ from urine gmis across groups. serum gmis of group dogs were higher than those of group and group dogs (p o . ). results from dogs in group did not differ from those in group (p . ). two dogs in group tested negative, but had localized pulmonary infections. one dog in group , which had paecilomycosis, tested positive. two dogs in group tested positive. one was being treated with plasmalyte. the other had a cutaneous opportunistic mycosis. these data support the utility of this assay to aid in the diagnosis of systemic aspergillosis in dogs. anaplasma phagocytophilum, an ixodes tick transmitted rickettsial bacterium has a wide mammalian host range that is not commonly reported in cats. clinical signs in humans, dogs and cats are often vague and include lethargy, anorexia and malaise. the purpose of this retrospective study was to describe the clinical signs, laboratory data and response to treatment in cats that tested positive for a. phagocytophilum on a commercially available pcr of peripheral blood (fastpanel tm ). this study describes and reports the appearance of intracellular morulae in feline neutrophils contributing to the diagnosis of a. phagocytophilum. the a. phagocytophilum real-time pcr (rt pcr) assay consists of four multiplexed primer systems designed to detect a total of three distinct genes. amplicons were confirmed as a. phagocytophilum by dna sequencing. clinicopathologic data was obtained by review of medical records and interview of primary veterinarians. complete blood counts were available from / cats and / blood smears were reviewed. the cats included in this study were all positive for a. phagocytophilum by real-time pcr. the cats ranged from months to years of age with an average age of . years. fifteen of cats had a history of tick exposure and lived in the northeastern region of the us, an ixodes endemic area. all cats presented with lethargy, / were anorexic and / had a fever (temperature o f). other clinical findings included hepatomegaly, splenomegaly, ataxia and ocular changes of conjunctivitis and elevation of the nictitating membrane. hematologic findings included leukopenia ( / ), neutropenia ( / ) and lymphopenia ( / ). thrombocytopenia was not noted in any case. morulae were seen within neutrophils in / cases. all cases in this report responded to treatment with doxycycline. this is the first report of the identification of morulae within neutrophils via peripheral blood smear review in cats confirmed by rt pcr to be infected with anaplasma phagocytophilum in north america. infection with anaplasma phagocytophilum should be considered in a clinically ill cat with tick exposure, living in an ixodes endemic area that presents to a veterinarian for lethargy, anorexia and fever. the spectrum of disease manifestations and the accompanying clinicopathological abnormalities indicative of bartonellosis in dogs have not been thoroughly characterized. the objective of this unmatched case-control study was to compare signalment, clinical and pathologic findings in clinically-ill dogs suspected of a tick-borne disease that were negative for bartonella sp. dna (controls) as were the dogs diagnosed with bartonellosis by pcr amplification, dna sequencing and the bapgm (bartonella alpha proteobacteria growth medium) enrichment culture approach. both groups were tested under the same laboratory conditions and in the same time frame. medical records were reviewed for information regarding signalment, medical history, physical examination findings, clinicopathological abnormalities, microbiological data and treatment. the study population consisted of bartonella-infected dogs and non-infected dogs. healthy dogs with no historical illnesses, such as blood donors, were excluded. the following species were amplified: b. henselae (n , . %), b. vinsonii subsp. berkhoffii (n , . %), b. koehlerae (n , . %), b. volans-like (n , . %), b. bovis (n , . %). nineteen ( . %) bartonella-infected dogs were febrile and lethargic and ten ( . %) had neurological signs. laboratory abnormalities for both groups are summarized below (number of affected dogs provided in parenthesis): multivariate logistic regression using confounding factors was performed to establish potential associations between specific variables and bartonella sp. infection. there were no differences in signalment, age, sex, body weight and duration of clinical signs between the two groups. compared to the control population, infection with the genus bartonella was associated with a diagnosis of endocarditis (p . , or . , %ci . - . ) and hypoglobulinemia (p . , or . , % ci . - . ). controls were more likely to have joint effusion (p . , or . , % ci . - . ) and azotemia (p . , or . , %ci . - . ) than were the bartonella sp. infected dogs. bartonella was detected in dogs with signs such as fever, anemia, thrombocytopenia, hyperglobulinemia and proteinuria that are typically associated with tick-borne diseases. when endocarditis or hypoglobulinemia are detected, testing for bartonella should be prioritized. likewise, the detection of bartonella should prompt further testing for endocarditis, if not already investigated. surveillance studies in other species depend on detection of antibodies to the highly conserved influenza a nucleoprotein (np); however, no such antibody detection assay is approved for canine use in the u.s. the purpose of this study was to determine the diagnostic accuracy of a commercial blocking elisa used for avian species in detecting influenza a np antibody in dogs. since the blocking elisa is not a species-specific or viral subtype-specific format, we hypothesized that it would detect np antibodies in dogs infected by influenza a virus. serum samples from uninfected dogs (n ) and dogs naturally infected with canine influenza h n (n ) were tested using the idexx flockchek blocking elisa for influenza a np antibody according to manufacturer instructions. the sample/negative control (s/n) absorbance ratios for infected dogs ranged from . to . compared to . to . for uninfected dogs. a receiver operating characteristic (roc) curve analysis determined optimum diagnostic sensitivity ( . %) and specificity ( . %) at a s/n cutoff ratio of . . using this cutoff ratio, the overall diagnostic accuracy was . %. coefficients of variation for intra-assay ( . %) and inter-assay ( . %) testing demonstrated good repeatability with canine sera. the excellent diagnostic accuracy of the commercial blocking elisa makes it a suitable tool for large-scale surveillance of influenza a virus exposure in dogs. upper respiratory disease (urd) can affect a majority of cats in shelters and is one of the leading reasons for euthanasia of otherwise adoptable cats. the purpose of this study was to determine prevalence and risk factors for upper respiratory pathogens in four different models for managing unowned cats: short-term animal shelters (shel), long-term sanctuaries (sanc), home-based foster care (fost), and trap-neuter-return (tnr) programs. conjunctival and oropharyngeal swabs were collected from cats, half of which had clinical signs of urd, and tested for feline herpesvirus (fhv), feline calicivirus (fcv), chlamydiophila felis, bordetella bronchiseptica (bb) and mycoplasma felis by real-time pcr. management model, vaccination, sex, age, body condition, and clinical signs were evaluated as risk factors for infection. a majority of cats in all management models carried one or more organisms capable of causing urd. in many cases, prevalence was similar in cats with or without clinical signs. unlike diseases that can be controlled by segregation of symptomatic animals, the lack of strong correlation between the presence of pathogens with the presence of clinical signs suggests that feline urd control should be managed by vaccination before or at the time of intake ,biosecurity protocols that presume all cats may be shedding pathogens, and minimizing stressful conditions that contribute to disease susceptibility. depending on geographical location, sex, age and environment, - % of cats worldwide are infected with the feline immunodeficiency virus (fiv). knowledge of the fiv status of cats is important to limit the spread of disease and to institute appropriate health management. however, like all lentiviruses, fiv is highly variable in nucleotide sequence, and viral load in cats is variable during different disease stages. detection of antibodies is the most widely employed diagnostic approach, but does not distinguish fiv-infected from fiv-vaccinated cats. in this study, samples from fiv-seronegative cats, fivseropositive cats, and fiv-historically seronegative but vaccinated cats, were analyzed by a commercial quantitative pcr (qpcr) assay and virus isolation. replicate blood samples were coded, and then submitted for ) qpcr (idexx); and ) mononuclear cell isolation with -day culture and viral p antigen detection by elisa. for the p antigen elisa, cutoff absorbance values were established from analysis of fiv-negative samples. fiv infection status was pre-determined based on antibody-elisa results and vaccination history. results indicated that qpcr had a sensitivity of % for samples from fiv-seropositive cats, and a specificity of % and . % for samples from fiv-seronegative and fiv-vaccinated cats, respectively. at a cutoff value of standard deviations above the mean absorbance for p antigen elisa, results from fiv-negative samples yielded a sensitivity of . % for samples from fiv-seropositive cats, and a specificity of . % and . % for samples from fiv-seronegative and fiv-vaccinated cats, respectively. conclusions from this study are ) the commercial fiv qpcr assay has high specificity but limited sensitivity for diagnosis of fiv infection; ) -day virus culture has limited sensitivity and specificity. hence, detection of antibodies remains the most reliable test for diagnosis of fiv infection, but qpcr may be suitable to rule out infection. oral disease is an important clinical problem in feline medicine and includes common painful conditions such as oropharyngeal inflammation (formerly known as gingivostomatitis) and tooth resorptive lesions. a number of infectious agents have been associated with private veterinary clinics in the u.s. were recruited to test feline patients presenting with oral disease. presenting cases included cats with plaque, calculus, gingivitis, stomatitis, periodontal disease, tooth resorptive lesions and other oral diseases as defined by the practitioner. all cats were tested using a commercially available point-of-care elisa test (idexx snap combo). confirmatory tests were not performed as part of the study. seroprevalence was calculated as the percentage of positive tests in the study population for each virus. a total of , cats were tested. seroprevalence for felv was . % and for fiv was . %. of these, cats ( . %) were infected with both viruses. seroprevalence was higher in cats with inflammatory oral disease than in cats characterized with other types of oral disease. of , cats with gingivitis, seroprevalence for felv was . % and for fiv was . %, with . % of cats co-infected. of , cats with stomatitis, seroprevalence for felv was . % and for fiv was . %, with . % of cats co-infected. the seroprevalence for felv and fiv reported in this population of cats with oral disease was higher than in a recent large study where samples from u.s. cats not specifically selected for oral disease were tested (felv . %, fiv . %). results of this study indicate that further investigation of the role of retroviruses in cats with oropharyngeal inflammation is warranted. reliable tests and preventive vaccines and medications for feline retroviral and heartworm (hw) infections are available, but compliance with protocols to reduce transmission is unknown. no largescale longitudinal studies evaluating prevalence over time have been reported. the purpose of this study was to determine the prevalence and risk factors for infection compared with a similar study completed for the first time years previously. veterinary clinics and animal shelters in the us and canada submitted results of testing using a point-of-care elisa for felv antigen, fiv antibody, and hw antigen (idexx snap triple) and risk factor information for cats tested during march-september . bivariable and multivariable analyses were used to evaluate risk factors for infections. a total of , cats were tested. only % of owned cats were prescribed hw preventive. risk of retroviral infections was increased by outdoor access, adulthood, and male gender. the most important risk factor associated with all infections was clinical disease; in particular, respiratory and oral diseases and abscesses or bite wounds. multivariate analysis revealed differences among geodivisions and across infection types. feline retroviral and heartworm infections are easily prevented, but difficult to treat. despite availability of effective management protocols, compliance remains inadequate to reduce the prevalence of these infections. improved use of preventive care and testing to identify and segregate contagious cats, particularly those at high-risk, is required to reduce the morbidity of these preventable infections. infectious disease outbreaks are common in animal shelters and are frequently managed by depopulation when risk-assessment tools are not available. during a canine distemper virus (cdv) and parvovirus (cpv) outbreak in sheltered dogs, we used a cdv/cpv point-of-care antibody titer elisa, a cdv quantitative rt-pcr test, and a cpv fecal antigen test as risk assessment tools to guide release of exposed dogs from quarantine and euthanasia of diseased dogs. serum samples (for antibody titers) and swabs of the conjunctiva and upper respiratory tract (for cdv pcr) were collected from asymptomatic dogs starting on day of the outbreak. dogs with positive cdv pcr tests were retested every weeks until euthanized for progressive disease or released following recovery from infection. dogs with clinical signs of parvoviral infection were tested using a cpv fecal antigen test. for dogs ! months old, protective antibody titers correlated with resistance to clinical disease, but % of dogs shed cdv. lack of protective cdv antibody titers correlated with susceptibility to clinical infection, but most dogs recovered. risk assessment and outcome in dogs ! months of age feline herpesvirus (fhv- ) is a common ocular and respiratory pathogen of cats that can have clinical illness exacerbated by stress. cyclosporine (csa) is commonly used for the treatment of a number of inflammatory diseases in cats and can induce immune suppression. a small number of cats administered csa to block renal transplant rejection have developed clinical signs of upper respiratory tract disease that may have been from activated fhv- . in this study, young adult cats experimentally inoculated with fhv- several months previously were divided into three groups and administered methylprednisolone acetate ( cats, mg/kg, im, day and day ), csa ( cats, . mg/kg, po, daily for days), or a placebo ( cats, corn syrup; . ml/kg, po, daily for days). each cat was assigned a daily individual clinical score by a trained, masked observer using a standardized score sheet during the initial pre-treatment time period (day - to day ) and throughout the day treatment period. each individual clinical score (conjunctivitis, blepharospasm, ocular discharge, sneezing, nasal discharge, nasal congestion, and body temperature scores), the total clinical score (sum of all parameters), the total ocular score (sum of conjunctivitis, blepharospasm, ocular discharge), and total respiratory score (sum of ocular discharge, sneezing, nasal discharge, nasal congestion) were analyzed using sas proc glimmix with 'treatment', 'time', and the two-way interaction 'treatment by time' all as fixed effects. statistical significance was defined as p o . . on day of the study, all of the csa treated cats had detectable concentrations of csa in serum (mean . ng/ml; standard deviation . ng/ml; median . ng/ml). when group mean values for clinical signs were compared over time as described, significant differences in individual clinical score measurements, in total score, total ocular score, or total respiratory score were not detected over time among any of the treatment groups. while clinical signs of activated fhv- occurred in some cats administered methylprednisolone or csa, disease was mild and self-limited in most cats and there were no significant csa sideeffects. these results suggest that the csa protocol described here is unlikely to reactivate latent fhv- infection and cause significant clinical illness. the purpose of this study was to determine the prevalence and risk factors for enteropathogens in four different models for managing unowned cats: short-term shelter, long-term sanctuary, home-based foster care, and trap-neuter-return (tnr) programs. fecal samples were collected from cats, half with diarrhea (d) and half with normal feces (n), and tested for a panel of feline and zoonotic enteropathogens by polymerase chain reaction, antigen, and fecal flotation. risk factors for infection evaluated include management practices, fecal consistency, and signalment. a majority of cats had at least one enteropathogen of feline or zoonotic importance, regardless of management model or preventive healthcare protocol. for most enteropathogens, the presence or absence of diarrhea did not correlate with infection, the exceptions being t. foetus in sanctuary cats and fcov in foster cats. prevalence of specific enteropathogens varied between management models, reflecting differences in preventive healthcare and housing conditions. management protocols for unowned cats were inadequate for elimination of infections present at the time of intake and for prevention of transmission of enteropathogens among shelter cats. improved compliance with effective vaccination, deworming, sanitation, and housing protocols is needed to reduce zoonotic and feline health risks. several allergic diseases of cats, including atopy and gingivostomatitis, can be resistant to glucocorticoids but responsive to cyclosporine. toxoplasma gondii infection occurs in approximately % of cats and the effect cyclosporine therapy has on the t. gondii oocyst shedding period is unknown. the objective of this study was to determine whether administration of cyclosporine before or after t. gondii infection influences the oocyst shedding period. the young adult cats were t. gondii seronegative when administered , t. gondii tissue cysts orally on day . group cats (n ) were never administered cyclosporine; group cats (n ) were administered cyclosporine ( . mg/kg, po) daily on days - ; and group cats (n ) were administered cyclosporine ( . mg/kg, po) daily from days - . available feces from individual cages were collected daily and fecal flotation by sugar centrifugation was performed for days after t. gondii inoculation. group shed oocysts for a significantly shorter period than groups or and had a significantly lower oocyst shedding scores than groups and on days - after t. gondii inoculation. group cats had completed the oocyst shedding period prior to being administered cyclosporine and repeat oocyst shedding was not detected during administration of the drug. administration of cyclosporine prior to t. gondii infection lessened oocyst shedding which is likely from the anti-t. gondii effects of the drug. administration of cyclosporine using this protocol is unlikely to induce repeat t. gondii oocyst shedding in client-owned cats. à group with diarrhea significantly different than group with normal feces p o . known about its metabolic pathways or mechanism of pathogenicity and whole genome sequencing of feline hemoplasmas has not yet been reported. the aim of this study was to completely sequence the genome of m. haemofelis to further characterise this important pathogen. mycoplasma haemofelis genomic dna was purified and subjected to whole shotgun roche sequencing. gaps were closed using targeted pcr and amplicon sequencing. ribosomal genes and potential open reading frames (orfs) were predicted in silico. putative orfs were annotated and orthologous groups identified. analysis showed a circular genome of . mbp with a gc content of . %. thirty-one transfer rnas (trnas) were identified, accounting for all amino acids, including a tryptophan trna for the opal codon (uga). of the , putative proteins identified, ( . %) matched to proteins from other bacterial species. in common with the pneumoniae group of mycoplasmas, the closest phylogenetic relatives of the hemoplasmas, genes involved in carbohydrate metabolism were limited to enzymes of the glycolytic pathway, with glucose appearing to be the sole energy source for m. haemofelis. the majority of the pentose phosphate pathway genes present in other cultivatable mycoplasmas appear to be incomplete or absent in m. haemofelis, suggesting an alternative mechanism for sourcing purine and pyramidine bases such as scavenging from the host. a gene encoding a glyceraldehyde- -phosphate dehydrogenase homolog of the immunogenic msg protein of mycoplasma suis was present. of the uncharacterized hypothetical proteins, , were arranged in series of orthologous repeats, or comprised fragments there-of, encoding putative proteins of approximately amino acids. the predicted motifs of the majority of these putative proteins were consistent with these proteins being presented on the cell surface; an n' terminal signal peptide or transmembrane region followed by a non-cytoplasmic tail. these data have provided valuable information as to why this pathogen remains highly fastidious; it lacks some of the metabolic pathways found in cultivatable mycoplasmas. we have also identified a homolog of a known m. suis immunogenic protein, and identified a potential mechanism for host immune system evasion by way of highly repetitive, putatively surface-expressed hypothetical proteins with variable sequences. canine leptospirosis has been recognized as a re-emerging disease in the u.s. over the past years, and several serosurveys of the prevalence of leptospiral antibodies in dogs have been published during that time. the role of cats in the epidemiology of leptospirosis has received little attention. serosurveys of cats for exposure to or infection with leptospires have been published from other geographic areas, but none for cats in the u.s. in the past four decades. the new england states have been found to have a high incidence of canine leptospirosis. the purpose of this pilot study was to determine the prevalence of leptospiral antibodies in a population of feral cats in central massachusetts. blood was collected from sexually intact feral cats presented to a spay and neuter program. microagglutination titers to leptospira serovars autumnalis, hardjo, bratislava, icterohaemorrhagiae, canicola, pomona, and grippotyphosa were determined. three of cats ( . %) had a positive titer to one or more serovars, with autumnalis being the most common. these results are consistent with previously published prevalence rates in feral cats. further studies are required to determine the role of leptosporosis in clinical disease in the domestic cat. since years the rivalta's test is routinely used in several european countries as a tool to diagnose feline infectious peritonitis (fip) in cats with effusion. it is inexpensive and easy to perform in private practice. there is, however, only little information about mode of action or its diagnostic value. the objectives of this study were to evaluate sensitivity, specificity, positive (ppv) and negative predic-tive values of the rivalta's test to diagnosis of fip and to examine if there is a correlation with any effusion or blood parameters. medical records of cats with effusion in which the rivalta's test was performed between and were reviewed concerning diagnosis, blood and effusion parameters, and survival time. effusion and blood parameters were compared between rivalta-positive and -negative effusions using the mann whitney u test. prevalence of fip in cats with effusion was . %. the rivalta's test showed a sensitivity of . %, a specificity of . %, a ppv of . %, and a npv of . % for the diagnosis of fip. the ppv improved, when cats with lymphoma or bacterial infection were excluded (ppv . %) and also, when only cats younger than years (ppv . %) or year (ppv . %) of age were included. the most important significantly different parameters between rivalta-positive and -negative effusions were specific gravity as well as cholesterol, triglyceride, and glucose concentration in the effusion. the rivalta's test in general is a useful tool to diagnose fip, but its sensitivity and specificity are not as high as previously assumed. if the rivalta's test, however, is performed in young cats or if certain diseases have been ruled-out, its diagnostic value is high. effusion total protein is not highly correlated with test outcome. therefore, it is still unclear, which components in the effusion of cats with fip lead to a positive rivalta's test. canine parvovirus (cpv) and canine distemper virus (cdv) infections are relatively common in animal shelters and are important population management issues since the immune status of incoming dogs is usually unknown. our study aimed to determine the antibody protection status of dogs at the time of admission into an animal shelter (pre-vaccination) and over the following weeks after vaccination. serum samples were obtained from incoming shelter dogs aged months and older with no known history of vaccination. immediately following serum collection, the dogs were vaccinated against cpv and cdv using a modified live vaccine (mlv). cpv and cdv antibody protection status was determined using synbiotics titerchek. dogs with unprotective serum antibody levels against cpv and/or cdv were retested at - days post-vaccination and again at - days post-vaccination, if antibody levels were still unprotective against cpv and /or cdv. at the conclusion of the study, stored duplicate sera were submitted for batch 'gold standard' testing to determine canine distemper virus serum neutralization and canine parvovirus hemagglutination inhibition antibody titers. based on the synbiotics titerchek results, / dogs ( . %) were protected against cpv and / ( . %) were protected against cdv at intake. older incoming dogs were more likely to be protected against cpv (p o . ) and cdv (p . ). dogs that were spayed/neutered were more likely to be protected against cpv on intake than intact animals, although this result was not statistically significant (p . ). the number of dogs with protective titers against cpv/cdv was increased at - days post-mlv (cpv - / , . %; cdv - / , . %) and further increased at - days post-mlv (cpv - / , . %; cdv - / , . %). we conclude that incoming shelter dogs often do not have protective antibody titers against cpv and cdv, but older shelter dogs are more likely to be protected against cpv. based on this population, we further conclude that a large percentage of dogs develop protective antibody titers to cpv and cdv within to weeks when vaccinated with a mlv. mycoplasma spp. are common inhabitants of the feline oral cavity and so likely contaminate many cat bite abscesses. mycoplasma spp. are cell-wall deficient and so do not respond to beta-lactam class antibiotics, the class most commonly use for the treatment of cat bite abscesses. the objectives of this study was to determine whether mycoplasma spp. are common contaminants of cat bite abscesses and are associated with beta-lactam resistant clinical disease. privately owned cats with clinical evidence of an acute abscess suspected to be from a cat bite were included in the study. participants were given a free aerobic and anaerobic culture as well as mycoplasma spp. culture and polymerase chain reaction using mycoplasma genus specific primers. mycoplasma spp. amplicons were sequenced to determine the species. all cats were initially treated with appropriate wound management, were administered an antibiotic in the beta lactam class (amoxicillin-clavulanate or cefovicin), and were rechecked in person or by phone days after beginning treatment. of the cats entered into the study to date, mycoplasma spp. were amplified from cats ( . %). of the positive samples with adequate dna for sequencing, one was consistent with m. felis and the other was consistent with m. equigenitalium. of the cats, responded by day to the initial treatment, including of the mycoplasma spp. positive cats. the cat that failed initial treatment was positive for m. equigenitalium on both day and day and ultimately responded to administration of a fluoroquinolone. the results suggest that while mycoplasma spp. commonly contaminate cat bite abscesses, routine wound management and antibiotic therapy is adequate for control. however, as mycoplasma spp. infections do not respond to beta lactam class antibiotic therapy, these organisms should be on the differential list for cats with abscesses that fail treatment with this antibiotic class. molecular diagnostic assays are frequently used in clinical practice to aid in the diagnosis of suspected infectious respiratory diseases in dogs. however, most currently available assays cannot distinguish strains of the organisms used in vaccines from naturally occurring strains. our prior studies demonstrated that previously immune adult dogs are unlikely to shed nucleic acids of vaccine strains of adenovirus , parainfluenza, or bordetella bronchiseptica. however, whether this is true for puppies is unknown. puppies (n ) at a breeding facility were moved into area without other dogs at weeks of age. swabs of the nasal and pharyngeal mucosa were collected prior to vaccination and on days , , , , , , , , , , , , , and after vaccination with an intranasal adenovirus , parainfluenza, and b. bronchiseptica vaccine (intratrac , schering plough). the swabs were shipped on cold packs by overnight express for dna/rna extraction and assay in the fastpanel tm pcr canine respiratory disease profile at antech diagnostics. all puppies were negative for the infectious agents prior to vaccination. after vaccination, positive assay results for parainfluenza and b. bronchiseptica were first detected on day and on day for adenovirus . by day , dna or rna of the agents were amplified from all puppies from both sample sites and most samples were positive for all agents through day . by day , only one dog was still positive for b. bronchiseptica. the results indicate that intranasal administration of adenovirus , parainfluenza, or bordetella bronchiseptica vaccines commonly leads to positive molecular diagnostic assay results for a short time period after primary vaccination. these findings should be considered when assessing the results of these assays in client-owned puppies with respiratory disease. antimicrobial resistance in escherichia coli is an increasing concern in both human and veterinary hospitals' patients. the choice drug for treatment in dogs is enrofloxacin, a second generation fluoroquinolone (fq) whose activity reflects, in part, ciprofloxacin. among the difficulties in effective e. coli treatment is rapid detection of fq resistance. the purpose of this study was to determine the specificity and sensitivity of a fret based assay for the rapid detection of urinary tract infections caused by fq associated multi-drug resistant e.coli. clinical e. coli isolated from canine urine and clinical veterinary urine samples being examined for e. coli were subjected to susceptibility testing for drugs representing drug classes. pure isolates were designated ndr (no drug resistance), sdr (single drug resistance) and mdr (multi-drug resistant) (n mdr, sdr and ndr). minimum inhibitory concentration (mic) for enrofloxacin ranged from . mg/ml to mg/ml, with high mic generally associated with mdr. extracted dna from culture and from urine were subjected to fret-pcr targeting single nucleotide polymorphisms in gyra. the resulting product was sequenced to detect other polymorphisms. further, to determine the level of detection, microbial free canine urine was inoculated with to cfu/ml of isolates characterized by variable susceptibility to enrofloxacin (mic enro . , . , . , , , , mg/ml). of pure isolates, were confirmed positive for enrofloxacin resistance (mic enro mg/ml), of which were positively identified by the fret-pcr assay giving a sensitivity of . %. only isolate that was resistant was not detected (specificity of . %). however, of the isolates expressing high level resistance (mic x breakpoint [ mcg/ml]), and mdr (n ), sensitivity . %. of the urine samples contained e. coli of which determined to be fq-resistant by the assay. colony dilutions of e. coli confirmed the assay able to detect enrofloxacin resistance at as low as cfu/ml. the relationship between cfus and the peak of the -(d/dt) fluorescence of the melting curve was r . . these results conclude that the assay is capable of detecting not only the presence of escherichia coli in clinical samples, but also detecting severity of fluroquinolone resistance and infection. the fluoroquinolones (fqs) are a key class of synthetic antimicrobial agents with an established history in both humans and companion animals of efficacy for treatment of urinary tract infections (utis) caused by e. coli, and fluoroquinolones are common therapy. among the commonly used fqs in dogs and cats are the nd generation drugs, enrofloxacin, marbofloxacin, orbifloxacin (all veterinary approved) and the human drug ciprofloxacin; no rd and th generation fq is routinely used. the purpose of this study was to assess the in vitro activity of different generation fqs toward e.coli uropathogens whose phenotype ranges from no resistance to multidrug resistance. a total number of canine uropathogenic canine or feline e.coli isolates had been subjected to susceptibility testing to drugs classes ( drugs) and phenotyped as to resistance: none (ndr, n ), single (sdr, n ), or multiple, mdr (resistance to - drug classes; n ). mdr included isolates susceptible (enr s -mdr, n ) or resistant (enr r -mdr) to enrofloxacin. the minimum inhibition concentrations (mics) for quinolones ( - st generation, - nd generation, - rd generation and - th generation) were determined for these isolates using broth microdilution methods according to clsi guidelines (e. coli atcc s served as a negative control). mic statistics were generated for each drug among phenotypes. the results showed that companion animal e. coli expressing ndr or sdr are largely susceptible to nd to th generation fqs. however, isolates expressing resistance to st or nd generation quinolone also express high level resistance based on the mic to rd and th generation fqs. the overall potency (mic) for the drugs for isolates not expressing enr resistance (that is, ndr, sdr and enr s -mdr) is gat canine leproid granuloma (clg) was first reported in brazil in . over the past years, cases of clg were diagnosed in sa˜o paulo, brazil, and clinical and epidemiological findings were similar to those reported in australia. all dogs presented with one or more, uni or bilateral, ulcerated or not, papular, nodular or tumoral lesions, mainly observed in the dorsal surface of the ear, site usually more affected. in general, the lesions are painless and confined to the subcutis and skin, and it does not involve regional lymph nodes, nerves or internal organs, and systemic clinical signs frequently are absent. short-coated breeds show a marked predisposition for this disease. the definitive diagnosis of clg was obtained by histological examination of skin biopsies that were stained with acid fast (ziehl-neelsen) and diffquik s . thirty one ( . %) of the dogs were purebred; in this study the breed pattern comprised ( . %) boxers, ( . %) german shepherd and labrador retriever, ( . %) dobermann, ( . %) brazilian terrier, ( . %) golden retriever, ( . %) bulldog, ( . %) american pitbull, ( . %) mastiff, ( . %) fila brasileiro and ( . %) cocker spaniel, ( . %) were of unknown breed. nineteen ( . %) of the thirty seven dogs were males. twenty ( . %) dogs were - years old. in most cases, dogs presented with unilateral or bilateral ear lesions, but rarely thoracic, foot and caudal lesions. the animals were successfully treated by use of rifampicin orally (''the brazilian protocol'') or enrofloxacin orally and topical rifamicin. anaplasma phagocytophilum is being recognized more frequently in dogs in endemic areas. currently, most suspected cases are evaluated for a. phagocytophilum antibodies by immunofluorescence assay (ifa) or elisa. since a. phagocytophilum is an acute disease, detection by antibody measurement may be negative on initial evaluation. it is possible that a. phagocytophilum dna can be amplified from blood or synovial fluid prior to seroconversion. wild caught ixodes scapularis adult ticks from rhode island were allowed to feed on young adult ( - years), mixed sex beagles for up to days. blood (weekly for weeks), serum (weekly for weeks), and synovial fluid (radiocarpal joint; alternating arthrocentesis weekly for weeks) were collected prior to tick attachment and then weekly after tick attachment. joint fluid cytology was performed and total dna was extracted from blood and synovial fluid and assayed in a proprietary real time pcr assay (fastpanel tm ) that amplifies the dna of anaplasma phagocytophilum, a. platys, ehrlichia canis, e. chaffeensis, and e. ewingii. serum was assayed for a. phagocytophilum antibodies by ifa. time to first positive results for serology and pcr were compared by paired student's t test. none of the beagles developed clinical evidence of disease, and no major changes in synovial fluid cytology were detected over time. of the beagles, were positive for a. phagocytophilum dna in blood or synovial fluid or ifa antibodies in at least one sample after tick attachment. antibody titers appeared in of dogs from weeks to (median to st positive weeks ae ). titer magnitude ranged from : to : , . anaplasma phagocytophilum dna was amplified from the blood of of dogs with positive test results ranging from to weeks (median to st positive weeks ae . ). anaplasma phagocytophilum dna was amplified from synovial fluid from of dogs between weeks to (median to st positive weeks ae ). of the dogs, were pcr positive for only one week and dog was pcr positive for two consecutive weeks. of the dogs, were positive for a. phagocytophilum in both blood and joints by dna analysis. anaplasma phagocytophilum dna was amplified from blood more quickly than seroconversion was detected by ifa antibody titer (t À . , p o . ) or dna was amplified from synovial fluid (t . , p o . ). anaplasma phagocytophilum dna can be amplified from the blood prior to development of detectable antibody titers by ifa. amplification of a. phagocytophilum dna from synovial fluid does not occur in all dogs, appears to be transient in most dogs, and a negative test result does not preclude a diagnosis of a. phagocytophilum infection. canine granulocytic anaplasmosis and granulocytic ehrlichiosis are tick-transmitted infections caused by anaplasma phagocytophilum (aph) and ehrlichia ewingii (eew), respectively. both organisms induce an acute clinical disease, frequently accompanied by fever, polyarthropathy and thrombocytopenia. however, aph and eew have different tick vectors, i.e. ixodes scapularis and ambylomma americanum, respectively, with different, but overlapping geographic distributions. in addition, infection outcome may be affected by other regional ticktransmitted pathogens, such as borrelia burgdorferi (mn) or ehrlichia chaffeensis (ar). therefore, we compared serology and pcr results derived from dogs examined at two private practices located in highly endemic areas for either aph or eew. serum collected between april-december, from minnesota dogs (n ) was tested by snap s dx s and whole blood was tested by aph pcr. serum collected from arkansas dogs (n ) for year beginning in august was tested using microtiter plate elisas for antibodies to eew, e. canis, and e. chaffeensis (ech) while whole blood was tested by ehrlichia pcr. comparisons were evaluated using chi square (Ã) and binomial (w) tests with an alpha of %. the above results indicated that dogs are frequently exposed to both aph and bb in mn, whereas ar dogs are often exposed to eew, but less frequently to ech. antibodies to e. canis peptides were found infrequently in both mn and ar with only seroreactive dogs detected in both locations. active eew infection, as determined by pcr, was four times more frequent in ar pet dog seroreactors as compared to active aph infections among aph seroreactors. although both organisms induce acute disease, the number of aph and eew pcr positive dogs that were also seropositive was relatively high suggesting that both organisms induce persistent infections or that dogs are frequently re-infected, despite the presence of a measurable humoral immune response. additional studies are needed to determine regional infection profiles in other areas that are endemic for these pathogens. anaplasma phagocytophilum and ehrlichia canis are two of the most common vector borne disease agents that infect dogs and cats. while pcr assays that amplify the dna of these agents from blood are currently available, there is minimal information concerning the performance of these assays in different commercial laboratories that utilize different techniques. the purpose of this study was to compare the e. canis and a. phagocytophilum results of two different laboratories on the same samples collected from client-owned animals. veterinarians in states (az, md, ct) were recruited to participate in the study based on high prevalence rates for e. canis or a. phagocytophilum infection. blood in edta was collected from dogs or cats with fever, thrombocytopenia, or clinical evidence of polyarthritis and an equal volume of the same blood sample was simultaneously shipped on cold packs by overnight express to colorado state and to antech diagnostics. standard operating procedures at each laboratory were followed for total dna extraction and amplification of gapdh as the dna control. at colorado state university, a previously published pcr assay that amplifies the dna of ehrlichia spp., anaplasma spp., neorickettsia spp., and wolbachia was performed on each sample with positive amplicons sequenced to determine the species. at antech diagnostics, a proprietary real time pcr assay (fastpanel tm ) that amplifies the dna of anaplasma phagocytophilum, a. platys, ehrlichia canis, e. chaffeensis, and e. ewingii was performed. in the study to date, samples from animals ( dogs and cats) have been assayed at both laboratories. dna of a. phagocytophilum ( cats and dogs) and e. canis ( dog) were amplified at both laboratories with a percentage agreement between laboratories of %. the results to date suggest that the assay results of the two laboratories for a. phagocytophilum and e. canis are comparable. ehrlichiosis and bartonellosis are zoonotic diseases caused by extremely small, obligate intracellular bacteria that require a mammalian reservoir and a blood sucking arthropod vector. human ehrlichiosis is present in peru, with a seroprevalence as high as % in the highlands. bartonella species in humans were also identified in peru since (b. bacilliformis). recently, a new species (b. rochalimae) was isolated from an american woman who became febrile after travelling to peru. dogs can become infected with the same ehrlichia species, and the majority of bartonella species that affect human beings. the role of dogs as reservoirs for human infections has not been clearly established, but exposure and/or infection in dogs has been used to monitor human exposure to tick-borne disease (tbd), since they share the same environment. the objective of this study was to determine the serological and molecular prevalence of anaplasmosis, ehrlichiosis and bartonellosis in rural dogs in the highlands of peru. a total of healthy adult dogs were enrolled in this study from four communities in the central highlands of peru: ondores, pachacayo, san juan de pachayo, and canchayllo. edta-blood samples were collected from dogs, whereas serum samples were available from dogs. serum samples were tested for ehrlichia canis, anaplasma, borrelia burgdorferi and dirofilaria immitis infections using a qualitative dot-elisa (snap s dx). the edta-blood samples were screened by conventional pcr for the groel gene of the genus anaplasma and ehrlichia, and for the intergenic transcribed spacer of the genus bartonella. speciation was conducted by nucleotide sequencing. bartonella genus dna was detected from seven of the dogs ( . %) and ehrlichia canis dna was detected and sequenced from one dog ( . %). four of the bartonella positive samples were identified by dna sequencing as b. rochalimae (genbank accession numbers hq and hq ). the other three bartonella positive samples were identified as b. vinsonii subspecies berkhoffii, the causative agent of endocarditis in dogs and humans. no dog was infected with anaplasma species by dna amplification, but one dog was seroreactive for this genus ( . %). no specific antibodies against ehrlichia canis and borrelia burgdorferi and no antigens of dirofilaria immitis were detected. this study expands the current knowledge about tbd in peru and describes for the first time the infection of b. rochalimae in dogs in peru. the results suggest that dogs may play an important role in the epidemiology of this infection in humans, since they can be asymptomatic but bacteremic. bartonella spp. dna is commonly amplified from the blood of cats exposed to ctenocephalides felis. in previous work, it was shown that cats administered imidacloprid and experimentally exposed to b. henselae infected cats and c. felis did not become pcr positive for b. henselae whereas untreated cats all developed infection. the purpose of this study was to determine if administration of imidacloprid to clientowned cats likely to be exposed to bartonella spp. and c. felis in the field lessens prevalence of bartonella spp. infection. veterinary students in tennessee and florida that owned cats that spent at least days per month outside and that were willing to apply imidacloprid to their cats monthly for six months were recruited for the study. blood for bartonella spp. pcr assay was collected from the cats seven months after starting imidacloprid administration and assayed at colorado state university. to serve as a control group that was unlikely to have been administered flea control products in the previous months, blood was collected from feral cats during tnr programs in each of the two cities and assayed for bartonella spp. dna. the bartonella spp. dna prevalence rates between the groups were compared by chi square analysis with significance defined as p o . . the overall prevalence rates for bartonella spp. dna in the blood of veterinary student cats ( . %) and the feral cats ( . %) were significantly different (p o . ). the distribution of results is shown in table . the results suggest that florida feral cats were more commonly exposed to c. felis than tennessee feral cats. while the cats in the groups were not exactly matched, the student cats were allowed outdoors for approximately days per month and lived in the same cities as the feral cats, so c. felis exposure rates were likely similar. as previously shown in experimentally-exposed cats, the use of imidacloprid monthly may influence transmission rates of bartonella spp. amongst naturally-exposed cats. in an endemic area for leishmaniosis and filariosis, coinfection can occur and immunomodulation produced by wolbachia might influence the clinical signs and progression of both diseases. the aims of the present study were ) to determine the prevalence of wolbachia in dogs infected with dirofilaria immitis (di) and other filarial nematodes, ) to evaluate the level of coinfection of leishmaniosis and filariosis by molecular assays and ) to evaluate any associations between leishmania infantum (li) infection, filariosis with or without wolbachia and clinical presentation and outcome. statistical differences between groups were tested for significance by the fisher exact test using spss v. . software (significance: p-value o . ). one-hundred and eighteen owned dogs from southeastern spain presenting for clinical evaluation were included in the study. criteria the results of this study highlight the increased sensitivity of pcr for diagnosis of filariosis, confirm the presence of wolbachia in dogs from the mediterranean basin, show the increased severity of hwd when li-filaria coinfection is present and suggest that wolbachia could play a protective role for leishmaniosis. wolbachia antigens can stimulate a th -type immune response, as has been previously described. however other factors (as treatment with doxycicline) might be responsible for the lower prevalence of wolbachia among filaremic dogs infected with li and further studies must be done to clarify this interaction. the purpose of the present study was investigate the occurrence of leishmaniasis in cats in the municipality of arac¸atuba, sa˜o paulo, brazil, an endemic area for canine visceral leishmaniasis. animals were evaluated by direct parasitological examination of lymphoid organs and serology for visceral leishmaniosis by immunosorbent assay (elisa) and indirect immunofluorescence (ifat). thirteen ( . %) out of cats studied were diagnosed with visceral leishmaniasis; eight ( %) by parasitological diagnosis through cytological examination of lymphoid organs, six ( %) were considered positive by elisa and one ( . %) by ifat. only two ( . %) out of the thirteen infected cats had clinical signs, characterized by the presence of crusty lesions on the dorsal cervical region and hepatosplenomegaly. regarding age five cats ( . %) had between six months and two years, being the others older than years ( . %). only one cat ( . %) was positive for the three employed methods. pcr confirmed leishmania sp infection in nine ( . %) cats, of which six were diagnosed previously by cytological examination, two by elisa and one by the three techniques employed. since its first description in feline leishmaniosis has been reported in several countries. the purpose of this study was to assess the prevalence of leishmania chagasi infection in cats showing dermatologic lesions from an endemic area for visceral leishmaniasis in brazil. animals were evaluated by direct parasitological examination of lymphoid organs, immunohistochemical technique for detection of amastigotes in lesioned skin and serology for visceral leishmaniosis by immunosorbent assay (elisa) and indirect immunofluorescence (ifat). twenty seven ( . %) out of the cats studied were diagnosed with visceral leishmaniosis. twelve ( . %) were positive by parasitological diagnosis; amastigote forms of leishmania sp were identified in lymphoid organs from / ( . %) infected cats, and immunohistochemical technique allowed the identification of nine ( . %) positive animals. the seroprevalence of leishmaniosis was . % ( / ) by elisa and . % ( / ) by ifat. fiv specific antibodies were found in / cats ( . %), of which / ( . %) had leishmaniosis. real time pcr confirmed leishmania chagasi infection in three cats. based on the evidence of the high occurrence of leishmaniosis in cats in this study, this disease should be included in the differential diagnosis of skin diseases of felines living in endemic areas. blastomyces dermatiditis is a dimorphic fungus that commonly affects large-breed hunting dogs. a recent advancement in diagnosis has come with the advent of a urine antigen screening test that has both high sensitivity and moderately high specificity. therapy for the disease involves use of antifungal agents, usually itraconazole, and length of treatment is based chiefly on resolution of clinical and radiologic signs. with the new urine antigen test, however, a noninvasive route of monitoring treatment progress is available and could be an adjunct device utilized to determine treatment efficacy and may even reveal a need for prolonged treatment. therefore, the purpose of this study was to determine if monitoring the blastomyces urine antigen test and comparing to pulmonary radiographic signs would elucidate the necessity for prolonged antifungal therapy, even after resolution of radiologic signs. to this end, a retrospective case review was performed that identified a series of client-owned animals with naturally occurring blastomycosis. the inclusion criteria were radiographic pulmonary parenchymal signs consistent with fungal disease and urine antigenconfirmed blastomycosis with repeated testing of both radiographs and urine antigen quantification as monitoring parameters until negative results achieved in each. ideally, intervals between testing dates would be between two and five months. radiographs were considered negative if all radiographic changes had resolved or if repeated radiographs separated by at least one month were considered static after documented improvement had occurred from original diagnostic radiographs (suspected scarring). urine antigen testing was considered negative if concentrations were less than . enzyme immunoassay units, a reference interval set by the testing laboratory. preliminary data analysis reveals resolution of radiographic signs of blastomycosis occurred earlier in many of the cases presented than did attaining a negative urine antigen concentration. ceasing treatment month after radiographic resolution of signs as has been recommended in the past might have resulted in premature discontinuation of therapy in many of the cases. monitoring of urine antigen concentrations may be of additional clinical use for determining when cessation of treatment should occur in cases of blastomycosis. persistent elevation of urine antigen concentrations after radiographic resolution of infection may account for apparent recrudescence of blastomycosis after suspected clinical resolution. giardia spp. and cryptosporidium spp. are both known to cause infections in dogs and humans in the united states. nevertheless, prevalence rates for dual infection in dogs had not been widely reported. in this study, fecal samples from dogs housed in a northern colorado animal shelter (n ), dogs owned by veterinary students in northern colorado (n ), and dogs from the pine ridge reservation in south dakota (n ) were collected. each sample was assayed with a commercially available fluorescent antibody assay that detects giardia spp. cysts and cryptosporidium spp. oocysts. those samples that were positive for giardia spp. or cryptosporidium spp. with adequate dna available for sequencing were genotyped by the glutamate dehydrogenase [gdh] and by the heat shock protein- [hsp- ] genes, respectively. overall, ( . %) of the dogs had current evidence of a protozoal infection ( table ). the dogs from pine ridge reservation had the highest prevalence rates for giardia infection and also for dual infections. from the student dogs, sequencing was successful for the three giardia isolates (assemblage d from dogs; assemblage c from one dog) and one cryptosporidium isolate (c. canis). from the reservation dogs, sequencing was successful for nine giardia isolates (assemblage d from dogs; assemblage c from dogs) and one cryptosporidium isolate (c. canis). cryptosporidium and giardia co-infections are commonly detected in dogs; in this study dual infections were more common than cryptosporidium infections alone. further studies will be required to determine the clinical importance of this finding. although the giardia and cryptosporidium isolates that were sequenced were the dog specific assemblages/genotypes, more samples should be analyzed in order to assess the potential for zoonotic transmission of either parasite. the current study was conducted to determine the prevalence of intestinal parasites in dogs visiting the veterinary teaching hospital, chiang mai university, northern thailand. fecal samples (n ) were collected and submitted by owners between august to february . demographic and geographic data were recorded. intestinal parasitic infection was diagnosed by both microscopic examination after zinc sulfate centrifugation flotation and commercially available ifa for giardia spp. and cryptosporidium spp. polymerase chain reaction and dna sequencing were performed on all giardia and cryptosporidium positive samples to provide genotyic information. overall prevalence of intestinal parasitic infection in dogs in chiang mai was . %. the most prevalent parasite was giardia spp. ( . %) followed by ancylostoma spp. ( . %), cryptosporidium spp. ( . %), cystoisospora spp. ( . %), toxocara canis ( . %), trichuris vulpis ( . %), coccidian-like ( . %), toxascaris leonina ( . %), and strongyloides spp. ( . %). the prevalence of having at least one parasite in dogs o year, - years, and years were . %, . %, and . %, respectively. of these infected dogs, . %, . %, . %, and . % were infected with one, two, three, and four organisms, respectively. available dna sequences from giardia spp. positive samples were shown to be dog specific. only one adequate dna sequence was available for cryptosporidium spp., which was shown to be c. canis. the findings suggested that intestinal parasitic infection was common in dogs in chiang mai, thailand. dogs could be potential source for zoonotic intestinal parasitic infection since dogs in this area are allowed for free roaming. regular deworming program is indicated to prevent not only transmission among dogs but also to human. a retrospective study was conducted on parasite positive fecal specimens consisting of canine, feline, equine and from other host species, comparing recovery of eggs, protozoan cysts and coccidian oocysts using standardized methods of parasite concentration: the formalin/ethyl acetate (f/ea) sedimentation concentration and the commercial fecalyzer (flotation) kit procedures. specimens were processed by each technique either according to manufacturer's instructions or according to standard laboratory procedures. formalin/ethyl acetate concentrations used at a ratio of ml normal saline to ml ethyl acetate for extraction of lipophilic material from pelleted stool samples, previously fixed in sodium acetate/acetic/acid/formalin (saf) solution. flotations with the fecalyzer kit were performed with concentrated zinc sulfate solution (s. g. . ) . the range of parasites recovered from these specimens included flagellate cysts ( total), coccidian oocysts ( total), ova and larvae of nematodes ( total), and ova of trematodes ( total) , and cestodes ( total). recovery rates by fecalyzer flotation were good for protozoan cysts, coccidian oocysts and nematode eggs and larvae, but very poor for cestode and trematode eggs. formalin/ethyl acetate concentration showed excellent recovery of all parasites and consistently outperformed fecalyzer in recovery rates. recoveries by f/ea concentrations were higher by . % for giardia, by . % for coccidia and by . % for nematode eggs and larvae. with the exception of coccidian oocysts, based on z-test analyses, recovery rates were significantly higher, at a confidence level of at least %, for all parasites, using formalin/ethyl acetate sedimentation concentration. although capc recommends the use of flotation with centrifugation methods for standard fecal ova and parasite examination for veterinary patients, sedimentation concentration methods are widely and effectively used in human diagnostic parasitology laboratories. these results provide good evidence for the use of f/ea concentration as a preferred method to flotation procedures for stool ova and parasite examinations in veterinary laboratories. cyclosporine and glucocorticoids are powerful immunosuppressive agents used to treat many inflammatory diseases. cyclosporine inhibits calcineurin-dependent pathways of t-cell activation and the resultant cytokine production, and glucocorticoids directly inhibit genes coding for cytokines. little work has been done comparing the effects of these agents on cytokine production in dogs. our study assessed these effects by measuring t-cell cytokine production using flow cytometry, and cytokine gene expression using quantitative reverse transcriptase polymerase chain reaction (qrt-pcr) in activated canine t-cells treated with cyclosporine and dexamethasone. for flow cytometric assays, peripheral blood mononuclear cells were separated using density gradients and cultured for hours in the presence of cyclosporine ( , , or ng/ml), dexamethasone ( À , À , À m), or cyclosporine plus dexamethasone. for qrt-pcr, whole blood was cultured for hours with the same drugs at the same concentrations, and rna was then extracted from leukocytes. expression of cytokines il- and ifn-g was analyzed in pma/ionomycinactivated t-cells by flow cytometry, and gene expression for il- and ifn-g in activated t-cell populations was assessed via qrt-pcr. flow cytometry and qrt-pcr both demonstrated inhibition of il- and ifn-g that was generally dose-dependent in response to both cyclosporine and dexamethasone. flow cytometry results from the average of samples collected from different dogs are shown in figure a . similar results were achieved using qrt-pcr ( figure b ). suppression of il- and ifn-g in activated t-cells has potential as an indicator of the efficacy of cyclosporine and glucocorticoids in suppressing canine t-cell function in vivo, and may therefore be of value for characterizing the immunosuppression induced by these drugs in clinical patients. idiopathic eosinophilic diseases are described in several breeds, but are over represented in rottweilers. the immunopathogenesis of idiopathic eosinophilic disorders is poorly characterised. studies in people highlight the importance of cytokines, particularly interleukin- (il- ), in mediating eosinophil maturation, differentiation, egress from the bone marrow, migration and polyclonal expansion. eotaxin- and eotaxin- also appear important for induction of chemotaxis and release of reactive oxygen species from eosinophils. the aim of the current study was to establish whether definable differences in specific cytokines associated with mediation of eosinophil production and survival are present between healthy rottweilers, non-rottweilers and rottweilers with non-parasitic eosinophilia. secondly, by evaluating cytokine profiles the study aimed to improve understanding of the pathophysiology of eosinophilia therefore assisting development of potential molecular treatment options. quantitative real-time reverse transcriptase polymerase chain reaction (qrt-pcr) assays were used to quantify messenger rna (mrna) encoding cytokines il- , il- , il- , il- p , il- p , il- p , il- , interferon gamma (ifn-g) and chemokines eotaxin- and eotaxin- from peripheral blood mononuclear cell (pmbc) samples obtained from healthy non-rottweiler dogs with normal eosinophil counts (n ) and rottweilers with normal (n ), mildly increased (n ) and high (n ) eosinophil counts. quantification of serum ifn-g was also performed using a commercially available canine-specific elisa. all samples were positive for housekeeping genes and all cytokines could be quantified with the exception of eotaxin- and - . results were normalised using three stably expressed housekeeper genes (rpl a, sdha and ywaz) and a relative copy number was calculated for each sample with the sample with the fewest copies given a value of . no significant differences were found between groups but there was a tendency for ifn-g mrna expression to be lower in the rottweilers with moderate to severe eosinophilia versus control dogs (p . ). this trend was not seen in the concentration of serum ifn-g quantified by elisa as there were no significant differences between normal and diseased animals. in conclusion, there were no significant differences in cytokine mrna profiles between normal dogs and rottweilers with varying degrees of eosinophilia. additional studies including larger numbers of affected dogs are warranted before any accurate conclusions can be made. the presence of large amount of antibody on erythrocyte membrane can accelerate red blood cell (rbc) removal process by the mononuclear phagocyte system. an antigenic stimulus such as the one promoted by vaccines, for example, can induce hypersensitivity reactions and may accelerate rbc destruction. the study objective was to evaluate the erythrocytic membrane potential in inducing lymphocyte proliferative response of recently immunized dogs. healthy adult dogs (n ) were immunized with multiple antigens (commercial vaccine with eight antigens: distemper virus, parvovirus, coronavirus, parainfluenza virus, adenovirus, infectious hepatitis virus and leptospire; and anti-rabies). blood samples from each animal were collected into edta tubes in two moments: pre (immediately before vaccination) and pos ( to days after vaccination). mononuclear cells were separated by gradient, marked with cfse-fitc and cultured. the stimuli for lymphocyte proliferation used were autologous erythrocytic membrane (aem) and concanavalin a (cona). aem was obtained by hypotonic lysis and tested in two concentrations (m : . ug/ ul; m : . ug/ ul). the proliferation assay was evaluated by flow cytometry and analyzed with specific software. the proliferation index (pi) was calculated dividing the fluorescence intensity of the basal sample by the stimulated one. statistical analysis was performed using paired t-test for parametric samples and wilcoxon test for non-parametric samples (a . ). the for the tested concentrations, autologous erythrocytic membrane does not constitute a stimulus for lymphocyte proliferation in vitro, either before or after vaccination procedure. additionally, there was no evidence of self-reagent lymphocytes to erythrocyte membrane after vaccination. e. coli is a common cause of canine urinary tract infection. current treatment emphasizes eradication of established infection rather than infection prevention but increased antibiotic resistance necessitates strategies to prevent infection. proanthocyanidins found in cranberry juice inhibit e. coli attachment to human uroepithelial cells, impairing bacterial adherence and colonization. we hypothesized that purified cranberry extract (ce) inhibits bacterial adhesion to canine uroepithelial cells. five healthy female dogs received an oral ce supplement (vetoquinol; mg ce/tablet) according to body weight for days. voided urine collected from each dog before (pre) and after ( -day) completion of the protocol was membrane filtered ( mm) and stored frozen (- c). bacterial adhesion was determined using an in vitro assay. briefly, urine samples were incubated with an uropathogenic e. coli strain that had been subcultured to promote fimbriae expression. urine samples containing e. coli were next incubated in -well plates containing methanol-fixed madin-darby canine kidney (mdck) cells for -hr ( c) to permit bacterial attachment. after incubation, plates were washed to remove nonadherent bacteria and fresh media added. plates were incubated ( c) for -hr to grow attached bacteria to detection level. bacterial concentration in each well was determined using a spectrophotometer ( nm). results were analyzed using the chi-square test. ce significantly reduced bacterial adhesion by % (n ; p . ) in -day urine samples compared with pre samples. the results show that ce supplementation can reduce adhesion of uropathogenic e. coli to canine uroepithelium and suggests one mechanism by which ce might improve urinary tract health. the purpose of this study was to determine prevalence of urovirulence factors (uvfs) and antimicrobial resistance in canine uropathogenic e. coli (upec) and to evaluate associations between uvfs and antimicrobial resistance. two hundred and twenty-one upec isolates from samples collected from different canine patients submitted to the university of tennessee microbiology laboratory in were evaluated. a multiplex pcr assay was used to detect cnf, hlyd, sfa/foc, and papgiii in dna lysate. in vitro susceptibility was evaluated and if the isolate was resistant to any antimicrobial in a class, it was considered resistant to that class. of the samples, the number of uvf expressed per isolate was: / ( %), / ( %), / ( %), - / ( %), and / ( %). expression of uvf was sfa ( %), hly ( %), cnf ( %), and pap ( %). presence of uvfs was associated with less resistance (p o . ). the combination of hly, cnf, and sfa was associated with less resistance (p o . ). when sfa was present alone, resistance was less (p o . ). average resistance to antimicrobial class by number of uvfexpressed was: uvf . ae . classes, uvf . ae . classes, uvf . ae . classes, uvf . ae . classes, and uvf . ae . classes. urovirulence factors were present in a moderate number of upec and correlated negatively with resistance. neither individual nor combinations of uvfs were associated with increased resistance. obesity is associated with several comorbidities in dogs including pancreatitis, osteoarthritis, oral disease, neoplasia, and lower urinary tract disease. investigator observations led to the hypothesis that morbidly obese dogs are more likely to have asymptomatic bacterial urinary tract infections (abuti) than overweight and moderately obese dogs. therefore, a pilot study was conducted to screen for abuti in obese dogs. urinalysis with urine culture and dual energy x-ray absorptiometry (dxa) were performed on fortythree dogs with body fat (bf) percentages ranging from to %. following dxa, subjects were categorized as obese (o)(bf - %, n ) and morbidly obese (mo)(bf %, n ). no dogs had owner-reported symptoms indicative of uti. the prevalence of abuti in o dogs was % (n ) and % (n ) in mo dogs. the dog in the o group with abuti was close to being mo with a bf equaling . %. of the nine dogs with positive cultures, were neutered males and were spayed females. the prevalence ratio of abuti in mo dogs was . , indicating dogs with % or greater bf are . times more likely to have the condition then dogs o % bf. the results of this pilot study coincide with other surveillance data describing an increased prevalence of lower urinary tract disease in obese dogs. in conclusion, dogs with body fat percentages greater than % are at risk for abuti, and veterinarians should consider screening all morbidly obese patients for urinary tract infections. calcium carbonate (cac) is recommended to decrease phosphate intake in chronic kidney disease. however, its effect is poorly documented in dogs. our objectives were to assess within-day, postprandial and cac effects on phosphatemia variations in healthy dogs. phosphatemia was measured every hours for hours in eight adult healthy beagle dogs in i) fasted condition and ii) a  crossover design. one group received cac mixed with maintenance diet ( . % phosphorus), while the second group received the diet alone. after a -week wash-out period, groups were switched. a general linear model was used to test the period, sequence, treatment, dog and time effects on phosphatemia and the area under the phosphatemia versus time curve (auc - ). a significant (p o . ) circadian variation existed in fasted dogs. the maximum difference (mean: À . mg/dl; % c.i.: À . mg/dl; À . mg/dl) was observed between a.m. and midnight. the auc - with cac ( ae mg.min/dl) was mildly but significantly lower (p . ) than without cac ( ae mg.min/dl). however, it was similar to the auc - in fasted conditions. feeding, with and without cac, has minor effect on phosphatemia. however, circadian variation of fasted phosphatemia might affect its interpretation. gfr measurement permits diagnosis of kidney injury prior to development of azotemia, and is the gold standard for kidney function assessment. accurate and rapid (o min) gfr measurement has been performed in rats by simultaneous transcutaneous assay of two intravascular fluorescently-labeled markers. a recently developed analyzer assays fluorescence via a fiberoptic cable introduced through a peripheral catheter, and thus should also allow rapid gfr determination in larger species. the purpose of this study was to determine correlation and agreement between fluorescent ratiometry (fr) and iohexol plasma clearance (ipc) in dogs over a range of gfrs. acute kidney injury (aki) was induced in female hound-type dogs ( mg/kg gentamicin iv q h), and fr and ipc gfr were simulta-neously determined on days , , and . a -sample, -hr protocol was used for ipc; fr was determined following bolus injection of a dextran conjugate mixture ( -sulfohexamine rhodamine-carboxymethyl kd dextran, -aminofluorescein-carboxymethyl kd dextran) with fluorescence measured over min. gfr was calculated using -compartment model concentration-vs.-time curves for both techniques. correlation was determined via spearman's rho; agreement was analyzed via bland-altman plots. ipc gfr and serum creatinine confirmed progressive aki in all dogs. correlation between fr and ipc was . (p o . ). bland-altman plots confirmed good agreement between techniques with slight underestimation of gfr by fr across most observed values. these results suggest fr is suitable for gfr determination in dogs with aki. importantly, the portable analyzer allowed for point-of-care gfr determination in o min using a peripheral vein. previously presented at the american society of nephrology renal week (related but not identical abstract). dogs with protein-losing nephropathy (pln) are at risk of thromboembolic disease, but the mechanism of hypercoagulability and the population of dogs at risk are unknown. the purpose of this study was to characterize thromboelastography (teg) in dogs with pln. twenty-eight client-owned dogs with pln (urine protein:creatinine ratio (upc) . ) and control dogs were enrolled. teg parameters, antithrombin activity, serum biochemical profiles, and upc were measured. teg analyses were run in duplicate with kaolin activation; reaction time (r), clot formation time (k), maximal amplitude (ma), and g (global clot strength) were analyzed. a wilcoxon sum rank test was used to evaluate differences between groups. twelve pln dogs ( . %) were azotemic. nineteen pln dogs ( . %) were hypoalbuminemic [serum albumin (salb) o . g/dl]; had salb o . g/dl. dogs with pln had higher k (p o . ), ma (p o . ) and g (p o . ) than controls. r was similar between the two groups. pln dogs with salb o . g/dl had higher g (p o . ) values than dogs with salb . g/dl; however, even pln dogs with normal salb ( . g/dl) had significantly higher g values than controls (p o . ). no significant relationship between upc and g, salb and g, antithrombin and g, or salb and antithrombin was noted using linear regression analysis. these results indicate that antithrombin, salb, and upc cannot be used alone to predict hypercoagulability as assessed by teg in dogs with pln. a comprehensive evaluation of the coagulation system in individual patients may be necessary to predict the point at which to initiate anti-thrombotic therapy. cystinuria is a hereditary renal tubular reabsorption defect of cystine, ornithine, lysine and arginine (collectively, cola). the low solubility of cystine in acidic urine predisposes to the formation of uroliths. type i cystinuria in newfoundland and labrador retriever dogs is an autosomal recessive trait caused by mutations in the slc a gene, whereas in other breeds, the cause of cystinuria has not yet been determined. we report here on the clinical, biochemical and molecular features of cystinuria in irish terriers. urine and edta blood were collected from irish terriers from europe and australia. a nitroprusside screening test was used to identify increased cystine in urine. urinary amino acid concentrations were determined by high-pressure liquid chromatography. cystinuric dogs were defined as having cystine calculi, a positive nitroprusside result, urinary cystine ( mmol/g creatinine) and/ or a cola concentration of mmol/g creatinine. all females tested nitroprusside negative and had normal urinary cystine (o mmol/g creatinine) and cola (o mmol/g creatinine) concentrations. the intact males that formed calculi as adults exhibited cystine concentrations ranging from - and cola from - mmol/g creatinine. an additional males had similarly high cola values with cystine levels from - mmol/g creatinine. among the affecteds tested, % were nitroprusside positive. the negative nitroprusside results and/or low urinary cystine levels of affecteds may be due to precipitation of cystine in acidic urine. sequencing the coding regions of the slc a and slc a genes from edta blood identified no mutations. the mode of inheritance remains undetermined. however, castration appears to lower the urinary cystine and cola concentrations and to prevent cystine calculi formation, while diet changes have lesser effects. in conclusion, non-type i cystinuria in irish terriers (and several other breeds like mastiffs and scottish deerhounds) is a unique form characterized by increased aminoaciduria only in males, with lower cystine and cola excretion and fewer and later urolith formation compared to type i cystinuria. castrating cystinuric irish terriers lowers their cystine and cola excretion and thus their risk for calculi formation. cats and dogs that are diagnosed with acute kidney injury (aki) and resultant uremia that is not responsive to standard medical therapy are likely to benefit from renal replacement therapies, such as intermittent hemodialysis (ihd). the purpose of this study was to evaluate the long-term outcome of patients with aki treated with ihd, and to establish whether renal function, as determined by serum or plasma creatinine concentrations, is associated with longterm survival. medical records of cats and dogs that were diagnosed with aki, treated with ihd, and survived longer than days following the last ihd treatment were retrospectively analyzed. standard methods of survival analysis using kaplan-meier product limit curves and the log-rank test were performed. for all-cause mortality, the median survival time was days ( % confidence interval: , ) for cats and days ( % confidence interval: , ) for dogs. when only renal-related causes of death were taken into account, the median survival time was not reached for cats or dogs. survival time for all-cause mortality was inversely associated with the lowest creatinine concentration within the to day period following the last ihd treatment (p o . for cats, p o . for dogs). this study demonstrates that veterinary patients that are diagnosed with aki, treated with ihd, and survive greater than days after the last ihd treatment have a good longterm prognosis and frequently die from causes that are unrelated to renal impairment. renal fine-needle aspiration (r-fna) is oftentimes attempted during evaluation of dogs and cats with renomegaly, mass lesions, or suspected infiltrative processes. diagnostic utility of fna is dependent upon the organ being sampled; additionally, in some organs, certain diagnostic imaging findings are associated with improved concordance of fna with final diagnosis. objectives of this study were to evaluate the diagnostic utility of r-fna and determine whether concordance with final diagnosis is associated with specific clinicopathologic or diagnostic imaging findings. we hypothesized that r-fna is most useful in patients with diagnostic imaging results suggestive of renal neoplasia (i.e. masses or suspected infiltrative processes). dogs and cats that had undergone r-fna from jan , to dec , were identified by database search. patient signalment, serum creatinine and blood urea nitrogen concentration, urine specific gravity, dipstick protein, r-fna result, and final diagnosis were recorded. patients were excluded if abdominal radiographs or sonographic images were not available for review, or if diagnostic test results were insufficient for determination of final diagnosis. a single coauthor blinded to final diagnoses interpreted all abdominal images using a pre-set list of descriptors and grading criteria. radiographic kidney shape, margin distortion, and ventrodorsal kidney-to-l ratio were evaluated. sonographic kidney margin distortion, cortical echogenicity, and corticomedullary junction distinction were described, and presence of nodules or masses, peri-renal effusion, or a peripheral sonolucent rim was noted. concordance of r-fna and final diagnosis was determined, and the chi-squared or fisher's exact test were used to determine association of concordance with the above variables; p o . was considered significant. dogs and cats ( animals) met all inclusion criteria. r-fna results were concordant with the final diagnosis in ( . %) patients, discordant in ( . %) patients, and inadequate for cytologic interpretation in ( . %) patients. neoplasia or fip were the final diagnoses in of ( . %) and of ( . %) patients with concordant results, respectively. renal lymphoma (p . ), renal carcinoma (p . ), and renal neoplasia in general (p . ) were not associated with a higher likelihood of r-fna and final diagnosis concordance. there was no association noted between likelihood of r-fna and final diagnosis concordance when patients were stratified by species, serum creatinine or blood urea nitrogen concentration, urine specific gravity, dipstick proteinuria, or any diagnostic imaging variables. this study failed to identify concurrent clinicopathologic or diagnostic imaging findings that enhanced the diagnostic utility of r-fna. future studies should use standardized criteria to prospectively identify patients in which r-fna will be performed, evaluate additional variables that may be associated with increased r-fna diagnostic utility, and directly compare the utility of r-fna with that of other diagnostic techniques. feline lower urinary tract disease (flutd) is a disease with increasing prevalence in private practices and veterinary teaching hospitals. although several underlying causes can cause the obstructive form in male cats, the idiopathic form (feline interstitial cystitis) often is diagnosed as underlying reason in cats o years. the goal of this retrospective study was to identify possible predisposing factors in order to optimize the therapy of these patients. as a study group, cats hospitalized with obstructive flutd at the veterinary university of vienna were examined during a year period ( ) ( ) ( ) . as a control group cats presented for other reasons were randomly chosen during the same time period. the data were examined concerning the signalment and history. furthermore, the long-term outcome was evaluated with a questionnaire. based on assumptions a student's t-test or a chi-square test was used. there were no significant differences in age and breed. the body weight was significant higher in the flutd group than in the control group (p o . ). we could observe a significant risk for the disease of a weight of kg (p o . ). there were significant less cat toilets in the flutd group compared to the control group (p o . ). furthermore we could observe that in the households of flutd cats there was significant less than one toilet per cat (p o . ) and more cats diseased on flutd lived strictly indoor than outdoor (p . ).there were no significant differences at the time of hospitalization in age, breed, number of cats per household or season of the year between the two groups. in summary, we could observe that cats over kg body weight kept indoor with less than one toilet per cat have a significant higher possibility to be affected by obstructive flutd. further studies with an extensive history of animal husbandry are needed to identify risks predispoing cats to this frequent and cost-intensive disease. although purine uroliths (ammonium urate, sodium urate, xanthine, uric acid, etc.) represent the third most common stone type in cats, purine uroliths have the highest rate of recurrence ( % in months). in dogs, mutation of the urate transporter (slc a ) and portovascular anomalies are common risk factors. however the underlying cause(s) for purine urolith formation in cats is unknown. the purpose of this study was to test the hypothesis that hyperuricosuria without alterations in liver function is common in cats with urate uroliths. urine concentrations of purine metabolites were measured by high-performance liquid chromatography in cats with ammonium uroliths (cases), clinically healthy, breed and gender matched cats (negative controls), and cats with naturally occurring xanthine uroliths (positive controls). prior to urine collection, all cats were fed a standard maintenance food (protein g/ kcal) for weeks. urinary xanthine, uric acid, and allantoin concentrations and concentration to creatinine ratios were calculated and compared between groups. also, serum pre-and post-prandial bile acid concentrations were measured. when compared to control cats, urinary uric acid concentration was significantly higher in case cats (p . ). xanthine was not detected in the urine of cases or negative controls. a significant difference in fasted and post-prandial serum bile acid concentrations was not detected in cases or controls (p . , . ).hyperuricosuria without increased concentrations of urinary xanthine or allantoin appears to be a risk factor for ammonium urate urolith formation in cats. an association between portovascular shunts and purine urolithiasis was not observed in this population of cats. studies indicate that proteinuria is predictive, on a population basis, of those cats at risk of developing azotemia. seldi-tof-ms is a sensitive, high-throughput, proteomic technique utilising chromatographic surfaces to facilitate separation and detection of proteins and peptides within biological fluids such as urine. individual low molecular weight (lmw) urinary proteins have been considered as potential biomarkers for renal damage but provide only a limited representation of the urinary proteome; seldi-tof-ms may provide a more global assessment. normotensive, non-azotemic geriatric cats ( years) were recruited prospectively from two first-opinion clinics for routine health screening. at entry cats received a full physical examination, plasma biochemistry, evaluation of total t concentration and urinalysis including urine protein to creatinine ratio. re-examination was offered at and months. cats were divided into two groups based on clinical status at the month re-examination (azotemic; creatinine concentration ! . mg/dl and non-azotemic). optimisation studies were performed to facilitate the automated preparation (biomek ) of cm (weak cation exchange) arrays for seldi-tof-ms analysis (ciphergen enterprise ) of urine samples from cats at entry to the study. results are reported as median [ th , th percentile]. mann whitney u-test and wilcoxon signed rank test were used to compare variables between groups and between timepoints, respectively. ciphergen express ( . ) software was used to analyse spectral data and a mann whitney u-test was used to identify clusters which differed significantly between groups (p o . ) at entry to the study. twenty non-azotemic cats were recruited, of which cats developed azotemia by months. no significant differences in age, body weight, biochemical or urinalysis variables were identified between groups at entry to the study. as might be expected creatinine increased significantly ( . mg/dl [ . , . ], . [ . , . ], p . ) between study entry and months in the cats that developed azotaemia and there was a commensurate increase in phosphate concentration ( . mg/dl [ . , . ], . [ . , . ], p . ). creatinine and phosphorus did not change significantly over time in the cats that did not develop azotaemia. seven clusters with m/z values of , , , , , were found to differ significantly between groups at entry to the study. the low protein concentration of feline urine makes the use of proteomic techniques challenging. however, this pilot study indicates that seldi-tof-ms can be utilised to examine the feline urinary proteome and that differences in low molecular weight protein patterns may be useful to differentiate those cats which are at risk of the development of azotemia. further work is necessary to identify these proteins/peptides. fibroblastic growth factor (fgf- ) is a phosphotonin with an important physiological role in the regulation of phosphorous and vitamin d metabolism, and may therefore play a part in the development of renal secondary hyperparathyroidism. previous studies in cats have shown parathyroid hormone (pth) to be elevated prior to the development of azotemia. the study objectives were to explore the hypothesis that fgf- is a mediator of the development of renal secondary hyperparathyroidism in the nonazotemic stages of feline ckd. healthy, non-azotemic (plasma creatinine concentrations (cr) o . mg/dl) geriatric cats were recruited into the study prospectively and followed for months. at the study end point cats were categorised into the following groups: group (n )-cr . mg/dl, group (n )-cr ! . mg/dl but did not meet the criteria for group and group (n )-cr . mg/dl in association with reduced urine concentrating ability (usg o . ) or demonstration of persistent azotemia (cr . mg/dl). plasma samples were subjected to routine biochemical analysis, intact pth, calcitriol and intact fgf- assay. variables were compared between the groups at the baseline time point. gfr was measured in an additional group of cats ( non-azotemic, iris stage ii, iris stage iii) using a corrected slope-intercept iohexol clearance method. relationships were explored using linear regression analysis and determining the coefficient of determination (r ). results are presented as median [range] . at the baseline time point fgf- concentrations were significantly higher in group ( . [ . - . ], p . ) and group ( . [ . - . ], p . ) compared to group ( . [ . - . ] ). weak positive relationships were identified between fgf- and pth (r . , p . , n ) and fgf- and cr (r . , p . , n ). however, the positive relationships between fgf- and phosphate (r . , p . , n ) and fgf- and calcitriol (r . , p . , n ) were not significant. the additional group of cats in which gfr measurement was performed there was an inverse relationship between fgf- and gfr (r . , p . ). in conclusion, fgf- was elevated in cats prior to the development of azotemia. the role of fgf- in the development of feline renal secondary hyperparathyroidism remains to be determined and should be explored through interventional studies. however, considering the relationship between fgf- and gfr, it cannot be excluded that the phosphotonin is simply a marker of reduced filtration. chronic kidney disease (ckd) is common in geriatric cats and hypoxia might contribute to the progression of this disease. the aim of this study was to evaluate urinary vascular endothelial growth factor (vegf) as a marker of renal hypoxia. cats were recruited through geriatric clinics held at two first opinion london practices. vegf was measured in stored samples using a canine elisa kit validated for use on feline urine and indexed to creatinine concentration to yield a vegf to creatinine ratio (vcr). two studies were undertaken -firstly a cross-sectional analysis of clinical variables associated with vcr in cats with ckd. diagnosis of ckd was based on concurrent findings of plasma creatinine ! mg/dl and usg . , with persistence of azotemia for ! weeks. only patients receiving no medical therapies were included. normotensive and (pre-treatment) hypertensive cats were included, but borderline cases (mean systolic blood pressure - mmhg on the date of sampling) were not. hyperthyroid cats were also excluded from this cross-sectional study. associations between vcr and clinical data were initially assessed using the spearman's coefficient and mann whitney test. linear regression was then used for multivariate analysis. the second study used samples from a trial in which hypertensive cats that had been treated with amlodipine for at least months were entered into a randomised cross-over study where they received placebo or benazepril ( . to mg/kg daily) for weeks in turn. vcr on placebo was compared with that on benazepril using the wilcoxon signed ranks test. cats with well controlled hyperthyroidism were included in this intervention study. results are reported as median [ th, th percentile]. vcr was higher ( . [ . , . ] vs. . [ . , . ] fg/g, p . ) in untreated hypertensives (n ) than normotensives (n ). vcr was correlated with pcv (r À . , p . , n ), upc (r . , p o . , n ), plasma phosphate (r . , p . , n ), and usg (r À . , p . , n ), but not plasma creatinine concentration. in the best multivariate model, pcv was associated with vcr independently of upc (r . , n ). vcr was significantly reduced by benazepril therapy ( . [ . , . ] fg/g) compared with placebo ( . [ . , . ] fg/g; p . , n ) with a reduction seen in % of cases. these results suggest urinary vegf excretion is associated with proteinuria in cats with ckd and might be a marker of renal hypoxia induced by low pcv. ace inhibitor therapy might reduce urinary vegf excretion because angiotensin ii causes constriction on efferent arterioles resulting in tubular hypoxia. fgf- is a phosphaturic hormone. fgf- concentrations increase with declining renal function in humans. the objectives of this study were to validate a method for fgf- quantification in feline plasma and to assess the association between fgf- concentration and plasma creatinine or phosphate concentration in cats with chronic kidney disease (ckd). non-azotemic and azotemic (plasma creatinine concentration (cr) . mg/dl) geriatric ( yrs) cats were recruited into the cross-sectional study from two london first opinion practices. cats were excluded from the study if they were fed a phosphate restricted diet, or had evidence of concurrent disease. the cats were categorized, using a modified iris staging system, into the following four groups: group (cr . mg/dl), group (cr . - . mg/dl), group (cr . - . mg/dl), group (cr . mg/dl). groups and were further subdivided based on the iris targets for plasma phosphate concentration (po ): group a (po . mg/dl), group b (po . mg/dl), group a (po mg/dl), group b (po mg/dl). fgf- concentrations were measured in feline edta plasma using a human intact fgf- elisa, validated by intraand inter-assay variability and assessment of dilutional parallelism. comparisons between groups were made using the kruskal-wallis test and mann-whitney u test, with statistical significance defined as p o . . bonferroni correction was applied where appropriate (statistical significance then determined as p o . ). results are reported as median [ th, th percentiles]. fgf- concentrations ! pg/ml (upper limit of quantification) were assigned the value of pg/ml. intra-and inter-assay variability of fgf- measurements were o . % and dilutional parallelism between feline samples and the calibration curve were demonstrated. plasma fgf- concentrations increased with increasing creatinine concentrations (group : [ , ] , n , group : [ , ] , n , group : [ , ], n , group : [ , ], n ). fgf- measurements were significantly different between all groups (p . to o . ) except between groups and (p . ). fgf- concentrations were significantly higher in cats with higher plasma phosphate concentrations (group a: [ , ] , n vs. group b: [ , ], n ; p . ) and (group a: [ , ] , n vs. group b: [ , ], n ; p . ). in conclusion, fgf- concentrations were higher in cats with more severe ckd or higher plasma phosphate concentrations as would be predicted from its known biological actions. further work is warranted to explore the role of fgf- in the development of renal secondary hyperparathyroidism by measuring parathyroid hormone (pth) and calcitriol in cats at different stages of ckd. progressive non-cardiogenic edema and lung dysfunction are common complications of acute kidney injury (aki) in people. pulmonary abnormalities have not been systematically reviewed in dogs with renal azotemia, but anecdotal reports of dogs with aki and concurrent non-cardiogenic pulmonary edema are suggestive of uremic pneumonopathy (up), a centrally-distributed pulmonary edema syndrome associated with kidney disease in people. we therefore hypothesized that pulmonary-associated clinical signs or thoracic radiograph abnormalities are more common in dogs with renal azotemia than in non-azotemic dogs, and that this association is more likely in dogs with aki than dogs with chronic renal failure (crf). our study objectives were ) to describe thoracic radiograph and lung histopathologic abnormalities in dogs with renal azotemia, ) to compare the occurrence of these findings in dogs with aki, crf, or non-systemic illness, and ) to determine if these abnormalities are associated with shorter survival times. records of dogs with renal azotemia evaluated from / / to / / were reviewed; dogs which could be classified as having aki or crf and which had complete thoracic radiograph studies available for review were included. dogs with primary intracranial disease and normal serum creatinine and a complete thoracic radiograph study were selected as controls. signalment, weight, presence of pulmonary-related clinical signs, azotemia duration and severity at time of radiography, and leptospirosis antibody titer were noted. alveolar, bronchial, interstitial, or nodular lesions were described using a -point scale, and lung tissue collected at time of necropsy was reviewed; both the radiologist and pathologist were blinded to final diagnoses. significance was p o . for all analyses. the final study population included aki, crf, and control dogs. crf dogs were older (p o . ) than aki and control dogs. pulmonary-related clinical signs were more commonly diagnosed at first evaluation in aki dogs ( / dogs, . %) than in crf ( / , . %; p . ) or control dogs ( / , . %; p o . ). presence of an alveolar pattern was the only radiographic finding which differed amongst groups (more common in aki [n , . %, p . ] and crf [n , %, p . ] dogs than in control dogs [n , . %]). there was no association between presence of an alveolar pattern and any other variable. alveolar mineralization was the most common lesion in aki dogs ( / dogs; . %), with concurrent alveolar space concretions or mineralization of vessels or bronchioles noted in dog each. necropsies had not been performed in any of the crf dogs, but mineralization was not seen in lung tissues from any control dogs (n ). neither pulmonary-associated clinical signs nor alveolar pattern were associated with median number of days from discharge until death in dogs with aki (p . and . , respectively) or crf (p . and . , respectively). in this group of dogs, presence and type of radiographic pulmonary abnormalities were associated with renal azotemia but not with median time until death. the association between and clinical relevance of alveolar mineralization in aki dogs were not determined, but both the radiographic and histopathologic abnormalities reported here differ from up in people. chronic kidney disease (ckd) is a common cause of morbidity and mortality in cats. the purpose of this study was to investigate the effects of chinese rhubarb (rheum officinale) supplementation on the progression of feline ckd. cats with stable iris stage ii or iii ckd and without comorbidity were included in the study. cats were divided into treatment groups and administered rhubarb extract (group , rubenal s , vetoquinol, mg tablet po q h), benazepril as a positive control (group , . mg/kg po q h), or both (group ). cats were fed a commercial renal specific diet and enteric phosphate binder as appropriate. body weight, laboratory data, and blood pressure were recorded every months for up to months. variables between groups at enrollment and within groups over visits were compared with anova and repeated measures ano-va, respectively. a treatment by visit interaction term was included in all repeated measures models. significance was set at p . . except for body weight there was no significant differences between treatment groups at enrollment. there was no significant change in body weight, hematocrit (hct), upc, or creatinine over time as compared to baseline within any group. there was no significant difference between groups over time in regards to change in weight, hct, upc, or creatinine. the treatment by time interaction was non-significant in all models. although there was no benefit associated with combination treatment, the results for rhubarb treatment alone were not different from benazepril treatment. azodyl, an encapsulated, enteric-coated probiotic/prebiotic nutraceutical, is marketed for reduction of azotemia (bun & creatinine) in dogs and cats. cat owners often sprinkle contents onto cat food to facilitate administration. however, exposure to air and stomach acid are thought to inactivate the lyophilized bacteria within the product. therefore, we examined the ability of foodsprinkled azodyl to reduce azotemia in cats with ckd. cats with ckd were enrolled in the study and randomized receive azodyl or placebo. owners were provided with - capsules of azodyl prior to enrollment to ensure compliance with administration. baseline blood samples were obtained month apart, and then & months after beginning therapy. clinicians and owners were masked as to medication assignment. we hypothesized that a % decrease in bun and/or creat in the azodyl group would be significant, and set a . . in order to maximize the probability of detecting a difference, we determined the % change as being the difference between the maximal baseline analyte concentration and minimal therapeutic concentration. we compared the % change between groups by mann-whitney u test. bun and creatinine did not differ between groups. based on these results, azodyl, applied by sprinkling onto food fails to reduce azotemia in cats with ckd. whether intact capsule administration reduces azotemia in cats with ckd remains unknown. lower urinary tract disease (lutd) occurs commonly in cats, and idiopathic cystitis (fic) and urolithiasis account for over % of cases in cats less than years of age. although several strategies have been recommended, a common recommendation is to induce dilute urine resulting in more frequent urination and to dilute calculogenic constituents. in addition to conventional therapy using modified diets, traditional chinese and western herbs have been recommended, although only one, chorieto, has published data. we evaluated commonly used herbal treatments recommended for use in cats with lutd including ( ) san ren tang, ( ) wei ling tang, and ( ) alisma. we hypothesized that these chinese herbal preparations would induce increased urine volume and decreased urine saturation for calcium oxalate and struvite. six healthy, spayed female, adult cats were evaluated in a placebocontrolled, randomized, cross-over design study. cats were randomized to of treatments including placebo (p), san ren tang (srt), wei ling tang (wlt), or alisma (a). treatment was for weeks each with a week washout period between treatments. at end of each treatment period, a -hour urine sample was collected using modified litter boxes. urine volume and biochemistries were measured, and urine saturation for struvite and calcium oxalate was estimated using equil . b. analysis of variance (anova) was used to analyze data statistically if distributed normally and kruskal-wallis was used to analyze data statistically if data were not distributed normally. a p o . was considered significant. body weights were not different between treatments. no differences were found in -hour urinary analyte excretions, -hour urine volume, urine ph, or -hour urinary saturation for calcium oxalate or struvite between treatments (table) . urolithiasis is a multifactorial disease, frequent and recurrent in dogs in the worldwide, in which breed, sex, age, diet, some anatomical abnormalities, urinary tract infection, urine ph and some geographical and hereditary features in the populations studied have been implicated as risk factors. the effective long-term management of urolithiasis depends on identification and control of the pathophysiological mechanisms involved, which, in turn, depend on accurate knowledge of the mineral composition of the uroliths. the aim of this study was to determine for first occasion the main epidemiological data of canine urolithiasis in mexico. this study was developed with dogs with urolithiasis from of the states of the country. chemical composition of the uroliths was determined by stereoscopic microscopy, infrared spectroscopy, scanning electron microscopy and x-ray microanalysis. urolithiasis affected nearly the same number of males and females; with ages ranging from two months to years with a median age of years. adult animals were the most affected. breeds more affected were schnauzer miniature, poodle, dalmatian, yorkshire terrier, scottish terrier, chihuahua and bichon frisee´. uroliths were found in the lower urinary tract in . % of the cases. mineral composition of the uroliths was: struvite . %, followed by calcium oxalate . %, purines . %, silicate . %, others . %, mixed . % and compound uroliths . %. struvite uroliths affected females in most cases, whereas calcium oxalate, purines and silicate uroliths, were mainly observed in males. our results are similar to studies developed in other countries and continents, though we found a higher frequency of uroliths containing silicate, either pure, mixed or compounds uroliths ( . %); in mexico city the frequency reached %. this high frequency may be due to high consumption of silicate in home-made food or in the groundwater derived from aquifers. acknowledgments: this work has been partially supported by a project of waltham foundation in mexico and the consejo nacional de ciencia y tecnologı´a (conacyt) of mexico. voiding urohydropropulsion is a non-invasive method for removing small urocystoliths from the dog, most commonly used in females due to the relatively wider and shorter urethra. this procedure is typically performed under general anesthesia to allow complete relaxation of the urethra, however, anesthesia results in longer procedure times and difficult endotracheal tube stabilization due to the vertical positioning of animals, especially in larger dogs. the aim of this study was to devise a novel injectable sedation protocol for urohydropropulsion when cystoscopy was not concurrently required. an intravenous catheter was placed, and a combination of medetomidine ( to mg/kg iv) and hydromorphone ( . to . mg/kg iv) was administered, with the addition of ketamine ( mg/ kg iv) in fractious animals; atipamezole (double volume of medetomidine, administered im) was used as a reversal agent upon procedure completion. this protocol was considered in cardiovascularly healthy, non-diabetic dogs without evidence of urinary obstruction. monitoring equipment included electrocardiography, blood pressure measurement, and pulse oximetry, and supplemental flowby oxygen was provided. two dogs received the proposed sedation protocol in order to perform urohydropropulsion. dog one was a year old female spayed shih tzu cross, and dog was a year old female spayed standard poodle. ultrasonography revealed a moderate number of urocystoliths present in both dogs, measuring up to mm in dog and . mm in dog . urohydropropulsion was performed and resulted in retrieval of urocystoliths in dog , and approximately urocystoliths in dog . repeat ultrasonography revealed no uroliths present after urohydropropulsion in both dogs. the time from administration of sedation to administration of reversal agent was minutes for dog , and . minutes for dog . records were obtained from dogs that had traditional general anesthetic protocols for urohydropropulsion with cystoscopy for confirmation of urocystolith removal, performed within the last years, and the average anesthetic time was minutes. subsequent to the use of medetomine-based sedation protocols for the above dogs, cystoscopy was performed in a year old neutered male golden retriever with prostatomegaly. medetomidine ( ug/kg iv) and butorphanol ( . mg/kg iv) were administered; atipamezole (double volume of medetomidine, administered im) was used as a reversal agent upon procedure completion. this sedation allowed adequate immobilization for cystoscopy of the urethra and urinary bladder, and endoscopic biopsying of the prostatic urethra and urinary bladder. the time from administration of sedation to administration of reversal agent was minutes for this dog. in conclusion, a novel sedative protocol for urohydropropulsion is proposed which allows for an appropriate level of sedation along with a short procedure time and rapid recovery. this sedation protocol may also be useful for certain cystoscopic procedures. analysis may be delayed for a variety of reasons, including the need for sample batching within the laboratory or shipping to an outsourced location. therefore, it is important to know how storage of the sample may affect enzyme activity. we hypothesized that urinary nag and ggt activity would be affected differently in samples stored by refrigeration vs. freezing. thirty-four canine urine samples submitted to the clinical pathology laboratory at kansas state university were included. samples were collected from clinical patients with a variety of medical/surgical disorders and were selected based on the day of the week and a minimum volume of ml. a complete urinalysis was performed on each sample; however there were no exclusion criteria based on urinalysis results. nag and ggt activity in the urine supernatant was assessed by colorimetric assay. aliquots of each supernatant were refrigerated for days and frozen at À c for and days at which time enzyme activity was re-assessed. compared to baseline values, enzyme activity for both nag and ggt were stable after days of refrigeration, however there were significant (p o . ) declines in ggt and nag activity when urine supernatants were frozen for and days. treatment for canine urinary tract infections (uti) typically consists of - days of antimicrobial drugs in primary care veterinary practice. compliance with this drug regimen can be difficult for some clients. enrofloxacin is a veterinary approved fluoroquinolone antimicrobial and is useful for treatment of canine uti. fluoroquinolones are often used in human medicine to treat uncomplicated utis in women and can be prescribed for as little as days. the primary objective of this study was to determine if dogs with naturally occurring uncomplicated uti have equivalent microbiologic cure with a high dose short duration protocol of enrofloxacin, compared to a standard antimicrobial protocol. client-owned adult dogs with naturally occurring, uncomplicated uti were prospectively enrolled in a multi-center clinical trial and assigned to of groups in a randomized blinded manner. group received treatment with - mg/kg oral enrofloxacin once daily for consecutive days. group dogs were treated with . - mg/kg oral amoxicillin-clavulante twice daily for days. both groups had urinalyses and urine cultures submitted on day , , and . at the time of this interim analysis, thirty-six dogs have completed the trial. bacteriological cure was achieved in dogs ( %) treated with enrofloxacin and dogs ( %) treated with amoxicillinclavulante, respectively. these data suggest that the high-dose, short-duration enrofloxacin protocol was equally effective to the standard protocol in treating uncomplicated canine uti in the sample patient population. and may represent a viable alternative therapeutic regimen for similar patients. azotemia is frequent in dogs with dmvd (nicolle et al; jvim ; : - ) and could result from renal hemodynamic alterations. renal resistive index (ri) allows assessment of renal vascular resistance. the aim of this prospective study was to assess ri in dogs with different dmvd stages. fifty-five dogs with dvmd were used (isachc class (n ), (n ), and (n )). physical examination, renal ultrasonography and echo-doppler examinations were performed in awake dogs by trained observers. plasma creatinine, urea and nt-probnp were measured. statistical analyses were performed using a general linear model. whereas ri of renal and arcuate arteries were unaffected by isachc class, left interlobar ri increased (p o . ) from . ae . (mean ae sd) in class to . ae . in class . left interlobar ri was also higher (p o . ) in azotemic ( . ae . ) than in non azotemic ( . ae . ) dogs. similar findings were observed for right interlobar ri. a positive effect of nt-probnp (p . ), urea (p o . ), creatinine (p . ), urea-to-creatinine ratio (p o . ), left atrium-to-aorta ratio (p o . ), regurgitation fraction (p . ), systolic pulmonary arterial pressure (p o . ) and shortening fraction (p . ) on ri was also observed. in conclusion, interlobar ri increases with the severity of dmvd and azotemia. a cause-effect relationship remains however to be established. antibodies against alpha-enolase are associated with immunemediated nephritis in people. it was previously shown that vaccinated cats commonly develop antibodies against alpha-enolase. the purpose of this study was to assess for associations between alphaenolase antibodies and azotemia in privately-owned cats. clinically stable privately owned cats ! years of age, with and without azotemia (creatinine mg/dl), and with an available vaccine history for ! years were recruited for the study. sera were assayed for creatinine concentrations and alpha-enolase antibodies by use of previously validated techniques. results from cats with and without azotemia were compared by student's -tailed t test or fisher's exact test with significance defined as p o . . median ages were years (range: - ) and years (range: - ) for cats with (n ) and without azotemia (n ), respectively. there was no significant difference in vaccine events (number, type, or route of administration) between groups. azotemic cats ( . %) were more likely than normal cats ( . %) to be positive for antibodies against alpha-enolase (p . ). in addition, alpha-enolase antibody concentrations were greater (p . ) in azotemic cats (mean % elisa . %) than cats with normal creatinine concentrations (mean %elisa . %). results of this study suggest that antibodies against alpha-enolase in cats may be associated with renal disease. additional prospective evaluation in a larger number of cats is indicated. aki is used in human medicine as a predictor of mortality based on the akin (acute kidney injury network) scoring system which utilizes relative increases in creatinine to determine stage. with this scheme, mortality has been shown to increase as the stage of kidney injury (indicated by akin score) increases. accordingly, we hypothesized that this system would improve predicting prognosis in dogs and cats. we retrospectively evaluated dogs and cats ( ) ( ) ) that had ! creatinine measurements within days, and whose first creatinine was o . mg/dl. patients were categorized as: level (no aki); level (second creatinine value o . mg/dl, but creatinine increased ! . mg/dl); or level (second creatinine . mg/dl with a creatinine increase ! . mg/dl). thirty and day survival for each level was compared to level . adjusted odds ratio (or) in dogs for day survival was . for level (ci %, . - . ) and . (ci %, . - . ) for level ; or for day survival was . for level (ci %, . - . ) and . (ci %, . - . ) for level . for cats, or at days was . (ci %, . - . ) for level and . (ci %, . - . ) for level ; or for day survival was . (ci %, . - . ) for level and . (ci %, . - . ) for level . thus, detecting increasing stage of aki helps predict mortality in dogs and cats. abstract n/u- feline urate urolithiasis: cases ( - . j dear , r shiraki , a ruby , j westropp . william r pritchard veterinary medical teaching hospital, university of california, davis, ca, gerald v. ling urinary stone analysis laboratory, university of california, davis, ca and the department of veterinary medicine and epidemiology, university of california, davis, ca. feline urate urolithiasis accounts for % of the feline stones our laboratory analyzes each year; little information is known about this disease, particularly the incidence of those cats with hepatopathies. the objective of the study was to characterize the signalment, clinicopathologic data, and diagnostic imaging of cats with this disease as well as the salts of uric acid present. a retrospective analysis of feline urate uroliths submitted to the stone lab between january -december were included. from these data, primary veterinarians were solicited to submit records. furthermore, all records from cats with urate uroliths from the vmth were analyzed separately. records were received from the primary care veterinarians. sixteen cases were identified from the vmth. median values for the cbc and chemistry panels available were within the reference ranges provided, with only a few outliers present. of the cats with radiographic reports, ( %) had visible evidence of uroliths. two external cases had confirmed pss; five cases from the vmth had a pss. cats with urate uroliths and pss were younger than cats without a documented hepatopathy ( years vs. years). the siamese breed was overrepresented. all stones were ammonium hydrogen urate. the pathogensis of urate uroliths in cats is poorly understood. most cats were not completely evaluated for pss, however, there were few clinicopathologic parameters which indicated hepatopathies were present. further studies are warranted to evaluate genetics and purine metabolism in cats with urate uroliths to help tailor proper management and breeding strategies. -indoxyl and p-cresyl sulfate (is, and cs, respectively), small protein-bound molecules derived from gastrointestinal protein metabolism, are among the most important uremic solutes affecting morbidity and mortality in human chronic kidney disease (ckd). in the blood stream, these compounds are predominantly bound to protein, but their debilitating effects on prognosis and quality of life in ckd appear to be driven by the free fraction. the objectives of the present study were to assess the normal, physiological levels of is and cs in healthy cats and to evaluate the correlation of the respective free and protein-bound levels. blood samples were taken from clinically healthy adult cats enrolled at five participating veterinary practices in germany. after centrifugation, the serum was deep frozen until transport on dry ice to the analytical laboratory. serum creatinine and urea levels were quantified by vettest s (idexx laboratories, inc.). total and free is and cs, respectively, were quantified by turbulent flow chromatography coupled with a tandem mass spectrometry detector. statistical analysis of the results comprised i) a descriptive report of the median with upper and lower bounds of the % confidence interval for reference values of is and cs, ii) a calculation of various pearson correlation coefficients r, also tested with reference to the null hypothesis of no relationship, and iii) wilcoxon-mann-whitney utest for an estimation of the effect of hemolysis on serum is or cs levels. six animals with serum creatinine or urea levels outside the reference range were excluded from the calculation of reference values. median levels of is in cat serum were . mg/l with upper and lower bound % confidence intervals at . and . mg/l, respectively. the corresponding median levels of cs were . mg/l (median) and . vs . mg/l (upper vs lower bound levels, respectively). these values showed a low, non-significant correlation with serum creatinine or urea levels. however, is and cs serum levels were moderately correlated (total levels r . , p o ). their respective free levels constituted about % of the total serum levels (r ! . , p o . ). non-hemolytic samples tended to yield lower values than hemolytic samples. due to the low number of hemolytic samples (n ) , the group difference could, however, not be statistically confirmed. the results indicate that it is sufficient to determine total levels of either is or cs in serum while studying the effects of therapeutic or dietetic interventions on the evolution of these parameters in feline ckd. reference values are provided for orientation towards clinically relevant changes. disrupted urothelial differentiation has been implicated in the pathogenesis of feline idiopathic cystitis (fic). studies of cultured human urothelium have shown that abnormalities in urothelial differentiation and repair may be mediated by persistent -hydroxy-prostaglandin dehydrogenase (pgdh) activity and subsequent metabolism of cytoprotective prostaglandins. the goal of this study was to confirm persistent pgdh expression in fic bladders compared to desmoplakin i ii expression, a marker of urothelial differentiation. urinary bladder biopsy specimens were obtained by cystotomy from symptomatic cats with chronic fic. cats with a history of another major disease, previous cystotomy, or recent treatment with corticosteroids, nsaids, antihistamines, antidepressants, or glycosaminoglycans were excluded. urinary bladder tissue specimens were also obtained from untreated clinically normal specific-pathogen-free cats. tissue specimens were fixed in buffered % formalin and embedded in paraffin. tissue sections were deparaffinized and subjected to citrate buffer microwave antigen retrieval. tissues were stained for pgdh using a rabbit anti-pgdh antibody, an isotype negative control or goat anti-desmoplakin i ii and developed using the avidin-biotin peroxidase complex method. all fic ( / ) and normal ( / ) cat bladder samples showed similar staining of urothelial cytoplasm for pgdh. however, desmoplakin i ii staining, found on the luminal cell surface in / normal tissues, was disrupted in / fic bladder samples. desmoplakin i ii staining confirmed altered urothelial differentiation in fic cats. however, pgdh expression remained intact in fic samples. we hypothesize that pgdh expression in fic may contribute to its pathophysiology due to breakdown of prostaglandins essential for urothelial healing. additional studies will explore this hypothesis. the university of tennessee college of veterinary medicine's picture archiving and communication system was searched over a month period for cats that had undergone both abdominal radiographs and ultrasound during the same visit. one hundred and three cats were identified (age range o to yrs; median yrs). kidney size was determined based on radiographic and ultrasound findings. of the included cats, . % had two normal sized kidneys, . % had one small and one normal, . % had one large and one normal, . % had two small, . % had two large, and . % had one small and one large kidney. the presence of mineralization, uroliths and hydronephrosis was also noted. medical records were reviewed for clinical chemistry data and historical information concerning previous urinary disease. no significant differences were found between kidney size and renal function, kidney size and the presence of uroliths, renal mineralization and function or the presence of uroliths and function. the presence of uroliths was significantly associated with hydronephrosis. of the cats with at least one large kidney, ( %) had hydronephrosis. of the cats with current or previously diagnosed uroliths, urinary tract infections or other uropathies, ( . %) had at least one small kidney. small kidneys were commonly found in older cats, however, this correlation was not statistically significant. based on these findings, small kidneys are more likely to be the result of urinary disease as opposed to being either congenital or due to aging. this study aimed to evaluate ife, which has been advocated for treatment of lipid-soluble drug intoxication, in the treatment of clinically-occurring canine ivermectin toxicosis. one australian shepherd and two miniature australian shepherds were included. all three dogs were homozygous for the mdr- gene mutation. two dogs roamed on horse ranches where ivermectin-based deworming products had recently been used. ivermectin was administered to the third dog ( mg/kg po). all three dogs exhibited tremors, ptyalism, and cns depression, which progressed over several hours to stupor in two dogs, and to a comatose state requiring mechanical ventilation in the remaining dog. a % formulation of ife (liposyn ii, hospira) was administered as a bolus ( . ml/kg) followed by a slow iv infusion ( . - ml/kg over minutes). no change was observed in the neurologic status of any patient. lipemia visible upon blood sampling persisted for hours in one dog. no other adverse effects were noted. serum ivermectin levels confirmed ivermectin exposure in each case. in this study, ife administration did not result in clinical benefit in cases of ivermectin toxicosis. brain ivermectin concentrations in mdr mutant/mutant genotype dogs may be too high to be overcome by ife. additionally, these dogs may lack p-glycoprotein-mediated biliary clearance mechanisms needed for optimal ife function. further investigation is needed to determine the utility and optimal dosing of ife in canine toxicoses, to characterize its safety, and to determine how mdr- status may alter the efficacy of ife in treatment of canine ivermectin intoxication. rufinamide is a recently approved antiepileptic drug used for the treatment of seizure disorders in human patients. rufinamide is administered at a dose of mg/kg divided twice daily to achieve therapeutic concentrations of mg/ml. the objective of this study was to determine the pharmacokinetic properties and short-term adverse effects of single-dose oral rufinamide in healthy dogs in preparation for a possible clinical trial evaluating the efficacy of rufinamide in the treatment of canine epilepsy. six healthy adult dogs were included. the pharmacokinetics of rufinamide were calculated following administration of a single mean oral dose of . mg/kg (range . - . mg/kg), extrapolated from the dose used in human patients. dogs were monitored by repeat physical examinations, electrocardiograms and blood pressure assessments during the course of the study. plasma rufinamide concentrations were determined using high-performance liquid chromatography. pharmacokinetic data were analyzed using winnonlin version . . no adverse effects were observed. the mean terminal half-life was . /À . hours. the mean maximum plasma concentration was . /À . mg/ml and the mean time to maximum plasma concentration was . /À . hours. mean clearance was . /À . l/hr. auc inf was . /À . mgÃh/ml. results of this study suggest that rufinamide given orally at mg/ kg twice daily in healthy dogs should result in a plasma concentration and half-life sufficient to achieve the therapeutic level extrapolated from humans without short-term adverse effects. further investigation into the efficacy and long-term safety of rufinamide in the treatment of canine epilepsy is warranted. the aims of this study were to investigate the abg for (i) the prevalence of skull abnormalities; (ii) the prevalence of sm; (iii) an association between lateral ventricular size, cerebellar size and sm; and (iv) associations between sm, skull abnormalities, csf pleocytosis and clinical signs. seventy-six abgs, recruited as part of a larger epidemiological and genetic study, underwent brain and spinal mri evaluation ( . t general electric signa hdx, milwaukee, wi). all dogs were evaluated neurologically, recording deficits and the presence of spinal pain. sequences acquired included t w, t w pre-and postcontrast, and t w flair, sagittal and transverse. cervical spinal cord central canal (cc) and or syrinx size and its percent area of spinal cord was measured using osirix s . the presence of chari-like malformation (cm) was assessed by recording the presence of caudal cerebellar deviation and/or foramenal vermal herniation. lateral ventricle and cerebellar volume was expressed as a percent of the cerebrum and intracranial volume qa respectively. forty-five dogs underwent atlanto-occipital cerebrospinal fluid tap at the time of mri and the white blood cell (wbc) count was recorded. student's t-tests were used to compare the measured variables between groups with and without skull abnormalities, spinal pain and neurological signs. the mean age of the males ( intact) and the females ( intact) was . months (range - ; median months). neurological deficits and neck pain were noted in ( %) and ( . %) of dogs respectively; dogs ( . %) exhibited both. cerebellar deviation and vermal herniation were present in ( . %) and ( . %) dogs respectively; twenty-three dogs ( . %) had both. mean height of the cc was . mm ( - . mm). forty ( . %) ccs were greater than mm in height; the mean length of these lesions was . vertebrae ( . - ). mean csf wbc count was . /ml ( - ). syrinx height and extent were significantly higher in dogs with neurological signs (size p . ; extent p . ). there were no significant differences in syrinx sizes and extent in dogs with or without skull abnormalities or spinal pain. there were no associations of syrinx height or extent with csf wbc count or age of dog. intact females had a significantly lower syrinx extent than intact males (p . ). there were no significant differences in presence of spinal pain or neurological signs between dogs with or without skull abnormalities. there was a significant negative association of ventricular percentage and cerebellar percentage (p o . ). there was a significant association of ventricular percentage with syrinx percentage (p . ) and height (p . ). this study suggests that sm and cm are prevalent in abgs. syrinx size and extent are associated with neurological signs and ventriculomegaly is associated with both small cerebellar size and large syrinx size. however, sm may not be associated with cm as defined by cerebellar herniation and deviation and is not associated with csf inflammation. the power tissue resection device (ptrd) is a hand-piece comprised of an outer cannula with motor driven vacuum-assisted inner cutting blade. this device was designed and is marketed for human neurosurgical brain/spinal cord tumor resection. the purpose of this study is to describe the use of the ptrd for intervertebral disc fenestration and to compare the effectiveness of manual fenestration to that of the ptrd. fifteen cadaveric lumbar spines were randomly placed into three study groups: group was the control group on which no fenestrations were performed, group was the manual fenestration group and group was the ptrd fenestration group. the effectiveness of fenestration via both manual and ptrd was assessed by calculating the ratio of remaining nuclear weight post fenestration to total nuclear volume. discs with lower ratios were more effectively fenestrated. results showed a smaller ratio of post fenestration remaining nuclear weight to nuclear volume following fenestration with the ptrd ( . ae . ) as compared to manual fenestration ( . ae . ). these results did not show statistical significance. when fenestrated samples were compared to control samples ( . ae . ), there was a statistically significant reduction in ratios. in conclusion, the ptrd is easy to use and is as effective as the manual technique for canine intervertebral disc fenestration. according to the human who classification gliomatosis cerebri (gc) is a rare astrocytic tumor affecting at least three lobes of the brain with extensive infiltration, but relative preservation of brain architecture. gc has not been reported to occur as a hereditary disease, neither in man nor in animals. here, we report the temporally clustered occurrence of gc in a family of bearded collies. a years old female bearded collie with forebrain signs was presented. differentials included inflammatory/ infectious, metabolic/ toxic, and neoplastic diseases. within a time period of months, offspring of this bitch were presented with similar clinical signs. two dogs were full siblings ( males). the remaining female dog originated from a match with a different male dog. mri was performed in all dogs and revealed a diffuse and extensive intra-axial lesion with moderate mass effect and midline shift. the ill defined lesion showed mainly a white matter distribution with hyperintense signal in t -w and flair images and iso-to hypointense signal in t -w images without contrast enhancement. the lesion was bilateral in all cases, continued along the white matter extending partially into the gray matter with contact to the brain surface. neuropathology revealed a diffuse and extensive infiltration of the brain and spinal cord by a neoplastic glial cell population involving white and gray matter of both hemispheres, thalamus, brainstem and cerebellum in all dogs. based on the cell morphology and immunoexpression of glial fibrillary astrocytic protein by neoplastic cells diagnosis of gc was made. this is the first report of familial occurrence of gc, which is likely the result of a germ-line mutation. several human hereditary cancer syndromes are associated with cns tumors including amongst others the li-fraumeni cancer family syndrome (p mutation), neurofibromatosis (type and ) (neurofibromin, merlin mutation), and tuberous sclerosis (hamartin, tuberin mutation). furthermore, familial clustering of human gliomas unassociated to the known inherited cancer syndromes has been described. in the dog, hereditary cns tumors are not known. the exact mode of inheritance and putative gene mutations of gc in this bearded collie family are currently under investigation. preliminary results are consistent with a monogenic autosomal dominant mode of inheritance, although a recessive inheritance cannot be completely ruled out at this time. mutations in the tp gene were not found following amplification and sequencing of exons - in affected dogs. previously presented at the ecvn annual meeting in cambridge, uk. the gm gangliosidoses are characterized by a deficiency of bhexosaminidase. there are two isoforms: hex a composed of an a and b subunit encoded by hexa and hexb genes respectively and hex b with two b subunits. hex a requires an activator encoded by gm a. two japanese chin dogs with confirmed gm gangliosidosis showed elevated total hexosaminidase and normal hexosaminidase a activity, a pattern associated with the ab variant in humans and consistent with prior reports in the breed. this study was performed to identify the mutation responsible using resequencing with an applied biosystems xl dna analyzer as previously described (awano ). mutations in gm a cause the ab variant in humans, but resequencing gm a revealed no mutation that could account for the disease. resequencing hexa and hexb revealed a c. g a mutation in hexa which was homozygous in both affected dogs. sixty-five normal japanese chin dogs were screened for the mutant allele; were homozygous for the ancestral allele and heterozygous. this mutation predicts a p. e k substitution affecting one of two primary active-site amino acids that participate in the hydrolysis of gm ganglioside. substitution of a lysine residue at this site is likely to eliminate subunit a enzymatic activity. the apparently normal levels of hexosaminidase a activity in affected dog samples may be a result of b subunit overexpression. human hex b possesses low levels activity against the artificial substrate used to assess hex a activity, but specificity of activity of the canine enzyme is not known. previously presented at the american society for neurochemistry: additional data in this abstract. phenytoin (pht) is the intravenous drug of choice in humans for seizure emergencies following benzodiazapines. iv fosphenytoin (fos) is a pht pro-drug which causes less administration related adverse events. while the short half-life of pht is not suitable for chronic oral therapy in dogs, iv fos has not been studied. two dogs received mg/kg phenytoin equivalent (pe) and two dogs received mg/kg pe of fosphenytoin intravenously at a rate of mg pe/min. blood for plasma levels were drawn at time-points over hours; total and unbound drug levels were measured by hplc. vital signs including ekg, blood pressure, and neurological examination were monitored. the half-life of metabolism of fos to pht was $ min, with % of fos metabolized to pht by minutes. eighty to % of pht was protein-bound during the first minutes after dosing, compared to - % in humans. the elimination half-life for total pht ranged from . - . hours and for unbound pht ranged from . - . hours. dogs receiving mg/kg pe intravenously achieved unbound pht plasma maximum concentrations of . - . ug/ml at minutes, consistent with human loading dose levels. adverse events observed in some dogs included vomiting, mild ataxia, and short lived tremors, the severity of which appeared dose dependent. all dogs were clinically normal within minutes of all doses. a mg/kg pe dose of iv fos appears adequate for production of pht levels predicted to be effective for the treatment of canine seizure emergencies. further studies in clinical canine patients are warranted. acquired myasthenia gravis (mg) is caused by antibodymediated inactivation of the acetylcholine receptor on the neuromuscular endplate causing focal, regional or generalized muscle weakness. many medical treatments have been reported; however, responses to therapy and outcomes are unpredictable and death often results from aspiration pneumonia. therapeutic apheresis is an extracorporeal procedure that separates blood into its components for removal or specific alteration prior to return to the patient. therapeutic plasma exchange (tpe) is an apheresis treatment in which plasma (containing pathologic antibodies) is removed and exchanged with donor plasma. tpe is used routinely to treat mg in human patients with severe disease or disease unresponsive to conventional therapy. we report the successful use of tpe to treat large breed dogs with confirmed mg (aceytlcholine receptor antibody concentration: . and . nm/l, respectively; normal concentration: o . nm/ l) that was severe and not adequately responsive to traditional therapies. both dogs were non-ambulatory, recumbent, and demonstrated megaesophagus and aspiration pneumonia. three tpe treatments ( plasma exchange each) were performed over and days, respectively, in each dog without complication. both dogs became ambulatory within days of starting tpe treatment with subsequent resolution of regurgitation and megaesophagus. pyridostigmine was continued during tpe sessions and discontinued in both dogs within - months. both dogs remain asymptomatic and have had no recurrence of mg during and months of follow-up, respectively. tpe is a viable treatment option for dogs with mg that have severe disease, life-threatening complications or that remain unresponsive to traditional therapies. tpe may alleviate clinical signs more rapidly, and improve long-term outcomes when compared to historical experiences in patients with comparable disease. clinical findings, clinicopathologic data, imaging features, and treatment of canine spinal meningiomas have been described in the veterinary literature, but histological characteristics and tumor grading have less commonly been reported. the aims of this retrospective case series were to describe the clinical, imaging, and histologic features of seven canine spinal meningiomas including a cervical spinal cystic meningioma that had imaging and intraoperative features of a subarachnoid cyst. medical records from dogs with a histopathological diagnosis of spinal cord meningioma presented to the veterinary teaching hospital between and were reviewed. signalment, presenting clinical signs, physical and neurologic examination, clinicopathologic data, surgery reports and available images were reviewed. all meningiomas were histologically classified and graded following the international who human classification for cns tumors. seven dogs were included, males and females. median age at presentation was . years (range, . - . years), and median weight was kg (range, - kg) . median time between onset of clinical signs and diagnosis was days (range, days - year). cerebrospinal fluid (csf) analysis was performed in dogs, showing increased protein concentration in cases, and being normal in the other . spinal radiographs revealed vertebral canal widening in one case. myelography ( / ) showed intradural/extramedullary lesions in three cases, one of them consistent with a csf-filled subarachnoid cavity, and an extradural lesion in one case. magnetic resonance imaging (mri) was performed in all cases and revealed mild to marked hyperintensity on t w and precontrast t w images and homogeneous contrast enhancing (ce) intradural/extramedullary masses ( cervical and thoracic) in six cases, with one of these showing an additional intramedullary ce pattern. a dural tail was identified in two dogs. one dog had a fluid-filled subarachnoid enlargement located dorsally to the spinal cord. this lesion was hyperintense on t w, hypointense on t w and flair images, and did not enhance. it was diagnosed as a spinal subarachnoid cyst, but the histopathological study of the surgically resected mass revealed a grade i cystic meningioma. five other cases underwent cytoreductive surgery, two transitional meningiomas (grade i) that survived (alive at the time of writing) and months; and three anaplastic meningiomas (grade iii) that survived - . months before neurological deterioration and euthanasia. another anaplastic meningioma was euthanized right after diagnosis. there are few reports grading canine spinal meningiomas, with most being grade i or ii. of the few grade iii tumors reported, only one had been treated surgically and was euthanized days later because of neurological deterioration. we report four grade iii (anaplastic) meningiomas, three of which surgically treated and with longer survival times. finally, cystic meningioma should be considered in the differential diagnosis of cases with imaging features consistent with arachnoid cyst because of their similar appearance, making histopathological analysis essential for a definitive diagnosis. head trauma is a common veterinary emergency, but few prognostic indicators have been studied in dogs, making it challenging for clinicians to counsel clients about the odds of recovery. a recent meta-analysis showed that higher plasma glucose, lower plasma ph and lower hemoglobin at admission were associated with increased risk of death in human head trauma. the goal of this retrospective study was to investigate the association between admission point of care blood gas parameters and survival to discharge in dogs with head trauma. fifty one dogs presenting to the cornell university hospital for animals with head trauma from to that had a blood gas analysis done within hour of presentation were eligible for inclusion. parameters assessed included glucose, base excess (be), anion gap (ag), ph, hemoglobin, and sodium. biochemical data were found to be normally distributed using the kolmogorov-smirnov test. t-tests or welch tests were used to compare parameters between survivors (s,n ) and non-survivors (ns, n ). of glucose, be, ag, ph, hemoglobin, and sodium, only mean glucose (s mg/dl, ns . mg/dl, p . ) was significantly different between groups, although there was a trend for a difference in mean be (s À . , ns À , , p . ). logistic regression analysis showed that of the parameters, only be was independently associated with outcome (odds ratio . , % ci . - . , p . ). these results suggest that two easily measured biochemical parameters (glucose and be) may yield useful prognostic information in dogs with head trauma, but further studies are needed to further elucidate these findings. type i intervertebral disc disease (ivdd) commonly affects chondrodystrophic dogs. neurological recovery and outcome following surgical decompression may be unpredictable due to suspected ischemic neuronal injury. hyperlactatemia has been associated with spinal cord injury in humans and experimental animals. the purpose of the study was ) to determine the relationship between serum and csf lactate levels and ) to compare lactate levels with neurological outcome following decompressive surgery in dogs with ivdd. healthy, chondrodystrophic dogs diagnosed with ivdd localized to the t -l spinal cord were included. serum lactate levels were obtained at: anaesthetic induction, skin incision, muscle dissection, and extubation. in patients with hyperlacatemia at extubation, additional samples were obtained. csf was analyzed for lactate concentration. neurological status was recorded at presentation and multiple times during the recovery period. dogs were included in the study ( - years old). / dogs had normal lactate levels throughout the study. / dogs had serum hyperlactatemia prior to anaesthetic induction; / dogs returned to normal during anaesthesia and / dogs had continued hyperlactatemia until the end of the observation period. neurological status of the dogs varied similarly between all groups. in / dogs where csf lactate levels were measured, initial serum levels were lower than csf lactate levels; in / dogs where csf and serum were collected simultaneously, serum lactate concentration was consistently lower than csf lactate. no association between presenting neurological status or neurological outcome and serum or csf lactate concentration was made. neither serum nor csf lactate concentration is useful for predicting neurological outcome in dogs with ivdd. chiari-like malformation (cm) has been associated with syringomyelia (sm) in cavalier king charles spaniel (ckcs) and is postulated to result from a mismatch between the volume of the caudal cranial fossa and the brain parenchyma contained within. the objective of this study was to assess the role of cerebellar volume in caudal cranial fossa overcrowding and syringomyelia. three dimensional models were created using t -weighted transverse magnetic resonance images in the commercial software package mimics s . volumes of cerebellar parenchyma were analyzed as percentages of caudal cranial fossa volume (cerebellar caudal cranial fossa percentage) and total brain parenchyma volume (cerebellar brain percentage). data was assessed for normality and the appropriate statistical test was used to compare means/medians between groups. forty-five small breed dogs (sb), ckcs and labradors (ld) were compared. as sm is thought to be a late onset disease process, two subgroups were formed for comparison: ckcs younger than years with sm (group ) and ckcs older than years without sm (group ). ckcs had a larger cerebellar caudal cranial fossa percentage than the other groups . %] vs. sb . % [ . - . %] and ld . % [ . - . %]; p o . ). the cerebellar brain percentage was also larger in ckcs compared to the other groups (ckcs . % [ . - . %] vs. sb . % [ . - . %] and ld . % [ . - . %]; p o . ). group had a significantly larger cerebellar caudal cranial fossa percentage than group ( . % ae . vs. . % ae . , p . ) and a significantly larger cerebellar brain percentage ( . % ae . vs. . % ae . , p . ). our findings show that the ckcs has a relatively larger cerebellum than small breed dogs and labradors and there is an association between increased cerebellar volume and sm in ckcs. chiari-like malformation (cm) is nearly omnipresent in the cavalier king charles spaniel (ckcs) breed. the mis-match of the caudal cranial fossa and the parenchyma within is thought to lead to syringomyelia (sm). there is currently a lack of information if the morphological changes seen in ckcs with cm are progressive or non-progressive. in this retrospective study we used established measurements of cerebral volumes, foramen magnum height and cerebellar herniation length to assess if there is a significant difference between subsequent magnetic resonance (mr) imaging of the brain of the same dog. electronic patient records were reviewed for ckcs with cm which had two separate mri scans, which were a minimum of months apart. ckcs with diseases affecting measurements were excluded. for the volumetric measurements three-dimensional models were created using t -weighted transverse mr images in the medical imaging software (mimics v . , materialise n.v, ) . volumes of the caudal cranial fossa parenchyma were analyzed as percentages of caudal cranial fossa volume and caudal cranial fossa volume was analyzed as a percentage of total cranial cavity volume. the volume of the ventricular system was recorded as a percentage of total parenchymal volume. data was assessed for normality and the appropriate statistical test was used to compare means/medians. twelve ckcs were included with a median scan interval of . months ( - months). the size of the foramen magnum increased significantly between the first and second scan ( . ae . cm vs. . ae . cm; p . ), as did the length of cerebellar herniation ( . ae . cm vs. . ae . cm; p . ) and the caudal cranial fossa percentage ( . % [ . - . %] vs. . % [ . - . %]; p . ). there was no significant difference noted between the two time points in any of the other volumetric measurements ( this work could suggest that overcrowding of the caudal cranial fossa in conjunction with the movements of cerebrospinal fluid and cerebellar tissue secondary to pulse pressures created during the cardiac cycle causes pressures on the occipital bone. this leads to a resorption of the bone and therefore an increase in caudal cranial fossa and foramen magnum size allowing cerebellar herniation length to increase. the cord dorsum potential (cdp) is a stationary potential arising in dorsal horn interneurons after stimulation of sensory nerves. cdps have been recorded in normal anesthetized dogs previously, and normal latency values have been determined for tibial and radial nerves. this study was undertaken to determine whether cdps could be reliably recorded from the caudal nerves in normal dogs, thus allowing electrophysiological assessment of the cauda equina, and whether neuromuscular blockade improved recording quality. ten adult dogs weighing from . to . kg were anesthetized and cord dorsum recordings were compared before and after administration of atracurium. recording needles were placed onto the dorsal lamina at intervertebral sites from l /s to l / . stimulations were made on the lateral aspect of the caudal vertebrae approximately - cm from the tail base. recordings from stimulations were averaged. cdps were recorded successfully in all dogs. onset latency varied from . to . ms. the cdp was largest when recorded closest to the site of entry of the stimulated nerve into the cord, as determined by post-mortem examination immediately after testing in dogs. administration of atracurium did decrease muscle artifact, and in some cases helped isolate the origin of the cdp. these data show that cdps can be readily assessed from the caudal nerves of anesthetized dogs, with or without atracurium. cord dorsum potentials from caudal nerves may add important information about the integrity of the cauda equina in dogs with suspected degenerative lumbosacral stenosis. canine intracranial glial tumors and many human brain tumors express heat shock proteins (hsps) associated with their degree of malignancy. the up-regulation of hsps during tumor cell growth helps keep tumor proteins stable and therefore makes them a reasonable target for therapy. ki expression and ec have been strong indicators of cell proliferation and dedifferentiation, respectively.the aims of this study were to determine (i) if canine meningiomas express hsp and/or hsp ; (ii) whether the expression of the hsps was associated with ki and/or e-cadherin (ec) expression; and (iii) whether peritumoral edema was associated with hsp, ki and/or ec expression. forty-one formalin-fixed, paraffin-embedded canine intracranial meningiomas underwent immunohistochemical staining using anti-hsp , or antibodies. these tumor samples were also immunohistochemically stained for ki and ec expression. canine mammary carcinoma and squamous cell carcinoma tissues served as the control samples, as both have previously been shown to express hsps. skin was used as control for ki and ec. four non-overlapping high power fields of each stained sample were selected and cell staining was analyzed using a semi-quantitative method for hsps and ki ; a qualitative assessment was used for ec. all analyses were performed using sas v . (cary, nc). descriptive statistics of staining percentages were calculated for all tumors tested. simple pearson's correlation was used to test for correlations of ec area with hsp areas and ki- percent positive cells and of ec intensity with hsp intensities and ki- percent positive cells. all hypothesis tests were sided and the significance level was a . . thirteen meningiomas had mr images quantitatively evaluated for peritumoral edema using t flair sequences. the edema index (ei) was evaluated for an association with hsp , hsp , ec and ki expression. hsp was expressed in % (mean . % of cells; range - %), hsp in % (mean . % of cells; range - %) and ec in % of meningiomas. there was no association demonstrated between either hsp expression variable and ec or ki- expression. there was also no association between the ec expression variables and ki- . however, there was a significant negative association between hsp extent (p . ) and area (p . ) with ei. in conclusion, hsp and expression was demonstrated in canine intracranial meningiomas but was not associated with ki- or ec expression. this study suggests that hsps may not have a significant role in the maintenance of canine meningiomas and so do not represent a novel treatment target for this group of tumors unlike canine glial cell tumors. however, hsp may be involved in the pathogenesis of peritumoral edema in meningiomas and warrants further investigation. an extended release (xr) formulation of levetiracetam, a second generation antiepileptic drug, was recently approved for human use on a once daily basis. although levetiracetam is clinically effective for seizure control in dogs, it requires a three times daily administration. the potential benefits of the xr formulation include reduced daily dosing leading to improved compliance and relatively constant plasma concentrations. the aim of this study was to compare the pharmacokinetics of levetiracetam xr tablets with immediate release (ir) tablets following single dosing in dogs. five clinically and neurologically normal mixed breed dogs were used in a cross-over design. all dogs (mean body weight . kg; range . - . ) had normal hematology, serum chemistry and urinalyses. following a hour fast, each dog was administered oral ir levetiracetam ( mg; mean dose . mg/kg; range . - . ). heparinized blood for drug analysis was taken from each dog prior to administration and . , . , . , , , and hours after. blood was immediately centrifuged and supernatant plasma was stored at À c until analysis. after a day wash-out period, each dog was administered mg oral xr levetiracetam and blood samples were taken at identical timings. plasma samples were thawed at room temperature before preparation by solid phase extraction for hplc analysis. reverse phase chromatographic separation was performed. levetiracetam and an internal standard were detected using ultraviolet spectroscopy at nm. concentrations of levetiracetam were determined by peak area comparison to the internal standard. mean data were fit to a one compartment pharmacokinetic model with first order elimination and absorption and included a lag-phase for xr formulation. no adverse clinical effects were noted in any of the dogs. the auc associated with xr was hr ug/ml, a . fold increase over that with ir ( . hr ug/ml). the absorption half-life was . hr with xr and . hr with ir, a . fold difference. the elimination halflife was . hr with xr and . hr with ir, a . fold difference. the tmax associated with xr . hr and . hr with ir, a . fold difference. the cmax associated with xr was . mg/ml and . mg/ml with ir, a . fold difference. the plasma concentration of ir levetiracetam was not detectable at hr after administration whereas it was greater than mg/ml at hr after xr administration. based on the auc data, there is an approximately fold increase in bioavailability of the xr compared to the ir formulation. the cmax was approximately times greater following xr administration and a high plasma level in excess of the suggested canine therapeutic concentration ( mg/ml) for at least hours. although specific dosing recommendations cannot be made from this data, the favorable pharmacokinetics of xr over ir suggests that single, daily administration could be efficacious. thoracic and lumbar vertebrae are frequently affected by fractures and or luxations in dogs following trauma. surgical repair is part of the emergency treatment described for this disorder but does not guarantee improvement of the associated clinical signs. multiple surgical repair techniques have been described but have not been compared in terms of their success and the factors associated with a positive outcome. the aims of this study were to retrospectively evaluate the effect of different types of vertebral repair, injury type and injury location on outcome in dogs with thoracolumbar (tl) and lumbosacral (ls) spinal trauma. medical records were searched for dogs with radiographic evidence of a tl or ls vertebral fracture and or luxation ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ; signalment, body weight and duration of disease were recorded. dogs were retrospectively scored neurologically ( - ; normal to plegic with absent pain perception) on admission and at re-evaluation following surgery. lesion location was classed as t -l and l -s ; dogs were evaluated as one group and as two separate groups with respect to outcome. a subset of lesions were classed as cord compression or not based on advanced imaging. three repair techniques were evaluated (i) pins and polymethylmethacrylate (pmma); (ii) screws and pmma; and (iii) spinal stapling. regression analysis was applied to test for an association between the type of surgery and a successful outcome (non-painful and ambulatory). simple bivariate analyses were performed to investigate for variables predictive of a successful outcome. fifty-nine dogs were included. twenty-eight dogs were classed as t -l and were l -s . there were dogs with fractures and with luxations; dogs had both. thirty-one of dogs evaluated had spinal cord compression. ten dogs were repaired with im pins and pmma, dogs with screws and pmma and dogs with spinal stapling. overall, there was a . % success rate; there was no significant difference in outcome between the anatomic sites (p . ). all dogs initially graded as - pre-operation were classed as a successful outcome after at least one week following surgery; % of dogs initially graded as (plegic with pain perception) were classed as successful recovery. one dog ( . %) initially as graded as (plegic with no pain perception) had a successful outcome. a low admission score was statistically predictive of a successful outcome (p o . ). surgery type was not associated with a successful recovery (p . ). signalment, body weight, location of injury, injury type (fracture, luxation or both), presence of compression, and duration of disease did not predict outcome. from this study, the successful recovery of dogs following surgical fixation is high and is only dependent on the neurological score at the time of admission. the choice of surgical technique does not seem to influence outcome although a prospective study comparing two surgery types is warranted to further investigate this issue the results of which can be confounded by surgeon experience and variable follow-up. cranial thoracic intervertebral disc disease (ivdd) is extremely rare due to the presence of the intercapital ligament, although anecdotic data suggest german shepherd dogs (gsd) can share some predisposition for this disorder. the objective of the study was to retrospectively evaluate through mri if cranial thoracic ivdd is significantly more common in gsd compare to other large breed dogs. a search was done through database of the ontario veterinary college. any gsd were a spinal mri including t -t spine was performed was recruited. a group of large-breed non-gsd was used as a control. in the midsaggital t wi plane, three variables were assessed and graded for each intervertebral disc space t -t : spinal cord compression (scc), disc degeneration (dd), and herniation. wilcoxon sign rank test was used to assess if scores were different between groups. exact conditional logistic regression was used to determine whether any intervertebral disc space was a risk factor. gsd and large breed non-gsds were recruited. the gsd group had significantly higher scores than the non-gsd for scc, and herniation. regarding the individual intervertebral discs, in the gsd group t - , t - , t - discs had significantly an increased risk for scc, and t - for herniation. the results of this study show that gsd have a higher risk of cranial thoracic disc ivdd than other large breed dogs. that risk was higher in discs t -t , t - , and t - , particularly in t - . genetic and/or conformational factors, such as weakness of the intercapital ligament, may predispose gsd to this lesion. diskospondylitis is a common disease of the canine spine; however, few reports of mr imaging findings in dogs are available. the purpose of this study was to describe the signalment, clinical and mr imaging features in affected dogs. twenty-three dogs with a diagnosis of diskospondylitis based on clinical signs, mr imaging, and urine, blood, csf and/or intervertebral disk cultures were included. large breed dogs ( kg) accounted for of the cases. the mean age was . years with males and females equally represented. most dogs ( / ) were ambulatory with varying degrees of pain and paresis. mr imaging characteristics of sites were reviewed. on t w images, vertebral endplates were of mixed signal intensity ( / ) while the vertebral body was hypointense ( / ). the intervertebral disk space was hyperintense on t w ( / ) and stir ( / ) images and mixed signal intensity ( / ) on t w images. paravertebral soft tissue hyperintensities were noted on / t w and / stir images. contrast enhancement occurred at / endplates and / intervertebral disk spaces. only / vertebral bodies and / parvertebral soft tissues contrast enhanced. intramedullary spinal cord t w hyperintensity was noted at / sites. spinal cord or cauda equina compression occurred at / sites. based on the spearman correlation coefficient, a significant direct correlation was found between the degree of spinal cord or cauda equina compression and the patient's neurologic status (p . ). the incidence and severity of spinal cord compression in canine diskospondylitis may have prognostic value and may have been previously underestimated using other imaging modalities. hemilaminectomy and pediculectomy are both well described and commonly utilized techniques to access the spinal canal. these procedures are most often performed to approach a compressive lesion, such as intervertebral disc disease and neoplasia, the goal being adequate visualization of the spinal canal and access to the offending lesion. a proposed benefit of pediculectomy is preservation of the articular facets and thus better maintaining stability of the vertebral column, but at the cost of reduced access to the spinal canal. the purpose of this study was to describe standardized anatomical limits of each technique and report any observed differences that could be considered during presurgical planning. ten canine cadavers had both procedures performed on opposite sides to access the t - , t -l , and l - spinal canal. measurements were obtained after performing a computed tomography study of the spine and recorded from the transverse slice most representative of the defect. the surgical technique, vertebral site, and side of vertebral column were compared with the mean spinal canal and defect height using a covariate model. dorsal and ventral remnant lamina heights were also compared. the height of the defect relative to the spinal canal was - % with hemilaminectomy and - % with pediculectomy. the observed difference in defect height of - % (p o . ) and varied with spinal canal height. dorsal remnant lamina height was . - . % of spinal canal height with hemilaminectomy and - % with pediculectomy. ventral remnant lamina height ranged from - % and . - . %, respectively, though the difference was not statistically significant. while a larger defect is expected with a hemilaminectomy procedure, our results demonstrate that this difference increases with increasing spinal canal height. interestingly, the proportion of exposed spinal canal decreases with increasing canal height for both procedures. the difference in defect height between techniques was due to greater removal of the dorsal spinal canal, possibly making the hemilaminectomy technique better suited for more dorsal lesions, while no statistically difference in access to the ventral canal is observed. no effect of vertebral site was detected. of note was the involvement of articular facets in half of the pediculectomy defects, involving an average of % of the articular facet height. this result questions the suggested benefit for the vertebral stability, but further biomechanical studies would be required. low level laser therapy (lllt) is a treatment used in human and veterinary medicine for a variety of clinical syndromes. some uses in human medicine include acute pain associated with osteoarthritis, rheumatoid arthritis, tendonitis, tmj disorders, chronic joint disorders, and wound healing. research is currently on-going to determine the adequate wavelengths to promote effective treatment results with lllt in these conditions. it is purported that lllt acts via the mitochondria to increase cellular metabolism promoting wound healing and a decrease in pain and inflammation. in this study, we hypothesized that dogs treated with lllt in conjunction with hemilaminectomy would display quicker recovery times regardless of the presence or absence of deep pain sensation. seventeen dogs ( dachshunds, chihuahuas, french bulldogs, lhasa ahpsos, and each of a pembroke welsch corgi, and a miniature poodle) were selected and divided into two groups. the dogs ranged in age from to years old, weighed between and pounds, and underwent hemilaminectamies after acute onset of paraplegia secondary to intervertebral disc disease (surgically confirmed). one group received laser treatments on days through of hospitalization. the second group did not receive lllt, but followed the same peri-operative medication protocol. the laser used in this study was an erchonia laser model pl ( nm). the hertz setting was similar for each patient using the previously established protocol for intervertebral disc disease (ivdd) with pulse rate ranging from hz to hz. all dogs received advanced imaging pre-operatively with myelogram or mri. results of the study revealed that treatment with lllt of nm wavelength did not shorten or improve recovery times for dogs with acute onset paraplegia secondary to ivdd after hemilaminectomy procedures. dogs that showed recovery to ambulation at the two week recheck were consistently dogs that were deep pain positive on presentation. a lengthened recovery time or no recovery was seen in the majority of those dogs with absent deep pain on presentation as has been revealed historically in past studies. lllt did not appear to have an effect on this result. however, there are few data describing normal glucose uptake of the canine brain for comparison with suspected or confirmed disease. thus the purpose of this study was to assess the normal distribution of fdg uptake of canine brain structures using a high-resolution research tomography-pet and t-magnetic resonance imaging (mri) fusion system. fdg-pet and t -weighted mr imaging of the brain were performed on healthy laboratory beagle dogs. acquired pet and mr images were automatically co-registered by the image analysis software. on mr images, regions of interest (roi) were manually drawn over intracranial structures, including gross structures (whole brain, telencephalon, diencephalon, mesencephalon, dorsal metencephalon, ventral metencephalon and myelencephalon). a standard uptake value (suv) and relative suv ratio (rsuv suv of roi/suv of whole brain) were calculated for each roi. t-mr images compensated the low anatomical resolution of pet qj;by proving good spatial and contrast resolution for the identification of the clinically relevant brain anatomy. among gross structures, mesencephalon and ventral metencephalon had the highest (suv: . ae . ; rsuv: . ae . ) and the lowest (suv: . ae . ; rsuv: . ae . ) fdg uptake respectively. when suvs were calculated on detailed regions, rostral colliculus and corpus callosum had the highest (suv: . ae . ; rsuv: . ae . ) and the lowest (suv: . ae . ; rsuv: . ae . ) value respectively. these data acquired from normal dog brain will be used in clinical neurology to investigate various intracranial diseases such as inflammation, neoplasm and behavioral disorders. degenerative lumbosacral stenosis (dlss) is a multifactorial condition affecting predominantly large breed dogs. the combination of stenosis and compressive neuropathy cause lumbar pain, lameness and neurologic dysfunction. previous reports describe urinary and fecal incontinence in severely affected dogs. the objectives of this retrospective case series were to describe the clinical signs associated with dysuria and eventual diagnosis of dlss in dogs, and to describe factors associated with regained micturition following prompt diagnosis and treatment. medical records from the university of georgia and the university of missouri between and of dogs were reviewed. inclusion required observation of dysuria, urine retention, absence of structural lower urinary tract disease and concurrent presumptive diagnosis of dlss. dysuria was defined as inability to initiate or sustain a urine stream. urine residual volume was evaluated postvoiding. dysuria was further evaluated using urethral contrast studies, urodynamic testing (urethral profilometry ( ) and cystometry ( )), ultrasonography ( ), and urine culture ( ). presumptive diagnosis of dlss was based on imaging using plain radiography and epidurography ( ), computed tomography ( ) or magnetic resonance imaging ( ). breeds represented included the german shepherd dog (n ), golden retriever (n ), burnese mountain dog (n ), and each labrador retriever, weimaraner, rottweiler and mixed-breed. all dogs were male. were intact at onset of clinical signs. median body weight was . kg (range . - ) and median age was years (range - ). median duration of clinical signs prior to admission was months (range . - ). other pertinent presenting clinical signs included dyschezia ( ), fecal incontinence ( ), general proprioceptive ataxia ( ), weakness ( ), and difficulty rising ( ). physical examination findings included pelvic limb muscle atrophy ( ) and prostatomegaly ( ). abnormal neurologic examination findings included postural reaction deficits ( ), hyporeflexia ( ), decreased tail tone ( ) and lumbosacral hyperesthesia ( ). neurologic examination was normal in dogs. dorsal laminectomy was performed and diagnosis confirmed in dogs; recovery was monitored for a median of . months (range . - ). three of the dogs ( %) regained normal micturition within . - . months of surgery. though not statistically significant, dogs that regained micturition tended to have a shorter duration of clinical signs (median . months, range . - ) versus dogs that remained dysuric (median months, range - ). two of the dogs that regained micturition were neutered at the onset of clinical signs, but only of dogs that remained dysuric was neutered. signs improved in all dogs with postural reaction deficits and decreased tail tone. hyperesthesia resolved in of dogs ( %) and fecal continence returned in of dogs ( %). these findings suggest that following prompt diagnosis and surgical decompression, normal micturition could be regained in dlss affected dogs presenting with signs of dysuria. glycogen storage disease type ia (gsdia; von gierke disease) is an inherited metabolic disorder resulting from a deficiency of glucose -phosphatase-a (g pase). previous reports indicate that clinical manifestations of gsdia occur only in individuals with homozygous expression of a p.i l mutation. heterozygote dogs (het) have been previously reported to exhibit an overall normal outward phenotype. the purpose of this report is to briefly describe some differences that have been observed between het and homozygous wild type (wt) dogs. a colony of dogs at the university of florida contains a mix of affected, wt, and het individuals. in the course of studies designed to determine the effectiveness of gene therapy for correction of gsdia in dogs, both wt and het dogs have been utilized as controls. available information about body weights, clinical pathology tests, fasting studies, and liver biopsies was retrieved from records for both wt and het dogs and compared. although birth weights are similar, het dogs have a slower average rate of weight gain than wt dogs and this difference is especially prominent during the first few months of life (figure ) . in contrast to affected dogs, both wt and het dogs are able to maintain normal blood glucose concentrations for up to - hours of fasting, however, after longer fasts of - hours, het dogs have lower glucose and higher lactate concentrations (table ). in addition, liver biopsy samples from het dogs had greater apparent levels of glycogen suggested by pas staining than did samples from wt dogs, and this correlated with the results of proton magnetic resonance spectroscopy which demonstrated . times greater glycogen content in a liver biopsy sample from a het dog compared to a sample from a wt dog. together, these findings suggest that the level of g pase activity in heterozygote dogs does not provide a completely normal physiological, biochemical, or histological phenotype as previously reported. the glucokinase gene (gck) encodes an enzyme involved in cellular glucose-sensing mechanisms in pancreatic beta cells and hepatocytes. gck mrna is present in feline pancreas but the gene is not expressed in feline liver. hepatic gck expression is abundant in omnivores so its absence may reflect an evolutionary adaptation of strict carnivores, like feline species. we hypothesized speciesspecific features in the gck hepatic promoter may underlie the gene expression pattern observed in cats. the putative feline gck (fgck) promoter region was located using bioinformatic software to identify homology with human gck (hgck). genomic dna from a dsh cat was subjected to direct sequencing using a series of pcr reactions with speciesspecific primers. dna clones thus obtained were aligned to generate the feline sequence. direct sequencing yielded . kb of genomic dna sequence with high homology with sequences (acbe , acbe ) archived in the feline genome project. the feline sequence had six regions homologous with non-coding regions of hgck; four of these conserved regions are upstream of the putative fgck start. a . kb segment immediately upstream of feline hepatic exon is not present in hgck. the . kb insert is the reverse complement of a conserved sequence located downstream of exon in feline and human sequences. in conclusion, the putative hepatic promoter of fgck shares extensive homology with the hgck promoter but contains a . kb insert not found in hgck. functional studies are needed to confirm the role of the unique insert in regulation of fgck gene expression. deuterium oxide (d o) dilution has been proposed for quantifying body water content, but remains difficult to perform routinely. the objective of this study was to assess if the volume of distribution (vd) of creatinine could be proposed as an alternative in dogs for such a measurement. creatinine and d o vd were measured before (c) and after induction (o) of obesity (by giving an hypercaloric diet ( kcal/ kg) for months) in six healthy adult beagle dogs. creatinine ( mg/kg) and d o ( mg/kg) were simultaneously injected by bolus iv. blood was collected before administration and then at , , , , , , , , and min post-injection (creatinine), and , , , , , , and min (d o) . plasma concentrations of both markers were determined. vd was calculated using pharmacokinetic equations. the body weight increased from . ae . (c) (mean ae sd) to . ae . kg (o). d o vd decreased from ae (c) to ae (o) ml/kg. similarly, creatinine vd decreased from ae (c) to ae (o) ml/kg. the individual difference between creatinine and d o vd (expressed in % of d o vd) ranged from À . to . % (c) and from À . to À . % (o). in conclusion, creatinine vd provides a good estimate of d o vd in both normal and obese conditions. a wk double blinded study was conducted comparing the affect of two foods on mobility in dogs. all work was approved by an iacuc. healthy beagle dogs ( - years old, mixed gender) were used. affected (a) and non-affected (na) dogs were identified based on orthopedic examination and radiography as having or not having evidence of naturally occurring joint pathology (presence of osteophytes, dysplasia, effusion, pain on manipulation etc) in one or more joints. a and na dogs were evenly distributed between two locations. foods had nutrient profiles adequate for maintenance according to the aafco official publication. the test food contained greater amounts of methionine, manganese, carnitine, vit. e,, vit. c, alpha linolenic acid (ala), and eicosapentaenoic (epa) acid: the food provided mg n fatty acids and mg n fatty acids per kcal. all dogs were fed the control food for wks followed by a wk feeding period where a and na dogs consumed the test food and a and na dogs the control. blood and urine were collected at weeks , , and and analyzed for serum fatty acids and urine thromboxane:creatinine ratios were determined. evaluators in this study were different than those making the original diagnosis and so were blinded as to treatment and diagnosis. orthopedic exams were performed by two veterinary surgeons at each site on weeks , , and . the same two evaluators examined the same dogs throughout. the data was evaluated for the difference between a and na dogs and between foods with age, gender and location as covariates. body weight, disease status, age and gender were blocked. analysis included anova repeated measures mixed procedure (sas version . ) to determine treatment effects over time.serum epa was greater and arachidonic acid lower at weeks , and in the test food fed dogs (p o . ). urine thromboxane:creatinine ratios were decreased in the a dogs fed the test food compared to the a dogs fed the control food at wks (p o . ). lameness score was significantly improved (p o . ) within and between groups of dogs fed the test food. a significantly greater proportion of a dogs fed the test food had improvement in total het (n ) blood glucose (mg/dl) (ae ) (ae ) blood lactate (mmol/l) . (ae . ) . (ae . ) joint score, lameness, functional disability and overall assessment score at wks compared to a dogs fed the control food. % of a dogs had an improved overall assessment score on the test food after wks and at wks compared to % at wks and % at wks of a dogs consuming the control food. this study shows that a food with moderate amounts of added linolenic acid and epa can have a positive impact on systemic inflammation and mobility in - weeks. a similar abstract will be presented at the orthopedic research society meeting in january to an audience largely of orthopedic researchers interested in human orthopedics. fat is an important dietary component, serving both as a source of energy and as a supplier of essential fatty acids (fa). medium-chain triglycerides (mct) contain intermediate length fa that do not rely on l-carnitine for transport across the inner mitochondrial membrane, bypassing this rate-limiting step in fa oxidation. longchain (n- ) polyunsaturated fatty acids (pufa) from fish oil (fo), and in particular eicosanoids derived from eicosapentaenoic acid (epa), may protect against excessive inflammatory reactions, which may be exacerbated by eicosanoids derived from (n- ) arachidonic acid (aa). this study investigated the effects of adding mct:fo and l-carnitine to a control diet (prescription diet s k/d s ) on lean body mass, and serum fa and metabolites. forty healthy beagles ( . to . y) were fed one of three foods (n to dogs each) for mo. the study protocol was reviewed and approved by iacuc, hill's pet nutrition, inc. all foods were complete, balanced, and sufficient for maintenance of adult dogs; and had similar concentrations of moisture, protein, and fat (approx. . %, . %, . %, respectively). composition of serum fa was determined by gas chromatography of fa methyl esters. metabolomic profiles of serum samples were determined from extracted supernatants that were split and run on gc/ms and lc/ ms/ms platforms, for identification and relative quantification of small metabolites. body composition was determined by dual energy x-ray absorptiometry. serum concentrations of lauric and myristic fa increased; epa and dha increased in a dose-dependent manner; and aa decreased in dogs fed treatment food (proc-mixed procedure in sas; all p . ) when compared to dogs fed treatment foods or . serum concentrations of acetylcarnitine and succinylcarnitine increased, indicating lcarnitine incorporation, in dogs fed treatment foods and . thus, a diet enriched with mct:fo significantly altered serum fa composition, enriching (n- ) pufa and lowering aa concentrations. there was no change in lean body mass for any of the diets compared to baseline values, and no difference between treatments, showing that all three treatment foods met protein requirements. ten owned dogs, obese for more than months (body condition score [bcs] of ; fat mass [fm] . ae . %) were studied. these dogs had their weight reduced by % (bcs ; fm . ae . %; p o . ) being designated weight reduced (wr) group and then were fed to maintain constant body weight during days (bcs . ; p . ), designated maintenance (main) group. a control (ct) group of beagles was also included (bcs . ; fm . ae . %; p o . ). in all groups the glucose postprandial response test was performed after hours of fasting. blood samples were taken prefeeding and after , , , , , , , , , and minutes of the consumption of cooked rice enough to the ingestion of g of starch/kg body weight. tnf-a and il- were dosed in milliplex tm map panel, insulin and leptin by radioimmunoassay. statistical analysis included paired or non-paired t-tests and wilcoxon (p o . ). the regimen normalized meal glucose response, the area under the curve (auc) of glucose for wr was lower than for obese (p o . ) and similar to main and ct (p . ). insulin secretion did not normalize immediately, as obese and wr exhibited similar auc of insulin and higher values than for ct (p o . ). main, however, presented similar auc of insulin than ct, with lower values than obese and wr (p o . ), suggesting that dogs require some time to adapt their metabolism. leptin, tnf-a, and il- presented significant reductions after weight loss (p o . ), without differences between wr, main and ct (p . ), suggesting an improvement of the pro-inflammatory state consequent to obesity. studying food base excess (be) modification, methionine intoxication was described. in a basal kibble dog diet (be meq/kg; . g/kg of s) two dosages of ammonium sulphate and methionine was added, resulting in diets with be of meq/kg ( . g/kg of s) and À meq/kg ( . g/kg of s), or be of meq/kg ( . g/kg of s) and meq/kg ( . g/kg of s), respectively. a  factorial plus a control diet design, resulting in five treatments, and adult health beagle dogs were used, in a completed randomized design with six dogs per diet. a -d adaptation phase followed -d of total urine collection (in bottles with mg of thymol). urine were pooled by dog and analyzed for density, volume and ph. food macroelements were determined by standards methods (aoac, ) and used for be calculation. dog's acid-basic status was studied by blood gas analysis of venous blood, at : h (pre feeding) and hours after meal. a dose-dependent reduction of urinary ph was verified for both compounds (p o . ). blood bicarbonate (r . ; p o . ), and blood base excess (r . ; p o . ) were highly correlated with food be. acidemia and reduced blood be were verified in diets with be close to zero (higher dose of both compounds, or . g/kg of s), resulting in daily or each other day vomiting episodes in the dogs. ataxia, seizures, and vomiting were previously describe in dogs fed g/kg of methionine, but our results suggest that a much lower value ( . g/kg) was toxic and that the safe upper limit should be between this value and . g/kg (the lower evaluated dose). in people with chronic kidney disease and heart failure, obesity is associated with longer survival times. this association, called the "obesity paradox," also has been recognized in dogs and cats with heart failure. excess weight appears to modulate the serious deleterious effects of muscle loss in these diseases. the purpose of this study was to determine the effects of body condition and body weight changes in dogs with naturally-occurring chronic kidney disease (ckd). dogs diagnosed with ! iris stage ii ckd between and at iowa state university and tufts cummings school of veterinary medicine were eligible for the study. dogs o year of age and those with acute renal failure or suspected congenital renal diseases were excluded. medical records were reviewed using a standardized data form, and data were collected for initial body weight and body condition score (bcs, - scale), clinicopathologic values, changes in body weight and bcs, comorbidities, and treatments. dogs were classified as underweight (bcs - ), moderate weight (bcs - ), or overweight (bcs - ). a change in body weight was defined as . kg. survival times were determined for all dogs that were discharged from the hospital and lived day. associations between survival and bcs or body weight changes were analyzed using cox proportional hazards models. one hundred two dogs were enrolled in the study. at the time of diagnosis, dogs were classified as iris stage ii, dogs were stage iii and dogs were stage iv. median body weight at baseline was . kg (range, . - . kg). for dogs with body condition scores recorded (n ), were underweight ( %), were moderate ( %), and were overweight ( %). for dogs that had at least two body weights recorded over the course of their disease, gained weight, lost weight, and had no change in weight. changes in body weight were not associated with survival; however, bcs at the time of diagnosis was significantly associated with survival. dogs classified as underweight had a significantly shorter survival time compared to both moderate (p o . ) and overweight dogs (p o . ). these results suggest that body condition is an important consideration in dogs with acquired chronic kidney disease. further studies are warranted to evaluate the relationship between obesity and longer survival in dogs with ckd. protein restriction is the cornerstone of dietary management of kidney disease. the national research council recommends % crude protein and the american association of feed control officials (aafco) recommends a minimum of % crude protein for maintenance for healthy adult cats. protein requirement is unknown for adult cats with kidney disease. most commercially produced cat foods for adult maintenance contains % or more crude protein on a dry matter basis. a typical therapeutic food for cats with kidney disease contains about % crude protein. the objective of the present study was to investigate whether dietary crude protein at . % would be adequate for the maintenance for adult cats with impaired kidney function. seven adult cats, female and male, with age ranging from to . years old (mean: . years) were used in the study. all cats had elevated serum creatinine concentration ( . mg/dl, range: . - . mg/dl) and reduced glomerula filtration rate (mean: % reduction; range: - % reduction) during the study. they did not have other systematic diseases, e.g., hyperthyroidism, at the beginning of the study. cats were fed an expanded dry food made with ingredients commonly used in commercial dry cat foods. the food contained . % crude protein (chemical analysis) and kcal/kg (calculated) on a dry matter basis, or . g protein/ kcal. each essential amino acid in the food was at least % of that recommended by aafco. other nutrients in the food also exceeded aafco's recommendations for maintenance for adult cats. cats were fed the food for weeks. lean body mass (dual x-ray absorptionmetry; hologic, hologic, inc, ma) and serum albumin concentration were measured periodically to monitor protein status of cats. the average lean body mass (mean ae sd) was . ae . kg, . ae . kg, . ae . kg, and . ae . kg in weeks , , , and of the study, respectively. paired t-test did not detect statistical difference (p . ) when comparing the lean body mass in weeks versus weeks , , and , respectively. serum albumin concentration were within the normal reference range during the study (mean ae sd: . ae . %, . ae . %, . ae . %, and . ae . % in weeks , , , and , respectively) . these data show that . % dietary crude protein in a dry food with kcal/kg on a dry matter basis, or . g protein/ kcal, is adequate for maintenance for cats with impaired kidney function. in humans, several disease conditions exist that involve abnormal patterns of polyunsaturated fatty acids and similar abnormalities may be present in companion animals. indeed there have been reports of decreased plasma arachidonic acid and reduced delta- desaturase activities in dogs with atopy and other skin disorders. the present study investigated serum fatty acid profiles in dogs and cats presented to the texas a&m university veterinary teaching hospital, clinical pathology laboratory over the past one year period. results were compared with normative data generated among dogs and cats from earlier feeding studies. sera used were residual samples submitted to the laboratory for other diagnostic procedures and stored frozen for no more than months after collection. the samples were grouped according to presenting disorders involving liver, kidney, digestive, and cardiac diseases. total lipids were extracted using chloroform:methanol ( : v/v) and fatty acid methyl esters were prepared for capillary gas chromatography. relative percentage distribution of individual serum fatty acids for each animal were then compared with average normative serum phospholipid fatty acid values (dogs, n ; cats, n ) by calculating the ratio of the value in the diseased individual to the normal mean value and used as an index of normalcy. normalcy ratios were then plotted on a logarithmic scale with normal at . . the ratio was then compared to changes greater than , and standard deviations of the normal mean values. in this way a graphical presentation of resultant values was obtained. although the animals had been fed various commercial diets and some home-prepared foods, a number of noteworthy patterns emerged from this analysis. dogs showed increased linoleic acid, decreased arachidonic acid, increased total monounsaturated and decreased saturated fatty acids at p o . ; oleic acid was increased at p o . . remarkably, these findings were similar for all canine disease categories evaluated (n , heart; n , kidney; n , liver, and n digestive disorders). in cats, a slight decrease in arachidonic acid and large decrease in : was observed but only in heart disorders. by contrast, modest elevations of arachidonate were observed in kidney, liver, and digestive disease groups but at p o . . sample sizes of the feline sera were considerably smaller (range of - per group). a limitation of this analysis is that variability of normal data may exist depending on diet fed making comparisons less reliable however, these preliminary data suggest that metabolic diseases of dogs may depress plasma arachidonic acid independent of diet fed suggesting either reduced conversion from linoleic acid or increased utilization of arachidonate for eicosanoid production during times of metabolic stress. conversely, in cats, increases in arachidonic acid may be associated with diet arachidonate or other mechanisms. additional studies to verify these findings are warranted. the objective of this study was to determine whether or not lalanyl-l-glutamine (ala-gln) supplementation in dogs with parvoviral enteritis improves the survival rate and ameliorates clinical signs without side effects. this randomized, double-blinded, placebo-controlled clinical trial included client-owned dogs. the dogs were randomly assigned into two groups and administered ala-gln solution (dipeptiven; . g/kg) or an equivalent volume of placebo orally twice a day. all of the dogs (ala-gln group [n ] and placebo group [n ]) received standard treatment while hospitalized and were monitored daily according to a clinical scoring system and diagnostic evaluation for days. among the dogs, (ala-gln-treated group [n ] and placebo group [n ]) were vaccinated and (ala-gln-treated group [n ] and placebo group [n ]) were not vaccinated. the population consisted of purebreds and mixed breed dogs, with a mean age of . ae . weeks. the survival data were compared statistically by means of a log-rank test for the kaplan-meier survival curves. the clinical scores of ala-gln-treated dogs improved significantly relative to the placebo group. there was a significant difference between the two groups in the survival distribution (p . ); specifically, of the ala-gln-treated dogs ( . %) died, whereas of the dogs in the placebo group ( . %) died. no side effects were associated with the administration of ala-gln. these results suggest that the oral administration of ala-gln is effective in improving clinical signs and survival rate in dogs with parvoviral enteritis. bleeding disorders, thrombocytopenia and alterations in platelet function have been documented in humans receiving lipid-containing parenteral nutrition formulations. despite a lack of evidence in the veterinary literature, it is believed that parenteral lipids are contraindicated in critical illness when the development of bleeding disorders is likely. the objective of this study was to determine if there is an in vitro effect on platelet function and thromboelastography (teg) in normal dogs with varying concentrations of a % soybean oil emulsion (intralipid s ). twelve clinically healthy dogs were used for this study. whole blood platelet aggregation, using adp and collagen agonists, was measured using multiple electrode aggregometry in hirudinated blood with final lipid concentrations of , , , and mg/ml. the teg parameters r, k, a-angle, and maximum amplitude (ma) were evaluated from citrated whole blood with equivalent final lipid concentrations as platelet aggregation. there was no significant difference between groups with collageninduced platelet aggregation. there was a significant increase in the area under the curve (auc) with adp-induced aggregation at a lipid concentration of mg/ml (p . ). the ma was significantly reduced at both the mg/ml (p o . ) and mg/ml (p o . ) lipid concentration. there was no statistical difference between groups evaluating the other teg parameters. while platelet aggregation appeared enhanced at the highest concentration evaluated, this concentration is not clinically relevant. the reduction in ma seems discordant but both fibrinogen and platelets contribute to the ma. therefore the higher lipid concentrations may be interfering with fibrinogen kinetics or fibrinogenplatelet interaction. in vivo studies are indicated to determine if any of these changes are clinically significant. rosiglitazone is a peroxisome proliferator-activated receptor gamma (pparg) agonist and an fda-approved anti-diabetic agent in humans that has been investigated for its ability to reduce tumor cell growth. specifically, the combination of rosiglitazone and carboplatin has demonstrated enhanced tumor control. the purpose of this study was to determine the peak plasma concentrations and side effect profile of rosiglitazone after oral administration in dogs with spontaneously occurring cancer. all dogs received carboplatin intravenously concurrently with oral rosiglitazone. ten cancer-bearing dogs with normal pre-treatment hepatic and renal function were enrolled. complete pre-treatment hematological and biochemical parameters were available in ten dogs and post-treatment parameters in nine dogs. peak plasma concentrations varied with dose and ranged from . - . ng/ml and occurred between minutes and hours post administration and rapidly declined after the peak. the dose limiting toxicity was hepatic at a dose of mg/m . there was one grade iii, two grade i alt, and one grade iii ast elevations noted. no changes in total bilirubin, alkaline phosphatase, or ggt values were noted. blood glucose values remained within normal limits. mild, self-limiting gastrointestinal and hematologic toxicities were observed when rosiglitazone was administered in combination with carboplatin. based on this study, the recommended dose of rosiglitazone in cancer-bearing dogs with normal hepatic function is mg/m orally once daily. side effects of the combination appear similar to side effects noted with carboplatin alone. further study is needed to determine efficacy of this combination and if more frequent dosing is required to maintain plasma concentrations. carboplatin has shown little activity as a single agent for the treatment of canine transitional cell carcinoma (tcc). however, gemcitabine has shown synergism with carboplatin in human cell lines. the purpose of this study was to evaluate the activity of gemcitabine against canine tcc cell lines alone or in combination with carboplatin. we hypothesized that gemcitabine in combination with carboplatin would have synergistic effects in vitro. the results of this study could provide a rationale for treatment of canine tcc with the combination of these drugs. tcc cell lines tcc-kiss, tcc-knapp-js, tcc-axa, tcc-hxc, and tcc-sh were treated with gemcitabine, carboplatin, or the combination. cell proliferation was assessed using cyquant assay, cell cycle was evaluated using propidium iodide staining, and apoptosis was assessed by measuring caspase- / activation. synergy was quantified by combination index analysis using compusyn software. treatment of canine tcc cell lines with carboplatin or gemcitabine decreased cell proliferation, induced cell cycle arrest, and apoptosis. when tcc cell lines were treated with gemcitabine and carboplatin in combination at a therapeutically relevant concentration (gemcitabine o um, carboplatin o um), a significant decrease in cell proliferation was observed compared to gemcitabine or carboplatin alone, and the drug combination was synergistic in of cell lines, and additive in the remaining lines. gemcitabine exhibits biologic activity against canine tcc cell lines and carboplatin combined with gemcitabine exhibits synergistic activity at biologically relevant concentrations. our results support further evaluation of these drugs in dogs with tcc to determine the clinical efficacy of this combination. metronomic chemotherapy has been shown in murine models and humans to improve tumor control by inhibiting tumor angiogenesis and suppressing regulatory t cells (treg). treg are a subset of t lymphocytes demonstrated to be increased in humans and dogs with cancer and are thought to suppress cellular immune responses against tumors. the purpose of this study was to determine whether metronomic cyclophosphamide therapy depletes treg and/or exhibits antiangiogenic activity in dogs with soft tissue sarcoma. client owned dogs with histologically confirmed grade i or ii soft tissue sarcoma were administered cyclophosphamide at . mg/m or mg/m orally once daily for days. whole blood and tumor biopsies were obtained on days , , and . flow cytometric analysis of blood was performed to assess changes in t lymphocyte subsets, including cd and cd cells as well as cd foxp treg. tumor microvessel density (mvd) was assessed by performing immunohistochemistry for cd . five dogs were enrolled in the . mg/m /day dose cohort and six dogs were enrolled in the . mg/m /day dose cohort. in patients that received cyclophosphamide at . mg/m /day, the mean number of treg decreased from day to but there was no change in the mean percentage of treg or mvd. for patients that received . mg/m /day, both the mean number and percent of treg as well as mvd decreased over the day time period. cyclophosphamide at . mg/m /day or greater selectively depletes treg and inhibits angiogenesis in dogs with soft tissue sarcoma. arsenic trioxide (ato) is used to treat leukemias, multiple myeloma, and relapsed lymphoid malignancies in humans; its use has not been explored in veterinary oncology. prior therapy with glucocorticoids decreases likelihood and duration of remission for dogs with lymphoma treated with chemotherapy. we hypothesized that ato will re-sensitize glucocorticoid-resistant canine lymphoma cells to glucocorticoid-induced apoptotic death. the osw canine lymphoma cell line was cultured with um dexamethasone. remaining viable dividing cells were considered resistant. resistant cells were exposed to . um and . um of ato without dexamethasone, after which cells were washed and re-exposed to um dexamethasone. after , and hours of dexamethasone exposure, cells were counted using trypan blue stain. apoptosis was assessed by tunel assays on cytospin preparations collected at , , and hours from ato-exposed and control groups. statistical analysis was performed using way anova and tukey's test. the proportion of dead cells increased over time in both . um and . um ato exposed groups. the proportion of dead cells was greater for . um ato (p o . ) and . um (p o . ) groups compared to control. apoptosis increased with increasing ato concentration and duration of dexamethasone exposure compared to control. these results support the effectiveness of ato at re-sensitizing glucocorticoid-resistant canine lymphoma cells to apoptotic death following re-exposure to glucocorticoids. ongoing gene expression studies aim to elucidate this mechanism. additional studies to determine if this effect is seen with other chemotherapeutic agents are warranted. lymphoma is the most common hematopoietic tumor of dogs. protein disturbances may be associated with this disease including monoclonal gammopathies in a low percentage of cases. serum protein electrophoresis (spe) is routinely used to aid diagnosis of various canine diseases including lymphoma when total protein concentration is elevated. the purpose of this study was to compare spe changes in lymphoma patients without elevated total proteins with a population of healthy dogs. agarose gel electrophoresis was performed on residual serum from healthy control dogs and untreated dogs with multicentric lymphoma (stage iii -v) after measuring total protein (tp) using the biuret method. densitometric traces of the protein bands were obtained using computer software (totallab ) and the albumin, alpha- , alpha- , beta- and gamma globulin subfractions were identified by visual inspection. the total protein concentration, the number of subfractions and the relative and absolute protein subfraction concentrations were then compared statistically between the two populations. in lymphoma dogs, tp, absolute albumin, beta- and gamma globulin concentrations and both relative and absolute concentrations of the alpha- globulins were significantly lower however relative and absolute alpha- globulin concentrations were significantly elevated. no monoclonal gammopathies were identified in any of the dogs and not every patient with lymphoma had the above changes in their electrophoretogram. this study has demonstrated that significant changes occur in the albumin and globulin fractions of canine lymphoma patients despite no obvious increase in tp. further investigation is required to identify the proteins responsible for these changes. it is well known that immunophenotype has a prognostic value for the outcome of canine lymphoma, with t-cell lymphomas having a worse prognosis than b-cell lymphomas. the recent advent of flowcytometric techniques allowed easy detection of many different markers on lymphoma cells and therefore, not only distinguish between t and b cells, but also estimate possible aberration on immunophenotype. in human oncology, although some controversy persists, it seems that non-hodgkins lymphoma and acute leukemia carrying aberrations have a worse prognosis. the aim of this study was to evaluate the role of immunophenotype aberration in canine high-grade lymphoma considering outcome and time span to achieve complete response under chemotherapy. samples of bone marrow, blood and lymph node suspensions from twentythree dogs were evaluated with flow-cytometry. eleven dogs had aberrant expression of neoplastic lymphocytes and twelve were non-aberrant. the most common aberrations found were: positivity to cd , biphenotypes, double expression of tantigens (cd , cd ), diminished expression of cd . all dogs were treated with a chop-based protocol. there was a significant difference for the time to achieve response to chemotherapy (partial or complete). / non aberrant lymphomas went into cr or pr after the first treatment (l-asparaginase), while aberrant lympho-mas needed more than treatment to reach cr or pr. there was a trend for a prolonged disease free interval with non-aberrant versus aberrant, although it was not statistically significant. aberration of immunophenotype may be a prognostic factor for canine lymphomas, but further studies with larger groups are needed. class ii major histocompatibility expression is a significant and independent predictor of prognosis in human b cell lymphoma. low class ii mhc is consistently associated with poorer outcome. the mechanism underlying this relationship is not clear, but one hypothesis is that high class ii mhc allows for better antigen presentation and tumor-specific immune responses. in the this study, we investigated whether that class ii mhc expression in canine b cell lymphoma was associated with remission and survival times. a total of patients were categorized by level of class ii mhc,expression of cd and cell size for on neoplastic b cells. multivariable cox-proportional hazard analysis was used investigate this research question using a randomly selected subset of the data, and the predictive ability of this model was validated on the remaining / of patient data. results suggested that low class ii mhc expression was associated with decreased times to relapse and death as is seen in human b cell lymphoma, and that large neoplastic cells were associated with decreased survival time. cd expression was not associated with patient outcomes. these findings have implications for the use of dogs to model human lymphomas, for the study of tumor vaccines, and for prediction of mortality in dogs with b cell lymphoma with a high level of specificity. one of the reasons for the failure of canine lymphoma treatment is related to the resistance of tumor cells against chemotherapy drugs. the major form of this resistance is provide by multidrug resistance abc transporters. abc transporters proteins comprise a large superfamily of transmembrane proteins, atp-dependent, that extrude a large variety of drugs from the cells. multidrug resistance phenotype in cancer cells is associated with overexpression of these transmembrane proteins. abcg , also known as bcrp, is a residue half-transporter protein that protect hematopoetic stem cells against toxic compounds. the aim of this study was to investigate the expression of bcrp (abcg ) in canine multicentric lymphoma. samples were collected by fine needle aspiration of an enlarged lymph nodes, from dogs with multicentric lymphoma (stage iii to v) at diagnosis, and normal lymph nodes (control). dogs that were previously treated with prednisone or chemotherapy were excluded from the study. quantitative rt-pcr was used to measure the mrna expression level of bcrp and flnb expression as a endogenous reference canine gene. a widely range expression value for abcg expression was found for canine multicentric lymphoma. high gene expression was observed in % ( / ) canine lymphoma, but % of dogs had a lower expression when compared with normal lymph node. gene expression was not associated with clinical staging, complete or partial remission, relapse and survival time. in conclusion, abcg was expressed in canine lymph node and canine multicentric lymphoma at the diagnosis, and it was not correlated with clinical response. osteosarcoma (osa), the most frequent primary malignant bone tumor of dogs, is both locally aggressive and highly metastatic. prognostic factors for canine osa include tumor location, distant metastatic disease, and serum alkaline phosphatase (alp) concentration. an increased serum alp concentration is associated with poor prognosis; however the mechanisms underlying this phenomenon are currently unclear. during normal bone development alp may be used as a marker for osteoblasts. additionally, alp is a downstream target of activated canonical wnt/b-catenin signaling. therefore, we hypothesized that increased serum alp would be associated with increased expression of b-catenin in canine osa. the goals of this study were: ( ) characterize and compare cellular alp expression in osa tissue from patients with normal and high serum alp; and ( ) assess b-catenin expression in those same patient populations. we used frozen osa samples collected from patients with either high alp (n ) or normal alp (n ). total rna was isolated from the frozen tissue, converted to cdna, and analyzed using quantitative reverse-transcriptase polymerase chain reaction (qrt-pcr) with either target gene alp (aim ), or target gene b-catenin (aim ). additionally, b-catenin expression was analyzed by western blot. qpcr data for bcatenin and alp expression were normalized to s, and relative expression was calculated by the ddct method. the relative expression of cellular alp was higher in high serum alp samples compared to normal serum alp samples: . ae . (mean relative expression ae standard deviation; p o . ). further, the relative expression of b-catenin was also increased; b-catenin expression of high serum alp samples relative to low serum alp samples was . ae . (p o . ), which is also seen by western blot. this study begins to clarify the mechanism behind high serum alp in canine osa, and suggests the wnt signaling pathway may be active in this population of patients. further work will focus on elucidating the role active wnt signaling plays in the biology of osa. in the future, serum alp status of osa patients may help identify patients that would benefit from therapies targeting this pathway. accurate assessment of abdominal lymph node status is of vital importance for appropriate treatment planning and determining prognosis in dogs with apocrine gland adenocarcinoma of the anal sac (agaas). pretreatment knowledge of lymph node status is helpful for determining prognosis and planning the optimal extent of lymphadenectomy. in addition, pretreatment knowledge of lymph node status may help in selecting patients who might benefit from adjuvant chemotherapy and radiation therapy. abdominal ultrasound is currently the most commonly employed test to screen for abdominal lymphadenopathy in dogs with agaas. imaging studies in people indicate that magnetic resonance imaging ( to determine and compare the plasma concentration of cyclophosphamide and its metabolite -ohcp, within the plasma of lymphoma bearing dogs being treated with either oral or intravenous cyclophosphamide. in this prospective study, patients were randomly assigned to either receive oral or intravenous cyclophosphamide, at a dose of mg/m . based on a priori power calculation eight patients per treatment group were enrolled. plasma was obtained at times , , , minutes, and then at , , , , hours post administration for evaluation of -ohcp concentrations by liquid chromatography-dual mass spectrometry (lc/ms/ms). average values were obtained for both cyclophosphamide and -ohcp concentrations within the plasma of both groups. the following values were obtained, half life (hl), time to maximum concentration (tmax), maximum concentration (cmax), and area under the curve (auc). the mann-whitney statistical test was used to compare the groups. the auc for cyclophosphamide was statistically significant (p o . ) when compared between the two groups. the auc for -ohcp was not statistically significant between the groups. the difference between cmax for cyclophosphamide and -ohcp was statistically significantly (p o . ) between the groups. although the auc for cyclophosphamide was statistically significant between the two groups, the auc for the active metabolite -ohcp was not different when administered intravenously or orally. thus drug exposure to the active metabolite of cyclophosphamide is the same when administered intravenously or orally. previously the percentage of successful intraosseous (io) catheter insertions, insertion times, and ''ease of use'' scores using the ez-io g power driver by a wide spectrum of novice participants in feline cadavers were evaluated. novice users' mean io catheter insertion time using the ez-io g driver was also compared to the mean iv catheter insertion time in normovolemic feline and canine patients presented to the western college of veterinary medicine (wcvm) small animal hospital. novice users included wcvm personnel ( technicians, veterinary students, interns, residents, clinicians). after watching a -minute ez-io g training video, each participant inserted io catheters using the ez-io g driver. site (proximal humerus or trochanteric fossa of the femur) and side of cat (right or left) were randomized for each attempt for each participant. a gauge x mm long needle and a gauge x mm needle were used for io catheter insertion in the humerus and femur, respectively. participants then graded the ''ease of use'' of the ez-io g device on a visual analog scale (vas) that was converted to a -point scale. twenty-six iv catheter insertions in normovolemic feline and canine patients performed by wcvm small animal hospital personnel ( technicians, veterinary students, intern, resident, clinicians) were then timed and compared to the mean io catheter insertion time in feline cadavers by study participants using the ez-io g device. the io catheter was inserted correctly on every attempt by % ( / ) of participants. no difference was found between participant groups for mean io catheter placement confirmation percentage (p . ). percentage of io catheter ''slippage off the bone'' at the time of placement did not vary across participant groups (p . ). mean io catheter insertion times were all less than seconds and did not differ significantly as a function of attempt number (p . ) or as a function of participant group (p . ). participants rated the ez-io g 's ''ease of use'' favorably and subjective scores did not differ across participant groups with varying levels of clinical experience (p . ). compared to the mean insertion time for iv catheterization ( sec), mean io catheter insertion by participants using the ez-io g ( sec) was significantly faster (p . ). regardless of their level of clinical experience, participants rated the ez-io g device favorably in terms of its ''ease of use'' and their willingness to use the device in the future. regardless of their level of clinical experience, study participants successfully placed io catheters using the ez-io g device and did so significantly faster than the reported iv catheter insertion time in normovolemic feline and canine patients in the wcvm small animal hospital. intraosseous catheterization using the ez-io g has the potential to provide very rapid vascular access and is a skill that can be easily learned. previously presented at the western college of veterinary medicine undergraduate poster competition. multicavitary effusion is a common cause of presentation for dogs to emergency medical centers. the goal of this study was to identify common underlying causes of multicavitary effusion as well as determine their relative importance. a retrospective analysis of cases of multicavitary effusion admitted to the icu of a tertiary referral center (ontario veterinary college) from to was performed. twenty-three different breeds, with golden and labrador retrievers ( . % and %, respectively) being most commonly seen, were included in the study. ages ranged from to years with a median age of years and a mean of . years. . % of cases were males ( / cases). most common presenting signs included lethargy ( . %), anorexia ( . %), vomiting ( %) and dyspnea ( %). cavitary effusion was detected by either ultrasonography (pericardial, pleural or abdominal) or radiographs (pleural). bicavitary effusion was present in cases ( . %) whereas cases ( . %) had tricavitary effusion. neoplasia was found to be the most common underlying cause overall ( . %), with hemangiosarcoma being the leading type ( . % of neoplasia cases), followed by congestive heart failure ( . %), gastrointestinal lymphangectasia ( . %), peritonitis/pancreatitis ( . %), cirrhotic liver disease ( . %) and acute renal failure ( . %). in cases ( . %), no underlying cause could be found. of these, ( . % of all cases) were diagnosed as having idiopathic pericardial effusion. taken together, these findings suggest a strong association between multicavitary effusion and diseases carrying a guarded prognosis in dogs. infection control practices in veterinary clinics and hospitals are becoming increasingly important, with rising client expectations, growing concern about the spread of antimicrobial-resistant pathogens, and the potential for zoonotic transmission of disease. surgical patients are at increased risk of developing infections, and can serve as sources of these pathogens for other animals and people with whom they have contact within and outside the clinic. taking all reasonable precautions to reduce the risk of surgical site infections, beginning with preoperative preparation of the surgeon and patient, is therefore an important part of any infection control program. while guidelines are available for preoperative preparation procedures, there has been no objective investigation of compliance with these guidelines in veterinary practices. the objectives of this pilot study were to describe a range of preoperative hand scrub and surgical site preparation practices in veterinary clinics, and to determine if there were any areas that consistently require improvement. observation of preparation practices was performed in each of ten clinics over - days using - small wireless surveillance cameras. data was coded for surgical patients, and surgeons performing a total of hand scrubs. patient hair removal was most commonly performed after induction of the animal ( / , %) and using clippers ( / , %) . steps in surgical site aseptic preparation ranged from - . contact time with soap ranged from - s (mean s, median s), and with alcohol from - s (mean s, median . s). application of alcohol or antiseptic using a ''cleanest to dirtiest'' pattern was infrequent ( / ( %) and / ( %), respectively). potential contamination of the surgical site occurred most frequently when the animal was moved to the surgery table after initial preparation ( / , %). preoperative alcohol hand rub was used in / facilities, but soap and water hand scrub was still more commonly used even at these clinics. proximal-to-distal scrubbing was noted in / ( %) of soap and water scrubs. contact time during surgeon hand preparation ranged from - s (mean s, median s) for soap and water and from - s (mean s, median s) for alcohol-based hand rub. approximately % of the variation in contact time was due to inter-surgeon variation. no significant changes in practices were identified over the course of the observation period. some preoperative preparation practices were fairly consistent between clinics in this study, while others varied considerably. contact times with preparatory solutions were often far shorter than recommended, and there was a high frequency of non-sterile contact with the surgical site during movement of patients to the surgical suite. the camera system used to perform this study did not have a significant time-dependent effect on the behavior of participants, and could be useful for performing similar field-based observational studies in the future. this prospective randomized study compared the percentage of successful intraosseous (io) catheter insertions, insertion times, and ''ease of use'' scores using the ez-io g power driver to manual io catheterization in feline cadavers. the io catheter insertion time in cadavers using the ez-io g device was also compared to iv catheter insertion time in normovolemic feline and canine patients. after a purposely limited training period, a preclinical veterinary student was timed and video-recorded as she performed io catheter placements in feline cadavers ( io insertions by placing an illinois needle manually and io insertions using the ez-io g ). order of technique (manual or ez-io g ), site of io placement (proximal humerus or trochanteric fossa of the femur), and side of cat (right or left) were randomized for each attempt. when using the ez-io g , a gauge x mm long needle and a gauge x mm needle were used for io catheter insertion in the humerus and femur, respectively. after each attempt, the student graded the ''ease of use'' of each technique on a visual analog scale (vas) that was converted to a -point scale. twenty-six iv catheter insertions in normovolemic feline and canine patients performed by western college of veterinary medicine (wcvm) small animal hospital personnel ( technicians, veterinary students, intern, resident, clinicians) were then timed and compared to the student's mean io catheter insertion time using the ez-io g .median io catheter insertion times for the techniques were significantly different (manual io technique sec; ez-io g sec) (p o . ); the manual method took seconds longer ( % confidence interval of to seconds) than the ez-io g method. insertion time was more variable for the manual technique than for the ez-io g . percentage of catheter ''slippage off the bone'' and extravasation around the inserted catheter were significantly higher for placement of the manual io catheter compared with placement of the ez-io g catheter (p o . ). student's subjective ratings were more favorable and more consistent for the ez-io g technique compared to the manual technique for io catheter insertion. compared to the mean insertion time for iv catheterization in the wcvm small animal hospital, io catheter insertion by the student using the ez-io g was significantly faster (iv catheter sec; ez-io g io catheter sec) (p o . ). intraosseous catheter insertion using the ez-io g can be said to be significantly faster, less traumatic, more user-friendly, and as effective as io catheter placement using the manual technique. vascular access via io catheter insertion using the ez-io g device may be suggested to be faster than iv catheter insertion. previously presented at the western college of veterinary medicine undergraduate poster competition. computed tomography (ct) has been widely investigated and applied as a means for non-invasive quantitative bone mineral determination in human medicine. the aim of this study was to assess age-related changes and anatomic variation in bone mineral density (bmd) using quantitative ct in normal cats. seventeen normal cats were included in this study and divided into the following age groups: o year (n ); - years (n ); and years (n ). a computed tomographic scan of each vertebra from the th thoracic to the th lumbar spine, and the pelvis, was performed with a bone-density phantom ( , , and mg/cm , calcium hydroxyapatite, cirs phantom s ). on the central transverse section, the elliptical region of interest (roi) was drawn to measure the mean hounsfield unit value. those values were converted to equivalent bmd by use of the bone-density phantom and linear regression analysis (r . ). the mean bmd value of the thoracic vertebrae ( . ae . mg/cm ) was significantly higher than of the lumbar vertebrae ( ae . mg/cm ). the maximum bmd occurred at the t , t , and l levels in all age groups. there was a statistically significant difference in the mean bmd value among the age groups at the t (p o . ), t (p o . ), and l levels (p . ), respectively. in addition, there was no significant difference between the mean bmd value of the left and right iliac bodies ( . ae . mg/cm and . ae . mg/cm , respectively). the present study suggests that age-related changes and anatomic variation in bmd values should be considered when assessing bmd using quantitative ct in cats with bone disorders. dynamic contrast-enhanced computed tomography (dce-ct) is a rapid and widely available method of cerebral perfusion imaging. however, there is no established reference value of cerebral blood flow (cbf) measured by dce-ct according to a dog's age. the purpose of this study was to identify the correlation between regional cbf and aging in clinically normal dogs using dce-ct. fourteen dogs with no evidence of hemodynamic disorders and central nervous system dysfunction were included in this study. dogs were assigned to the following age groups: o year (group ); - years (group ); and o years (group ). dce-ct scans were performed at the level of the third ventricle and mesencephalic aqueduct. cbf in the gray and white matter was calculated using stroketool-ct s software. the overall mean ae standard deviation quantitative estimate for regional cbf in clinically normal dogs was . ae . ml/min/ g, . ae . ml/min/ g, and . ae . ml/min/ g in groups , , and , respectively. there was no significant regional cbf difference between the right and left sides of the brain in each group. also, a statistically significant difference in the regional cbf was observed between groups and (p o . ). thus, aging affects the regional cbf in normal dogs and the values should be considered assessing the results of dce-ct. according to several clinical behavior guidelines, ''toileting'' type inappropriate urination (i.e. large amounts of urine deposited in horizontal surfaces) can arise in cats suffering from a medical problem (typically lower urinary tract disease). by contrast, ''spraying'' type behaviour (i.e. possibly smaller amounts of urine deposited on vertical areas) is more typically associated with anxiety brought about by a threat to local resources, arising from either a change in the physical environment or threat to these resources from another cat. however, there is some evidence that ''sprayers'' may also be presented with a medical problem, which might be linked to the disease (e.g. painful voiding associated with crystalluria may lead to a standing posture being adopted and small amounts eliminated at a given time). this might be associated with an apprehensive state or simply a co-morbid state. as part of a larger research project aimed at investigating behavioral and physical aspects of cats presented with inappropriate urination, owners of ''spraying'' and ''toileting'' cats with appropriate control subjects from the same households were recruited throughout local media coverage and the internet. the case-control dyads were brought by the owners to the veterinary hospital of the university of sa˜o paulo, at the same time, for a medical work-up (i.e. physical examination, complete blood count, biochemical profile, urinalysis, urine culture and abdominal ultrasound). no significant differences between the ''sprayers'' and ''toileters'' regarding the occurrence of medical problems were found. both groups had a similar proportion of cats affected by medical illnesses (sprayers: . %, toileters: . %; chi , p . ), directly or indirectly relating to the urinary system (e.g. diabetes, chronic kidney disease). in both groups, control cats also had a relatively high occurrence of medical concerns ( . % and . %, respectively for each control group). these results emphasize the importance of careful medical evaluation of cats presented for a urinary housesoiling problem. the relatively high prevalence of medical concerns among apparently healthy cats in multi-cat households may have arisen, at least in part, as a result of an inability/failure of owners to monitor individuals, thus allowing some early signs to pass unnoticed. the way in which medical and behavioral elements are linked (if at all) remain unknown but deserve further investigation. considered as a semi-social species, domestic cats appear to be highly sensitive to the effects of social stress, especially when living in high density populations. cats are capable of adapting to live ingroup; nonetheless, they do not appreciate living in close proximity with others as result of an environment lacking of great opportunities of escaping and hiding. this study aimed at testing the following hypotheses: (a) owners' perceived quality of life affects cats' global levels of stress; (b) cats' global levels of stress are influenced by cats' personality; (c) cats' living style (single housing versus large group housing) does affect stress levels in cats. to our knowledge, this is the first study investigating stress levels of domestic owned cats, under natural conditions, throughout measurement of faecal glucocorticoids metabolites concentration, and taking into consideration cat personality, cat living style and owner's subjective life quality. in this study, adrenocortical activity, as a valuable physiological indicator of emotional stress, was evaluated throughout the measurement of faecal glucocorticoids metabolites in fourteen single and sixteen in-group housed cats. cat personality as well as owners life quality was evaluated by self reported questionnaires given to the owners to answer. significant differences in mean glucocorticoids metabolites concentrations (mgcm) between the two populations (i.e. single versus in-group cats) were not detected (random effect model, p . ). however, when mgcm were taken as a function of cat personality, there were differences regarding single catstimid cats showed higher levels in comparison to easy-going (random effect model, p . ) and bossy (random effect model, p . ) cats. as to owner subjective life quality, a direct association between the scores given by the owners to the social dimension and mgcm was found for single cats only (i.e. the better the owner felt itself social wise the higher the mgcm of the cat; random effect model, p . ). social stratification may compensate the stress resulting from spatial restriction in large in-group living cats. other underexplored factors such as feline personality and owner life style seem to play an equally important role in domestic cats' day to day levels of stress, especially in the cats kept as single pets. in dogs, raas activation is a major feature of congestive heart failure (chf). benazepril (fortekor s ) is a potent ace inhibitor with well-documented effectiveness in canine chf. although ace activity (ace a ) has been used in preclinical studies as a surrogate marker of efficacy, some authors have reported a poor correlation between plasma ace a and changes in angiotensin ii (aii) or aldosterone (al). the purpose of this study was to investigate the effect of benazepril on canine plasma renin activity/concentration (pra/prc), angiotensin i (ai), aii, al, and fractional excretion of potassium (ufek), sodium (ufena) and aldosterone (ufeal). sixteen beagle dogs were fed a low-sodium diet and dosed with placebo or benazepril tablets ( mg po, q h) for days. blood and urine samples were collected on day (d ) and day (d ) over -hour periods. data were analyzed by repeated measures anova of baseline corrected values, and anova of auc hours . compared with placebo, benazepril induced a significant increase in pra and ai at d (p-value [pra] : . , p-value [ai] : . ) and d (p-value [pra] : . , p-value [ai] o . ). no differences in prc were noticed. based on auc hours, aii levels were % lower in the benazepril group at d (p-value [aii] : . ). ufeal and al decreased by up to % and % at d and d , respectively, though differences did not reach statistical significance. benazepril markedly influences raas dynamics in dogs. decreased exposure to aii and al are likely to be the key events required to counteract pathological remodeling of the heart in chf. this study compared two intravenous anesthetic agents, alfaxalone (alf) (alfaxan s , jurox pty. ltd.) and propofol (ppf) (rapinovet s , schering plough animal health) and their effects on spontaneous ventilation after induction of anesthesia in dogs at various doses. this randomized, crossover, dose-escalation study used six dogs in weight and gender-matched pairs ( m- f). for each drug, each dog was dosed incrementally at , , , and times the labeled anesthetic induction dose rate (alf mg/kg, ppf . mg/kg) or until a dose was reached that rendered the dog apneic. a minimum of three days was allowed between doses. for each dose administration, the entire calculated dose was delivered constantly over min. the primary variable was apnea, defined as an absence of spontaneous ventilation for minute. apneic dogs were manually ventilated with oxygen until they resumed adequate spontaneous ventilation. once the apneic dose was determined for an individual dog for one drug, the dog began incremental doses with the alternate drug. for each anesthetic episode times were recorded from completion of induction dose to; removal of endotracheal tube, dog lifting head, dog attaining sternal recumbency and dog standing. pulse rate, respiratory rate, spo and etco were each measured every min. within-dog comparisons were made using the paired student's t-test. for both alf and ppf all dogs respired voluntarily at the labeled ( x) dose. for ppf at and x doses, and dogs respired voluntarily respectively. for alf at , and x doses, all , and dog respired voluntarily respectively. for all six dogs to become apneic required x dose of ppf and x dose of alf. the mean no observable adverse effect dose (noael) expressed as a multiple of the labeled dose was higher for alf ( . x) than for ppf ( . x) (p . ). there were no significant differences between times to extubation, head lift or attaining sternal recumbency after alf and ppf at , and x doses. at the x dose, dogs took longer to stand after alf ( . ae . min) than ppf ( . ae . min). we concluded that based on anaesthetic duration, the manufacturer's labeled dose rates of mg/kg for alf and . mg/kg for ppf were equivalent. however, based on the dose escalation, the number of dogs becoming apneic at each dose-multiple is consistent with ppf having a narrower safety margin, i.e., ppf caused more respiratory depression than alf. parenteral levetiracetam (lev) has been shown to rapidly attain therapeutic levels in dogs when given iv or im, and has been used offlabel for the treatment of seizure emergencies. the purpose of this study was to determine the safety and pharmacokinetics of subcutaneously administered levetiracetam in healthy dogs. potential application of these results would be use of sq lev instead of or in addition to rectal diazepam for the treatment of cluster seizures at home. lev was administered sq between the shoulder blades to healthy, purpose-bred hound dogs, at a dose of mg/kg (undiluted). blood samples were collected at , and minutes after lev administration via jugular venipuncture. plasma lev concentrations were measured by high pressure liquid chromatography. none of the dogs became sedated, nor was there pain evident on palpation of the injection site. mean (standard deviation) lev concentration was . ( . ), . ( . ) and . ( . ) mg/ml at , and minutes, respectively. administration of sq lev was well tolerated, and exceeded the suggested therapeutic range ( - mg/ml) within minutes of administration, and remained above the range for at least hours. these data indicate that sq lev administration may be an alternative for the at-home treatment of cluster seizures in dogs, and prospective studies in epileptic dogs are warranted. the purpose of this study was to assess the effects of cyp inhibitors (ketoconazole, chloramphenicol, fluoxetine, trimethoprim, cimetidine, and medetomidine) in varying combinations on the bioavailability of oral methadone in healthy greyhound dogs. the iacuc approved this study. cyp inhibitors were administered po for hours prior to methadone administration. methadone hydrochloride was administered po at a targeted dose of mg/kg. blood was obtained for the determination of methadone plasma concentrations by mass spectrometry. the area under the curve (auc) of methadone for each treatment group was compared statistically to the auc of methadone administered without inhibitors using the mann-whitney rank sum test. significant increases (p o . ) in the methadone auc occurred in all treatment groups which included chloramphenicol, including chloramphenicol as the only inhibitor. the magnitude of increase was at least fold. mean concentrations of methadone exceeded ng/ml for at least hours in all groups administered concurrent chloramphenicol. no significant increases in the auc occurred in any of the groups which did not include chloramphenicol. in conclusion, chloramphenicol significantly inhibits the metabolism of methadone in greyhound dogs. as a result, the oral bioavailability of methadone is significantly increased and plasma concentrations are achieved that are reported to be effective in humans for - hours after a single oral administration. doxycycline hyclate is used frequently in small animals, horses and exotic animals for treatment of a wide variety of infections. because doxycycline hyclate tablets may not be suitable for oral administration in some animals, particularly horses and cats, it has been compounded into liquid suspensions. the commercially available doxycycline calcium mg/ml oral suspension, vibramycin s (pfizer) is not suitable for use in animals due to its low concentration and flavoring that animals find unpalatable. because of the known inherent instability of doxycline in aqueous vehicles under storage, this study was conducted to determine the potency of two formulations stored in dark and light conditions. a high pressure liquid chromatography (hplc) assay with uv absorption at nm was developed for analyzing doxycycline in formulations, in comparison to a reference standard from the united states pharmacopeia (usp). doxycycline hyclate mg tablets were first tested for potency. the tablets were then crushed and mixed with a pharmaceutical vehicle to make two concentrations: . mg/ml and . mg/ml. the vehicle used was a : mixture of a vehicle for oral solution (ora-sweet, usp-nf) and vehicle for oral suspension (ora-plus, usp-nf). the suspensions were prepared in replicates of . each replicate was divided, with one aliquot stored at room temperature in lighted conditions, and the other aliquot stored at room temperature in the dark. doxycycline was extracted from the formulations and measured by hplc at day , , , , , , and . each replicate was tested and the potency reported as the percent doxycycline relative to the usp reference standard. on day , , , and , the potency of each formulation was within - % of the reference standard (range . - %). this value is within the accepted range cited in usp o on pharmaceutical compounding-non-sterile preparations. however, starting at day , the potency declined dramatically and remained low for the tests performed on day and . the potency on day , , and was below % of the reference standard (range - %). there was also a noticeable change in the quality of the formulation starting on day , and a change in the color of the formulation to a dark brown. these results indicate that when doxycycline hyclate tablets are compounded as a suspension in an aqueous vehicle as described in this study, at . and . mg/ml under the storage conditions used in this study, potency of the formulation cannot be assured beyond days. we recommend a beyond-use-day (bud) of days for formulations prepared and stored at room temperature in light or dark conditions. therapeutic options for multidrug resistance (mdr) escherichia coli urinary tract infections (uti) are limited. fosfomycin (fos) tromethamine is an oral, broad-spectrum, cell-wall active, bactericidal drug approved for treatment of uncomplicated uti in humans. the purpose of this study was to determine time dependency of fos and the disposition of fos tromethamine in dogs. using a randomized, double crossover design, client-owned dogs received fos sodium iv ( mg/kg) and fos tromethamine (po, mg/kg) either with (n ) or without food (n ). serum and urine were collected for hr; fos was quantitated with a bioassay (atcc e. coli , serum or atcc proteus vulgaris , urine). in-vitro killing curves (cell counts through hours) were performed at (control), . , , , , and x mic for mdr e. coli canine fos susceptible (e-test s ) uropathogens. killing curves indicated fos to be time dependent. after iv administration, clearance (mlÃkg/hr), volume of distribution (l/kg), elimination half-life (hl; hr) and mean residence time (mrt, hr) were (mean ae sd): ae , . ae . , . ae . , . ae . and . ae . , respectively. for po, c max , hl and mrt were ae , . ae . and . ae . , respectively. serum fos exceeded the mic reported for multidrug resistant (mdr) e. coli ( . mg/ml) for hr (iv; . mg/ml) and hr (po, mg/ml diminazene is an aromatic diamidine, anti-protozoal drug that has shown promise in a small number of cases of cytauxzoonosis. in a noncontrolled case series, of cats with clinical cytauxzoonosis given mg/ kg of diminazene aceturate survived infection. dosage frequency was two intramuscular injections given one week apart. commercial formulations contain the diminazene diaceturate salt. the active base is diminazene with the salt consisting of two aceturate molecules. currently there is no data available on the pharmacokinetics of either diminazene compound in cats. the objective of this study was to determine the pharmacokinetics of diminazene diaceturate in healthy cats. four purpose bred cats with normal physical examination, cbc, chemistry and urinalysis were used. a powdered commercial drug formulation (veriben s , ceva sanet animale) was freshly reconstituted with sterile water to a concentration of mg/ml prior to administration and sterile filtered solution. heparinized blood samples were collected just before (hour ) or . , , , , , , , , , , , , and hours after intramuscular administration of mg/kg ( . mg/kg of diminazene base) diminazene diaceturate. the plasma was separated by centrifugation within minutes of collection and frozen (À c) until analysis. concentrations of diminazene were measured by hplc analysis using uv absorption and ion-pairing conditions. the pharmacokinetic profile was analyzed using a simple one-compartment model. in these cats, diminazene had a mean terminal half life (t / ) of . ( /- . ) hrs and mean peak plasma concentration (c max ) . ( /À . ) mg/ml. the mean residence time (mrt) of diminazene was . hrs ( /À . ). systemic clearance (cl/f) was . ( /À . ) l/kg/hr. the volume of distribution per fraction absorbed (vd/f) was . ( /À . ) l/kg. a single intramuscular dose of diminazene diaceturate was well tolerated by all cats. without knowing the concentration required to inhibit or kill cytauxzoon felis, it is not yet possible to make suggestions regarding optimum dosing schedules for this drug. additional toxicology data and studies to assess clinical efficacy for the treatment of cytauxzoonosis are indicated before routine clinical use can be considered. meloxicam has been shown to accumulate in areas of inflammation in both the rat and human. the objective of this study was to investigate the concentration of meloxicam in synovial fluid of inflamed joints versus that of non-inflamed joints in dogs. eight male dogs were treated with . mg/kg of meloxicam on day one and . mg/kg of meloxicam on day two. all treatments were administered orally. on day three reversible acute synovitis was induced in one stifle by aseptic, intra-articular administration of ml sodium urate crystal suspension ( mg/ml). in four dogs synovitis was induced in the l stifle and in four dogs the same procedure was used in the r stifle. in each dog the stifle without induction of synovitis served as the ''normal'' joint sample. a synovial fluid sample was collected from both the r and l stifle of each dog. sample collection occurred eight hours after administration of sodium urate and twenty four hours after the last administration of meloxicam. synovial meloxicam concentration was analysed using high performance liquid chromatography-mass spectrometry (hplc/ ms-ms). the concentration of meloxicam in the inflamed versus non-inflamed joint in each dog was compared using the paired t-test. the results indicate that meloxicam preferentially accumulates in inflamed joints in the dog as meloxicam concentrations are statistically significantly higher in inflamed joints than in non-inflamed joints. no national surveillance system exists for monitoring emergent resistance in companion animals. however, e. coli resistance is an increasing therapeutic and public health concern in these in dogs and cats. the purpose of this study was to describe current resistance patterns of canine and feline pathogenic e. coli throughout the united states and identify risk factors of antimicrobial resistance. isolates (n ) of clinical e. coli collected from dogs or cats from may through may located in different regions. susceptibility was determined to drugs ( drug classes) by broth microdilution methods. pharmacodyamaic statistics were described regionally. phenotypes were determined and type of resistance was based on the number of drug classes to which resistance was expressed: none (ndr), single (sdr) and multi (mdr). the majority of isolates were from urinary tract ( . %) and dogs ( . %). the proportion of resistance type for each drug was: ndr ( . %), sdr ( . %) and mdr ( . %). the proportion of mdr was greatest in the southwest ( . %) and least in the northwest ( . %) (p o . ). for all regions, the proportion of resistance was: cephalothin (cph, . %) amoxicillin-clavulanic acid (amx, . %), ampicillin (amp, . %), tricarcillin-clavulanic acid (tcx, . %), doxycyline (dxy, . %) cefoxitin (cfx, . %), cefpodoxime (cpx, . %), chloramphenicol (chp, . %), enrofloxacin (enr, . %) , ciprofloxacin (cif, . %), trimethoprim-sulfamethoxazole (tmx, . %), ceftazidime(cfz, . %), gentamicin (gtm, . %), cefotaxime (cft, . %) meropenem ( . %) (p o . ). the mic exceeded the resistant breakpoint for amp, amx, cpx, cph, cif, cfx, dxy and enr whereas mic did not surpass the susceptible breakpoint. beta-lactams ( . %) was the most and aminoglycosides the least ( . %) sdr. the drug class most frequently involved in mdr was beta-lactams ( . %) and least, gen ( . %). resistance differs regionally, being greatest in the southwest. cph is the most and meropenam is the drug least associated with resistance; these patterns are consistent with current drugs used by veterinarians. the fluoroquinolones (fqs) are common choices for treatment of e. coli urinary tract infections (utis) in animals and humans. nd generation drugs approved in animals include enrofloxacin (enr), marbofloxacin (mar), orbifloxacin (orb); human drugs include ciprofloxacin (cip). rd and th generation fq for humans include moxifloxacin (mox), gatifloxacin (gat) and ofloxacin, (ofl]), its lisoform levofloxacin (lev). for animals, pradofloxacin (pra) is approved for use in europe. the purpose of this study was to assess the in vitro activity of st (naladixic acid [nal] through th generation fqs (n ) toward dog or cat e.coli uropathogens (n ). isolates were subjected to susceptibility testing to drugs classes ( drugs). isolate phenotypes included no (ndr; n ), single (sdr; n ) or multidrug (to more than drug classes; mdr; n ) resistance (including enr resistant [enr r -mdr; n ] or enr susceptible (enr s -mdr, n ). the minimum inhibition concentrations (mics) were determined for each isolates using broth microdilution (e. coli atcc s served as a negative control). mic statistics were generated for each drug among phenotypes. the overall potency (mic ) for all enr susceptible isolates (ndr, sdr and enr s -mdr) was gat pra, mox, mar, lev, cip sar, orb, ofl enr nal. each e. coli isolate expressing ndr or sdr was susceptible to all fq. however, isolates expressing resistance to st or nd generation fq were also resistance to later generation drugs. glucocorticoids (gc) are standard therapy for allergic asthma but do not reverse the underlying type i hypersensitivity. allergenspecific immunotherapy (asit), a process of ''desensitization'', is potentially curative but requires identification of offending allergens. the purpose of this study was to determine if oral or inhaled gc administered at routinely used dosages would interfere with allergen identification. we hypothesized that oral but not inhaled gc would interfere with accurate identification of allergen-specific ige using skin and serum testing in experimentally asthmatic cats. asthma was induced in eighteen cats using bermuda grass allergen (bga). cats (n /group) were randomized to receive oral gc ( mg prednisolone q hr po), inhaled gc ( ug budesonide q hr) or placebo (gelatin capsule q hr po) for one month. intradermal skin testing (idst) and bga-specific ige amounts were measured prior to, during (weeks one and four) and every two weeks after treatment until both tests were positive. a paired t test was used to compare serum ige among groups pre-and post-treatment (p o . significant). idst reactivity was eliminated in / cats on oral gc, / on inhaled gc, and / placebo-treated cats. within two weeks after stopping treatment, idst was again positive in all cats. contrary to our hypothesis, serum ige reactivity to bga was not significantly diminished by any treatment. in conclusion, a two week withdrawal from gcs is adequate for idst identification of allergen but no withdrawal is required prior to serum ige testing to identify the sensitizing allergens. previously in people, increasing severity of asthma is associated with low serum concentrations of -hydroxyvitamin d ( -oh-d). -oh-d is thought to ameliorate lower airway inflammation primarily by decreasing the production of pro-inflammatory mediators, and by increasing the production of the anti-inflammatory cytokine il- . in people, serum -oh-d concentration is associated with sunlight exposure as well as dietary intake. cats do not rely on sunlight for vitamin d synthesis; all vitamin d comes from dietary intake. cats have a naturally occurring lower airway disease syndrome (lad) that shares many features with human asthma. the goal of this study was to evaluate serum -oh-d concentrations in cats with lad. cats with naturally developing lad were enrolled. criteria for a diagnosis of lad included a history of cough, wheeze or respiratory distress, radiographic evidence of a bronchial pattern and hyperinflation, negative heartworm antigen and antibody test, and a resolution of clinical signs in response to glucocorticoids. dietary history was obtained. -oh-d concentrations were determined on serum samples by a commercial laboratory. twelve cats with lad were enrolled. all cats ate commercial cat food. the median -oh-d concentration was nmol/l with a range of - nmol/l which is within the reported reference range of - nmol/l. in contrast to human asthma, lower airway disease in cats is not associated with low serum concentrations of -oh-d. interstitial lung diseases (ild) are uncommon in dogs, with the most commonly recognized ild idiopathic pulmonary fibrosis (''westie fibrosis''). in human medicine, ild represent a large umbrella of pulmonary diseases, with ipf only a subset. other, more treatable, ilds are also identified, and may respond to either the removal of a stimulus (hypersensitivity) or steroid therapy. the goal of this report is to describe the clinical course, including outcome, computed tomography and histopathology of dogs affected with an ild. the computed tomography (ct) log was reviewed for dogs that underwent thoracic ct scanning for evaluation of respiratory signs, and had changes consistent with ild as the primary abnormality, including the presence of diffuse disease in all lobes, and at least of the following: reticulation, ground glass opacity, consolidation, or traction bronchiectasis. survival time from ct date was calculated. the presence of moderate pulmonary hypertension [phtn] ( mmhg) as estimated by tricuspid regurgitant jet, was also reported and survival times were compared with a mann-whitney rank sum with p o . considered significant. thirteen dogs were identified. terriers and chihuahuas were the most commonly affected breeds. two dogs were adolescents, the remaining dogs ranged from - years, with a median of years. histopathology results (n ), including moderate to severe interstitial fibrosis ( ) alveolar proteinosis with fibrosis ( ), and interstitial eosinophilic pneumonia ( ). one had suspected cryptogenic organizing pneumonia and had a good response to glucocorticoids. eight dogs died of respiratory failure, with a median post ct survival time of days (range - ), two dogs died of non-pulmonary disease, dogs had severe lower respiratory infections as puppies with persistent respiratory signs, and both are still alive at years since diagnosis, terrier is alive at months and was lost to follow up. dogs had phtn, with a median survival of days ( - ), while the dogs without had a survival of days (range - ), [p . ]. interstitial lung disease in dogs is not just idiopathic pulmonary fibrosis. following respiratory infection, young dogs may develop an ild with a relatively indolent course and rare ild is steroid responsive. ct is useful to identify ild but further research correlated with echocardiography and histopathology is advised to use it to prognosticate. idiopathic pulmonary fibrosis (ipf) is an interstitial pulmonary disease, mainly described in west highland white terriers (whwt). identification of molecular pathways important in the pathogenesis of ipf would improve our understanding of this disease and may help identify therapeutic targets. the aim of the present study was to investigate gene expression in lungs of whwt with ipf using oligonucleotide microarray. total rna was extracted from post-mortem pulmonary samples from five whwt with ipf and five control dogs (ctrl) without pulmonary disease. the rna was pooled from each group (ipf and ctrl) and analysed using the canine specific affymetrix microarray technology. genes with a minimum of a two-fold difference in expression between the two groups were selected for further analysis. the most significant biological functions for these genes were identified using ingenuity pathways analysis. more than genes were identified as having greater than twofold difference in expression. the significant biological functions associated with these genes were related to cellular movement, cellular proliferation and apoptosis. most notable among these were genes encoding the leukocyte chemotactic proteins: ccl (fold change . ), ccl ( . ) and il ( . ); the proteins involved in fibroblast migration; and the matrix metalloproteinases (mmps) involved in matrix degradation: mmp (À . ), mmp (- . ), mmp (À . ). this study has identified genes which may be important in pathogenesis of ipf, e.g. proteins involved in leukocytes chemotaxis, fibroblast recruitment and activation, regulation of apoptosis, and extracellular-matrix turn-over. however, real-time quantitative rt-pcr studies are needed to confirm these results before any definitive conclusions can be drawn. idiopathic pulmonary fibrosis (ipf) is an interstitial disease, mainly described in west highland white terriers (whwt). defini-tive diagnosis ultimately relies on lung histopathology. identification of specific biomarkers would be very helpful. expression microarray is a powerful screening tool to study local gene expression in a disease state. the aim of the present study was to measure gene expression profiles in lungs of whwt with ipf to identify potential blood or bronchoalveolar lavage fluid (balf) biomarkers. total rna was extracted from post-mortem pulmonary samples from five whwt with histopathologically confirmed ipf and five control dogs (ctrl) without pulmonary disease. the rna was pooled from each group (ipf and ctrl) and analysed using the canine specific affymetrix microarray technology. ipa-biomarkers analysis (ingenuity system) was used to filter and prioritize biomarkers candidates using the three following criteria: a minimum of a two-fold difference in expression between ipf and ctrl; expression of the gene in lung tissue; possible detection of the protein in blood or in balf. fifty-four molecules met all the criteria. based on difference in expression, promising proteins included ccl (fold change . ), a -actinin ( . ), ccl ( . ), serum amyloid a ( . ), il ( . ), plunc (À . ), mmp (À . ). some are well-known biomarkers of ipf in humans either for diagnosis (mmp , il ) or prognosis (ccl ). these results provide novel potential biomarkers of canine ipf. measurement of these proteins in blood and balf of healthy dogs, dogs with ipf and with other respiratory diseases is needed to assess their use as biomarkers of canine ipf. heliox is a mixture of helium and oxygen that has been used therapeutically in human medicine for treatment of airway obstruction. helium's low density and other physical properties have been shown to reduce the work of breathing by limiting turbulence. the purpose of this study was, therefore, to evaluate respiratory parameters in response to inhaled heliox in dogs with meso-and brachycephalic conformation. eleven healthy dogs were recruited, five were mesocephalic and six were brachycephalic. flow-volume loops were collected using commercial software (buxcor) while breathing : helium: oxygen (heliox) and : nitrogen:oxygen (nitrox) in a randomized order via a low dead-space face mask. due to the intrinsic gas properties, gas flow rates and volumes were corrected in-vitro by a conversion factor for the effect of helium on the pneumotachograph. respiratory rate, tidal volume (ml), minute ventilation (l), inspiratory time (ti), expiratory time (te), peak inspiratory flow (pif) and peak expiratory flow (pef) were recorded while breathing heliox or nitrox. values were compared using a paired sample t-test, with p o . considered significant. all dogs cooperated with testing. there was no significant difference in respiratory rate, tidal volume, minute ventilation, inspiratory or expiratory times, or peak inspiratory flow. peak expiratory flow was significantly higher (p . ) while breathing heliox than when breathing nitrox in brachycephalics but not in mesocephalics (p . ). heliox is well-tolerated in healthy dogs and results in an increased expiratory flow rate in brachycephalic dogs. further investigation of heliox is warranted in dogs with airway obstruction. of this prospective multicentric study is to assess the effects that surgical correction has on the severity of clinical signs and levels of acute phase proteins (c-reactive protein [crp] , haptoglobin [hp]) and cardiac troponin i (ctni). thirty three brachycephalic dogs with boas were included and evaluated before and, approximately two months, after surgical correction. the most common components of boas found were elongated soft palate ( / ; %), stenotic nares ( / ; %) and everted laryngeal saccules ( / ; . %). staphylectomy was performed by means of two different surgical techniques: laser (n ) or electrical scalpel (n ). there were significant differences between dogs depending on the surgical technique used, with a higher reduction of respiratory signs (p o . ) and a better postsurgical improvement (p o . ) with the use of laser. the levels of crp, hp and ctni were categorized into normal or elevated. before surgical treatment three ( . %), six ( . %) and thirteen ( . %) dogs had elevated values of crp, hp and ctni, respectively. two months after surgical correction, five ( . %), eleven ( . %) and fourteen ( . %) dogs had elevated values of crp, hp and ctni, respectively. there were no statistical differences between values of crp and ctni before and after surgical correction but the levels of hp increased significantly after surgical treatment (p o . ), probably due to postsurgical treatment with corticosteroids. as previously suggested by others, there was a statistically significant reduction of respiratory and gastrointestinal signs in dogs with boas submitted to surgical correction (p o , ). according to the results obtained in the present study, the determination of crp, hp and ctni before and two months after surgical treatment do not have a prognostic value in dogs with boas. even though, near half of the dogs studied had elevated levels of ctni ( . %) that persisted after surgical treatment ( . %), suggesting some degree of myocardial damage is present. further studies are needed considering the influence of breed and age. to the authors' knowledge, this is the first description of crp, hp and ctni determination in dogs with boas. overweight and obesity are common conditions that lead to alterations in respiratory mechanics, airway resistance, pattern of breathing and gas exchange in humans. the objective of the present study was to investigate if there are significant differences on respiratory parameters and arterial gas analysis of obese and overweight cats, in conscious state and under general anesthesia. twenty nine adult cats were arranged in three groups: obese (n ), overweight (n ) and with ideal body score index (bsi) (n ). mean of bsi in the groups were: , (obese), , (overweight) and , (ideal bsi). cats did not had respiratory, cardiac or others systemic diseases. the respiratory parameters were evaluated with a ventilometer equipment coupled to facemasks in conscious cats and directly to the endotracheal tube in anesthetized cats under spontaneous respiration. the anesthesia was performed with propofol ( ae , ml/kg) and the cats were maintained in the same anesthetic plan. the three groups were compared by analysis of variance followed by tukey's test and conscious and anesthetized cats were compared by student's t test, with a % significance level. there were not observed differences on the respiratory parameters evaluated on ventilometry (tidal volume, expiratory and inspiratory times and peak pressures, respiratory rate and partial pressure of end tidal co (petco )) and on arterial gas parameters (pao e paco ) in the three groups. the pao of cats with ideal bsi was , ae , mmhg, although was not significantly different (p , ) from overweight ( , ae , mmhg) and obese cats ( , ae , mmhg). comparison of anesthetized to conscious cats, it was detected decreases in tidal volume, expiratory and inspiratory times and peak pressures and increase in petco in respiratory rate in the anesthetized cats. only petco , inspiratory time and respiratory rate in overweight cats did not differ in anesthetized cats. these results suggest that obesity and overweight did not result in impairment of respiratory function in cats and propofol induced respiratory depression. osteosarcoma (osa) is the most common bone tumor in dogs, however, little is known regarding the mechanisms underlying malignant transformation in these tumors. breeds such as rottweilers and greyhounds are at higher risk for developing osa, suggesting that heritable factors play a role in this disease. mirnas have tumor/tissue specific roles in regulating gene expression and dysregulated mirna expression is found frequently in cancer. we hypothesize that canine osa is characterized by a unique mirna expression profile(s) with dysregulation of some mirnas being associated with specific breeds. mirna expression profiling of primary osa tumors from greyhounds and rottweilers was performed using the nanostring technologies ncounter mirna expression assay kit, interrogating the mirna expression profile of human mirnas, of whose mature sequences are % conserved between human and dog. mirnas were differentially expressed in greyhound versus rottweiler tumors (p o . ), suggesting that breed-specific dysregulation of mirnas may contribute to the development and progression of spontaneous osa. hierarchical clustering revealed distinct mirna expression signatures in greyhound osa tumors as compared to rottweilers. based on these preliminary results, we are evaluating a larger cohort of osa tumor samples including greyhounds, rottweilers, golden retrievers, and a mixed population of other breeds. statistical analysis will be performed to determine the association of mirna transcript levels with specific breeds and overall outcome. characterization of mirna expression in canine osa will facilitate our understanding the biology of this disease and has the potential to identify targets for therapeutic intervention. originally combination therapies using drugs with documented single-agent activity and lack of overlapping toxicities could potentially improve outcome. the hypothesis intended to be tested is that palladia s can be safely administered concurrently with a standard weekly protocol of vinblastine (vbl), at dosages known to have activity against mast cell tumors. dogs with histologically confirmed measurable mast cell tumors were evaluated for eligibility to enter a standard phase i dose-finding trial ( cohort), at a starting dose of . mg/m iv vbl (weekly for a total of treatments) and . mg/kg po palladia s eod, concurrently. dose escalation of palladia s was scheduled in . mg/kg increments until mtd was established or fda label dose completed ( . mg/kg). safety evaluation was performed weekly throughout the week study period. dose-limiting toxicities were described following established vcog-ctcae(v . ) criteria. while antitumor response is not a primary endpoint of phase i trials, activity was documented prior to vbl treatments - , and monthly thereafter, based on recist criteria. nine dogs have been enrolled; cohort is filled and approaching completion of the evaluation period. hematologic dose limiting toxicity led to de-escalations of vbl. the current safe combination appears to include vbl at . mg/m every other week and palladia s at . mg/kg eod. response was seen in all but one dog. without head to head trials comparing efficacy of bi-weekly vbl combined with palladia s and vbl alone, choice of therapy should remain at the clinician's discretion. originally prostate specific membrane antigen (psma) is a transmembrane protein expressed by tumor-associated neovasculature, but not normal blood vessels. based upon its selective expression in endothelial cells associated with cancer, psma may serve as a conserved angiogenic target shared by macroscopic solid tumors of various histologies. to investigate the feasibility of targeting a homogenous population of psma-expressing endothelial cells as a novel anticancer strategy, we have investigated psma expression in several canine hemangiosarcoma (chsa) cell lines, and subsequently developed self-assembling nanoparticles containing diagnostic (near infrared dyes) and therapeutic (doxorubicin) cargo which selectively bind to psma by means of the a aptamer, a commercially-available oligonucleotide. the expression of psma by chsa cells was confirmed transcriptionally and translationally by real time pcr and immunohistochemistry, respectively. selective binding and endocytosis of a decorated nanoparticles was studied by fluorescent microscopy. the ability of a decorated nanoparticles encapsulating doxorubicin to exert in vitro cytotoxic effects in chsa cells was assessed by colony forming assays. using a chsa xenograft murine tumor model, clinically-relevant anticancer effects of a decorated nanoparticles encapsulating doxorubicin were tested. all chsa cell lines expressed psma mrna and protein. a decorated nanoparticles were selectively endocytosed by psma-expressing cells, and when these nanoparticles encapsulated doxorubicin, significant cytotoxic effects were exerted in vitro. finally, a decorated nanoparticles encapsulating doxorubicin significantly reduced the size of macroscopic chsa tumor burdens in transplanted mice. diagnostic and therapeutic nanoparticles can be targeted to psma-expressing endothelial cells, and chsa provides a comparative model for the future study of nanoparticle therapeutics. canine transitional cell carcinoma (tcc) is the most common tumor of the urinary tract, and is similar to human invasive tcc in histopathologic characteristics, molecular features, sites of metastasis, and response to medical therapy. prevalence is increasing, and novel therapies and strategies are needed to effectively treat this aggressive form of cancer in both species. personalized medicine techniques intend to improve treatment outcome by using patient tumor profiling to identify potential and individualized therapeutic targets. a genomic algorithm has been developed termed ''coexpression extrapolation'', or coxen, that aims to use expression microarray data to predict drug activity in patient tcc samples. the utility of this predictive methodology has been established in other types of cancer in vitro, however its clinical utility has not yet been determined. validation studies of coxen in canine tcc cell lines were conducted. the goal was to determine the value of coxen in predicting baseline sensitivity of canine tcc to chemotherapy agents (gemcitabine, mitoxantrone, carboplatin, vinblastine and cisplatin) that would then be used in a proposed clinical trial. additionally, expression data from canine treatment-naı¨ve primary tumor samples were generated on an affymetrix array platform (canine genome v . ). both the expression data and tcc cell line data (antiproliferative effects, % growth inhibition or gc ) were used to establish a canine specific predictive coxen algorithm. coxen scores for canine tcc cell-line drug activity were then analyzed. scores predicted the activity of cisplatin, gemcitibine, and mitoxantrone in all cell lines, and of carboplatin in cell lines. because all of the cell lines were sensitive to vinblastine (gi o . mm), the coxen score was not predictive of its potency. interestingly, coxen fails to predict vinblastine response in human tcc cell line data as well. in concurrent work, comparative genomic studies to define and compare the gene expression signatures of tcc in dogs and humans provides further evidence that canine tcc is a valuable genomic model of the human disease. current studies involve testing the chemo-predictivity of this derived canine coxen algorithm in additional canine tcc cell lines. canine tcc offers an excellent model for in vitro and in vivo studies of the coxen approach. this preclinical work will be used to guide the feasibility of future coxen clinical trials in dogs and humans with tcc. a small molecule complex (aminoact) isolated from bovine milk is a natural peptide mixture with multi-kinase inhibitory effects against epidermal growth factor receptor (egfr) and insulin-like growth factor receptor- (igfr- ). ingestion of aminoact in people with cancer results in lower serum tnf-alpha, an increase in antioxidant superoxide dismutase (sod) enzyme activity, and subjects' blood serum causes apopsotis in cancer cell lines. this study was designed to first assess safety and secondly the efficacy of three dosage levels of ax- in sustaining progression free survival (pfs) for dogs with refractory advanced and/or metastatic cancer. the prospective, open label study included dogs of different breeds with naturally occurring histologically confirmed malignancies. the first dogs received aminoact at g/m ; the second group of dogs subsequently received the same dosage mg of aminoact; and the third group of dogs subsequently received g/ m . each dog was treated orally daily for six weeks along with mg betaine hcl, that aids in peptide absorption. all patients were evaluated for toxicity using the vcog-ctcae and efficacy using the recist criteria via assessment of clinical parameters, blood work and client questionnaires. no toxicity other than mild, transient (grade i) nausea was noted, nor were there any changes in hemograms or biochemical profiles in any patient. dogs with tumors that were confirmed as responders ( % reduction in size) include pulmonary adenocarcinoma, mast cell tumor, trichoepithelioma and soft tissue sarcoma. it appears in limited studies that the response rate may be more durable at higher dosages. the response to aminoact is dose dependent and only transient mild toxicity was observed, which suggest the maximum effective dosage has not been reached. further clinical studies will be valuable in determining the effective dosage and response duration. treating cancer in dogs with aminoact offers a unique opportunity as a model for human cancer biology and translational cancer therapeutics. stereotactic radiation therapy (srt) combines patient immobilization, image guidance, and intensity modulated delivery to achieve ablative radiation doses within the tumor, while preferentially sparing surrounding normal tissues. the purpose of this study was to evaluate the efficacy of srt as a means of achieving local tumor control for canine nasal tumors. retrospective analysis was performed on dogs with a nasal tumor confirmed by histopathology and computed tomography, no previous surgical or radiation therapy, at least six months of follow-up, and completion of three fractions of srt at csu.srt was administered via the varian trilogy linear accelerator once daily for three consecutive days. the varian eclipse treatment planwas reviewed to determine the planned target volume (ptv) and dose to % of the ptv. kaplan-meir survival analysis was performed for disease free interval (dfi) and overall survival (os). sixteen patients with nasal tumors ( adenocarcinoma/carcinomas, squamous cell carcinomas, chondrosarcomas, osteosarcomas, and undifferentiated sarcoma) were treated with srt. a median dose of . gy was administered to % ptv with a median ptv of . cc. srt was well tolerated by the normal tissues with minimal, manageable side effects. to date, the median dfi is days, while the median os is days. based upon the initial clinical experience, stereotactic radiation therapy is an emerging modality in the management of canine nasal tumors. canine leptospirosis can vary from subclinical infection to illness that ranges from mild to severe, including death, depending on the susceptibility of the dog, virulence of the organism, and route and degree of infection. the objective of this study was to evaluate the ability of a canine leptospira bacterin to prevent infection and disease following challenge with virulent leptospira canicola, l. pomona, l. grippotyphosa, or l. icterohaemorrhagiae. groups of week-old beagles were vaccinated (day ) and boosted (day ) with placebo (n ) or the -way bacterin (n ! ) and subsequently challenged with each serovar. the results demonstrated that blood and various tissue samples from placebo-recipients became reliably infected, and the dogs developed typical clinical signs of leptospirosis including loss of appetite, ocular congestion, depression, dehydration, jaundice, hematuria, melena, vomiting, petechiae, and death. in addition, placebo-recipients developed kidney and liver dysfunction. in contrast, some vaccine-recipients became infected, but the organisms were cleared quickly from the blood. vaccinated dogs failed to develop severe clinical disease requiring medical intervention, and no animals died (p ! . ). a few of the vaccinated dogs developed clinical abnormalities, but the clinical signs remained mild and were self-limiting (p o . for each serovar). administration of the bacterin also prevented thrombocytopenia ( ciprofloxacin, a synthetic fluoroquinolone antimicrobic, is not fda-approved for veterinary use. however, due to recent availability of less expensive generic formulations, extra-label use of ciprofloxacin by veterinarians appears more common. although ciprofloxacin crystalluria and uroliths have been reported in humans, we are unaware of any published reports in dogs. this is surprising since mean urine ciprofloxacin concentration ( . mg/ml) in dogs following a modest iv dose ( mg/kg) was times higher than the solubility of ciprofloxacin in water ( . mg/ml). to identify the occurrence of ciprofloxacin uroliths in dogs, records from the minnesota urolith center were reviewed. between january and december , ciprofloxacin was identified in uroliths from dogs; uroliths were composed of % ciprofloxacin in , mixed uroliths containing ciprofloxacin were identified in , a shell of ciprofloxacin was observed in , and ciprofloxacin surface crystals were identified in . based on an experimental study in which % of human volunteers consuming mg of ciprofloxacin with nahco exhibited ciprofloxacin crystalluria (urine ph . ), while no volunteers consuming mg of ciprofloxacin and nh cl to acidify urine formed crystals; we postulated that ciprofloxacin uroliths could be dissolved in acidic urine. to test this hypothesis, canine uroliths composed of % ciprofloxacin from a single source ( -yr-old male, english bulldog receiving mg/kg of ciprofloxacin po, q hr to manage superficial pyoderma; turbulent flow chromatography/tandem mass spectrometry detected mg of ciprofloxacin/g of urolith) were incubated in urine at selected ph's and monitored for dissolution. urine obtained from multiple dogs not receiving fluoroquinolones, was pooled and divided into aliquots. aliquots were adjusted with hcl or naoh to a ph of , , , , or . aliquots were capped and preserved by refrigeration; ph was monitored and readjusted weekly. ten uroliths of approximately equal weight were randomly assigned to individual flasks containing mls of urine. flasks were constantly agitated and maintained at c. every hours, urine was discarded and replaced with mls of urine of identical ph until stone dissolution was complete. ciprofloxacin urolith dissolution times at each urine ph are reported below. ciprofloxacin uroliths are a newly recognized disease and a potential adverse effect of ciprofloxacin administration in dogs. in vitro dissolution of ciprofloxacin uroliths was achieved in canine urine, supporting the premise that in vivo dissolution is possible. urolith dissolution times were shortest at lower and higher ph's, which is consistent with the pka ( . and . ) of this amphiprotic antimicrobic (more soluble at ph below the acidic pka and above the alkaline pka). foods designed to promote struvite urolith dissolution may be designed for short term feeding facilitating rapid dissolution or may be formulated with a more moderate target urine ph to allow for dissolution and then life-long maintenance feeding minimizing recurrence. the purpose of this study was to compare the efficacy and rate of dissolution of a maintenance food with a struvite dissolution food. sixteen client-owned adult cats ( fs, mc) with naturally occurring struvite urocystoliths (mineral composition based on history, radiographs, urinalysis, urine culture and physical examination) were randomized to either a dry maintenance food (test) or a dry food known to dissolve struvite uroliths (control). the clinical care team and owner were blinded to treatment assignment. the test food was formulated to provide . % mg (dm), . % p, % protein, and a calculated target urine ph value (uph) of . - . . the control food was formulated to provide . % mg (dm), . % p, % protein, and a targeted urine ph of . - . . owners were advised to feed the assigned diet exclusively in an amount to maintain body condition. after diet assignment radiographs were performed at eight weekly intervals until there was no evidence of uroliths or until there was evidence that the uroliths were the same size or larger. a physical examination, complete blood count, serum chemistry profile, urinalysis and urine culture were repeated at the conclusion of the study. statistical analysis was by anova. all uroliths dissolved in all cats and both foods were palatable. radiographs of cats fed the control food indicated the uroliths dissolved in a significantly shorter time (mean ae std dev of . ae . weeks) compared to cats consuming the test food (mean . ae weeks; po . ).). cats in the control group finished the study at (n ), (n ) and weeks. cats in the test group finished the study , , (n ), , , , and weeks. all the minnesota urolith center occasionally receives uroliths for analysis that are immersed in formalin. results of quantitative analysis of these uroliths revealed that some submitted in formalin consisted of newberyite (magnesium hydrogen phosphate trihydrate). because newberyite is uncommonly found in uroliths formed by cats and dogs, we hypothesized that this mineral was an in vitro artifact caused by exposure of struvite (magnesium ammonium phosphate hexahydrate) to formalin. the purpose of this study was to determine if formalin alters the mineral composition of uroliths. urolith submissions containing stones of either % struvite (n dogs and cats), % calcium oxalate (n dogs and cats), % calcium phosphate apatite (n dogs and cats), % cystine (n dogs and cats), % ammonium urate (n dogs and cats), and % silica (n dogs) preserved by only air drying were tested. one urolith from each submission was quantitatively analyzed by polarized light microscopy or infrared spectroscopy. a subsequent urolith from the same submission was immersed in ml of % buffered formalin for hours at room temperature. uroliths were then air dried for minutes and the analysis was repeated. after exposure to formalin, portions of all struvite uroliths were transformed into newberyite. three ( dog and cats) of ammonium urate uroliths were completely dissolved. newberyite was not detected in any of the remaining uroliths. likewise quantitative mineral analysis of non-struvite uroliths remained unchanged. to avoid misdiagnosis of mineral composition, uroliths should not be immersed in formalin prior to analysis. we previously reported that transfusion to normal dogs of autologous erythrocyte concentrates (prbcs) that had been stored for days causes a profound inflammatory response ( x increase in leucocyte count and fibrinogen, x increase in c-reactive protein). we speculated that inflammation was due to cytokines produced during the storage period, and hypothesized that transfusion of fresh (f) prbcs would elicit less inflammation than would stored (s) prbcs. a whole blood unit was collected from healthy dogs (n ) for prbcs on day , then again on day . on day dogs received an autologous transfusion of prbcs stored for either days (s, n ) or days (f, n ). cbcs and in-tem thromboelastometry (ct:coagulation time, cft:clot formation time, a:alpha, mcf:maximum clot firmness) were evaluated on blood samples collected at (pre) and , , , , and hours after transfusion. fresh prbcs did not elicit any change in leucocytes, platelets, or thromboelastometry. stored prbcs elicited a degenerative left shift ( hr) followed by a regenerative left shift ( - hr), thrombocytopenia ( % decrease at hr), and marked hypocoagulability characterized by prolonged ct ( , , hr) and cft ( , hr), and decreased a ( , hr) and mcf ( , , hr). data are mean(sd). a: p o . between groups f and s by t test. b: p o . compared to '' '' by rm anova. transfusion of autologous stored prbcs elicits a greater inflammatory response than fresh prbcs, and results in hypocoagulability on thromboelastometry. clopidogrel is a potent antiplatelet drug that is gaining popularity in veterinary medicine for antithrombotic therapy. the parent molecule is an inactive prodrug that must be converted by hepatic isozymes to an active metabolite. the majority of the parent molecule is directed to the formation of inactive metabolites with only an extremely small proportion of parent molecule directed to the formation of the active metabolite. there are multiple hepatic isozymes responsible for the formation of the active metabolite. a non-specific hepatic isozyme inducer such as rifampin could increase the formation of the active metabolite of clopidogrel thereby increasing the pharmacodynamic response which may allow a reduced drug dose to achieve a clinical effect. we have previously presented data supporting the increased pharmacodynamic response of clopdiogrel after rifampin therapy. the goal of this study was to demonstrate an increased pharmacokinetic response of clopidogrel after rifampin induction of hepatic isozymes. six healthy, purpose-bred dogs were used for this study. the pharmacokinetics of clopidogrel were determined by measuring the parent molecule, primary inactive metabolite and active metabolite through lc/ms/ms. the pharmacodynamics of clopidogrel were determined by measuring collagen-induced whole blood aggregation. blood samples were collected prior to clopidogrel administration (baseline), after days of mg/kg clopidogrel po q hrs, and after days of mg/kg clopidogrel po q hrs mg/ kg po q hrs rifampin. given the absence of a known standard for the active metabolite, only a semi-quantitative assessment of active metabolite concentration can be made. there was no identifiable active metabolite peak noted at baseline or after clopidogrel treatment. however, with clopidogrel and rifampin combined administration there was an active metabolite peak identified in all dogs with a mean area of . ae . . the development of the active metabolite peak was associated with an increase in the pharmacodyamic response of clopidogrel in the dogs. this is the first study in any species to document the increased formation of the active metabolite of clopidogrel in response to a strong, non-specific hepatic isozyme inducer. this increased pharmacokinetic response was associated with an increased pharmacodynamic response of clopidogrel. this data provides supportive evidence to develop therapeutic protocols to improve the pharmacodynamic response to clopidogrel in dogs that may reduce dosing requirements or correct subtherapeutic pharmacodynamic response. critical illness-related corticosteroid insufficiency (circi) has been identified in humans, foals, dogs and cats with lower-thanexpected circulating cortisol concentrations, and/or by a blunted cortisol response to acth stimulation. our purpose was to determine if circi exists in critically ill horses. endogenous plasma acth and serum cortisol concentrations, and cortisol at t and t min after . mg/kg cosyntropin, were measured by radioimmunoassay from horses with colic or systemic illness on admission, and days , and of hospitilization. horses were divided into mild, moderate, or severe illness groups based on clinicopathologic data. inappropriately low cortisol was defined as endogenous cortisol o mean- sd achieved after administration of . mg/kg cosyntropin to normal horses ( o nmol/l). inadequate delta cortisol was defined as o mean delta cortisol in normal horses after . mg/kg cosyntropin ( o nmol/l). cortisol, acth and delta cortisol were compared using anova between groups, with p o . considered significant. fifty-eight horses classified as having mild ( ), moderate ( ) and severe ( ) disease at admission had survival rates of %, % and % respectively. admission acth and cortisol concentrations were highest in severely ill horses ( ae pg/ml, ae nmol/l) compared to moderate ( ae , ae ) and mildly ill horses ( . ae . , ae ). admission cortisol concentrations were higher overall in severely ill horses (p . ), but were low in % ( / ). admission delta cortisol was low in % ( / ) of severely ill horses, and was associated with marked adrenal hemorrhage in non-survivors. severely ill horses have high cortisol and acth, but low cortisol and delta cortisol may indicate circi secondary to adrenal hemorrhage. equine pituitary pars intermedia dysfunction (ppid) is a common endocrinopathy of aged horses that results from neurodegeneration of the dopaminergic periventricular neurons that innervate the intermediate lobe of the pituitary. factors that initiate spontaneous dopaminergic neurodegenerative disease remain elusive, however accumulation of misfolded a-synuclein protein and dysfunctional protein clearance have been implicated. misfolded protein accumulation occurs due to increased protein production or decreased clearance of damaged macromolecules through the process of autophagy. while have previously demonstrated that horses with ppid have increased asynuclein in the periventricular neurons compared to controls, it remains unknown whether the protein accumulates due to increased production or decreased clearance. we hypothesized that autophagy is decreased in the pituitary neurointermediate lobe from horses with ppid compared to controls. neurointermediate lobe pituitary tissue was from collected from horses with ppid (n ) and healthy horses (n , - years). realtime pcr was used to determine the relative expression of autophagy genes (mtor, beclin , atg , atg , atg , pink, lamp ) and a-synuclein relative gene expression from horses with ppid were compared to healthy horses by t-test following log transformation. a pearson coefficient of correlation was calculated comparing a-synuclein expression with autophagy gene expression. the expression of a-synuclein, autophagy-related genes (atg , beclin, lamp ), and mtor was greater in horses with ppid than in healthy horses. age was not correlated to a-synuclein or autophagy gene expression. there was a significant positive correlation between expression of a-synuclein and beclin , atg , atg , atg , and pink, but not mtor expression. accumulation of a-synuclein protein in horses with ppid may result from increased a-synuclein expression. autophagy genes are upregulated in horses with ppid, suggesting a compensatory response, although these findings need to be confirmed by demonstrating an increased functional response. asynuclein expression was positively correlated to expression of autophagy genes except mtor, suggesting a-synuclein may stimulate autophagy in an mtor independent manner. acvim forum session a efficacy of delayed antiviral therapy against ehv- challenge. lk maxwell , ll gilliam , n pusterla , r carmichael , rw eberle , jw ritchey , tc holbrook , t gull , gb rezabek , d mcfarlane , cg macallister . oklahoma state university, stillwater, ok. university of california, davis, ca. equine herpes virus type- (ehv- ) outbreaks are often not recognized until exposed horses are at immediate risk for developing equine herpes myeloencephalopathy (ehm). the objective of this study was to determine whether delayed therapy with the antiviral drugs valacyclovir or ganciclovir could protect those horses most at risk for ehm. eighteen aged ( years) mares were randomized to treatment: no therapy (control), oral valacyclovir therapy, or intravenous ganciclovir therapy. drug administration was initiated at the onset of the second febrile phase, between days - after ehv- inoculation (pi), and continued for one week. neurological examinations were performed prior to the study and for three weeks pi. one horse was excluded from the study for failure to become febrile. body temperature was significantly lower in the ganciclovirtherapy horses as compared to control horses on days - pi (p o . ), whereas valacyclovir-therapy horses did not differ from control horses. viremia in whole blood, as determined by pcr, was also lower in the ganciclovir-therapy horses on days - pi and on day pi in the valacyclovir-therapy horses (p o . ). although antiviral drug administration did not reduce the risk of ataxia (p . ) or nasal shedding, ganciclovir therapy did decrease the severity of ataxia (p o . ) as compared to valacyclovir-therapy and control horses, where / , / , and / horses, respectively, developed at least a two grade change in ataxia. in summary, ganciclovir administration provided better protection against ehm than did valacyclovir when therapy was initiated just prior to the onset of neurological disease. equine vaccination is amongst the most important method of prophylaxis against equine influenza virus (eiv), a pathogen in which continuous antigenic drift can lead to vaccine failure. a month duration of immunity (doi) challenge infection study was conducted using commercial inactivated vaccines containing different strains of a/equine/ /influenza virus's, including innovator tm , containing kentucky/ (pfizer animal health, new york, ny), and calvenza, containing a combination of ohio/ , kentucky/ , and newmarket (boehringer ingelheim vetmedica, st. joseph, ms) . the challenge virus strain was colorado/ , the most contemporary challenge strain currently in use. the study design was a blinded, randomized challenge trial. three groups of yearling ponies, with no history or serological evidence of eiv infection were established. each group received one of three treatments: vaccination with innovator tm ; vaccination with calvenza tm ; or injection with a saline placebo. each treatment was administered times, at intervals of month between the first two treatments, and months between the second and third treatments. all ponies were challenged by nasal nebulization of x eid influenza virus a/eq/ /colorado/ months after the third treatment. clinical signs of disease, including rectal temperature, nasal discharge, anorexia, coughing, and depression, were recorded daily for days prior to challenge infection, and days post-challenge. nasal shedding of eiv was measured on the same days, using a realtime pcr test procedure. eiv-specific antibody responses were measured by elisa. differences between groups were analyzed by non-parametric repeated measures anova, and differences were declared significant when p o . . all control group ponies demonstrated clinical signs of disease consistent with eiv infection post-challenge infection, including pyrexia, nasal discharge, inappetance and partial anorexia. these signs were significantly lower in both vaccine groups; mean body temperature was elevated ( . f) for days in controls, but only days in vaccine groups. nasal shedding of eiv was detected in all ponies in all groups: over the duration of the study the calvenza group shed significantly less virus than innovator and control. over time antibody titers were significantly higher in the calvenza than the innovator group, and both were significantly greater than controls. this study demonstrated that both current commercial inactivated eiv vaccines have a duration of clinical protection of at least months after a highly pathogenic challenge with a recent eiv isolate. both antibody responses and virological protection differed between the vaccines. formulation difference between the vaccines, including the eiv antigens employed, may have contributed to this performance difference. degenerative myelopathy (dm) may be homologous to a form of amyotrophic lateral sclerosis in humans which has excitotoxic and immunologic pathogeneses described. the aims of this study were to determine (i) presence or absence of abnormalities in concentrations of csf amino acid (aa) neurotransmitters (glutamate, glycine and gÀaminobutyric acid (gaba)) and cytokines in dogs with dm and if present (ii) investigate associations with disease severity. twenty-two dogs histopathologically confirmed for dm and dogs with suspected dm based on thorough diagnostic investigations and clinically normal age-matched control dogs were included in the study. the neurological severity of the dm dogs was graded ( - ) using an established scale. csf was evaluated for presence of glutamate, glycine and gaba by high performance liquid chromatography and for gm-csf, ifn-g, il- , il- , il- , il- , il- , il- , il- , il- , ip- , kc (keratinocyte chemoattractant), mcp- (monocyte chemotactic protein- ) and tnf-a using a commercially available, canine multiplex immunoassay (millipore, billerica, ma). all data analyses were performed using sas v . (cary, nc). analyte levels were compared between dm confirmed, dm suspected and control dogs by an analysis of variance (anova). spearman correlation was used to test for correlations of analyte levels and neurological grades. all hypothesis tests were -sided with a . . there were no significant differences between individual csf analytes in dm confirmed and dm suspected dogs. glutamate levels were not significantly different between dm affected (mean . mg/ ml; range . - . ; sd . ) and control dogs (mean . mg/ ml; range . - . ; sd . ). control dogs (mean . mg/ml; range . - . ; sd . ) had significantly higher levels of gaba (p o . ) than dm dogs (mean . mg/ml; range . - . ; sd . ). control dogs (mean . mg/ml; range . - . ; sd . ) also had significantly higher glycine concentrations (p o . ) than dm dogs (mean . mg/ml; range . - . ; sd . ). dm-affected dogs also had significantly higher levels of il- (p . ), kc (p o . ) and mcp- (p . ) than control dogs. neurotransmitter levels were not significantly associated with neurological grade. kc levels were significantly higher in the least affected dogs (p . ). there were no associations with disease severity and analyte concentrations. dm affected dogs have an imbalance of csf aa concentrations creating a relatively excitotoxic environment. reports in human als confirm an imbalance between csf excitatory and inhibitory aas suggesting a pathogenic role for excitotoxicity in als. it also appears that dm affected dogs have increases in csf cytokines and chemokines suggestive of an immunologic component to the pathogenesis as is similar to als. further prospective analysis of dm is warranted to evaluate the role of treatment on csf variables. the pathogenesis of neuropathic pain (np) and syringomyelia (sm) in association with chiari-like malformation (clm) in dogs has focused on the anatomical anomalies and secondary cerebrospinal fluid (csf) flow abnormalities. neuropathic pain in humans has been associated with abnormalities of neurotransmitters such as glutamate and serotonin as well as immunologic mechanisms. the aim of this study was to investigate the csf neurotransmitter and cytokine levels in brussels griffon dogs (bgs) with clm, sm and np. as part of an mri study investigating the prevalence of sm in bgs, atlanto-occipital csf was acquired from dogs and stored at - c until analysis. all dogs underwent a neurologic exam prior to mri; osirix s software was used to measure sm and the presence of cerebellar herniation and deviation were recorded. deproteinized csf samples were analysed for presence of serotonin (ng/ml), glutamate, glycine and gaba (mg/ml) by high performance liquid chromatography. all csf samples were evaluated simultaneously for gm-csf, ifn-g, il- , il- , il- , il- , il- , il- , il- , il- , ip- , kc, mcp- and tnf-a. a commercially available, canine multiplex immunoassay (millipore, billerica, ma) was used for the cytokine analysis (pg/ml). student's t-tests were used to compare the means of neurotransmitter and cytokine values between groups with and without skull abnormalities or spinal pain. simple pearson's correlation was used to test for correlations of neurotransmitter and cytokine values with syrinx dimensions and correlations of neurotransmitter with cytokine values. all hypothesis tests were -sided and the significance level was a . . np was detected in dogs ( %); sm was present dogs ( %); and cm was detected in dogs ( %). ifn-g levels were significantly lower in dogs with np than without (p . ). there were significant positive correlations between syrinx size and il- (p . ), kc (p . ) and mcp- (p . ). there were significant negative correlations between ifn-g and syrinx height (p . ) and extent (p . ). there was a significant negative correlation between il- and syrinx height (p . ). neurotransmitter levels were not associated with skull abnormalities or spinal pain, but there was a positive correlation of glycine with il- (p . ) and mcp- with glutamate (p . ) and serotonin (p . ). the size of the syrinx in bgs with sm is associated with several cytokine elevations but only a decrease of ifn-g was associated with np. based on this study it does not appear that excitotoxicity plays a role in either sm development or np. further work is justified on the role of the immune system in cm, sm and np. current knowledge about the conservative management of disk associated cervical spondylomyelopathy (da-csm) is rather limited and mainly based on retrospectively retrieved data. the goals of this study were to prospectively evaluate the evolution of clinical signs in dogs treated conservatively for da-csm. additionally, several potential prognostic parameters and the correlation of initial clinical signs with magnetic resonance imaging (mri) and transcranial magnetic stimulation (tms) were investigated. twenty-one dogs were included. after neurological evaluation, neurological status was graded from ( normal) to ( tetraplegia). all animals underwent low-field mri and tms with measurement of onset latencies and peak-to-peak amplitudes from the extensor carpi radialis and cranial tibial muscles. from the mr images, the following dimensions were calculated: remaining spinal cord area; compression ratio; vertebral occupying ratio of the spinal cord; canal height to body height ratio (cbr); canal height to body length ratio (cblr); and the canal compromise ratio. intraparenchymal intensity (isi) changes were graded from to . all dogs were reevaluated by the same person after , , , , and months. eight of dogs ( %) experienced a positive clinical evolution with improvement of clinical signs or stabilization of mild clinical signs. all dogs with a negative clinical evolution month after diagnosis experienced a further progression of clinical signs resulting in a poor outcome. the opposite was true for all dogs with a positive clinical evolution after month. outcome was further significantly associated by the remaining spinal cord area and the vertebral canal compromise ratio. prognosis was not significantly affected by clinical presentation or tms. progression of clinical signs, in unsuccessfully treated dogs, was generally characterized by a rapid and dramatic deterioration of neurological status. there were no significant correlations between clinical presentation, mri and tms. two dogs underwent necropsy and histopathological examination. this revealed in both cases chronic wallerian degeneration and segmental myelomalacia. the results of this study suggest that conservative treatment of da-csm is associated with a rather guarded prognosis. clinical evolution month after diagnosis and selected mri parameters can be considered as prognostic indicators. the lack of correlation between clinical presentation and outcome, medical imaging and electrophysiological evaluation is disturbing and warrants further investigation. a mri-guided stereotactic brain biopsy system has not been clinically evaluated in dogs. the purpose of this study was to determine the ability of the brainsight tm system to obtain histologically diagnostic samples and access the impact of this procedure on neurologic status for hours after the biopsy. five dogs with mri definable lesions in the brain have been enrolled. breeds included a pitbull mix, pembroke welsh corgi, french bulldog, border terrier and west highland white terrier. age ranged from - years. weight ranged from . - . kg.dogs presented with seizures (n ), ambulatory paresis(n ), unilateral blindness(n ) and head tilt(n ). one dog had a normal neurologic exam. lesions chosen for biopsy were in the olfactory and/or frontal lobes (n ), parietal lobe(n ), and pyriform lobe(n ). lesions were between - mm in diameter. all lesions were well-circumscribed and contrast enhancing except for one. histologic diagnosis of meningioma(n ) and granulomatous meningoencephalitis(n ) were made. the poorly-circumscribed, non-contrast enhancing frontal mass yielded non-specific necrosis. following biopsy, three dogs returned to pre-biopsy neurologic status within hours. the french bulldog took hours to return to previous neurologic status due to brachycephalic syndrome that required oxygen support. one dog had acute respiratory arrest hours post-biopsy. necropsy is pending. these results suggest that this mri-guided biopsy system can provide an accurate histologic diagnosis of brain lesions. biopsies of poorly-circumscribed and non-contrast enhancing brain lesions may be less diagnostic. further evaluation is on-going to determine the true diagnostic yield and complication rate of this procedure. concurrent malformations of the craniocervical junction are commonly identified in humans with chiari type i malformation. recent evidence suggests such craniocervical junction abnormalities (cjas) also occur in dogs suspected of having chiari-like malformation (clm). the purpose of this study was to objectively describe morphometric features of the craniocervical junction region of dogs with suspected clm and to investigate for associations between these features and the occurrence of other malformations in this region. magnetic resonance (mr) and computed tomographic (ct) images from dogs with clm were evaluated. three regions of neural tissue compression were assessed: cerebellar compression (cc); ventral compression at the c /c articulation, termed ''medullary kinking'' (mk); and dorsal compression (dc) at the c /c articulation. a compression index (ci) was calculated for all abnormal regions for each dog. multiple logistic regression analysis was performed (p o . ) to ascertain whether ci values for the different regions of compression were associated with the incidence of other craniocervical junction abnormalities. % of dogs had mk and % of dogs had dc. % of dogs also had evidence of atlanto-occipital overlapping (aoo medical infrared imaging (mii) is a non-invasive diagnostic imaging technique that measures skin surface temperature and generates thermal pattern maps based on predetermined color scales. because skin temperature, dependent on regional perfusion, is under direct control of the sympathetic nervous system, mii provides information about the function of the autonomic nervous system. because of recent advances in technology and lack of sedation needed to image patients, mii has potential use as a screening test for a variety of conditions that may result in autonomic dysregulation like chiari-like malformation in dogs (clm). the purposes of this study were to establish a mii protocol for dogs suspected of having clm, to identify thermal imaging patterns for various regions of interest (roi), to evaluate changes in thermal patterns and compare the results to those of mri findings, considered the standard for diagnosing clm in dogs. one hundred and five cavalier king charles spaniel dogs with clinical signs attributable to clm and confirmed clm with mri were evaluated with a complete blood count and chemistry profile, examination by a board certified surgeon/neurologist, multidetector ct scan of the craniocervical junction, whole body mri and mii. the protocol for thermal imaging included cranial and caudal views of the body, full lateral right and left body views, dorsal views of the head and body, and right and left lateral views of the head. thermal patterns were assessed with custom image recognition software. after each dog was imaged awake, general anesthesia was administered and the dogs re-imaged using the same protocol. mri findings in dogs with severe or moderate cerebellar compression and cerebellar herniation were compared with mii results. the top of head and front of head roi were . % and . % successful in identifying dogs with clm. based on these preliminary findings, mii may be a viable screening tool to detect clm in dogs. medical infrared imaging (mii) is an imaging technique that measures skin surface temperature derived from cutaneous perfusion and generates thermal pattern maps based on color scales. mii has been used as a test for a variety of conditions that cause autonomic dysregulation resulting in altered cutaneous perfusion. acute thoracolumbar intervertebral disk disease (tlivdd) is common in dogs. the purpose of this study was to: ) determine the success of mii in identifying dogs with tlivdd, ) compare the mii localization with mri results and surgical findings ) determine if the mii pattern returns to that of normal dogs following decompression surgery. small breed chondodystrophic dogs with tlivdd confirmed with mri and dogs with no tlivdd were evaluated with mri and mii. regions correlating with the intervertebral disk spaces were analyzed for average temperatures and thermographic patterns. thermal patterns were assessed with computer recognition pattern analysis (crpa) software. dogs were re-evaluated weeks after surgery using the same protocol. when analyzing temperature averages over a region, no significant difference was found between control and affected dogs. crpa was % successful in differentiating normal from affected dogs. crpa was % successful in identifying the intervertebral disk space when compared with mri and surgical findings. based on these findings, mii may be a viable screening tool to detect tlivdd in dogs. microglia physiologically shows regional topographical differences in immunophenotype and function within the central nervous system indicating the endowment for a prompt response to pathological stimuli such as trauma. spinal cord injuries (sci) consist of a primary injury encompassing the mechanical impact and the ''secondary wave'' of injury occurring minutes to weeks later and comprising various consecutive effects such as increased production of free radicals, excessive release of excitatory neurotransmitters and inflammatory reactions. activated microglia has the potential to perform some of these reactions, their contribution to the secondary wave is therefore controversially discussed. it has to be considered a double-edged sword as both, beneficial and deleterious effects have been attributed to these cells. the purpose of the presented study was to assess microglial involvement, particularly in the ''secondary wave'' following sci. microglia from dogs with sci was isolated and characterized ex vivo in terms of morphology, immunophenotype, and function by flow cytometry. the results were compared to region-specific findings obtained from healthy control dogs (n ). the histopathological exam confirmed the diagnosis of sci in the cervical (n ) and thoracolumbar (n ) spinal cord, and revealed a significant activation of microglia/ macrophages and upregulation of myelinophagia in dogs with sci days or longer prior to euthanasia. microglial ex vivo examination showed significantly increased expressions of b - , b - , mhc ii, cd c, icam- , cd , cd , and cd , and significantly enhanced phagocytosis and generation of reactive oxygen species (ros) in sci compared to healthy controls. microglial cells seem to be highly activated following sci with an immunophenotype indicating their active role in co-stimulation of t cells, in leukocyte adhesion and aggregation, and in lipid and glycolipid presentation. microglial phagocytosis might play a pivotal role in removal of injured or damaged cells and initialize subsequent healing processes. however, as ros can be directly neurotoxic an enhanced microglial generation might lead to bystander damage of the traumatized spinal cord and might therefore add to the deleterious effects of the secondary wave. modulating the microglial response in sci might be a valuable novel therapeutic strategy alleviating further damage to the spinal cord. thymidine kinase (tk) is a soluble biomarker present in s-phase of a salvage pathway for dna synthesis, and can be measured in serum. tk activity correlates with stage, prognosis, and relapse in canine and human lymphoma. we previously reported the results of a pilot study evaluating tk activity in archived canine osteosarcoma, transitional cell carcinoma, and hemangiosarcoma (hsa) sera, and found elevated tk activity in % of canine hsa sera evaluated. the purpose of this study was to prospectively evaluate serum tk activity in a large number of dogs presenting to emergency clinics with hemoabdomen and a splenic mass, to determine if tk activity could be used as a noninvasive means to distinguish hsa versus benign conditions in this population. dogs presenting with hemoabdomen and a splenic mass identified on ultrasound examination were studied. serum was collected prior to anesthesia, euthanasia or surgical intervention and frozen until batch analysis. tissue from all patients was evaluated histologically by a single pathologist. sera from age-matched normal dogs comprised a control population. an elisa using azidothymidine as a tk substrate was used. comparisons between groups were made using -tailed student t-tests, and receiver-operator characteristic (roc) curves were generated. sixty-two patients and normal controls were studied. there were dogs with hsa, dogs with other splenic neoplasia, and dogs with benign diseases. using a training set of normal dogs, a cutoff of . u/l was established from the roc curve. tk activity was significantly higher (p o . ) in dogs with hsa than in the validation set of normal dogs (mean /Àsd . /À . and . /À . respectively), but not between dogs with hsa and benign splenic disease (mean /Àsd . /À . , p . ). using a cutoff of . u/l, tk activity demonstrated a sensitivity of . , specificity of . , positive predictive value of . and negative predictive value of . for distinguishing hsa versus benign splenic disease. when interval thresholds of o . and . u/l were used together, diagnostic utility was markedly increased for distinguishing both hsa versus normal and hsa versus benign disease. in conclusion, serum tk evaluation may assist in detection of canine hsa, and may also discriminate between benign disease and hsa in dogs with hemoabdomen and a splenic mass. t cell chronic lymphocytic leukemia (cll) is a heterogeneous disease that affects a number of dog breeds. cll patients have variable disease outcomes. the objectives of this study were to use gene expression profiling of cd t cell leukemias with variable outcomes in order to identify markers that can be used in routine diagnostic tests to distinguish good from poor prognosis disease, and to identify potential targets for novel therapy. gene expression profiling of cd t cell leukemias ( good, poor prognosis) was conducted. samples from normal dogs were also profiled. several differentially expressed genes were found including cd , cd , and cd . these were selected for further study using flow cytometry to determine expression of protein on the cell surface. seventy nine cases of cd t cell leukemia were screened for cd expression. forty seven had associated outcome information. based on analysis to date, cd expression as assessed by flow cytometry does not appear to provide prognostic information. a monoclonal antibody to cd was recently made available. to date patients with cd t cell leukemia have been profiled. cd is variably expressed on t cell leukemias compared to normal cd t cells. cd is the receptor for interleukin . cyclosporin, a commonly used immunosuppressive drug, inhibits il- production, and has been used to treat a subset of t cell leukemias in people. thus, the finding that cd is up regulated on t cell leukemias compared with normal t cells suggests a possible new therapeutic avenue. recent molecular studies have revealed a highly complex bacterial microbiota in the intestine of dogs. there is mounting evidence that microbes play an important role in the pathogenesis of acute and chronic enteropathies of dogs, including idiopathic inflammatory bowel disease (ibd). similarly, compositional changes of the intestinal bacterial ecosystem have been associated with ibd in humans. the aim of this study was to characterize the bacterial microbiota in dogs with various gastrointestinal disorders using a next generation sequencing technique. fecal samples were obtained from healthy dogs (n ), dogs with acute uncomplicated diarrhea (n ), dogs with acute hemorrhagic diarrhea (ahd; n ), and dogs with active (n ) and therapeutically controlled ibd (n ). the bacterial composition was analyzed by massive parallel s rrna gene -pyrosequencing. differences between groups were analyzed using mann-whitney u tests and kruskal-wallis tests followed by dunn's multiple comparison tests. statistical significance was set at p o . . significant differences in the proportions of several bacterial groups were identified between healthy and diseased dogs. dogs with gastrointestinal disease had significantly higher proportions of proteobacteria (p o . ). proportions of firmicutes were lower in diseased dogs, but this difference did not reach significance (p . ). within the firmicutes the most notable findings were decreases in bacterial groups belonging to clostridium clusters iv and xiva (i.e., ruminococcus, dorea, and faecalibacterium spp.; p o . for all). dogs with ahd had the most profound changes of the microbiota, followed by dogs with acute uncomplicated diarrhea, and dogs with active ibd. faecalibacterium spp. was the bacterial group most prominently depleted in dogs with active ibd, but was not significantly different between healthy dogs and dogs with therapeutically controlled ibd (p . ). results of this study revealed bacterial dysbiosis in fecal samples of dogs with various gi disorders. bacterial changes were more profound in dogs with severe disease, but were not identified in dogs with therapeutically controlled ibd, suggesting that the microbiota is stable in non-active disease. the bacterial groups identified are considered to be important short chain fatty acid producers and may serve as candidates for the diagnosis or therapeutic monitoring of gi disease. future studies are necessary to determine if these microbial changes correlate with functional changes in the intestinal microbiota. ciprofloxacin oral tablets, available in a generic formulation for people, are widely used for treatment in dogs. oral absorption data for ciprofloxacin in dogs has been variable, and too limited to guide accurate dosing. subsequently, published doses for dogs in veterinary formularies have varied from to mg/kg. this study was undertaken to explore the factors that may affect oral absorption of generic ciprofloxacin in dogs, and to derive a pharmacokinetic-based dose for treating susceptible bacteria. six healthy adult beagle dogs were used for the study ( . kg mean weight). after placing jugular vein catheters for collecting blood samples, these dogs were administered either a single oral dose of ciprofloxacin ( mg tablet; mean dose mg/kg), or an intravenous (iv) dose ( mg/kg; mg/ml solution). a randomized crossover design was used with a washout time between treatments. blood was collected for plasma drug analysis for hours. ciprofloxacin concentration in plasma was analyzed using high pressure liquid chromatography (hplc) and pharmacokinetics analyzed using a computer program. oral absorption was also evaluated via deconvolution analysis. the oral dose was well-tolerated, but the iv dose produced transient vomiting and depression in some dogs. after the oral dose, the peak plasma concentration (c max ) was . mg/ ml (cv . %), terminal half-life (t / ) . hr (cv . %), auc . mg Á hr/ml (cv . %), and systemic absorption (f) . % (cv . %). after the iv dose, the t / was . hr (cv . %), systemic clearance . l/kg/hr (cv . %), and volume of distribution . l/kg (cv . %). after examining the pharmacokinetic results from the oral dose, it was apparent that oral ciprofloxacin was absorbed well in some dogs (approximately %), but poorly in others (approximately %). to explore the factors that may have affected oral absorption, two high absorbers and two low absorbers were administered an additional oral dose as a mg/ml solution ( mg total dose) via gastric tube. after administration of the oral solution, the plasma concentrations were more uniform and consistent among dogs. absorption of the oral solution of ciprofloxacin was . % (cv %) with a t / of . (cv . %) hr and c max of . mg/ml (cv . %). therefore, it appears that inconsistent oral absorption of ciprofloxacin in some dogs may be formulation-dependent, and affected by tablet dissolution in the canine small intestine. doses were calculated using the data for oral tablets in these dogs. the pharmacokinetic-pharmacodynamic (pk-pd) target was an auc/ mic ratio of . because of the wide range in oral absorption of tablets, a dose to reach the pk-pd target ranged from canine distemper (cd) is a highly contagious, acute or subacute systemic viral disease of dogs and other carnivores which can be controlled efficiently by the use of modified live-virus (mlv) vaccines. however, mlv strains do cross-react with molecular diagnostic tests and cause significant confusion for clinicians. the purpose of this study was to use quantitative real-time pcr viral load information to differentiate between vaccine virus used in mlv vaccines and wildtype infections in dogs. a real-time pcr test for cd virus (cdv) based on the p gene for phosphoprotein was used to determine viral loads in vaccinated and wildtype infected animals. a total of respiratory mucosal swab samples from mlv vaccinated and asymptomatic dogs were obtained within the first weeks after mlv vaccination. based on the viral load in vaccinated animals, a cutoff value was established for the differentiation of dogs with clinical signs of respiratory distress and presumably infected with a wildtype strain of cdv. two hundred clinical cases with known clinical and vaccination histories were analyzed to validate the cutoff value. the cdv real-time pcr proved to be of high analytical and diagnostic sensitivity: a standard curve was established using known numbers of cdv molecules to allow absolute quantitative cdv viral load data. the limit of detection was in the single molecule range while the limit of quantitation was established at around molecules per pcr reaction. a comparison to ifa showed real-time pcr to be % more sensitive. the cdv viral load in vaccinated animals averaged , viral particles per swab. a cutoff value of , viral particles was calculated by adding standard deviations to the average value. this cutoff value correctly detected . % of the vaccinated samples. acutely infected dogs with cdv compatible clinical signs have high viral loads normally several logs higher than the cutoff value. in dogs with clinical distress, recent cdv mlv vaccination but viral loads below the cutoff value, other infectious agents were detected by using a panel of real-time pcr tests. testing additional infectious agents in clinical settings is important in order to explain clinical signs when viral loads below cutoff values indicate that cdv is not the cause of clinical signs. in conclusion, quantitative real-time pcr is a sensitive, rapid and reliable test regardless of recent vaccination. the use of a cutoff value will be of significant help to discriminate between vaccine interference and wildtype infection in clinical settings. feline ureteral obstructions are a common urinary dilemma and traditional therapy is associated with substantial morbidity/mortality. feline nephrostomy tubes are reported as being effective when pelvic drainage is required. the biggest limitation is externalized drainage, requiring careful management to prevent infection/dislodgement. the development of an indwelling ureteral bypass using a combination locking-loop nephrostomy/cystostomy tube was modified from humans, resulting in permanent indwelling drainage, reduced complications, and improved quality of life. the objective is to describe the technical and clinical outcome using a novel device called a subcutaneous ureteral bypass (sub) in cats with ureteral obstructions. fifteen cats ( kidneys) had a sub placed for: ureterolithiasis ( ), ureteral stricture ( /À stones) ( ), and ureteral stent rejection ( ). the median pre-and post-procedure creatinine was mg/dl (range: . - ) and . mg/dl (range: . - ), respectively. the median pelvis diameter pre and post-procedure were (range: - ) and mm (range . - ), respectively. six french tubes were placed in , and fr. in . the bypass remained indwelling for a median of days (range - ). there were major complications resulting in nephrostomy tube dislodgement ( ) and port leakage ( ) days after surgery. one patient with severe coagulopathy developed a clot which resolved with tpa infusion through the port. no sub got occluded/obstructed long-term. overall, the use of a sub for cats with ureteral obstructions can be considered a functional option when other therapies have failed or are contraindicated, but shtime. oxidative stress is considered central to the pathogenesis of many systemic diseases. in humans, biomarkers of oxidative stress, antioxidant depletion and lipid peroxidation, have been correlated with disease severity and associated with poor clinical outcomes. therapeutic antioxidant supplementation with nac in glutathione (gsh)-deficient patients has shown clinical benefits, including repletion of intracellular gsh levels. we have shown that clinically ill dogs are gsh-deficient, and that gsh deficiency correlates with mortality, but it is not clear whether there are direct benefits of antioxidant intervention in these patients. the purpose of this randomized, investigator-blinded, placebo-controlled, prospective study was to evaluate the effect of nac to normalize blood antioxidants (rbc reduced gsh (rbc gsh), plasma cysteine (cys), serum vitamin e (vit e), and whole blood selenium (se)), reduce lipid peroxidation (urine isoprostane/creatinine ratio (u i/ c)), and improve illness scores (spi ) and outcome (survival to discharge) in clinically ill dogs. clinically ill client-owned dogs, admitted to the uw veterinary medical teaching hospital that did not receive blood transfusions, tpn, vitamins, or antioxidants were eligible for the study. dogs enrolled in the study were randomized to receive iv infusions q. h. of either nac (  mg/kg and  mg/kg) or equal volumes of % dextrose (placebo) over hours. at the time of enrollment, and hours following the final hour infusion, blood and urine were collected to quantify rbc gsh, cys, vit e, and se concentrations; u i/c ratios; and calculate spi scores. rbc gsh and cys concentrations were quantified by hplc. commercially available hplc, atomic absorption spectroscopy, and eia were used to quantify vit e, se, and u i/c ratios, respectively. nonparametric statistical analyses were used, with results reported as medians and p o . considered significant. sixty-one ill dogs were randomized to either nac (n ) or placebo (n ). overall this group of ill dogs had significantly decreased rbc gsh ( . vs. . mm; p . ), vit e ( vs. mg/ml; p . ), and se ( . vs. . mg/ml; p . ) levels and elevated u i/c ratios ( vs. pg/mg; p . ) in comparison to healthy control dogs. dogs in the placebo group showed a significant further decrease in rbc gsh over the next hours ( . to . ; p . ). nac supplementation significantly increased plasma cys levels ( . to . mm; p o . ), and prevented a further decline in rbc gsh ( . to . mm; p . ). however, serum vit e ( vs. mg/ml), se ( . vs. . mg/ ml), u i/c ratios ( vs. pg/mg), spi scores ( . vs. . ), and outcome ( % vs. %) were not significantly different between the nac and placebo groups after treatment. the results of this study further support that clinically ill dogs experience oxidative stress, and suggest that antioxidant supplementation with nac within the first hours of hospitalization prevents further rbc gsh depletion. further studies are necessary to investigate whether longer duration or combined antioxidant supplementation normalizes the redox state and impacts long-term outcome. diabetes mellitus in cats is very similar to type ii diabetes in humans, preceded by a period of insulin resistance. evaluating insulin resistance in a cat is a time consuming, expensive, and difficult procedure. there is a need for a simple biomarker based test predictive of insulin resistance. there is a biomarker based assay predicative of insulin resistance in humans. the purpose of this study was to evaluate the utility of this assay in overweight cats and show improvement in insulin sensitivity following weight loss and weight maintenance. the insulin resistance assay is based on the quantitative analysis of metabolites ( -hydroxybuterate, creatine, palmitate, decanoylcarnitine, and oleoyl-lpc). a proprietary algorithm (metabolon, inc, durham nc) was used to generate a predictive rd (rate of disposal) value (normal range in cats . - . ). individuals with an rd value less than will have a greater than % chance of being insulin resistant and an rd value less than will have a greater than % chance of being insulin resistant. initial studies demonstrated that the rd values indicating insulin resistance in cats correlated with age, obesity and severity of diabetes as determined by histopathology and blood glucose levels. in a feeding study of cats ( % vs. o % body fat) rd values improved from . ae . to . . (p . ). during weight maintenance, % body fat for months, further improvement was observed (rd, . . (p . e- )). these results demonstrate that long term weight maintenance following weight loss is critical for increasing insulin sensitivity in cats. the use of monoclonal antibodies and antibody fragments to directly target tumor antigens and neutralize their growth factors has shown promising results in human clinical trials. however, these targeted approaches have not been possible in dogs since specific tumor antigens have not been identified, monoclonal antibodies of canine origin are not available and the efficacy of xenogeneic antibodies in the dog is limited by neutralizing antibody responses. to overcome these obstacles, we have generated canine antibody phage display libraries from canine splenocytes. these libraries consist of single chain variable fragments (scfv) comprised of canine variable heavy (vh) and variable light (vl) immunoglobulin chains displayed on the surface of bacteriophage (fig. ) . the antigen specificity within these libraries is diverse and recapitulates the antigen-experienced immunoglobulin repertoire of the dog. we can now use simple panning techniques to isolate scfv of canine origin that bind to either known targets or unknown targets which can then be identified using standard molecular techniques. canine hsa is a highly aggressive malignancy of vascular endothelial cells that affects large breed dogs. although there are no confirmed immunological targets for hsa, serum levels of vascular endothelial growth factor (vegf) are elevated in these patients and, as in many human cancers, vegf may represent an important therapeutic target for neutralization. we used simple panning techniques to screen canine scfv libraries generated from the spleens of dogs with hsa against canine vegf and successfully isolated scfv clones that bind and neutralize canine vegf in vitro. these scfvs are now being taken into a murine model of canine hsa to determine whether they can inhibit tumor growth and metastases. in addition, we have panned the same antigen-experienced scfv phage display libraries against allogeneic primary canine hsa cells of low passage number to isolate canine-derived antibody fragments that can target malignant endothelial cell surface molecules. early results demonstrate enrichment of scfv phage libraries for malignant endothelial cell binders. these scfv can be readily linked to chemotherapeutic agents or other toxins and used to deliver high doses directly to the malignant cell. this novel approach aims to reduce side effects of systemic chemotherapy and augment therapeutic response. calcitriol, (vitamin d ), has antineoplastic activity and acts synergistically to potentiate the antitumor activity of a diverse array of chemotherapeutics. ccnu, vinblastine, corticosteroids, and tyrosine kinase inhibitors, are used to treat canine mast cell tumors (mct). vitamin d receptor is expressed in the majority of canine mcts, suggesting a role for calcitriol in the management of dogs with these tumors. the purpose of our study was to examine the in vitro effects of calcitriol in combination with ccnu, vinblastine, imatinib, or toceranib on canine mastocytoma c cells. also, we evaluated the antitumor activity of dn , a highly concentrated oral formulation of calcitriol, as single-agent treatment in dogs with naturally occurring mcts. c cells were incubated with serial dilutions of calcitriol ( . - nm). twenty-four hours later, cells were then treated with vehicle control or serial dilutions of ccnu ( . - um), vinblastine ( . - nm), imatinib ( . - . nm), or toceranib ( . - nm). cell viability was assessed with an mtt assay after hours and data was used to derive a combination index (ci: values o , , indicate synergism, additivity, antagonism, respectively). in the phase ii clinical trial, dogs were eligible if they had at least measurable, histologically confirmed, mct. calcitriol was administered orally. recist criteria were used to assess tumor response. calcitriol, ccnu, vinblastine, imatinib, and toceranib each suppressed c cell viability in a dose-dependent manner. ci values o were obtained for calcitriol ( . - . nm) combined with ccnu ( and um), vinblastine ( . and nm), imatinib ( . - . nm) and toceranib ( . - . nm). due to the occurrence of toxicity (vomiting, anorexia, hypercalcemia), the phase ii trial was terminated early; only of planned patients were treated. one dog with a metastatic muzzle mct had a complete response that lasted days. three dogs achieved partial response lasting from - days. in summary, our in vitro data demonstrate that calcitriol combined with ccnu, vinblastine, imatinib or toceranib has synergistic effects on c mastocytoma cells. antitumor responses were observed in dogs with spontaneously occurring mcts treated orally with single-agent calcitriol, but the frequency of adverse effects was high. together these results suggest calcitriol combination therapies might have significant clinical utility in the treatment of canine mcts but refinement of the calcitriol-dosing regimen must be done. cyclosporine is a potent immunosuppressive agent used to treat many canine inflammatory and immune-mediated diseases. cyclosporine has gained popularity as an immunosuppressive agent because of a favorable toxicity profile compared to many other immunosuppressive agents. optimal dosing regimens for cyclosporine in the dog remain unclear, primarily because standard methods that monitor effectiveness of immunosuppression have not been established. pharmacokinetic testing is currently used during treatment with oral cyclosporine to adjust doses based on measurement of blood drug levels. individual patients, however, often demonstrate marked variations in blood drug levels while on similar oral doses of cyclosporine, and can also demonstrate different clinical responses even at comparable drug levels, making correlation of blood cyclosporine levels and degree of disease control extremely difficult. pharmacodynamic testing offers an alternative method for regulating cyclosporine dosing by objectively measuring the effects of cyclosporine on t-cells, the drug's main cellular target in the body. our acvim foundation-funded research has focused on developing and evaluating a comprehensive panel of biomarkers of immunosuppression that can be utilized for pharmacodynamic monitoring during treatment with cyclosporine and other immunosuppressive agents that affect t-cell function. we have completed several studies using flow cytometry to evaluate activated t-cell expression of surface molecules (cd & cd ) and cytokines (il- , ifn-g & il- ) as potential biomarkers. our first study was an in vitro study evaluating expression of surface molecules and cytokines in canine t-cells exposed to varying concentrations of cyclosporine. this study established consistent drug-associated suppression of the cytokines il- , ifn-g and il- . our second study was an in vivo study in normal dogs evaluating the effects of two doses of oral cyclosporine, a high dose considered to be reliably immunosuppressive (starting dose mg/kg bid, titrated upwards as needed to attain trough drug blood levels of at least ng/ml) and a lower dose used to treat atopy ( mg/kg sid), on t-cell expression of these three cytokines. significant suppression of il- and ifn-g expression was seen at the high cyclosporine dose, while at the lower dose only ifn-g expression was suppressed. because tcell expression of il- was not significantly suppressed at the high cyclosporine dose, il- was not evaluated at the lower drug dose. because of specialized sample handling requirements, flow cytometry is not as practitioner friendly as other assays (such as pcr) for routine use in pharmacodynamic testing. we have therefore conducted an in vitro study comparing the effects of cyclosporine on activated t-cell expression of il- and ifn-g using flow cytometry and qrt-pcr, and demonstrated dose dependent and comparable suppression of il- and ifn-g using either methodology. we are currently evaluating, using qrt-pcr, the effects of oral cyclosporine on t-cell expression of il- and ifn-g in normal dogs prior to moving on to pharmacodynamic trials in our clinic patients. effect of hypothyroidism on reproduction in bitches. dl panciera , bj purswell , ka kolster , sr werre . departments of small animal clinical sciences, large animal clinical sciences, and laboratory for study design and data analysis, virginia-maryland regional college of veterinary medicine, virginia tech, blacksburg, va. numerous reproductive abnormalities, including irregular interestrous period, anestrus, and infertility have been attributed to hypothyroidism. we previously documented reduced fertility and lower birth weight and increased periparturient mortality in pups born to bitches with experimentally-induced hypothyroidism for a median duration of weeks. the purpose of this study was to evaluate reproductive function in these same bitches after hypothyroidism was treated with a replacement dose of levothyroxine. twelve multiparous bitches were studied. hypothyroidism was induced in dogs by administration of mci/kg i. hypothyroidism was confirmed by finding serum t concentrations before and hours after iv administration of human recombinant tsh that were o nmol/l. levothyroxine ( . mg/kd q h) was administered to all hypothyroid bitches. six bitches served as euthyroid, untreated controls. dogs were evaluated daily for signs of estrus and were bred by of males when serum progesterone was ! ng/ml. interestrous interval, gestation length, strength and duration of contractions during whelping, time between pups, number of live pups and stillbirths, viability of pups at birth, weight of pups, and periparturient mortality were recorded. the student's t-test and anova were used to compare differences between control and hypothyroid bitches for continuous, normally distributed data. the wilcoxon rank sum test was used to analyze data between groups that was not normally distributed. the mean duration of hypothyroidism prior to levothyroxine administration was ae . weeks. breeding took place after levothyroxine treatment for ae . weeks in the hypothyroid group. all dogs in the hypothyroid group and / control dogs were pregnant, while / hypothyroid and all control bitches became pregnant prior to levothyroxine administration. no difference in interestrus interval or gestation length was noted between groups. during whelping, no difference in strength of contractions, contraction duration, interval between pups, or viability scores of pups was found between groups. litter size, birth weight and peirparturient mortality were similar between groups. levothyroxine administration reverses the detrimental effects of hypothyroidism on fertility and neonatal health. racing sled dogs have a high prevalence of exercise-induced gastric erosions/ulcers, with reports ranging from - % of dogs running at least miles in a day or less. omeprazole reduces the severity of, but does not completely prevent, gastritis under racing conditions, and can be difficult to administer under these conditions. famotidine can be administered in food, but has only demonstrated efficacy under less intense training conditions. the purpose of these studies was to evaluate different acid suppression strategies under racing conditions for the prevention of exercise-induced gastritis. experiment # was a randomized placebo-controlled study using sled dogs ( - years) competing in a mile race over - h. treatment groups were famotidine (approx mg/kg qd) or no treatment, beginning days prior to the start of the race and proceeding until gastroscopy was performed h after the race. experiment # was a randomized positive-control study using sled dogs ( - years) running a mock race of miles in h. dogs were divided into omeprazole (approx mg/kg qd, administered min prior to a meal) or famotidine (approx mg/kg bid) groups beginning days prior to the exercise challenge and continuing for h after completion. gastroscopy was performed immediately prior to the start of dosing and h after completion of the exercise. in all cases, mucosal appearance during gastroscopy was blindly scored using previously described scoring system. famotidine ( mg/kg qd) reduced the prevalence of clinicallyrelevant, exercise-induced gastric lesions compared to no treatment ( / vs / , p . ). compared to famotidine at mg/kg bid, omeprazole significantly decreased the severity ( . vs . , p . ) and prevalence ( / vs / , p . ) of gastric lesions. although famotidine provides some benefit in the prevention of exercise-induced gastric lesions, neither the recommended dose nor the higher dose were considered acceptable in the prevention of exerciseinduced gastritis as between - % of the dogs receiving famotidine had clinically significant lesions. a previous study examining omeprazole under racing conditions, but without careful administration on an empty stomach, resulted in a % prevalence of clinically significant gastric lesions. however, the bioavailability of omeprazole is reduced in the presence of food, and when the daily administration of the drug is carefully scheduled to coincide with an empty stomach, the resulting prevalence of clinically significant lesions induced by racing-intensity exercise is reduced to just over %. the conclusions of these studies are that omeprazole is superior to famotidine in preventing gastritis in racing sled dogs during competition. routine administration of omeprazole is recommended to prevent stress-associated gastric disease in exercising and racing alaskan sled dogs. mares may be an important source of environmental contamination with rhodococcus equi on breeding farms. attempts to reduce fecal shedding of r equi by the mare and the effects of the mare's fecal r equi concentration on airborne concentrations in the foaling stall have not been previously reported. twenty-one arabian mares were treated daily with either oral gallium nitrate or placebo in a randomized double-blind study. fecal samples were collected at day of gestation (time ), the week before foaling (time ), and the week after foaling (time ). airborne concentration of r equi were measured in the stall within hours post foaling using a microbial air sampling system into which standard ( -mm) culture plates with a media selective for r. equi have been loaded. concentration of total r equi were determined by morphological characteristics. the concentration of virulent r equi was determined using a modified colony immunoblot method. concentrations of total and virulent r equi were compared among mares to examine effects of treatment, time, and treatment by time interaction. there were significant (p o . ) effects of treatment that depended on time of sample collection. at sample times and there were no significant differences between groups in the fecal concentration of virulent r equi. at time concentrations of virulent r equi were significantly lower among mares in the treatment group (p o . ) compared to control. effects of time depended significantly on groups: for the control group, there were no significant effects of time. for the treatment group, concentrations tended to decrease over time, and concentrations at time were significantly (p o . ) lower than those at time . no other differences among times for concentrations in the treatment group were statistically significant. there were no significant effects of treatment, sample time, or their interaction on the concentration of total r equi between groups; however, the pattern for these data was similar to that observed for the virulent isolates. no significant differences were determined between treatment groups for airborne concentrations of virulent or total r equi. treatment of mares with oral gallium nitrate significantly reduced the fecal concentrations of virulent r equi over time, but had no impact on the airborne concentration of r equi shortly after foaling. the purpose of this study was to evaluate the protein profile of bronchoalveolar lavage fluid (balf) in horses affected with recurrent airway obstruction (rao) and in control horses using proteomics and western blot techniques. rao-affected (n ) and control horses (n ) were subjected to an experimental exposure trial; when the rao-affected horses showed clinical signs of disease, balf was collected from all horses. balf was also collected from client-owned rao-affected horses (n ) with naturally-occurring clinical signs of disease and client-owned control horses (n ) from the same environments. the balf from the experimental exposure trial horses was subjected to trypsin digestion and proteomics analysis with mass spectrometry (ms). peaks detected with ms were identified using tandem ms analysis and database searches. western blot was used to confirm the identity and expression levels of two proteins identified using proteomics techniques in the balf of all horses. data from ms experiments were analyzed with the student's t-test to compare peak intensity between rao-affected and control horses. western blot band density data was analyzed with the kruskal-wallis anova for comparison between groups of horses. significance level was set at p o . . with ms proteomic analysis of the balf from the experimental exposure trial horses, total peaks (peptides) were identified. of these peaks, were differentially expressed between the rao-affected ( over-expressed) and control horses ( over-expressed). identifications were made for balf proteins. transferrin and secretoglobin were chosen for validation with western blot. proteomics indicated that secretoglobin was not differentially expressed between the experimental exposure trial group; this was confirmed with western blot analysis. western blot also showed that clientowned rao-affected horses had lower secretoglobin expression than client-owned control horses and control horses before experimental exposure. according to the proteomics data, transferrin was over-expressed in control horses after experimental exposure compared to rao-affected horses. while the western blot analysis did not show a statistically significant difference in this comparison, transferrin was significantly over-expressed in control horses before experimental exposure compared to client-owned rao-affected horses. in addition, both secretoglobin and transferrin band densities on western blot were negatively correlated with airway obstruction and neutrophilic pulmonary inflammation. this study demonstrates that proteomics techniques can be used in the investigation of equine balf proteins. the proteins identified as differentially expressed between rao-affected and control horses in this study including, but not limited to, secretoglobin and transferrin should undergo further evaluation for their use as biomarkers of rao, and as potential targets of new therapeutic agents for rao. cardiotoxic effects of rattlesnake venom in the horse are not well defined. the first aim of this study was to document cardiac damage in naturally envenomated horses. twenty horses with clinical diagnosis of snake bite were included. a snake venom elisa was utilized to confirm envenomation when possible. serum and plasma were collected at selected intervals. plasma was assayed for cardiac troponin i (ctni) using a flurometric assay (stratus cs s , dade behring). holter monitors (zymed s , philips) were placed at admission, week and month post presentation. echocardiography was performed on available horses - months after envenomation. the second aim of this study was to investigate potential mechanisms of the cardiac damage. serum samples were assayed for tnfalpha using a commercial assay (endogen). antibody titers to crotalus atrox venom were measured at admission, week and month after natural envenomation and compared to titers in vaccinated horses (crotalus atrox toxoid, red rock biologics). a significant number of horses showed elevations in ctni (p o . ) at one or more time point indicating myocardial damage. holter readings revealed the presence of arrhythmias or persistent tachycardia in horses. five of twenty horses were available for echocardiography; no abnormalities were noted. horses with increased ctni tended to have greater tnfalpha concentrations compared with horses without increased ctni. peak venom titers in bitten horses were significantly higher than peak titers in vaccinated horses (p o . ). rattlesnake envenomation was associated with evidence of cardiac damage in a significant proportion of bitten horses. further studies are needed to determine the cause as well as mechanisms to treat and/or prevent its occurrence. little is known about the gastric mucosal flora in healthy horses and its role in gastric disease has not been critically examined. our laboratory previously reported that a diverse microbial flora with a predominance of streptococcus spp. and lactobacillus spp. exists in healthy horses using fluorescence in situ hybridization (fish). the present study sought to further characterize the gastric mucosal flora of healthy horses using massive parallel srrna bacterial tag encoded flx-titanium amplicon pyrosequencing (btefap). biopsies of the squamous, glandular, antral and any ulcerated mucosa were obtained from healthy horses via gastroscopy after a -hour fast and horses immediately post-mortem. dna was extracted from the mucosal biopsies and btefap and data processing was performed. hierarchical cluster analysis based on relative abundance data on the genus level were performed to look for trends in bacterial diversity among the individual horses. pyrosequencing yielded between , and , reads per horse with , , reads in the antrum, squamous and glandular regions, respectively. the microbiome segregated into two distinct clusters: cluster comprised of horses that were stabled, fed hay and sampled at post-mortem and cluster consisted of horses that were pastured on grass, fed hay and biopsied gastroscopically after a -hour fast. samples from different antomic regions clustered by horse rather than region. despite being very similar at the higher taxonomic level (phyla) differences in the distribution of bacteria were seen at the genus and species level. the dominant bacteria in cluster horses were firmicutes ( % reads/sample) consisting of mainly streptococcus spp., lactobacillus jensenii, l. fornicalis and sarcina maxima. cluster had more diversity with a predominance of proteobacteria, bacteroidetes and firmicutes and genera identified such as streptococcus spp., moraxella spp., actinobacillus spp., and others. though the relative abundance of the individual taxonomic groups was significantly different between individual horses, no significant differences in the overall diversity could be found (as assesed by shanon weaver, ace and choa i diversity indices). helicobacter spp. sequences were not identified in any sample (out of , reads). the ulcerated mucosa from horse (group ) had lower diversity and higher numbers of bacteria predominated by lactobacillus equigenerosi. this data shows that the equine gastric mucosa harbors an abundant and diverse microbiome which is unique to each individual and differs by sampling method, fasting prior to sampling and diet. seasonal pasture myopathy (spm; atypical myopathy [am] in europe), typified by nonexertional rhabdomyolysis, occurs in pastured horses during autumn or spring. clinical signs rapidly progress from muscular weakness to recumbency and frequently death. extensive myonecrosis and intramyofiber lipid storage occur in highly oxidative respiratory and postural muscles. recently, a defect of lipid metabolism called madd has been identified in european horses with am. this report documents the first cases of equine madd in the united states. six midwestern us horses suspected of having spm in the spring or fall of were evaluated for madd by urine organic acids, plasma acylcarnitines and/or muscle carnitine and histopathology. five horses had clinical signs and clinicopathologic data consistent with severe rhabdomyolysis. one horse was found dead on pasture after days of rear limb stiffness and inappetance. urinary organic acid profiles revealed markedly elevated ethylmalonic and methylsuccinic acids, butyrylglycine, isovalerylglycine, and hexanoylglycine, consistent with equine madd. plasma acylcarnitine profiles from horses had marked elevations of short chain acylcarnitines, while the third horse and only survivor had minor elevations of short chain acylcarnitines. affected muscle showed extensive degeneration with intramyofiber lipid accumulation, a marked decrease in free carnitine, and high levels of carnitine esters. spm appears to be a highly fatal emerging disease of pastured horses in the us characterized by weakness, colic-like signs and myoglobinuria. the disease is associated with a defect in muscular lipid metabolism that can be diagnosed by performing lipid staining of muscle samples and urine organic acid profiles. candidatus mycoplasma haemolamae (cmhl) is a common red blood cell parasite of new world camelids. the high degree of parasitemia that develops in an infected splenectomized animal allows for the efficient collection of parasitic dna. this dna can then be used in the development of genetically-derived tools such as pcr and in-situ hybridization. thus, one splenectomized animal can replace many immunologically intact animals within a research setting. the purpose of this study was to track the natural progression of cmhl parasitemia and associated clinical signs in a splenectomized alpaca. an intact, -month-old, . kg male alpaca was used in this study. he had tested positive via pcr for cmhl on three different occasions, although no organisms were seen on peripheral blood smears. the alpaca was placed under general anesthesia and a ventral midline incision was made. the spleen was located, the vessels ligated, and the organ removed. buprenorphine and flunixin meglumine were given for and days after surgery respectively. body weight, attitude, rectal temperature, blood glucose, and pcv were recorded daily. in addition, a peripheral blood smear was examined daily and the percent of red blood cells that were infected with mycoplasma organisms was determined. the alpaca was not parasitemic prior to surgery. one percent of the rbc's contained mycoplasma on days and after splenectomy. parasitic bloom developed on day with % of the red blood cells infected, and over % containing or more organisms. the alpaca was treated with mg/kg oxytetracycline i.v. on day . on postoperative day no parasites were seen in the peripheral blood. the peripheral blood remained free of parasites for days. on the morning of the th day, % of the peripheral red blood cells contained mycoplasma. by late that afternoon, % of the observed rbc's contained - organisms. the alpaca again received oxytetracycline. there were no more parasites observed from that time until the alpaca was euthanized days later. the alpaca lost . kg between days À and after surgery. his weight fluctuated between . and . kg for the remainder of the study period. blood glucose ranged between and mg/dl there was no major change in pcv (range - %), a finding that was expected as the spleen was not available to remove infected red blood cells. body temperature ranged between . and degrees celsius except for days and when more than % of red blood cells contained parasites. on those days rectal temperature reached . and . degrees respectively. this study confirmed that a non-parasitemic, yet pcr positive alpaca did indeed harbor cmhl. the time from splenectomy to parasitic bloom was shorter, and the length of oxytetracycline suppression longer than has been observed in other species. gastro-intestinal (gi) disease frequently results in increased wall thickness in many species. identification of changes in gi wall thickness using ultrasound has proved to be a useful diagnostic tool and is widely used in human patients, small animals and horses. although gi motility has been evaluated in cattle, normal reference ranges for wall thickness has not been reported in ruminants. the aims of this study were to report normal values for wall thickness of various gi structures and to assess the repeatability of this technique in adult dairy cows, sheep and goats. eight healthy adult holstein friesian (hf) cattle ( ae kg), eight jersey (j) cattle ( ae kg), thirteen adult sheep ( ae kg) and eleven adult goats ( . ae kg) were recruited and examined on three consecutive days. ultrasonographic images were optimised for the structure of interest. structures were identified based upon appearance and anatomical position. a minimum of three cineloops were obtained of the abdominal organs per intercostal space (ics) and three along the ventral midline in each ics; images were analysed offline. data were analysed using anova and post-hoc bonferoni, student's ttest and intra-class correlation coefficients. each structure was measured per ics per species; if no differences were noted for structures in different ics, then measurements were pooled. no differences were noted between hf and j cattle so data were pooled. data are displayed in table . good repeatability (icc . ) was obtained for all measurements and no differences were noted between animals of the same species or between days. these measurements for assessment of normal gi thickness are repeatable and may allow valuable additional information to be gained from ruminants with gi disease. ocular infections with the infectious bovine keratoconjunctivitis (ibk) agent moraxella bovis (m. bovis) are associated with significant economic loss in the cattle industry.although antibiotic therapy is the treatment of choice for ibk, treatment failures are common and current vaccines are not optimally effective mainly due to antigenic variation. as a result, our laboratory has been actively investigating the therapeutic potential of bdellovibrio bacteriovorus j (b. bacteriovorus); a predatory bacterium capable of attacking and inducing lysis of gram-negative bacteria, as a new treatment for ibk. we have previously shown that b. bacteriovorus can reduce the number of m. bovis attached to bovine epithelial cells in an in vitro model of ibk and that b. bacteriovorus can be trained to kill m. bovis as effectively as e. coli using serial passages. in this study, we hypothesized that b. bacteriovorus can remain viable in bovine tears without its prey for up to hours. this hypothesis was addressed by incubating inocula of active b. bacteriovorus in its preferred media peptone yeast extract (pye) and comparing b. bacteriovorus viability in bovine tears or phosphate buffered saline (pbs) at time , , , , and hours. using a plaque assay to quantify the mean amount of plaque forming units (pfus) of b. bacteriovorus exposed to each treatment, it was determined that viability of b. bacteriovorus over time was comparable between treatment groups. overall, the results supported that b. bacteriovorus can remain viable in tears for up to hours in the absence of prey bacteria. further studies are needed to determine the therapeutic potential of b. bacteriovorus in an in vivo model of ibk. correction of the measured ionized calcium concentration (cca ) to a ph . is routinely applied in experimental studies in order to assist in the interpretation of measured values relative to a reference range. the equation most commonly used for ph correction in bovine plasma is: cca ph . cca  (- . Â{ . -ph}) . the validity of this equation for bovine plasma is unknown. accordingly, our first objective was to characterize the in vitro relationship between cca and ph for bovine plasma. feeding rations with a low dietary cation-anion difference (dcad) during late gestation mitigates periparturient hypocalcemia in dairy cows, particularly when chloride containing acidodgenic salts are fed. the mechanism for this beneficial effect remains unclear. our second objective was to determine whether hyperchloremia displaces calcium from binding sites to albumin, thereby increasing cca . the in vitro relationship between plasma log(cca ) and ph in was investigated using lithium heparin anticoagulated blood from healthy holstein-friesian calves. plasma was harvested and tonometered with co at c over a ph range of . - . . plasma chloride concentration (ccl -) was altered by equivolume dilution of plasma with electrolyte solutions of varying ccl -( ae , ae , and ae meq/l; mean ae sd). the slope of the linear regression equation relating log(cca ) to ph for tonometered plasma samples from the calves was - . ae . at normal values for cca ( . ae . meq/l), albumin concentration ( . ae . g/l), and ccl -( . ae . meq/l). the experimentally-determined value for the slope for bovine plasma was identical to that determined previously for human plasma. the formula for correcting cca in bovine plasma for change in ph from . is therefore: cca ph . cca  (À .  { . -ph}) . this equation is only valid at normal concentrations of albumin and chloride in plasma. equivolume dilution of plasma by electrolyte solutions of varying cclindicated that cca ph . increased by . meq for every meq/l increase in ccl -. in other words, plasma cca at a given ph increases directly in response to an increase in plasma ccl -, presumably because the additional chloride displaces calcium that is electrostatically bound to albumin. furthermore, the increase in cca is independent to the change in plasma ph induced by an increase in ccland decrease in plasma strong ion difference. our finding that hyperchloremia directly increases plasma cca provides an additional mechanism by which ingestion of high chloride (acidogenic) rations prevents the clinical signs of periparturient paresis. our finding is consistent with the results of other studies that indicate acidogenic salts that contain chloride as the predominant anion (ie, nh cl, cacl ) are more effective in increasing cca than equimolar quantities of acidogenic salts such as mgso . coagulase negative staphylococci (cns) are among the most common bacteria isolated from the bovine mammary gland. historically, these bacteria were lumped together as minor mastitis pathogens. modern molecular techniques have allowed accurate speciation and fingerprinting of the cns species. these methodologies have recently been applied to the study of cns in bovine mastitis. the aim of the studies presented here was to evaluate the role of individual cns species on milk somatic cell count (scc) and duration of intramammary infection (imi). in the first study, mammary quarter foremilk samples were aseptically collected from all lactating cattle ($ head) at the university of missouri dairy research center once monthly for months for bacterial culture and milk scc. staphylococcal isolates were speciated by sequencing the rpob gene and strain-typed using pulsed-field gel electrophoresis (pfge). using species and fingerprint data along with published definitions for staphylococcal imi, cns imis were identified. overall, species of cns were identified with staphylococcus chromogenes, s. cohnii, s. epidermidis, and s. simulans being most prevalent. duration of imi and scc data were analyzed using regression models accounting for repeated measures. mean milk scc and duration of imi were found to differ between cns species (p o . ). although most imis were of short duration ( month), staphylococcus capitis and s. chromogenes imis had longer mean durations of infection than or more of the other species isolated. mean sccs were under , cells/ml in most cases. however, staphylococcus simulans and s. xylosus imis were more inflammatory (mean , cells/ml) and had a higher mean scc than s. cohnii, s. epidermidis, and s. haemolyticus. to examine the relationship between cns imi and milk scc in a larger population of cattle, cns isolates from the canadian bovine mastitis research network (cbmrn) culture collection were obtained for speciation. speciation and fingerprinting were performed as above. isolates were from subclinical imi from before and after the dry period and from subclinical imi during lactation. data associated with each isolate were obtained from the cbmrn database. nine-hundred-thirty-eight isolates from mammary quarters in herds were successfully speciated. twenty-two different species of cns were identified. staphylococcus chromogenes was the most frequent species identified accounting for % of the infections. three species, s. chromogenes, s. xylosus, and s. simulans accounted for % of all infections. data were analyzed using a linear hierarchical repeated measures mixed model. differences in mean scc were found between some cns species and culture negative control quarters and also between different species of cns (p o . ). overall, our data demonstrate potential differences in pathogenicity between strains of cns that cause bovine mastitis. passive transfer of maternally derived antibodies via ingestion of good-quality colostrum within the first hours of life is crucial for the health and future productivity of dairy calves. however, infectious diseases can be transmitted via colostrum feeding, which may require use of a colostrum replacement product or pasteurization to decrease disease transmission. while pasteurization of colostrum is effective for sterilization, heating during pasteurization can alter the viscosity of colostrum, destroys important nutritional biomolecules, and has been shown to decrease colostral igg concentrations. the purpose of this study was to investigate the effect of high pressure processing (hpp) on the viscosity, igg concentration, and bacterial contamination of bovine colostrum. first milking colostrum samples were collected from cows from different farms, and ml aliquots of each sample were pooled for analysis. pooled colostrum was processed in triplicate using an isostatic press at mpa ( , psig) for , , , , and minutes. samples were tested for the effects of hpp on the viscosity, bacterial load (cfu/ml), and igg concentration. there was a significant decrease (p o . ) in bacterial load at each time point when compared to time . no significant difference in igg concentration was found between any time points. subjectively, the colostrum viscosity appeared to increase with the processing time, though the rheologic assessment has not been completed at this time. hpp appears to be an effective method to decrease bacterial contamination of colostrum while maintaining appropriate igg concentrations. minimizing the processing time or pressure may be necessary to maintain an acceptable viscosity of the colostrum. based on these results, additional studies are justified in order to determine the optimum combination of processing time and pressure and the effect of hpp on specific bovine pathogens. the heme-associated iron-binding apoprotein lactoferrin (lf) is known for its, anti-inflammatory, anti-parasitic, antimicrobial and bactericidal effects. lactoferrin demonstrates ubiquity throughout mammalian host biological fluids: saliva; tears; mammary secretions, as well as at mucosal surfaces. it is also released from immune cells under pathogenic stimulation. the purpose of this study which has been approved by western university's institutional animal care and use committee is to further characterize the mechanisms through which lf modulates inflammation in the face of bacterial endotoxin. it was hypothesized that lf would inhibit p phosphorylation. numerous studies speak to the ability of lf to alter leukocyte function, inhibit cytokine production, and bind lipopolysaccharide (lps); mechanisms through which it is believed to achieve its anti-inflammatory effects. recently, investigators demonstrated its ability to interact with host dna while others describe regulation of granulocyte adhesion and motility; elucidating its roles in the apoptotic signaling. in earlier studies, dawes me, et al. demonstrated lactoferrin's ability to limit the expression of inducible cyclooxygenase- and the gelatinase, matrix metalloproteinase - by lps-induced macrophages. the generation of these inflammatory mediators is modulated by pro-inflammatory cytokines such as interleukin- b (il- b) and tumor necrosis factor-alpha (tnf-a), the production of both being dependent on signaling through the p mitogenactivated protein kinase (mapk) pathway. peripheral mononuclear cells (  )isolated from buffy coat cells of healthy neonatal to -month old holstein calves were cultured in the presence and absence of lf ( ng/ml), lps ( mg/ml), anisomycin ( mg/ml), a known p activator -the positive control, and mm of sb , a known p inhibitor -the negative control. sample lysates obtained post culture was subjected to immunoprecipitation and kinase reactions. reactions were terminated under reducing conditions and evaluated using western immunoblotting. phosphorylation of activated transcription factor- (atf- ) by phosphorylated p served as the marker of investigation. immunologically reactive atf- expression by lps and anisomycin-treated cells was compatible with a prominent band at kd. evidence of lf-induced inhibition of lps-induced p- activation was observed in lanes representative of co-cultures of lf lps; lf anisomycin; and anisomycin sb , which was demonstrated by decreased immunological reactivity at kd. the findings here, suggest that lf interferes with lps-induced p- activation of transcription factor atf- , in vitro. this serves as additional proof of its potential use in attenuating the systemic effects of lps. six ( ) clinically normal, purpose-bred cats of similar age and body condition were imaged with [ f] fluorodeoxyglucose ([ f]fdg) and [ f]ftha by using dynamic cardiac-gated fused pet/ct for kinetic assessment of myocardial glucose and fatty acid uptake and metabolism, respectively. kinetic tracer uptake within the myocardium was achieved by initiating image data acquisition simultaneously with tracer injection. pet data were acquired over a hour period with the heart in the center of the scanner field of view. regions of interest were drawn in the left ventricular wall and thoracic aorta for the purpose of measuring the kinetics of tracer redistribution. serial blood samples were also taken during pet imaging for comparison with image data. the equilibrium biodistribution of both tracers was documented hour post-injection in a whole body pet/ct image. standard echocardiographic examination of cardiac structures was also performed. both radiotracers remained in the plasma fraction; however, [ f]ftha was cleared from the more rapidly than [ f]fdg (t / $ and $ min, respectively). the tracers were readily visualized within the feline myocardium in dynamic pet images and analysis of the blood pool clearance from the kinetic image data agreed with blood sampling data. myocardial uptake of each tracer was best described by a double exponential analysis and was rapid but variable among animals (range - bq/cc/min), although blood glucose levels were similar in all cats during image acquisition. physiologic [ f]fdg was observed in the brain, salivary tissue, gastrointestinal tract, renal pelves and urinary bladder, with [ f]ftha seen in the myocardium, liver and renal cortex. all cats were normotensive with normal echocardiographic parameters. this study demonstrates the utility of kinetic imaging using the left ventricle (lv) shape has been suggested to change from elliptical to more globular in response to chronic volume overload. real-time three-dimensional echocardiography (rt de) offers new modalities for lv assessment. the aim of the study was to investigate left ventricular changes in shape and volume occurring in response to different severities of naturally acquired myxomatous mitral valve disease (mmvd) in dogs using rt de. privately owned dogs were classified by standard echocardiography into: healthy ( ), mild ( ), moderate ( ) and severe mmvd ( ). a lv cast was obtained using semi-automated endocardial border tracking from rt de dataset, from which global and regional (automatically acquired basal, mid, and apical segments based on lv long-axis dimension) end-diastolic (edv) and endsystolic volumes (esv), lv long-axis dimension and rt de sphericity index, were derived. global and regional edv and esv increased significantly with increasing mmvd severity, assessed by mmvd group-wise comparisons and linear regression analyses using left atrial to aortic root ratio, and lv end-diastolic and end-systolic dimensions. all three segments contributed to the overall increased global volumes, but the mid edv segment was strongest associated with increasing lv end diastolic dimension (p . ). furthermore, lv long axis distance and lv sphericity index increased with increasing mmvd severity. the basal and apical edv segments were strongest associated with sphericity index (p o . ). in conclusion, this rt de study showed that increased lv edv, primarily in the mid segment, leads to rounding of lv apical and basal segments in response to increasing mmvd severity in dogs. dogs from shelters in florida with naturally acquired di infection were euthanized and necropsied. all adult di in each dog were sexed using morphological features. total worm burdens and numbers of males and females were recorded. no other information was available for any dog. all data, raw and transformed, were examined visually and descriptively. raw numerical data were further examined by a paired t-test; log-odds transformed data were examined by logistic regression. we also conducted a binomial distribution goodness of fit analysis assuming a null hypothesis of a m:f . . worm intensities ranged from to di per dog. eight dogs had unisex infections: / had all-female infections. dogs with lowintensity dual-sex infections were more likely to have greater numbers of female di. overall, sex ratios were equal (paired t-test, p . ). however, logistic regression demonstrated that the probability of being female is strongly affected by the total worm intensity, with lower intensities increasing the probability of having a predominance of female worms. our data show that di sex ratios in naturally-infected dogs equal when examining the entire dog population, but deviate to favor female worms at low worm intensities. these data could impact adulticide treatment strategies. the reasons for sex ratio distortion in di are unknown. we evaluated cardiac reverse remodeling after mitral valve repair under cardiopulmonary bypass (cpb) for mitral regurgitation in small breed dogs. fifty dogs (body weight . - . kg, age - years) with mitral regurgitation were treated between august and november . the cardiac murmur was grade / - / . the preoperative chest x-rays showed cardiac enlargement (vertebral heart scale (vhs) . - . ). echocardiography showed severe mitral regurgitation and left atrium enlargement (la/ao . - . ). after inducing anesthesia, a thoracotomy was performed in the fifth intercostal space. cpb was started by using a cpb circuit connected to carotid artery and jugular vein catheters. after inducing cardiac arrest, the left atrium was sectioned and chordae tendineae rupture confirmed. the chordae tendineae were replaced with expanded polytetrafluoroethylene. a mitral annulus plasty was also done, and the left atrium was closed. after de-clamping for restarting the heart, the chest was closed. heart rate decreased from - bpm to - bpm. the grade of cardiac murmur was reduced to / - / three months postoperatively, and the heart shadow was reduced (vhs . - . ) in the chest x-rays. echocardiography confirmed the marked reduction in mitral regurgitation and the left atrial dimensions (la/ao . - . ). mitral valve repair reduced enlarged cardiac size by reduction of regurgitant rate. pulmonary arterial hypertension (pah) is a well recognized condition in dogs leading to considerable morbidity and mortality. the majority of therapeutics has focused on endothelial dysfunction causing reduced production of vasodilators, such as nitric oxide and prostacyclin, coupled with overproduction of vasoconstrictors, such as endothelin- . more recently, it has been shown that the mitochondria play an important role in the development of pah as oxygen sensors and regulators of cellular proliferation. in pah, pulmonary artery smooth muscle cells undergo a metabolic shift from oxidative phosphorylation in the mitochondria to glycolysis in the cytoplasm as the major energy source and this leads to suppression of apoptosis and increased proliferation. dichloroacetate (dca) inhibits pyruvate dehydrogenase kinase to activate pyruvate dehydrogenase which catalyzes the rate limiting step for entry of pyruvate into the krebs cycle, thus increasing mitochondrial respiration. in three different rat models of pah, dca has been shown prevent and reverse pah by normalizing molecular pathology, stimulating apoptosis of pulmonary artery smooth muscle cells, and reducing pulmonary artery hypertrophy. dca has known toxic effects, including reversible hepatotoxicity and peripheral neuropathy, and has not been studied in any species with naturally occurring pah. the objective of this open label pilot study is to evaluate the therapeutic and toxic effects of dca in naturally occurring canine pah. three dogs with pah diagnosed by doppler echocardiography and no correctable underlying cause are enrolled in the study. dogs are orally administered mg/kg of dca divided daily for weeks, and then . mg/kg of dca divided daily for the remainder of the study. at baseline, , , , and weeks, an echocardiogram, cbc, serum chemistry profile, urinalysis, nt-probnp, blood uric acid, blood lactate, noninvasive blood pressure, nerve conduction study, and trough dca level ( hr post-dose) are obtained. the measured echocardiographic parameters include peak and mean tricuspid regurgitant flow velocity and pressure gradient, peak and enddiastolic pulmonary regurgitant flow velocity and pressure gradient, pulmonary valve flow velocity acceleration time and ejection time, pulmonary valve flow velocity time integral, right ventricular myocardial performance index, tricuspid annular plane systolic excursion, and systolic tricuspid annular tissue velocity. variables are inspected for normalcy and equality of variances and a two-sided paired t-test is used to compare the variables before and after treatment at each evaluation time. the basis for the role of the mitochondria in pah and the results of this pilot study will be presented to determine if dca warrants further study as a therapy for dogs with pah. study produced the strongest associations between the ncl phenotype and cfa markers. all ncl-affected tibetan terriers were homozygous for the same haplotype which extended for consecutive snps spanning . mb. none of the annotated genes within this target region had previously been associated with human or rodent ncl. we used dna from ncl-affected tibetan terriers to resequence the coding regions and intron-exon borders of several genes harbored within the target region and found a single base pair deletion, c. delg, in exon of positional candidate atp a . this deletion produces a frame shift and a predicted premature termination codon. we genotyped all tibetan terrier dna samples in our collection and found all ncl-affected tibetan terriers to be homozygous for the c. delg allele. eleven additional c. delg homozygotes were either less than years old, or lost to follow up. there were no known cases of ncl in the remaining tibetan terriers which were either heterozygous (n ) or homozygous for the ancestral allele (n ). atp a is a member of group of ion transport genes and has been associated with lysosomes. mutations in human atp a cause kufor-rakeb syndrome (krs), a rare neurodegenerative disorder with clinical features that include parkinsonism plus spasticity, supranuclear upgaze paresis, and dementia. post-mortem findings in krs have not been reported. we conclude that ncl in tibetan terriers is caused by a mutation in atp a . our results suggest that krs may be a form of adult onset ncl in humans. niemann-pick type c (npc) disease is a progressive neurological disorder characterized by dementia and ataxia, hepatic and pulmonary disease, and death typically within the first or second decade. despite the identification of causative mutations, the pathogenesis is not clear and therapies to successfully treat npc disease have been ineffective to date. the recent use of intravenously administered -hydroxypropylbeta-cyclodextrin (hpbcd), an fda-designated orphan drug (may ), in a small number of children with npc disease is based on favorable treatment outcome data in subcutaneously treated mouse and cat models. to rigorously evaluate the mechanistic, pharmacologic, and toxicity issues associated with hpbcd therapy in npc disease, we have utilized the spontaneous feline npc model harboring a missense mutation in npc (pc s), orthologous to the most common mutation in juvenile-onset patients. the feline npc model has clinical, neuropathological and biochemical abnormalities similar to those present in juvenile-onset patients making this model homologous to the most common disease form seen in human patients. we identified that intrathecal administration of hpbcd ameliorated all clinical aspects of neurological disease at least up to weeks of age (an age when untreated cats die) but had no effect on hepatic disease. we identified that while subcutaneous therapy with hpbcd at all doses ameliorated liver disease, only mg/kg substantially affected neurological disease but also resulted in early death due to pulmonary toxicity. finally, we identified a dose-related toxic effect of hpbcd on hearing function that had not been described in any other species. leukodystrophies are disorders of myelin synthesis and maintenance that affect cns myelin. they are subdivided as leukodystrophies, hypomyelinating disorders and spongy degenerations. although infrequently seen, several forms have been described in various dog breeds. we present a novel form of complex leukodystrophy consisting of hypomyelination and spongy degeneration that presents primarily with hind end tremors in border terrier puppies. three border terriers from two different litters (and lineages) are described here that presented with a history of shaking movements. the youngest dog was a -week old male. it was the only dog affected in the litter. the other two dogs were -week old female littermates. there were two unaffected males in the same litter. physical examination revealed no abnormalities. on neurological examination, the affected dogs displayed severe hind end tremors, with a characteristic swinging side-to-side movement (best described as ''rumpshaker''). the tremors also involved the head and thoracic limbs but to a lesser degree, and disappeared when the dogs were asleep or at rest. severe cerebellar ataxia was observed when the dogs ambulated. proprioceptive positioning was delayed in the pelvic limbs of all dogs. spinal reflexes and nociception appeared normal. necropsy was performed in all puppies. no macroscopic changes were observed. histologic evaluation of the cns revealed spongy degeneration and hypomyelination in all funiculi of the cervical and thoracic spinal cord. white matter of the frontal, temporal and parietal cortices had mild multifocal spongy degeneration and hypomyelination, whereas white matter of the cerebellum, medulla and pons showed severe diffuse spongy degeneration and hypomyelination with gliosis. the combination of reduced myelin formation combined with spongiform white matter changes in the absence of microglial responses suggest a complex pathogenesis affecting both oligodendrocytes' capacity to synthesize myelin and the stability of the myelin that was formed. the number of oligodendrocytes and axons appeared subjectively normal indicating a primarily hypomyelinating process. the clinical and pathological features of this disease have not been described in any other canine leukodystrophy. the primary and most striking clinical feature is the presence of severe tremors in the hind end, causing the ''rumpshaker'' pheynotype. genetic studies are underway to determine if the disease is inherited and the inheritance mode. a syndrome of border collie collapse (bcc) appears to be common in dogs used for working stock. this syndrome has also been called malignant hyperthermia, heat intolerance, exerciseinduced collapse and ''wobbles''. a presumptive diagnosis of bcc can only be made by eliminating other causes of exercise intolerance and weakness. the purpose of this study was to describe the clinical features of collapse in affected dogs and determine if there were characteristic clinical or laboratory features at rest or after exercise that could aid in diagnosis. seven adult border collies with a history of collapse during sheep herding (affected) and adult border collies regularly used for sheep herding but showing no signs of exercise intolerance (normal) were evaluated before and after participating in a videotaped minute exercise protocol consisting ofa series of continuous short outruns and fetches of three sheep in an outdoor pen. exercise was halted at minutes or earlier if there were signs of gait or mentation abnormalities. pre-exercise evaluation included physical examination, orthopedic and neurological exam. pre and immediate post exercise rectal temperature, pulse and respiration, patellar reflexes, ecg, cbc, serum biochemistry profile, cortisol, arterial blood gas and plasma lactate and pyruvate concentrations were measured. clinical parameters (gait, temperature, reflexes) and lactate and pyruvate concentrations were evaluated at intervals up to minutes after exercise. additional testing in affected dogs included measurement of acetylcholine receptor antibodies (achrab) and dna testing for dynamin-associated exercise induced collapse (deic) and the ryanodine receptor mutation associated with canine malignant hyperthermia(mh). one week after exercise affected dogs had thoracic radiographs and echocardiography performed and were anesthetized for emg and muscle biopsies. there were no significant differences in temperature, pulse, respiration, or any laboratory parameter at any time point between normal and affected dogs. no arrhythmias were detected. affected dogs were negative for the dna mutations tested and for achr ab. thoracic radiographs, echocardiograms, emgs and muscle biopsies were normal. the normal dogs had no alterations in mentation or gait during or after exercise. three of the affected dogs had exercise halted early ( min- min) because of altered gait or mentation. all of the affected dogs were abnormal in the minutes following exercise. abnormalities seen in all dogs included disorientation, dull mentation, swaying, falling to the side, exaggerated lifting of limbs each step, choppy gait, delayed limb protraction, scuffing of rear and/or forelegs, and crossing legs when turning. all dogs returned to normal by minutes. bcc appears to be an episodic nervous system disorder that can be triggered by exercise. genetic testing excluded deic and the described canine mh mutation. common causes of exercise intolerance were eliminated, but the cause of collapse in bcc was not determined and no clinical or biochemical marker to aid diagnosis was established. equine cushing's disease (ecd) is common in older horses. the purpose of this study was to determine the frequency of diagnosis, identify prognostic factors and assess owner satisfaction with treatment. the study was a retrospective cohort design evaluating equine accessions reported to the veterinary medical data base (vmdb) and the ohio state university from - . proportional accessions, annual incidence and demographic characteristics of horses with ecd were compared with all accessions in the vmdb. medical records for a subset of horses were extracted and owners contacted to obtain long-term follow up information. two hundred seventeen new cases of ecd were reported to the vmdb. incidence increased from . / , in to . / , in . eighty-one percent of horses were ! years of age. average delay from onset of signs to diagnosis was days (range to , days). hirsutism ( %) and laminitis ( %) were the most common clinical signs. improvement in one or more signs months after diagnosis was reported by / ( %) of horse owners. none of the clinical or laboratory data were associated with survival and, % of horses were alive, . years after diagnosis. / ( %) of horses were euthanatized and / ( %) were euthanatized due to conditions associated with ecd. twenty-eight of ( %) of horse owners said they would treat a second horse for ecd. ecd is becoming a more frequent diagnosis. fifty percent of horses survived . years after diagnosis and owners were satisfied with the horse's quality of life. supported by centers of excellence in livestock diseases and human health, college of veterinary medicine, university of tennessee. the role of the hypothalamic-pituitary-adrenal (hpa) axis in sepsis has been a subject of a great deal of research. the role that the somatogenic axis plays in sepsis is less well understood and how these two axes interact during critical illness is not clear. the purpose of this study was to assess inter-relationships of adrenocorticotropin (acth), cortisol, and insulin-like growth factor-i (igf-i), in septic and non-septic term foals. blood samples were obtained from term septic foals less than days of age (n ) admitted to texas a&m university veterinary medical teaching hospital or mid-atlantic equine hospital. the foals were classified as septic by a sepsis score ! and/ or a positive blood culture. non-septic term foals less than days of age (n ) and having a sepsis score o and a negative blood culture, were obtained from texas a&m university veterinary medical teaching hospital and mid-atlantic equine hospital. plasma and serum were processed from whole blood collected by jugular venipuncture upon admission, at hours post admission and at days post admission or at the time of discharge. plasma concentrations of acth, and serum concentrations of cortisol and igf-i were determined by specific rias. data were analyzed using linear mixed-effects modeling with foal modeled as a random effect and day of admission modeled as an ordered categorical variable; post-hoc testing of pair-wise comparisons was made using the method of sidak. significance was set at p o . , and analyses were performed using s-plus software (tibco, inc., seattle, wa). plasma concentrations of acth were not significantly different between septic and non-septic foals whereas septic foals had greater serum cortisol ( ae ng/ml vs ae ng/ml) but lower serum igf-i ( ae ng/ml vs ae ng/ml) relative to non-septic foals pooled overall sampling times. the positive association of the peripheral blood concentrations of acth and cortisol depended on disease status of the foals. specifically, cortisol and acth were positively correlated for the septic foals (p . ) but not significantly correlated in the non-septic foals. peripheral concentrations of acth and igf-i were not significantly correlated whether data were pooled overall or stratified by sepsis status. however, peripheral concentrations of cortisol and igf-i were negatively associated (p . ); disease status did not influence this association, although it appeared to be a stronger association for the septic than the non-septic foals. the negative correlation between serum concentrations of the adrenal axis steroid cortisol and the somatogenic axis peptide igf-i may reflect interactions of these homeorhetic hormones. further studies of these and other metabolic hormones in a greater number of foals are warranted to better understand how these factors contribute to survival or non-survival of critically ill foals. botulism is a potentially fatal paralytic disorder which definitive diagnosis is difficult. the purpose of this study was to investigate if repetitive stimulation of the common peroneal nerve will aid in the diagnosis of suspected botulism in foals. four healthy foals were used for its comparison with foals with suspected botulism. controls were anesthetized and affected foals were sedated to avoid risks of anesthesia. the common peroneal nerve was chosen for its superficial location and easy access. stimulating electrodes were placed along the common peroneal nerve. for recording, the active and reference electrodes were positioned over the midpoint and distal end of the extensor digitorum longus muscle, respectively. repeated supramaximal stimulation of the nerve was performed utilizing a range of frequencies ( to hz). amplitude, area under the curve and percentages of decrement or increment for each m wave over subsequent potentials for each set of stimuli were analyzed. baseline m waves were decreased in affected foals compared to controls. a decremental response was seen at all frequencies in control foals. decremental responses were also observed in affected foals at low frequencies. however, an incremental response in amplitude and area under the curve was seen in all affected foals at hz. reduced baseline m waves with incremental responses at high rates are supportive of a presynaptic neuromuscular disorder which botulism was the most likely cause in these foals. repetitive nerve stimulation is a safe, simple, fast, and non-invasive technique that can aid in the diagnosis of suspected botulism in foals. this study examined the frequency with which dogs are exposed to e. chaffeensis and e. ewingii relative to e. canis, which is transmitted by the more ubiquitously distributed brown dog tick (rhipicephalus sanguineus). a total of , canine serum samples, ranging from to from each of the participating institutions, collected at random from clinical accessions, diagnostic laboratories and/or shelters were evaluated. all serum samples were tested by three microtiter plate elisas using species-specific peptides for antibodies to e. canis, e. chaffeensis and e. ewingii. zip code information for sample origin was provided by the collaborator and was used to assess seroprevalence by region. comparisons were evaluated using the chi-square test. seroreactivity for at least of ehrlichia spp was found in samples from every institution both mississippi and oklahoma had greater than a % samples from ohio had the lowest aggregate seroprevalence ( . %) with only dogs e. canis seropositive, one e. ewingii seropositive and no e. chaffeensis seroreactors. the geospatial pattern of e. chaffeensis and e. ewingii seropositive samples was similar to that previously reported based on modeling seroreactivity to e. chaffeensis in white-tailed deer as well as the distribution of human monocytic ehrlichiosis (hme) cases reported by the cdc. this study provides the first large scale regional documentation of canine exposure to these three ehrlichia spp., highlighting where infections most commonly occur and thus identifying areas where heightened awareness about these emerging vector urinary incontinence (ui) occurs in approximately % of spayed female dogs. the most common cause is urethral sphincter mechanism incompetency (usmi). pharmacological agents are effective, however, not all dogs respond, and dogs may become refractory to treatment over time. urethral bulking, where a compound is injected submucosally in the urethra, has been used in women and in female dogs with urinary incontinence. new synthetic compounds have been used in human medicine; the most promising is polydimethylsiloxane (pdms), which has been shown to be more effective than glutaraldehyde cross-linked collagen. the purpose of this descriptive clinical trial is to evaluate the safety and effectiveness of pdms urethral bulking agent (pdms uba) in client-owned, spayed female dogs with naturally-occurring ui due to usmi.twenty-two, spayed female dogs were included. dogs had a median age of years ( to years). eighteen dog breeds were represented, and dogs weighed a median of . kg ( . to . kg). average length of time of ui was . ae . years; / dogs had been treated medically, of which / were continent, / were improved, and / had no improvement. dogs were deemed healthy based on results of physical examination, complete blood cell counts, plasma biochemical analysis, and urinalysis; urine cultures were negative.dogs were anesthetized, positioned in dorsal recumbency, and cystoscopy performed using a . mm, -or -degree, cm rigid cystoscope. urethral bulking was performed with pdms uba. on average, . ae . ml were injected in to locations approximately to . cm distal to the trigone submucosally in the proximal urethra. good coaptation was achieved in all dogs. the procedure took on average . ae . minutes. one dog experienced urethral obstruction after the procedure; a foley catheter was inserted for approximately hours and removed at which time she urinated normally and was continent. three dogs experienced an acute allergic reaction characterized by blepharedema and urticaria treated successfully with diphenhydramine. dogs were discharged on day of procedure except for the one dog that experienced urethral obstruction. all dogs were treated with meloxicam ( . mg/kg po q h for days).owners were contacted on day after discharge and / dogs were continent; / dogs was improved. dogs were re-evaluated week after discharge and / dogs were continent and / dogs polyneuropathy in large breed dogs is a relatively common clinical problem for which the genetic basis is generally unknown. the first cases of polyneuropathy in the leonberger breed (leonberger polyneuropathy or lpn) were identified in by one of the authors (gds) and a report published in (musclenerve : - ) . in this report a spontaneous, distal and symmetrical polyneuropathy with onset between to years of age was described and characterized clinically, electrophysiologically, histologically and morphometrically. there were striking similarities between lpn and the charcot-marie-tooth group of human inherited sensory and motor polyneuropathies, which have many known genetic mutations.a genome-wide case-control association study for lpn was performed with cases and controls on high-density k canine snp arrays and revealed a significantly associated region on cfa (p raw .  À p genome .  À ). a clear association of an approximately mb cfa haplotype with cases (p .  À ) was observed, particularly with those cases that were affected more severely and at a younger age (p .  À ). a positional candidate gene, arhgef , which has previously been associated with peripheral nerve abnormalities in humans, was sequenced, revealing a deletion that results in a frame shift and premature stop codon. of all leonbergers with young onset lpn (before years), . % ( of ) have two copies of this deletion, and, of all young onset leonbergers that are nerve biopsy positive for lpn, . % ( of ) have two copies of this deletion. importantly, nearly all dogs carrying two copies of the deletion ( of or . %) are affected with lpn by the age of years.the leonberger breed was generated from crossing several breeds, including the st. bernard, and a polyneuropathy clinically and histologically similar to lpn occurs in this breed. to determine if the arhgef mutation was associated with polyneuropathy in the st. bernard, dna was extracted from archived frozen muscle biopsy specimens from clinical cases (n ). the identical arhgef startle disease or hyperekplexia is caused by defects in mammalian glycinergic neurotransmission resulting in an exaggerated startle reflex and extensor hypertonia triggered by noise or touch. in humans and animals, startle disease is typically caused by mutations in one of three genes (glra , glrb, and slc a ) encoding postsynaptic glycine receptor subunits (a and b) or a presynaptic glycine transporter (glyt ). a litter of seven irish wolfhounds was recently identified in which two puppies developed muscle stiffness and tremor beginning at - days of age post-partum. signs were dramatic when the puppies were handled and resolved when the puppies were relaxed or sleeping. both puppies were euthanized due to ongoing stiffness, tremor and breathing difficulties. necropsies were performed, but no microscopic pathological abnormalities were identified in the peripheral or central nervous system.based on the clinical signs, exons from the three candidate genes were amplified from genomic dna isolated using pcr and directly sequenced. no deleterious polymorphisms were identified in either glra or glrb. however, difficulties were experienced in amplifying slc a exons and from affected animals, although control samples were positive, suggesting that the pcr primer designs and conditions were not at fault. further pcrs revealed that the reason for this anomaly was the presence of a homozygous . kb deletion encompassing exons and of the glyt gene in both affected animals. this deletion is predicted to result in the loss of part of the large cytoplasmic n-terminus that is vital for trafficking of glyt to synaptic sites, and a loss of all subsequent transmembrane domains via a frameshift. this genetic lesion was confirmed by defining the deletion breakpoint, southern blotting and multiplex ligationdependent probe amplification (mlpa). this analysis enabled the development of a rapid genotyping test that revealed heterozygosity for the deletion in the dam and sire and three other siblings, suggesting recessive inheritance of this disorder. wider testing of related animals has identified a total of carriers of the slc a deletion and enabled the identification of non-carrier animals to guide future breeding strategies. insulin resistance (ir), obesity, and type diabetes affect glucagon-like peptide (glp- ) concentrations in humans and rodents, but this incretin hormone has not been examined in horses. we therefore hypothesized that glp- concentrations would change in horses as obesity and ir were induced or exacerbated by overfeeding. six horses previously diagnosed with equine metabolic syndrome were provided with twice the amount of digestible energy required for maintenance as sweet feed and hay for weeks. intravenous and oral glucose tolerance tests (ogtts) were performed at and weeks. effects of time and period ( and weeks) were assessed by repeated measures anova.mean body weight increased from ae kg (range, to kg) to ae kg (range, to kg) over weeks, with individual horse weight gain varying from to %. mean body condition score increased (p . ) from ae (range, to . ) to ae (range, to ). three horses developed mild laminitis. glucagon-like peptide concentrations increased over time during ogtts (p . ), but the period  time effect was not significant (p . ). area under the glp- curve remained unaffected by weight gain, whereas area under the insulin curve increased (p . ) over time, indicating a reduction in insulin sensitivity. obesity and ir were induced or exacerbated when horses previously diagnosed with ems were overfed, but glp- concentrations did not change as a result. hypertonic saline solution ( . %) (hss) is an intravenous fluid used for the emergency treatment of intravascular volume deficits. the use of this fluid in horses with severe dehydration is controversial. the purpose of this study was to compare the use of hss and isotonic saline solution ( . %) (iss) for the emergency treatment of endurance horses.endurance horses eliminated from competition and requiring intravenous fluid therapy were eligible for enrollment in the study. twenty-two horses were randomly assigned to receive ml/kg of either hss or iss along with l lactate ringer's solution (lrs). following this bolus, all horses were treated with an additional l of lrs. blood and urine samples were collected before, during and after treatment. data was compared using two-way anova with repeated measures.as compared to iss, hss horses showed a greater decrease in pcv (p . ), total protein (p . ), albumin (p . ), and globulin (p . ). hss horses showed a greater increase in sodium and chloride (p o . ) as compared to iss horses. horses receiving hss had a shorter time to urination (p . ) and lower specific gravity (p o . ) than those receiving iss.results of this study indicate that hss may provide faster restoration of intravascular volume deficits than iss in endurance horses receiving emergency medical treatment. more profound electrolyte changes should be expected with hss however. b -adrenergic receptor agonists have been shown to increase erythrocyte carbonic anhydrase activity, which may stimulate the jacobs-stewart cycle and increase pulmonary circulation transvascular fluid fluxes during exercise. increase in pulmonary transvascular fluid fluxes (j v-a ) and consequent increase in the pulmonary interstitial fluid would be detrimental for alveolar o exchange during the fast erythrocyte transition time across the pulmonary capillaries. therefore, we hypothesised that treatment with inhaled b -adrenergic receptor agonist will increase j v-a and the alveolar-arterial po difference (aado ) during exercise.six stb horses were exercised on a high-speed treadmill at % vo peak until fatigue. horses were randomly assigned to treatment with salbutamol (sal: mcg) or placebo (control: con) inhalation via aeromask à min prior to exercise, with cross over treatment used at the repeated exercise test ( days later). arterial and mixedvenous blood, as well as co elimination and o uptake, were sampled simultaneously at rest, during exercise at sec intervals until fatigue, and into recovery. blood gases were analyzed. aado was calculated using the inspired po ( mmhg), and blood partial pressure of o and co . blood volume (%) changes across the lung were calculated from changes in hemoglobin and hematocrit values in venous and arterial blood. cardiac output (q) was calculated using the fick equation. j v-a was calculated using q and blood volume changes across the lung. variables were analyzed using two-way repeated-measures anova (p o . ).the duration of exercise to fatigue was . ae . min and . ae . min in both con and sal, respectively. at rest sal had no effect on j v-a , oxygen consumption (vo ), blood oxygen saturation (so ) or aado (p . ). at the onset of exercise j v-a increased in con and sal (p o . ) and at fatigue reached . ae . l/min and . ae . l/min, respectively. treatment with sal had no effect on j v-a during exercise (p . ). at the onset of exercise so and vo increased in con and sal (p o . ). treatment with sal had no effect on so or vo during exercise (p . ). aado increased during exercise in con and sal (p o . ) and at fatigue reached . ae . mmhg and . ae . mmhg, respectively. treatment with sal had no effect on aado during exercise (p . ).inhaled b -adrenergic receptor agonist salbutamol at the dose of mcg given min before exercise did not affect the duration of exercise to fatigue, j v-a , vo , so or aado . therefore, it had no detrimental effect on alveolar-capillary diffusion distance and the ventilation/perfusion mismatch in exercising horses. inflammatory airway disease (iad) and recurrent airway obstruction (rao) represent two classes of equine lung inflammatory diseases that may share some similar immunologic mechanisms. there is evidence that th cytokines and il- play some role in rao. iad is a common condition in horses, but its pathophysiology is still not understood. the aim of the present study was therefore to determine the mrna expression of th , th and th inflammatory cytokines, to understand the immunological mechanisms of iad.the mrna expression of ten inflammatory cytokines and chemokines was measured in the bronchoalveolar fluid (balf) of seventeen horses with iad and compared with ten control horses. the horses were selected based on -their clinical signs, -the inflammatory cells count in the balf, -their physical examination and -their medical history. the mrna expression of il- , il- b, il- , il- and il- was significantly up-regulated in balf from horses with iad.furthermore, the balf samples were subdivided in two groups based on the differential cells count -balf with increased mast cells (iad-mast) and -balf with increased neutrophils (iad-neutro). il- was significantly down-regulated in the iad-neutro group compared to the iad-mast group. il- , il- and il- were significantly up-regulated in the iad-neutro group compared to the iad-mast group.the present study shows that iad in horses is characterized by a th and a th mrna inflammatory expression profile and that different immunological mechanisms are involved in mast cells or neutrophils accumulation in the balf of horses with iad. b -adrenoreceptor (b -ar) agonists are a class of medications that promote smooth muscle relaxation and bronchodilation in horses and humans with airway disease. activated human peripheral blood lymphocytes (pbls) also respond to b -ar agonist stimulation by attenuating the production of cytokines associated with the pathogenesis of asthma and recurrent airway obstruction (rao). the aim of this study was to develop an in vitro technique for measuring the response of equine pbls to stimulation with salbutamol, a b -ar agonist. this method was then used to compare the response of pbls from rao-affected and non-affected horses to b -ar agonist stimulation. pbls from rao and nonaffected horses were cultured ( x /ml) in rpmi complete media with concanavalin a (cona, ug/ cells) for , , or days then stimulated with salbutamol ( minutes). using flow cytometric techniques, response was measured by detecting protein kinase a phosphorylation of vasodilator stimulated phosphoprotein (vasp). results were verified by western blot analysis. activated pbls were then incubated with cona for one day were pre-incubated with b or b-adrenoreceptor antagonist (ici , , sigma s ; atenolol, sigma s ) for minutes, followed by minutes salbutamol ( nm) stimulation. results were analyzed by anova or ancova and differences were considered significant when p o . .response to b-antagonist was only observed in activated pbls (pre-cultured with con a) and was greater in cells from rao horses as compared to cells from non-affected horses. the addition of b-antagonist attenuated the response of pbls to salbutamol while the addition of a b -antagonist had no effect. these findings indicate that activated pbls from rao-affected horses have a greater response to salbutamol as compared to pbls from non-affected horses, and this response is mediated mainly through the b -ar.human b -ar are known be polymorphic and this polymorphism results in a variable response to b -agonist binding that affects long term outcome in human asthmatics. further studies are required to determine if the difference in response of pbls from rao affected as compared to non-affected horses is due to genetic polymorphism in the equine b -ar, and whether this difference is associated with a propensity for horses to develop equine rao. key: cord- -ilhr iu authors: nan title: isev abstract book date: - - journal: nan doi: . / . . sha: doc_id: cord_uid: ilhr iu nan introduction: primary tumours secrete large amounts of extracellular vesicles (evs), which play critical roles in preparing distant sites for a pre-metastatic niche formation, thereby promoting metastasis and even determining metastatic organotropism. whether biogenesis, secretion rates and organotropism of evs are linked remains unknown. we have recently shown that ral gtpases control evs secretion in nematodes as well as in mouse mammary tumour cells (hyenne et al. jcb ) . since both rala and ralb are overexpressed or over-activated in various human cancers, we aimed to investigate the mechanisms by which these two gtpases control evs secretion and to determine how this affects metastatic progression, with a focus on breast cancer. methods: we used t mouse mammary carcinoma cells knocked down for either rala or ralb and determined their ability to induce orthotopic tumours and metastasis in a syngeneic mouse model. in vitro, we investigated ev secretion mechanisms using confocal and electron microscopy (em). evs were isolated either by uc or sec and characterized by nta, em, rna sequencing and mass spectrometry. the function of evs was assessed using a transwell assay. finally, we tracked the organotropism of fluorescently labelled evs and their capacity to induce pre-metastatic niches in mice. results: we show that rala and ralb promote lung metastasis of breast cancer cells in mice without affecting their invasive behaviours. we found that rala and ralb control the biogenesis of exosomes, by acting on the formation of multi-vesicular bodies though the phospholipase pld . as a consequence, knock down of rala or ralb reduces the levels of secreted evs and modifies their rna and protein contents. these differences alter the pro-tumoural function of evs, as demonstrated with an in vitro permeability test. importantly, we show in vivo that evs from rala or ralb depleted cells have a decreased lung organotropism and, as a consequence, are less efficient in priming lung metastasis. finally, we show that high expression of rala or ralb is associated with a bad prognosis in human breast cancer patients. summary/conclusion: altogether, our study identifies ral gtpases as central molecules linking the mechanisms of evs secretion, their dissemination and their capacity to promote metastasis. nuclear proteins are recruited into tumour-derived extracellular vesicles upon expression of tetraspanin tspan introduction: tetraspanin tspan is a transmembrane protein that exhibits a unique expression pattern, being overexpressed in many cancer types, but undetectable in most healthy tissues. although there is increasing evidence of an effect of tspan in invasion, metastasis, and regulation of extracellular vesicle cargo, the molecular mechanisms of tspan are yet not fully understood. methods: to study the function of tspan , we have established a fibrosarcoma model consisting of the parental cell line (ht ) and its derivatives expressing tspan (ht -tspan ) either fused with different fluorescent tags or tag-free. life imaging, sted and storm microscopy were used to determine the intracellular localization of tspan . co-immunoprecipitation from nuclei lysates was performed to detect direct and indirect interacting partners of tspan . small evs were purified from cell-conditioned media using sec and subjected to mass spectroscopy and ngs for a comprehensive comparative analysis of the proteome and transcriptome of the evs. results: the results of the proteome analysis showed a strong effect in the protein cargo of evs upon tspan expression. remarkably, among of the most regulated targets, several histones and ribosomal proteins were enriched in the evs derived from ht -tspan cells. in line with this finding, life imaging and super-resolution microscopy revealed that, while a majority of the intracellular tspan is located on the cell membrane or intracellular membranes, -as it is known for other tetraspanins-, a portion of tspan is located on the nuclear envelope. in fact, several histones co-immunoprecipitated with tspan , indicating their interaction. summary/conclusion: our data show that the expression of tspan in the tumour cells greatly impacts ev cargo. moreover, localization of tspan on the nuclear envelope, together with the enhanced recruitment of nuclear and ribosomal proteins to the evs, suggests a new mechanism of action of tspan . introduction: extracellular vesicles (evs) modulate tissue development, regeneration and disease through the transfer of proteins, nucleic acids and lipids between cells. currently, the mechanism of cytosolic delivery of ev cargo is largely unknown. here, we unravel how evs release their cargo in recipient cells. methods: evs were isolated from gfp-cd and cd -rfp expressing hek t cells by ultracentrifugation. gfp-cd and cd -rfp evs were added to hek t cells stably expressing anti-gfp fluobody and fluorescently tagged galectin- , respectively. clem and fluorescence microscopy were employed to visualize fluorescent markers in recipient cells. bafilomycin a and u a were used to inhibit endosomal acidification and cholesterol export from lysosomes, respectively. results: fluorescent galectin- which binds to betagalactosides present at the luminal side of endosomes was used to detect endosomal permeabilization. the absence of galectin- recruitment to endosomes in presence of cd -rfp evs showed that endosome permeabilization is not the mechanism behind ev cargo release. gfp-cd ev addition to cells expressing anti-gfp fluobodies resulted in the formation of fluobody punctae, reflecting cytosolic exposure of ev cargo. subsequent clem of the fluobody punctae revealed endosomes as the underlying cellular compartments from where cargo release takes place. neutralization of endosomal ph and accumulation of endosomal cholesterol blocked cargo release, showing that ev cargo release is dependent on endosomal ph and cholesterol level. summary/conclusion: we show that genetically encoded cytosolic probes and clem offer an excellent approach to study both the mechanism and efficiency of ev cargo release in cells. we provide experimental evidence that ev cargo release occurs from endosomes. funding: the research was supported by dutch technology foundation ttw and netherlands organization for scientific research nwo, de cock-hadders stichting, and erasmus mundus namaste scholarship. healthy humans. to determine cut-off values for diagnoses, reference intervals of evs in plasma are needed. to establish such reference intervals, ( ) a significant number of healthy donors should be included, ( ) the presence of non-ev particles, residual platelets, lipoproteins, and haemolysis should be quantified, ( ) flow cytometry signals should be in si units. the long-term aim of this study is to determine reference intervals of ev concentrations in human plasma within known dynamic ranges of the detectors. methods: ( ) to establish a clinical reference, we collected blood from healthy volunteers and prepared platelet-free plasma. ( ) we performed quality control measurements including residual platelet count, serum index, and lipid spectrum. ( ) we measured all samples by flow cytometry (apogee a -micro) and used custom software (matlab r b) to automate calibration of all signals and data processing. scatter signals were calibrated in comparable units of scattering crosssection (nm ) and diameter (nm). fluorescence signals were calibrated in units of molecules of equivalent soluble fluorophores (mesf). results: the quality controls showed that most residual platelet concentrations ranged from ^ to ^ per ml except for one outlier, while the serum index and lipid spectrum were normally distributed. preliminary results of the first donors analysed, show that within the ev size range of - , nm, the median concentration of cd + evs is . • ^ per ml (apc> mesf), cd p+ evs is . • ^ per ml (pe> mesf), cd a+ evs is . • ^ per ml (pe> mesf), and cd + evs is . • ^ per ml (apc> mesf). summary/conclusion: we have developed reliable procedures for establishing reference intervals of ev concentrations, within a well-defined size and fluorescence intensity range, in human plasma by flow cytometry. we are currently applying these procedures to samples to obtain, for the first time, ev reference intervals for human plasma. funding: pol, e. van der is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . introduction: the use of extracellular vesicles for diagnostic and therapeutic applications has seen a major interest increase in recent years because of their capacity to exchange components such as nucleic acids, lipids and proteins between cells. isolation of a pure population of evs is the first step in studying their physiological functions since contamination of ev preparations with non-ev proteins can lead to incorrect conclusions about their biological activities. we have developed a new method termed tangential flow for analyte capture (tfac) using ultrathin nanomembranes to purify extracellular vesicles from pure, highly complex biological fluids such as blood plasma, resulting in a new method for extracellular vesicle purification. methods: the tff microfluidic devices are assembled through a layer stack process using patterned polydimethylsiloxane (pdms) sheets with the membranes sandwiched between top and bottom channels. undiluted plasma was tested in both normal flow filtration (nff) and tangential flow filtration (tff) modes on ultrathin nanomembranes. we have utilized a pore patterning technique called nanosphere lithography (nsl) that uses close-packing of nanoscale beads to pattern pores in an ultrathin membrane. results: nff of undiluted plasma resulted in a protein cake of~ μm on the membrane, which prevented further transport across the membrane and evs were buried in the formed cake that were impossible to identify. however, tfac as a modified version of tff, led to capturing cd positive evs on the pores of the membrane with little evidence of protein fouling. nsl allows us to fabricate nanopockets (bowls with a single pore at the base) with various diameter, depth and pore diameter. using nsl, we further utilize nanopocket membranes to purify ev samples in tfac devices. this nanomanufacturing technology will allow us to pattern nanopockets with various diameter, depth and pore diameter which increases the efficiency of capturing of evs. furthermore, nanopockets can be modified and coated by specific ev markers to capture different subpopulation of evs based on size and affinity and further allows identifying the phenotypic subsets of evs by combining both size and affinity-based techniques. summary/conclusion: we have developed a method for the capture and release of nanoparticles such as evs called tfac using ultrathin nanomembranes. nsl technology can be applied to fabricate nanopockets with different physical and biochemical properties. utilizing nanopocket membranes in tfac system will allow us to separate different subpopulations of evs based on size and affinity. funding: this project was supported in part by the national science foundation (iip ) to j.l.m and t.r.g., department of defence (ca ) to j. l.m., and the national institutes of health (r gm ) to t.r.g. the addition of a size exclusion chromatography step to various urinary extracellular vesicle concentrating methods reveals differences in the small rna profile introduction: urinary extracellular vesicles (evs) and their rna cargo are a novel source of biomarkers for various diseases, however non-vesicular rna (e.g. associated with proteins) is also present within urine. this study aimed to identify the optimal method for isolating and enriching evs from human urine prior to small rna analysis. methods: three ev concentration methods, ultracentrifugation (uc); a precipitation-based kit (pk); and ultrafiltration (uf), were compared using ml aliquots of pooled healthy volunteer urine. evs were then separated from protein contaminants by size-exclusion chromatography (sec). presence of evs was confirmed by transmission electron microscopy and western blotting, and evs were quantified using nanoparticle tracking analysis (nta). small rna content of concentrated urine and fractions obtained by sec (evs and proteins) were evaluated with the agilent bioanalyzer small rna chip. results: ev recovery following sec of concentrated samples was - %, however particle: protein ratio (indicating ev purity) was approximately x greater after sec, regardless of the concentrating method used. uf+sec yielded the highest number of evs (per ml of urine) compared with pk+sec and uc+sec. small rna analysis from uf-concentrated urine (prior to sec treatment) identified peaks at nucleotides (nt) and nt. following sec, rna analysis indicated that ev fractions contained mostly small rna of~ nt, whereas the protein factions contained small rna of nt in size (consistent with mirnas). summary/conclusion: uf+sec provided the best balance between ev recovery (per ml urine) and particle: protein ratio. these data indicate that most of the nt sized rnas, presumably mirnas, are not within evs in urine. ev preparations obtained after uc, pk and sec (regardless of concentrating method) contain pre-dominantly~ nt sized small rna. these data outline the importance of removing non-vesicular proteins and rna from urine ev preparations prior to small rna analysis. funding: this research has been funded by petplan charitable trust. the use of rev for the optimization of ev separation and characterization by af introduction: the reproducibility of extracellular vesicle (ev) research has been hampered by the infinite number of separation and measurement techniques and the lack of appropriate reference materials (van deun et al., nat methods, ) . recombinant extracellular vesicles (rev) were developed as a biological reference material to overcome these limitations (geeurickx et al. nat comm ) . since rev have ev-like physical an biochemical characteristics and as they are trackable and distinguishable from sample ev they can be used as a spike-in material for data normalization and method development, and as a quality control. we used rev to optimize ev separation by asymmetrical flow field-flow fractionation (af ). methods: an af long channel column with a frit inlet driven by the eclipse system (wyatt) was coupled to a uv detector (shimadzu), mals dawn helios-ii (wyatt) and fluorescent detector (agilent). a spacer of µm and a regenerated cellulose membrane of kda were used. pbs supplemented with . % nan was used as a running buffer. light scatter profiles and uv profiles were analysed as well as the fluorescent emission spectrum as the rev are gfp positive. fractions were collected and analysed by nanoparticle tracking analysis (nta) and western blot. we also estimated the repeatability and reproducibility of the af technique by light scatter and fluorescence profiles as well as the recovery efficiency by nta. results: in a first step * ^ rev isolated from conditioned medium by a velocity gradient were injected in the af system to optimize the ev characterization protocol. later concentrated conditioned medium was spiked with * ^ rev and injected in the af column to optimize ev separation from non-ev contaminants. the most optimal separation protocol was obtained by varying detector and cross-flow settings. this protocol shows elution of monodisperse particles at each time point and size distribution estimations by af correspond to size determination by nta and electron microscopy. summary/conclusion: we were able to optimize the af protocol for characterization of ev by af as well as for separation of ev from crude conditioned medium samples by using rev. we demonstrate that rev are suitable for method development and that af has high potential as an ev separation technique. comparative evaluation of ev isolation methods for ev subpopulation analysis in human urine, plasma and cell culture media liang dong, richard zieren, kengo horie, sarah amend and kenneth pienta the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: extracellular vesicles (evs) are membrane-enclosed particles of variable sizes that are released by any cell types to the extracellular space and are identified in all body fluids. a shortcoming in ev research is the lack of standardized isolation protocol for various sample types, resulting in heterogeneous outcomes in downstream analyses. in this study, we compared the ev isolation purity and efficiency among ultracentrifugation (uc), precipitation, sizeexclusion chromatography (sec) and a microfluidic tangential flow filtration device (exodisc) in human plasma, urine and cell culture media (ccm). methods: all evs were isolated by different isolation methods and characterized per misev guidelines. single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence and nanoflow (nfcm) were used for single particle analysis. results: in ccm, total particle yield of exodisc was about times higher than those of the rest three methods. size distribution differed per sample, but the ranges were comparable between the different isolation methods. the total protein amount of sec, precipitation and exodisc were similar which were - times higher than that of uc. uc had the highest particle-toprotein ratio followed by exodisc. precipitation and sec had low ratios. when loading ug of total protein for western blot, cd , cd , cd and flot could only be detected in uc and exodisc samples, but not precipitation or sec. sp-iris and nfcm demonstrated consistent purity findings. in urine, total particle yields of exodisc and sec were about times higher than those of the rest two methods. the total protein amount of precipitation was times higher than exodisc and sec, times higher than uc. sec had the highest particle-to-protein ratio followed by uc and exodisc. precipitation had low ratios. in plasma, total particle yields of exodisc and precipitation were times higher than those of the rest two methods. and so were the total protein amount. sec had the highest particle-to-protein ratio followed by uc. exodisc and precipitation had low ratios. western blot, sp-iris and nfcm demonstrated consistent purity findings in urine and plasma. to evaluate particle capture efficiency, we spiked a known number of density-gradient uc purified evs to each method and the recovery rate of uc, precipitation, exodisc and sec was . %, %, . % and %, respectively. summary/conclusion: the order of ev isolation purity in ccm is uc, exodisc, sec and precipitation. in urine it's sec, exodisc, uc and precipitation. and in plasma, this order is sec, uc, exodisc and precipitation. exodisc and sec have similar high isolation efficiency followed by precipitation. uc has low efficiency for ev capture. a capillary-channelled polymer (c-cp) fibre spin-down tip approach for the isolation and biomarker characterization of extracellular vesicles of ovarian cancer origin kaylan d. kelsey, rhonda r. powell, terri f. bruce and r. kenneth marcus clemson university, clemson, usa introduction: extracellular vesicle (evs) profiling has shown promise for disease detection through less invasive sampling (liquid biopsies). current diagnostic tools for ovarian cancer are invasive or only semiinformative. thus, use of evs could prove useful in early disease detection. demonstrated is a hydrophobic interaction chromatography (hic)-based capillarychannelled polymer (c-cp) fibre tip spin-down process for the isolation of ovarian cancer evs for use in diagnostics. methods: polyester c-cp fibre micropipette tips are employed in the isolation of evs from biological matrices including cell culture media, urine, and blood plasma in a spin-down solid-phase extraction (spe) approach. evs were isolated from standards of healthy urine origin and from skov cells (human ovary adenocarcinoma). the c-cp fibre isolation method (taking less than mins and μl sample volumes) preserves the morphology and functionality of evs as confirmed by sem, tem, and confocal fluorescence microscopy. results: the dynamic binding capacity of ev standards on a cm pet c-cp fibre tip was found to be~ e particles ( %). the release of evs was confirmed using dot blot analysis for cd , cd , and cd tetraspanin proteins. immobilized evs were subjected to immunolabeling to allow the positive identification of a profile of ovarian cancer biomarker proteins (her , cd , egfr, epcam, ca ). summary/conclusion: this new ev isolation method introduces a simple capture mode, allowing for direct immuno-characterization and imaging on the fibre surface. this offers a unique and cost-effective opportunity for clinical analyses related to early detection and diagnosis of ovarian cancers (and others). the longterm goal is the creation of a rapid ev isolation and biomarker detection platform. funding: support from the national science foundation, eppley foundation for scientific research, gibson foundation, prisma health system and itor biorepository are gratefully acknowledged. development and optimization of purification method of exosomes by tangential flow filtration and ion-exchange chromatography approach tek lamichhane, ali navaei, sandeep choudhary, yonatan levinson and senthil ramaswamy cell & gene therapy r&d, lonza inc, rockville, usa introduction: extracellular vesicles (evs) such as exosomes have significant therapeutic potential, however, translation of ev-based therapies has been slowed down because of the biomanufacturing challenges. the isolation of evs, especially exosomes, is inherently challenging due to their small size, and heterogeneity in the mixture. the current isolation methods either have low recovery rate, aggregation, damaging the structure, time consuming or co-precipitation of contaminants. specially, it is difficult to process larger sized samples by centrifugation-based or immunoaffinity based methods because of the time and cost associated with these methods. methods: to overcome these roadblocks, we developed and optimized alternative purification techniques to isolate evs with higher purity and yield by using tangential flow filtration (tff) coupled with ion-exchange chromatography. we used bioreactor platform to produce evs from serum-free medium using bm-msc and hek s cells. bm-mscs were cultured on stirred tank bioreactors using microcarriers which provide a high surface area to volume ratio for the optimal cell growth and evs production. impellers were used to enhance mixing and maintain homogeneous culture conditions that can be easily monitored and controlled. results: depth filtration was applied for clarification of conditioned medium. we screened different types of filters during depth filtration for the best recovery of evs. tff membranes with different pore sizes were used to optimize the purity and yield of evs. because of the negatively charged nature of evs, anion exchange chromatography was chosen to capture and separate tff purified vesicles by their surface charge characteristics. we compared monolith based and membrane-based anion exchange columns to remove contaminants and purify exosomal fractions. the purity, size and presence of exosomal markers in isolated evs at each step of purification was evaluated by f-nta, nano-fcm and tetraspanins based elisa kits. summary/conclusion: in summary, our optimized methods improved the speed of isolation and purity of evs to the clinical grade. the production and isolation methods of exosomes that we developed here will be easily expandable to support large-scale and cgmp compatible bio-manufacturing in the future. use of an alternating current electrokinetic microelectrode chip to positively identify oncology, neurology, and infectious disease samples through plasma extracellular vesicle analysis juan pablo hinestrosa, jean lewis, david searson, orlando perrera, alfred kinana, heath balcer and rajaram krishnan biological dynamics, inc., san diego, usa introduction: cancer, neurological, and infectious diseases are leading causes of death, with early detection needed to improve outcomes. extracellular vesicles (evs) in the blood contain disease biomarkers, but current methods do not allow rapid analysis, and are often limited to one biomarker type. methods: we developed methods using alternating current electrokinetics (ace) to isolate evs from bloodbased samples and analyse the evs in situ with downstream assays for protein and nucleic acid biomarkers. we investigated if we could identify tuberculosis (tb) donor samples, protein and nucleic acid biomarkers in evs derived from cancer cell lines, and alzheimer's disease (ad) protein biomarker levels. results: ev isolation was confirmed by positive identification of the proteins cd , cd , and cd and measurement of ev mrnas using a direct rt-ddpcr assay. different disease models were analysed following method development. tb was used as a model for infectious disease, with tb positive and tb negative samples isolated on ace chips and analysed for levels of lipoarabinomannan and ag . using a cut-off above the negatives, the auc of roc curves were . and . , respectively. for oncology, cancer cell lines were cultured and evs isolated from supernatants were spiked into human plasma for analysis. levels of pd-l or glypican- on evs were able to be measured following ace capture. additionally, dna and rna mutations known to be present in the cell lines were able to be detected using ngs and qrt-pcr, respectively. using ad samples as a neurological disease model, tau and phospho-tau t (p-tau t ) in human donor plasma were detected. in ad and healthy donor samples, p-tau t signal increased % in diseased versus healthy donors. summary/conclusion: ace chips are an innovative ev isolation and analysis platform that allow rapid disease sample detection in a wide range of studies with high sensitivity and specificity. introduction: colorectal cancer (crc) is one of the most frequent causes of cancer-related death. in the majority of crc patients, mutation in the apc gene is among the first genetic events. it leads to uncontrolled activation of the wnt pathway, and thus, to adenoma formation. some of these adenomas may then further progress to crc with the accumulation of other mutations. the d organoids maintain the cellular and genetic heterogeneity of in vivo tissues and haves proved to be so far the best ex vivo model of human cancers. here we analysed the ev-based communication between cancer cells and fibroblasts by i) identifying factors that substantially increase ev release from intestinal cancer cells and ii) by determining cargo components of evs that enhance tumour cell proliferation. methods: we used commercially available and patientderived fibroblasts and crc organoids. the medical research council of hungary approved all experiments with human samples and informed consent was obtained from patients. evs were studied by using antibody-coated beads, trps, nta, tem and western-blotting. we introduced apc mutation into wild type murine small intestinal organoids by crispr-cas . results: we found that in crc patient-derived organoid cultures, small evs were preferentially secreted. we observed that apc mutation and the accumulation of the extracellular matrix component collagen critically enhanced ev secretion in intestinal organoids. furthermore, we showed that amphiregulin, present on fibroblast-derived ev, contributed to the maintenance of the intestinal stem cell pool and to cell proliferation in epidermal growth factor-dependent crc organoids. summary/conclusion: by proving the key role of mutations, collagen deposition and ev-bound amphiregulin in the release intensity and functions of the evs, we identified novel mechanisms in the progression if crc. funding: this work was funded by otka-nn , by the national competitiveness and excellence program nvkp_ - (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). prostate cancer-derived evs induce a pro-inflammatory phenotype in the stroma blandine f. victor a , dolores di vizio b , andrew chin c , tatyana vagner b , javier mariscal b , mandana zandian a , catherine grasso a , roberta gottlieb a and helen goodridge a a cedars-sinai medical center, los angeles, usa; b cedars-sinai medical center, west hollywood, usa; c cedars-sinai, los angeles, usa introduction: since % of patients with metastatic prostate cancer (pc) develop bone metastasis, identifying the mechanism that drives this process is essential. most ev research has been focused on the role of exosomes in mediating the pre-metastatic niche formation. however, most of these studies do not separate exosomes from large evs. our preliminary studies have demonstrated that a subclass of evs known as large oncosomes (lo) can reprogram prostate fibroblasts, at the primary tumour site, promoting angiogenesis and enhancing the migration and invasion of pc cells in vitro and tumour growth in vivo. the bone marrow is the initial site of entry into the bone microenvironment for disseminating tumour cells (dtcs) and is a rich source of nutrients that houses various cells types including bone marrow derived mesenchymal stem cells (bm-msc) and immune cells such as neutrophils, which have been implicated in metastasis. here we investigate the role of lo in reprogramming bm-mscs and driving bone metastasis in pc. methods: differential centrifugation, density gradient centrifugation, trps, rna sequencing, qpcr, migration assay, invasion assay, chemotaxis assay. results: we report that pc-derived evs induce distinct gene expressions changes in bm-mscs. rna-seq analysis identified inflammatory and immune regulating cytokines as top differentially expressed genes (deg) in bm-msc. moreover, lo induced a more potent response in bm-msc in comparison to exo and to non-treated controls. the genes enriched in lo treated bm-msc were associated with tumour cell motility. in agreement with the gene expression data, lo-treated bm-msc attracted migration and invasion of significantly more pc cells than exo -treated bm-mscs. in addition, the top deg expressed in ev treated bm-msc were identified as potent neutrophil chemoattractant proteins. in line with the rnaseq findings, the lotreated bm-msc demonstrated enhanced chemotaxis of neutrophils towards them in comparison with exo or vehicle-treated bm-msc. finally, we show that the observed differences in bm-msc's response to lo and exo may be mediated by distinct molecular pathways. summary/conclusion: the results from this study provide novel insight into how tumour derived evs alter the bone marrow microenvironment and how they may drive bone metastasis in prostate cancer. the αvβ integrin in cancer cell-derived small extracellular vesicles enhances angiogenesis introduction: prostate cancer (prca) cells crosstalk with the tumour microenvironment by releasing small extracellular vesicles (sevs). sevs isolated from prca cell media, express the epithelial-specific αvβ integrin, a surface receptor for fibronectin and vitronectin. the αvβ integrin is not detectable in healthy prostate tissues but is highly expressed in prca. in this study, we hypothesized that αvβ in cancer sevs plays a crucial role in angiogenesis. methods: the sevs isolated from prca cell media were characterized by nanoparticle tracking analysis, iodixanol density gradients and expression of sev markers. the αvβ -negative endothelial cells (hmec ) were incubated with αvβ -positive sevs from prca cells to evaluate the transfer of αvβ by immunoblotting (ib) and facs. the effect of αvβ -positive sevs on motility, tube formation and angiogenic signalling were assessed by boyden chamber, angiogenesis assays and ib in hmec . results: we demonstrate for the first time that the αvβ is de novo expressed on endothelial cell surface by sevmediated protein transfer. prca cell-derived αvβ -positive sevs, significantly promote the motility and the formation of nodes, junctions and tubules by hmec . mechanistically, we demonstrate that hmec treatment with sevs from pc cells that endogenously express αvβ , decreases pstat (y ), a negative regulator of angiogenesis, while upregulating survivin, an inducer of angiogenesis. hmec treatment with sevs isolated from pc cells harbouring crispr/cas -mediated downregulation of β , or shrna-mediated downregulation of β , results in increased levels of pstat (y ). this sev treatment also results in a decrease of survivin in sevs and hmec . summary/conclusion: overall, our findings show that αvβ in prostate cancer sevs regulates a novel proangiogenic signalling pathway. funding: this study was supported by nci r - (lrl); p - (lrl and dca). introduction: advanced prostate cancer (pca) is asso-introduction: extracellular vesicles (evs) are secreted from cells, and carry bioactive proteins and rna cargoes. increasing numbers of studies have identified key roles for exosomes in driving aggressive tumour behaviours, including metastasis. however, the detailed mechanisms and responsible factors in the ev cargo are still unclear. recently, immune system has been considered as an important factor in establishing and maintaining metastasis. our goal is to identify the role of head and neck squamous cell carcinoma (hnscc) derived small evs (sevs) in tumour metastasis from the study analysing the effects of sevs on metastasis and tumour immunity. methods: sevs were collected from the conditioned media of hnsccs and purified through cushioned density gradient ultracentrifugation. an orthotopic mouse model was used for the assessment of tumour angiogenesis and metastasis. moc (inflammationinducing rarely metastasizing murine hnscc line) and moc (highly metastasizing murine hnscc line) were used for this study. moc and moc cells were transplanted into mice tongues orthotopically, and moc /moc derived sevs or pbs were injected into the tumour twice in a week. two weeks after tumour transplantation, mice were sacrificed and tumours were sectioned for pathological analysis and facs analysis. in facs analysis, the number and species of tumour-infiltrated immune cells were measured. results: injection of sevs from moc into moc tumours suppressed frequency of lymph node metastasis. on the other hand, injection of sevs from moc into moc tumours didn't promote metastasis. cd positive t-cell distribution in moc tumour was significantly changed by moc sev injection. t-cell deprivation treatment using anti-cd antibody increased the frequency of metastasis in moc -sev treated moc tumours. from the result of proteomics analysis on moc and moc sevs, immune-regulated proteins and metastasis-suppressing proteins were observed in moc sevs. summary/conclusion: we find that low aggressive hnscc sevs affect metastasis of highly metastasized hnscc, and also find that changing immune cell distribution may be related to the result. this mechanism and finding contributes to understanding the possible role of hnscc sevs on metastasis as well as on the tumour immune microenvironment. funding: this work was supported by the nih under award numbers r ca and r ca to aw. desmoglein enhances squamous cell carcinoma tumour development through extracellular vesicles in an il- /mir- a-dependent mechanism introduction: the cadherin dsg is a stem cell marker that is upregulated in many different cancers, including sccs, and its expression correlates with poor prognosis. dsg activates mitogenic signalling and plays a key role in cell proliferation, migration, and survival. we recently showed that dsg enhances ev release, but the mechanism by which these evs modulate tumorigenesis is not fully understood. methods: we established scc cell lines stably expressing wildtype dsg or a palmitoylation deficient mutant, dsg cacs. evs were isolated by sequential ultracentrifugation, iodixanol gradient separation, or qev izon column, and analysed by nta and bca. tumour xenografts were established by subcutaneous injection of cells in scid mice and monitored up to weeks. cytokine profiling was determined by antibody array. mirna expression was analysed by rnaseq and confirmed by qpcr. results: dsg enhanced ev release by % and promoted a~fivefold increase in tumour size in xenograft models. tumour growth was increased when control cells were treated with a single µg dose of evs. loss of palmitoylation, which altered membrane trafficking of dsg , reduced ev release (~ %) as well as tumour development. plasma evs from xenograft mice reflected in vitro particle counts from scc cell lines. a cytokine array analysis was performed revealing that dsg -evs were enriched with pro-inflammatory cytokines including il- , a potent chemotactic and angiogenic factor. most importantly, il- was surface-bound on evs. furthermore, rnaseq revealed mir- a, a negative regulator of il- , to be significantly downregulated in response to dsg . treatment with mir- a mimic or mir- a inhibitor decreased or increased, il- expression in scc cells, respectively. summary/conclusion: in summary, dsg plays a key role in scc tumour development by increasing ev biogenesis and downregulating mir- a, which in turn upregulates il- synthesis and release which can promote invasion, angiogenesis and metastasis. funding: nih r introduction: stem-and progenitor cell transplantation therapy holds great promise for regenerating damaged heart tissue. several lines of evidence suggest that its efficacy is mainly caused by secreted extracellular vesicles (evs). indeed, cardiac progenitor cell (cpc)-derived evs have been shown to protect the myocardium against ischaemia/reperfusion injury in several preclinical models. however, the underlying mechanisms for cpc-ev-mediated cardioprotection remain elusive. here, we utilized the proteomic composition of cpc-evs released during different culture conditions, to unravel protein-mediated effects of cpc-evs on the endothelium. methods: cpcs were stimulated with calcium ionophore (ca ion-evs) or vehicle (control-evs) for hours and evs were isolated from serum-free conditioned medium using size exclusion chromatography. ev concentration and size was assessed using nta. evs were functionally characterized based on endothelial cell activation by western blotting and an endothelial cell scratch assay. the proteomic composition of both ev conditions was profiled using mass spectrometry. cpc-ev knockouts for specific proteins were generated using crispr/cas technology. results: we found enhanced phosphorylation of erk / and akt in endothelial cells and increased wound closure after stimulation with control-evs, but not after stimulation with ca ion-evs. proteomic analysis identified a total of ev-associated proteins, with proteins uniquely expressed in control-evs. another proteins were revealed as candidate proteins, based on their relative enrichment in control-evs compared with ca ion-evs. go analysis demonstrated that differentially expressed proteins were involved in vascular endothelial growth factor signalling, extracellular matrix organization and angiogenesis. to investigate the involvement of the individual candidate proteins on endothelial cell activation, knockout evs of multiple proteins were generated and functionally characterized. summary/conclusion: a specific set of ev proteins is identified that may be functionally responsible for the activation of endothelial cells upon exposure to cpc-evs. generating knockout evs for each of these proteins will help to investigate their individual roles. this may lead to a better mechanistic understanding of the use of cpc-evs as therapeutics for cardiac repair. funding: erc- -cog- evicare grant. hypoxia enhances the therapeutic potential of human cd + stem cell exosomes in ischaemic hindlimb repair ischaemic cardiovascular disease. we have previously shown that human cd + cell-derived exosomes (cd exo) improve perfusion and function of the ischaemic tissues. hypoxia is shown to modulate the secretion and content of exosomes in both cardiovascular and cancer research. therefore, we hypothesized that hypoxia can modulate the content and regenerative efficacy of human cd exo. methods: cd exo were isolated from primary human cd + stem cells cultured under hypoxia ( . % o , or normoxia ( % o , n-cd exo) using density gradient ultracentrifugation. cd exo size was measured using trps, nta, and dls and surface protein expression was determined using imaging flow cytometry. function of cd exo was assessed using cell viability, migration and matrigel tube formation assays in vitro and a mouse hind limb ischaemia model (hli) in vivo. protein content of hypoxic or normoxic cd exo was evaluated via lc-ms/ms and -d -dige followed by lc-ms/ms. results: we did not observe any significant differences in size or in quantity of exosomes secreted from h-or n-cd cells. both h-and n-cd exo expressed cd , cd and cd surface markers. interestingly, h-cd exo significantly improved cell viability, migration and tube formation of huvecs in vitro compared to n-cd exo. in the same line, h-cd exo also significantly improved perfusion (ratio: . ± . v . ± . ) and prevented ischaemic limb amputation ( % v . %) as compared to n-exo (p < . ; n = - ) in a murine (balbc nude) model of hind limb ischaemia. flow cytometry and confocal microscopy indicated that h-exo was uptaken by endothelial cells in the ischaemic limb. remarkably, we detected several proteins (including a fragment of hemopexin) and mirnas (mir- ) that could be responsible for the proangiogenic and beneficial function of h-cd exo. we have also demonstrated that removal of surface proteins diminished the pro-angiogenic function of cd exo. summary/conclusion: hypoxia enhanced the proangiogenic and regenerative potential of cd exo, and thus, may represent a more efficient clinical strategy for cd exo therapy. our research is clinically important to improve therapeutic angiogenesis in diabetic and cardiovascular patients with compromised stem cell populations. hyun-ji park a , jessica r. hoffman b and michael davis b a emory university, decatur, usa; b emory university, atlanta, usa introduction: exosomes, a subset of membrane nanovesicles, transfer cellular information by passing proteins and nucleic acids between cells. exosomes have been implicated as the mechanistic unit in stem cell therapy, as inhibition of exosome synthesis abrogates the effects of cell therapy following cardiac injury. more importantly, increasing evidence indicates that mirnas (mirs) within exosomes serve as important signalling molecules to regulate inflammation, recruit stem cells, and repair diseased tissue. among exosomal mirs, mir- and − are known to decrease angiogenesis, cell migration, and increase inflammation in various types of cells. here, we investigated the inhibition of these negative mirs as a means to improve the reparative capacity of c-kit+ progenitor cell (cpcs) exosomes. methods: cpcs were isolated from three paediatric patients using magnetic-bead sorting. ʹ-o-methylated rna duplexes inhibited mir- and − expressions in cpcs. exosomes (inhexos) were isolated from mirinhibited cpc conditioned medium. mir expression in exosomes and cpc was quantified by qrt-pcr. migration and proliferation of mesenchymal stem cells (mscs) were assessed two days post-exosome treatment. for inflammation analysis, thp cells with/without tnfα exposure were treated with exosomes and the expression of il- , − , and − was quantified by qrt-pcr. finally, the angiogenic potential of inhexos was tested by tube formation of cardiac endothelial cells. results: inhibitor treatment of cpcs decreased exosomal mir- and − expression. treatment with inhexos enhanced msc migration and proliferation compared with normal cpc exosome (norexo). moreover, inhexos showed promising results for immune regulation, as tnfα-induced inflammation was decreased in thp exposed to inhexos for h. however, tube formation capacity is slightly decreased (~ %) by inhexo compared to norexo. summary/conclusion: exosomes from mir- and − -depleted cpcs may be a promising strategy for the treatment of various cardiac diseases, as they enhanced stem cell recruitment and proliferation, and regulated inflammation and angiogenesis. while other studies focus on boosting the reparative potential of exosomes by increasing positive mir and mrna cargo, the inhibition of negative mir in exosomes could be an overlooked strategy for the treatment of cardiac disease. endo-lysosomes as an alternative intracellular location for ev cargo delivery with disease relevance introduction: extracellular vesicles (ev) are lipidbilayer nanovesicles that carry macromolecules and act as paracrine vectors for cell-to-cell communication. the processes regulating ev biogenesis are largely known, whereas how ev cargo is delivered to recipient cells remains poorly understood. a simple mechanism proposed is direct ev fusion with the cell membrane that liberate cargo into the cytosol. in this study, we observed that cargo release occurs also at an alternative intracellular location and that this acquires a disease relevance. methods: ev were isolated by serial centrifugation and characterized. for uptake studies, ev were traced by labelling donor cells with a lipophilic dye or by overexpressing gfp-cd . uptake was assessed by cytofluorimetry or by live confocal imaging. co-localization studies were performed with ectopic marker expression or by immune staining. protein-protein interaction was analysed by bi-molecular fluorescence complementation (bifc). prion-like transmission was studied using a pro-fibrillogenic tau fragment in donor cells and full-length tau in recipient cells. for quantification of subcellular localization, an automated algorithm based on machine learning was developed. lysosomal stress was monitored by nuclear translocation of tfe and lysotracker staining. antibodies directed against pathogenic epitopes of tau were employed to assess prionlike transmission. results: ev were taken up by recipient cells through an endocytic process and accumulated in endo-lysosomes (el). when cells were exposed to ev carrying a profibrillogenic tau, recipient cells accumulated tau within el by an autophagic process. direct interaction of ev-tau and cellular tau in el favoured the appearance of pathological epitopes. cells displaying this condition showed an increased el stress and cytotoxicity. summary/conclusion: in this study, for the first time we report that el represent a critical subcellular location where transcellular prion-like transmission mediated by ev of a neurodegeneration-associated protein occurs. thus, the degradative pathway most likely involved in the recycle of ev and endogenous proteins is highjacked in disease. these findings represent a novel mechanism for ev acting as vector for transcellular propagation of tau, which opens up new therapeutic interventions trying to halt the disease. funding: supported by gelu foundation. anti-human fab fragment of cd antibody prevents the endocytosis of melanoma and colon cancer-derived extracellular membrane vesicles and nuclear transfer of their cargos introduction: interfering with the mechanisms regulating intercellular communication mediated by extracellular membrane vesicles (evs) may find relevance especially in oncology where cancer cell-derived evs have an implication in the malignant transformation of tumour microenvironment. our laboratories recently demonstrated a novel intracellular pathway in which a fraction of endocytosed ev-associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of rab + late endosomes entering into the nucleoplasmic reticulum. here, we have investigated the effect of a monovalent fab antibody against the tetraspanin cd (referred hereafter as cd fab), on the internalization of evs and nuclear transfer of their cargo proteins. chair: david r f. carter -oxford brookes university chair: neta regev-rudzi -weizmann institute of science methods: to monitor the intracellular transport of ev-associated proteins, we used bioengineered fluorescent evs containing cd -gfp fusion protein from femx-i melanoma, sw colorectal cancer and bone marrow-derived mesenchymal stromal cells (msc) as donors and the same cell types as recipients. evs were enriched by differential centrifugation from h serum-free conditioned media and characterized by zetaview nanoparticle tracking analysis, zetapotential and immunoblotting. cd fab was prepared from h hybridoma cells using the pierce fab purification kit. results: we previously demonstrated that silencing cd both in evs and recipient cells strongly decreased the endocytosis of evs and abolished the nuclear transfer of their cargos. here we show that cd fab significantly reduced the cellular uptake of cd -gfp+ evs and the nuclear transfer of their proteins in melanoma, colorectal cancer and msc used as receptor cells in a dose-dependent manner. the effect on the nuclear transfer is probably a direct consequence of the endocytosis inhibition of evs. in contrast, the divalent, intact cd antibody stimulated both events. summary/conclusion: the effect of cd fab appears independent of the used ev-donor cell types or receptor cells, probably due to the widespread expression of cd both at plasma membrane and ev surface. in conclusion, by impeding intercellular communication in the tumour microenvironment, cd fab-mediated inhibition of ev uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti-cancer therapeutic strategies. a bright, versatile reporter for multivesicular body trafficking and exosome secretion and uptake bong hwan sung, ariana von lersner, jorje guerrero, evan krystofiak, david inmann, roxanne pelletier, andries zijlstra, suzanne ponik and alissa weaver vanderbilt university, nashville, usa introduction: live imaging of exosomes is one of the required tools to understand the function of exosomes. our previous live-cell reporter, phluorin-cd allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. however, there were some caveats to its use, including dim fluorescence and the inability to make cell lines that stably express the protein. methods: a stabilizing mutation, m r is incorporated in the phluorin moiety and now exhibits stable expression in cells and superior monitoring of exosome secretion. a dual-tag reporter was created by incorporating a further ph-insensitive red fluorescent protein, mscarlet to the c-terminus of phluo_m r-cd . cancer cells stably expressing the constructs were imaged using a variety of microscopy techniques in vitro as well as in vivo. purified small evs labelled with phuo_m r-cd were imaged using immunogold transmission electron microscopy (tem) and quantitated for the half-life in the blood circulation using flow cytometry. results: phluo_m r-cd and phluo_m r-cd -mscarlet are exclusively detected in exosomeenrich small ev preparations. immunogold tem visualizes the phluo_m r tag is located on the surface of small evs. live cell imaging reveals phluo_m r-cd -positive puncta left behind migrating cells suggesting the deposition consists of exosomes. those puncta and trails are not only positive for exosome markers such as cd , alix, and tsg but also correspond to small evs observed by a scanning electron microscopy. the dual-tag reporter allows visualization of the exosome lifecycle, including multivesicular body (mvb) trafficking, mvb fusion, exosome uptake and endosome acidification. summary/conclusion: using phluo_m r-cd construct, we demonstrate superior visualization of exosome secretion in multiple contexts and a role of exosomes in promoting leader-follower behaviour in collective migration by observing that exosomes are secreted at the front of migrating cells and left behind in exosome trails. the dual-tag reporter allows visualization of the entire exosome lifecycle. we anticipate that these reporters will be broadly useful to investigate regulation and functions of exosome secretion and uptake in diverse physiological conditions. funding: r gm , r ca , u ca - s , r ca . uncovering novel genes regulating ev-mediated functional rna transfer using a crispr/cas -based reporter system introduction: extracellular vesicles (evs) play a pivotal role in intercellular communication through functional transfer of bioactive cargo, including rna molecules. despite increasing interest in ev-mediated rna transfer, our understanding of the pathways and mechanisms regulating ev-mediated rna delivery and processing is limited due to a lack of suitable readout systems. we recently developed a novel crispr/cas -based reporter system that allows study of ev-mediated rna transfer at single-cell resolution. here, we further validate this system by studying the role of known targets involved in ev uptake and intracellular membrane trafficking, and subsequently employ this system to uncover various novel genes that play a regulatory role in functional rna transfer. methods: we employed a novel crispr/cas -based stoplight reporter system, in which egfp expression is activated upon functional delivery of targeting single guide rnas (sgrnas) stably expressed by donor cells. intercellular functional rna transfer was assessed by measuring egfp expression in acceptor cells using fluorescence microscopy and flow cytometry after direct co-culture, transwell co-culture, and upon addition of isolated evs. potential roles of various genes in intercellular rna transfer were assessed by rnaimediated target knockdown in acceptor cells, prior to co-culture experiments. rnai knockdown was confirmed by qpcr analysis. results: a significant activation of egfp expression was observed in acceptor cells after direct co-culture and transwell co-culture with donor cells expressing sgrnas, as well as after addition of evs from cells expressing sgrnas. reporter activation was substantially decreased after knockdown of multiple targets involved in ev uptake through endocytosis and/or intracellular membrane trafficking. based on these results, a potential role of various novel genes in intercellular rna transfer was studied in acceptor cells. these experiments uncovered various novel targets involved in ecm binding, endocytosis, intracellular membrane trafficking, as well as various rho gtpase interactors. summary/conclusion: we previously demonstrated a crispr/cas -based reporter system that allows the study of functional delivery of small non-coding rnas with single-cell resolution. here, we show that this novel approach allows the study of specific genetic targets and pathways in ev-mediated functional rna delivery, and unravel the regulatory pathways that dictate the underlying processes. quantitative characterization of extracellular vesicle uptake and content delivery within mammalians cells gregory lavieu a , emeline bonsergent b , eleonora grisard c and clotilde théry d introduction: extracellular vesicles (evs), including exosomes, are thought to mediate intercellular communication through the transfer of biomolecules from donor to acceptor cells. occurrence of ev-content delivery within acceptor cells has not been unambiguously demonstrated, let alone quantified, and remains debated. methods: we developed a cell-based assay in which evs containing luciferase-tagged cytosolic cargo are loaded on unlabelled acceptor cells. measurement of luciferase activity associated with acceptor cells revealed ev uptake efficacy. additional cell fractionation procedure that separates membranes from cytosol revealed the occurrence of ev-content release within the cytosol of acceptor cells. results: results from dose-responses, kinetics, and temperature-block experiments suggest that ev-uptake is limited ( % spontaneous rate at h), does not depend on bona-fide ev-receptor, at least for the tested acceptor hela cells. yet, further characterization of this limited ev-uptake, through cell fractionation that separates membranes from cytosol, revealed the occurrence of ev-content release within the cytosol of acceptor cells. cytosolic release is inhibited by bafilomycin-a and overexpression of ifitm proteins, which prevent virus content delivery. summary/conclusion: our results show that ev-content release requires endosomal acidification and suggest the involvement of membrane fusion. funding: anr -ce - - and arc pja and pga rf . introduction: glioblastoma is a highly malignant brain tumour with a poor prognosis. its ability to develop therapeutic resistance result in devastating clinical outcomes. to solve the intractable problem, we need highly sensitive diagnostics that can detect the molecular changes during treatments. extracellular vesicles (evs) can be a potential biomarker to monitor treatments and the host cell ev mapping can better reflect molecular changes in the tumour immune microenvironment. we have developed a droplet-based single ev protein sequencing platform that overcomes limitations of current bulk measurement technologies, which make it difficult to discover a rare ev population in the presence of high background. methods: we multiplex protein measurements to profile hundreds of proteins at a time by using an antibody-dna conjugate and sequencing. we barcode each ev in droplets and make amplicons that are comprised of both ev barcodes and antibody barcodes for sequencing. barcoded antibodies are made using tco-tetrazine click reaction and evs are labelled with these barcoded antibodies. the labelled evs are encapsulated into droplets with barcoded beads that serve as a template for ev barcodes. we then perform extension to make amplicons that contain both ev barcodes and antibody barcodes for sequencing. results: we successfully fabricated barcoded beads using a split-pool approach and validated by observing a fluorescence decrease of the sybr green after dna strand denaturation. we used a -channel droplet maker to encapsulate barcoded beads, single ev, and master mix into droplets. close packing of barcoded beads allowed > % encapsulation into droplets. both droplet and tube-based methods achieved a similar high amplification efficiency (ct < for evs). we confirmed the amplicon size by running a gel, which showed the right amplicon size (~ bp) from the droplet and tube prepared samples and no signal from the negative control. summary/conclusion: the droplet-based single ev profiling platform has the ability to identify rare immune ev subtypes in the peripheral blood, which would otherwise be impossible to detect due to the copresence of abundant normal evs. this cutting-edge technique has the potential to revolutionize treatment monitoring of high-cost immunotherapies, avoid unnecessary toxicities, and enhance personalized medicine capabilities. funding: schmidt science fellows, in partnership with the rhodes trust po ca , ro ca , r ca quantbio graduate student award at harvard university. introduction: in this study, we compared four orthogonal technologies for sizing, counting, and phenotyping of evs. the platforms were: single-particle interferometric reflectance imaging senor (sp-iris) with optional fluorescence, nanofcm nanoflow (nf), nanoparticle tracking analysis (nta) with fluorescence, and microfluidic resistive pulse sensing (mrps) . results from these platforms were compared with results from standard ev characterization techniques such as transmission electron microscopy (tem) and western blot (wb). methods: human t lymphocyte h (high cd , low cd ) and promonocytic u (low cd , high cd ) cells were chosen for their distinct tetraspanin profiles without abnormalities that might result from genetic manipulation. evs were isolated from culture conditioned medium (ccm) by differential ultracentrifugation (duc) and size exclusion chromatography (sec) and characterized per misev guidelines. synthetic particles (silica and polystyrene spheres) with known concentrations and mixed size distributions were also tested. results: particle counts from nf and mrps were consistent, while nta detected approximately one order of magnitude lower for ccm derived evs, but not for synthetic particles. sp-iris events could not be used to estimate particle concentrations. for sizing, nf, mrps, and sp-iris returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of nm. nta detected a population of particles with a mode diameter above nm. additionally, sp-iris, nf, and mrps were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. finally, for tetraspanin phenotyping, the sp-iris platform in fluorescence mode and nf were able to detect at least two markers on the same particle. summary/conclusion: based on the results of the study, we can draw conclusions about existing singleparticle analysis capabilities that may be useful for ev biomarker development and mechanistic studies. funding: this project is funded by mh and ug ca . importance to ev organotropism. yet, most techniques rely on bulk characterization, or are severely restricted by the diffraction limit. the exoview r (nanoview biosciences) combines interferometry, immunocapture, and immunofluorescence, introduced as an alternative technique to multiplex protein detection on single evs below the limit of diffraction. here, we use this technique to characterize tetraspanin multiplexing on evs and to identify spatial patterning of tetraspanins using steric hindrance of antibodies (abs). methods: evs were isolated from conditioned media from skov- cell culture or human serum. evs were incubated overnight on chips to allow immunocapture by anti-cd , anti-cd , or anti-cd . chips were then incubated with three fluorescent abs against the same epitopes and imaged on the exoview r . following concentration optimization, evs were tested after preincubating with carboxy-fluorescein diacetate succinimidyl ester (cfse) or fluorescent abs against tetraspanins. results: using different concentrations of evs, binding curves could be fit to characterize binding kinetics of abs. maximum concentration of evs could be identified that minimized fluorescent overlap. bright-field interferometry (detection limit~ nm) distinguished x fewer bound evs than fluorescent detection, while pre-labelling evs with cfse produced x more detectable evs than immunofluorescence. interestingly, evs captured by one tetraspanin did not necessarily show high fluorescent detection of the same tetraspanin. upon pre-incubating evs with a single ab, vastly different expression profiles were identified, indicating significant steric hindrance between abs. furthermore, pre-incubating evs with anti-cd ab significantly decreased detection of cd with less impact on cd . this discrepancy indicated possible spatial patterning of tetraspanins with cd and cd closely colocalizing on the ev surface. summary/conclusion: this combination of interferometry, immunocapture, and immunofluorescence produces unique information about size distribution of evs and single ev protein profile. this data corroborates that evs have distinct subpopulations of tetraspanins and indicates that tetraspanins may be spatially patterned. regulation of liver homoeostasis, regeneration and diseases by mesenchymal stem cell-derived apoptotic extracellular vesicles university of pennsylvania, philadelphia, usa introduction: billions of cells undergo apoptosis and produce apoptotic extracellular vesicles (apopevs) each day, whereas the roles of apopevs in regulating the organismal health and disease remain poorly understood. mesenchymal stem cells (mscs) emerge as critical contributors to tissue homoeostasis, while mscs suffer from apoptosis in regenerative transplantation. in this study, we investigated the function and mechanisms of msc-derived apopevs in regulating the organismal homoeostasis. methods: fas mutant (fasmut) and caspase knockout (casp -/-) mice were applied for apoptotic and apopev deficiency. mouse bone marrow mscs were cultured and apoptosis was induced by staurosporine (sts). msc-derived apopevs were collected by serial centrifuges and were infused into mouse circulation via caudal vein. tracing of apopevs were performed by radioisotope or fluorescent labelling. liver homoeostasis was evaluated at the histological and functional aspects. liver regeneration was induced by partial hepatectomy (phx). acetaminophen (apap) was used to establish acute liver drug injury. high-fat diet (hfd) was used to establish type diabetes (t d) and non-alcoholic fatty liver disease (nafld). results: after systemic injection, msc-derived apopevs migrate to liver and can be uptaken by liver macrophages and hepatocytes. fasmut and casp -/mice develop hepatomegaly with structural disorders, which particularly reveals hepatocyte polyploidization. furthermore, fasmut and casp -/-mice demonstrate liver glucose and lipid metabolic disorders. importantly, msc-derived apopev infusion significantly rescues structural and metabolic dysfunction in fasmut and casp -/-mice. mechanistically, apopevs use the soluble n-ethylmaleimide-sensitive fusion protein attachment protein receptor (snare) protein for interactions with recipient organelles thus transferring signalling molecules. moreover, msc-derived apopev infusion promotes liver regeneration after phx, prevents apap-induced liver injury, and ameliorates nafld in t d. summary/conclusion: msc-derived apopevs serve as crucial regulators of liver homoeostasis, regeneration and diseases. these findings indicate potential significant roles of apopevs in maintaining the organismal health and in developing therapeutics for diseases. (msc-sevs) mediate osteochondral regeneration in rats. however, the therapeutic effects of these msc-sevs/exosomes in restoring the mechanical competence of the repaired cartilage for joint function in a clinically relevant animal model remain to be addressed. to investigate this, we compared the structural and mechanical properties of the repaired cartilage in a rabbit model after intraarticular administration of msc-sevs and hyaluronic acid (ha) with that of ha alone, which is widely used as visco-supplementation. methods: bilateral osteochondral defects were surgically created on rabbits. immediately after surgery and at days and post-surgery, rabbits received ml injections of µg msc-sevs and ha in both knees, and rabbits received -ml injections of ha in both knees. at and weeks, macroscopic evaluation, histological scoring and compressive testing at different points on the repaired cartilage were performed. results: defects treated with msc-sev/ha showed improvements with time in macroscopic and histological scores and mechanical properties than defects treated with ha alone. in contrast, ha treated defects showed some repair at weeks, but this was not sustained, as evidenced by significant deterioration of histological scores and a plateau in mechanical properties from to weeks. by weeks, the msc-sev/ha repaired tissues demonstrated significantly better macroscopic score ( . vs . ; p < . ) and histological score ( . vs . ; p < . ). mechanical strength as measured by the young's modulus was significantly higher in the msc-sev/ha repaired cartilage than that in ha repaired tissues [defect centre ( . vs . mpa; p = . ) and overall periphery ( . vs . mpa; p = . ], and approximated that of the adjacent native cartilage. summary/conclusion: our findings demonstrated that msc-sevs and ha not only improved tissue morphology of the repaired cartilage but also promoted functional mechanical competence. this study establishes a clinically translatable protocol for use of msc-sevs for cartilage repair. introduction: mesenchymal stromal/stem cell (msc)exosome (mex) treatment has shown considerable promise in experimental models of bronchopulmonary dysplasia (bpd) and pulmonary hypertension (ph). mechanisms by which mex afford their beneficial effects remain incompletely understood and here, we embark into investigating them through assessment of mex biodistribution and impact on immune cell heterogeneity. methods: newborn fvb mice were exposed to hyperoxia (hyrx, % o ) at birth and returned to room air at postnatal day (pn) . mice received a bolus mex dose at pn . adoptive transfer studies were used to determine the role of mex-educated myeloid cells in vivo. mice were harvested at pn , , , or to characterize mex biodistribution and for assessment of pulmonary parameters. results: mex therapy effectively ameliorated core features of hyrx-induced neonatal lung injury, improving alveolar simplification, pulmonary fibrosis, vascular remodelling and blood vessel loss. exercise capacity testing and assessment of ph showed functional improvements following mex therapy. biodistribution studies demonstrated that mex localize in the lung, where they interact with lung monocytes/macrophages. whole lung mass cytometry (cytof) revealed that mex treatment promotes a pro-homoeostatic shift in lung immune cell apportion, replenishing the early hyrx-induced depletion in pulmonary cd + immune cells, restoring alveolar monocyte and macrophage populations and suppressing cellular inflammation. ex vivo and in vivo analysis showed that mex promotes a "pro-resolving" ccr -monocyte phenotype. notably, adoptive transfer of mex-educated bone marrow-derived myeloid cells (bmdmy), but not naïve bmdmy, restored alveolar architecture, blunted fibrosis, improved vascular remodelling and pulmonary blood vessel loss. summary/conclusion: mex treatment ameliorates core features of experimental bpd, restoring lung architecture, decreasing pulmonary fibrosis and vascular muscularization, ameliorating ph and improving exercise capacity. the beneficial actions of mex are associated with modulation of immune cell phenotypes, arising from mex-monocyte interaction. furthermore, adoptive transfer of mex-educated bmdmy rescued, at least in part, alveolar architecture, reduce fibrosis, improve vascular remodelling and pulmonary blood vessel loss. funding: this work was supported in part by an american thoracic society foundation grant (grw); the little giraffe foundation (grw); charles h. hood foundation major grants initiative (sk), nih r hl (sk) and united therapeutics research grant (sk and sam). immunomodulatory small extracellular vesicles derived from mesenchymal stem cells: a potential cell-free therapy for acute and chronic pulmonary vascular diseases introduction: vascular inflammation plays a critical role in acute respiratory distress syndrome (ards) and pulmonary arterial hypertension (pah). despite decades of research, there is no curative therapy for either condition. mesenchymal stem cells (mscs) have shown preclinical efficacy, mediated by release of extracellular vesicles. hence, msc-derived small extracellular vesicles (sevs) can harness the benefits of mscs with advantages in cost and safety. this study aims to evaluate the immunomodulatory effects of sevs in preclinical ards and pah. methods: msc-sevs were characterized by nanoparticle tracking analysis, electron microscopy and western blot. live fluorescence imaging measured in vitro and in vivo distribution of sevs. using a lipopolysaccharide (lps)-induced mouse model of acute lung injury (ali), a time course study of inflammatory response guided endpoint analyses. cell count and cytokines were measured in bronchoalveolar lavage fluid (balf) and histological lung injury was assessed. in ali mice, saline, mscs, msc conditioned media or sevs were administered . h post-lps. using a monocrotaline (mct)-induced rat model of pah, animals received saline or sevs at day . haemodynamic changes and right ventricular hypertrophy were evaluated at weeks. results: msc-sevs were nm in size with cd / expression. pkh -labelled sevs were taken up by endothelial cells. in the ali time course study, cell count and il b in balf peaked at h post-lps, whereas il peaked at h. histology showed significant intra-alveolar cell infiltrate at h. msc conditioned media attenuated il b in balf, whereas a trend towards reductions in il b and cell count were seen from delivery of mscs and sevs. using fluorescence imaging, lung accumulation of dir-labelled sevs was highest when administered h post-lps as compared to h, h or h. for pah rats, sevs reduced right ventricular systolic pressure ( . ± . mmhg) as compared to control ( . ± . mmhg; p = . ), whereas no changes were observed for right ventricular remodelling. summary/conclusion: these findings demonstrate the potential of msc-sevs to be used as a cell-free immunomodulatory therapy for acute and chronic lung vascular diseases. additional live and ex-vivo biodistribution studies will determine optimal timing of sev administration, tissue distribution and clearance in both ali and pah. changes in extracellular vesicle protein cargo after pro-inflammatory priming of umbilical cord mesenchymal stem cells (ucmscs) have been shown to suppress inflammatory responses in studies of autoimmune diseases. these therapeutic effects can be attributed to paracrine signalling, by which extracellular vesicles (evs) are one of the essential components. this study looks at how the culture conditions of ucmscs affects the type of evs they secrete. it also aims to identify an ev population with an anti-inflammatory potential for the treatment of autoimmune diseases. methods: ucmscs were isolated and culture expanded in a quantum® cell expansion system, then grown at %o , %o and primed with a pro-inflammatory cocktail. evs were isolated from ucmsc conditioned media by differential ultracentrifugation using a sucrose cushion and characterised by transmission electron microscopy and nanoparticle tracking analysis. ev markers were analysed using a europium-based immunoassay, macsplex exosome detection kit and immunoblotting. a proximity-based extension assay was used to identify inflammatory proteins in the evs. results: there was no difference in evs cultured at %o , %o or with pro-inflammatory conditions when analysed for size and morphology. all evs displayed the tetraspanin markers (cd / / ) and internal proteins (alix, hsp ). evs from primed cells showed a > twofold increase of cc chemokines and a > sixfold increase in cxcl and csf- . protein cargo did not differ in evs from %o and %o . summary/conclusion: this study showed that proinflammatory culture conditions alter ev protein cargo, evidenced by the increased production of chemotactic and angiogenesis associated proteins. upcoming rnaseq analysis will show if ucmsc culture conditions also affect mirna expression in evs. ongoing functional studies will determine how changes in ev cargo correlates with changes in t-cell proliferation and polarisation. funding: this work is fund by the orthopaedic institute ltd, keele university and the rjah orthopaedic hospital charity. alzheimer's disease biomarkers in plasma extracellular vesicles of neuronal origin correlate with brain pathology in mice introduction: multiple studies have shown that neuronal-derived extracellular vesicles (ndes) in blood contain alzheimer's disease (ad) biomarkers, especially tau. however, the convergent validity of tau in blood ndes in relation to brain pathology is yet to be determined. to address this, we measured total and phosphorylated tau levels in matched nde and brain tissue samples from ad mouse models. methods: we collected the cortex, hippocampus and plasma of xtg-ad, xtg-ad, and wild type (total of mice; female, male; age: mean = . , sd = . , - months) . plasma samples were collected retro-orbitally for weeks and at euthanasia via heart puncture. ndes from the pooled serial blood collections (nde ) and the single endpoint (nde ) were immunocaptured by targeting the neuronal marker l cam. we measured human total tau and pthr -tau (p-tau) in ndes and cortex and hippocampus homogenates using a luminex multiarray. results: overall, there were strong positive correlations for both total tau and p-tau between ndes and brain tissues across mice types. total tau in ndes showed positive correlations with levels in the cortex and hippocampus (r = . and . , p < . , cortex vs nde and nde ; r = . , p = . , hippocampus vs nde ; r = . , p = . , hippocampus vs nde ). levels of p-tau in nde showed positive correlations with levels in the cortex (r = . , p = . ) and hippocampus (r = . , p = . ); however correlations were not observed for nde (r = . , p = . vs cortex; r = . , p = . vs hippocampus). summary/conclusion: tau levels in circulating ndes reflect levels in cortex and hippocampus across ad model mice, supporting their convergent validity as "liquid biopsy" biomarkers for ad. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. exosomal ceramide mediates neurotoxicity of amyloid beta (aβ) in alzheimer's disease. ahmed elsherbini a , simone crivelli b , alexander kirov c , michael dinkins d , zhihui zhu a , haiyan qin a , sanjib karki a , priyanka tripathi a and erhard bieberich a a university of kentucky, lexington, usa; b university of kentucky, lexington, usa; c augusta university, augusta, usa; d augusta university, augusta, usa introduction: amyloid beta is a pathologic hallmark of alzheimer's disease (ad), however, the mechanism of aβ neurotoxicity is not fully understood. it has been reported that exosomes associate with aβ, but it is not clear how this association affects aβ neurotoxicity. methods: here we utilized several techniques to isolate exosomes from the sera of wild type (wt) and ad transgenic mouse model. ( xfad) as well as alzheimer's patients and healthy controls. we used exoquick, exoeasy, sequential ultracentrifugation, and size exclusion chromatography. particles' size and number were characterized by nanoparticle tracking analysis (zetaview). results: we report that the sphingolipid ceramide mediates neurotoxicity of aβ. we show that sera from ad transgenic mouse model ( xfad) and ad patients, but not the wt or healthy controls, contain a subpopulation of astrocyte-derived exosomes that are enriched with ceramide and are prone to aggregation (termed astrosomes) as confirmed by nanoparticle tracking and cluster analyses. when taken up by introduction: multiple sclerosis is the most common chronic inflammatory demyelinating disease of the central nervous system, affecting more than million people worldwide. ms is a multifactorial, immunemediated disease caused by complex genetic and environmental interactions. in recent years, extracellular vesicles (evs) have been described as powerful mediators of the modulation of biological processes (e.g. inflammatory and immune response) following environmental exposures such as particulate matter (pm), and have been described altered in ms. we characterized evs in patients with ms and healthy subjects matched for age and gender and evaluated the effects of pm exposure on ev release patients with ms compared with controls. methods: evs isolated from blood samples were characterized by nanotracking analysis and by flow cytometry after labelling with the following markers: cd + (monocyte), cd + (platelet), cd + (neutrophil), cd + (t-reg), and cd + (endothelium). pm and pm . concentrations at the residency of each subject were obtained from the regional air quality monitoring network. results: we observed decreased concentrations of cd + (p < . ), cd + (p < . ), cd + (p < . ), cd + (p < . ), and cd + (p < . ) in patients compared with controls. in cases, pm was inversely associated with cd + evs (pm . , β = − . ; p < . ), cd + evs (pm . β = − . ; p < . ), and cd + evs (pm β = − . ; p < . ; pm . , β = − . ; p < . ). on the contrary, in controls pm was positively associated with cd + evs (pm β = . ; p < . ; pm . , β = . ; p < . ). summary/conclusion: our findings showed a different composition of blood-derived ev subpopulations in patients compared with controls. moreover, we observed that patients and controls react differently to pm exposure in terms of blood-derived ev release, suggesting the involvement of this mechanism in the modulation of both inflammatory and immune responses, and thus in ms pathogenesis. plasma neuronal and astrocyte-derived exosomes serve as biomarkers of neurodegeneration and systemic bioenergetic effects in male cynomolgus monkeys self-administrating oxycodone ashish kumar a , yixin su a , david soto-pantoja a , jingyun lee a , ravi singh a , cristina furdui a , michael nader b and gagan deep a a wake forest baptist medical center, winston-salem, usa; b wake forest baptist medical center, winston-salem, usa introduction: opioid use disorder (oud) is currently a health emergency in the usa affecting millions of people. oud is a complex issue requiring a multipronged strategy. at the biological level, there is an urgent need to understand the dynamic molecular changes and adverse effects associated with opioid addiction. here, we aimed at identifying the biosignature of brain cells-derived exosomes associated with opioid addiction in a non-human primate (nhp) model of oud in which cynomolgus monkeys perform cognitive tasks and self-administer (sa) intravenous oxycodone daily. we also characterized the systemic adverse effects of the brain cells-derived exosomes from drug-naïve and oxycodone sa monkeys. methods: we isolated total exosomes (te) by ultracentrifugation and exoquick methods from the plasma of male monkeys self-administrating oxycodone for years and naive monkeys. subsequently, from the te population, we isolated neuron-derived exosomes (nde) and astrocytes-derived exosomes (ade) using surface biomarkers l cam (l cell adhesion molecule) and glast (glutamate aspartate transporter), respectively. this novel method involved streptavidin coated magnetic beads and photo-cleavable (pc) biotin, providing us biologically intact exosomes useful for co-culture studies. we characterized the exosomes by nanoparticle tracking analyses (nta), western blotting, flow cytometry, immunogold labelling, transmission electron microscopy (tem), elisas and mass spectrometry. respirometric profiling in cardiac myoblasts and monocytes following exosomes treatment was performed by seahorse xf. results: the quality of isolated exosomes (te, nde, and ade) was confirmed by nta (size distribution and concentration), western blotting (e.g. cd ) and tem (size and shape). nta did not show any significant difference in exosomes size and concentration (number per ml) between control and oxycodone sa groups. flow cytometry (e.g. l cam and glast) and immunogold labelling (cd , cd and l cam) confirmed the purity of nde and ade isolated from te. proteomics analyses of te, nde and ade identified several unique proteins present in exosomes from the oxycodone sa group. interestingly, we observed significantly higher expression of neurodegeneration markers neurofilament light protein (nfl) and alpha-synuclein in nde and ade of oxycodone sa group compared to controls. furthermore, te treatment of h c cardiac myoblasts and raw . monocytes significantly compromised their mitochondrial metabolism (basal and maximum respiratory capacity). summary/conclusion: these results suggest the utility of plasma exosomes as biomarkers for better understanding of the neurodegenerative and systemic effects of oxycodone addiction. funding: da , da . vesicles released during mycobacterium tuberculosis infection: immunomodulatory (glyco)lipids and role in host-pathogen interactions emilie layre, pierre boyer and jerome nigou cnrs-université paul sabatier, toulouse, france introduction: the tuberculosis disease remains one of the top causes of death worldwide. mycobacterium tuberculosis (mtb) has evolved strategies to evade immune responses and to persist within the hostile intracellular environment of alveolar macrophages. the current lack of efficient anti-tuberculosis strategies is largely due to our incomplete understanding of the host-pathogen interactions of mtb infection. vesicles released by the bacillus itself (bacterial membrane vesicles, bmv) and by infected cells (host extracellular vesicles, hev) have immunomodulatory properties in vitro and when administered to animals. if vesicles likely play key role in host-pathogen interactions of the tuberculosis infection, their content in bacterial factors, their uptake, trafficking and interaction with host cells receptors remain incompletely deciphered. methods: bmv and hev have been purified by combining differential centrifugation, density gradient and exclusion chromatography. after characterization by microscopy, nanosight and western blot, their content in bacterial (glyco)lipids has been characterized by the use of high sensitivity mass spectrometry-based lipidomic approach. bmv have been tested for their capacity to activate reporter cell lines of pattern recognition receptors. in addition, fluorescent-labelled bmv have been used to study their uptake by host cells thank to super-resolution microscopy. results: we have undertaken to characterize the content, the trafficking and interaction with pattern recognition receptors of bmv and hev released during infection by mycobacteria of variable virulence. we have importantly optimized the purification of bmv showing that lipoproteins aggregates are co-purified with vesicles on density gradient. sfc-ms lipidomic analyses allowed the characterization of the repertoire of immunomodulatory bacterial lipids released by bmv and hev, which excluded a continuum between these two release pathways. preliminary, assays have shown that these vesicles are capable to interact with different pattern recognition receptors including tlr and lectins. finally, we have been able to visualize fluorescent-labelled vesicles uptake by macrophages using superresolution microscopy. summary/conclusion: during m. tuberculosis infection, the bacillus as well as infected cells release vesicles that harbour different content in immunomodulatory bacterial (glyco)lipids, including strain-specific lipids. these vesicles likely play important role in host-pathogen interactions by modulating immune response beyond the infected cells, in part through their interaction with different pattern recognition receptors. funding: fondation pour la recherche medicale, fondation fonroga. introduction: conventional diagnoses of mycobacterium tuberculosis (mtb) rely on quantifying bacteria in sputum samples, which make it incapable of measuring the body's total bacterial load and diagnosing patients that have difficulty producing sputumsuch as children and those that are hiv-positive. nanoscale ( - nm) outer membrane vesicles (omvs), which are shed from their bacterial cells of origin and circulate in the bloodstream, have been found to contain rich molecular information from their mother cells. despite the diagnostic potential, their nanoscale size in the presence of high background has complicated the use of these promising biomarkers for clinical diagnosis of tuberculosis. chair: amy buck -the university of edinburgh chair: cherie blenkiron -the university of auckland methods: here we report two complementary approaches to systematically discover and clinically detect mtb-derived omvs using protein and rna biomarkers. first, we employ a digital droplet elisa on whole, unprocessed samples to detect and quantify the presence of these omvs using surface protein markers. second, we have developed a platform to specifically enrich for mtb-derived omvs using our previously developed magnetic nanopore platform, wherein millions of nanofluidic devices are operated in parallel, increasing throughput relative to a single nanofluidic device by a million-fold. using this approach, we identify rnas that are specifically enriched in mtb-derived omvs and can be used to identify tb strain, infectious activity, and total body burden. results: using these platforms, we enriched for mtbderived omvs from plasma and profiled their cargo, both proteins and rna. we first determined a panel of protein biomarkers for multiplexed detection of omvs through a digital droplet sandwich elisa. we then tested our protein markers on spiked plasma samples as models for clinical tb samples. simultaneously, we performed rna sequencing and discovered a panel of rna biomarkers that are preferentially enriched in omvs. we picked ten of the most highly-expressed rna biomarkers and also tested for them on spiked plasma samples using our magnetic nanopore platform. summary/conclusion: these results demonstrate the capability of omv biomarkers in the development of novel liquid biopsy based mtb diagnostics. building on this work, we are working with clinical collaborators to test our assays on clinical samples from philadelphia and west africa. funding: nih ot . introduction: a dearth of knowledge exists regarding the molecular mechanisms by which host exosomes regulate immune response to infections caused by gram-negative pathogens. to address this gap in knowledge, our laboratory has been using two wellestablished model organisms; yersinia pestis (yp), and burkholderia pseudomallei (bp). yp and bp cause the emerging human diseases plague and melioidosis respectively. currently, no licenced vaccines or highly effective therapeutics are available for either disease. methods: ex were purified from naïve u monocytes (exu) and infected u (exi) by serial centrifugation followed by sucrose density gradient purification, and characterized by tem, zetaview nanoparticle tracking, and exosome markers (cd , tsg , . immune responses of naïve u cells and response mechanisms were analysed following treatment with equivalent amounts of exi or exu (as control). these included macrophage differentiation assays, multiplex measurements of inflammatory cytokines, bacterial clearance assays, quantitative protein microarray analysis of host signalling proteins, sirna knockdown of exi-induced cytokines in recipient cells, and mass spectrometry (ms) analysis of exi contents. for all assays, at least four biological replicates were performed. results: exi induce monocyte differentiation to macrophages and dramatic release of il- , il- and il- cytokines, effects that are also seen when monocytes are infected with the bacteria. the exi also induce a substantial increase in the capacity of the recipient monocytes to clear bacteria in an il- -dependent manner. specific host signalling molecules are strongly modulated by the exi, including p , jak and alk for exi-yp, influencing the observed phenotypes. ms analysis showed lack of lps in exi-yp and demonstrated the presence of specific bacterial proteins that have antigenic properties. summary/conclusion: we have identified some of the molecular mechanisms by which exi assist the host in clearing infection. exi prime distant naïve monocytes through modulation of distinct pathways such as p to mount immune responses similar to when they become infected. these include differentiation to macrophages and migration to infection site for increased il- -dependent bacterial clearance. introduction: recruitment of monocytes to sites of infection is important in restricting growth and invasion of various microorganisms such as pathogenic fungi c. albicans. beside complement supported phagocytosis and extracellular trap formation, human monocytes secrete extracellular vesicles which are crucial in cellular communication in physiology and pathophysiology as they transfer proteins, lipid, and nucleic acids. the current study attempts to shed light on immune evasion mechanisms by c. albicans via extracellular vesicles. methods: human monocytes were isolated by magnetic beads technique and extracellular vesicles were isolated using polymer precipitation or ultracentrifugation or size exclusion chromatography. vesicles were characterized by elisa, lc/ms-based proteomics, confocal laser scanning microscopy (clsm) as well as electron-and dynamic light scattering microscopy, etc. crispr-cas based genome editing was performed to knockout cd b in human monocytic thp- cells. effect of isolated vesicles were determined by using proximity ligation assay (pla), elisa, western blot, next generation rna sequencing, qpcr, immunohistochemistry, etc. results: here we show for the first time that human blood derived monocytes alone and in a whole blood model strongly induced and released extracellular vesicles in response to the pathogenic fungus c. albicans. one induced population carried the anti-inflammatory cytokine tgfβ- . release of these vesicles is triggered by binding of soluble β-glucan from c. albicans to the cr receptor on monocytes as demonstrated by crispr-cas based cr genome editing in thp- cells, and by using cr knock out mice. isolated tgf-β -transporting vesicles reduced the inflammatory response in human m macrophages and in a whole blood model. the anti-inflammatory effect by tgf-β transporting vesicles is investigated in detail and results in inhibition of il- β gene transcription. summary/conclusion: showing that human apoptotic bodies similarly induced tgf-β -transporting vesicles from human monocytes we hypothesize that c. albicans hijacks this new cr -dependent anti-inflammatory vesicle pathway for immune escape. funding: this work was supported by the "deutsche forschungsgemeinschaft" transregio funginet projects c , c , c and z . introduction: to date, most research involving extracellular rnas has focused in rnas encapsulated inside extracellular vesicles (evs) or in total unfractionated biofluids. it is known that exrnas also exist outside vesicles or in lipoprotein particles. however, nonvesicular exrnas remain widely uncharacterized despite being a feasible source of contaminants in ev preparations. our interest in nonvesicular exrnas arises from the observation that some small rnas, such as specific trna-derived fragments, have much higher relative representation in this extracellular fraction. at least in part, this enrichment seems to be a consequence of their differential extracellular stability. methods: to get a representative picture of the whole set of rnas released to the extracellular nonvesicular space by cultured human cells, we inhibited extracellular degradation by adding recombinant ribonuclease inhibitor to the cell-conditioned media and studied the kinetics of rna release and degradation. high-resolution iodixanol gradients were used to separate evs from extracellular rnps or vesicle-free rna. the conversion rate between parental ncrnas and their fragments was studied by high-throughput sequencing and northern blot. results: the inhibition of extracellular rnase activity revealed the presence of full-length trnas and ribosomes in the extracellular space of a variety of malignant and non-malignant cell lines. extracellular ribosomes co-isolate with evs purified by ultracentrifugation or size-exclusion chromatography, but not with evs purified by density gradients.these ncrnas are substrates of extracellular rnases, demonstrating an extracellular biogenesis route for the formation of ncrna-derived fragments, some of which achieve remarkable stability and can be detected in biofluids. we also highlight the immunoregulatory potential of purified rna-containing extracellular complexes. summary/conclusion: in conclusion, ribonuclease inhibition dramatically shapes extracellular rna profiles and uncovers a population of extracellular ribosomes, trnas and other coding and noncoding rnas which exists outside evs. although these rnas are prone to degradation, some of their fragments can accumulate in cell culture media and in biofluids. this dynamic view of exrnas impacts our understanding of rna secretion mechanisms and may offer a window to new molecules with biomarker potential. in contrast, evs confer an rnase-protected environment and contain more full-length ncrnas (trnas, yrnas, sl rnas, rrnas depending on vesicle size) than their fragments. introduction: cd is a ubiquitously expressed membrane protein that functions as a receptor for thrombospondin- and the counter receptor for signal regulatory protein-α in phagocytes. high expression of cd is associated with a poor prognosis for some cancers. conversely, cd blocking agents are in clinical trials for enhancing innate and adaptive antitumor immunity in cancer patients. these studies suggest utility of cd as a diagnostic and prognostic biomarker and as a therapeutic target. cd is also expressed on extracellular vesicles (evs), and we reported that cd expression identifies a distinct population of evs from those that express the traditional ev markers cd or mhc . cd -, cd -and mhc -enriched vesicles contain distinct small rna populations (pmid: ), and these differ in rna content from evs that lack any of these markers. the mechanisms by which cd directly or indirectly regulates which rnas are packaged into ev remain unknown. methods: to elucidate the mechanism by which cd regulates ev rna composition and function, we performed global mirna microarray analysis between evs produced by wild type and cd -deficient t cells. results were further validated using real-time pcr and rna-immunoprecipitation. interactions between cd and exportin- /ran complex was identified by mass spectrometry and confirmed by using co-immunoprecipitation, subcellular localization, flow cytometry, and confocal and electron microscopy. results: ev released from cd -deficient human t cells and in cd -/-mouse plasma were enriched in ʹ- -methylguanosine-capped micrornas and mrnas that depend on the exportin- /rangtp pathway. knockdown of cd in wild type cells or thrombospondin- treatment correspondingly enhanced levels of capped-rnas released in ev and re-expressing cd in null cells decreased their levels. mass spectrometry and co-immunoprecipitation identified specific interactions of cd with components of the exportin- / ran nuclear export complex and its known cargos and between the cd cytoplasmic adapter ubiquilin- and the exportin- /ran complex. interaction of cd with exportin- was inhibited by leptomycin b, which inactivates exportin- and increased levels of cap-dependent rnas in ev released from wild type but not cd -deficient cells. summary/conclusion: these findings indicate that cd -dependent thrombospondin- signalling regulates cytoplasmic levels of cap-dependent rnas in t cells at least in part through ubiquilin- -and gtpdependent physical interactions with the exportin- / ran transport complex, which regulate levels of specific pre-mirnas and mrnas available for sorting into evs. funding: this work was supported by the intramural research program of the nih/national cancer institute (zia sc ). role of membrane protein palmitoylation in extracellular vesicle biogenesis in squamous cell carcinoma introduction: desmoglein (dsg ), is a palmitoylated cadherin that is involved in cell-cell adhesion. interestingly, dsg promotes mitogenic cell signalling and is upregulated in many cancers, including scc, contributing to poor prognosis and survivability. we recently demonstrated that dsg promotes ev release, but the mechanism by which dsg enhances ev biogenesis and role of palmitoylation is poorly understood. methods: pharmacological drug inhibitors -bromopalmitate, gw , and bafilomycin a were used. stable scc cell lines were established by retrovirus infection expressing gfp, wild type dsg /gfp, or palmitoylation deficient dsg cacs/gfp. evs were isolated by sequential ultracentrifugation and iodixanol gradient separation and analysed by nta. proteins associated with the endocytic pathway were analysed by immunofluorescence and imaged by confocal microscopy or immunoblotting and signals were quantitated using imagestudio. results: here we demonstrate that the effect of dsg on ev release was reduced by the palmitoylation inhibitor -bromopalmitate. furthermore, mutations that prevented palmitoylation (dsg cacs) dramatically abrogated ev release by targeting of un-palmitoylated dsg to the lysosomes for degradation. dsg increased expression and subcellular localization of flot , a membrane lipid raft protein critical for membrane invagination. dsg also altered membrane localization of several early (eps and eea ), but not late (rab , rab , and hrs), endocytic pathway proteins. loss of palmitoylation in the dsg cacs mutants abrogated these effects. finally, dsg -induced ev release was abrogated by the sphingomyelinase inhibitor gw or augmented by the v-atpase inhibitor bafilomycin a . summary/conclusion: the combined results of the drug treatments and functional mutations of dsg suggest that dsg plays a critical role in ev biogenesis by modulating proteins involved in early endosome sorting and is dependent on post-translational palmitoylation. introduction: the translation initiation factor eif e ( e) is an oncogenic protein that is upregulated in % of cancers including a subgroup of acute myeloid leukaemia (aml) patients. eif e regulates post-transcriptional rna processing including the nuclear export and/or translation of mrna transcripts. in particular, it selectively increases the expression of genes that have a prominent role in cancer progression such as myc, cyclin d , and mcl . furthermore, our lab pioneered studies demonstrating that a subset of ehigh aml patients is clinically responsive to treatment with a e inhibitor (ribavirin) indicating the importance of e in aml progression and its relevance as a therapeutic target. we investigated an as yet unexplored perspective of e-whether the oncogenic role of e is in part mediated by its function as a master regulator of vesiculation. methods: to assess mrna export and identify ebound mrna targets that correspond to vesiculationrelated genes and associated cargo, we used cellular fractionation and rna immunoprecipitation techniques. to determine whether e regulates the number of extracellular vesicles (evs) released as well as their protein and rna cargo we used nanoparticle tracking analysis (nta) as well as mass spectrometry, antibody microarrays, and rna sequencing technologies. results: eif e upregulates cellular protein levels of the vesiculation marker cd by increasing its nuclear export. in addition to increased cellular expression, cd , cd , cd , and flotillin- proteins are elevated in evs released from e-high cells. this is also associated with an increased release of vesicles that are - nm in size. currently, we have validated the upregulation of several receptors and cytosolic proteins in evs isolated from e-overexpressing cells that function in cell growth, migration, invasion, and stemness. the most abundant rnas in our ev preparations are micrornas (mirs) and we have confirmed downregulation of several of these. summary/conclusion: our work shows that e reprograms the vesiculation of cancer cells changing the release and cargo of evs. this may impact cellular communication and tumour biology, which we are currently addressing in functional studies. we hope that these studies will highlight novel therapeutic strategies for aml patients. intranasal administration of neural stem cells-derived extracellular vesicles promotes neurogenesis and reduces neuroinflammation and amyloid plaques in a mouse model of alzheimer's disease introduction: cognitive and memory impairments worsen with time in alzheimer's disease (ad), likely due to a progressive loss of hippocampal neurogenesis, and escalation of neuroinflammation. these changes are also accompanied by increased deposition of amyloid plaques in the brain. methods: in this study, using the xfad mouse model, we examined the efficacy of extracellular vesicles (evs) shed from the rat subventricular zone neural stem cells (svz-nscs) for disease modification. we first purified evs from the rat svz-nsc cultures through ion-exchange chromatography and then administered intranasally to -months old xfad mice (~ billion/week for two weeks). two months later, the functional effects of ev treatment were quantified through a series of behavioural tests, and animals were euthanized for quantification of hippocampal neurogenesis, oxidative stress, neuroinflammation, and amyloid plaque deposition. results: in comparison to ad mice receiving vehicle, ad mice receiving nsc-evs displayed improved cognitive function to discern minor changes in the environment in an object location test, better spatial recognition memory in an object-in-place test, and improved pattern separation ability in a pattern separation test. besides, ev-treated ad mice displayed no anhedonia in a sucrose preference test. analyses of neurogenesis using the birth-dating marker ʹ-bromodeoxyuridine and the newly born neuron marker doublecortin revealed maintenance of a higher level of hippocampal neurogenesis in ad mice receiving evs, in comparison to vehicle-treated ad mice. moreover, analyses of brain tissues from ev-treated ad mice revealed decreased concentrations of oxidative stress markers malondialdehyde and protein carbonyls and elevated levels of antioxidants catalase and superoxide dismutase. also, the concentration of proinflammatory cytokines tumour necrosis factor-alpha and interleukin- beta and the extent of amyloid plaques were significantly reduced in ev treated ad mice. immunohistochemical analysis showed reduced hypertrophy of astrocytes. summary/conclusion: intranasal administration of nsc-derived evs restrains the deterioration of cognitive and mood dysfunction of ad by maintaining higher levels of neurogenesis and curtailing the progression of neuroinflammation. funding: supported by a grant from the national institute of neurological disorders and stroke ( r ns - to a.k.s.) ot . the case of mesenchymal stromal cells, opening new perspectives in the use of ipsc in regenerative medicine. the aim of this study is to evaluate the potential of ipsc-ev in the treatment of kidney disease. methods: the ipsc were generated from skin fibroblast after informed consent of healthy donors (cytotune®-ips . sendai reprogramming kit -protocol: clementino fraga filho uh . . . / . ). the ev were isolated from ipsc supernatants (cultured h in mtesr- medium) by ultracentrifugation ( , g for h at °c). characterization of ipsc-ev was performed using zetaview, tem, exoview™ tetraspanin kit and macsplex exosome kit. for in vitro injury, renal epithelial cells were cultured under hypoxia ( % o ). for in vivo injury, male wistar rats were submitted to bilateral renal arterial clamping ( min) followed by reperfusion without or with injection of ipsc-ev (protocol approval: federal university of rio de janeiro - / ). kidney damage was assessed by histological and immunohistochemistry analyses (pcna, tunel and ed- ). modulation of rna levels was assessed by rt profiler pcr array. results: the results show that ipsc-ev reduce renal cell death, tissue damage, macrophage infiltration, promote mitochondria protection and ameliorate renal function. the ipsc-ev mechanism of action is related to the regulation of key genes known to prevent damage caused by oxidative stress like gstk , sod , sod , txn and txnrd . characterization of ipsc-ev showed that ipsc-ev can carry important molecules that can support renal recovery as epcam and prominin- . summary/conclusion: ipsc-ev presents renoprotective properties, acting on different aspects of aki. this presents a new relevant application of ipsc as a source of ev for therapeutic purpose in kidney diseases. the hospital for sick children, toronto, canada introduction: incomplete lung development, also known as pulmonary hypoplasia (ph), is a recognized cause of neonatal death. we have previously shown that experimental ph can be rescued by the administration of extracellular vesicles derived from amniotic fluid stem cells (afsc-evs) through an rna-mediated mechanism. this effect was not observed with evs derived from mesenchymal stromal cells (msc-evs) . the aim of this study was to ) evaluate which rna species were responsible for ph rescue, and ) to define the mechanism behind this effect. methods: evs were isolated and characterized from conditioned medium of rat afscs and rat mscs (control group) using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd , hsp , flo- , and tsg (western). to identify the mediators of afsc-evs, we used deseq (fdr< . ) to differentially analyse rna from: a) asfc-ev and msc-ev cargo, isolated with seramir and sequenced with nextseq. b) lung epithelial cells from rat ph lungs treated with vehicle or afsc-evs. epithelial cell rna was isolated with mirvana and sequenced with nextseq. we correlated afsc-ev cargo mirna with validated mrna targets that were downregulated after ev conditioning in lung epithelial cells. results: of the rna species contained in asfc-ev and msc-ev cargo, mirnas were the most proportionally different between the two ev populations. afsc-evs were enriched for mirnas that are critical for lung development, such as mir ~ and their paralogues that control lung branching morphogenesis. afsc-ev administration to ph lung cells significantly downregulated genes, which formed mirna-mrna reported interactions. summary/conclusion: afsc-evs contain many rna species in their cargo, but mirnas are the main effectors of their ability to rescue underdeveloped foetal lungs. we have identified for the first time that afsc-ev biological effect on underdeveloped foetal lungs is in part due to the release of mir ~ cluster. funding: cihr-sickkids foundation grant. bottom-up assembly of fully-synthetic extracellular vesicles oskar staufer a , franziska dietrich a , jochen hernandez a , martin schröter a , sebastian fabritz b , heike böhm a , ilia platzman a and joachim spatz a a max planck institute for medical research, department for cellular biophysics, jahnstraße , heidelberg, germany, heidelberg, germany; b department for chemical biology, max planck institute for medical research, jahnstraße , heidelberg, germany, germany, germany introduction: extracellular vesicles (evs) are considered as key elements for future therapeutic and diagnostic procedures. however, despite enormous research efforts to understand their physiological relevance and several greatly successful clinical trials, evs are currently not authorized for clinical routines by american or european regulation and approval agencies. this is especially because therapeutic evs are produced or isolated from cell cultures or biofluids, both of which are subjected to batch-to-batch variations and ill-defined contaminations. therefore, complementary technologies that produce evs as reproducible and defined as state of the art nanotherapeutics, would revolutionize the application of evs in clinical settings and provide the scientific community with a holistic understanding of ev-mediated signalling processes. in our study, we achieve de novo bottom-up assembly of fully synthetic evs (fsevs) that comprise identical physiological and therapeutic functionalities to natural evs. methods: we applied droplet-based microfluidic synthesis to sequentially amalgamate synthetic lipids, proteins and nucleic acids into defined vesicles that display analogous therapeutic capabilities to natural evs. fsevs were characterized by electron and confocal microscopy, dynamic light scattering and mass spectrometry and tested on organotypic models or in vivo. results: using previously described evs as "naturegiven" blueprints, we assembled several fsevs in their exact molecular composition. in particular, we produced wound-healing promoting evs composed of several exosomal proteins, lipids and micrornas and showed that their therapeutic performance on human skin wounds is equivalent to that of natural evs. besides their high molecular complexity, being composed of dozens of different molecular building-blocks, the presented fsevs are completely defined on a quantitative level. based on this, we achieved a stoichiometric understanding of cell-vesicle interactions. summary/conclusion: by applying bottom-up synthesis of fsevs for quantitative studies on ev signalling, we not only provide innovative and safe compounds for ev-therapeutics but also a vastly new perspective on the application spectrum of extracellular vesicles in fundamental research. introduction: small extracellular vesicles (sevs) contain functional molecules from their cell of origin and can enter recipient cells for intercellular communication. ifnβ has been shown to induce some lncrnas to regulate host immune response and play a major role in the positive regulation of the activity of natural killer (nk) cells. here, we aim to clarify whether ifnβ induced sevs can regulate the cytotoxicity of nk cells by transferring specific lncrnas into nk cells. methods: evs were purified from a with/without ifnβ treatment by serial centrifugation followed by sucrose density gradient purification. elisa assay were performed to demonstrate the cytotoxicity of nk cells. qpcr and western blot were used to verify the expression of nkp . results: surprisingly, ifnβ induced sevs can strengthen the cytotoxicity of nk cells. through human transcriptome array (hta) we found the expression levels of lncrnas were significantly changed within sevs isolated from a cells following ifnβ treatment. additionally we found a specific sev cargo, linc-epha - , acted as a competing endogenous rna (cerna) for hsa-mir- which subsequently up-regulate the natural cytotoxicity receptor (nkp ) expression. furthermore, we verified over-expression of linc-epha - significantly enhance the cytotoxicity of nk cells against zika virus-infected a cells. summary/conclusion: our results demonstrated that ifnβ-induced linc-epha - wrapped in sevs can regulate the cytotoxicity of nk cells. our study provides a novel link between type i ifn and nk cells, which are two major players for the host innate immunity against pathogen infections. introduction: hiv-infected t cells release simultaneously viral particles and small extracellular vesicles (sevs) including mvb-derived exosomes and plasma membrane-derived evs. sevs and hiv share many physical and chemical characteristics, which makes their separation difficult. although several approaches have been used to obtain sevs free of virus they leave a majority of sevs within hiv preparations. for this reason, the function of sevs during hiv infection remains unclear. methods: we have developed a novel un-biased proteomic profiling approach to identify specific markers of the virus or sev subtypes released by a human t lymphoma cell line. our approach was to combine differential centrifugation of medium/small evs contained in the ccm with quantitative mass spectrometry to generate protein abundance profiles across the different sub-fractions. we generated an interactive database to define groups of proteins with similar profiles, suggesting their release in the same evs. results: we thus identified different categories of evs, which bear different surface proteins, e.g. different combinations of t cell surface markers, integrins or tetraspanins. in evs released by infected cells, we identified cellular proteins behaving like hiv proteins, and several that changed behaviour after infection, either moving towards or away from the hiv cluster. we identified two cell-derived proteins that are included in the viral particles and one that is specific of non-viral sevs that are modified by infection, and analysed their respective roles in controlling ev composition or virus infectivity. summary/conclusion: our approach presents a powerful tool for identification of common cargoes of given ev subtypes, and could be now used to identify modifications of ev composition in any given physiological or pathological situation. the encephalomyocarditis virus leader modulates autophagic pathways to promote the release of virions inside extracellular vesicles introduction: recent data indicate that naked viruses belonging to the picornaviridae family can be released from host cells via enclosure in extracellular vesicles (ev). ev cloak virus particles in a host-derived "envelope" and can thereby affect antiviral immune responses and disease severity. a better understanding of the formation and function of ev-enclosed viruses is therefore required. previously, we showed the presence of the autophagosome marker lc in ev isolates from encephalomyocarditis virus (emcv) infected cells, suggesting the involvement of a secretory autophagy pathway in ev-mediated virus release. however, little is known about the viral and host factors that regulate this process. here, we have assessed the role of the emcv leader, a viral protein that is dispensable for replication but is required for symptomatic disease. methods: cells were infected with wildtype virus or a mutant carrying an inactive leader. ev produced during the infection were isolated using differential ultracentrifugation and density gradient purification. ev were characterized by high resolution flow cytometry and their infectivity determined using end-point dilution assay. in addition, the fate of autophagosomes in infected cells was monitored using a reporter assay for autophagosome-lysosome fusion and analysis of the secretion of autophagosomal proteins. results: inactivation of the emcv leader strongly reduced the release of ev-enclosed virus. whereas autophagosomes are typically degraded, we show this is blocked by the leader. instead, autophagosomes fuse with the plasma membrane, as indicated by the secretion of autophagy marker lc during infection with wildtype but not the mutant virus. pharmacological reactivation of degradative autophagy in infected cells resulted in a strong reduction in the release of ev and ev-enclosed virus. similarly, the reduction in evenclosed virus release in the absence of the leader could be partially reversed by drugs that promote the secretion of autophagosomes. summary/conclusion: our data supports a role for secretory autophagy in the release of viruses in ev, a pathway that is regulated by the emcv leader. these findings highlight an unconventional route for ev formation that intersects with autophagosomal compartments and contributes to viral pathogenesis. introduction: zika virus (zikv) causes a public health emergency of international concern because of its correlation with microcephaly. during viral infection, the innate immune response quickly to produce some endogenous functional molecules which can prevent viral invasion or replication. extracellular vesicles (evs) contain molecules from their cell of origin under virus infection and can enter recipient cells for intercellular communication. here, we aim to clarify whether zikv induced evs can regulate viral pathogenicity by transferring specific rna. methods: evs were purified from a with/without zikv infection by serial centrifugation followed by sucrose density gradient purification. human transcriptome array (hta) was used to found rna expression within evs. flow cytometry was used to determine cell cycles. zikv replication was assayed by qpcr and western blot. flow cytometry was used to determine cell cycles. results: through hta we found the defensin alpha b (defa b) expression was significantly increased within evs isolated from zikv infected a cells. additionally, we found that the extracellular defa b but not the intracellular defa b exerts anti-zikv effect mainly before entry step. surprisingly, up-regulate defa b can retard cell cycles of host cell. we verified defa b could bind with the origin recognition complex (orc ) which is required to start dna replication during the cell cycle. furthermore, up-regulate defa b decreased the orc level in nuclear. interestingly, evs with defa b can internalize into recipient cells and inhibit their cell cycles. summary/conclusion: together, our results demonstrated that zikv infection can induce defa b wrapped in evs, and defa b not only exerts anti-zikv effect but also regulate cell cycles which may affect neurodevelopment. our study provides a novel viewpoint that defa b act as first-line anti-viral molecules during zikv infection also correlate with neurodevelopment by retarding cell cycles. extracellular vesicles mediate bacterial-immune cell interactions during respiratory viral-bacterial co-infections sidney w. lane a , matthew hendricks b and jennifer bomberger a a university of pittsburgh, pittsburgh, usa; b university of washington, seattle, usa introduction: respiratory infections are a major cause of morbidity and mortality worldwide and host-derived extracellular vesicles (evs) play important roles in mediating these infections. during respiratory infection, evs are shown to have a modulatory effect: promoting or suppressing infection dependent on the pathogen and cell type. in the age of next-generation sequencing, we now appreciate that many respiratory infections are polymicrobial in nature, with viral-bacterial co-infections correlating with worse disease outcomes. epidemiological studies correlate acute viral infections with the increased likelihood and severity of both acute and chronic secondary bacterial infections; however, the exact mechanisms of these interactions remain poorly understood. evs have been understudied in the context of respiratory viral-bacterial coinfections; thus, their role in mediating these infections is relatively unknown. unpublished data from the lab shows that in airway epithelial cells (aecs), viral infection induces the release of evs that associate with pseudomonas aeruginosa (pa) and promote biofilm growth. here, we aim to expand upon these findings and determine how aec evs mediate pa-immune cell interactions during respiratory viral-bacterial co-infection. methods: to determine how exposure to evs impacts pa-immune cell interactions, evs were isolated from the apical secretions of aecs and co-cultured with pa. ev-treated pa was then co-cultured with macrophages to evaluate ev impact on pa uptake and survival. results: in preliminary experiments using control evs, we observed that evs associate with pa. interestingly, during co-culture with macrophages, ev-treated pa are more susceptible to phagocytosis in comparison to non-treated pa. however, after hours of co-culture with macrophages, ev-treated pa are able to survive and replicate, while nontreated pa are effectively controlled by the macrophages. summary/conclusion: these findings suggest that while pa-ev association promotes pa uptake, it may ultimately enhance pa immune evasion and survival. ongoing experiments in the lab are evaluating the mechanism of pa-ev association and how evs from virus-infected aecs affect the phenotypes observed with control evs. notably, this is one of few reports of a mammalian ev influencing the pathogenesis of a bacterium; thus, results from these experiments will define the function of aec evs in regulating bacterial-immune cell interactions during respiratory co-infections. using machine learning with neuronal ev target proteins and clinical data to predict cognitive impairment in hiv infection lynn pulliam a , michael liston b , bing sun c and jared narvid d a university of california, san francisco, san francisco, usa; b veteran affairs, san francisco, usa; c ncire, san francisco, usa; d ucsf, san francisco, usa introduction: objective biomarkers are needed to assess and predict neuronal function and cognitive impairment. in people ageing with chronic infections, such as hiv, determining the mechanism of impairment will be important when therapies are available. methods: sixty plasma samples from hiv-infected people were obtained from nih-sponsored aids banks. clinical and epidemiological data were collected. all underwent neuropsychological testing and were considered impaired. neuronal extracellular vesicles (nevs) were isolated from plasma and assayed for high-mobility group box (hmgb ), neurofilament (nf-l) and phosphorylated tau- (p-tau) proteins. results: using different algorithms, support vector machines (svm) performed the best with an area under the curve (auc) value of . ± . . using different combinations of clinical data and the nev protein targets, selected clinical data and hmgb best predicted cognitive impairment (auc = . ). the most important features included cd count, hmgb , nf-l and education. summary/conclusion: specific clinical features plus nev hmgb , an inflammatory marker, were the best predictors of cognitive impairment. previous published data showed nev p-tau- elevated in alzheimer's disease and in this study p-tau had no importance in assessing hiv-associated cognitive impairment. nev target discovery can be improved to better identify neuronal damage, possibly to differentiate other neurodegenerative diseases and hopefully recovery after therapies are identified. in recent years, we have been able to separate and characterize extracellular vesicles (evs) from several different viruses including hiv- , htlv- , rift valley fever virus and ebolavirus. however, to date it is not clear whether there is a timing difference between ev and virus release from infected cells. methods: ev isolation by nanoparticle capture and differential centrifugation, ev quantification by nanoparticle tracking analysis, western blot, rt-qpcr, virus rescue assay. results: we have attempted to address the kinetics of ev and virus release from multiple-infected cells using serum starvation experiments from infected ( %) cells. these infected cells were initially put in g quiescent stage using serum starvation. both supernatants and cell pellets were collected postinduction release ( % fbs + pma/pha) at , , , , and hours and examined for the presence of ev, autophagy and viral proteins as well as viral rna expression. results from supernatants of uninfected cells showed a peak of tetraspanin proteins (cd , cd , and cd ) at hours and a gradual decrease of all ev associated proteins by hours. however, the ev from hiv- infected cells showed all three tetraspanins present at hours and expression gradually increased up to hours. when compared to htlv- infected cells, the three tetraspanin proteins peaked at hours and expression continued to decrease up to hours. htlv- infected cells also showed a unique pattern of cd expression. autophagy associated proteins (lc a, lc b and p ) from uninfected cells and htlv- infected cells plateaued at hours, whereas in hiv- infected cells their expression continued to increase and peaked at hours. hiv- viral proteins (p , gp , nef) expression was present at hours and continued to increase and peaked at hours. htlv- proteins (p and gp / ) peaked at hours and gradually decreased overtime. hiv- and htlv- rna gene expression analysis was performed, and data correlated with viral protein expression. additionally, evs release was quantified and showed significant increase of ev concentration overtime in both uninfected and infected samples. finally, experiments of infectivity from -and hour supernatants were performed on three naive cells. hiv- supernatant -hour sample was found not to be infectious. however, hiv- was successfully rescued from -hour sample. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = ). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine distinct uev surface markers of cd +/cd +/cd + vesicles in a multiplexed bead-based manner including respective controls. the accuri c (bd) was utilized for data acquisition. for further misev -compliant characterization, cd +/cd +/cd + uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd , all other surface markers could be identified. the most abundant markers were cd and cd , which were detected in % of samples, followed by cd / ( %), cd ( %), cd and cd ( %, each). cd ( %), cd , cd ( %), cd e ( %) and cd showed similar relative median fluorescent intensities (rmfi), while cd yielded significantly higher (p = . ) and all other markers significantly lower rmfi (p < . ). tem and f-nta revealed cup-shaped vesicles ( ± nm) with . ± . e + particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg , cd , cd and cd without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a ,esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = ) aged - yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a -colour panel, which included annexin v (for the majority of circulating evs), cd (for platelet-derived evs) and cd (for endothelial-derived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and -yr cvd risk detected by qrisk . results: ev numbers, as determined by nta, were strongly associated with bmi (r = . , p < . ), blood pressure (systolic bp: r = . , p = . ; diastolic bp: r = . , p < . ) and plasma triacylglycerol levels (r = . , p < . ). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = . , p = . ). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining . % of the variance for total ev numbers. an additional . % of the variance in total ev numbers was predicted by diastolic bp. ancova of the -yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with -yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd + t cells were isolated from secondary lymphoid organs of foxp -rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th , th , and th ), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, -day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th , th , th ) showed either no change in proliferation (th ) or decrease in proliferation (th , th ), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < . ). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th ), il and il (th ), or il a and il f (th ). in contrast, exposure of tregs to cdc-evs resulted in~ -fold increase in expression of il , a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il- in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il- . this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t hl . prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from patients treated with prostanoids (study group; n = , ± years, % female) and patients not treated with prostanoids (control group; n = , ± years, % female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; . mm), adenosine diphosphate (adp; . µm) and thrombin receptor-activating peptide (trap; µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd + and cd p+; pevs), leukocytes (cd +, levs) and endothelial cells (cd +, eevs) were measured in plateletdepleted plasma by flow cytometry (a -micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = . ) and lower concentrations of pevs and levs (p ≤ . ). furthermore, thrombus formation was delayed (p ≤ . ) and thrombus size was decreased (p = . ) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd + pevs (p = . ). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ . ) and the concentrations of pevs (p ≤ . ). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ . ). introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from cf children (< years of age). for nanoflow cytometry, evs were stained with di- -anepps, and with epcam, cd b and cd (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. results: the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = . , spearman's rho = . ). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = . , rho = . ). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv a ), emory pediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: individuals were enrolled into our study, subdivided into a training (volunteer n = , pneumonia n = , sepsis n = ) and testing cohort (volunteer n = , pneumonia n = , sepsis n = ). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq ) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative realtime pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed significantly regulated mirnas in pneumonia compared to volunteers, and mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: . , pneumonia: . , sepsis: . ). mir- a- p (log fc = . , padj = . e- ) and mir- - p (log fc = . , padj = . e- ) differentiated between pneumonia and volunteers and mir- (log fc = − . , padj = . e- ) between pneumonia and sepsis. expression levels of mir- a- p and mir- were related to disease severity. mir- - p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was . %. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study, healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd and cd were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and mirnas (mir- a-p, mir- - p, mir- a, mir- b- p, mir- - p, mir- - p, mir- - p, mir- - p, let- g- p) , were evaluated by rt-qpcr. after the optimization of the methodology, healthy adults subjects and patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd and cd . however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna- a showed a significant increased (p = . ) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir- a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf -active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds joseph kuhn a , absara hassan b , sonali sharma b , jennifer kwong b , montaha rahman b , salma adam b , jasmine lee b , alvaro villarreal ponce b and piul rabbani b a nyu langone health, new york, usa; b nyu langone health, new york, usa introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid -related factor (nrf ) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf -active exosomes, mscs were incubated with potent nrf activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express > % positive msc markers (cd , cd , cd , and cd ) and < % express negative markers (cd , cd , cd , cd , or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd , cd and tsg . nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of . ± . nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf -active human mscs increase exosome secretion by %, compared to nrf -baseline mscs (p < . ). both nrf -baseline and nrf -active exosome treatment significantly reduced closure time to . and days respectively, compared to . days for vehicle-treated wounds (p < . ). this reduction eliminated the delay in closure time compared to wounds of c /b mice. nrf -active exosome treatment of db/db wounds reduced closure time by a further . days compared to untreated c /b wounds. at day , exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd + vessels compared to vehicle-treated wounds. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist is a key mediator of tumour cell metastasis.. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist . methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc -ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc -ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc -ml lines stably overexpressing or deficient in twist . results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc -ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc -ml overexpressing twist showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. funding: american heart association of symposium introduction: as researchers continue to explore the therapeutic potentials of extracellular vesicles (evs) for the treatment of many diseases, there is a growing unmet need for real-time in vivo monitoring of these therapeutic evs after they are injected into a subject to understand their safety, targeting, and effectiveness. while current optical imaging solutions like bioluminescence and fluorescence are useful for ev tracking studies in animal models, there is limited utility in clinical applications. here we present a novel ev tracking solution utilizing clinically applicable mri technology. methods: to generate trackable evs, cells were labelled with a clinically applicable novel magnetic agent. evs secreted by the labelled neural stem cells and amniotic fluid stem cells (afscs) were isolated by differential ultracentrifugation. the viability and morphology of labelled-cells were evaluated, and the in vitro mr properties of their derived evs were analysed by magnetometer. a proof of concept in vivo biodistribution study was conducted by injecting labelled evs into wt and alport mice (a model of chronic kidney disease) via retro-orbital and intra-cardiac routes and tracking them via mri at min and hr postinjection. results: the magnetic label did not affect the physiological characteristics of the cells. the mr detectability of labelled-evs was confirmed by in vitro/ex vivo mri phantoms. mri studies showed that homing of afsc evs to the kidney injected intra-cardiacally into alport mice were more efficient versus the retro-orbital route, and prussian blue staining of kidney sections confirmed the mr findings. introduction: a central question in ev biology is the fate of circulating ev. this can be evaluated by developing non-invasive ev bioimaging techniques in mice in order to benefit from transgenic and knock-out models. recent reports described ev biodistribution in vivo using optical (fluorescence) and nuclear imaging. but the physicochemical properties of the probes impact ev integrity, labelling efficiency, background signals and observation timecourse. methods: we developed the radiolabeling of red blood cells (rbc) and ev with [ f]fluorodeoxyglucose ( f-fdg). we used rbc-derived ev in their native, intact form, without pre-experimental processing (no centrifugation or filtration). we tracked f-fdg in vivo by pet-scan, within seconds of ev, rbc or free f-fdg injection, and during their dissemination in blood and recruitment by organs over one hour. ev and rbc biodistribution were confronted to the kinetics of free f-fdg. results: we collected images of the biodistribution of rbc, and rbc-derived ev. nuclear imaging was well suited for accurate studies of ev organotropism, with high sensitivity, excellent signal-to-noise ratio, very low signal absorption by tissues and an inherent quantitative tomographic nature. ev-specific signals were mostly accumulated within minutes of injection (tail vein), in the spleen and liver, with a small part in the bone marrow (femurs). signals in other compartments were largely transient and linked to tissue perfusion and blood volume. we selected the most drastic control conditions to secure a correct interpretation of the data. this made kidneys, hearts and brains unavailable for analysis. hence the new approach came with limitations, but we describe how "free" f-fdg signals can be used to draw sound conclusions for ev. summary/conclusion: we propose that three types of compartments coexist in control mice at rest: active ev-capturing organs with high capacity and specificity including the spleen, and to a lesser degree the bone marrow; passive ev-retaining organs with high capacity, including the liver; and ev-neutral organs where transient signals only mirror tissue perfusion. we also report how ev biodistribution patterns are altered in ageing animals, as an example. we hope that this novel, non-invasive, quantitative, dynamic wholebody imaging approach will help characterize native cell-derived ev and help set standards for the reproducibility of ev bioimaging in mice. funding: frm grant "biface", inserm copoc, cnrs. introduction: extracellular vesicles (ev) are important mediators of intercellular communication; however, basic principles of ev biogenesis and loading remain largely unknown. a limited repertoire of tools has thus far made these processes challenging to research. the development of an ev-transfer reporter in a genetically tractable organism such as drosophila has allowed us to study mechanisms of cargo loading in vivo and has provided us with a platform to explore fundamental aspects of ev biology. methods: we have developed a bioinformatic pipeline to analyse the properties embedded in the ʹutr of mrnas enriched in evs released by drosophila cells. in parallel, we have adapted a cre-loxp system for use in fruit flies that appears to be proficient to reveal the exchange of bioactive molecules between secretory and recipient cells. results: taking advantage of computational methods, we uncovered sequence motifs that preferentially appear in combinations along the ʹutr. these sequence motifs occur within characteristic secondary structures, in a way that is more variable and motif dependent than previously reported. identified motifs also show similarities to known binding sites for rna binding proteins; a feature potentially important for ev-loading. in parallel, we developed a drosophila in vivo system to detect cell communication in complex tissues and between different cell types. using this system, we studied the biological significance of specific sequence motifs and identified their ability to modulate mrna ev-transfer in a context dependent and evolutionarily conserved manner. summary/conclusion: in summary, we have developed a novel tool to study cell communication in complex tissues, and shown its effectiveness to study principles of ev biogenesis and loading. beyond improving our understanding of ev biology and providing a novel tool to the scientific community, we hope this knowledge will pave the way to harnessing evs as a means of remotely manipulating cell communication in many biological contexts. introduction: the idea of cross-kingdom, species and inter-individual transfer of bioactive compounds via extracellular vesicles (evs) is a recent avenue. however, the bioactivity and bioavailability of these dietary compounds upon consumption is highly debated. it has been proposed that evs from diet can absorbed by consuming organisms, be bioavailable in various organs and exert phenotypic changes. milk is the most vastly consumed beverage and is an abundant source of evs that may act as signalosomes. whether these milk-derived evs can serve as cross-species messengers and have a biological effect on host organism has been poorly understood. methods: bovine milk-derived evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. the evs were characterised by tem, nta, quantitative proteomics and rna-seq. evs were orally administered to various mice models of colorectal, breast and pancreatic cancer. primary tumour burden was monitored, and the rate of metastases was measured by imaging and qpcr. immune cells were analysed by facs. mechanistic insights were obtained using quantitative proteomics, confocal microscopy and biochemical experiments. results: we demonstrated that upon oral administration, bovine milk-derived evs were able to survive the harsh degrading conditions of the gut and be bioavailable in peripheral tissues. interestingly, oral administration of milk-derived evs reduced the primary tumour burden in various cancer models and attenuated cancer cachexia. intriguingly, despite the reduction in primary tumour growth, milk-derived evs accelerated metastasis in breast and pancreatic cancer mice models. timing of ev administration was critical as oral administration after resection of the primary tumour reversed the pro-metastatic effects of milkderived evs in breast cancer. biochemical and quantitative proteomics analysis highlighted the induction of epithelial-to-mesenchymal transition and senescence upon treatment with milk-derived evs. summary/conclusion: taken together, we were able to demonstrate the capacity of bovine milk-derived evs in mediating cross-species communication and regulating cancer progression in a context-dependent manner. bacterial membrane vesicles (mvs)a bacterial innate defence system against viral infection xiaomei yan, qian niu and ye tian xiamen university, xiamen, china (people's republic) introduction: in order to survive the constant onslaught of phage, bacteria have evolved diverse defence mechanisms that act at every stage of the phage life cycle. it has been suggested that bacterial membrane vesicles (mvs) may play a key role in innate bacterial defence against phage infection by acting as a decoy to prevent phage adsorption. nearly a decade has passed since mvs were first proposed as a decoy, but details of how bacteria utilize mvs to defend against phages remain poorly understood. here we use the laboratory-built nano-flow cytometer (nfcm) to reveal details of the interaction between mvs and phages at the single-particle level, and to provide new insights into innate defence mechanisms of mvs. methods: s. typhimurium was used as the model system. differential ultracentrifugation and density gradient centrifugation were used to isolate and purify mvs and bacteriophage p . cryo-tem was used to determine the morphologies of mvs and phage p . the purity of mv isolates was validated by measuring the particle concentration before and after triton x- treatment. monodisperse silica nanoparticles were used as the size reference standards to measure the size distribution of mvs via single-particle light scattering detection. the purity of phage p was verified by concurrent detection of side scatter and fluorescence signals of single phages upon nucleic acid staining by syto . results: by incubating mvs and af -labelled p , the number of phages adsorbed on single mvs were accurately quantified. we found that s. typhimurium and mvs it secretes express different affinity for phage p attachment. the binding ability of p to mvs is greater than that of bacteria. we confirmed that p can inject their nucleic acids into mvs, and these nucleic acids can be degraded by non-specific nucleases inside mvs for the first time. besides, by labelling the nucleic acids of mvs with syto , we were able to distinguish three different subpopulations of mvs. summary/conclusion: taking advantage of the superior sensitivity of nfcm in single-particle analysis, we developed a novel approach to the characterization of the interaction between mvs and phages. our study revealed that bacteria produce mvs as bait to attract viral adsorption and nucleic acids injection. funding: this research was supported by the national natural science foundation of china (grants , and ). introduction: the development of evs for therapeutic applications requires an in-depth understanding of their in vivo biodistribution and pharmacokinetic profile. in this study, we have made a comprehensive comparison of nuclear, fluorescent, and bioluminescent imaging technologies to identify the most suitable in vivo ev tracking method. methods: evs were purified from expi f cell supernatant by differential centrifugation followed by iodixanol density gradient separation and further characterized following misev guidelines. engineered expi f cells were used to generate evs carrying mcherry or nanoluc (nluc) proteins. the membrane of naïve ev was labelled with indium (in )-dtpa or xenolight dir post-ev isolation. ct tumour-bearing balb/c mice were intravenously dosed with × evs followed by imaging at h, h and h using spec/ct and ivis systems. tissue distribution and blood circulation profile of evs were analysed from ex vivo samples up to h post-injection. results: xenolight dir and (in )-dtpa were the most suitable ev labels for live whole-body animal imaging, ex vivo organ imaging, and tissue lysate quantification. nluc was appropriate for ex vivo imaging and tissue lysates quantification, but suboptimal for live imaging with limited sensitivity. mcherry evs were found not suitable for in vivo tracking studies due to high background signal fluorescence. ex vivo organ quantification of in -dtpa and dir showed that naïve evs mainly accumulate in liver, followed by spleen, kidneys, and lungs at h post-dose, with less than % ev exposure to the tumours. interestingly, nluc-evs accumulated mainly in the lungs, regardless of the small size of the particles injected and the absence of aggregation. blood circulation profile of in -dtpa and nluc evs showed rapid clearance of vesicles from circulation, with % of injected dose detected in blood after min and less than % after h. summary/conclusion: radionuclide imaging is an excellent technology to detect evs in vivo and ex vivo with high resolution and sensitivity but requires advanced infrastructure for radiolabeling. the optical methods have limited tissue penetration and sensitivity but can be improved with the right selection of the dye. these results contribute to the understanding of the biodistribution and pharmacokinetics of evs and are highly relevant to exploiting their potential for targeted delivery to diseased tissues in vivo. symposium introduction: new methods for quantifying extracellular vesicles (evs) in complex biofluids are critically needed. we report the development of a new technology combining size exclusion chromatography (sec), a commonly used ev purification technique, with fluorescence detection of specifically labelled evs (flu-sec). methods: flu-sec was validated using red blood cell derived evs (revs). size and concentration measurements were performed by microfluidic resistive pulse sensing (mrps) using the ncs instrument (spectradyne llc, usa). pe-cd a (anti-glycophorin a) and alexa -wga (wheat germ agglutinin) were used to label revs. flu-sec experiments were performed on a liquid chromatography system using a tricorn / glass column filled with sepharose cl- b gel (ge healthcare). results: a log-normal size distribution was obtained for revs with a mean diameter of . ± . nm and standard deviation of . ± . nm. the concentration of revs measured by mrps was . * e particles/ ml. the fluorescence chromatograms of the rev samples labelled with pe-cd a and with alexa -wga show the typical features of the separation of evs from soluble proteins with sec and enables the determination of the labelling efficiency of the markers. the linear range for quantification of evs in our experiments spans over two orders of magnitude ranging from e particles/ml to e particles/ml. the lod depends on the type of the label. in our experiments the lowest lod was e particles/ml for alexa -wga. summary/conclusion: the results indicate that flu-sec is a quantitative technique with very good linearity over a wide range of concentrations, though the limit of detection depends largely on the employed label (sci. rep. , , ) . moreover, the ratio of ev-bound and free-antibody molecules can be also determined by flu-sec, which can be used to calculate the labelling efficiency of the used marker. funding: this work was supported by the national research, development and innovation office (hungary) under grant numbers pd and nvkp_ - - - . zv was supported by the janos bolyai research fellowship. the conan assay: purity grade and concentration of ev microlitre formulations by colloidal nanoplasmonics. (evs). control over such properties is constantly experienced by researchers to be critical for ev proper manipulation, engineering and translation. however, the need for characterization methods that strike the balance between robustness, working volume, cost and accessibility remains unmet. methods: the colorimetric nanoplasmonic (conan) assay we developed consists of a solution of gold nanoparticles (aunps) into which - μl of the ev formulation is added. the solution turns blue if the formulation is pure, while stays red if soluble exogenous single and aggregated proteins (saps) are present. the colour shift is visible by the naked eye and can be quantified by conventional uv-vis spectroscopy, providing a quantitative index of purity and an estimation the ev molar concentration (particle number). results: the assay specifically targets saps, and not the ev-related proteins, with a detection limit < ng/μl (an order of magnitude higher resolution than the bradford protein assay). for pure solutions, the assay also allows for determining the ev number, as the colour shift is linearly dependent to the aunp/ev molar ratio. instead, it automatically reports if the solution bears sap contaminants, thus avoiding counting artefacts. experiments, conducted on ev separated from milk and ascaris suum culture medium, are repeatable, with an error below %. summary/conclusion: conan proves to be robust and reliable, while displaying appealing performances in terms of cost (inexpensive reagents, run by standard microplate reader), working volumes ( - μl) and time (the procedure takes less than one hour). the ability to assign a quantitative purity grade is, up to date, a unique peculiarity of this assay. finally, the assay is potentially extendable to all classes of natural and artificial lipid micro-and nanoparticles. funding: evfoundry project, horizon -future and emerging technologies (h -fetopen), id: . marina cretich a , roberto frigerio b , alessandro strada b , greta bergamaschi b , marcella chiari c and alessandro gori c a consiglio nazionale delle ricerche (cnr), istituto di scienze e tecnologie chimiche (scitec), milano, italy; b consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy; c consiglio nazionale delle ricerche (cnr); istituto di scienze e tecnologie chimiche (scitec), milano, italy introduction: small extracellular vesicles (sev) present fairly distinctive lipid membrane features in the extracellular environment. these include high curvature, lipid packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sev membrane could be then considered as a "universal" marker, alternative or complementary to traditional characteristic surface-associated proteins. here we introduce the use of membrane sensing peptides as new, highly efficient ligands to directly integrate sev capturing and analysis on a microarray platform. methods: we designed and synthesized membranesensing peptide ligands as molecular baits for small ev and we demonstrate their use in a microarray platform as valuable alternative/complement to antibodies. evs from blood serum and plasma were isolated by ultracentrifugation, characterized by tem, nta, wb. samples were analysed by label-free, single particle counting and sizing on peptide microarrays coupled to fluorescence co-localization immune staining with fluorescent anti-cd /anti-cd /anti-cd antibodies. results: peptide microarrays were realized using a click-chemistry strategy for optimal peptide surface orientation and used to analyse evs from human blood. membrane sensing peptides showed a capturing capacity higher than anti-tetraspanin antibodies. in addition to purified vesicles, peptide ligands were tested with pure serum showing capacity to capture evs even from complex samples. in order to get insights into the ev-peptide binding mechanism and verify whether it is directly mediated by the lipid membrane, trypsin-treated evs were captured on peptide microarrays demonstrating that binding is not directly mediated by surface associated proteins. summary/conclusion: we introduced the use of membrane sensing peptides as a novel class of molecular ligands for integrated sev isolation and analysis, reporting for the first time on peptide microarrays for extracellular vesicles. given their affinity to the membrane of small ev, these molecules can serve as general baits, enabling vesicles capturing unbiased by differential surface protein expression. these new class of molecular probes may be integrated with the use of protein markers towards improved small ev isolation and characterization. compared to proteins and antibodies, peptides are characterized by low cost of preparation, remarkable stability and ease of chemical manipulation, offering virtually unlimited possibilities for experimental design. we anticipate that this new class of ligands, may greatly enrich the molecular toolbox for ev analysis. funding: hydrogex (regione lombardia&fondazione cariplo, grant n. - ) and index (european union's horizon research and innovation programme under grant agreement n° ) projects are acknowledged for partial financial support. high-resolution size-based profiling and morphological analysis of extracellular vesicles by scanning electron microscopy sara cavallaro a , federico pevere b , petra hååg c , kristina viktorsson c , rolf lewensohn c , jan linnros a and apurba dev b a kth royal institute of technology, stockholm, sweden; b uppsala university, uppsala, sweden; c karolinska institute, stockholm, sweden introduction: extracellular vesicles (evs) have been found to mediate intercellular communication in physiological and pathological processes. nevertheless, the understanding of evs bio-functionality remains elusive, mainly because of their high heterogeneity in molecular content, but also in size ( - nm) . therefore, accurate size measurements of evs are highly desired, particularly for exploiting their full diagnostic/therapeutic potential. currently available techniques, such as nanoparticle tracking analysis (nta), cannot accurately measure evs smaller than nm and are not capable to distinguish them from protein aggregates. on the contrary, electron microscopy (em) techniques allow high-resolution size-profiling and morphological analysis of evs over their whole size range. however, their low throughput combined with several long preparatory steps have prevented em from being routinely used for ev size profiling. methods: we shall present a method improvement in throughput and reproducibility of ev size-analysis by scanning em (sem). the technique is based on covalent ev capture onto a silicon wafer, using the protocol reported by cavallaro et al. up to the glutaraldehyde step. after immobilization, critical point drying (cpd) is performed to dehydrate evs before sem, while preserving their shapes. results: sem images, showing the comparison in densities of evs prepared by covalent and non-covalent coupling to substrate, indicated a good capture efficiency of our covalent protocol. the size distribution analysis showed good agreement between nta and sem for evs > nm. for smaller evs, sem is more sensitive than nta, thus more suitable to check the purity of ev-isolation techniques. last, atomic-force microscopy (afm) measurements was also used to validate our measurements. introduction: extracellular vesicles (evs) are membrane vesicles secreted into extracellular space, by almost all cellular populations, playing a major role in cell-to-cell communication. it has been already demonstrated that changes in luminal or surface protein cargos of these vesicles, may reflect the status of producing cells. for this reason, evs are considered as potential biomarkers in several types of diseases ranging from cancer diagnosis to heart rejection. periostin (postn) is a matricellular protein associated with evs, and its level is considered a possible biomarker, which indicate malignancy and poor clinical outcome in different types of cancer. here we extensively characterize the presence of postn associated on evs, showing how different isolation methods can drastically affect the amount of postn content in extracellular vesicles fraction. methods: serial ultracentrifugation steps or size exclusion chromatography were used to isolate evs from primary culture of cardiac progenitor cells. evs were characterize, according to misev guidelines, by western blot, nta, facs and cryotem analysis methods. postn amount, associated with evs, was analyses by western blot and elisa. furthermore, functional tests were performed on h c cardiomyoblast cell line, treated with the same amount of evs from different isolation methods; cells response were analysed by western blot. results: evs, from both the isolation methods, showed tsg , syntenin , cd positivity while grp was absent. nta showed no differences, in terms of amount and size of particles. by facs analysis evs resulted enriched with cd , cd and cd . cryotem showed a similar morphology in the two preparations with presence of protein contaminant in the ultracentrifuge pellet. in vitro, h c treated with evs showed activation of pfak after ʹ of treatment, this induction was . times higher in cells treated with evs isolated with ultracentrifuge compared to evs isolated with sec, confirming a drastic effect of postn protein contamination. furthermore, by phospholipase-c treatment, we found that postn is bound to evs surface through a gpi anchor. summary/conclusion: these results suggest that selection of a proper isolation method is critically relevant in evs studies, in particular when protein analysis is considered. different isolation methods dramatically influence protein amount in extracellular vesicles and consequentially their function. furthermore, in this study we show for the first time, that postn is actually bound to evs surface and not carried in their lumen as previously believed. members of the y-rna family have been detected in ev from various cell types and are among the most abundant non-coding rna types in plasma. we previously showed that shuttling of full-length y-rna into ev is modulated by tlr-activation of ev-producing immune cells. this suggested that y-rnas may have potential as biomarker for immune-related diseases. methods: we separated rna-containing structures in plasma based on differences in size, density, and resistance to protease/rnase treatment. using rt-qpcr, we quantified full-length y-rna subtypes (y , y , y ) in ev from various blood-related cell types cultured with or without lps-stimulation. inflammationinduced changes in y-rna were assessed in plasma samples from a human endotoxemia study. results: full-length y-rna in plasma was mainly found in ev (early sec-fractions, density . - . g/ml). in contrast, specific mirnas were either enriched in lpp (e.g. mir- ), in both ev and lpp (e.g. mir- and mir- ), or in ev (e.g. mir- ). evenclosed full-length y-rna was resistant to enzymatic degradation, while lpp-bound mirnas were degradation sensitive. we discovered that ev released by different blood cell types varied in y-rna subtype ratios. these ratios remained stable upon lps-stimulation of the ev-producing cells. in endotoxemia plasma samples, the neutrophil-specific y /y ratios and pbmcspecific y /y ratios changed significantly during systemic inflammation. importantly, the plasma y-rna ratios strongly correlated with the number and type of circulating immune cells during the inflammation process. summary/conclusion: cell type specific "y-rna signatures" in plasma ev can be determined without prior ev-enrichment, and may be further explored as biomarkers to diagnose inflammatory responses or other immune-related diseases. mining public ev small rna-seq data with mirev -insights into potential reference transcripts and abundant mirnas recently, extracellular membrane vesicle (mv) production has been proposed as a general secretion mechanism that could facilitate the delivery of functional bacterial nucleic acids into host cells. s. aureus produce membrane-bound, spherical, nano-sized, mvs packaged with a select array of bioactive macromolecules and they have been shown to play important roles in bacterial virulence and in immune modulation through the transmission of biologic signals to host cells. the present study sought to examine the nature of the association between nucleic acids and mvs produced by s. aureus. we also sought to analyse the immunostimulatory potential of mvassociated rna and dna, and to evaluate receptormediated recognition of mv-associated rna and dna molecules by innate immune cells. methods: by following a stringent purification protocol, we characterized the rna and dna content of mvs produced by actively growing s. aureus. nuclease protection assays were performed to determine whether mv-associated nucleic acids are protected from degradation. we assessed the immunomodulatory potential of mv-associated rna and dna by treating cultured mouse macrophages with mvs and measuring the induction of interferon-β mrna using qpcr. introduction: urinary extracellular vesicle (uev) transcriptome could potentially reflect the kidney gene expression profile and serve as virtual/liquid biopsy. in order to explore this possibility, we performed mrna sequencing of uevs from individuals with type diabetes to assess whether it can capture a "kidney enriched genes" expression signature that could lead to novel biomarker discovery for diabetic kidney disease. methods: the study included type diabetic individuals ( normoalbuminuric, microalbuminuric and macroalbuminuric). urine samples were collected either overnight (n = ) or during -hours (n = ) and uevs were isolated from - ml of urine by differential centrifugation. the evs quality was ensured by electron microscopy (em), western blotting and ev-rnasprofiling with the bioanalyzer. isolated rnas were subjected to rna sequencing after cdna library preparation (ultra-low amount protocols) using hiseq (illumina) pair-end protocol. the association between kidney specific gene expression levels (> fold higher compared to other tissues, n = ) and degree of albuminuria or glomerular filtration rate was explored. results: isolated ev quality appeared good by em and western blotting. rna quantity and quality were sufficient for sequencing of all samples with > million pair end reads. we detected on average expression of , genes. principal component analysis (pc) of the expression of all genes did not reveal any systematic batch differences between the overnight and -hour urine collections. comprehensive look-up of kidney-enriched genes revealed expression of > % (total ) of these genes in urine evs with high expression of five kidney-specific genes (slc a , slc a , nphs , aqp and slc a ). pc analysis combining the impact of kidney-enriched genes revealed that most macroalbuminuric patients clustered together along the pc axis, and the axis also correlated with the albumin-to-creatinine ratio (p = . ) explaining % of the variance (p = . ) in the whole data set. the pc axis also showed correlation with hba c (p = . ), but not with diabetes duration, bmi, age and egfr. introduction: due to their safety profile, tissue tropism and long-term transgene expression, adeno-associated viruses (aavs) have become the vector of choice for human gene therapy. however, pre-existing neutralizing antibodies (nabs) to many aav serotypes pose a critical challenge for the translation of gene therapies to clinic. here, we describe the use of exosomal aavs (eaav) as a robust cardiac gene delivery system that enhance transduction efficiency while shielding from pre-existing humoral immunity to the viral capsid. methods: we developed an ultracentrifugation-based purification strategy to obtain eaav specimens from aav-producing hek- t cells, and used electron microscopy-based visualization, confocal microscopybased colocalization studies, qpcr, immunoblotting, dynamic lights scattering, exoview technology and protease assays to characterize eaav morphology, contents and mechanism of action. we then evaluated efficiency of heart targeting for eaav or eaav and standard aav or aav encoding for egfp, mcherry or firefly luciferase in different human cell lines in vitro, in black mouse and in passive immunity nude mouse model in vivo using flow cytometry, confocal microscopy, langendorff perfusion system and methods: hlhs patients (n = ) after glenn procedure and swine (n = ) after pab were given rv injections of allogeneic/xenogeneic mscs. donor-specific, hla-i+, exosomes were isolated from plasma. in swine, exosomes were collected and rv fractional area change (fac) was measured post-msc-injection. in the elpis patients, exosomes were collected and outcome measurements (fac, stroke volume (sv), rv mass) were recorded and -months post-injection. exosomal mrna, microrna (mirna), and proteins were quantified and partial least squares regression (plsr) reduced the dimensionality of the datasets to build a swine model, upon which elpis outcome predictions were made. results: multiomics analysis of swine exosome cargo revealed mirna to be the largest contributor to overall variance. in swine and elpis patients, mirnas were similarly expressed ( %, fold-change< ). plsr reduced the dimensionality of the swine mirna dataset to mirnas with the highest weighted coefficients for changes in fac. pathway analysis of mirna targets revealed links to smooth muscle cell proliferation and cardiac chamber development. importantly, the swine mirna plsr model predicted elpis patient improvements in fac, sv, and rv mass with strong correlation (r > . ). summary/conclusion: these findings support the use of: ( ) swine pab model for rv failure in hlhs, ( ) circulating donor-specific msc-exosomal mirna as a novel, non-invasive biomarker of patient outcomes, and ( ) introduction: evs have been shown promising potential as a drug delivery vehicle, especially nucleic acid therapeutics. however, the overall short of specificity to target cancer cells has led to low therapeutic efficacy and potential toxicity. rna nanotechnology is the bottom-up self-assembly of nanometre-scale rna architectures. we previously discovered a stable phi prna three-way junction ( wj) motif and used it to construct multivalent rna nanoparticles with high chemical and thermodynamic stability. the resulting arrow-shape rna nanoparticles are homogenous, uniform in size and shape, and can harbour different functionalities while retaining their tertiary folding and independent functionalities both in vitro and in vivo. this flexible platform using rna nanotechnology to achieve tumour-specific targeting has been demonstrated over the last decade. here we introduce a strategy to take advantage of both evs and rna nanotechnology to develop a versatile platform for efficient target-specific delivery of sirnas for cancer treatment. methods: we design membrane-anchoring arrowtail wj rna nanoparticles to display tumour targeting ligand (psma rna aptamer or egfr rna aptamer or folate) on birc sirnas loaded evs (fig. ). nanoparticles were characterized by nanoparticles tracking analysis (nta), transmission electron microscopy (tem), dynamic light scattering (dls) and atomic force microscopy (afm). evs were produced by hollowfiber bioreactor and purify by tangential flow filtration (tff) follow by ultracentrifugation. cell binding were evaluated by flowcytometry and confocal microscopy and gene knockdown effect were assay by quantity reverse transcription-pcr (qrt-pcr). formulated evs were introduced to tumour (prostate, triple negative breast cancer, colon pdx) xenograft mice by tail-vein injection and evaluate in vivo tumour inhibition. results: ) we found the orientation of arrow-shaped rna can be used to control ligand display on evs membranes for specific cell targeting. ) by placing membrane-anchoring cholesterol at the tail of the arrow results in display of rna aptamer or folate on the outer surface of the evs and enhance cancer cell binding and uptake. ) taking advantage of the rna ligand for specific targeting and evs for efficient cytosolic delivery, the resulting ligand-displaying evs or plant derived evs-like nanovesicles were capable of specific delivery of sirna to cells, and efficiently blocked tumour growth in three cancer models. summary/conclusion: we developed an rna-evs based nanoparticles platform and shown the flexibility for different cancer type treatment. related publications: pi f et.al. nature nanotechnology. , : . li z et.al. sci rep. introduction: extracellular vesicles (evs) contain plasma membrane surface markers that provide insights into their cell source. until now, our understanding of the circulating ev-biome has been limited by the lack of celland size-specific ev quantitation methods. we have developed and validated a multiplex nanoscale flow cytometry approach to image cell-and size-specific ev populations using a novel human "ev-lyoplate" with differently coloured monoclonal antibodies per well in a well plate format (n = separate antibodies with isotype, stained pbs, unstained plasma, and quant-beads controls per plate). we hypothesized that platelet poor plasma samples from patients diagnosed with pancreatic cancer would have significantly different ev-biome profiles than screen negative study subjects. methods: study subjects were enrolled and sampled before clinically scheduled endoscopic ultrasoundguided biopsy (eus-fna) procedures to screen patients with symptoms of pancreatic duct obstruction who later had at least two years of clinical follow up, including surgical resection in cases of pancreatic neoplasia (n = ) or at least one follow up clinic visit to confirm resolution of symptoms. blood samples were uniformly collected, processed, and banked per isev recommended guidelines. uniform machine (facsymphony) settings to standardize light scatter and fluorescence detection were based on commercially available beads (eg. megamix). samples were coded and randomized for testing and results were reported as the mean cell-and size-specific ev events/ul of plasma. results: clinical outcomes confirmed cases of cancer and screen negative controls. principle component analysis suggested that a number of different celland size-specific evs were significantly more common in the cancer cases (adjusted p-value < . , with aucs > . ), including epcam+/cd + events likely from cancer cells and cd +/cd p+/cd + microvesicles from platelets, among others. summary/conclusion: in this proof of principle study employing an ev-lyoplate design and nanoscale flow cytometry, we could reliably discriminate the ev-biomes in patients with cancer from negative controls. ongoing studies will determine whether these discriminators will be validated in larger cohorts and provide at least noninferior predictive value compared with the current gold standard clinical testing assay (eus-fna introduction: small cell lung cancer (sclc) is an aggressive tumour type, usually metastatic at diagnostic leading to poor overall survival. interestingly, sclc tumours are composed by distinct subpopulations of cells that cooperate as an ecosystem to drive tumour survival. since the subtype of sclc may have prognostic significance, the aim of this study was to identify surface marker proteins as biomarkers of sclc. methods: a linear discriminant analysis (lda) model, implemented in python via sci-kit learn, was used to choose the best markers for distinguishing subtypes. this analysis was based on rna-seq data from a previous study. in order to identify ev-based biomarkers that would identify sclc evs and not normal evs, we excluded from this analysis proteins without a verified transmembrane domain and proteins associated with evs expected to be present in white and red blood cells, and endothelial cells (according to exocarta and vesiclepedia databases). we also prioritized proteins that could be pan markers for sclc and that might have prognostic significance. to validate our findings, we performed western blotting and flow cytometry in sclc cell lines from different subtypes. results: our rna analysis indicated that the best surface markers to distinguish sclc subtypes were ceacam , fam a, lrfn , epha . immunoblot analysis validated ceacam and epha but not fam a or lrfn . we also found that ncam , a commonly used sclc marker, only marks some of the subtypes. for further analysis, we chose proteins with antibodies validated for flow cytometry as our chosen biomarker platform. flow cytometry analysis of cd is suitable as a pan-sclc marker. however, the expression of non-ne cell lines was decreased compared to rna-seq data. summary/conclusion: protein analysis of ceacam and epha corresponded to rna-seq data. ncam was not detected as a pan marker for all sclc subtypes. however, we could see cd expression in all sclc subtypes, indicating it may be a useful pan marker for sclc. future studies will be performed to validate the expression of other surface markers in cells, purified evs, and plasma of sclc patients. funding: nih u ca and nih u ca . leukobiopsyexploiting extracellular vesicle-mediated leukocyte sequestration of cancer-specific signatures introduction: in cancer, extracellular vesicles (evs) act as a unique exit mechanism for mutant and oncogenic macromolecules (proteins, rna and dna) en route from malignant cells to blood . while this process has inspired major liquid biopsy efforts, the biology of circulating evs that carry oncogenic mutations (oncosomes) is still poorly characterized. it is also unclear what part (if any) of the tumour-related cell free dna (ctdna) , , a major liquid biopsy analyte, is linked to circulating evs and what is their fate, receptacles and biological activity. methods: we employed as series of cancer cell lines carrying mutations in major oncogenes (hras, her , egfrviii). ev-dna was analysed by digital droplet pcr (ddpcr), along with nuclear anomalies in donor cells (dapi, electron microscopy) and transfer of dna to recipient cells of endothelial (huvec, mmbec), astrocytic (nha) or myeloid (hl ) origin. blood underwent fractionation into red blood cells (rbc), white blood cells (wbc), platelets (plt), evs ( , g ultracentrifugation) and soluble plasma (sup) . results: hras-mediated cellular transformation (in ras- cells) triggers profound changes in the structure of nuclear chromatin, which is driven into the cytoplasm and released as cargo of evs. oncogenic dna is detectable in blood fractions of tumour bearing mice. while evs, ctdna and plts contain intermediate levels of mutant dna, rbcs contain only traces of this material. the highest hras copy number per ml of blood is found in wbcs (monocytes and neutrophils), which contain more cancer dna/cell than liver, spleen and bone marrow. depletion of neutrophils using anti-ly g antibody results in an increase in ev-and ctdna-associated mutant dna in blood, suggesting the role of these cells in regulating the circulating levels of cancer cell-derived particles. uptake of dna-containing evs impacts the phenotype of myeloid cells, which adopt thrombo-inflammatory properties. these cells also retain cancer-specific transcripts and other cargo. finally, normal astrocytes treated with oncogenic evs also exhibit phenotypic changes and signs of genomic instability including formation of micronuclei. summary/conclusion: we propose that the process of leukocyte sequestration of circulating particles containing tumour-related nucleic acids renders these cells potentially usable as a novel liquid biopsy platform (leukobiopsy) in cancer. introduction: early diagnosis of colorectal cancer (crc) and precancerous adenoma patients is of vital importance. previously we profiled small extracellular vesicles (sevs) derived mirnas isolated from plasma, proposed a new promising biomarker category of crc patients. here we further gave a full landscape of circulating sevs derived rnas to explore and evaluate sevs based rna biomarkers for early detection of both crc and adenoma patients. methods: plasma sevs were isolated from participants, including early-stage crc patients, adenoma patients, and normal controls (nc), and characterized according to misv guideline. the total sevs derived rna expression profile of all participants was investigated by next-generation sequencing (ngs). weighted gene coexpression network analysis (wgcna) was performed to categorize differentially expressed rnas, and t-distributed stochastic neighbour embedding (tsne) was adopted to distinguish crc, adenoma, from nc samples with the top-ranked genes in wgcna modules. rt-qpcr validation was performed in a cohort of additional participants. results: a total of rna species (including mirnas, mrnas, and lncrnas) were found differentially expressed between plasma sevs in crc and nc participants. additionally, rna species were differentially expressed between plasma sevs in adenoma and nc participants. rna species were differentially expressed between plasma sevs in crc and adenoma participants. wgcna categorized all rnas into modules, which exhibited different expression trends during the carcinogenesis of crc. a -gene combined tsne model consists of the top genes in each module could perfectly classify crc, adenoma, and nc samples. a -gene combined tsne model consists of the top gene in each module could roughly distinguish crc and adenoma from nc, with only sample misclassified. rt-qpcr assays also confirmed the potential classification ability of those genes in another validation cohort of participants. introduction: although the concept of systematic "liquid biopsy" using bodily fluids is simple and elegant, the path of clinical reality has been challenging. recently, numerous tissue-specific biomarkers have been discovered in evs derived from blood, urine, cerebrospinal fluid, cell culture media, and a variety of other fluids. however, tracing the lineage of evs to their tissue of origin remains challenging due to their minute amount of cargo and unavailability of matching biopsied tissue and bodily fluids from the same patient. we recently demonstrated in three separate publications (dogra et. al; smith et. al; murillo et. al) , a new device (nanodld) for ev isolations, it's comparison with current technologies, bioengineered vesicles, and a detailed study of rna types present in small/ large vesicles, lipoproteins, and ago protein in different biofluids. in the present study, we aim to investigate the lineage of prostate derived evs in biofluids. methods: using our chip technology, we have isolated exosomes from prostate cancer cell lines and patient tissue, blood and urine samples. after exosome isolation, small rna libraries were prepared, and sequencing is carried out at icahn school of medicine and new york genome center using illumine sequencer hiseq . our nanofluidic pillar array is manufactured in an sio mask using optical contact lithography and deep ultraviolet lithography. results: our study revealed i) rna markers, which are exclusive to their prostate tissue of origin and are secreted in evs; ii) approximately - % of prostate tissue-specific rna were discovered in evs; iii) over % ( of rna) of literature curated prostatespecific rna signatures were detectable in serum and urine evs from pca patients; iv) evs contained over - % of noncoding rna ( - % was mirna), while tissue predominantly yielded rrna (> %); v) finally, gene set analyses generated that over % of evs rna were enriched for signalling pathways, yielding mirna-associated, non-canonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively monitor prostate tissue-specific biomarkers, identify tumour-specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. summary/conclusion: in summary, we have investigated patient matched tissue, serum, and urine derived evs in prostate cancer. we present a set of prostatic rna in evs, which are enriched in noncanonical wnt signalling, and androgen receptor pathways. this study enables us to noninvasively track prostatic biomarkers, identify tumour specific rna, and potentially may benefit in liquid biopsy by avoiding unnecessary surgical procedures. a multi-model, liquid biopsy approach for diagnosing and staging pancreatic adenocarcinoma introduction: pancreatic ductal adenocarcinoma (pdac) is the third largest contributor to cancerrelated death in the usa. since there is not yet a feasible technology to diagnose pdac early in the disease, % of patients are diagnosed at an advanced stage. moreover, for patients with confirmed pdac, standard imaging method has low sensitivity to detect early metastatic disease, which complicates the selection of therapy. to address these challenges, there has been great interest in developing minimally-invasive, extracellular vesicle (ev) based blood tests for pdac. to this end, we have integrated measurements of tumour derivedev rna cargo with circulating proteins and cell free dna (cfdna), and use machine learning algorithms to distill this multiplexed diagnostic to . diagnose pdac patients from healthy and disease controls and . distinguish pdac patients with distance sites of metastasis to guide their treatment. we make use of our lab's magnetic nanopore isolation technique to specifically enrich for tumour derived evs directly from patient plasma. methods: we have developed a high throughput nanofluidic sorting platform, which immunomagnetically isolates individual evs from plasma using magnetic nanostructures. however, our architectures is uniquely designed for massive parallelization allowing high throughput, robust processing of ml of plasma in minutes. we performed sequencing on a discovery set of patients and controls (n = ). subsequently, we trained our panel of biomarkers using a training set of n = . finally, we validated the performance of our platform using an independent blinded test set of n = . results: the results of a blinded test set achieved an accuracy = % and an auc = . on binary classification of pdac patients versus those that were healthy or disease controls. in addition, we achieved an auc = . and accuracy = % with sensitivity of % and specificity of % on detecting occult metastasist. summary/conclusion: we developed a highly sensitive pancreatic cancer diagnostics by combining our nanomagnetic isolation platform for tumourderived ev isolation, rna sequencing, and machine learning. we isolated tumour-derived evs and profiled their rna cargo, combined with cfdna and ca - for pancreatic cancer diagnosis. the predictive panels successfully distinguished non-cancer patients from pdac patients, and nodistant metastasis patients(m ) from distant metastasis patients(m ) for appropriate treatment. the resulting auc and accuracy from the independent blinded test set outperformed any individual biomarker, showing both the benefits and the robustness of combining multiple orthogonal biomarkers for pdac diagnosis. introduction: both hypertension and diabetes exhibit significant molecular changes to the vasculature that are associated with increased cardiovascular risk. here we examined the protein composition of large evs (l-evs) isolated from the plasma of hypertensive, diabetic and healthy mice to identify common and diseasespecific molecular changes. methods: we examined circulating l-evs isolated from transgenic mice expressing active human renin in the liver (ttrhren, a model of hypertension), ove type diabetic mice, and their wild-type (wt) littermates. at weeks of age mice were sacrificed and blood samples were obtained by cardiac puncture. l-evs were isolated from platelet-free plasma via differential centrifugation and protein content was assessed via mass spectrometry (ms). results: ttrhren mice exhibited increased blood pressure compared with ove mice or their wt littermates. ( . ± . vs . ± . [ove ] vs. . ± . mmhg [wt], p < . ). ms identified independent proteins with at least peptides per protein. of these, proteins were found in all groups studied, were exclusive to wt mice, were exclusive to ove mice and were exclusive to ttrhren mice. in addition, proteins were observed with > . fold change (fc) compared to wild-type mice, and proteins were reduced by > %. amongst the top ten differentially expressed proteins, fibrinogen was upregulated in both ove and ttrhren mice compared with wild-type controls. similarly trem-like transcript , sarcoplasmic/endoplasmic reticulum calcium atpase and junction plakoglobin were all downregulated in both ove mice and ttrhren mice suggesting molecular changes common to both conditions. conversely, arginase was up-regulated in diabetic, but not hypertensive mice while carboxypeptidase was upregulated in hypertensive but not diabetic mice. summary/conclusion: taken together, these results show that the protein composition of circulating l-evs is altered in diabetes and hypertension and that both common and disease-specific changes may be detected. further analysis of these changes may lead to the identification of novel pathways associated with the pathogenesis of vascular injury in hypertension and diabetes. funding: this study was supported by grants (to db) from the canadian institutes of health research, an ontario early researcher award, and the canada foundation for innovation. understanding the role of endothelial cell-derived apoptotic bodies in inflammatory signalling and cell clearance in an atherosclerosis model of inflammation. introduction: apoptotic bodies (apobds) are a class of large (~ - um) evs formed during apoptotic cell disassembly, that are becoming increasingly recognized as potential mediators of intercellular communication, e.g. via the transfer of proteins and other cargoes to target cells. during the inflammatory vascular disease atherosclerosis, endothelial cell (ec) apoptosis contributes to loss of barrier function and promotes the formation of plaques in regions of ec damage. although, experimentally, ecs generate an abundance of apobds, a specific role for ec-derived apobds (ec-apobds) in the progression of atherosclerosis remains poorly defined. methods: in the present study, a detailed in vitro characterization of ec disassembly was performed via flow cytometry, confocal live cell imaging and cytokine profiling, followed by function analyses of ec-apobds using a murine in vivo model of dead cell clearance. results: characterization of ec disassembly revealed that apobd formation in ecs is regulated by rho-associated, coiled-coil-containing protein kinase (rock ), a process that can be pharmacologically inhibited using a rock- inhibitor, thereby providing tools for functional in vivo studies. the specific cargo and role in clearance of ec-apobds were then investigated. profiling of ec-apobds was performed via cytokine antibody array to reveal that ec-apobds generated under inflammatory conditions contain high levels of pro-inflammatory cytokines including mcp- and il- , suggesting a potential role for ec-apobds in the propagation of inflammation during vascular disease. furthermore, the ability of ec-apobds to be cleared from the vasculature via phagocytosis was investigated, revealing that ec-apobds can travel to distal organs to undergo clearance. summary/conclusion: these findings provide important insights into the potential functions of ec-apobds generated under both non-inflammatory and inflammatory conditions and may contribute to future studies involving the therapeutic targeting of ec disassembly for the treatment of atherosclerosis. funding: this work was supported by grants from the national health & medical research council of australia (gnt , gnt ) adipose mesenchymal stromal cell derived evs foster cardio-renal protection in the doca-salt hypertensive rat model introduction: cardio-renal syndromes (crs) are disorders of the heart and kidneys whereby "acute or chronic dysfunction in one organ may induce dysfunction of the other". stem cell-derived extracellular vesicles (evs) mediates the protection of the kidney from development of chronic kidney disease (ckd). we here investigated the potential of adipose-mesenchymal stromal cells derived evs (asc-evs) as therapeutic tools for the treatment of crs. methods: adult wistar rats were uninephrectomized and treated with a high-na+ diet and deoxycorticosterone-acetate (doca-salt) for -weeks ( / ; a / - - ). evs were isolated by ultracentrifugation method. ev dimension, concentration and surface markers were characterized by nta, cytofluorimetric analysis and transmission electron microscopy. to characterize the role of evs in crs, doca-salt rats were injected weekly with asc-evs. systolic blood pressure was measured by the tail-cuff method. plasma creatinine and urinary protein excretion were determined by colorimetric assays and microalbuminuria by immune turbidimetric assay. qrt-pcr and western blot were conducted to evaluate fibrosis and inflammatory-related genes and proteins in the kidney and heart of doca-salt rats. immunohistochemistry was used to confirm matrix accumulation (a-sma) and immune infiltrate (cd + cells). results: multiple administration of asc-evs in doca-salt rats induced a protective effect on the kidney, by reducing tubular and vascular damage. kidney function was also conserved by ev treatment as detected by the normal glomerular filtration rate and the absence of proteinuria with respect to doca-salt untreated rats. ev administration significantly decreases the pro-inflammatory molecules mcp- and pai and reduce the recruitment of macrophages in the kidney. the mitigation of the inflammatory response by asc-ev infusion consequentially affected the development of fibrosis, as detected by the decrease in collagens (col a , col a ) and fibronectin (fn) expression in respect to doca-salt animals. asc-evs were able to act in multiple organs, preventing fibrosis and inflammation also in the heart, therefore alleviating blood pressure rise during the -weeks of treatment in doca-salt rats. summary/conclusion: our results indicate that asc-ev administrations in hypertensive-induced ckd rats promote protection from renal damage, reduction of the inflammatory response and prevention of interstitial fibrosis in the kidney. asc-evs are also able to protect the cardiac tissue and to control blood pressure increase, displaying complex and multiorgan beneficial effects. introduction: alveolar macrophages (ams) tonically secrete extracellular vesicles (evs) containing suppressor of cytokine signalling (socs ) protein. uptake of socs -containing evs by alveolar epithelial cells is critical for restraint of cytokine-induced janus kinasesignal transducer and activator of transcription (jak-stat ) signalling to promote homoeostasis in the distal lung. at steady state, ams exhibit suppressed glycolytic activity, a metabolic phenotype that promotes homoeostatic function. whether this glycolytic restraint is critical for am secretion of socs is unknown. in fact, to our knowledge, metabolic control over release of any ev cargo has never been explored in any cellular context. methods: immortalized mouse ams (mh-s) were treated with various doses of -deoxy-d-glucose ( -dg) and oligomycin, inhibitors of glycolysis and oxidative phosphorylation, respectively. primary rat ams collected by lung lavage were treated with an aqueous extract of cigarette smoke (cse) with or without -dg. metabolic activity was measured by seahorse assay, evs were quantified by nanoparticle tracking analysis, and vesicular (> -kda) socs secretion was determined by western blot of conditioned medium. additionally, ams collected from wild-type (wt) and lsl-krasg d mice bearing lung tumours weeks after intrapulmonary ad-cre were cultured ex vivo in the presence or absence of -dg. vesicular (> -kda) socs secretion was measured by elisa. results: in a dose-dependent manner, oligomycin inhibited, whereas -dg enhanced, socs and ev release by mh-s cells. treatment of rat ams with cse ( %) attenuated secretion of socs , an effect that coincided with increases in glycolytic activity, and co-treatment of ams with -dg abrogated the inhibitory effect of cse on socs release. finally, ams collected from lsl-krasg d mice exhibited a deficiency in socs secretion relative to wt ams, an effect that was reversible by overnight culture in the presence of -dg. summary/conclusion: in tandem, our data generated using in vitro and in vivo approaches demonstrate that am secretion of vesicular socs is down-regulated by glycolysis. we speculate that metabolic control over release of ev cargoes is a phenomenon of broad biologic relevance within and outside of the lung. introduction: bacterial extracellular vesicles (ev) are described to play roles in defence and resistance, pathogenesis and stress responses. cyanobacteria pioneered oxygenic photosynthesis, and are the ancestors of modern chloroplasts. we previously described that by deleting the gene encoding tolc (Δtolc) in the model cyanobacterium synechocystis sp pcc (s ), a key player in protein-mediated secretion systems, a hyper-vesiculating phenotype could be obtained. the goal of this work was to understand why Δtolc hyper-vesiculates. methods: isobaric tag for relative and absolute quantitation (itraq) was used for quantitative proteomic analyses of total cell extracts. ev were isolated as follows: cells were separated from the extracellular medium (em) by centrifugation ( g, min) and filtration ( . µm pore-size filters). cell-free em was concentrated using centrifugal filters (mwco of kda), and later ultracentrifuged for h at g. the final ev fraction was suspended in growth medium. ev characterization was performed using tem, dls, nanosight, and by the detection and quantification of lps (lipopolysaccharides). detection of specific proteins in ev was carried out by western blot. copper (cu) levels were quantified by atomic absorption spectrometry (aas). results: a large-scale quantitative proteomic analysis was performed, resulting in the identification of several metal-related proteins with differential regulation in s Δtolc. both wild-type (wt) and Δtolc cells were then challenged with different metals. compared to the wt, Δtolc showed impaired growth only when exposed to cu, a co-factor for several proteins with roles in primary metabolism. the intracellular cu levels were quantified and Δtolc accumulates threefold more cu than wt cells. we then asked whether the hyper-vesiculating phenotype observed could be linked to the stress induced by cu accumulation. in ev isolated from Δtolc we detected the metallochaperone copm, a periplasmic cu-binding protein involved in cu-resistance mechanisms in s . in addition, cu could also be detected in isolated Δtolc-ev. in addition, more ev were detected when s wt cells were challenged with cu, in a cu-concentration dependent manner. summary/conclusion: these results support the idea that bacterial ev represent an alternative cu-secretion mechanism to deal with cu-induced stress. funding: fct phd grant sfrh/bd/ / ; feder-compete -poci-fct project: poci- - -feder- . juan wang and maureen barr rutgers university, human genetics institute of nj, piscataway, usa introduction: extracellular vesicles (evs) function in intercellular communication. despite their physiological importance and biomedical relevance, knowledge of ev fundamental biology is not well understood, in part due to a lack of tractable animal systems. our analysis of environmentally-released c. elegans ciliary evs provides strong evidence that nematodes package cargo in evs that mediate inter-organismal communication, in analogy to intercellular signalling in mammals. we predict that conserved mechanisms underlie ev cargo sorting, biogenesis and signalling. cilia act as cell towers to both receive extracellular signals and to send information via ciliary evs. ciliary defects result in human ciliopathies including autosomal dominant polycystic kidney disease (adpkd). adpkd is a life-threatening disease that affects / and is caused by mutations in pkd and pkd , which encode polycystin- and − . in c. elegans and humans, the polycystins are architecturally similar, act in the same genetic pathway, function in a sensory capacity, localize to cilia, and are shed in evs, suggesting ancient conservation. moreover, ciliary ev biogenesis and shedding is an evolutionary conserved process from algae to worms to humans. by studying how cilia make and receive evs, we aim to uncover fundamental principles of how cells communicate using evs. methods: to study ciliary ev cargo sorting and biogenesis, we use genetically-encoded fluorescent-tagged ev cargo and superresolution zeiss airyscan confocal microscopy in living animals. results: we find that cargoes are sorted into distinct populations. in cilia, kinesin- motors and kinesin- klp- /kif transport different ev cargoes to the ciliary tip and generate an ev cargo enrichment zone. from here, evs are shed and released into environment in a spatially and temporally regulated manner. ciliary ev biogenesis and release is regulated by mechanical pressure and ph. our work revealsat the single cell levelthat different evs are made in response to environmental stimuli, which may be important for ev signalling properties. summary/conclusion: cells exploit the spatiallyrestricted cilium and its sophisticated transport system to generate distinct populations of ciliary evs. how these ciliary ev communicate cellular messages awaits decoding. introduction: we recently demonstrated that recycling endosomes marked by rab a generate exosome subtypes distinct in cargos and functions from late endosomes, which we collectively term rab -exosomes. these exosomes are preferentially released from cancer cells in response to metabolic stress and promote adaptive changes in a xenograft model. here we use comparative ev proteomics in hct colorectal and hela cervical cancer cell lines to identify rab -exosome signature proteins and screen for functional effects. methods: we analysed ev preparations by mass spectrometry using tandem mass tag® labelling to identify changes in ev protein cargo in response to glutamine depletion. candidate genes were subsequently knocked down in drosophila secondary cells, which permit visualisation of rab -exosome biogenesis using fluorescence microscopy, and in human cancer cell lines. results: we show that accessory escrt-iii proteins, chmp , chmp and ist , are enriched on glutamine-depletion-induced evs and play a selective and conserved role in generating rab -exosomes. they are, however, not required to traffic ubiquitinated cargos into late endosomes and lysosomes. escrt- components, thought to regulate trafficking of ubiquitinated cargos into intraluminal vesicles, are also required to make rab -exosomes. in flies the escrt- , hrs, localises to the limiting membrane of rab -endosomes. comparative proteomics reveals other proteins enriched in rab -exosomes, which also appear to be needed to mediate this novel exosome formation mechanism. summary/conclusion: we conclude that rab -exosome subtypes are formed via a distinct mechanism requiring accessory escrt-iii components, suggesting a route to selectively target these exosomes. introduction: the tumour microenvironment consists of a complex network of host cells embedded within extracellular matrix. communication between these cellular compartments is critical for tumour progression and exosomes have emerged as important regulators of intercellular communication. while a number of studies have implicated exosomes in cancer progression, mechanisms controlling exosome transfer are not well understood. we developed three-dimensional ( d) culture models to evaluate the role of cues provided by the extracellular matrix in exosome release and uptake. methods: exosomes were isolated from cells in two-and three-dimensional culture via ultracentrifugation and characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. exosomes were labelled with fluorescent lipophilic dyes and uptake in recipient cells quantified by flow cytometry. results: cells cultured in d display decreased exosome release and increased uptake compared to d cultured cells. exosome release in d culture was inhibited with the exosome release inhibitors brefeldin a and gw , but was not significantly altered by knockout of rab b. in addition, disruption of polarity signals provided by d culture did not impact exosome release or uptake in d, but induction of oncogenic hras increased both secretion and uptake of exosomes through activation of pi k signalling. summary/conclusion: release and uptake of exosomes is altered in d environments. these studies help provide insight into exosome production and uptake in vivo and have potential implications for therapeutically targeting exosome release and the development of exosome based therapeutic delivery vehicles. introduction: previous studies in our lab found that expression of r w-fibulin- induces rpe to undergo emt. the purpose of current study was to characterize the extracellular vesicles (evs) in rpe cells expressing wt-fibulin- versus rpe cells expressing r w-fibulin- and investigate the effects of these evs on rpe cell differentiation. methods: arpe- cells were infected with lentivirus with luciferase-tagged wild-type (wt)-fibulin- or luciferase-tagged r w-fibulin- . evs were isolated from the media of arpe- cells by conventional ultracentrifugation or density gradient ultracentrifugation. transmission electron microscopy (tem) and cryogenic electron microscopy (cryo-em) were performed to study the morphology of the evs. the amount and size distribution of evs were analysed by nanosight tracking analysis (nta). ev protein concentrations were quantified using the dctm protein assay (bio-rad). ev cargo were analysed by unbiased proteomics using lc-ms/ms with subsequent pathway analysis (advaita). migration ability was evaluated in arpe- cells with or without the exposure of evs by conducting scratch assays. results: morphologically, tem imaging showed concave-appearing vesicles and cryo-em imaging showed spherical vesicles with two subpopulations of evs: a small group with diameters around nm and a large group with diameters around nm. moreover, tem and cryo-em showed an increased amount of small evs (~ nm) in the mutant group compared to the wt group. this result was further confirmed by nta showing that, in the mutant group, the particle size distributions were smaller than the wt evs. no significant differences were shown in ev protein concentrations per particle between wt and mutant groups. our previous data suggest that the expression of r w-fibulin- causes rpe cells to undergo emt as evidenced by upregulated emt drivers and an increased migration ability. proteomic studies showed that evs derived from arpe- cells overexpressing wt-fibulin- contain critical members of sonic hedgehog signalling (shh) and ciliary tip components, whereas evs derived from rpe cells overexpressing r w-fibulin- contain emt mediators, indicating that ev cargo reflects the phenotypic status of their parental cells. ev transplant studies showed that exposing native rpe cells to mutant rpe cell-derived evs containing emt drivers, including tgf-β-induced protein (tgfbi), vim, and smad , leads to an enhanced migration ability of rpe cells in a dosedependent manner. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms ( d static tissue culture flask vs d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in d t-flasks and d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both d and d culture systems. introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies methods: pca patients and age-matched healthy controls (hc) plasma (n = in each group) were used to isolate sevs with different isolation methods including commercial exoquick ultra kit, qev and qev size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd and cd commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev . furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods summary/conclusion: qev method demonstrated better performance in isolating relatively pure sevs from human plasma; qev has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev but with the highest non-sev protein contaminations. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd , cd and cd , it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: ) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and ) the assessment of subpopulation target enrichment relative to the population average by leveraging tim as an unbiased, lipidbased ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasisassociated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr (her +), t d and mcf- (er+pr+), bt and mda-mb- (triple negative) breast cancer cell lines, as well as five mda-mb- -derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her was broadly detected in her + skbr evs. interestingly, her -t d and mcf- evs also expressed her where it was highly enriched in its epcam+ subpopulations. itg α , β and β were only found in triple negative and organotropic evs with itg β and β differentially enriched based on the organotropism. the population average of mda-mb- and lung-tropic evs had high expression of itg β , where subpopulations of cd + evs showed positive enrichment while cd + and cd + evs showed negative enrichment. itg α , β and β were absent in the bone-tropic cd + ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb- evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. op . = pf . heparan sulphate proteoglycans are required for ev-mediated delivery of multiple growth factors sara veiga, alex shephard, alex cocks, aled clayton and jason webber cardiff university, cardiff, uk introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified targets that bind to hs-gag chains, and also different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of ev- introduction: methamphetamine (ma) and related amphetamine compounds, which are potent psychostimulants, are among the most commonly used illicit drugs. neuroimaging studies have revealed that chronic ma abuse can indeed cause neurodegenerative changes in the brains of human ma abusers including prominent microglial activation throughout the brain. it is still unclear how chronic inflammation caused by ma abuse leads to long-term damage to the brain. with this in mind, we are particularly interested in studying the role of extracellular vesicles (evs) in eliciting chronic inflammation in ma exposed brains. in the present study, we focus on the role of a mirna, mir- a- p (mir- a) in chronic ma exposure. here, we present novel data that shows for the first time how chronic ma impacts not only the biogenesis but also the ev associated mirna cargo thereby affecting the overall health of the neurons and glial cells in the brain. methods: -density gradient centrifugation for isolation of brain-derived vesicles -characterization of bdes by western blotting, nanoparticle tracking analysis and transmission electron microscopy -quantitative rt-pcr -digital droplet pcr -confocal imaging of dendritic spines and synapses results: in the present study, we show from both in vivo and in vitro studies that chronic methamphetamine (ma) treatment alters ev biogenesis and microrna (mirna) cargo. brain-derived evs (bde) isolated from frontal grey tissue of rhesus macaques that were administered ma in a chronic regimen revealed a significant increase in both number and size. further analysis revealed increase in biogenesis genes and increased levels of mirna, mir- a- p (mir- a). in situ hybridization of the frontal brain area revealed that mir- a was exclusively expressed in microglia and neurons. further, in vitro studies revealed that ev associated mir- a elicited not only neuronal damage but also was able to activate microglia to release pro-inflammatory cytokines thereby inducing a chronic inflammatory cycle. finally, we show that an anti-inflammatory drug was able to rescue inflammation, mir- a levels and synaptodendritic injury. summary/conclusion: in summary, our results present for the first time show that chronic ma exposure in the brain affects ev biogenesis and mirna expression. we further confirm that mir- a can serve as potential marker to diagnose synaptic deficits for chronic ma addiction in humans. finally, we reveal that anti-inflammatory drug could rescue the ev biogenesis and reduces the secretion of mir- a, thereby rescues synaptodendritic injury. our data further supports the use of the anti-inflammatory drugs as therapeutic interventions for ma addiction. funding: nida funding # r da blood-borne and brain-derived ectosomes/microparticles in morphineinduced anti-nociceptive tolerance deepa ruhela, veena bhopale, ming yang, kevin yu, eric weintraub, aaron greenblatt and stephen r. thom university of maryland school of medicine, baltimore, usa introduction: opioid pain treatment is impeded because chronic administration decreases analgesia, a condition called tolerance that prompts dose escalation contributing to morbidity and mortality. inflammatory interleukin (il)- β is required for tolerance development, so we hypothesized that pro-inflammatory extracellular vesicles (evs) play a role. methods: evs with opioid administration were assayed in mice and humans. annexin v-positive, . - µm diameter microparticles (mps) were assessed by flow cytometry in murine and human blood and in murine deep cervical lymph nodes that drain brain glymphatics. blood-borne exosomes (< nm) were assayed by tunable-resistance pulse sensing (trps). anti-nociceptive tolerance following morphine administration to mice was assessed by speed of tail removal from warm water. results: repetitive morphine dosing of mice to induce anti-nociceptive tolerance increased blood-borne mps by eightfold, and by tenfold in cervical lymph nodes. mps expressed proteins specific to neutrophils, microglia, astrocytes, neurons and oligodendrocytes. il- β content of mps increased -fold. administration of an il- β antagonist to mice diminished blood and glymphatic mps elevations and abrogated tolerance induction. intravenous polyethylene glycol telomer b that lyses mps and intraperitoneal methylnaltrexone that binds peripheral opioid-mu receptors and myeloid differentiation factor- to inhibit toll-like receptors, inhibited mps elevations and tolerance. neutropenic mice did not develop anti-nociceptive tolerance, elevations of blood-borne mps or cervical node mps expressing microglial proteins. elevations of blood-borne exosomes were not identified based on trps analysis. patients entering treatment for opioid use disorder exhibited similar mps elevations as do tolerant mice. summary/conclusion: neutrophil-derived mps containing il- β are required for morphine-induced antinociceptive tolerance. funding: this project was supported by grant n - - - from the office of naval research and an unrestricted grant from the national foundation of emergency medicine. evs are a conveyor of toxic dipeptide repeat proteins in c orf als/ ftd models thomas jefferson university, philadelphia, usa introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterized by loss of motor neurons. in als, motor symptoms initiate focally and then progress gradually, distal from the initial focus. abnormal forms of als-associated proteins are physically exchanged between neuronal cells. pathogenic als proteins like sod , fus and tdp are transmitted between cells by assisted mechanisms, mainly extracellular vesicles (evs), spreading toxicity and misfolding of native proteins within the recipient cells. an intronic g c aberrant nucleotide repeat expansion in c orf gene is the most common genetic cause of als. translation of this expanded region occurs by a process called repeat associated non-aug (ran) translation that produces five dipeptide repeats proteins (dprs), polyga, polygp, polygr, polypa and polyga. polyga, polygr and polypr are associated with toxicity in neurons. in this work we study the recruitment of these aberrant proteins into extracellular vesicles (evs) and the potential role of these evs in spreading toxicity between cells of the central nervous system. methods: to isolate the evs from cell culture media we isolated by ultracentrifugation the larger vesicles at , xg and the smaller evs at , xg. number, size and fluorescence of the vesicles were analysed by fluorescent nanotrack analysis (f-nta) and by cytoflex. the protein content of the vesicles was analysed by western blot (wb). to evaluate the potential toxicity of the evs, a transwell system (tw) was employed. neuron viability was assessed using live imaging techniques. results: nsc were transfected with reporter constructs expressing dprs tagged with gfp protein. by f-nta, cytoflex and wb analysis we assessed that all the five dprs were loaded in both the large and the small vesicles isolated from cell culture medium. by tw, nsc transfected with the dprs were put in contact with primary cortical neurons (cns) transfected with synapsin driven td-tomato for live imaging purposes. we observed that polygr+ nsc were able to cause a significant decrease in cns viability. we also observed that polygr+ evs associated toxicity was directly dependent on polygr length. this effect was reverted reducing the number of polygr+ evs treating nsc with gw . to understand the downstream effect of polygr+ evs in recipient cells we studied tdp mislocalization, ran-translation and activation of the integrated stress response, finding a dysregulation of all these potentially toxic pathways in neurons treated with polygr+ vesicles. summary/conclusion: concluding, dprs are actively secreted in evs and polygr+ vesicles cause the activation of toxic mechanisms in the recipient cells, possibly contributing to the spreading of als introduction: pregnancy is the a condition that profoundly mitigates symptoms of multiple sclerosis (ms) a complex disease characterized by immune dysfunction and neurodegeneration affecting . million people worldwide. serum exosomes, released by specific cells during pregnancy, modulate the immune and central nervous system function and contribute to pregnancy-associated suppression of experimental autoimmune encephalomyelitis (eae), an induced preclinical model of ms. extracellular vesicles (evs) are the new means for communication among cells. the aim of our study was to characterize the ability of human amniotic fluid stem cells-derived evs (hasc-evs) to antigen presenting cell function thus correcting immune dysfunction in eae. methods: amniotic fluids were obtained from human - -week pregnant women. hasc-evs were collected by ultra-centrifugation. evs were characterized for their specific proteins, lipids and nucleic acids expression. the ability of evs to modulate immune responses was performed in vitro, testing the ability of evs to induce a tolerogenic phenotype in mouse bone marrow derived dendritic cells, and in vivo for their potential to suppress eae, induced by immunization c /b female mice with mog - peptide. results: we found that hasc-evs expressed high levels of galectin- and promoted a significant increase of the immunoregulatory enzyme indoleamine , dioxygenase- enzyme in dcs. moreover in in vivo experiments administration of hasc-evs significantly reduced disease severity in eae. such effect was associated with reduced neurological deficits and suppression of pathogenic t helper (th ) cells, and increased percentage of regulatory t cells (treg-foxp +) cells. summary/conclusion: our findings unravel immunoregulatory effects of evs secreted by hascs. evs may represent a novel cell-free immune regulatory and regenerative therapeutic approach that can potentially mitigate immune dysfunction and promote remyelination. association of neuronal-derived extracellular vesicles cargo with cognitive decline in late middle life introduction: alzheimer's disease (ad) is characterized by a long preclinical stage during which phosphorylated tau pathology spreads in the brain leading to clinical symptoms. pathogenic tau spreads, in part, via extracellular vesicles (evs). we and others have demonstrated that tau cargoes of neuronal-derived evs (nevs) from blood can serve as biomarkers for ad. we aimed to examine whether nev tau cargo can predict cognitive decline in late middle age by leveraging samples from participants in the wisconsin registry for alzheimer's prevention (wrap) study. methods: we blindly immunoprecipitated nevs using antibody against neuronal l cell adhesion molecule (l cam) from serum samples of wrap participants who were cognitively unimpaired at baseline (mean age . ± . years old; . % females; . % apoe carriers), of whom half subsequently developed cognitive decline. we measured phosphorylated (p and p ) and total tau in nevs using electrochemiluminescence assays. we used linear regression models to identify differences between cognitive status groups including age, sex apoe status and the cognitive status*age interaction in the model. results: at baseline, we found trends for higher p -(p = . ) and p -tau (p = . ) levels in future decliners compared to stable participants. further, there were significant cognitive status*age interactions for ptau (p < . ), total tau (p < . ) and ptau (p < . ) with higher levels with increasing age in future decliners summary/conclusion: nev tau cargo differs between late middle-aged individuals at risk for ad with and without future cognitively decline even before decline occurs, presumably due to subclinical spread of tau pathology. further nev biomarker development may allow preclinical ad diagnosis. introduction: in the brain, circulating extracellular vesicles (evs) in the cerebrospinal fluid (csf) contain a variety of signalling factors, including proteins, enzymes, and rna transcripts. while evs have been implicated in many cell-to-cell signalling contexts, the vast majority of these studies are based on findings derived from cell culture conditions. thus, the ability to identify cell typespecific ev release from cellular subpopulations within the brain represents a critical barrier in the field. methods: to address this knowledge gap, we utilized a novel transgenic mouse model to determine the release of cell-type specific evs. here we report the exomap- mouse, which is designed to express an exosomal green fluorescent protein in response to expression of cre recombinase. specifically, the exomap- transgene was inserted at the mouse h locus and consists of (i) a broadly expressed cag promoter/enhancer, (ii) a floxed orf encoding mts-tdtomato, (iii) an orf encoding the exosomal protein acyltya fused to mneongreen (mng), and (iv) a ʹ utr containing the wpre element and polyadenylation signal from the bovine growth hormone gene. results: intracranial ventricular injections of the viral vector aav-ttr-cre, which drives cre recombinase expression from the choroid plexus-specific promotor of the transthyretin gene, leads to acyltya-mng expression in the choroid plexus. moreover, we observed that these mice released mneongreen-positive evs into the cerebrospinal fluid and also visualized the vesicles in the blood. furthermore, these mice displayed an accumulation of acyltya-mng fluorescence in the medial habenula. summary/conclusion: the results indicate that choroid plexus-derived evs are trafficked to the csf and the medial habenula, and more generally, that the exomap- mouse can be used to follow the trafficking of tissuespecific evs into biofluids and between tissues in vivo. introduction: large-scale colorectal cancer (crc) sequencing studies have shown that % of all tumours had at least one mutation in proteins implicated in the wnt signalling pathway. mutations in β-catenin have often been associated with the constitutive activation of wnt signalling pathway and has been established as a major driver of crc. one of the proposed mechanisms of activating wnt signalling involves extracellular vesicles (evs) as cellular couriers to transfer wnt ligands from one cell to another. however, the association of oncogenic mutant β-catenin with evs has not been studied. subpopulations of cancer cells with different mutational loads and behavioural variations lead to intra-tumour heterogeneity methods: integrative proteogenomic analysis showed the secretion of mutant β-catenin via evs. evs were isolated by ultracentrifugation and optiprep density gradient centrifugation. silac-based quantitative proteomics analysis, immunofluorescence, biochemical analysis, qpcr and xenograft models were employed to unveiling the role of evs carrying mutant βcatenin. results: an integrative proteogenomic analysis identified the presence of mutated β-catenin in evs secreted by colorectal cancer (crc) cells. follow up experiments established that evs released from lim crc cells stimulated wnt signalling pathway in the recipient cells with wild type β-catenin. silac-based quantitative proteomics analysis confirmed the transfer of mutant β-catenin to the nucleus of the recipient (rko crc) cells. in vivo tracking of dir labelled evs in mouse implanted with rko crc cells revealed its bio distribution, confirmed the activation of wnt signalling pathway in tumour cells and increased the tumour burden. introduction: there has been a significant increase in incidence of human papillomavirus (hpv ) driven oropharyngeal cancer (opc) in developed countries. there is evidence that hpv alters the molecular cargo of exosomes released by opc. emerging evidence suggests that hpv integration within the human genome is associated with both genomic and transcriptomic alterations. consistent with previous studies, the genomic viral-cellular junctions were identified using dips-pcr method in ( %) saliva samples collected from hpv -driven opc. methods: morphology and molecular features of exosomes derived from three different saliva sampling methods: unstimulated saliva; acid-stimulated saliva; and salivary oral rinses were examined using transmission electron microscopy (tem), nanoparticle tracking (nta) and western blot analysis. hpv- dna detection in salivary exosome was determined by using qpcr method. proteome profile of salivary exosomes derived from both cancer-free controls and hpv -driven opc patients was characterized using liquid chromatography-electrospray ionization-tandem mass spectrometry (lc-ms/ms). results: we demonstrate that unstimulated saliva had greater abundance of exosomes when compared to the other sampling methods. three common exosome markers (cd , cd and cd ) were higher in unstimulated saliva. only salivary exosomes derived from hpv-driven opc patients had a detectable level of hpv- dna. the proteomic signature of salivary exosome was significantly (p < . ) different between cancer-free controls and hpv-driven opc. we found elevated protein abundance of five main glycolytic enzymes (i.e. phosphoglycerate kinase (pgk ), glyceraldehye- -phosphate dehydrogenase (gapdh), aldolase (aldoa) and lactate dehydrogenase a (ldha) in salivary exosomes derived from opc patients, suggesting a functional role of salivary exosome in the reciprocal interplay between hpv-driven opc and glucose metabolism. summary/conclusion: our data suggest that the development of a low-cost non-invasive saliva-based test using both salivary exosomal dna and protein may offer an opportunity to detect hpv-driven opc, that may be clinically useful in managing these patients. continuous in vivo release of mast cell derived extracellular vesicles from an implanted device spreads pro-inflammatory response in mice introduction: mast cells are important players of the immune system and they secrete a wide range of mediators during bacterial infections. mast cells are also able to release extracellular vesicles (evs). here, we report that mast cells communicate with each other in vivo by evs. methods: we isolated bone marrow-derived and peritoneal mast cells from gfp-transgenic and wild type mice. evs were separated from the conditioned media of these cells cultured in the presence or absence of lipopolysaccharide (lps). evs were characterised according to the misev guidelines by flow cytometry, electron and fluorescent microscopy, trps, the spv lipid and the bca protein assays. separated ev-s were cultured with naïve mast cells, and tumour necrosis factor (tnf)-α production was tested by elisa and intracellular flow cytometry. gfp+ mast cells were seeded in diffusion chambers which were implanted into the peritoneal cavities of mice enabling us to investigate the continuous in vivo release of evs. uptake of gfp+ evs and tnf-α expression of peritoneal mast cells were tested by flow cytometry and fluorescent microscopy. results: here, we showed that bacterial lps-sensing mast cells release evs that in turn, induce tnf-α expression in resting mcs in vitro. moreover, we confirmed that evs are transmitted to other peritoneal mast cells in vivo spreading the pro-inflammatory response by inducing tnf-α secretion in peritoneal mast cells. summary/conclusion: ev communication between members of the mast cell network, play an important role in spreading and escalating pro-inflammatory responses to immune stimuli. our data may provide an explanation how the relatively rare tissue resident mast cells can play key roles in diseases such as autoimmune arthritis. the ability of small extracellular vesicles (sevs) to reprogramme cancer cells is known. integrins, receptors for extracellular matrix proteins, are major players in mediating sev functions. previously, we have reported that the αvβ integrin is detected in sevs of prostate adenocarcinoma (prca) cells and transferred into recipient cells in a paracrine fashion; however, its role and expression have never been explored in the most aggressive forms of prca, such as neuroendocrine prca (neprca). neprca does not express androgen receptor (ar) but does express neuron-specific proteins, such as aurora kinase a, synaptophysin and neuron specific enolase, that activate pro-tumorigenic pathways independently from the ar. methods: we isolated sevs from prca c - b cells using iodixanol density gradients and characterized them by immunoblotting and exoview. the experiments were performed in vivo by injecting subcutaneously, in nude mice, du cells treated with sevs expressing or lacking the αvβ integrin, and in vitro, by testing anchorage-independent growth of different cell lines treated with the same sevs. discarded human tissues from prca metastasis were analysed by immunohistochemistry (ihc). results: we demonstrate that a single treatment of prca cells with sevs significantly stimulates tumour growth and anchorage-independent growth. moreover, we show that one treatment with sevs, shed from c - b cells that express αvβ , but not from the control cells that lack αvβ , induces differentiation of prca cells towards a neuroendocrine phenotype and downregulates ar. finally, our ihc analysis shows coexpression of αvβ integrin and synaptophysin in neprca metastatic lesions. summary/conclusion: in conclusion, our current study shows, for the first time, that αvβ integrin expression in donor cells generates sevs that reprogramme recipient cells towards an aggressive tumour phenotype. funding: this study was supported by nci-p - , r - to lrl. introduction: exosomes are small extracellular vesicles (sevs) that carry a variety of cargoes and have been shown to promote tumour cell motility and metastasis. cell motility is influenced by dynamic formation and stability of filopodia: actin-rich protrusions that extend from the leading edge and perform directional sensing. filopodia regulators such as fascin are upregulated in multiple epithelial cancers and can promote invasive phenotypes. however, how filopodia are induced and controlled by extracellular factors is poorly understood. here, we describe a role for sevs in regulating filopodia formation and tumour cell motility. we utilized b f melanoma cells and ht fibrosarcoma cells for fixed-and live-cell imaging to quantify filopodia numbers and dynamics in control and exosome-deplete conditions. itraq proteomics was used to identify sev protein cargoes that contribute to filopodia formation. in vivo experiments were performed using a chick embryo model for metastasis. results: inhibition of exosome secretion in cancer cell lines, via rab a or hrs knockdown, led to decreased filopodia numbers. specificity to sevs was demonstrated by rescue experiments in which purified sevs but not large evs rescued the filopodia phenotypes of exosome-inhibited cells. live imaging of hrs-kd cells revealed that exosome secretion regulates formation and stability of filopodia. proteomics data and molecular validation experiments identified the tgf-beta coreceptor endoglin as a key sev cargo regulating filopodia formation, cancer cell motility, and metastasis. summary/conclusion: in this study, we identified exosomal endoglin as a regulator of filopodia formation and in vivo metastasis. these data are relevant to cancer as endoglin expression is altered in many cancers. in addition, endoglin is the disease gene for hereditary haemorrhagic telangiectasia, and may influence angiogenesis. overall, our data implicate sev-carried endoglin as a key cargo regulating filopodia. astrocyte-derived ev-mediated blood-brain barrier disruption shilpa buch, ke liao, susmita sil, fang niu and guoku hu university of nebraska medical center, omaha, usa introduction: the breach of the blood-brain barrier (bbb), resulting in ensuing neuroinflammation, is a key feature of hiv-associated neurological disorders (hands). while combination antiretroviral therapy (cart) has successfully suppressed peripheral viraemia, cytotoxicity associated with the presence of viral tat protein in tissues such as the brain, remains a significant concern. our previous study has demonstrated that hiv- tat can induce disruption of bbb by downregulation of tight junction (tj) proteins in human brain microvascular endothelial cells (hbmecs) and that this is regulated by the autophagic pathways. methods: evs were isolated from hiv tat-stimulated mouse/human primary astrocytes using the standard differential ultracentrifugation method and characterized by transmission electron microscopy, nanosight & western blot analyses. among the various mirs dysregulated in hiv tat -stimulated astrocyte ev cargo, mir- was found to be upregulated by realtime pcr. confocal microscopy identified uptake of astrocytic evs by hbmecs. functional assessment of astrocytic ev uptake by hbmecs involved cell permeability using transepithelial electrical resistance as well as trans-well endothelial cell monolayer permeability assays. results: hiv- protein tat-mediated induction of micrornas (mirs) in astrocyte-derived extracellular vesicles (adevs) regulated the permeability of bbb by targeting the expression of tj proteins in the hbmecs. exposure of hbmecs to tat-adevs resulted in down-regulation of the tight junction protein claudin , resulting in increased endothelial cell monolayer paracellular permeability. microarray data of tat-adevs demonstrated upregulation of several mirs compared to that of controls, among which upregulated mir- was identified to target the tj proteins using ingenuity pathways analysis. increased expression of mir- was validated in tat exposed astrocytes and tat-adevs. adevs loaded with mir- oligos showed similar effects as that observed with tat-adevs in inducing permeability in hbmecs. increased expression of mir- with downregulation of claudin- was also recapitulated in microvessels isolated from the brains of doxycycline-inducible hiv- tat transgenic mice (itat) mice and in lysates isolated from the frontal cortices of siv+ macaques/hiv+ autopsied brains. summary/conclusion: our findings demonstrated that tat-adevs containing mir- as an important mediator underlying tat-mediated disruption of the bbb. introduction: endogenous exosomes and related extracellular vesicles (evs) are potent nanoparticles released by all cells tested to date. the exploitation of their unique scaffolding for engineering next-generation drug delivery systems represents a major area of academic and commercial interest. the lag in exploiting this potential is in part due to our inability to measured extent and efficiency of modification, e.g., composition and drug loading. here we report a robust pipeline of optical tweezing combined with raman spectroscopy to molecularly characterize engineered evs and quantitatively assess extent of drug loading at single particle resolution. methods: evs derived from cell culture and isolated by ultracentrifugation were fused with synthetic liposomes to create engineered evs (eevs). these eevs were formed via well-established vesicle fusion techniques, namely ( ) mechanical extrusion, ( ) freeze-thawing, or ( ) probe-tip sonication. prior to formation, calcein was encapsulated in the liposomes and used as a surrogate for soluble drug loading. laser trapping raman spectroscopy (ltrs) was used to optically trap single evs, before and after synthetic manipulation. raman spectral analysis was used to assess trapped eevs compared to pure standards to quantify ratiometric variation in chemical composition. results: raman laser trapping experiments confirmed that each formation method results in largely varying ( ) extent of fusion between evs and synthetic calceinloaded liposomes, ( ) efficiency of calcein loading, and ( ) particle size. we could also quantify the molar amounts of liposome vs. ev molecules for single particles, revealing a great amount of variation from particle to particle. functional membrane proteins we left intact to varying degree across fusion methods. summary/conclusion: given the rising importance of analytical tools able to characterize extent of molecular loading for engineered evs, we believe this technology will be very useful, thus warrants further investigation for eev characterization across a variety of clinical applications. funding: randy carney, phd was supported by a research scholar grant, rsg- - - -cdd, from the american cancer society. extracellular vesicles containing host restrictive factor ifitm inhibited zika virus infection of foetuses in pregnant mice through trans-placenta delivery allen z. wu nanjing university, nanjing, china (people's republic) introduction: zika virus (zikv) infection can lead to neurological complications and foetal defects, and has attracted global public health concerns. effective treatment for zikv infection remains elusive and a preventative vaccine is not available yet. therapeutics for foetus need to overcome blood brain barriers to reach placenta and require higher safety standard. methods: in the present study, we engineered mammalian extracellular vesicles (evs) to deliver a host restrictive factor, interferon-induced transmembrane protein (ifitm ), for the treatment of zikv infection. results: our results demonstrated that the engineered ifitm -containing evs (ifitm -exos) were overall safe to the animals and suppressed zikv viraemia by log s in the pregnant mice. moreover, the engineered evs effectively delivered ifitm protein across placental barrier and suppressed overall zikv viraemia in the foetuses to the basal level with significant reduction of viraemia in key foetal organs as measured by q-pcr. mechanistic study showed that ifitm was delivered to the endosomes/lysosomes where it inhibits viral entry to the host cells. summary/conclusion: our study demonstrates that exosomes can act as a cross placenta drug delivery vehicle to foetus and ifitm , an endogenous restriction factor that is highly expressed in placenta, is a potential treatment for zikv infection during pregnancy. introduction: extracellular vesicles (ev) are natural and abundant nanoparticles capable of transferring complex molecules between neighbouring and distant cell types. translational research efforts have focused on co-opting this communication mechanism to deliver exogenous payloads to treat a variety of diseases. important strategies to maximize the therapeutic potential of evs include payload loading, functionalization of the ev surface with pharmacologically active proteins, and delivery to target cells of interest. methods: through comparative proteomic analysis (lc/ms) of purified evs, we identified several highly enriched and ev-specific proteins, including a transmembrane glycoprotein (ptgfrn) belonging to the immunoglobulin superfamily. leveraging ptgfrn as a scaffold for surface display, we generated evs with functional targeting ligands, including single domain antibodies (sdabs), single chain variable fragments (scfvs), single chain fabs (scfabs), and receptor ligands, on the surface to direct ev uptake to cell types of interest. biological activity of these engineered evs was assessed in an array of in vitro and in vivo assays and compared to untargeted controls. results: we engineered evs displaying anti-clec a scfabs to target conventional type dendritic cells (cdc s), anti-cd scfabs to target t cells, and cd ligand to target b cells. in mice, systemic administration of anti-clec a evs resulted in a % increase in the percentage of cdc cells that take up evs over controls. anti-cd evs resulted in both an increase in the percentage of ev positive t cells ( . and -fold for cd + and cd +) and the number of evs per cell ( and -fold for cd + and cd +) in the blood. furthermore, in primary mouse dendritic cells, anti-clec a evs loaded with sting agonist achieved a fold greater pathway induction compared to untargeted controls. preliminary in vivo data suggest that anti-clec a evs reduce the required sting agonist dose -fold to achieve efficacy and induce anti-tumour responses, compared to control evs. summary/conclusion: these results demonstrate the potential of our ev engineering platform to generate novel ev therapeutics targeted to cell types of interest for pharmacologic payload delivery. a novel method for the delivery of cell-free therapy to foetuses with congenital anomalies: a proof of principle study lina antounians, louise montalva, gabriele raffler, maria sole gaffi and augusto zani the hospital for sick children, toronto, canada introduction: antenatal cell-based therapies are currently considered invasive for the foetus. a promising cell-free strategy that holds great regenerative potential for several organs is the administration of stem cell derived evs, whose cargo contains bioactive molecules that epigenetically regulate target cells. herein, we aimed to ) assess the ability of evs to reach foetal organs when administered to the mother intravenously or intra-amniotically; ) compare these administration routes on normal foetuses and foetuses with a congenital anomaly. methods: evs were isolated from rat amniotic fluid stem cell conditioned medium using ultracentrifugation. evs were assessed for size (nanoparticle tracking analysis), morphology (tem), and expression of cd , hsp , flo- , and tsg (western). we injected rat dams with evs stained by exoglow™-vivo or saline (control) via maternal tail vein (iv) or intra-amniotically (ia) at e . . ia and iv injections were performed on dams carrying normal foetuses or foetuses exposed to nitrofen to induce congenital diaphragmatic hernia. after h, dams and pups were sacrificed. d high-sensitivity optical reconstructions of whole foetuses or micro-dissected foetal organs were imaged using the ivis® spectrum imaging system. ev fluorescence signal was compared between normal (n = ) and nitrofen-exposed (n = ) foetuses. results: both iv and ia injection routes were successful in delivering evs to foetal organs. no fluorescent signal was detected in saline only control. ia injections yielded higher signal than iv, and evs reached more organs with ia than iv injections. ia injected evs were detected in the lungs, gastrointestinal, and urinary tract of normal and nitrofen-exposed foetuses. nitrofen exposed foetuses had higher signal than normal foetuses. summary/conclusion: this proof of concept study shows that antenatal administration of stem cell evs is feasible with different routes. although maternally administered evs cross the placenta, ia injection is more effective at reaching foetal organs. further studies are underway to reproduce these findings in experimental models of various congenital anomalies. funding: cihr-sickkids foundation grant os . introduction: safe, efficient and specific nano-delivery systems are essential to the current cosmetic, nutraceutical and therapeutic medicine sectors. the ability to optimise the bioavailability, stability, and targeted cellular uptake of bioactive molecules while mitigating toxicity, immunogenicity and off-target/side effects is of the utmost priority. ves us is a european project, which aims to develop an innovative platform for the efficient production of extracellular vesicles (evs) from microalgae, which constitute a promising renewable bioresource (www.ves us.eu). here we present characteristics of evs from several microalgal lineages, which offer the opportunity for a potentially developing a new and scalable tailor-made biogenic nanotechnology. methods: we cultivated a number of ev-producing microalgal species and developed protocols for ev isolation both at laboratory (differential ultracentrifugation) and pilot scales (tangential flow filtration). the physico-chemical characterization of microalgal evs was carried out according to the minimal information for studies of extracellular vesicles (misev- guidelines): biochemical methods to verify the presence of specific ev-biomarkers, tuned for microalgal evs; dynamic light scattering (dls) and nanoparticle tracking analysis (nta) to assess the particles number and size distribution; electronic scanning microscopy (sem), atomic force microscopy (afm), and cryo transmission electron microscopy (cryo-tem) for imaging analyses; bilayer-specific fluorescence staining (f-nta) to test the purity of ev preparation. results: we identified microalgae as a novel natural source of evs that could constitute a cost-effective and sustainable way of mass-producing them. we screened strains of microalgae and generated an "ev identity card" for each, which contained a variety of ev features relating to their biophysical, biochemical and biological characteristics in line with the misev- . our approach will next focus on the scalable production, surface functionalization and bio-engineering of selected microalgal evs. at the same time, their bioactivity will be explored using both in vitro and in vivo biological models. summary/conclusion: the ves us consortium is investigating the potential of microalgae as novel ev bioresources. this research will attempt to bioengineer novel naturally-derived nanocarriers, microalgal evs, suitable for the development of future cosmetics, nutraceutical or therapeutic formulations. funding: this project has received funding from the european union's horizon research and innovation programme under grant agreement no . sequence-specific rna trafficking to extracellular vesicles is conserved across cell types several sequences have been identified that act as a zipcode for preferential rna targeting into ev (evtropic) or for retention in parental cells (cell-tropic) . in this work, we aimed to compare the ev-tropic capacity of specific rna sequence motifs in promoting loading into ev, across different cell models representing the main cell types found in the body. methods: immune, epithelial and mesenchymal cell lines were transiently transfected with xenogeneic c. elegans micrornas (mirnas) containing ev-tropic or cell-tropic sequences and grown in culture. ev were isolated from the supernatant by differential (ultra)centrifugation. rna was extracted from both cell pellets and isolated ev fraction, and target mirnas were quantified by digital droplet pcr. distribution of cargo mirna across cells and ev was also analysed for chimeras of ev-and cell-tropic sequences. results: the mirnas containing an ev-tropic sequence were highly enriched on the ev fraction, with - , higher levels than in parental cells. contrarily, cell-tropic mirnas were only - times higher in ev. no significant differences were observed in the ev loading efficiency for the various ev-tropic motifs tested. mutations in the ev-sorting motif resulted in reduced ev loading. ev-tropic sequences consistently promoted mirna loading into ev across all the cell models evaluated, suggesting conserved biological mechanisms. summary/conclusion: we showed that rna loading into ev is dependent on the presence of defined evtropic rna motifs, and that sorting mechanisms are conserved across the major cell types tested. the highest loading efficiencies resulted in . mirna copies per particle on average, suggesting a limited scope for ev-tropic motifs for therapeutic rna loading into ev. funding: as, os and eli are fellows of the astrazeneca postdoc programme. introduction: coordinated activity between pancreatic islet cells is critical for the regulation of glucose homoeostasis. chronic exposure to diabetogenic factors such as pro-inflammatory cytokines, perturb islet cell crosstalk and β-cell function in diabetes. extracellular vesicles (evs) derived from cytokine-exposed β-cells modulate physiological and pathological responses to β-cell stress. however, the mechanisms governing this process remain largely unknown. we set out to test the hypothesis that β-cell failure in diabetes is mediated in part through β-cell autocrine release of pro-inflammatory evs which promote inflammation and inhibit βcell function. methods: pro-inflammatory cytokine-exposed evs (cytoevs) were generated using conditioned media from mouse min β-cell line treated with diabetogenic cytokines (tnfα, il- β, ifnγ, h). evs were also isolated from human type diabetic (t dm) and lean non-diabetics (lnd) plasma. gw (n-smase inhibitor) was used in the presence of cytokines to determine the effect of reduced ev concentrations on the restoration of β-cell function. proteomic and rna-seq analysis was conducted on min β-cell cytoev (vs. control ev) and cytoev treated mouse islets, respectively. results: assessment of ev concentrations from cytoev and human t dm plasma revealed a~twofold increase (p < . , vs. control (ctl) and lnd ev). immunofluorescence staining of cd and cd expression was significantly elevated in human t dm pancreas (p < . , vs. lnd). while acute inhibition of ev formation with gw ( µm) showed significant restoration in β-cell function (glucose stimulated insulin secretion assay, gsis) in cytokine-exposed mouse and human islets (~ and fold vs. cytokines alone, p < . ). moreover, functional assessment of mouse islets exposed to cytoev ( h) resulted in suppression of gsis (~ %, vs. untreated, p < . ). identification of cytoev content through proteomic analysis revealed a significant upregulation of the chemokine, cxcl (~ fold vs. ctlev) and rna-seq analysis of cytoev treated mouse islets depicted a marked upregulation of transcripts associated with cxcl -cxcr signalling (p < . ) and downstream pathways (e.g. nfκb; p = . and jak/stat; p = . ). furthermore, inhibition of cytoev (gw ) with cytokines markedly decreased cxcl (~ %) and cxcr receptor (~ %) expression in min β-cells. summary/conclusion: these data suggests that cytokines elevate cxcl expression in β-cell ev to enhance inflammation-induced diabetes. this is mediated through ev-autocrine release of cxcl consequently activating cxcr signalling and downstream pathways to impair β-cell function in diabetes. synergy between -lipoxygenase and secreted pla promotes inflammation by formation of tlr agonists from extracellular vesicles introduction: damage associated molecular patterns (damps) are endogenous ligands that induce innate immune response, thus promoting sterile inflammation. during oxidative stress, stress-derived evs (stressevs) were found to activate toll-like receptor (tlr ), but the activating ligands were not fully determined. additionally, several enzymes, among them -lipoxygenase ( -lo) and secreted phospholipase a (spla ) are induced during inflammation and were suggested to promote damp formation. methods: stressevs were produced from hek cells exposed to um a and isolated with ultracentrifugation. : lysopi was oxidized for min with -lo. additionally, synevs were prepared from phospholipids (pls), oxidized with -lo and hydrolysed with spla . activity was measured by qpcr and elisa on wt and tlr -ko macrophages. -lo oxidized : lysopi was analysed by mass spectrometry. spla activity was measured in synovial fluid from rheumatoid and gout patients using fluorometric assay. k/bxn serum transfer induced arthritis model on wt and tlr ko mice (c bl/ mice) with spla -iia injection was used (approval no. u - / / by mkgp of slovenia). results: stressevs released after oxidative stress were found to activate tlr with a gene profile different from bacterial lipopolysaccharide (lps). stressevs, -lo oxidized synevs, but only -lo oxidized lysopls activated cytokine expression through tlr /md- . hydroxy, hydroperoxy and keto products of : lysopi oxidation were determined by ms and they activated the same gene pattern as stressevs. furthermore, spla activity, which we detected in the synovial fluid from patients, promoted formation of tlr agonists after -lo oxidation. injection of spla -iia into mice promoted k/bxn serum induced arthritis in tlr -dependent manner. summary/conclusion: both -lo and spla are induced during inflammation, therefore these results imply the role of oxidized lysopls in stressevs in promoting sterile inflammation through tlr signalling. the formation of tlr agonists is enzyme driven so it provides an opportunity for therapy without compromising innate immunity against pathogens. funding: h -msca-itn project tollerant (grant no. ), slovenian research agency (project no. j - to mmk, research core no. p - to rj). monocytes traffic extracellular vesicles to damaged muscle and adopt a novel immunophenotype to support muscle regeneration russell g. rogers, akbarshakh akhmerov, weixin liu, lizbeth sanchez and eduardo marbán smidt heart institute, cedars-sinai medical center, los angeles, usa introduction: extracellular vesicles (evs) are secreted membrane vesicles that carry bioactive molecules such as mirnas, mrnas, proteins, and lipids to modify recipient cell behaviour. we recently demonstrated evs secreted by cardiosphere-derived cells (cdc-evs) augment endogenous muscle regeneration in mdx mice, a model of duchenne muscular dystrophy, when delivered intravenously. in parallel, macrophages preferentially accumulate surrounding small regenerating myofibers in cdc-ev treated mdx muscle. however, it is currently unclear how intravenous cdc-evs home to dystrophic muscle and exert their therapeutic bioactivity. methods: fluorescently-labelled and unlabelled cdc-evs were infused into the contralateral femoral vein of wild-type mice with unilateral muscle injury induced by bacl . injured and uninjured muscles were dissected h following infusion and subjected to optical imaging, immunohistochemistry, and confocal microscopy. this experiment was repeated using clodronate liposomes to deplete endogenous monocytes/macrophages. next, rna-seq was preformed on bone marrow-derived m , m , and cdc-ev (mcdc-ev) polarized macrophages from mdx mice. conditioned media (cm) from these macrophages were tested in an in vitro model of myogenesis. lastly, small rna-seq was performed on evs secreted by m , m , and mcdc-ev macrophages. results: when delivered intravenously, cdc-evs naturally home to injured, but not uninjured, skeletal muscle. cdc-evs were detected in the interstitium adjacent to non-muscle cells, macrophages, and within surviving myofibers. after depletion of monocytes/ macrophages by clodronate liposomes, the presence of cdc-evs in the injured muscle was attenuated. bioinformatic analyses indicate cdc-evs confer a novel immunophenotype to mdx macrophages with features of both m and m . indeed, mcdc-ev cm promotes myoblast proliferation and supports myogenic differentiation. interestingly, mcdc-ev evs have a unique mirna signature and contain several mirnas with known roles in myogenesis. summary/conclusion: these data indicate circulating monocytes traffic cdc-evs to damaged muscle where they adopt a novel immunophenotype to support muscle regeneration. we propose mcdc-ev macrophages mediate their pleiotropic effects via paracrine factors, possibly including evs. introduction: microglia, the immunocompetent cells of the cns, play an important role in maintaining cellular homoeostasis in the cns. these cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. the contribution of microglial-derived extracellular vesicles (m-evs) to the maintenance of cns homoeostasis is unclear. in addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to m-evs is scarce. methods: here, we analysed the effects of m-evs produced in vitro by either tnfα-activated or non-stimulated microglia bv cells. we showed that m-evs are internalized by both mouse bv and human c microglia and that the uptake of m-evs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. consistently, exposure of microglia to m-evs increased the protein expression of the autophagy marker, lc b-ii, and promoted autophagic flux in live cells. to elucidate the biological activities occurring at the transcriptional level in c microglia exposed to m-evs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted rna sequencing. results: inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia exposed to m-evs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. summary/conclusion: we demonstrate that in vitro produced microglial evs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homoeostasis. funding: this work was financed by hasselt university and by efro through the interreg v grensregio vlaanderen nederland project trans tech diagnostics. evaluation of plasma extracellular vesicles as biomarkers for longevity xin zhang a and virginia kraus b a laboratory medicine center, nanfang hospital, southern medical university, guangzhou, guangdong, , p. r. china, guangzhou, china (people's republic); b division of rheumatology, duke molecular physiology institute, duke university school of medicine, durham, usa introduction: extracellular vesicles (evs) have emerged as key indicators and effectors of ageing. although plasma concentrations of evs decline with age, the ev biomarkers associated with ageing and longevity are not fully understood. recently, our group found an age-related decline of plasma evs associated with immune cells during normal human ageing. our study aims to evaluate the association of plasma evs with longevity. methods: plasma samples were selected from the established populations for epidemiologic studies of the elderly study subjects (n = ): half dying within years (short-lived group) and half surviving ≥ years (long-lived group) after the blood draw; all matched for age (median age . ± . years, range - ), gender ( % female), and race ( % white/ % black). the samples were acquired under donor consent and irb approval of duke university. evs were separated from the plasma samples, and profiled based on the surface markers of haematopoietic stem cells (hscs), mesenchymal stem cells, immune cells, skeletal muscles, cardiac muscles and adipocytes (cd , cd , cd , cd , cd , cd , cd , cd , cd , cd , cd , cd a, cd a, cd , cd , hla-abc, hla-g, hla-drdpdq, cd , cd , cd , m cadherin, ryr , ryr , fabp , dlk ). the percentages of evs expressing each tested molecule were determined using a high-resolution multicolour bd lsr fortessa x- flow cytometer as we recently reported. graphpad prism . software was used for statistical analysis. results: we found significantly increased percentages of cd +, hla-abc+, cd + and cd a+ large evs ( - nm) in the long-lived compared to the short-lived group. none of the tested surface marker expressing medium ( - nm) or small (< nm) evs showed differential percentages between the shortand long-lived groups. summary/conclusion: evs carry surface markers from their parent cells. cd is expressed by hscs and immune cells. cd regulates homing of human cord blood cd + hscs, and delivers a potent cd independent costimulatory signal to activate t cells. hla-abc, the key human immunogen, is expressed by nucleated cells and platelets. cd is expressed by hscs, immune cells and epithelial cells, and cd + plasma evs declined with age in healthy people. cd a is expressed by hscs, megakaryocytes and platelets, and is functionally relevant for hsc maintenance and haematopoietic homoeostasis. our preliminary data suggest that hscs and immune cell associated plasma evs (cd +, hla-abc+, cd +, cd a+ large evs) inform on health status related to longevity. introduction: it is anticipated that stem/progenitor cells-derived extracellular vesicles (spc-evs) will rapidly progress towards clinical studies, and the development of reproducible, efficient, scalable and costeffective process for their production is expected to boost the therapeutic applications of evs-based products. in addition, the use of defined serum-/xenogeneic(xeno)-free culture medium formulations could result in substantial improvements for spc-evs production in terms of reproducibility, stability and quality, while ensuring the approval of regulatory agencies. the main goal of this work is to develop a full-controlled manufacturing platform for the spc-evs production. methods: human mesenchymal stromal cells (msc) were expanded in a xeno-free microcarrier-based bioreactor culture system operating in fed-batch feeding mode and after days the conditioned medium was collected. different methods for spc-ev isolation/purification from the msc-derived conditioned medium, including chromatography were compared and the the quality of the final product obtained was characterized by different methods according to misev, including nanoparticle tracking analysis, lipidomics and western blot. moreover fourier-transform infrared (ftir) spectroscopy was evaluated in terms of its implementation as a standard technique for the identification and characterization of evs. results: after days of msc expansion under dynamic conditions, we collected . l of conditioned medium with approximately . million evs/msc. a combination of a pretreatment with a nuclease for the digestion of dna/chromatin with a purification using strong anion exchange chromatography led to the best results so far in terms of evs isolation. of notice, by ftir spectroscopy, it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture conditions tested. summary/conclusion: the platform established herein could be applied to the production of wellcharacterized spc-evs targeting their biomedical use in different settings (e.g. as drug delivery systems), as well as evs from other parental cells lines (i.e. dendritic cells) in therapeutic settings as cancer. ultrasensitive protein detection for quantification of extracellular vesicles in human biofluids enables comparison of isolation techniques dmitry ter-ovanesyan, maia norman, wendy trieu, roey lazarovits, george church and david walt wyss institute, boston, usa introduction: extracellular vesicles (evs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. one of the main challenges in studying evs and using them in diagnostics is a lack of suitable methods to quantify evs that are sensitive enough and can differentiate evs from similarly sized lipoproteins and protein aggregates. we propose using ultrasensitive single molecule array (simoa) assays to quantify evs by immunoisolating and detecting ev transmembrane proteins in microwell arrays. we developed single molecule array (simoa) assays using the quanterix hd-x analyser for the quantification of evs using the tetraspanins cd , cd , and cd . simoa allows for the detection of single proteins using arrays of femtoliter wells, turning elisa into a digital immunoassay. we then used these assays, together with an additional assay for albumin, to compare commonly used ev isolation methods from plasma and cerebrospinal fluid (csf): ultracentrifugation, precipitation (exoquick), and size exclusion chromatography (sec) using the izon qev columns. we further used these assays to rapidly optimize and improve sec by comparing different sec resins and column dimensions in both plasma and csf. results: in comparing our simoa assays to traditional elisa with the same antibodies, we found that the simoa assays were more than times more sensitive, detecting the tetraspanins in samples where the proteins were undetectable by elisa. given the high dynamic range and high-throughput capabilities of simoa, we were able to comprehensively compare relative ev yields and ev purity for different isolation methods of evs from plasma and csf. we provide average tetraspanin and albumin levels to directly compare the methods. we also tested different sec resins and provide data for custom sec columns that outperform izon qev and allow for fine tuning of different ratios of evs to albumin. summary/conclusion: our results highlight the utility of quantifying evs using ultrasensitive simoa assays for tetraspanins. we were able to rapidly simoa to rapidly evaluate different ev isolation methods in csf and plasma. in general, the experimental framework we present could be easily applied to evaluate new ev isolation methods, or applied to any other biological fluid. thus, we think simoa is a powerful new tool for relative ev quantitation. introduction: the protein profile of extracellular vesicle (ev) subpopulations has been shown to contain valuable disease information, notably in cancer. currently, techniques aiming to find ev proteins that associate together mainly focus on transmembrane proteins, while methods that also probe cytosolic proteins generally resort to a combination of affinity capture, elution, and lysis, which limits throughput. to allow the high-throughput analysis of both membrane and cytosolic ev proteins, we optimized a total extracellular vesicle antibody microarray (tevam) incorporating fixation and heat-induced epitope retrieval (hier), then leveraged it to perform combinatorial protein profiling of evs from colorectal cancer (crc) cell lines ht and sw . methods: arrays of iggs targeting surface protein markers were incubated overnight with evs purified from cancer cell line supernatants. hier optimization was carried out through variation of buffer contents, presence or absence of prior permeabilization, as well as incubation time and temperature, for a total of conditions. a evs, previously profiled with other methods, were used as a model during the optimization. cytosolic protein hsp and membrane marker egfr, both with high expression in a evs, were probed and the results used to compare hier conditions. following hier treatment, protein targets were detected through incubation with primary antibodies and fluorescent secondary antibodies or streptavidin. the resulting optimized tevam workflow was used to phenotype ht and sw evs through probing of trios of surface ( ) and internal ( ) protein targets. results: the selected tevam protocol successfully maximized hsp signal while minimally affecting egfr detection, enabling simultaneous analysis of surface and internal proteins. profiles of more than combinations, featuring integrins, claudins, cytokines, and other key actors of cancer-relevant pathways, were obtained for ht and sw evs, revealing coexpression patterns that highlight the biomolecular heterogeneity both within and between crc cell line evs. summary/conclusion: using tevam, intra-and extravesicular proteins can be detected simultaneously in evs immobilized based on surface protein content, yielding extensive combinatorial protein profiles with significance for health and biomarker research. characterization of evs using orthogonal techniques identifies discrete ev populations from a mouse dendritic cell line bryce killingsworth a , timothy traynor b , joshua a. welsh c , aleksandra dakic a , jason savage a , kevin camphausen d , kenneth aldape a and jennifer jones a a laboratory of pathology, national cancer institute, national institutes of health, bethesda, usa; b laboratory of pathology, national cancer institute, national institutes of health, gaithersburg, usa; c laboratory of pathology, national cancer institute, national institute of health, bethesda, usa; d radiation oncology branch, national cancer institute, national institutes of health, bethesda, usa introduction: extracellular vesicles (evs) have the potential to serve as valuable biomarkers for patient response to cancer therapy. however, development of robust ev-based clinical assays relies on knowledge of ev concentration and diameter distribution. many different methods exist to measure the size and concentration of evs, and each method exhibits strengths and limitations. it is important to use orthogonal methods for determination of these important properties of ev preparations. here, we use dendritic cellderived evs to demonstrate that some ev analysis methods can give a biased interpretation of both diameter and concentration. through comparison, we highlight why orthogonal assays are essential in providing measurement reliability. methods: dc . mouse dendritic cells were cultured in flasks containing a total of . l of ev-depleted media ( % fbs, centrifuged hr. x , g.) when cells reached % confluency, conditioned media was collected, depleted of debris with two min. x , g spins, and concentrated down to~ ml using a pall jumbosep kda mwco filter. the ev concentrate was purified from protein using an izon qev- column, with ml fractions collected. the protein content of the ev-containing fractions was analysed by a , pierce bca, and bioanalyzer. the diameter distribution of the evs was determined by nanoparticle tracking analysis (nta), resistive pulse sensing (rps), flow cytometry (fcm), and electron microscopy (em.) concentration was compared using nta, rps, and fcm. evs were further analysed by protein mass spectrometry and rna sequencing. results: we have identified two distinct populations of evs with our dc . preparation, one highly abundant population with a power-law distribution, whose peak diameter is below nm, and a second, less abundant population with a peak diameter at approximately nm. these two distinct populations and their relative concentration were not detectable with all analysis techniques. based on cross-platform measurements, these populations appear to have distinct compositions that warrant further investigation. summary/conclusion: the use of orthogonal methods allowed the detection of two discrete populations of evs which was not possible on some platforms and would have resulted in a biased perspective of the sample composition. this work has highlighted the need for orthogonal measurements to be conducted by pairing techniques that do not have the same biases. introduction: extracellular vesicles (evs) are nanosized vesicles shed by all cells that serve vital roles in cell-to-cell communication. tumour-associated ev subpopulations vary in molecular content (lipids, proteins, nucleic acids, small molecules), enabling minimally invasive spectroscopic analysis for a wide variety of cancers. here, we use surface-enhanced raman spectroscopy (sers) in combination with a novel plasmonic substrate for global chemical composition analysis of cancerous and non-cancerous populations of evs to determine distinguishing surface characteristics. methods: evs were isolated from ovarian cancer (ovca) patient serum samples by differential ultracentrifugation. a new hybrid nanoplasmonic scaffold comprised of a microscale biosilicate diatoms embedded with silver nanoparticles (agnps) was used for sers measurements. the substrate was incubated with cysteamine to positively-charge the agnps (responsible for the sers enhancement) so that evs could attach (evs are naturally anionic). in a typical experiment, μl of~ particles/ml evs per sample were incubated with the porous substrate surface, which was inverted on a glass cover slip for raman interrogation. principle component analysis (pca) was used to compare the spectra and determine distinguishing characteristics between populations from tumour and non-tumour sources. we also trypsinized evs before sers analysis to see the extent of influence the surface molecules play in localizing the evs to the agnp "hot spots." results: a total of clinical samples ( ovca and non-malignant control) were tested in combination with ovca skov- cell line evs. simple pca was able to separate clinical samples according to disease subtype and major peaks were identified to provide chemical content analysis. each sample exhibited inherent heterogeneity but clustered together in a distinguishable way from the others. summary/conclusion: despite innate heterogeneity within single samples (i.e., evs isolated from a single patient sample), evs isolated from clinical samples could be easily distinguished from each other using our hybrid sers substrate, with minimal sample processing, a label-free approach, and only a few microlitres of sample. our study using this novel plasmonic material demonstrates its potential for use as a component in next-generation diagnostic platforms. introduction: single-particle analysis is critical for understanding extracellular vesicle (ev) heterogeneity. yet such techniques remain technically challenging due to low detection sensitivity and presence of variable amounts of "contaminants," including lipoproteins. the high degree of structural similarity between evs and lipoproteins in size, density, and chemical composition, results in their co-isolation using any of the standard ev isolation techniques. here we introduce laser trapping raman spectroscopy (ltrs) as a wellsuited, label-free, and non-destructive tool to distinguish evs from various lipoprotein species at single particle resolution. methods: ev samples were isolated from skov- cell culture supernatant by differential ultracentrifugation and their raman spectra measured. as the most abundant lipoproteins in ev isolations from human biofluids are sub-micron low density lipoprotein (ldl), very low density lipoprotein (vldl), and high density lipoprotein (hdl) particles, these were purchased as pure components and also measured by ltrs. ldl and vldl were then spiked-in to isolated evs to mimic "contaminated" post-isolation ev samples. raman spectra were analysed by principal component analysis (pca) using a custom matlab script. results: ldl and vldl have been observed to adhere to ev surfaces in vitro after standard isolation techniques. we could readily distinguish pure vldl, ldl, and hdl standards according to their raman spectra. pca revealed distinction of skov- evs from both ldl and vldl. pca also differentiated skov- evs incubated with ldl from skov- evs incubated with vldl. extent of ldl and vldl adherence to evs could be observed and quantified. summary/conclusion: through raman and pca, classes of lipoprotein and evs can be identified and quantified when co-incubated. ltrs is a quantitative single-ev analysis technique that can be used to differentiate between lipoprotein classes and evs when incubated together. this technique allows for analysis of evs where standard isolation methods fall short. introduction: extracellular vesicles (evs) are endogenous membrane-derived vesicles that shuttle lipids, proteins or nucleic acids between glia and neurons, thereby promoting neuronal survival and plasticity in the cns and contributing to neurodegenerative conditions. although evs hold great potential as cns theranostic nanocarriers, the specific molecular factors that regulate neuronal ev uptake and release are currently unknown. methods: we used a combination of patch-clamp electrophysiology and ph-sensitive dye imaging to examine stimulus-evoked ev release in individual neurons in real time. results: whereas spontaneous electrical activity and the application of a high-frequency stimulus (hfs) induced a slow and prolonged fusion of multivesicular bodies (mvbs) with the plasma membrane (pm) in a subset of cells, the neurotrophic factor bfgf (basic fibroblast growth factor) greatly increased the rate of stimulusevoked mvb-pm fusion events and, consequently, the abundance of evs in the culture medium. proteomic analysis of neuronal evs demonstrated bfgf to increase the abundance of the v-snare vesicle-associated membrane protein (vamp , cellubrevin) on evs. conversely, knocking-down vamp in cultured neurons attenuated the effect of bfgf on ev release. introduction: heat shock proteins (hsps) function as chaperones under both normal and pathologic conditions. as chaperones they assist in protein folding, in holding protein complexes for current or future activation, and in degradation of senescent proteins for recycling of components and display for immune surveillance. during stressful situations, hsp quantities and/or activities increase as cells and tissues seek protection from insults. these insults can result in the cell surface display of hsps, which can then lead to the surface display of hsps on extracellular vesicles (evs). hsps present on the cell surface or in the extracellular space are regarded as "danger signals" in an ancient biologic paradigm. hsp-accessorized evs may act as "danger boli", carrying not only the hsps, but hundreds of components of the stressed parental cell, capable of prompting differential responses depending on the status of the recipient cell. methods: clarified/filtered plasma from patients suffering from neurologic maladies (cancer, brain injury, multiple sclerosis) was incubated with peptides designed to bind hsps. the evs congeal under these conditions and are pelleted (microfuge) and washed with increasing-stringency buffers. we lysed the evs and subjected them to metabolomic analyses (focused on lipids) or assayed them on phosphokinase arrays. results: we show that evs from the blood of patients suffering from brain tumours, or from tbi, or from ms, possess distinct metabolomes compared to blood evs from healthy donors. we found hundreds of differentially-expressed lipids amongst the patients vs the healthy donors. the levels of annotation and identification for these compounds ranges from level (low, no matches in databases) to level (high, annotation matches to known database components). in addition, we found differences in phosphorylated kinases as cargo in these evs between patients with matched primary vs recurrent gliomas, and among tbi/stroke patients compared to healthy donors. summary/conclusion: hsp-accessorized evs present different metabolomic and phosphokinase content which may serve as biomarkers in a "liquid biopsy" setting, but may also play roles in the pathobiology of neurologic diseases. introduction: methamphetamine (ma) has deleterious effects to both peripheral organs and the central nervous system. the rewarding properties and addictive potential of ma are correlated with increased synaptic dopamine availability following alterations in dopamine and vesicular monoamine transporter function. in rodents, ma alters brain mirna expression and the mirna content of serum extracellular vesicles (ev). here we examined plasma evs isolated from human subjects actively using ma (ma-act) for size, concentration, protein markers, and mirna content. methods: plasma samples from ma-act, and controls (ctl) were obtained from the methamphetamine abuse research center. plasma evs were evaluated by vesicle flow cytometry (vfc) for size, concentration, and surface protein markers. vfc antibodies included markers for a pool of tetraspanins (cd , cd , and cd ), platelet evs (cd ), pro-coagulant evs (annv), and red blood cell evs (cd ). next plasma ev isolated by size exclusion chromatography were analysed by qpcr on taqman® array human microrna a + b card set v . . fold change was calculated by ΔΔcq between ma-act and ctl for mirna expressed in ≥ % of samples in at least group. we identified the top % of ranked mirna by fstatistic; of these, the mirna of interest for ma-act were identified by at least a (i) . fold change in expression, (ii) area under the receiver operating characteristic curve of . , and (iii) glass's Δ of . for mirna of interest correlations to additional ma variables were conducted, along with ingenuity pathway analysis of predicted gene targets. tobacco use was controlled for. results: vfc data show that the size (~ nm) and concentration (~ . x particles/ml) of all plasma evs is comparable between ma-act and ctl groups. in addition, the plasma evs primarily consist of tetraspa-nin+, annv+, or cd + evs, and to a much lesser extent cd + evs. of the mirna expressed in ma-act and/or ctl plasma evs, there were mirna that have at least a . fold increase or decrease in ma-act. mirna were identified to be of interest in ma-act based on fold change, effect size and diagnostic potential, compared to ctl. further, of the mirna correlate with a ma associated variable, including frequency of use and age of first use. together the mirna best cluster subjects based on ma-act status, not tobacco use. finally, the predicted gene targets of the mirna are associated with canonical pathways linked to ma. summary/conclusion: ev mirna expression in ma-act subjects was unique to ctl participants, suggesting that ma may affect ev communication among cells. the differential mirna expression also implicates a role for evs in behavioural and physiological effects specific to ma, and suggests that there may be changes in expression of mirna that are relevant to specific drugs of addiction, as well as to a spectrum of drug-mediated addiction disorders. bone marrow-derived extracellular vesicles may alter the ageing phenotype of murine haematopoietic stem cells. sicheng wen a , jill kreiling b , mark dooner c , elaine papa c , michael del tatto c , yan cheng c , mandy pereira c , peter quesenberry c and laura r. goldberg d in natural ageing of haematopoietic stem cells (hscs) is unclear. we tested the hypothesis that bone marrowderived evs (bm-evs) can modulate the ageing hsc phenotype. methods: we flushed bone marrow from old ( - month old) and young ( - -week old) c /bl mice and collected bm-evs by differential centrifugation ( × g for min, supernatant collected and centrifuged , × g for hour, bm-ev pellet collected and quantified by nanoparticle tracking analysis). we injected old mice with x ^ young bm-evs via tail vein, daily x days. control mice were injected with age-matched bm-evs or vehicle alone. we euthanized the mice one month post-injection, harvested whole bone marrow (wbm) and highly purified hscs (lineage negative/c-kit+/sca- +/cd +; lsk-slam) and tested stem cell function in competitive bone marrow transplants ( - recipients/group). results: at months post-transplant, wbm from old mice exposed to young bm-evs exhibited a statistically significant decrease in engraftment when compared to wbm exposed to age-matched bm-evs (percent average donor chimerism ± sem: % ± % (young evs) vs. % ± % (old evs)). for lsk-slam from old mice, we observed a trend towards decreased engraftment when exposed to young bm-evs and a trend towards increased engraftment potential when exposed to old bm-evs (percent average donor chimerism ± sem: % ± % (young evs), % ± % (old evs), % ± % (vehicle)). these findings are consistent with our previous data showing that, in contrast to highly purified hscs which develop impaired stem cell function with ageing, total un-separated old wbm actually has increased engraftment capacity when compared to young wbm. of note, we found that the classic myeloid skewing by old lsk-slam was partially reversed by exposure to both young and old bm-evs. finally, consistent with the known increase in highly purified hscs with age, our preliminary data showed that old mice exposed to young bm-evs had an approximately -fold decrease in the number of lsk-slam cells in marrow, indicating that bm-evs may influence agerelated changes in hsc number. summary/conclusion: these preliminary data suggest bm-evs may play a role in modulating hsc ageing phenotypes, potentially altering engraftment capacity, lineage fate, and lsk-slam population size. future studies delineating the molecular mechanisms underlying these ev-mediated effects could provide key insights into normal haematopoietic ageing. funding: this work was supported by the nih grants p gm , dk - a and by the savit foundation. oral with poster introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr. results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom . mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the biogenesis of evs is neutral sphingomyelinase (nsmase ), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles. methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-( -( -( , -dimethoxyphenyl)- , -dimethylimidazo [ , -b] pyridazin- -yl) pyrrolidin- -yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated xfad mice with mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay. results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting % of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human post-mortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of mdd subjects and hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed. results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd . tem images showed the typical cup-shaped morphology with sizes mostly between and nm. preliminary sequencing results revealed that mir- a- p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir- a- p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. op . = ps . combining nanomagnetic isolation and artificial intelligence to guide the treatment of traumatic brain injury zijian yang a , kryshawna beard b , david meaney b , danielle sandsmark b , ramon diaz-arrastia a and david issadore c a university of pennsylvania, philadelphia, usa; b university of pennsylvania, philadelphia, usa; c department of bioengineering, university of pennsylvania, philadelphia, usa introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores ( nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the protein biomarkers (tau, uchl- , nfl, gfap, il , il , and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl were elevated in mtbi patients compared to controls (p < . ), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il and tnf-with tau from glur + evs showed % accuracy with % sensitivity and % specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. l cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd , cd and cd ) as well as l cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l cam appears. we also immunocapture l cam from csf and plasma and perform western blots for the internal and external domains of l cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l cam in plasma is likely not coming from the brain. this data call into question the utility of l cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in -month-old wt mice, (n = /groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for % of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy y cells with n-myc amplified sk-n-be cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. op . = ps . introduction: quantification and characterization of single extracellular vesicles (sevs) based on surface markers can aid in dissecting the heterogeneous landscape of ev subpopulations. we and others have demonstrated the potential of imaging flow cytometry (ifc) to perform sev characterization. we recently showed release of protoporphyrin (ppix) positive sevs by -aminolevulinic acid ( -ala) dosed glioma cells, in vitro and in vivo. rickfels et al. also used ifc to demonstrate the enrichment of cd +/cd + evs in the plasma of glioma patients. herein, we performed in vitro studies to characterize ev subfractions using -ala as well as ev and cns specific surface markers. methods: we use ifc to characterize evs released by glioma using -ala, fluorescently labelled ev (cfda-se, cd ) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd ) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that % are tenascin c positive, . % are egfrviii positive and . % are -ala positive. there was only a minor overlap (< %) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs ( . -fold), tumour specific evs ( . -fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: f. graminearum (fgr) and f. oxysporum f. sp. vasinfectum (fov) are severe fungal pathogens of cereals and cotton, respectively. fgr and fov cause economic losses and threaten food and fibre supplies worldwide. understanding host-pathogen interactions is crucial for developing new strategies for disease control. we are determining whether extracellular vesicles (evs) have a role in the interaction between fungal pathogens and their host plant. methods: we isolated evs from fgr and fov by sizeexclusion chromatography and characterized them by nta and tem. evs from fgr and fov are between - nm and have morphology similar to evs reported for other fungi. we performed label-free quantitative proteomics to describe the protein cargo of evs from fgr and fov, including a comparative study of evs from fov grown on different media: czapek dox (cd) and saboraud's dextrose broth (sdb). results: a total of proteins were detected in fgr evs and, according to prediction software effectorp, . % of these were potential effectors. similarly, % of ev proteins do not contain signal peptide indicating that packaging into evs is a novel mechanism of secretion for these proteins. notable fgr ev proteins include lipases, proteases and synthases for toxins and chitin. fov produced evs in similar quantities in both growth media tested, but ev protein cargo differed between them. there was a % overlap in proteins identified in the cd and the sbd ev proteins. in general, ev proteins were involved in metabolism, cell wall architecture and oxidoreduction, with . % and . % of potential effectors, respectively. polyketide and toxin synthases, proteases and effectors were present in both types of fov evs. summary/conclusion: this new fungal ev isolation method was rapid, yielded high-quality evs, and did not submit particles to high centrifugal forces. our data revealed that both fgr and fov produce evs enriched with proteins that could alter host immune responses or facilitate fungal infection. furthermore, the protein composition of fov evs was dependant on culture conditions. this supports a potential role for fungal evs in disease progression in plants and provides the foundations to pursue the role of evs in plant-fungal interactions with the potential to identify new targets for disease control. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll receptor (tlr ) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il- and il- , which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells. objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow fieldflow fractionation (af ) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp- ) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk- epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with to nm (ev ) and another with to nm (ev ). ev induced more tnf-alpha, il- , ip- and ccl than ev . it was also more effective in promoting t. cruzi infection in epithelial cells. summary/conclusion: t. cruzi released two ev populations that affects differently host cells. identification of these evs composition might help to better understand the role of evs in the modulation of t. cruzi infection funding: fapesp, cnpq and capes op . = ps . commensal bacterial extracellular vesicles act as carriers for norovirus sutonuka bhar, melissa jones, annalise galbraith and mariola edelmann university of florida, gainesville, usa introduction: human norovirus (hunov) are one of the most common causes of gastroenteritis and, along with inducing morbidity and mortality by diarrhoea, have a massive economic impact resulting in approximately usd billion each year in healthcare costs and missed worker productivity. development of anti-viral therapies for hunov has been hampered by the lack of robust in vitro cultivation systems. several cell types support viral replication but only produce modest amounts of virus due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays. noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr. isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and . um filtration. co-purification of norovirus with the evs was checked. ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems. detection of bacterial extracellular vesicles in blood from healthy volunteers kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of . µg/ml of plasma, as geometric means ≥ . µg/ml were nearly equal. hevs were detected at µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f x electron microscope and measured between - nm, and hevs were between - nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: new zealand (nz) has a population of just . million people with a remote geographical location in the pacific ocean. its unique culture, food-based industries and ethnic population make nz an invaluable place for extracellular vesicle research into all areas. however, as for many places in the world, standardization of methodologies, training and access to appropriate equipment is challenging. methods: the hub for extracellular vesicle investigations (hevi) is a virtual research centre established in with three-year seed funding from a university of auckland strategic research initiatives fund. two staff members are employed to support training, education and optimization of methods. the hevi is guided by a governance group providing valuable input from australasian experts in evs. results: since the hevi has organized research symposia, hands-on training days, hosted international students as well as providing one-on-one training for individuals from universities and institutes across nz. training is provided on multiple isolation and characterization methods and tailored to individuals access to essential equipment without bias towards individual manufacturers or techniques. travel funding has supported people to attend conferences and workshops for the purposes of education, networking and research dissemination. the hevi also provides support for project design with grants awarded to hevi members and a number of manuscripts in submission for publication. the embo practical course "extracellular vesicles: from biology to biomedical applications" is organized each year by a group of researchers active in the ev field in collaboration with the embl advanced training center in heidelberg. the course focuses on training phd students and postdoctoral researchers who enter or are already active in the field of ev research. given the large number of methods and protocols that is being used by researchers in the ev field, the organizers aim to provide practical guidance to new researchers and teach them appropriate skills. methods: participants obtain theoretical knowledge and hands-on experience on different ev purification and characterization techniques, such as fluorescent labelling, density gradient centrifugation, size exclusion chromatography, electron microscopy, flow cytometry and nanoparticle tracking analysis and on databases like ev-track and funrich. in addition, the organizers and invited lecturers from several different research areas explain which strategies are used to understand the role of ev in biomedical applications and give an overview of the current state of the field of ev research. results: the course therefore covers a broad range of topics important in the ev field, including heterogeneity in ev subpopulations, mechanisms of ev cargo selection, ev biogenesis, pre-analytical variables, therapeutic and diagnostic use of ev, and in vivo functions of ev. group discussions are facilitated and stimulated via assignments to analyse data obtained during the practicals and to critically evaluate literature. participants also have the opportunity to present their own research during poster presentations and ask for feedback from organizers and invited lecturers. summary/conclusion: among the participants selected for the course, a large geographical distribution is reached, including researchers from the newer eu member states and from outside of europe, to ensure a broad geographical distribution of the knowledge gained during this course. introduction: on october , we organized the st lugano exoday, first initiative in the southern switzerland to bring together resident researchers and european experts in the field of extracellular vesicles (evs). the workshop, centred on "technical challenges of extracellular vesicle research" aimed to highlight technical requirements and advances in the evs area, focusing on isolation, characterization and tracking. methods: the workshop started with a lecture by dr. cecilia lässer, from the university of gothenburg. the rest of the workshop was divided in two working groups (wg), each introduced by a keynote lecture followed by presentations by young researchers and a round-table discussion. wg , introduced by dr. mercedes tkach, from the institute curie in paris, focused on recent advances on evs characterization and isolation. wg was centred on evs tracking and introduced by dr. frédérik verweij, from the institute of psychiatry and neuroscience of paris. results: dr. lässer opened the workshop with a comprehensive review and introduced recent developments in the evs field. the first wg discussed different isolation methods, focusing on ultracentrifugation, size exclusion chromatography and immunoprecipitation-based techniques. supported by the keynote speakers, the participants agreed that the best approach to optimize the isolation process consists in the combination of different techniques. wg shared insights about new strategies to visualize and tracking evs, focusing on how to improve the routinely approaches used, defining optimal criteria for evs labelling and imaging. all the participants had an in-depth overview on the requirements and the state-of-the-art techniques currently in use for the isolation, characterization and tracking of evs. summary/conclusion: the transferable knowledge acquired during the workshop ensures participants to remain up-to-date with the advances in the field of evs. as our ultimate goal is to create a competence centre in southern switzerland around the field of evs, the workshop was an invaluable opportunity to intensify collaborations between resident laboratories and broaden the scientific exchange with laboratories of the experts hosted during the event. given the success of this first workshop we are already working to prepare the second edition and make the event a recurring appointment. funding: supported by the swiss national science foundation the role of core facilities and emerging technologies in maximizing rigour and reproducibility of ev quantification and characterization and following misev guidelines rachel derita a and andrew hoffman b a thomas jefferson university, philadelphia, usa; b university of pennsylvania school of veterinary medicine, philadelphia, usa introduction: it remains very clear in the field of extracellular vesicle (ev) research that the rapid rate of increase in publications and expansion of interdisciplinary clinical ev interest has created the need for increased standardization and access to the appropriate technologies to uphold these standards. as the first core facility in the usa with the sole intention of creating a space where users can both isolate and characterize evs, we provide a central location for the facilitation of ev research via access to multiple technologies (both established and emerging) such as resistive pulse sensing, nanoparticle tracking analysis, ultracentrifugation, high-performance liquid chromatography, flow cytometric analysis of evs and additional immune or fluorescence-based ev characteri zation techniques. methods: we surveyed a group of leading scientific investigators and researchers in varying stages of their scientific careers in the mid-atlantic region of the us. the survey data demonstrate applications of greatest current and future interest to be employed in a shared lab resource. results: the current demand is highest for isolation services, ultracentrifugation and nta, with a gradually increasing demand for immunophenotying analyses such as the exoview chip array, fluorescent nta and flow cytometry. we additionally present strategies and data-based examples of how shared resource facilities can facilitate multifactorial and rigorous ev characterization in accordance with misev guidelines, and encourage collaboration among ev researchers. summary/conclusion: in order to answer the larger remaining questions in the ev field such as the isolation of specific ev subsets, ev tracking between cells and the use of evs for biomarker discovery and drug delivery, it is essential that shared resource facilities interact not only with investigators, but with each other to integrate the necessary resources to progress. programme to assess the rigour and reproducibility of extracellular vesicle-derived analytes for cancer detection national cancer institute, rockville, usa introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par - , is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par - / , successfully funded r and r grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, ); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, ). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, ). summary/conclusion: drs. sudhir srivastava and matthew young are the program directors for the par which began accepting applications on january . this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute introduction: early detection of cancer as well as monitoring cancer treatment are important to improve cancer care. diagnostics for cancer are mainly based on tissue biopsies and re-biopsy during treatment is challenging. moreover, current diagnostics are expensive, time-consuming and have low-throughput. therefore, liquid biopsies are expected to bring the next breakthrough in cancer diagnostics. in liquid biopsies tumour-secreted material is isolated from body fluids and subsequent analyses thereof allow for non-invasive diagnostics. one type of tumour-secreted materials are extracellular vesicles (evs), which are schedded from tumour cells. evs are surrounded by a lipid bilayer, whichs composition resembles the plasma membrane of their parental cell. as many tumours are driven by over-expression or upregulation of transmembrane proteins e.g. growth factor receptors, detection of the later on evs holds promise for early tumour detection and treatment monitoring. methods: for the immuno-pcr evs were first affinitycaptured on magnetic beads, allowing immobilization of purified evs as well as evs secreted into cell culture medium or spiked into plasma. afterwards each sample was divided and affibody-dna-conjugates directed against different targets were added. affibodies are small affinity proteins, which often are developed as high affinity binders for tumour imaging, making them suitable probes in the presented assay. after washing, the bead-ev-affibody-dna-complexes were analysed for the immobilized dna-amount via qpcr. results: via the presented immuno-pcr evs secreted from the non-small cell lung cancer cell line h as well as the ovarian cancer cell line skov were analysed. the immuno-pcr method allowed the detection of the tumour-associated membrane receptors epidermal growth factor receptor (egfr), receptor tyrosineprotein kinase erbb /her and insulin-like growth factor receptor (igf r). different levels of membrane receptors depending on the ev source and concentration were detected. summary/conclusion: the presented immuno-pcr showed to be a comparably fast and robust method for detection of tumour-associated membrane receptors on evs derived from cancer cell lines with medium through-put and is currently further developed into a method for liquid biopsy for non-small cell lung cancer patients. introduction: introduction. evs produced by cells can originate from different cellular compartments and evs in complex biofluids may originate from many different cell types. traditional biochemical analysis, which reports on the total composition of all evs in a sample can't adequately resolve this heterogeneity. single vesicle analysis methods can, if they have the necessary specificity, sensitivity and speed. flow cytometry (fc) is capable of rapid and quantitative analysis of individual particles, but conventional fc-based assays lack the specificity and sensitivity to measure individual evs. assays that combine sensitive instruments with ev-selective sample staining can measure individual evs with accuracy and precision. to better understand the nature and origins of ev diversity, we used single vesicle fc (vfc) to quantitatively measure vesicle number, size, and surface cargo expression on individual evs. methods: methods. evs in culture supernatants ( t, a , u , thp- , sh-sy y) were used neat or enriched by standard methods including differential centrifugation or ultrafiltration. evs from platelets (plt) and red blood cells (rbc) were induced by ionophore treatment of washed cells, and measured in diluted supernatant. evs were stained with a membrane-selective dye and fluorescence-labelled antibodies using a commercial vfc assay kit (cellarcus biosciences), measured using a commercial flow cytometer (cytoflexs, beckman coulter), and data analysed using fcs express v (de novo software). vesicle size, fluorescence intensity, and antibody binding were calibrated using appropriate vesicle and beadbased standards and essential controls performed as recommended by the miflowcyt-ev reporting guidelines. results: results. to assess the compositional heterogeneity of evs, we first characterized the expression of tetraspanins (tss; cd , cd , cd , cd , cd , cd , cd ) on evs released from cultured cell line and primary cell cultures. we find quantitative differences in the expression of ts on evs from different cell types that generally reflected the expression on the cell of origin, with most ev types expressing detectable amounts at least one of the common ts molecules (cd , cd or cd ) but generally not all three. in evs from some cell types, ts expression was uniform across the ev population (cd on evs), but evs from other cell types differentially expressed tss, with some evs expressing no detectable ts (rbc evs). intracellular cargo labelled genetically using fluorescent proteins (egfp or mneongreen) or fluorogenic enzyme substrates (cfse) were measured in individual evs and revealed distinctive associations between ev surface and internal cargo. summary/conclusion: conclusions. high resolution measurement of cargo on/in individual evs can help interpret ev heterogeneity in terms of cell of origin, signals carried, and effects on target cells. integrated omics reveal conserved and divergent modulation of cardiovascular disease by tissue-entrapped extracellular vesicles introduction: fewer than % of patients develop both vascular and valvular calcification, implying differential pathogenesis. while circulating extracellular vesicles (evs) act as biomarkers of cardiovascular diseases, tissue-entrapped evs are implicated in early mineralization but their contents and function are unstudied. we developed an innovative method to isolate and study evs from fibrocalcific tissue and investigated entrapped ev cargoes in human cardiovascular diseases. methods: human carotid artery endarterectomies and stenotic aortic valves were obtained from donors under irb-approved informed consent. tissues underwent enzymatic digestion, ultracentrifugation, and a -fraction optiprep density gradient. global proteomics was performed on intact tissue, each optiprep fraction, and ev-enriched pooled fractions; the latter also underwent mirna-seq. fractionated samples were also studied by cd immunogold electron microscopy (tem) and nanoparticle tracking analysis (nta). high confidence mir targets were predicted by targetscan, pathway analyses utilized the biocarta/kegg/reactome databases, and protein-protein interaction networks were built in string. results: vesicle-associated pathways were increased . x (p < . ; / vesicle-related top go terms) in proteins common to intact arteries and valves (n = , ). proteomics found ev markers to be highly enriched in the four least-dense optiprep fractions of arteries and valves, while extracellular matrix and mitochondria were predominant in denser fractions, as confirmed by tem/nta. proteomics and mirna-seq of tissue evs quantified , proteins and mir cargoes linked to , target genes. pathway networks of proteins and mir targets common to artery and valve tissue evs revealed a shared regulation of rho gtpase and mapk intracellular signalling cascades. proteins and mirs were significantly altered between artery and valve evs (q < . ); multi-omics integration found that evs differentially modulated cellular contraction and p mediated transcriptional regulation in vascular and valvular tissue. summary/conclusion: our findings delineate a novel strategy for studying tissue-entrapped ev protein and mir cargoes and identify critical roles that tissue-resident evs play in mediating cardiovascular disease. funding: this study was supported by a research grant from kowa company (ma) and nih grants r hl , r hl and r hl (ea). mir- a regulates exogenous cd expression on proliferation, invasion, migration and angiogenesis of gastric cancer zhengzhou university, zhengzhou, china (people's republic) introduction: to investigate the possible mechanism of mir- a regulating the expression of exosome cd on proliferation, invasion, migration and angiogenesis of gastric cancer, and to study the application value of cd in the early diagnosis and prognosis of gastric cancer. methods: the gastric cancer cell line mgc- was used as the research object. the exosomes were extracted from the culture supernatant of mgc- by exosome extraction kit. the extracted exosomes were identified by transmission electron microscopy and western blotting. the expression of cd in exosomes was detected by elisa. the expression of cd in exosomes and cd in whole blood and serum were detected by western blot. they were randomly divided into blank group (mock) and mir- a lentivirus experimental group (mir- a group). the lentivirus control group (mir- a/con) was transfected into cells. qrt-pcr was used to verify the status of mir- a after transfection; western-blot was used to detect the expression of cd and downstream erk / , akt and m tor proteins; mtt assay, cell colony formation assay, transwell migration assay for cell proliferation, invasion, and migration. a nude mouse xenograft model was constructed to observe the growth of transplanted tumours,microvessel density (mvd) was detected by immunofluorescence, and distant metastasis was recorded. results: the expression of cd in exosomes was detected by elisa and western blot. the expressions of cd , akt, erk / and m tor in mir- a group were significantly lower than those in mir- a/con and mock groups. cd protein is positively correlated with downstream protein levels.the growth rate and cell invasion ability of mir- a group were significantly lower than those of mir- a/con group and mock group. the weight of the nude mice in the mock group and the mir- a/con group decreased, while the weight loss in the mir- a group was not significant. the tumours in the mir- a/con group and the mock group showed invasive growth accompanied by abundant microvessels, while the mir- a group had smaller tumour volume and uniform cell distribution. only a small amount of microangiogenesis was observed, and no obvious necrotic area was observed. summary/conclusion: mir- a affects the proliferation, invasion, migration and angiogenesis of gastric cancer mediated by akt/erk/m tor signalling pathway by regulating the expression of exosome cd . streamlined detection and quantification of plasma extracellular vesicles and their protein cargo by high-performance nanoscale flow cytometry and label-free mass spectrometry introduction: nanoscale flow cytometry (fc) and mass spectrometry (ms) are useful for profiling ev surface proteins and performing bulk ev proteomics, respectively. this study sought to develop pre-analytical and analytical pipelines for ev protein profiling that are applicable to clinical studies. methods: to optimize plasma ev detection and quantification by fc, modifications of instrument settings and serial dilutions of platelet-free plasma (pfp) and antibodies were tested for improved separation of signal from noise and reduction of event coincidence and swarming. the high-performance flow cytometry (hpfc) platform was used to assess the effect of time ( , , , , , , or hrs) between blood draw (into acd, nacit, edta or heparin) and blood processing, on ex-vivo release of evs from blood cells. label-free ms was used to examine the intensity and breadth of identified proteins in plasma evs purified using several density and size separation methods, either manually or automated, along with various buffer conditions. results: ev event aborts were minimized at a pfp dilution, prior to staining, of : and by using a narrow cytometer window extension. target ev signals were distinct from noise and were triton x- labile. the most significant changes in plasma evs were associated with platelet-derived fractions, use of heparin and > -hour delay before blood processing. yet, platelet ev numbers did not significantly change for up to hrs in citrated and edta plasma. higher overall coverage of known ev proteins and a fivefold increase in number of uniquely identified proteins were observed in ms profiling of evs prepared by a combination of ultracentrifugation (uc) and manual size-exclusion chromatography (sec) compared to preparation by fplc on capto core /superose resins. uc/sec was better than direct sec at reducing contamination by excipient plasma proteins. column buffers with trehalose increased ev protein recovery while adding protease inhibitors had minimal effect. summary/conclusion: with our optimized hpfc protocol, we established that blood ev numbers remain stable for up to hrs in acd or edta and that uc+sec with trehalose-containing buffer result in high canonical ev protein recovery. we are applying these workflows to investigate cancer-associated changes in plasma ev protein cargo. the value of exosomes as a potential biomarker for devil facial tumour disease. university of tasmania, hobart, australia introduction: the tasmanian devil (sarcophilus harrisii), the largest living carnivorous marsupial is endangered because of two transmissible cancers: devil facial tumour disease (dftd) one and two. current efforts to manage dftd are hindered by the lack of a preclinical diagnostic test for dftd. detecting dftd infection is only possible once tumours are noticed, too late to stop dftd progression. a preclinal test could tell us about unknown components of dftd pathogenesis, such as latent period and host-tumour dynamics. exosomes are extracellular vesicles released by most types of cells under both physiological and pathological conditions. exosomes have utility as diagnosis and prognosis biomarkers in a range of diseases, including cancers. the aim of this study is to investigate exosomes-based approaches towards a preclinical and progression biomarker for dftd and in tasmanian devils. methods: exosomes were isolated from three different dftd- , dftd- and devil fibroblast cell lines by sizeexclusion chromatography. likewise, exosomes were isolated from plasma of healthy and diseased devils. to determine the size and morphology of exosomes, samples were imaged with transmission electron microscopy. exosomes isolated from cell lines and devil plasma were analysed with mass spectrometry to characterise proteins and determine their differential expression between the cell origins, and healthy and diseased animals. results: this study identified the presence of myelin proteins in exosomes from dftd cells relative to fibroblasts, which are diagnostic of dftd. additionally, we found that exosomes derived from dftd- abundantly express the inhibitory checkpoint molecule cd relative to exosomes from dftd- cells and devil fibroblasts, indicating a potential candidate for a differential diagnosis between tumours. moreover, exosomes from dftd cells present a greater amount of proteins related with metastasis in comparison with fibroblast exosomes, such as integrins. finally, we report the protein expression profile of exosomes from healthy and diseased devils, showing clear differences between them and the presence of immunosuppressive and metastasis proteins in animals in late stages of the disease. summary/conclusion: dftd-exosomes may provide a non-invasive diagnosis tool to detect early stages of dftd in tasmanian devils to facilitate the prevention of the disease. furthermore, dftd-exosomes may have utility as a prognosis biomarker, determining late stages of the disease using a simple a blood test, which would facilitate monitoring of wild populations. this project will provide long-term benefits for the future of the devils and encourage exosome-based solutions for other future wildlife disease outbreaks. introduction: despite the increased understanding of evs, from involvement in disease pathophysiology to therapeutic delivery, improved molecular tools to track biodistribution are largely lacking. current approaches used for ev labelling lacks sensitivity and specificity. here, we have explored bioluminescent labelling of evs to achieve a highly sensitive system for absolute in vivo quantification and tracking of exogenous evs at low cost and in a high throughput manner. methods: ev-producing cells were genetically engineered to express various tetraspanin-luciferase fusion proteins. evs purified by uf-sec from these cells were characterized by nta, multiplex bead-based array, tem and wb, followed by luciferase assay to determine the labelling efficiency. for in vitro applications cell lysate from treated cells or the conditioned medium were subjected to luciferase assay. for in vivo applications two different methodologies were applied to determine biodistribution; either by non-invasive real time in vivo imaging using ivis or by luciferase assay on harvested tissues for absolute quantification of injected evs. results: we initially performed a systematic comparison of five different luciferases for endogenous labelling of evs and identified nanoluc and thermoluc as lead candidates. we applied this technology to monitor in vitro cellular uptake and observed cell type differences in cellular uptake of engineered evs. in addition, we also observed an effect of different culturing conditions on exocytosis kinetics. for in vivo application, we applied the nanoluc labelling strategy to determine the pharmacokinetics and effect of different routes of injection on ev distribution. our results indicated a rapid uptake profile of administered evs in different tissues with liver, spleen, and lungs being the primary recipients. we also observed similar results upon tracking in vivo biodistribution in real time immediately after administration. finally, we show how different subpopulations of evs differ in their in vivo biodistribution. summary/conclusion: overall, nanoluc and thermoluc labelling of evs holds great potential for various in vivo and in vitro applications. in addition, it can enable the simultaneous detection of different subpopulations of evs in vivo, which may aid in our understanding of different sub-populations and their behaviour in vivo. apart from monitoring therapeutic evs, with one simple modification this platform offers great potential for tracking tumour derived evs both in vivo and in vitro and thus could aid in the development of anti-tumour therapies. biofunctional peptide-modified extracellular vesicles for cell targeting, macropinocytosis induction, and effective intracellular delivery ikuhiko nakase department of biological science, graduate school of science, osaka prefecture university, sakai-shi, japan introduction: [introduction] in our research group, developing therapeutic techniques based on extracellular vesicles (exosomes, evs) by effective usage of peptide chemistry to deliver therapeutic/diagnostic molecules into targeted cells has been focused. in this presentation, modification techniques using biofunctional peptides such as arginine-rich cell-penetrating peptides [ ] , artificial coiled-coil peptides with receptor targeting [ ] , and cell-penetrating sc peptides [ ] derived from cationic antimicrobial protein, cap for cancer targeting with macropinocytosis induction, on the ev membranes will be introduced. i will also show effects of lyophilization of the peptidemodified evs on their biological activity [ ] . methods: [methods] cd (ev marker)-gfpfusion protein expressed evs were used for cellular evs uptake assessments. all biofunctional peptides were synthesized by fmoc solid-phase method. results: [results] macropinocytosis with actin reorganization has been shown to be crucial for cellular ev uptake [ ] . we developed the methods for modification of arginine-rich cpps or sc peptides on ev membranes using chemical linker techniques, and for example, arginine-rich cpps modification can induce proteoglycan-clustering (e.g. syndecan- ) and macropinocytosis signal transduction [ ] . the artificial leucine zipper peptide-modified evs recognize the peptide-tagged epidermal growth factor receptor (egfr) on targeted cells, leading to macropinocytotic cellular ev uptake [ ] . in addition, lyophilization is a useful technique for long term storage, however, we found that lyophilization negatively affected biological functions of encapsulated proteins in the evs after their cellular uptake [ ] . summary/conclusion: [conclusion] these techniques and findings will contribute to development for the ev-based intracellular delivery systems. reference: [ ] sci. rep. , ( ), [ ] chem. commun. , ( ), [ ] chrmmedchem. , ( ) [ ] anticancer res. , ( ), [ ] sci. rep. , ( ) os . multi-compartmented microvesicles: novel extracellular secretory organelles that release exosomes and extracellular vesicles introduction: extracellular vesicles (ev) bud from the plasma membrane (pm) as microvesicles (mv) or arise from the fusion of multivesicular bodies (mvb) with the pm to release intralumenal vesicles (ilv) as exosomes. the variety of bioactive molecules carried by ev imparts diverse functionality to ev in intercellular signalling. the biogenesis and extracellular release of these specialized messenger organelles is not well understood. to investigate, we studied endothelial cells that line the inside of blood vessels, known to release ev that support angiogenesis. methods: cultured human umbilical vein endothelial cells (huvec) were examined by thin-section electron microscopy (em), serial sectioning and immunogold labelling to study the structure and composition ev release sites. to obtain optimal views of cellular ultrastructure, cells were preserved by fast-freezing and a freeze-substitution. results: a potential release site was identified in em thin sections as a discrete domain, up to several microns long, on the otherwise smooth huvec pm, where numerous bulbous membrane protrusions with thin necks were clustered. the cytoplasm in these protrusions was enriched with mvb and other vesicles and appeared to be on the verge of pinching off to release multi-compartmented mv (mcmv). consistent with this notion, in the neighbouring extracellular space, a plethora of mcmv of - nm with ultrastructural features matching the bulbous protrusions were observed, supporting the concept that mcmv bud from the release site. serial sections confirmed that these extracellular mcmv were independent of cells and not linked by nanotubes or other processes. remarkably, fusion of mvb with the mcmv membrane was directly observed, presumably caught in the act of releasing ilv (exosomes) from the mcmv. immunogold labelling for ev markers is being used to identify proteins enriched at release sites and on released mcmv. summary/conclusion: in summary, ) mcmv bud from localized sites on the endothelial pm, ) mcmv contain mvb, and ) fusion of mvb to mcmv to release exosomes occurs extracellularly. mcmv can now be evaluated as a potential source of exosome and ev release that occurs after budding from the cell of origin, adding new layers of regulation to when, where and how ev are assembled and released. funding: this work was supported by the division of intramural research of the nih. one size does not fit all: overcoming barriers to successful discovery and scaled manufacturing of therapeutic extracellular vesicles jieun lee a , wei guo b , hal sternberg c , mike west d and dana larocca d a stem cell team, seoul, republic of korea; b university of pennsylvania, philadelphia, usa; c agex therapeutics inc, alameda, usa; d agex therapeutics inc., alameda, usa introduction: extracellular vesicles have tremendous intrinsic therapeutic potential. however, the limited availability of production cell lines presents a barrier to scaled ev production and novel ev discovery. indeed, ev sources have been largely confined to a handful of cell types with the vast majority consisting of msc evs. to overcome this limitation, we developed a diverse library of hundreds of clonally pure and scalable progenitor cell lines that provides an alternative resource for ev drug discovery and production. methods: we harnessed the capacity of human pluripotent stem cells (hpsc) to differentiate into virtually any cell type by subjecting hpsc to a wide variety of media and culture conditions to maximize the diversity of partially differentiated cells. the resulting heterogeneous "candidate cultures" were plated at clonal density and further selected for self renewing and scalable clones. transcriptomic analysis indicated > distinct progenitor lines. cell fate potentials were mapped by screening for cell type specific marker expression in various differentiation conditions. evs were produced using cgmp methods (tff and sec) and characterized by nta, trps, surface marker analysis, rna and protein content. bioactivity assays included proliferation, migration, vascular tube network formation, senolysis, and oxidative stress. results: the progenitor library contained > distinct lines with diverse lineage fates including various types of bone, cartilage, muscle,and fat cells, as well as all blood vessel cell types. the lines displayed much longer replicative lifespans ( - pd) than primary cell lines like msc. clonal purity minimized phenotypic drift resulting in maintenance of cell identity, genome integrity, differentiation capacity and bioactive ev production over extended culture. evs were highly diverse in their rna and protein cargo and bioactivity displaying various degrees of migratory, proliferative, angiogenic and senolytic activity. library screening identified evs with higher angiogenic potency than primary adult stem cell evs. summary/conclusion: we demonstrated scalable and stable production of bioactive evs from a large progenitor cell library. library screening resulted in discovery of novel angiogenic and senolytic evs having diverse rna and protein cargo. we are currently creating a corresponding library of progenitor cell evs to accelerate discovery of novel evs and their production cell lines. funding: the initial establishment of the cell library was funded in part by grants from the california institute of regenerative medicine and national institutes of health. introduction: besides extreme potential in biomedical applications, extracellular vesicles (evs) are also promising candidates to expand biophysical understanding of membrane active biomolecules. their complex bilayer composition allows the better understanding of adsorbed proteins and protein coronas as well, which sets of macromolecules will likely be key for advanced ev targeted delivery. considering cargo, membrane active peptides are interesting as these can be both drugs to be delivered, but can also facilitate cargo insertion through lipid bilayers. however, at present very little is understood regarding interactions between the peptides and the ev lipid bilayer, and between peptides and membrane associated proteins on evs. methods: we have recently demonstrated, that ev membrane adsorbed proteins and their interactions can be studied by techniques such as polarized light spectroscopy, microfluidic resistive pulse sensing measurements and freeze-fraction transmission electron microscopy [ ] . furthermore, initially we studied several peptides with known antimicrobial properties and found that these strongly interact with the ev surface proteins, resulting in efficient removal of some from the lipid bilayer [ ] . results: here we present investigation of further evpeptide interactions also focusing on anticancer peptides, which may be promising drug candidates for targeted delivery. these studies allowed to gain insight to novel functions of several peptides, such as melittin, magainin, buforin, lasioglossin, temporin, but also provide a more detailed understanding on how ev protein coronas, or ev bilayers are affected, to such extent that they cannot exert their potential function as delivery systems. summary/conclusion: the above interactions are expected to be interesting both for applicability, i.e. for selecting suitable compounds for ev processing, and also for curiosity-driven understanding of peptide functions, and ev-biomolecule interactions. based on these we promote that peptide -ev interactions will receive increased focus in ev-engineering. introduction: our late-breaking finding is the identification of a non-coding rna (ncrna) in extracellular vesicles (evs) from neuronal cells that is a natural antisense transcript for the dbh gene and associated with epigenetic changes and gene silencing. dna methylation in neurons is involved in memory and neurological disorders (science ( )). earlier work found that during chronic brain infection with toxoplasma gondii induced a decrease in norepinephrine levels and expression of the host dbh gene; and the decrease is correlated with behaviours linked to noradrenergic signalling (infect immun. ( ); infect immun. ( )). dbh catalyzes the production of norepinephrine from dopamine in noradrenergic neurons. we found that evs from infected cultures suppress transcription of the dbh gene and hypermethylation of the gene in noradrenergic cells in vitro. in this study, we identify a ncrna in the evs from infected neuronal cells. methods: neuronal cells were induced by infection with toxoplasma gondii and evs purified on sucrose gradients. evs were characterised by electron microscopy and used to treat rat and human neuronal cells and expression levels of dbh mrna and nascent dbh gene transcription were measured. induced evs were injected into the locus coeruleus of rats and dbh gene expression was monitored. rna purified from evs was screened for natural antisense transcripts (nats) by strand-specific rt-pcr. results: we found that evs purified from infected neuronal cultures induced transcriptional gene silencing (tgs) and dna methylation of dbh in recipient neuronal cells. the induced evs down-regulated dbh gene expression > -fold and induced dna hypermethylation of the dbh gene. this could be induced in the brains of recipient rats by intracerebral injection of evs. using a panel of strand-specific primers, antisense transcripts for the dbh gene were identified in infected cells. this permitted us to examine the rna in purified evs and identify a lncrna in evs selective for evs from infected cultures. summary/conclusion: this is the first study to find a specific neurotransmitter antisense lncrna in evs associated with transcriptional gene silencing and epigenetic changes in the gene. this represents a different type of neuron-to-neuron signalling than the classic chemical and electrical neurotransmission. the findings will enhance our understanding of neurological disorders (ie. schizophrenia, epilepsy, drug addiction) and how memory works. human cd + t regulatory-derived extracellular vesicles and associated micrornas: role in cell-to-cell communication and involvement in the loss of immune tolerance during multiple sclerosis introduction: an impairment of immune tolerance is a determining factor in multiple sclerosis (ms) and dysregulation of cd + t regulatory (treg) cell function is believed to be a major pathogenic factor. micrornas (mirnas) released by treg cells in association with extracellular vesicles (evs) have been shown to participate in the block of pathological immune responses by inhibiting the growth and cytokine production of cd + t conventional (tconv) cells, but the molecular mechanism is still poorly characterized. aim of the present work was to evaluate whether treg cell-derived ev-associated mirna signature is dysregulated in ms and whether this defect may play a role in the development of autoimmunity. methods: human treg cells isolated from blood of naïve to treatment relapsing-remitting ms patients and healthy controls were in vitro stimulated and released evs were isolated by size exclusion chromatography and characterized by nanoparticle tracking analysis, electron microscopy and flow cytometry. evassociated mirnas were quantified by traditional rt-qpcr and droplet digital pcr for absolute quantification. the actual ev-mediated passage of rna molecules from cell to cell was followed through rnaspecific fluorescent staining and treg-derived ev effect on tconv cell transcriptome was evaluated by rnaseq. results: in healthy conditions, the treatment of tconv cells with treg-derived evs was shown to cause the specific repression of genes involved in the proteasome-dependent proteolytic process, known to be crucial for t cell activation. in ms, treg-derived evs may have lost this capability as a direct consequence of a significantly decreased expression of mir- - p, able to target key factors of the proteasome system. summary/conclusion: our results unveil a novel molecular mechanism for treg-mediated maintenance of self-tolerance based on ev-associated mir- - p and its potential alteration in human autoimmunity. funding: fondazione italiana sclerosi multipla, fism, # /r/ and # /r/ revealing the proteome of brain derived extracellular vesicles isolated from human amyotrophic lateral sclerosis post-mortem tissues. introduction: amyotrophic lateral sclerosis (als) is a neurodegenerative disease characterised by the deposition of misfolded proteins in the motor cortex and motor neurons. although a multitude of als-associated proteins have been identified, few have been associated with extracellular vesicle (ev) trafficking, a form of inter-cellular communication. additionally, the role of evs in als is undetermined, specifically in relation to pathogenic stress granule formation, a response to cellular stress involving aggregation of non-coding rnas and their rna binding proteins. therefore, this study aimed to determine the proteome of brain derived small extracellular vesicles (bdevs) isolated from als subjects and identify novel alsassociated deregulated proteins and their potential contributions to pathogenic pathways in als. methods: bdevs were isolated from human post-mortem als (n = ) and control (n = ) motor cortex brain tissues through an ultracentrifugation protocol (vella et al., ) . following thorough characterisation, bdevs that successfully met the minimum criteria required by the international society for extracellular vesicles were classified as evs. the bdevs subsequently underwent mass spectrometry analysis on the thermo scientific q-exactive hf with ultimate rslcnano. proteins identified to be statistically significant differentially expressed then underwent validation by western blotting. results: a panel of statistically significant differentially packaged proteins were identified in the als bdevs. this included several up-regulated rna binding proteins and a down-regulated cell adhesion molecule; dhx , stau and vcam , respectively. pathway analysis revealed that the bdevs were enriched in proteins associated with stress granule dynamics, exosomal and lysosomal pathways. summary/conclusion: the identification of the rna binding proteins in the als bdevs suggests there may be a relationship between als-associated stress granules and als bdev packaging. the packaging of stress granule associated rna binding proteins into als bdevs may be an attempt by the cells to compensate for lysosomal dysfunction caused by stress granule accumulation, a feature of als. thus, these results highlight a potentially novel role for evs in the pathogenesis of als for long-term cultivation . the whole cultivation process of tissue preparation, cultivation, and cryopreservation has been established using strict serum-free conditions under a good manufacturing practice. long-term-cultivated hmnpcs retained stemness and hmnpcs have excellent differentiation efficiency into dopaminergic neurons. hmnpcs reversed impaired motor function in a rodent model of parkinson's disease (pd). based on the promising results in animal experiments, the clinical trial is under way (nct ). multiple-system atrophy (msa) is one of fatal neurodegenerative diseases with a combination of progressive autonomic nervous system disorders, parkinson's syndrome, and cerebellar pyramid syndrome. there are three types of msa such as msa-a, msa-c, and msa-p. in case of a msa-p type, it is difficult to diagnose due to the similarity of symptoms with parkinson's disease (pd). methods: in vitro and in vivo animal msa model were established and rotational behavioural was performed. npc cells were isolated and cultured based on moon et al. mirna sequencing (bgi) was performed and several bioinformatics analyses were done. results: based on the finding that hmnpcs exhibited therapeutic effects on pd, we hypothesize that hmnpcs will have a therapeutic effect on msa-p, where sympotoms are largely common with pd. as expected, transplanted hmnpcs survived, integrated, and differentiated in to dopamine neurons in the host brain, consequently leading to the functional recovery in the msa-p model. to further investigate the therapeutic key factors of hmnpcs in msa-p, mirna sequencing of the extracellular vesicles (evs) secreted from hmnpcs was performed. we found that mir- a highly expressed in the npc-derived evs is one of key regulators of inflammatory response via nfkb pathway. we further experimentally demonstrated that mir- a had anti-inflammatory effect on cells of msa-p condition such that the level of cx cl expression and its receptor, cx cr were both decreased in the msa-p modelled cells and in severe inflammatory environment in msa brain. summary/conclusion: our study first showed that mir- a in hmnpcs-evs is one of key therapeutic factors for the recovery of brain damage through immuno-modulation in msa-p. introduction: oxidative insults are known to be involved in the pathophysiology of alzheimer's disease (ad). we have previously demonstrated that some blood-based redox-signature were associated to the cognitive scores in mild cognitive impairment patients and in ad (perrotte et al., ) . the aim of this study was ( ) to evidence the presence of some oxidative markers in circulating extracellular vesicles (evs), and ( ) to compare to their plasma levels. methods: plasma samples from healthy, mci and ad patients were from the memory clinic of sherbrooke (québec, canada). ad patients were stratified in three groups (moderate, mild and severe) according to the mmse and moca scores. total plasma extracellular vesicles (pevs) were isolated from plasma with the total exosome isolation reagent (invitrogen™ by life technologies inc.). pevs were then characterized by electronic microscopy, nta, dls and western blot. antioxidants apolipoprotein j, d (apo j, apod), the glyoxalase- and protein carbonyls were determined by western blot. results: in pevs, we found that apo d levels were higher in mci patients but not in ad patients. protein carbonyls levels were higher later, in pevs from moderate and severe ad while apo j levels were not different in pevs from the five groups of patients. in plasma, the pattern of apo j and apo d was different. the levels of apo d was not different in the five groups of patients while apo j levels were elevated in mci and in all ad groups. protein carbonyls were higher earlier from mild ad group, earlier than in pevs. the levels of the detoxifying enzyme glyoxalase- were higher in pevs than in plasma and were significantly decreased in early ad as compared to control subjects and mci summary/conclusion: these results demonstrate a differential regulation of redox homoeostasis in plasma and in pevs from ad patients. funding: acknowledgements: this work was supported by the chaire louise & andré on alzheimer's disease, foundation armand-frappier (cr) and cihr grant (tf). carlos j. nogueras-ortiz a , pavan bhargava b , sol kim b , francheska delgado-peraza a , peter calabresi b and dimitrios kapogiannis a a laboratory of clinical investigation, national institutes of ageing, baltimore, usa; b department of neurology, johns hopkins university school of medicine, baltimore, usa introduction: multiple sclerosis (ms) is a neurological disorder characterized by white matter demyelination and extensive synaptic pathology. recent studies have shown synaptic loss in the grey matter of ms brains in the absence of demyelinating lesions which could account for disease progression independent of demyelinating episodes. opsonization of synapses with complement components is a mechanism by which phagocytic cells normally prune synapses, but, when occurring in excess, it may underlie pathologic synapse loss. we sought to identify blood-borne biomarkers of hypothesized complement-mediated synaptic loss in ms using circulating neuronal-enriched and astrocytic-enriched extracellular vesicles (nevs and aevs). methods: nevs and aevs were immunocaptured in parallel from the plasma of ms patients ( with relapsing remitting, with progressive ms) and healthy controls, targeting the neuronal-specific marker l cam and the astrocyte-specific marker glast, respectively. we measured the protein levels of preand post-synaptic proteins synaptopodin and synaptophysin in nevs using elisas and multiple complement cascade components (c q, c , c b/ic b, c , c , c a, c , factor b, factor h) in aevs using a luminex array. results: synaptopodin and synaptophysin protein levels in nevs of ms patients compared to controls were markedly reduced ( . -fold; p < . for both), whereas multiple complement components in ms aevs were markedly increased (c q: . -fold change; c : . -fold change; c b/ic b: twofold change; c : . -fold change; c a: . -fold change; factor: . -fold change; p < . ); differences were not observed in total circulating evs or neat plasma. strikingly, we found the nev-associated synaptopodin/synaptophysin and the aev-associated complement levels to be negatively correlated in people with ms (synaptopodin vs: c q, r = − . and p < . ; c , r = − . and p < . ; factor h, r = − . and p < . /synaptophysin vs: c q, r = − . and p < . ; c , r = − . and p < . ; factor h, r = − . and p < . ), but not in controls. summary/conclusion: circulating evs provide markers of synaptic loss and complement activation in ms and suggest a link between astrocytic complement production and synaptic decline. funding: this research was supported in part by the intramural research program of the national institute on ageing, national institutes of health. methylglyoxal and glyoxal affect the protein cargoes in neuronal-derived extracellular vesicles introduction: advanced glycation end-products (ages) and their receptor rages are known to be involved in the pathogenesis of alzheimer's disease (ad). methylglyoxal (mg) or glyoxal (go) are the precursors of ages and particularly n-( -carboxymethyl)-l-lysine (cml), the most abundant ages. mg induced tau hyperphosphorylation and causes hippocampal damage and memory impairment in mice. the aim of our study was to analyse the effects of mg and go on the neuroprotective, neurotrophic factors, inflammatory and neurodegenerative markers in the human cell line sk-n-sh and their release into the neuronal derived-evs. methods: briefly, sk-n-sh cells were incubated in fbs free media with mg and go ( . mm) for hours. neuronal derived-evs (nevs) from culture media were isolated as previously described (haddad et al. ). nevs were characterized by electronic microscopy, nta and by western blot. cellular and nevs concentrations of bdnf, prgn, nse, app, mmp , angptl- , lcn , ptx , s b, rage, dj- and alpha synuclein were determined by a luminex assay from r&d systems, inc. aβ - , aβ - , ptau t and total tau levels were measured also with luminex assay from emd millipore corp. results: we found that both ages precursors, at non toxic concentration, reduced the neuronal levels of nse with no effect on bdnf, ptrx- , lcn- , dj- , on neurodegenerative markers and on cml. go decreased the levels of prgn, app, angpl- while the expressions of mmp- and angpl- were, respectively lower and higher in the presence of mg. mg and go greatly reduced the release of lcn- by neuronal cells in nevs. bdnf and prgn in nevs were reduced in the presence of go. both mg and go did not modify the release of nse, app, mmp , agntl- , ptx- , dj- , aβ, ptau and cml in nevs. summary/conclusion: our data demonstrated that mg and go differently affect the content of some protein cargoes in nevs and suggest that targeting mg and go may be a promising therapeutic strategy to prevent neurodegeneration. introduction: peripherally circulating brain-derived extracellular vesicles (evs) and their encapsulated rnas may serve as biomarkers for hiv-associated neurocognitive disorders (hand). however, rates of cigarette smoking are significantly higher in hiv+ individuals than the general population, and smoking can modulate the expression of these markers. to better understand how cigarette smoke might modulate rna expression and ev release, we examined several cnsderived cell lines, representing astrocytes (u mg), microglia (sv ), and oligodendrocytes (hog). methods: cigarette smoke extract (cse) was prepared by bubbling through culture medium using a standardized and published method. all cell types were exposed to either % or % cse for hours. cell viability was assessed by musetm cell analyser, and evs were isolated from culture conditioned media (ccm) by size exclusion chromatography. the void (fractions - ), ev ( - ), and protein ( - ) enriched fractions were pooled and concentrated. evs were characterized by transmission electron microscopy (tem), microfluidic resistive pulse sensing, and western blotting. total rna was isolated from cells and circular rna (circrna) expression was assessed with a circrna microarray. results: in response to cse exposure, cell viability was only slightly reduced for all cell types. tem images validate the presence of vesicles in the ev fractions, and their absence in the void and protein fractions. spectradyne particle counts indicated cse exposure substantially increased the ccm particle count in the ev fraction when compared with control. the presence of expected ev markers (cd , cd , and tsg ) in the ev fractions, and their absence in the void and protein fractions was observed via western blot. intracellular circrna expression was significantly altered in all three cell lines. summary/conclusion: cns cells display physiologic responses to cse that include vesiculation pathways and significant alterations in circrna expression. we are now studying the effects of cse exposure on circrna expression in released evs. funding: this work is supported by da , da , and ai . a method for exosomal rna extraction from paired human brain and blood specimens emily n. moya a , lillian wilkins a , esther cheng a , lisa linares b , brian kopell b , navneet dogra c , bojan losic a and alexander charney a a icahn school of medicine at mount sinai, new york, usa; b mount sinai hospital, new york, usa; c department of genetics and genomic sciences, department of pathology, icahn school of medicine, mount sinai, new york, usa introduction: diagnosis and treatment of neuropsychiatric disorders has made little progress in the last half-century likely in large part due to the absence of a scalable technique to profile the complex biological activity of the brain in a living person. exosomes are nanovesicles - nm in size that mediate intercellular communication and contain proteins, lipids, and nucleic acids. it has been shown that brain derived exosomes can be found in peripheral blood, but determining whether peripheral exosomes truly reflect ongoing brain processes has to date not been possible due to the absence of paired living brain and blood specimens. here, we present a novel method for paired sampling of the dorsolateral prefrontal cortex (dlpfc) and peripheral blood from living human subjects for exosomal rna profiling. methods: informed consent, approved by the irb at the icahn school of medicine at mount sinai, was obtained for patients undergoing deep brain stimulation (dbs). paired brain and blood specimens were collected from patients at two deep brain stimulation (dbs) electrode implantation procedures: left hemisphere followed by right hemisphere (total of samples). we developed protocols to profile rna from exosomes of brain tissue extracellular matrix (ecm) and peripheral blood. exosomes were isolated via our in-house protocol using ultracentrifugation. rna was then extracted from the exosomes using the qiagen mirneasy mini kit protocol. quality control (qc) was performed to determine whether rna obtained was sufficient for next-generation sequencing. results: we demonstrate the safety of a novel procedure to sample the brain in living human subjects. bioanalyzer traces and qc data show a mean total rna of . ng (range . - . ng) and no samples fell below the threshold required for library preparation and sequencing ( pg) determined by inhouse optimization on the smart-seq v ultra low input kit. summary/conclusion: to our knowledge, we have performed the first study to sample pairs of dlpfc and blood from living human subjects for exosomal rna for subsequent next-generation sequencing. ongoing analyses by our group promise to establish peripheral exosomal rna transcripts reflective of brain activity. this non-invasive approach to probing neurobiology in the living human brain may facilitate the development of exosome-based diagnostics for neuropsychiatric disorders. introduction: the relationship between obesity and dementia is complex. while obesity in middle age triples the risk of dementia years later, many patients with alzheimer's disease (ad) are cachectic, and a decline in adiposity portends progression of dementia. this suggests adipose-derived factors are important to nervous system homoeostasis. we previously showed that adipocyte-derived small extracellular vesicles (ad-sevs) induce pathologies critical to developing obesity-related diseases and may provide a mechanistic link between adiposity and dementia. we hypothesized that altered expression of ad-sev micrornas involved in neurodegenerative pathways is associated with more severe cognitive impairment methods: we studied serum and cerebrospinal fluid (csf) from participants with ad and non-ad controls. ad-sevs were isolated from samples by precipitation and immunoselection. ad-sev microrna expression was profiled in both biofluids and compared. results: serum and csf microrna expression correlated strongly (r = . ). in serum, micrornas were differentially expressed by a fold change ≥| . | in the ad and control groups (p ≤ . ) and micrornas were differentially expressed in csf. using ingenuity pathways analysis, we identified mrnas expressed in nervous system tissue that are targeted by the differentially expressed micrornas. the mrnas map to diseases and functions; neuronal cell death, neurodegeneration, and neuronal growth and developmental pathways are highly represented. of the differentially expressed micrornas in serum, were moderately correlated with participants' score on the mini-mental state exam, a test of cognitive function (rs = | . - . |). as validation, rencell cx cortical derived neuronal stem cells had decreased doubling time when exposed to ad-sevs from obese adipose tissue in vitro. summary/conclusion: these findings support our hypothesis that altered expression of circulating ad-sev micrornas are involved in neurodegenerative pathways associated with cognitive impairment. these findings support using serum ad-sevs as a surrogate for csf ad-sevs. functional validation is underway to define the connection between ad-sevs and ad. understanding the link between obesity and ad is crucial as the population ages and the global obesity epidemic grows. funding: supported by uw adrc (nih:p ag ) expression of extracellular vesicles after acute traumatic brain injury: an exploratory flow-cytometry study introduction: coagulation derangements related to disseminated intravascular coagulation (dic) are common after tbi and contribute to secondary neural injury. extracellular vesicles (evs) are released from all cell types, including platelets, endothelium, and lymphocytes, which are responsible for dic. we hypothesized that specialized flow cytometry techniques could identify a unique ev signature of dic in acute tbi. methods: using a modified flow cytometry instrument for detection of small particles, fluorescence panels were created to assess for evs from endothelial cells (cd , cd ), platelets (cd , cd p, cd a, cd b), and erythrocytes (cd ) as well as brain biomarkers (s b, uchl- , gfap, tau and nse) and t-lymphocytes (cd , cd , cd , cd ). samples were prepared in trucount tubes to determine volume and treated with triton to confirm presence of evs. results: / study patients and / controls were male. % of study patients presented with a glasgow coma scale of . in the hypercoagulability panel, of the subsets with statistically significant differential expression, involved s b+ and were elevated in patients. platelet-derived cd a evs and uch-l evs were significantly elevated in controls in ev subsets identified in the brain-specific panel. finally, cd +/ + evs, derived from t-cells and identified in the endothelial/t cell panel, are significantly lower in patients suggesting cns recruitment. summary/conclusion: endothelial and platelet/erythrocyte evs may be elevated early after tbi. s bcarrying evs are significantly elevated in circulation of tbi patients; if reproducible, this signature profile may be informative for diagnosis and risk stratification. further study is warranted to evaluate whether this expression correlates with secondary microvascular brain injury. funding: intramural award from the university of pennsylvania enrichment of mir- a in cns extracellular vesicles following impairment of the blood brain barrier nasser nassiri koopaei a , ekram-ahmed chowdhury b , lais da silva a , jinmai jiang a , behnam noorani b , ulrich bickel b and thomas d. schmittgen a a university of florida, gainesville, usa; b texas tech university, amarilo, usa introduction: extracellular rnas (exrnas) are present in essentially all biofluids and include all types of rna including mirna. to enhance their stability outside of the cell, exrnas are bound within ribonucleoprotein complexes or packaged into extracellular vesicles (evs). the blood brain barrier (bbb) is a dynamic interface between the systemic circulation and the cns and is responsible for maintaining a stable extracellular environment for cns cells. the intent of this study was to determine if evs and their contents are transferred from the peripheral circulation to the cns under conditions of an impaired bbb. methods: the bbb of mice was disrupted by hyperosmolar mannitol injections. to validate that the bbb has been disrupted with mannitol, intravenously-dosed [ c]-sucrose was increased in the forebrain by fold with mannitol compared to sham treated mice. evs were isolated from the forebrain, hindbrain and spinal cord following gentle tissue lysis and differential ultracentrifugation. evs were validated by nta, tem and western blotting. mir- a, a mirna that is highly abundant in erythrocytes, was measured in the evs by qpcr. results: qpcr showed that mir- a in cns tissue evs increased with mannitol treatment in the forebrain, hindbrain and spinal cord by -, . -and twofold respectively. qpcr analysis of mrna from reported mir- a target genes showed reduced target gene expression with mannitol. summary/conclusion: we demonstrate that evs containing mir- a, a highly abundant mirna present within erythrocytes and erythrocyte evs, is enhanced in the cns upon bbb disruption. astrocyte-derived extracellular vesicles in morphine tolerance guoku hu, rong ma, naseer kutchy, yuetong zhao, susmita sil and shilpa buch university of nebraska medical center, omaha, usa introduction: opiates, such as morphine are used extensively in the clinical setting owing to their beneficial effects. paradoxically, however, the prolonged use of morphine often results in the development of tolerance, drug addiction, and ultimately leading to various comorbidities associated with drug abuse. although great efforts have been made, at present there is no treatment. the sonic hedgehog (shh) plays a key role in brain development, and brain cells fine-tuning processes such as their proliferation, patterning, and fate specification recent findings have demonstrated that inhibition of the shh signalling prevents morphine tolerance in rodent models. we thus hypothesize that extracellular vesicles (evs) derived from morphine exposed astrocytes and their cargo such as shh are critical for the development of morphine tolerance. methods: mice were received either saline or chronic morphine injection with escalating doses of morphine for days (subcutaneously; mg/kg, day , mg/kg days - , and mg/kg days [ ] [ ] . the development of tolerance was assessed by measuring the tail-flick latency using tail flick analgesia metre (le , harvard apparatus). evs were isolated using either differential ultracentrifugation from astrocyte conditioned media or gradient ultracentrifugation from brain tissues. western blotting and qpcr were performed to determine the expression/activation of shh signalling pathway components. results: our data showed that the levels of shh protein were upregulated in morphine exposed astrocytederived extracellular vesicles (morphine-adevs). furthermore, shh containing morphine-adevs activated shh signalling in astrocytes. our in vivo study further demonstrated the upregulation of shh, as well as the activation of shh signalling, in astrocytes of morphine-administered mice. summary/conclusion: these findings thus demonstrated an autocrine mechanism for shh pathway activation in astrocytes associated with morphine tolerance. these findings could pave the way for the development of shh signalling pathway targeted strategies in the prevention and treatment for substance use disorders. biophotonics-based platforms for the evaluation of circulating extracellular vesicles as biomarkers of neurodegeneration in alzheimer's disease silvia picciolini a , cristiano carlomagno a , alice gualerzi a , monia cabinio a , francesca baglio a and marzi bedoni b a irccs fondazione don carlo gnocchi, milan, italy; b irccs fondazione don carlo gnocchi, milano, italy introduction: in the search for novel and non-invasive biomarkers of alzheimer's disease (ad), both circulating brain-derived extracellular vesicles (evs) and whole serum represent a valuable integration of the currently used classification system. to face the technological challenge of evs and serum analysis, we propose the use of biophotonics techniques as reliable, sensitive, fast and label free methods, potentially useful in tailoring pharmacological and rehabilitation treatments. methods: circulating evs, isolated by sec, and serum samples were collected from healthy subjects (hc) and ad patients. all subjects were asked to complete montreal cognitive assessment scale and mri examination. surface plasmon resonance (spr) was performed in order to detect evs coming from neurons, astrocytes, oligodendrocytes and microglia and to characterize each of them for the amount of ganglioside m (gm ), aβ and tspo expressed on their surface. serum analysis was performed using a raman microscope through the surface enhanced raman spectroscopy (sers) effect by mixing serum with ag nanoparticles. the pearson's correlation index was used to assess the linear correlation between spri data and clinical, mri data and data obtained from multivariate analysis (mva) of sers spectra. results: the spr analysis of evs showed that the selected bioactive molecules are differently loaded on neural ev populations and that their amount is increased on total evs in ad patients compared to hc. we observed a significant correlation between mva data from sers and the presence of aβ on neuronal and microglial evs and of tspo on neural evs, measured with the spr array. summary/conclusion: thanks to our methodological innovation we have verified the potentiality of evs as ad biomarkers, correlating biophotonics blood-based analysis with clinical data. this platform could provide a powerful tool for the evaluation of ad neurodegeneration. funding: the study was supported by the italian ministry of health (ricerca corrente - to irccs fondazione don carlo gnocchi). raman profiling of extracellular vesicles as new blood-based biomarker for brain disorders: focus on parkinson's disease introduction: extracellular vesicles (evs) play a pivotal role in brain homoeostasis and intercellular communication in both physiological and pathological conditions. in parkinson's disease (pd), evs are key players in the transfer of α-synuclein, with blood evs reported to undergo proteomic modifications. nonetheless, the detection and characterization of the ev cargo is technologically challenging, limiting the use of evs as biomarkers so far. herein, we propose raman spectroscopy for the label-free, bulk characterization of blood evs in pd patients. methods: evs were isolated by sec and ultracentrifugation from the serum of healthy subjects (hc) and pd patients. in all patients, the severity of pd was evaluated with the unified parkinson's disease rating scale (updrs) part iii and with hoehn and yahr scores (hy). after proper ev characterization following misev guidelines, raman analysis was performed. the raman microspectroscope was used with a nm laser in the spectral ranges - cm- and - cm- . data from hc and pd patients were compared by multivariate statistical analysis (pca-lda). results: the raman analysis of evs highlighted differences in the biochemical profile of the two groups, with the main variations in the spectral regions related to proteins, lipids and saccharides. a preliminary estimate of the accuracy of raman profiling of blood evs for pd diagnosis was obtained, demonstrating an accuracy of %. even more interestingly, we demonstrated the correlation between the raman spectra and the clinical scales (updrs and hy) used to stratify pd patients. summary/conclusion: in conclusion, the biochemical signature of blood evs can be detected by raman spectroscopy in pd patients and the ev spectral modifications can be related to their clinical status. these data suggest the possibility to use the raman profile of circulating evs as a biomarker for brain disorders, complementary to other specific molecular markers. funding: the study was supported by the italian ministry of health (ricerca corrente to irccs fondazione don carlo gnocchi) impact of circulating extracellular vesicles on brain functions and behaviours introduction: peripheral immune alterations have been described in psychiatric disorders such as schizophrenia, depression, and autistic spectrum disorders. in addition, behavioural changes have been observed in various immunodeficient animal models. however, the mechanisms by which peripheral immune system influences brain development and function are not well understood. in this study, we explored the mechanisms by which circulating extracellular vesicles (evs) mediate immune-brain communication and influence mouse behaviours. methods: mice deficient for rag or rag gene (rag ko mice) were used as a model to study the effects of loss of adaptive immune cells (t and b cells) on brain cellular phenotypes and behaviours. circulating evs were collected from their sera and analysed by using electron microscopy, nanoparticle tracking assay, and western blotting. brain cellular phenotypes were assessed by immunofluorescent staining and gene expression analysis. behavioural phenotypes of rag ko and wt mice were examined in social interaction test. in vivo transfer of evs was performed to see its effects on behavioural alterations of rag ko mice. results: rag ko mice displayed social behavioural deficits, accompanying by enhance c-fos immunoreactivity and altered microglia morphology in the medial prefrontal cortex (mpfc). circulating evs were also affected in these mice and lacked the expression of markers for t cells. a set of micrornas (mirnas) in circulating evs were diminished in rag ko mice. in vivo transfer of circulating evs rescues the social behavioural deficits of rag ko mice and ameliorate the c-fos immunoreactivities in mpfc of rag ko mice. summary/conclusion: our data showed that circulating ev profiles were altered in mice lacking adaptive immune cells and, accordingly, showing social behavioural deficits. notably, our in vivo experiments suggest that circulating evs may contribute to social behaviours. further study will provide a novel biological insight into the mechanisms underlying peripheral-to-brain immune communication via evs. introduction: the involvement of neuroinflammation on ageing process is widely recognized. extracellular vesicles (evs), such as exosomes, are able to cross the blood-brain barrier and were related to neuroinflammation. in this context, evs have been considered a potential mechanism of spreading molecules, including micrornas (mirnas) that can promote mrna degradation or inhibit translation of their targets. our aim was to investigate the mirna profile of circulating total evs during ageing process and their impact on canonical pathways. methods: the local ethics committee (comissão de Ética no uso de animais -ufrgs; n ) approved all animal procedures and experimental conditions. plasma was obtained from wistar rats ( and months-old) and total evs were isolated. ev microrna isolation and microarray expression analysis was performed to determine the predicted regulation of targeted mrnas. results: the analysis of global microrna expression revealed differentially expressed micrornas (p < . ; fold change of ≥ | . |); mirnas were up-regulated and were down-regulated in circulating total evs from aged animals compared to youngadult ones. a conservative filter was applied on ingenuity pathway analysis (ipa) and only experimentally validated and highly conserved predicted mrna targets were used. ipa showed that neuroinflammation signalling is ranked among the top canonical pathway impacted by differentially expressed micrornas and is upregulated in aged animals (p < . ; z-score: . ). the differentially expressed mirnas impacted molecules in the neuroinflammation pathway. interestingly, the ion channel grin b is predicted to be up regulated and is a target of many evs mirnas; in accordance with our results grin b was previously related to neurodegenerative diseases. moreover, let- a- p is predicted to be downregulated and target all the molecules of the neuroinflammation signalling pathway. previous studies have correlated let- a- p and neurodegenerative diseases. summary/conclusion: our data suggest that circulating total evs cargo, specifically mirnas, are altered by ageing and impact neuroinflammation pathway, suggesting the involvement evs mirna on ageinginduced susceptibility of neurodegenerative diseases. introduction: bidirectional cell-cell communication via paracrine mechanisms is critical for wound healing. a new paradigm involving exosome-borne distinctive repertoire of cargo such as mirnas has emerged as a predominant mechanism of cellular communication at the site of injury. unlike other shedding vesicles of similar size, exosomes selectively package mirna by sumoylation of heterogeneous nuclear ribonucleoprotein (hnrnp). methods: keratinocyte-derived exosomes (exoκ) were genetically labelled with fluorescent reporter (gfp) using tissue nanotransfection. purified, gfp-labelled exoκ were isolated from dorsal murine skin and wound-edge tissue by differential ultracentrifugation followed by affinity selection using magnetic beads. distributions of intact exosome were analysed using a prototype jarrold-geometry charge-detection mass spectrometer to directly measure differences in particle mass and charge distributions. complementary ms and ion mobility spectrometry (ims)-ms experiments have been used to characterize surface glycans and glycopeptides. to selectively inhibit mirna packaging within the exoκ in vivo, ph-responsive targeted sirna functionalized lipid nanocarriers (tlnκ) were designed using materials that have prior history of fda approval for human use. results: an increase in mass/charge ratio with glycan binding sites on the surface of wound-edge exoκ were observed compared to dorsal skin exoκ. wound-edge exoκ were selectively taken up by the macrophages in the granulation tissue (n = ). keratinocyte targeting sirnahnrnp functionalized lipid nanocarriers (tlnκ) were designed with encapsulation efficiency of . %. application of tlnκ encapsulating sirna of hnrnp (tlnκ/si-hnrnp) to murine dorsal woundedge significantly inhibited the expression of hnrnp by % in epidermis compared to control (tlnκ/sicontrol)(n = ). moreover, mice treated with tlnκ/si-hnrnp showed impaired barrier function, with significant presence of macrophage in granulation tissue at day , suggesting impaired conversion of macrophage in the granulation tissue. summary/conclusion: this work provides a novel insight wherein exosomes of keratinocyte lineage are recognized as a major contributor that directs macrophage conversion in granulation tissue for wound healing. multifaced effects of milk-exosome (mi-exo) as modulator of scar-free wound healing gna ahn, hyo-won yoon, yang-hoon kim and ji-young ahn chungbuk national university, cheong-ju, republic of korea introduction: recently, milk exosome (mi-exo) has been focused particularly on the possibility of oral distribution for therapeutic agents. however, studies related to the cosmeceutical effects associated with mi-exo are fairly limited. the purpose of this study is to suggest the anti-oxidant and antiinflammatory effect of mi-exo and possibility that can be induced by scar free healing by micro rna in mi-exo. methods: the characteristics of the extracted mi-exo were verified by size measurement, morphological characteristics through cryo-em and western blot. for antioxidant experiments, an abts assay was performed. next, mrna expression through four major cytokines (tnfα, il- , cox- , inos) was used to evaluate anti-inflammatory effects. finally, cell migration assay was performed to confirm the effect of scar-free healing and the detection of mir- b in mi-exo and vegf mrna expression confirmed. results: mi-exo using % acetic acid extraction showed the highest yield. the average size of the exosomes is approximately nm, confirmed the typical double membrane vesicle. as a result of antioxidant experiments, it was confirmed that the treatment of exosomes of ^ particles showed about % antioxidant activity. when ^ particles were treated, rna expression of cytokines showed about times more inhibitory effect than control. elisa test results also confirmed that the concentration-dependent decrease. the activation of the raw cell less proceeded as the treated mi-exo increased. the cell scratch assay cells did not close the cells as the number of milk exosomes increased (wound closing % of ^ particle = . %). and mir- b in milk exosomes was detected at ct value = . summary/conclusion: the antioxidant and antiinflammatory effects of mi-exo showed the greatest efficacy when ^ particles were treated. in addition, it induced to scar free healing rather than wound healing. mi-exo has great potential as a superior natural material in the future cosmeceutical field. extracellular vesicles in human milk expose tissue factor and promote coagulation introduction: tissue factor (tf), a transmembrane protein, initiates coagulation by binding and activating coagulation factor vii (fvii). tf is associated with extracellular vesicles (evs) in saliva and urine, but it is unknown whether also human milk (hm) contains evs exposing coagulant tf. methods: hm was collected from six healthy nursing women with informed consent. evs were isolated by ultracentrifugation and size exclusion chromatography (sec). the presence of tf antigen exposing evs was studied by western blot, flow cytometry, cryo-electron microscopy (cryo-em), and surface plasmon resonance imaging (spri). the ability of tf exposing evs to trigger coagulation was investigated with a plasma fibrin generation test (fgt), performed in the absence or presence of antibodies against tf or fvii(a). results: addition of hm to plasma shortened the plasma clotting time, even when hm was highly diluted. after ultracentrifugation of hm, both tf antigen and tf activity were detected in the ev-containing pellet. after sec, tf antigen and tf activity were present in the ev-containing fractions and . the presence of tf-exposing evs in these sec fractions was confirmed by western blot (cd , cd and tf), flow cytometry, spri, and fgt. in addition, the presence of evs in hm was confirmed by cryo-em. scalable isolation of evs from different probiotic strains with potential as cosmetic ingredients laura soriano-romaní, joaquin espí and begoña ruiz ainia, paterna, spain introduction: extracellular vesicles (evs) are increasing their application in a number of fields. recently, it has been shown that skin health may be affected not only by commensal skin bacteria, but also by the evs that they secrete. however, because most of the efforts have been directed to the characterization and evaluation of evs, the scaling up of the production process remains a bottleneck at the industrial level. in this work, the goal was to evaluate the potential applications of evs produced by different probiotic strains commonly used in the cosmetic field, considering the economic and technical viability of the process. methods: to meet our goal, a standardized workflow was defined to isolate evs from probiotic strains such as lactobacillus and bifidobacterium species, that have demonstrated cutaneous immuno-regulatory effects. the different bacterial strains were produced under standard culture conditions. to isolate the secreted bacterial evs, different chromatographic techniques were performed starting from clarified growth medium. then, evs were evaluated in vitro for a number of biological effects related with skin health. results: the ev yields obtained after downstream processing were calculated for each strain and isolation technique by means of nanoparticle tracking analysis (nta) and total protein content. moreover, evs were visualized by electron microscopy. the in vitro evaluation of isolated evs was based on changes in the expression of five biomarkers related with anti-ageing, anti-inflammatory and whitening effects using distinct skin cell types to identify possible cosmetic claims that could be associated to each probiotic source. summary/conclusion: the potential of evs obtained from probiotic strains as cosmetic active cell-free ingredients was preliminarily assessed with this work, where the process yield and cosmetic function were evaluated. however, additional experiments will be needed in order to increase and optimize the productivity of each step of the ev manufacturing process. acerola derived exosome-like nanovesicles enhances the repair of ultraviolet b-induced dna damage in cultured skin fibroblasts tomohiro umezu, masakatsu takanashi, yoshiki murakami and masahiko kuroda tokyo medical university, shinjyuku, japan introduction: acerola (melpighia emarginata dc.) is a fruit is known to contain not only high amounts of ascorbic acid but also various nutritional components such as carotenoids and polyphenols. previous reports showed the acerola juices are able to confer protection against ultraviolet radiation b (uvb), to improve barrier function of skin. uvb is the main cause of dna damage in epidermal cells, generating several types of pro-mutagenic lesions, like cyclobutene prymidine dimers (cpds) and prymidine ( - ) prymidinone photoproducts ( - pps): if not repaired, this dna damage leads to skin cancer. in this study, we investigated the biological property of the acerola derived exosome-like nanovesicles (adens), aiming to clarify the involvement of adens in repair of uv-induced dna damage. methods: normal human dermal fibroblasts (nhdfs) were purchased from lonza inc. the exosome-like nanovesicles were isolated from acerola juices using exoeasy maxi kit (qiagen). the morphology and size distribution of adens were checked by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta, nanosight lm , malvern). nhdfs were exposed to uvb ( mj/cm ) with pre-or post-adens. effect of uvb was assessed by examining cell viability, cell morphology, and dna damage levels through biochemical assays, microscopy and protein expression studies. results: purified adens were compatible with nta or tem for assessing the nanovesicle size range and concentration ( - nm). when nhdfs were added with adens and incubated at °c for h, there was no effect of adens on cell proliferation of nhdfs. we found that adens treatment to uvb exposed nhdfs significantly reduced cpds and - pps dna adduct formation. present results showed that aden treatment prevented uvb induced dna damage in nhdfs. summary/conclusion: we confirm that adens have the effect of repairing dna damage caused by uvb. these results provide that adens can be a new source to protect human skin from uv-induced skin cancer. introduction: introduction: despite the development of a variety of therapies, complex wounds resulting from disease, surgical intervention, or trauma remain a major source of morbidity. extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) have been shown to improve wound healing, especially via enhanced wound angiogenesis. however, despite their clearly established potential, evs have limitations that may limit clinical relevancy, such as low potency. hypothesis: increased expression of pro-angiogenic lncrna hotair within msc evs enhances their proangiogenic effects and thus their wound healing properties. methods: methods: hotair was overexpressed in human dermal microvascular endothelial cells (hdmecs) to determine any molecular or functional pro-angiogenic effects. anti-angiogenic mirnas and angiogenic mrna levels were quantified by rt-qpcr. effects of hotair on proliferation of hdmecs was also determined. hotair was then loaded into msc evs by delivering a cmv-based hotair plasmid to mscs for endogenous loading via a concentration gradient. evs were collected by differential centrifugation. hotair content within evs was confirmed by gel electrophoresis and rt-qpcr. effects on migration of hdmecs by hotair-loaded msc evs were determined using a scratch assay. results: results: overexpression of hotair decreased mir- c and mir- , while increasing vegf and hif- a. hdmec proliferation was also increased in hdmecs overexpressing hotair (p < . ). hotair was visually confirmed in hotair-loaded msc evs by gel electrophoresis, but was undetectable in unmodified msc evs. rt-qpcr confirmed a -fold increase of hotair compared to control msc evs. hdmecs showed a more statistically significant rate of gap closure when treated with hotair-loaded evs (p < . ) than compared to control msc evs (p < . ). summary/conclusion: summary: loading lncrna hotair into msc evs is achievable by a concentration gradient-dependent method and offers potential to enhance the angiogenic properties of msc evs. nanomaterial labelling of exosomes for cell biology introduction: exosomes are vesicles secreted by many, if not all, cell types and have been known about for decades. among larger micro vesicles that are produced directly from the cell membrane, the small ( - nm), exosomes are similar in size to a virus surrounded by a lipid bilayer. we and others have demonstrated that exosomes contain proteins, lipids, rna, and dna, making them promising materials for diagnosing and treating diseases, including many cancers such as brain cancer. in addition, exosomes from neurons and glial cells represent a novel type of intercellular communication. however, their size makes them hard to track with traditional fluorescence microscopy. to address this, we developed photothermal microscopy (ptm), which uses gold nanomaterial labelling to track exosomes' interaction with and effect on cells/tissue. methods: exosomes secreted by tumour cells and general exosomes found in the blood were isolated using differential ultracentrifugation or a commercially available kit (invitrogen). next, the exosomes were characterized by (tem), (nta), and western blotting to determine shape, size, morphology and the protein profile in the exosomal membrane. after characterization, the exosomes were labelled with gold nanoparticles via sonication. next, the samples were washed, and the exosomes were labelled with fluorescence dye to stain the membrane. after staining and labelling, the exosomes were added to u cells in culture and incubated for h. they were then fixed by % paraformldehyde and imaged by ptm. results: ptm found that exosome-cell interactions are exosome-type dependent, as u cells took up exosomes from other u cells but not human serum exosomes. this suggests that exosome uptake is a selective process and depends on the source of the donor cells. summary/conclusion: exosomes can be labelled with gold nanoparticles via sonication then successfully tracked by ptm to study the effect of exosome source on exosome-cell interactions and communication. cells incubated with u exosomes took the vesicles up rapidly, while cells incubated with serum exosomes had little uptake. ptm will help us design selective exosome-based strategies to treat different conditions, including brain cancer and cns damage. funding: nsf epscor riii award . loading of goat´s whey extracellular vesicles with spiked microrna and curcumin as an strategy for developing new nanocarriers for acellular therapies introduction: extracellular vesicles (ev) are involved in cell signalling and are present in a variety of cell secretions such as milk, from which enormous amount of ev can be purified, thus milk is an attractive raw material for scaling up ev production for therapeutic, cosmetic or other uses. here we isolated evs from the whey fraction of goat´s milk and demonstrated that such evs can be loaded with molecules like polyphenols and mirna. methods: to achieve this, milk was collected from lactating goats and fractionated by acidification and centrifugation into whey and caseins. evs were purified from the former fraction by serial centrifugation and precipitation with commercial kit (total isolation/ thermo fisher) and characterized by electron transmission microscopy (tem), western blot to identify surface markers and measurement of size through nanotracking analysis. once isolated, evs were loaded with different concentration of a spiked synthetic mir or with the polyphenol curcumin. mirna or curcumin were co-incubated over night with evs at oc, precipitated and purified as described above, with an additional washing and precipitation for curcumin. concentration of mirna uploaded by evs was quantified using mir specific qpcr. curcumin was measured using a spectrophotometer at nm. results: evs isolated from whey had an average size of nm, were positive for hsp , cd and alix. in tem, evs were identified with their natural conformation and corresponding size to exosomes. qpcr showed a significant difference of expression of mir in relation to control (loaded with shame) and the negative control (p < . ). curcumin presence was also confirmed after washing and precipitacion. summary/conclusion: in conclusion, milk evs and exosomes can be loaded with mirna and a polyphenol and can be used as alternative nanocarrier for acellular therapies. introduction: extracellular vesicles (evs) are cellderived lipid membrane nanoparticles that serve as messengers of intercellular communication, transferring bioactive molecules to recipient cells. evs have a natural therapeutic potential with high flexibility and biosafety for employing natural and synthetic biomolecules as therapeutic delivery vehicles. considering the importance of evs, their isolation methods are still a bottleneck. to get insights into the tissue-specific cargo in vivo for complete exploitation of evs as therapeutic, biomarker and diagnostic tools, ev purification methods are critical. the aim of the study was brought about to develop an efficient ev purification method both in vitro and in vivo and to further investigate function of evs in cellular senescence. methods: to isolate tissue-specific evs in vivo we developed recombinant evs by genetically fusing snorkel-tag to the cd . the snorkel-tag enables on-column protease treatment for purifying evs which does not rely on traditional immunoaffinity purification protocols using low ph or high salts solutions. results: we systematically evaluated the purification of evs harbouring snorkel-tag by employing different methodologies. our findings suggest that evs harbouring snorkel-tag indeed can be purified at high purity without altering ev biophysical properties. furthermore, we expressed cd -snorkel-tag under p ink a promoter and were able to purify evs derived from senescent cells. summary/conclusion: finally, we are developing an in vivo model with cd -snorkel-tag under p ink a promoter. this will provide us detail insights into the ev cargo secreted from senescent derived cells, by purifying evs harbouring snorkel-tag under pathophysiological conditions, allowing us to develop biomarkers and therapeutic tools. summarized, we have here developed novel tool for studying content and function of evs in the context of ageing and disease. this tool will now pave the way for studying the molecular mechanisms underlying these ev functions in vivo. funding: this work was funded by the austrian science fund phd program biotopebiomolecular technolgy of proteins (w ). engineering exosomes with gata- jie xu, christian paul, yi-gang wang and meifeng xu university of cincinnati, cincinnati, usa introduction: exosomes, are small vesicles ( - nm) secreted from cells that can transport and deliver of their components such as lipids, proteins, dna, mrna, and mirna to target cells. gata- , a cardiac transcription factor, has been shown to regulate differentiation, proliferation, and survival of a wide range of cell types. delivering gata- protein into ischaemic tissues may be one of the most straightforward approaches to improve cardiac function following myocardial infarction. here, exosomes were engineered with gata- by infusing gata- with exosome targeting peptide. methods: the open reading frame of mouse gata- cdna was ligated to xpack lentivirus vector (xpack-gata- ) and plvx-ef -ires-pouro lentivirus vector (plvx-gata- ), respectively. hek cells were transduced by lentivirus, then exosomes were isolated from conditioned medium of hek cells using ultracentrifugation. exosomes were identified using transmission electronic microscope (tem), and the expression of gata- was semi-quantified using western blot. the internalization of exosomes was tracked via treating bend cells with exosomes pre-labelled with pkh . introduction: chinese hamster ovary (cho) cells have dominated as the mammalian cell host for the manufacture of humanized biologics, in part owing to their genomic plasticity and robust growth in suspension culture. there is great interest surrounding the use of extracellular vesicles (evs) as novel therapeutics owing to their capacity to deliver bioactive molecules. however, much remains unknown about the mechanisms involved in ev cargo loading, limiting their development as novel biologics. to this end, we have engineered cho cells to stably express constructs enabling loading of gfp into evs. methods: tetraspanins are established markers of ev identity. accordingly, cd was selected as a tethering point to generate evs with gfp cargo and constructs were generated via golden gate assembly. cho cells were stably transfected by electroporation and expression was verified with fluorescence microscopy and western blotting. growth in batch culture was monitored to establish maximum viable cell densities for ev harvest and recovered evs were characterized by nanoparticle tracking analysis (nta). finally, uptake of gfp-evs was studied using time-lapse fluorescence imaging in co-culture experiments. results: strong localization of cd -gfp was observed at the cell membrane and blotting confirmed intact tetraspanin fusion present at the expected molecular weight. additionally, cells were confirmed to retain high gfp expression post-cryopreservation. stable cell pools were able to reach viable densities greater than million cells/ml in batch culture and nta allowed for detection of gfp cargo even prior to ev isolation. evmediated transfer of functional gfp to recipient cells was found to occur over a period of hours. introduction: extracellular vesicles (evs) are considered promising for therapeutic applications. evs resemble the cell membrane, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. despite the promising potential of evs for therapeutic applications, robust manufacturing processes that would increase the scalability and consistency of ev production are still lacking. methods: in this work, evs were produced by mesenchymal stromal cells (msc), isolated from different human tissue sources (bone marrow, umbilical cord matrix and adipose tissue). msc were selected as these cells allow for a scalable production of evs, while displaying low immunogenicity. a vertical-wheel™ bioreactor system was implemented for the production of msc-derived evs and compared with traditional static systems. the obtained ev products were characterized by nanoparticle tracking analysis, atomic force microscopy, zeta potential and western blot. results: the bioreactor system allowed to obtain evs at higher concentration and productivity, as well as more homogeneous size distribution profiles, when compared to traditional static culture systems. functional studies were performed using breast cancer and lung cancer cell lines. proliferation assays allowed to determine the dose-response profiles of these cell lines when exposed to msc-derived evs. a bell-shaped profile was observed for most cases, since raising the ev concentration lead to increased cell proliferation until a certain point ( - µg/ml), after which cell proliferation was attenuated with increasing ev concentrations. summary/conclusion: the bioreactor culture system allowed a substantial improvement in the production of msc-derived evs, while the obtained dose-response profiles will be valuable to determine the most appropriate ev concentrations for anticancer drug delivery. overall, we demonstrate that this culture system is able to robustly manufacture human msc-derived evs in a scalable manner towards the development of novel therapeutic products such as anticancer drug delivery systems. biodistribution and cellular location of inhaled exosomes and liposomes in the lung introduction: increasing evidence reveals the potential role of extracellular vesicles, such as exosomes and liposomes, in lung regenerative medicine for the treatment of lung diseases. encapsulation and delivery of potential rna and microrna targets into liposomes and exosomes are attractive drug delivery methods, but remain difficult to deliver to the pulmonary parenchyma to reach target lung cell types. here, we demonstrate effective delivery and cellular uptake of exosomes and liposomes to the pulmonary parenchyma via inhalation treatment in a murine model of idiopathic pulmonary fibrosis. methods: human lung stem cells (lscs) were generated and expanded from healthy whole lung donors. lsc-exosomes were purified via ultrafiltration and diilabelled using vybrant☐ labelling solution according to the manufacturer's instructions. dsred-labelled liposomes were generated using lipofectamine™ rnaimax transfection reagent and block-it™ alexa fluor™ red fluorescent control according to the manufacturer's instructions. lsc-exosomes and liposomes were delivered via nebulization to cd mice with bleomycin-induced pulmonary fibrosis. exosome and liposome delivery and biodistribution were visualized -and -hours post-treatment through histological analysis. the study was approved by the institutional animal care and use committee of north carolina state university and complied with all national and state ethical standards. results: exosome and liposome delivery to the pulmonary parenchyma was confirmed by the presence of dii and dsred fluorescence in lung histological sections that penetrated the mucus-lined respiratory epithelium. more exosomes and liposomes surpass mucus-lined surfaces -hours post-treatment compared to -hours post-treatment. fluorescent colocalization of exosomes and liposomes with alveolar type i cells, alveolar type ii cells, basal lung cells, and cd + macrophages was observed through immunohistochemistry analysis. more exosomes and liposomes colocalize with these cell types -hours post-treatment compared to -hours post-treatment. summary/conclusion: lsc-exosomes and liposomes penetrate the mucus-lined respiratory epithelium and reach the pulmonary parenchyma through inhalation treatment. lsc-exosomes and liposomes are uptaken by alveolar epithelial cells, basal cells, and interstitial macrophages with improved biodistribution -hours post-treatment. funding: this study was supported by the nc state chancellor's innovation fund. transfection reagent artefact accounts for some reports of extracellular vesicle function codiak biosciences, cambridge, usa introduction: extracellular vesicle (ev) functions are frequently investigated by transiently transfecting cells with plasmid dna to produce evs modified with protein(s) or nucleic acid(s) of interest. however, evs and the dna-complexes used to transduce cells are physically similar, raising the possibility that they may co-purify during differential ultracentrifugation, the most common ev isolation procedure. activities attributed to evs may therefore be due to contaminating dna -transfection reagent complex. methods: ev producing cells were transiently transfected with plasmid dna encoding gene-editing or split enzymes fused to ev-targeting protein sequences. differential and density gradient ultracentrifugation were used to purify evs from cell culture supernatant or dna lipoplexes from cell-free culture media. protein expression and localization to evs was confirmed by western blot. cell lines stably expressing fluorescent or luminescent reporters were used to assess functional enzyme delivery in recipient reporter cells. results: reporter cells treated with ultracentrifuge pellet material (ucp) from media of transiently transfected cells showed robust and reproducible signal, however fractionating the ucp with an iodixanol density gradient revealed that reporter activity was associated with high-density fractions that were depleted in evs. ucp isolated from identical transfection conditions, but lacking cells (and exosomes), showed identical biological activity levels and distribution in iodixanol gradients, suggesting that the activity was due to contaminating transfection reagent complexes and not evs. serial media changes on ev producing cells post-transfection did not significantly reduce ucp activity on reporter cells. treatment with nucleases did not digest complexed dna, did not significantly reduce dna levels in the ucp as measured by qpcr, and did not decrease activity in reporter cells treated with ucp from either transfected cells or no-cell controls. summary/conclusion: we find that dna-transfection reagent complexes are not separated from evs using differential ultracentrifugation and that common approaches to remove such complexes, including media exchanges and nuclease treatment, are ineffective. due to the pernicious nature of the dna-complex in these cellular assays, it is likely that some reports of ev function are likely artefacts produced by contaminating dna-complexes. we find that density gradient centrifugation can effectively separate evs and dnacomplexes, highlighting the importance of validating elimination of contaminating transfection reagent complexes when using transient transfection to interrogate ev function. chair: suresh mathivanan -la trobe university cancer stem cell-derived exosomes: potential biomarkers for early diagnosis and prognosis in pancreatic cancer introduction: pancreatic cancer (paca) is the most deadly manlignancy, due to late daignosis and early metastatic spread, which prohibits surgery. it is urgently for relaible, early detection. research shows that tumour-derived exosomes, which had been present in the blood in the early stage of tumour formation and before metastasis, is the vanguard forces of tumour formation and metastasis; cancer stem cell-derived exosomes (csc-exos) has stronger migration ability, so the detection of blood csc-exos for early diagnosis and monitoring of progress for paca has great research potential and the value of application. methods: protein markers were selected according to expression in exosomes of paca cell line culture supernatants, but not healthy donors' serum-exosomes. according to these preselections, serum-exosomes were tested by flow cytometry for the pancreatic cancer stem cell marker cd v and tspan . results: the majority ( %) of patients with paca and patients with nonpa-malignancies reacted with anti-cd v and anti-tspan . serum-exosomes of healthy donors' and patients with non-malignant diseases were not reactive. recovery was tumour grading and staging independent including early stages. introduction: chronic traumatic encephalopathy (cte) is a tauopathy that affects individuals with a history of mild repetitive brain injury frequently seen in contact sports. initial neuropathologic change of cte include perivascular deposition of phosphorylated tau (p-tau) in cortical neurons and, in later stages, the formation of neurofibrillary tangles in neurons throughout the brain. extracellular vesicles (ev) are known to carry neuropathogenic molecules in neurodegenerative disease and able to cross the blood brain barrier. we therefore examined the protein composition of ev separated from cerebrospinal fluid (csf) and plasma in former national football league (nfl) players with cognitive dysfunction, and an agematched control group with no history of contact sports. methods: evs were separated from csf and plasma from former nfl players (n = , ) and controls (n = , ) by affinity separation method or size exclusion chromatography, respectively. the ev protein profiling was characterized by simoa for tau and ptau and mass spectrometry. the protein data was analysed for ev enrichment, differentially expressed proteins, pathway analysis and correlation with cognitive function, head impact and tau/p-tau levels by biostatistics and bioinformatics. results: the level of total tau and p-tau in csf evs was not significantly changed, but significantly elevated in plasma evs from former nfl players. the proteins were commonly identified between the paired plasma-csf from the same patients, but there was no significant correlation with disease status. collagen alpha- (vi) chain (col a ), − (vi) chain (col a ) and reelin (reln) were differentially expressed in former nfl players' plasma evs. a combination of these proteins in plasma ev can distinguish former nfl players from controls with % accuracy by machine learning. summary/conclusion: the interacting plasma-csf ev proteomes provide an original resource to ev biomarker development for neurodegenerative disease, and col a , reln and col a in plasma evs can be potential biomarker for monitoring the cte development. density-based fractionation of urine to unravel the proteome landscape of extracellular vesicles in prostate cancer introduction: current diagnostic tests are unable to discriminate indolent from aggressive prostate cancer (pca), leading to overdiagnosis and overtreatment, and an intense interest in biomarkers to improve clinical decision making. urine is considered an ideal proximal fluid for biomarker identification in pca due to its direct contact with the urogenital system. the discovery and translation of extracellular vesicle (ev) content into pca biomarkers remains challenging due to the difficulty of obtaining urinary ev (uev) with high specificity. methods: we developed a step-by-step protocol to separate uev by orthogonal implementation of ultrafiltration and bottom-up density gradient centrifugation (bu-odg). we implemented complementary particle and protein measurements to identify uev (lower density) and protein rich fractions (higher density) and assess the performance of bu-odg (specificity, efficiency and reproducibility). using mass spectrometry-based proteomics we interrogated uev and protein rich fractions from matched urine and radical prostatectomy tissue samples from pca patients (n = ), and urine from men with pca prior to (n = ) and after local treatment (n = ), benign prostatic hyperplasia (n = ) and other urological cancers (n = ). results: bu-odg separated uev from soluble proteins and tamm-horsfall protein (thp) complexes with high specificity and reproducibility, outperforming differential ultracentrifugation, exoquick and size-exclusion chromatography. comparison of the uev proteome from men with benign or malignant prostate disease, allowed us to expand the known human uev proteome and identify a pca specific uev proteome not uncovered by the analysis of the protein rich fraction. proteomic analysis of ev separated from prostate tumour interstitial fluid and matched uev confirmed pca specificity of the uev proteome. analysis of the uev proteome from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uev reflecting their respective cancer tissues of origin. summary/conclusion: we identified hundreds of previously undetected proteins in uev of pca patients and developed a powerful toolbox to map uev and protein rich fractions, ultimately supporting biomarker discovery for urological cancers. immunoglobulin a coating of faeces-derived bacterial vesicles as a marker of inflammatory bowel disease in humans nader kameli a , frank stassen b , heike becker c , john penders c , daisy jonkers d and paul savelkoul b introduction: iga is the most abundant antibody in mucosal secretions and plays a crucial role in maintaining the balance between the host and the gastrointestinal microbiome. recent studies suggested that pronounced iga coating is especially prominent among inflammatory commensals which drive intestinal disease. membrane vesicles (mvs, nano-sized particles released by bacteria) have also been found to interact with the host and modulate development and function of the immune system. however, their interaction with iga has not been studied yet. here we developed a method to isolate and characterize the mvs from faecal samples and checked for possible differences in iga coating patterns of mvs in health and disease. methods: mvs were isolated by using a combination of ultrafiltration and size exclusion chromatography from faecal samples of healthy controls (hc), patients with active crohn disease (cd) and cd patients in a remissive state. quantification and verification have been done with tunable resistive pulse sensing (trpsbased analysis) bead-based flow-cytometer (bbfc) and transmission electron microscope (tem). mvs were selected with specific antibodies for capturing (gram +: lta, gram-: ompa) followed by pe-conjugated anti-human iga antibodies as detection. results: we could successfully isolate * - * particles/ml from mg of faeces. bbfc in combination with trps provide a valuable method for (semi-)quantitative measurements of mixed populations. intriguingly, remarkable differences were found between iga coating mvs derived from healthy controls and active and remissive cd patients as mvs derived from healthy controls were significantly more coated compare to both cd patient groups. in details, for selected g-ve derived mvs: % of the total population of mvs derived from hc were coated, % from remissive cd patients, and < % of active cd patients; and for selected g+ ve derived mvs: % of the total population of mvs derived from hc were coated, % from remissive cd patients, and % of active cd patients. (data are represented as the mean). summary/conclusion: here we demonstrate for the first time that mv isolated from the faecal samples are also coated with iga, and surprisingly mvs from healthy volunteers were more densely coated than mvs from diseased patients. the possible consequence of this difference remains to be determined in future studies. monitoring altered tetraspanin and psma expression in prostate cancer derived extracellular vesicles via advanced image flow cytometry (isx) lukas w. prause a , christopher millan b , natalie hensky c , tullio sulser c and daniel eberli c a universityhospital zurich, zurich, switzerland; b university of zurich hospital, schlieren, switzerland; c university of zurich hospital, zurich, switzerland introduction: new diagnostic and therapeutic options for patients with prostate cancer are urgently needed. prostate-specific membrane antigen (psma)-based imaging and therapy are increasingly used for prostate cancer management. unfortunately, as a membrane protein, psma is not found as a soluble protein in the blood and therefore has limited utility as a diagnostic biomarker. however, psma has reportedly been observed as a cargo protein of prostate cancer-derived extracellular vesicles (evs). we demonstrate altered psma expression on evs derived from prostate cancer cell cultures (c - , lncap) in response to novel next-generation androgen receptor inhibitor (enzalutamide), a standard chemotherapy agent (docetaxel), a novel experimental nonsteroidal antiandrogen (epi- ) that binds covalently to the n-terminal domain of the androgen receptor and dihydrotestosterone (dht). additionally, evs were isolated from the plasma of prostate cancer patients who participated in the prococ biobank campaign at the usz. plasma was taken and stored from patients both pre-and post-prostatectomy. results: transmission electron microscopy, nanoparticle tracking analysis and simple western (wes) analysis show stable size distribution and amount of evs produced by treated and non-treated cells. using advanced image-based flow cytometry, altered tatraspanin and psma expression could be detected in evs isolated from cell culture supernatants of lncap and c - prostate cancer cells following their treatment. summary/conclusion: measuring psma expression on extracellular vesicles might pave the way to use image flow cytometry of evs to develop a blood based diagnostic test for prostate cancer patients with a wide range of possible applications including: ) monitoring response to therapy and, ) early indications of potential relapse. funding: vontobel fondation. proteomic profiling of human neural cells derived extracellular vesicles to identify human brain cell-type specific markers introduction: alzheimer's disease (ad) is a common neurodegenerative brain disease which affects appropriately million patients worldwide. one of the major challenges in ad is to develop reliable biomarkers for early diagnosis and disease-modifying therapies, especially before the clinical symptoms. extracellular vesicles (evs) carry cargos of proteins, lipids and nucleic acids. there was no comprehensive characterization of evs isolated from specific brain cell types, which may be useful for cell type-specific biomarkers. the purpose of this study is to isolate evs from human induced pluripotent stem cell (ipsc)derived brain cells for proteomic profiling and characterization of cell type-specific molecules. methods: human ipscs-derived neurons, microglia and primary cultured astrocytes were differentiated in ev-depleted media. the evs were isolated by differential centrifugation combined with size exclusion chromatography, followed by characterization using nanoparticle tracking analysis and mass spectrometry. the proteomic data were subjected to bioinformatics analysis results: we identified proteins from neuronderived ev (nde), proteins from microgliaderived ev (mde) and proteins from astrocytederived ev (ade) by proteomics. gene ontology analysis indicated that most of these proteins are associated with evs. furthermore, , and proteins are present individually in ndes, mdes and ades. among them, high levels of atp a and syt in ndes, itgam and cd a in mdes, and eaat and gfap in ades were found, all of which are typically and highly expressed in the original cells. summary/conclusion: our results provide us the potential candidates for cell-type specific ev markers, which will be helpful to develop non-invasive tools to enrich ev originating from specific brain cells and may lead to the development of new biomarkers for neurodegenerative disorders. ) are a tremendous resource for extracellular vesicle (ev) research, but they are heavily focussed on mammalian evs, i.e. evs from humans and laboratory animals, where protein cargoes are well characterised, and a wide selection of antibodies are commercially available. protein markers can be used to identify and define the types of mammalian ev and to determine the presence of any contaminants that might confound functional studies. similar resources are not as readily available for bacterial evs as these are not as well characterised, commercially available antibodies are much less abundant and immunological variation between different bacterial species (and there are trillion bacterial species on planet earth!) means that each species, strain, or group of related species may require different antibodies. methods: to identify quality markers for bacterial evs, we have characterised the proteome of cells, crude evs (ultracentrifuge pellet from cell free culture supernatant) and size exclusion chromatography or density gradient centrifugation purified evs from two different (pathogenic vs probiotic) strains of escherichia coli grown under two different environmental conditions, and one strain of mycobacterium marinum grown in one medium. results: our results identify a selection of proteins enriched in purified ev preparations, and proteins that are depleted after purification steps. summary/conclusion: our results allow the identification of potential markers for ev purity and non-ev contaminants, but also highlight the variability in bacterial ev preparations and suggest potential targets that can be used to investigate the heterogeneity of bacterial ev populations. introduction: recent findings indicate an increase in mid-life mortality rates in the usa and persistent, significant race-related health disparities exemplified by differential mortality rates. this suggests that exploring new molecular markers that may be linked to mortality could provide novel insights into factors that are driving mortality rates. accumulating data suggests that extracellular vesicles (evs) circulating in blood may be potential biomarkers of age-related disease. evs are nano-sized membranous vesicles that bear molecular cargo and mediate intercellular communication between different cells and tissues. little is known about whether ev characteristics differ by race or whether evs are associated with clinically relevant mortality risk factors. methods: in this cross-sectional study, plasma evs were isolated from middle-aged african american (aa) and white males and females. results: we report no significant differences in ev size or concentration with race or sex. there were significantly higher ev levels of phospho-p , total p , cleaved caspase , erk / and phospho-akt in whites compared to aas. higher ev levels of phospho-igf- r were found in females compared to males. we examined ev characteristics and protein cargo in the context of well-established clinical mortality risk factors. ev concentration was significantly, and positively, associated with several mortality markers including, high-sensitivity c-reactive protein (hscrp), homoeostatic model assessment of insulin resistance (homa-ir), alkaline phosphatase, pulse pressure, body mass index, and waist circumference. the relationship of ev concentration and cargo with mortality markers differs by race. summary/conclusion: our data show that ev-associated proteins can differ by race and sex and are associated with mortality risk factors. this study provides insight into the characterization of evs in middle-aged aas and whites, which may aid in the development of ev-based diagnostics. funding: this study was supported by the national institute on ageing intramural research program of the national institutes of health. repurposing specialised cell-free dna blood collection tubes for extracellular vesicle isolation introduction: liquid biopsies offer a minimally invasive approach to patient disease diagnosis and monitoring. however, many plasma processing protocols have been designed with a single biomarker in mind. here we investigate whether specialised dna blood stabiliser tubes could be repurposed for the analysis of extracellular vesicles (evs). methods: peripheral blood (n = ) was collected into k -edta, roche or streck cell-free dna (cfdna) blood collection tubes and processed using sequential centrifugation immediately or after storage for days. microev were collected from platelet poor plasma by , g centrifugation and nanoevs isolated using size exclusion chromatography. particle size and counts were assessed by nanoparticle tracking analysis, protein by bca assay and dot blotting for blood cell surface proteins. results: major variations in micro and nanoevs were seen with delayed time to processing. nanoev counts did not change with processing delay or tube collection type but the associated protein amount increased, indicative of cell lysis or activation. the protein was predominantly derived from from platelets (cd ) and red blood cells (cd a). the increase in associated protein was seen more in the k -edta and streck tubes indicating that the roche tubes may offer improved cell stability. conversely, microevs increased in both quantity and protein content with delay to processing indicative of both lysis and cell activation, irrespective of tube type. epithelial cell surface marker epcam abundance remained the same across conditions in both micro and nanoevs demonstrating that epcam+ evs were stable. summary/conclusion: specialised cfdna collection tubes can be repurposed for micro and nanoev analysis, however simple counting or using protein quantity as a surrogate of ev number may be confounded by pre-analytical processing. the evs would be suitable for disease selective ev subtype analysis if the molecular target of interest is not present in blood cells. introduction: nutrigenomics and nutrigenetics have been defined as the effect of nutrients on gene expression and genetic variation on dietary response, respectively. here, we propose the isolation and characterization of exosomes from donors carrying different alleles of hla-dqa and hla-dqb , to investigate their involvement in coeliac disease (cd) management. methods: a chilean population (n = ) was investigated for snps mutations in hla class ii alleles associated to cd predisposition (as well as other mutations related to other food intolerances), using the genochip food technology. exosomes have been isolated from donors' serum by ultracentrifugation and characterized by sds-page, western blotting (cd and cd ), and transmission electron microscopy. exosomes were also studied for their interleukins (il- and il- ra) content. results: among the studied population, % present at least one of the alleles leading to cd development and % carry alleles encoding for αand β-chains heterodimers associated with very high risk to develop cd. in parallel, isolated exosomes from donors with low to extremely high risk for cd showed high il- ra content ( . ± . to . ± . ), as the persons were not following any treatment. however, values of il- ra decrease in exosomes isolated form persons receiving treatment for cd. a relationship between exosomes' content and genetic susceptibility for cd has been observed, which may suggest their possible use as biomarkers for cd as the diagnostic of this disease is still a big issue. summary/conclusion: until this point of this underway project, we demonstrate the existence of a relationship between the exosomes' content in il- ra and genetic susceptibility for cd. furthermore, the genetic predisposition to cd could also modulate the gut colonization process, another important player in intestinal homoeostasis. in the next step, extracellular vesicles from gut microbiota will be isolated and analysed to determine their role in cd management. nasibeh karimi a , razieh dalir fardouei a , jan lötvall a and cecilia lässer b a krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, göteborg, sweden; b krefting research centre, institute of medicine, sahlgrenska academy at university of gothenburg, gothenburg, sweden introduction: the ability to isolate extracellular vesicles (evs) from blood is vital in the development of evs as disease biomarkers. both serum and plasma can be used but few studies have compared them in terms of amount and type of evs. we have previously developed a method to isolate evs from plasma with minimal contamination of lipoprotein particles (karimi et al ) . the aim of this study was to compare the presence of different subpopulations of evs in plasma and serum. methods: blood was collected from healthy subjects, from which plasma and serum were isolated. evs were isolated using a combination of density cushion and size exclusion chromatography (sec) (protocol ) or a combination of density cushion and density gradient (protocol ) or immune-capturing (anti-cd , anti-cd and anti-cd beads) (protocol ). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, electron microscopy (em), exoview, flow cytometry and mass spectrometry (lc-ms/ms). results: as determined by nta and protein measurement more evs could be isolated from plasma with protocol and the majority of the vesicles were cd / cd a positive as determined with exoview and western blot. additionally, flow cytometry and western blot showed that more cd /cd a positive evs where also identified with protocol . furthermore, western blot showed increased amount of cd a in plasma samples in protocol . when labelled evs were spiked in freshly collected blood, no difference in recovery was seen for plasma and serum. summary/conclusion: this study shows that a larger amount of evs could be isolated from plasma compared to serum when three different isolation methods were used. firstly, this suggests that more evs are present in plasma. secondly, it suggests that these vesicles are probably released by platelets and that evs are not trapped in the clot during serum formation. future studies are needed to answer how this affects the use of blood-derived evs as biomarkers from serum and plasma. tumour-derived extracellular vesicles contain distinct integrin proteins stephanie n. hurwitz a and david g. meckes b a university of pennsylvania, philadelphia, usa; b florida state university, tallahassee, usa introduction: cargo profiling, including proteomic analyses, of tumour cell-derived extracellular vesicles (evs) may provide ripe opportunities for further understanding cancer growth, drug resistance, and metastatic behaviour. accumulating data suggest that cancer-derived evs contain membrane-bound integrin proteins which may aid in cell detachment, migration, and homing to future metastatic niches. we have previously published an extensive proteomic profile of secreted vesicles from the nci- panel of human cancer cells. methods: here, we further examine the distinct integrin components in these cancer-derived evs, and additionally profile evs released from benign epithelial cells by liquid chromatography and tandem mass spectrometry for comparison. results: we demonstrate the enrichment of integrin receptors in cancer evs compared to vesicles secreted from benign epithelial cells. total ev integrin levels, including the quantity of integrins α , αv, and β correlate with tumour stage across a variety of epithelial cancer cells. in particular, integrin α also largely reflects breast and ovarian progenitor cell expression, highlighting the utility of this integrin protein as a potential circulating biomarker of certain primary tumours. other integrins including α , αl, and β are enriched in vesicles derived from leukaemia cells, and may provide a means to distinguish haematopoietic cell-derived evs. summary/conclusion: this study provides preliminary evidence of the value of vesicle-associated integrin proteins in detecting the presence of cancer cells and prediction of tumour stage. differential expression and selective packaging of integrins into evs may contribute to further understanding the development and progression of tumour growth and metastasis across a variety of cancer types. effect of nicotine and menthol on cytochrome p and antioxidant enzymes in rat plasma-derived extracellular vesicles introduction: tobacco products such as e-cigarettes pose potential adverse health effects caused by direct exposure to aerosolized nicotine, flavorants such as menthol, and other particulates. here, we aimed to study the hypothesis that whether nicotine and menthol modulate nicotine-metabolizing cytochrome p a (cyp a ), antioxidant enzymes (aoes), sod and catalase in plasma extracellular vesicles (evs). modulation of these enzymes would eventually lead to nicotine-induced toxicity and hiv- pathogenesis via evs-based cell-cell interactions. methods: we isolated and characterized evs from rat plasma before and after nicotine self-administration (nic) with audiovisual cue (av) and menthol and characterized using ev markers according to the isev guidelines. protein associated with cyp a , sod , and catalase were quantified by western blot. results: we measured size, total protein, and acetylcholine esterase activity of evs and found no significance difference in these characteristics before and after nic. to investigate the effect av, menthol alone or in combination in the absence and presence of nic, first we evaluated the expression of ev markers cd and cd . the results showed menthol and av together increased the levels of cd (p ≤ . ), the marker of small vesicles, in the presence of nic. the nic with menthol and av showed a pattern of increased levels of small vesicle but could not reach to significance. next, we demonstrated that the nic with av increased the level of sod (p ≤ . ), which showed a pattern of increased levels of catalase and cypa , though statistically non-significant. the expression of nicotine receptor did not change under any conditions used. the results showed an increased level of cyp a (p ≤ . ), sod (p ≤ . ), and catalase (p ≤ . ) in plasma evs in the menthol-nic group compared to menthol group only. nic group with a combined av and menthol, showed further increase in the levels of cyp a (p ≤ . ), and catalase (p ≤ . ). further analysis of plasma evs on inflammatory cytokines/chemokines in these groups, and the effect of plasma evs on nicotine-induced toxicity and hiv pathogenesis are underway. summary/conclusion: nicotine administration increased, though not statistically significant, the levels of circulatory evs. moreover, the study provided evidence that nicotine in the presence of menthol, av, and/or menthol+av increased nicotine-metabolizing cyp a in all the groups and aoes in specific groups. funding: we thank the national institute on drug abuse (grant #da , da- ) for supporting our work. introduction: biomarker discovery in breast cancer (bc) is a clinical need for therapeutics and non-invasive diagnostics. tumour exosomes are involved in premetastatic niche formation and drug resistance and represent a source of non-invasive biomarkers. the identification of tumour exosomal biomarkers provides, not only, the possibility to discriminate patient groups also potential targets to control cancer progression that could be exploited to develop innovate bc therapeutic strategies. methods: we have performed a comparative differenti. al proteomic profile of four bc cell lines and their derived-exosomes, representative of the most relevant bc subtypes in clinic to search non-invasive biomarker candidates. then, we have carried on two bioinformatics approaches: ) protein association network analysis interaction (string) and ) pathway inference analysis (hipathia), to characterize the functional profiling for each bc subtype. results: we have found differentially-expressed proteins, in both cells and exosomes, that include indicators of invasion, metastasis, angiogenesis and drug resistance. exosome proteome profile reflects their different bc cell origin suggesting potential indicators of bc subtype. further, bioinformatics analysis reveals a differential role of exosomes in bc signalling pathways in recipient cells, according to their protein cargo and cell origin. summary/conclusion: our results show a set of cells and exosome proteins that highly discriminate bc subtypes and may significantly contribute to further studies for the design of bc biomarker predictor to stratify bc patients and the development of novel therapeutic strategies. funding: a set of potential biomarkers to discriminate breast cancer subtypes. circulatory evs as potential biomarkers of hiv-drug abuse interactions and neurological dysfunction in hiv-infected subjects and alcohol/ tobacco users sunitha kodidela a , kelli gerth a , namita sinha a , asit kumar b , prashant kumar a and santosh kumar a a uthsc, memphis, usa; b university of tennessee health science center, memphis, usa introduction: abuse of alcohol and tobacco can exacerbate hiv pathogenesis and its associated complications. further, the diagnosis of neurocognitive disorders associated with hiv infection and drug abuse using csf or neuroimaging are invasive or expensive methods, respectively. therefore, extracellular vesicles (evs) can serve as reliable non-invasive markers due to their bidirectional transport of cargo from the brain to the systemic circulation. hence, we aimed to study the specific evs proteins, which are altered in both hiv and drug abusers to identify a physiological marker to indicate the immune status and neuronal dysfunction of hiv-positive drug abusers. methods: evs were isolated from plasma of the following subjects: a) healthy b) hiv c) alcohol drinkers d) cigarette smokers e) hiv+alcohol drinkers f) hiv +cigarette smokers. quantitative proteomic profiling of evs was performed by mass spectrometry and potential ev proteins associated with neuronal dysfunction were quantified by westernblot. results: the evs were characterized according to the isev guidelines. a total of proteins were detected in evs of all the study groups. comparison of proteins among all the study groups revealed that hemopexin was significantly altered in hiv+drinkers compared to drinkers and hiv subjects. further, our study is the first to show properdin expression in plasma evs, which was decreased in hiv+smokers and hiv+drinkers compared to hiv patients. though we couldn't identify the few other cns-specific proteins, g-fap and l -cam, associated with neuronal dysfunction in plasma evs by mass spectrometry, we could detect those by westernblot. the protein expression of gfap (p < . ) was significantly enhanced in plasma evs obtained from hiv-positive subjects and drinkers compared to healthy subjects, suggesting enhanced activation of astrocytes in those subjects. the l cam expression was found to be significantly elevated in smokers (p < . ). both gfap and l cam levels were not further elevated in hiv+smokers compared to hiv+nonsubstance users. summary/conclusion: the present findings suggest that hemopexin, and properdin show potential as markers for hiv-drug abuse interactions. further, astrocytic and neuronal-specific markers (gfap and l cam) can be packaged in evs and circulate in plasma, which is further elevated in the presence of hiv infection, alcohol, and/or tobacco and thus may represent as potential biomarkers for neurological dysfunction in those subjects. funding: we thank the national institute on drug abuse (da ) for supporting our work. . electrochemical detection of mirna- - p introduction: micrornas (mirnas) are small, single-stranded, non-coding rna species that regulate gene expression post-transcriptionally, and are transported by extracellular vesicles (evs). they play an essential role in biological processes, such as development, cell proliferation, apoptosis, stress response and tumorigenesis. thus, mirnas are considered relevant biomarkers in health. more particularly, mirna- - p is expressed in neurons after traumatic brain injury, being expectably transported to peripheral fluids by brain evs that cross the blood-brain barrier. the main goal of this work is to develop an electrochemical biosensor for the detection of mirna- - p in serum. methods: overall, the experimental assembly of the biosensor was made in three stages. the first one consisted in the electrodeposition of aunps, the second one in the incubation of anti-mirna - p on the carbon screen-s printed electrodes and the final stage in the incubation of mercaptosuccinic acid for blocking unspecific bindings. the probe was hybridized with the target mirna - p by a consecutive incubation of several standard solutions. each modification was evaluated with cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis) and square wave voltammetry (swv). the electrochemical behaviour of the biosensor was followed in all steps by monitoring the electron transfer features of a standard redox system. the redox probe selected for this purpose was [fe (cn) ] -/[fe(cn) ] -. results: the results indicated that the electrodeposition of gold was more effective for − . v for s and could lead to better signals upon anti-mirna- - p hybridization. summary/conclusion: in general, the experiments showed increasing charged transfer resistance upon the incubation of higher concentrations of mirna- - p. in these experiments, ev concentration is a critical variable that must be carefully controlled to ensure scientific rigour and reproducibility: without controlling for concentration (dose), experimental outcomes will exhibit excess variability that could mask important biological discoveries. in this study, three orthogonal methods are compared for accuracy in ev quantification: microfluidic resistive pulse sensing (mrps) and nanoparticle tracking analysis (nta) were compared to each other and relative to the gold standard method, transmission electron microscopy (tem). the ability of nta to accurately measure particle concentration is shown to depend on the polydispersity of the sample itself. results validate the accuracy of mrps and emphasize the importance of using orthogonal techniques to quantify evs. methods: reference urinary vesicles were prepared and analysed with the three methods and the relative concentration accuracy of nta and mrps were compared as a function of particle size. the hypothesis that nta concentration accuracy was impeded by sample polydispersity was tested using polystyrene bead mixtures having a range of polydispersity. a theoretical argument based on fundamental physics explains the experimental observations. results: tem and mrps measurements of the evs were in excellent agreement and showed a broad, polydisperse particle size distribution with no peak on the measured size range ( nm - nm diameter). nta differed significantly from tem and mrps by reporting a steep decrease in measured concentration below about nm that resulted in a peak in the reported particle size distribution. bead measurements confirmed the hypothesis to be tested: sample polydispersity significantly affects the ability of the nta method to accurately measure concentration, even for particles as large as nm diameter. summary/conclusion: these experiments validate mrps as an accurate method for quantifying evs and highlight the importance of using orthogonal measurement methods in accordance with misev guidelines. clinically relevant synthetic reference materials to standardize concentration measurements of extracellular vesicles: state-of-the-art and future prospects introduction: there is an unmet need to standardize concentration measurements of extracellular vesicles (evs). flow cytometry is the clinically most applicable method, but the currently available reference materials for calibration are insufficient. for example, the refractive index (ri) between standard particles and evs substantially differs, whereas concentration and fluorescence calibration particles are too bright. the goal of this study is to ascertain the most desired properties of reference materials to standardise ev measurements. methods: an online survey was prepared within the meves ii project to measure the desired size, concentration range, optical properties, choice of fluorochromes, and stability of synthetic ev reference materials for flow cytometry (fcm) measurements. besides the desired properties of ev reference particles, also the available instrumentation was assessed in the survey, which was sent to the members of the stakeholder committee of metves ii project and members of the ev flow cytometry working group. results: the most desired size, concentration, and ri range for ev reference particles is nm to nm, e to e /ml, and . - . , respectively. based on mie-theory evaluation of the sensitivity of the available instruments, none of the respondents would be able to detect nm particles with ri = . with their current instruments. regarding fluorescence intensity, the most desired range according to the responses is from molecules of equivalent soluble fluorochromes (mesf) to mesf. considering the sizes of evs and fluorescent labels, the maximal mesf that can be obtained for ev reference particles with nm diameter and high molecular mass fluorescent dyes is in the range of several hundreds. typical antigen densities on evs fall below copies per ev with nm diameter, i.e. mesf values above are probably not physiologically relevant in this size range. summary/conclusion: a part of the desired properties of ev reference materials precludes either their physical feasibility of production or their detection at most currently available fcms, meaning that the intended reference materials will be future-proofed. funding: this work was supported under hlt metves ii project by the european metrology programme for innovation and research (empir). the empir initiative is co-funded by the european union's horizon research and innovation programme and the empir participating states. comparison of production and activity of amniotic fluid stem cell extracellular vesicles from d hollow fibre bioreactor and d culture. culture conditions may affect ev composition and potency. here we compare production, potency, identity and therapeutic potential of evs collected from cells grown in culture dish ( d) versus hfbr ( d). methods: human clonal afsc were derived from patient-consented amniotic fluids. x e hafsc were seeded in d ( cm ), and . x e hafsc on a small kd mwco hfbr (fibercell-c d, cm ) with fibronectin coating; both cultured in chang medium with % of es-fbs, starved for hr and then evs collected. the effect of harvest frequency was tested ( hrs, hr, hrs, wk). d-evs and d-evs were compared by nanosight, potency assay (by wb), identity (by exoview analysis) and therapeutic effect (in vivo in an animal model of kidney disease, alport syndrome). results: d production was~ . x e ev/ml/ hrs while d was~ . x e ev/ml (first four hrs) and . x e ev/ml (two days of hourly harvests). very little difference in ev concentration and very similar size distribution (~ nm) were observed during harvest intervals; possibly indicating either significant ev re-uptake or inhibition of ev secretion dependent upon free ev in the supernatant. d-evs trapped vegf (an in vitro established potency assay) as efficiently as d-evs, and expressed cd , cd , cd , cd , cd and vegfr as d-evs. summary/conclusion: d-evs had comparable properties and bio-activity to d-evs, but the hfbr produced x more evs. hfbr cell culture conditions for hafsc still need optimization, however an available . m cartridge provides a x scale up potential. the hfbr, a cgmp closed system, can produce sufficient numbers of ev to support pre-clinical and clinical applications with at least similar properties to evs produced by conventional d methods. funding: -intramural funding -intramural ev core pilot funding demonstration of high gain mode in combination with imaging flow cytometry for improved ev analysis luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. in recent years, the importance of evs has become apparent, as they are key mediators of intercellular communication. however, quantifying and characterizing evs in a reproducible and reliable manner is challenging due to their small sizeexosomes range from to nm in diameter. it is well-known that flow cytometers were originally designed to measure and detect cells, and due to the quantitative power flow cytometry offers, there has been a push to quantify and characterize evs using flow cytometric methods. however, these systems have not been designed to measure objects smaller than a cell. methods: here, we describe the use of high gain mode on the amnis® imagestream® imaging flow cytometer to address the challenges of measuring small particles. in this new high gain mode, the charge-coupled device (ccd)-camera is manually adjusted to higher gain settings, increasing the signal obtained from the ev. object thresholds and masking have also been adjusted to better identify and detect small particles. results: preliminary results using murine leukaemia virus-sfgfp reference particles have shown up to a fivefold increase in the number of gfp-positive objects collected in high gain mode, when compared to standard gain on the imagestream system. summary/conclusion: in this study, we demonstrate improved small particle detection, including evs, using this new high gain mode on the imagestream imaging flow cytometer. distance-controlled accelerated catalysed hairpin dna circuit for multiple and sensitive detection of exosomes-associated mirnas introduction: sensitive and simultaneous monitoring of multiplexed exosome-associated rnas is of great value for early cancer diagnosis remains a challenge. methods: here, we report a simple, multiple and sensitive exosomes-associated multiplex mirnas detection method that uses distance-controlled accelerated catalysed hairpin dna circuit (chdc) system without any complex operation or enzymatic amplification. the distance-controlled accelerated chdc can directly enter the plasma exosomes to generate fluorescent signal quantitatively by specifically targeting mirnas without any transfection means. results: we show that distance-controlled accelerated chdc strategy with signal amplification capability could selectively and sensitively identify low level rnas in serum evs, distinguishing patients with early-and late-stage breast cancer from healthy donors and patients with benign breast disease. summary/conclusion: this simple, accurate, sensitive, and cost-effective liquid biopsy by the distance-controlled accelerated chdc method is potent to be developed as a non-invasive breast cancer diagnostic assay for clinical applications. impact of isolation methods on biophysical heterogeneity of single extracellular vesicles university of california los angeles, ca, los angeles, usa introduction: current biophysical analysis of extracellular vesicles (evs) typically encompasses particle density and size distribution determinations using various techniques. however, variabilities in ev isolation methods and the structural complexity of these biological-nanoparticles (sub- nm) necessitate more rigorous nanoscale biophysical characterization of single evs to facilitate more reliable and comparable evbased assays. methods: combining atomic force microscopy (afm), super-resolution optical and conventional particle sizing light scatter and microfluidic techniques, we compared the unique sub-nanometre scale biophysical properties of breast cancer cell-derived ev isolates obtained using different isolation methods. results: afm and dstorm particle size distributions showed coherent unimodal and bimodal particle size populations in centrifugation and immune-affinity isolates respectively. more importantly, afm imaging revealed striking differences in nanoscale morphology, surface undulations, and vesicle-to-non-vesicle ratios among ev isolates from different isolation methods. our findings demonstrate the effectiveness of orthogonal high-resolution biophysical characteristics of single evs, not discernable via particle size distributions and counts alone. summary/conclusion: the identified nanoscale biophysical characteristics of ev isolates represent a strategic and complementary framework to resolve differences in the heterogeneity and purity of evs from introduction: extracellular vesicle (ev) concentrations measured by flow cytometry are incomparable. to improve comparability, the metves ii consortium is developing traceable reference materials and procedures, which require validation by test samples. in previous interlaboratory comparison studies, however, a main source of variation was introduced by pre-analytical variables and measurement artefacts introduced by test samples. to minimize variation introduced by test samples, our aim is to develop off-the-shelf biological test samples containing pre-labelled evs. methods: human urine and plasma were collected from healthy donors. evs were labelled with lactadherin-fitc, isolated by size-exclusion chromatography to remove free dye and minimize swarm detection, and mixed with dimethyl sulphoxide (dmso), exocap or trehalose, frozen in liquid nitrogen and stored at − °c . after thawing, ev concentrations were measured by a calibrated flow cytometer (apogee a -micro). results: compared to the ev concentrations measured in fresh plasma and urine, the concentrations decreased % in plasma (p = . ; mean of the cryopreservation agents) and % in urine (p = . ) after one day of storage. after months of cryopreservation, the concentration of plasma evs decreased % (dmso and exocap) and . % (trehalose) compared to one day of storage, whereas the concentration of urine evs decreased % (exocap) and % (dmso and trehalose). summary/conclusion: we have developed ready-touse, pre-labelled human evs that are stable up to months and dedicated for use in interlaboratory comparison studies. to further increase stability, other cryopreservation agents will be tested. our biological test samples will be key to validate the new reference materials and procedures developed by metves ii in . funding: this project has received funding from the empir program co-financed by the participating states and from the european union's horizon research and innovation program. understanding intracellular fate of ev-delivered content introduction: despite much work performed on evaluating the potential effects of extracellular vesicles (evs), the functional uptake of their cargo is still controversial. this project aimed to demonstrate that ev content (protein and mrna) is protected and can be subsequently transferred with functional activity into recipient cells, while also developing a tool to assess and quantify functional ev uptake. methods: fusion proteins used were mitochondrial localized coxviii-cfp-nanoluc(cox) and nuclear localized h b-rfp-nanoluc(h b). results: hek t cell-derived evs protected cox proteins from proteinase k digestion while demonstrating significantly improved efficiency of uptake when compared to free protein, as measured by bioluminescence that was still detectable in recipient cells hrs post-ev-exposure. to confirm functional uptake, recipient cells exposed to evs containing h b for hrs were imaged and some recipient cells manifested fluorescent red nuclei. to demonstrate the presence of functional mrna within evs, producer cells were transfected for such a duration as not to have detectable levels of protein in the evs while still containing detectable levels of mrna (qpcr) even after rnasea treatment. transfer of these evs to hela cells showed an increase in expression of h b which was blocked by cyclohexamide, confirming translation of the mrna ( . kb). to determine if recycling of ev delivered proteins occurs, recipient hela cells were exposed to evs containing cox for hrs. all extracellular evs were removed and cells were trypsinized ( . % for min) to remove any non-internalized cox protein. hrs later, evs (cd + and cd +) released from cells contained cox suggesting recycling of protein or possibly recycling of entire evs. lastly, an assay was developed to measure functional ev uptake. nanoluc protein was split in two and fused to mturquoise (n ) or mscarlet-i( c). expression of each fragment alone exhibited non-detectable levels of luminescence while expressing both together had a significantly increased signal. delivery of either fragment within an ev to a cell expressing the corresponding fragment worked as confirmation and quantification of ev uptake (hek , u , hela cells). summary/conclusion: this study robustly demonstrates ev delivery of functional mrna and protein to cells, while also establishing a simple assay to quantify and validate functional ev uptake. theoretical model of ev losses due to adsorption on the tube walls. application for immunomagnetic detection of the vesicles introduction: short-term storage of unfrozen samples of vesicles, mainly at °c, overnight or during a couple of days is rather common laboratory practice. however, it was found to lead to significant losses of vesicle concentration supposedly due to adsorption on the walls of the tube. the present work develops a theoretical model intended to describe the vesicle adsorption process. the experimental validation of the model was made using method of immunomagnetic precipitation. methods: the theoretical model considers the "diffusion-limited" case of vesicles storage. the maximal adsorption capacity of the surface of contact between the tube and the solution is given as the number of vesicles in hexagonally packed monolayer. for experiment, the vesicles were purified from ht cell culture supernatant by differential centrifugation, aliquoted and kept at − c. further the aliquots were consequently unfrozen, and placed into the tubes with different surface treatment and kept at + c. the kinetics of vesicles loss was measured by anti cd immunomagnetic capturing followed by cd , epcam and cd staining and flow cytometry. results: the model allows the estimation of the adsorption-associated losses as dependent on initial vesicles concentration, volume of the solution, tube geometry, the storage temperature and duration case of quiet vesicles storage (without mixing) and also accounts an expected effect of active agitation of the solution (ev-beads complexes formation). theoretical calculations were illustrated by analysis of ev at different storage conditions and during reaction of immunomagnetic precipitation of the vesicles. summary/conclusion: it was demonstrated that application of tubes surface treatment allows increasing sensitivity of immunomagnetic precipitation method to x ^ for cd +, x ^ for epcam+ and x ^ for cd + vesicles. introduction: it is now largely accepted that the intestinal microbiota plays a key role in intestinal bowel diseases (ibd). an imbalance in the composition and diversity of the intestinal microbiota (i.e. dysbiosis) of patients has been repeatedly pointed out by several teams. there are also indications that extracellular vesicles produced by bacteria and exosomes produced by epithelial cells might be increased in this family of diseases. methods: in order to differentiate healthy and ibd faecal samples on the basis of their vesicle profiles, we want to develop a means to enumerate rapidly particles in faecal samples, based on interferometric microscopy. the videodrop technology, developed by myriade, relies on the creation of single beam interferences between two signals from the same light path by nanoparticles such as small vesicles. it will permit to compare on large scales the viral load of healthy subjects and ibd patients. results: this fast and easy-to-use device was compared to the nta on several types of eukaryotic and prokaryotic vesicles and our preliminary results are encouraging. introduction: small extracellular vesicles (sevs) produced by mesenchymal stromal cells (msc-sevs) may be useful in cell-free therapies for immunomodulation and tissue regeneration. methods: to characterize msc-sevs produced ex vivo, human bone marrow mscs were cultured in mesencult-acf plus (macfp), an ev-free and animal component-free culture medium for days and spent medium collected to isolate sevs by ultracentrifugation (uc). analyses of sevs were performed by nanoparticle-tracking analysis (nta), western blot (wb), and human umbilical vein endothelial cell (huvec) tube formation assay. results: analysis of fresh uncultured macfp by uc, nta and wb for cd , cd , and cd confirmed the absence of sevs. msc-sevs isolated from spent macfp by uc ranged from - nm in size and were positive for cd , cd , and cd proteins. these sevs could be stored at − °c for > months in solution or lyophilized with minimal loss based on nta and wb analysis. the msc-sevs contained the msc-associated micrornas let a, mir , and mir a as per qpcr analysis. the biological function of ex vivo isolated msc-sevs was assessed using a human umbilical vein endothelial cell (huvec) tube formation assay. huvecs treated with msc-sevs generated tubes as early as h after seeding, which were not observed in control huvec cultures until h. moreover, the number of branch points present in such tube structures was >fourfold higher in huvec cultures (n = ) supplemented with msc-sevs versus control, with the former lasting > h and the latter lasting < h in culture. direct comparison of the performance of macfp medium to media containing non-depleted or ev-depleted foetal bovine serum demonstrated that only mscs cultured in macfp (n = ) were able to expand robustly with a doubling time of . , . and . days in these media, respectively. lastly, methods for isolating sevs using newly developed easysep-ev™ magnetic separation kits and size exclusion columns will be presented. summary/conclusion: taken together, these data demonstrate that msc-sevs can be produced in high yield in macfp medium and that these possess similar physical, phenotypic and functional characteristics as sevs in vivo. funding: this work was privately funded by stemcell technologies inc. introduction: extracellular vesicles (evs) are heterogeneous group of small vesicular structures released by different types of cells, including stem cells (scs). as recent studies demonstrate that they may enclose bioactive content and transfer it into the target cells, growing interest is placed on the utilization of evs in the field of biomedical research. however, there is still lack of standardized methods of evs characterization. as an example, typical flow cytometry-based protocols, commonly used for cells phenotyping, may be inadequate for the characterization of evs as particles with size close to the detection limit of conventional cytometers. thus, the aim of this study was to optimize and compare the use of different flow cytometry platforms for the multiparameter analysis of evs isolated from different types of scs populations. methods: ev samples were obtained by ultracentrifugation of conditioned media collected from selected scs types, including human induced pluripotent scs (ips) and mesenchymal scs (mscs). next, several high resolution flow cytometry systems: cytoflex, apogee (a and a micro-plus) and image stream mk ii were employed to compare their sensitivity and resolution, as well as influence of "swarm" effect. furthermore, we examined evs phenotype, including expression of tetraspanins and other surface markers. results: our results have revealed that tested flow cytometry systems may be utilized for the phenotypic characterization of evs secreted by scs populations. however, the conventional staining and gating strategy protocols have to be thoroughly optimized. additionally, depending on a type of tested cytometer, we have demonstrated the difference in a "swarm" effect and its influence on obtained results regarding evs phenotype. finally, imaging flow cytometry platform was also employed to visualize evs on the single particle level. summary/conclusion: in conclusion, we have demonstrated that tested high-resolution flow cytometry platforms are convenient methods for the multiparameter characterization of evs produced by different types of scs populations. however, careful selection of particular measurement parameters should be performed depending on a type of employed system. funding: this study was funded by ncbr grant strategmed iii (strategmed / / / ncbr/ ) to ezs. evaluation of atcc's exosomes from cell culture supernatant as reference standards in research and development. introduction: exosomes are subcellular particles - nm in size released from cells through a fusion of multicellular bodies with the plasma membrane. exosomes are stable carriers of cell-free cargo in the form of dna, rna, and proteins, thereby making them an attractive candidate for diagnostic and therapeutic applications. however, isolating a consistent population of exosomes can be challenging and there is an unmet need for highly characterized exosomes for use as reference standards in extracellular vesicle research (ev). methods: exosomes were isolated from cell culture supernatants of different atcc cell lines including stem cells and cancer cell lines representing the most prevalent cancer types -prostate, colorectal, breast, lung, cervical and glioblastoma, using tangential flow filtration (tff). these exosomes underwent sterility and mycoplasma tests as a part of their quality control. the morphology and size distribution of these exosomes were evaluated through multiple strategies including nanoparticle tracking analysis (nta), asymmetrical flow field-flow fractionation (af ), cryo-electron microscopy (cryo-em) and spectra dynetm particle analyser. exosome surface markers were also analysed through multiple strategies such as electro chemiluminescent elisa, flow cytometry and western blotting. also, stem cell exosomes and cancer exosomes were further evaluated for functionality through in vitro functional assays including migration assay, angiogenesis and anchorage independent growth assay. results: our optimized tff method resulted in high yields of > × exosomes/ml and average protein equivalent of more than mg/ml. more than % of the exosomes population had an average size distribution of - nm and median size of nm confirmed through a number of different size distribution instruments. although cell line dependent, we were able to obtain similar expression levels of different cell surface markers including tetraspanins (cd , cd , cd ) when evaluated through different methods. our functional data demonstrated stem cell exosomes were functionally active in promoting cell migration and tubule formation. additionally, cancer cell exosomes were found to promote a malignant phenotype in an anchorage independent growth assay. summary/conclusion: collectively, we demonstrated our ability to reproducibly manufacture production-scale batches of exosomes from multiple different cell types. our purified exosomes are of high yield, meet well-established quality control specifications, and are robust in maintaining size distribution, surface marker expression, and functionality in vitro. therefore, they can serve as ideal reference materials that can support different ev-based research applications. exo-cise: extracellular vesicles enriched from plasma post-exercise promotes myogenesis and neurogenesis bianca paris a , yaomeng liu a , vicente pagalday-vergara a , julie davies b , priya samuel a , ayman abu seer a , johnny collett a , laura gathercole a , ken howells a , karl j. morten b , zhidao xia a , daniel anthony b , david r f. carter a , helen dawes a and ryan c. pink a a oxford brookes university, oxford, uk; b university of oxford, oxford, uk introduction: physical activity brings about a widespread physiological response and elicits the beneficial adaptation of several tissues and organs. furthermore, regular participation in physical activity reduces the risk of developing major non-communicable diseases such as cardiovascular disease, diabetes, cancer, osteoporosis, and dementia. two important processes known to occur following physical activity are myogenesis and neurogenesis; both of which involve the activation and proliferation of specialised tissue-resident stem cells. the molecular mechanisms regulating these processes following exercise are poorly understood to date. here, we investigated the contribution of extracellular vesicles, which are released into the circulation after exercise, to benefit adult myogenesis and neurogenesis. methods: small extracellular vesicles were enriched from the blood of healthy participants before and following maximum and moderate intensity exercise. differentiation and proliferation using a range of methods was measured following vesicle treatment onto primary myoblasts and neuronal primary exvivo stem cells. activation of key cellular pathways were measured. results: we show significant proliferation and differentiation changes of both stem cell types. this is independent of extraction method, extracellular vesicle depleted fractions and is interestingly conserved across mammalian species. remarkably, we see an age-related effect. summary/conclusion: this advocates that short single bouts of exercise may promote myogenesis and neurogenesis via systemic signalling of extracellular vesicles which opens an interesting field in endogenous ev therapies. show promise as a cell-based therapy for retinal degeneration. while clinical trials are ongoing, the potential of extracellular vesicles (evs) as biomarkers for monitoring eye health and disease is not well studied. this study characterized the ev surface profile and cargo of hipsc-rpe to offer a baseline assessment in normal and disease conditions. moreover, we evaluated the importance of pnpla , a gene involved in membrane integrity and when mutated causes retinal degeneration, in ev biogenesis and secretion. methods: evs were isolated from serum-free culture medium of hips-rpe and identified with nanoparticle tracking analysis, transmission electron microscopy, and immunoblot analysis of exosomal markers, including alix, tsg , and cd . surface marker detection and proteomic profiling were completed using an ev surface marker kit and mass spectrometry, respectively. small interfering rna targeting pnpla was used to knockdown the expression in hipsc-rpe and evs were characterized. results: nanoparticle tracking analysis confirmed the presence of both microvesicles (> nm) and exosomes (< nm) by size distribution and the concentration of evs ( x particles/ml) from rpe. tem displayed typical morphological characteristics of evs. the presence of known ev markers, alix, tsg , and cd was confirmed via immunoblot and flow cytometry. surveillance of ev surface markers revealed enrichment of epithelial markers (cd ) and stem cell markers (cd / ) that depict donor cell origin and functional proteins including integrin-binding (cd ) and tgf-beta receptors (cd ). in addition, proteomic analysis revealed regulators of inflammation and rpe function, including hemopexin, clusterin, complement factor i, and pigment epithelium-derived factor. furthermore, reduction in pnpla expression reduced vesicle secretion and vesicle size compared to non-targeting controls. introduction: vascular endothelial growth factor (vegf) is a potent angiogenic factor and was first described as an essential growth factor for vascular endothelial cells. vegf plays a role in normal physiological functions such as bone formation, haematopoiesis, wound healing, and development. mesenchymal stem cell (msc) was found to secretes potential growth factors such as vegf when cultured in vitro. however there are some beliefs that foetal bovine serum (fbs) which usually used as serum in cell culture content vegf. methods: msc seeded in in -well plate in with concentration of , cell/well. cells were incubated for hours and fasted for another hours using only dmem. cells were treated with complete medium consist of dmem and % fbs. culture medium were collected after , , and hours after treatment. cell were culture in ºc dan % co . vegf concentration was detected using elisa technique. results: vegf concentration was not found in fbs which do not contact with msc. an increasing of vegf concentration in time-dependent manner was shown when culture medium was used in msc cell culture in normoxic condition. the result of vegf concentration when culture , , and hours were . pg/ml, . pg/ml, and . pg/ml, respectively. the mechanism of msc release growth factor is still under investigated. however, the classic growth factors and cytokines serves paracrine control molecules which were important in regenerative medicine. vegf was found to be an important molecules in angiogenesis process and determine the fate of cells. summary/conclusion: msc secreted vegf and concentration increased in time-dependent manner. isolation and characterization of exosomes from canine stem cells introduction: unlike induced disease models using laboratory animals, naturally occurring disease models display pathophysiologic attributes that are more similar to human diseases. unfortunately these models are underutilized in translational regenerative medicine research. this is partly due to the slow development of species-specific experimental therapeutics to investigate comparative efficacy. thus, we set out to isolate and characterize exosomes from canine adipose-derived mesenchymal stem cells (cad-msc) to use as a comparative therapeutic in dogs. to accomplish this, we optimized an isolation and purification strategy and characterized their molecular properties. methods: exosomes were isolated by sequential centrifugation and subsequent ultrafiltration. the proteome was characterized by tandem mass tag (tmt) mass spectrometry and the mirna cargo was identified using a canine specific pcr array with subsequent target and enrichment analysis using targetscan and the panther platform, respectively. also, nanoparticle tracking analysis and transmission electron microscopy were used to determine exosome size and structure. to investigate bioactivity, we measured the ability of exosomes to inhibit collagen production in an in vitro model of fibrosis. results: exosomes were purified by ultrafiltration using a kda cut-off. proteomic analysis by tmt mass spectrometry identified unique proteins. % of the exocarta top were identified from this list. additionally, we identified the mirna cargo within exosomes and found highly expressed mirnas. enrichment analysis identified multiple pathways of probable regulation including angiogenesis (fold enrichment = . ; p < . ) and transforming growth factor-beta (tgfb) signalling (fold enrichment = . ; p < . ). exosome size was quantified to be . ± . nm with a modal average of nm. lastly, in the presence of exosomes, tgfb stimulated fibroblasts deposited . % less collagen than vehicle controls (p = . ). summary/conclusion: in summary, cad-mscs exosomes display structural and functional features comparable to stem cell derived exosomes from other species. use of these exosomes in naturally occurring disease canine models may provide superior predictive value for human clinical trials. funding: support provided by the ccah, school of veterinary medicine, uc davis. mesenchymal stem cells-derived exosomes promote in vitro the progression of triple negative breast cancer cells introduction: mesenchymal stem cells (mscs) are multipotent stromal cells and have been described as key regulators of different aspects of tumour physiology. in tumour pathogenesis, mscs can integrate the tumour microenvironment after recruitment and are able to interact with cancer cells to promote tumour modifications by affecting epithelial-tomesenchymal transition (emt). it was revealed that exosomes derived from mscs are critical players in the tumour niche. exosomes are a novel way of cellto-cell communication and play crucial roles in the majority of pathways that contribute and affect response to therapy, cell-adhesion molecules and the progression of tumour cells. because of the known importance of this communication we decided to investigate the implication of mscs with triple negative breast cancer (tnbc) cell lines as well as exosomal profiles between the experimental conditions. methods: the interactions of mscs with triple negative breast cancer cell lines (mda-mb- and hs t) was performed by coculturing mscs (or tnbc cell lines) with exosomes derived from tnbc cell lines (or mscs). physical characterization of isolated exosomes was performed followed by their molecular investigations. cell proliferation was detected by mtt assay and migration was analysed by wound healing assay using d cultures. moreover, we also used d culture to assess the exosomes uptake and to observe their capability of internalization into a d structure. the alterations in expression level of some transcripts (mrnas and mirnas) and protein profile were investigated by qrt-pcr, western blot and immunofluorescence staining. results: we found that mscs-derived exosomes are actively incorporated by triple negative breast cancer cell lines ( d culture). in coculture, in tnbc cells the expression level of mesenchymal markers and emt markers (e-cadherin, vimentin) at mrna and at protein levels, as well as mirna-derived exosomes targeting mesenchymal genes were significantly affected. using bioinformatics tools, we highlighted the important biological processes which were activated by promoting tumour modifications. in addition, using d culture we provided a comprehensive understanding regarding exosomes internalization in d structures, which closely mimics in vivo conditions, compared to d culture. summary/conclusion: in this work, we focus on the investigation of mscs-derived exosomes in order to highlight their implication in several biological processes, including tumour proliferation and progression of triple negative breast cancer cells. all these alterations affect the response to therapy and should be considered for developing efficient therapeutic strategies. natural killer cell-derived extracellular vesicles have a potent anti-leukaemic effect and selectively target the cancer stem cell subpopulation introduction: natural killer (nk) cells of the immune system recognize and kill tumour cells. extracellular vesicles (evs) secreted from nk cells are capable of killing tumour cells independent of the cell to cell contact required for nk cell activation. cancer is a leading cause of death, primarily due to metastasis and recurrence. cancer stem cells (csc) within tumours are resistant to chemotherapy and immune attack, and cause metastasis and relapse. identification of the cancer types killed by nk evs is limited, and the effect of nk evs on cscs has not been described. here we determine whether nk-derived evs kill a myeloid leukaemia cell line and its csc subpopulation. methods: nk evs were isolated from our nk cell line, nk . , derived from normal human lymphocytes. nk . evs were characterized by immunoblotting, proteomics, and next generation rna sequencing. human k leukaemia cells were treated with nk . evs in vitro and analysed for proliferation and markers of cell death. results: nk . evs contain ev-associated proteins alix, cd , hsp , and tsg , nk effector molecules perforin, granzymes a and b, granulysin and nklam/ rnf b, an e ubiquitin ligase required for maximal nk cytotoxicity, and tumour suppressor mir- . nk . ev treatment of k significantly decreased its expression of proliferation markers cd and ki , and increased the frequency of apoptotic and necrotic cells, paralleled by elevated levels of active caspases − and − . non-tumorigenic cells were unaffected by nk ev treatment. most notably, nk . ev treatment significantly reduced the frequency of k cells highly expressing aldh, a csc marker. summary/conclusion: nk . -derived evs have a robust anti-tumour effect on k myeloid leukaemia cells and selectively target the csc population, suggesting they may circumvent the evasion and resistance mechanisms used by cscs. nk . evs therefore have introduction: due to their potential as a key bioactive agent in regenerative medicine applications, mscderived extracellular vesicles (msc-evs) are increasingly being investigated as a clinical therapy. manufacturing that generates enough evs for product development and clinical doses is currently a limitation in the field and clearly a scalable manufacturing solution will be necessary for successful translation. moreover, a complementary approach that increases the ev productivity, i.e. the number of evs produced per cell, could further help to accelerate the development of msc-evs as a therapy. methods: we developed a process that leverages a series of new cell culture reagents to couple to our established cell-media system for scalable manufacturing of msc-evs. briefly, human bone marrow-or umbilical cord-derived mscs were rapidly expanded under xeno-free conditions (i.e. > x expansion within days). cultures were then switched to our proprietary ev collection medium and evs were harvested for up to three additional days. at the end of culture, the evs in the conditioned media were concentrated using a tangential flow filtration (tff) system. to increase the productivity of mscs, two medium supplements were developed that increased ev yield by either increasing the number of evs generated per cell in a shortened culture process or increasing the number of collected evs by lengthening the ev collection culture period. results: this scalable msc-ev manufacturing method was implemented in both d flask and d bioreactor culture and generated over , particles per cell in d and over , particles per cell in d. with the addition of a medium supplement to increase evs produced per cell, the ev productivity was increased > x after hrs. alternatively, ev productivity was also increased > x by addition of the medium supplement that extended ev collection culture period. summary/conclusion: msc-ev success in clinical translation will be reliant on a manufacturing method that can scalably and reliably generate large amounts of evs. these results present one such solution. furthermore, increasing ev productivity, for instance by medium supplements that increase evs per cell or lengthen culture times could further address the limitation of generating the evs required for development and translation of clinical therapies. simplifying scalable msc ev production in a microcarrier-based bioreactor system divya patel, josephine lembong, katrina adlerz, jon rowley and taby ahsan roosterbio inc, frederick, usa introduction: the growpt ing numbers of msc-ev clinical applications drives the need for a scalable msc-ev production platform. while most msc-evs are generated while cells are attached to tissue culture plastic, such d cultures cannot be scaled up to meet the yields necessary for commercialization of ev-based therapeutics. we have shown that d bioreactors can be used to generate msc-evs and that paradigm can be scaled directly in terms of yield from the to l scales. the technical expertise of seeding cells onto microcarriers for expansion in bioreactors, however, requires technical expertise not available to all those in the ev field. therefore, our goal here is to simplify and expedite the ev collection process in bioreactors by cryopreserving cells on microcarriers, such that end users can merely thaw and then collect msc-evs. methods: mscs were expanded in d and then seeded on three different microcarriers and cultured in a bioreactor for days. when confluent, cells on microcarriers were cryopreserved. to evaluate the microcarriers and the cryopreservation protocol, the cells-microcarriers were thawed, cultured in a bioreactor in growth media for hours, then in ev collection media for additional days. cell recovery and ev production upon thaw was evaluated and compared to ev collection from fresh, non-cryopreserved cells. results: total cell counts hrs post thaw were comparable to those before cryopreservation and to fresh samples prior to ev collection. following -day ev collection, concentration of particles collected from cryopreserved cells on microcarriers were similar to those collected from the fresh cells ( e particles/ml). this process was validated for two different microcarriers using two separate cryopreservation solutions. summary/conclusion: our results show that cryopreserved hmscs on microcarriers can support ev collection in a d bioreactor process with a particle yield that is comparable to those collected from fresh cells. this cryopreserved product can simplify ev production, reducing cost and time by removing process steps associated with the hmsc expansion, with in a paradigm suitable for scale-up. the whitening, anti-wrinkle, and wound-healing effects of extracellular vesicles from orbicularis oculi muscle-derived stem cells. introduction: skeletal muscle-derived stem cells possess potent therapeutic activities in the treatment of muscle-related disorders. in our study, we tried to isolate and characterize orbicularis oculi muscle (orm)-derived stem cells (orm-scs) from the discarded human tissues which were obtained from the ocular surgery-subjected patients. we also prepared the natural extracellular vesicles (evs) from the cultured orm-scs and assessed the their therapeutic actitities including the skin whitening, anti-wrinkle, and wound healing effects. methods: we isolated the orm-scs from the patients subjected to ocular surgery and characterized the orm-scs by analysing cell morphology, proliferation, expression levels of the cell surface and stemness-associated markers, and tri-lineage differentiation and colony-forming capacities, confirming the stemness properties of the orm-scs. then, we prepared the natural evs from the orm-scs via the centrifugation and filtration of the media supernatants and their therapeutic activity was investigated. results: the isolated orm-scs showed spindle-like morphology and positive expression of cd , cd , and cd , but they were negative in expression of cd and cd . the orm-scs showed the capacity of osteogenic, adipogenic, and chondrogenic differentiations. the evs from orm-scs (orm-sc-evs) possessed the apparent inhibitory effect on the melanin synthesis in b f cells by blocking the tyrosinase activity, although orm-sc-evs treatment did not dramatically change the expression level of melanogenesisrelated genes, such as microphthalmia-associated transcription factors (mitf), tyrosinase (tyr), tyrosinaserelated protein (tyrp- ), and tyrp- . in addition, we confirmed that orm-sc-evs could stimulate skin cell migration and increase the expression level of antiwrinkle related genes and wound-healing properties. summary/conclusion: this study revealed the stem cell property of orm-scs and the whitening, antiwrinkle, and wound healing effects of orm-sc-evs, suggesting that orm-scs and orm-sc-evs can be successfully used for stem cell-based ev therapy and cosmetics, by regulation the melanogenesis, wrinkle, and wound. funding: this work was supported by grants from the national research foundation (nrf) funded by the korean government ( m a h ). use of stem cell extracellular vesicles as a holistic approach towards cns repair introduction: neurological diseases and disorders are leading causes of death and disability worldwide. many of these pathologies are associated with high levels of neuroinflammation and irreparable tissue damage. we have previously shown that extracellular vesicles (evs) from infected cells contain viral by products (noncoding rnas and proteins) and that these evs can exert deleterious effects on recipient cells - . therefore, in the context of neurotrophic viruses evs may contribute to or perpetuate processes relating to neuroinflammation and neurodegeneration. due to their multipotent properties, stem cells have broad applications for tissue repair; additionally, stem cells have been shown to possess both immunomodulatory and neuroprotective properties. in recent years it has been well-established that stem cell evs play a critical role in the functionality associated with stem cells. the diverse biological cargo contained within these vesicles are proposed to mediate their effects and, to date, the reparative and regenerative effects of stem cell evs have been demonstrated in a wide range of cell types. while a high potential for their therapeutic use exists, there is a gap of knowledge surrounding their characterization, mechanisms of action, and how they may regulate cells of the central nervous system (cns). methods: we have isolated and recovered high yields of evs from large scale cultures of both induced pluripotent stem cells (ipscs) and mesenchymal stem cells (mscs) using tangential flow filtration. our ev characterization includes both phenotypic (size, tetraspanin expression) and biochemical assays. ev functionality has also been assessed in vitro utilizing several cellbased assays related to cellular viability, migration, angiogenesis, and immunomodulation in both healthy and damaged recipient cells with relevance to the cns. results: our data suggests that evs from different sources of stem cells display unique phenotypes, exhibit differential association with various cytokines, proteins, and long non-coding rnas, and have the ability to significantly enhance processes that are critical for cellular repair . lastly, utilizing an ipsc-derived neurosphere model, we have observed a robust uptake of stem cell evs and have found that these evs are able to effectively penetrate these d structures. summary/conclusion: collectively, these results highlight the "holistic" properties of stem cell evs by demonstrating their ability to partially reverse or reduce damage in various cell types. funding: this work was supported by national institutes of health (nih) grants ai , ai , ai - , ai , and ns to fk and r ca and r ar to lal. the effect of cell culture media on extracellular vesicle secretion from mesenchymal stem cells and human pluripotent stem cell-derived neurons introduction: cell culture media and its supplements are known to affect the secretion and isolation of extracellular vesicles (evs) from cell cultures. identification of these effects is crucial especially when planning to use evs as therapeutic agents. here, we investigated the effect of cell culture media on ev yield from human mesenchymal stem cells (mscs) and human pluripotent stem cell (hpsc)derived neurons. methods: evs were collected from cell-conditioned media (ccm) and no cell control (ncc) media using size-exclusion chromatography (sec). mscs were cultured in dmem/f :neurobasal medium or in opti-mem reduced serum medium, both supplemented with exosome-depleted foetal bovine serum (fbs). the ev yield from hpsc-derived neurons was compared at two maturation time points (day and ), in dmem/f :neurobasal or in opti-mem, with and without -hour kcl stimulation. sec fractions were analysed by nanoparticle tracking analysis (nta), protein concentration assay and blinded transmission electron microscopy (tem). results: ccm samples had a clear peak of evs in sec fractions - , which was not detected with ncc. interestingly, a second population of evs eluted in sec fractions - in both ccm and ncc, indicating presence of evs in exosome-depleted fbs. moreover, this second population differed largely between used media batches. culture medium had no significant effect on msc ev yield (dmem: . e+ particles/ ml, opti-mem: . e+ particles/ml). with neuronal cultures, no significant differences in ev yield were found between culture media or cell maturation time points. in contrast to earlier findings, -hour stimulation of neurons by kcl resulted in significantly smaller ev yield compared to non-stimulated controls (stimulated: . e+ particles/ml, non-stimulated: . e+ particles/ml, p < . ). summary/conclusion: our results indicate that exosome depleted-media are not entirely devoid of vesicles, which can cause bias in downstream analyses. however, sec is a good method to separate cellsecreted evs from the contaminating medium-derived evs. culture medium did not affect the number of evs secreted by mscs or neurons; instead, we observed larger differences between media batches. this data emphasizes the importance of analysing the ncc as negative control in all cell culture experiments. mouse mesoangioblast stem cell extracellular vesicles are able to influence macrophage cell activity maria magdalena barreca a and fabiana geraci b a dept stebicef university of palermo, palermo, italy, palermo, italy; b dept stebicef, university of palermo, italy, palermo, italy introduction: it is largely demonstrated that stem cells release extracellular vesicles (evs) that are able to modify target cell behaviour. interestingly, there is a bidirectional signalling exchange between stem cell evs and damaged cells. moreover, it is well known that macrophages, could also play a role in wound repair and tissue regeneration. it was also demonstrated that stem cell evs are involved in immune cell regulation. for this reason, today takes hold the idea that evs could replace stem cells in regenerative medicine. the aim of our work was to evaluate if evs released by mouse mesoangioblast stem cells (a ) could have a role in immune cell regulation. specifically, we have investigated the possible a ev effect on murine macrophages (raw . ) in terms of cell proliferation, migration and phagocytic ability, and cytokines/chemokine release. methods: a evs were collected from conditioned milieu by ultracentrifugation. raw . cell proliferation with or without a evs was evaluated via cfse assay. scratch test was performed to assay their migration ability. to study raw . cell phagocytosis they were treated with μm beads. finally, cytokine array was used to monitor their secretion after ev treatment. results: we have found that a evs inhibited macrophage proliferation as proved by a proliferation index significantly reduced after ev treatment. simultaneously, we have noticed that evs increases raw . migration ability. furthermore, a evs are able to increase macrophage phagocytic activity. as it is known that hsp is involved in for macrophagic activity increase and a evs express hsp on their surface, we performed phagocytosis assays assay by blocking the protein or its receptor tlr , tlr and cd . our data demonstrated that a evs increase phagocytosis through hsp and its receptors. we have also proved that a evs modify the expression pattern of cytokines/chemokines released in the extracellular milieu by raw . cells. in particular, we observed an increase in anti inflammatory cytokines, and a decrease in some inflammatory ones, suggesting that evs could polarize macrophages towards an anti inflammatory m phenotype. summary/conclusion: in conclusions, our data show that a evs influence macrophage activity and additional studies could provide a new insight into understanding the underlying potential of evs in tissue regeneration. and ) . in a healthy kidney, the polycystins localize to renal cilia. mutations that abrogate ciliary localization of pkd (yet preserve its channel function) also cause cysts. besides cilia, pkd is also found in other subcellular locations including extracellular vesicles (evs) of human urine. how dysfunction of pkd trafficking and localization leads to the kidney pathology remains unknown. pkd is evolutionarily conserved across all members of eumetazoa. in c. elegans, pkd- is exclusively expressed in ciliated male-specific neurons, where it is trafficked to cilia and evs. gfp-tagged pkd- -containing evs play a signalling role in inter-organismal communication between animals. conservation of polycystin- cellular localization between worm and human suggests that their network of molecular interactions may also be conserved. we propose that pkd- plays distinct roles in cilia versus ciliary evs. methods: to understand the role of evs in c. elegans inter-organismal signalling, we aim to identify the pkd- -associated ev proteome, transcriptome, and metabolome. we established a pipeline for fluorescent labelling and tracking specific ev cargoes in a living animal using super-resolution microscopy. we used fluorescence of the pkd- carrying evs to optimize biochemical procedures for their enrichment. results: our initial analysis revealed two populations of pkd- -carrying evs that differ in their densities: . - . versus . g/ml. we are currently characterizing these two distinct populations using transmission electron microscopy and refining our enrichment procedure for protein identification by mass spectrometry, sequencing of their rna cargoes and metabolome analysis. summary/conclusion: what function human pkd plays within the cilia and within the urinary evs is not well understood. identification of molecular mediators of c. elegans pkd- ev signalling will inform on the interactome of human pkd and its function in cilia versus evs. introduction: ectosomes play roles in many physiological and pathophysiological processes, and their precise is dependent on molecular cargo and parent cell type. a single cell can release distinct subpopulations of evs enriched with different molecular cargo, which adds complexity to elucidating cargo sorting and biogenesis mechanisms. in the nematode c. elegans, ectosomes bud from sensory neuron cilia and are released into the environment to modulate animal behaviour. methods: c. elegans is genetically tractable and optically transparent, allowing for live imaging of fluorescently tagged ev cargo. we express all tagged cargo at endogenous levels, adding physiological relevancy. results: we discovered that the calcium homoeostasis modulator ion channel clhm- localizes to cilia of ev-releasing neurons and observed gfp-tagged clhm- in ciliary evs. using super resolution microscopy, we imaged evs released from animals coexpressing tdtomato-tagged clhm- and gfp-tagged pkd- (another vesicle cargo) in the same neurons. while the two proteins colocalize in the cilia, clhm- ::tdtomato and pkd- ::gfp rarely colocalize in evs. this indicates that separate subpopulations of evs are being released from the same neurons. to determine how the clhm- subpopulation is formed, we are investigating candidate genes. anoh- , a homolog of the ca + scramblase tmem f, localizes to neuron cilia and induces phosphatidylserine exposure on the outer membrane leaflet. in anoh- mutants, the number of clhm- ::gfp evs released is significantly decreased but the number of pkd- ::gfp evs does not significantly change. in addition, i am using facs to isolate clhm- and pkd- containing evs and analysing the respective proteomes with lc-ms/ms. summary/conclusion: we are elucidating mechanisms that give rise to distinct subpopulations of ciliary evs in c. elegans and defining cargoes being enriched in these ev subpopulations to gain insight into ev cargo sorting and biogenesis mechanisms in ciliated neurons. ceramide accumulation induces exosome secretion through lysosomal protein laptm b kohei yuyama, hui sun and yasuyuki igarashi hokkaido university, sapporo, japan introduction: exosomes, a type of extracellular vesicles originated from multivesicular bodies (mvb), are important carriers of cellular molecules and have critical roles in intracellular communication in both health and disease. ceramides (cer) are implicated in biogenesis of exosome, however the molecular machinery that mediates exosome secretion remains obscure. lysosome-associated protein transmembrane- b (laptm b) is a lysosome/late endosome-resident transmembrane protein, which has been reported to bind cer. we demonstrate here that laptm b is involved in the exosome secretion, which are induced by exogenous cer treatment or lysosomal ceramidase inhibition in cultured neuronal cells. methods: neuroblastoma sh-sy y cells were treated with cer (porcine brain-derived cer or synthetic d : /c : ~c : cer) for h. exosomes were isolated from the culture supernatants by sequential centrifugation and their amounts were measured using ps capture exosome elisa kit. to analyse mvb transport, mvb and recycling endosomes are visualized with gfp-cd and rab immunostaining, respectively. results: we found that exogenous treatment of cer, especially those with c and c fatty acids, resulted in a marked increase in exosome secretion. in addition, lysosomal cer accumulation induced by acid ceramidase inhibition also accelerated exosome production. knockdown of laptm b significantly prevented the ceramide-dependent exosome release. in addition, we showed that these cer loading promoted colocalization of cd -positive mvb with rab -positive recycling endosomes, further demonstrated that laptm b knockdown cancelled the cer-dependent increase of the colocalization. summary/conclusion: these data suggest that lysosomal cer binds to laptm b and promote the transport of mvb to plasma membrane, resulting in an increase of exosome secretion in neuronal cells. chloroquine-mediated lysosomal inhibition alters composition and function of cancer-derived extracellular vesicles jing xu a , kevin yang a , shane colborne a , elham hosseini-beheshti b , gregg morin a , emma guns b and sharon m. gorski a a bc cancer, vancouver, canada; b the vancouver prostate centre, vancouver, canada introduction: small extracellular vesicles (sev) are signalling entities released by many types of eukaryotic cells. sev are of special interest in cancer due to their reported roles in modulating the cancer microenvironment and facilitating cancer cell invasion. macroautophagy (hereafter autophagy) is a catabolic process well-known for the recycling of cytosolic cargos through lysosome-mediated degradation. in this study, we profiled the changes in sev content and function under lysosome inhibition and investigated the involvement of autophagy machinery in sev content. methods: chloroquine (cq) was used to inhibit lysosomal degradation and autophagy turnover in triplenegative breast cancer (tnbc) cell lines. sev were collected via precipitation after pre-clearing and concentration of conditioned media. western blotting, nanosight and transmission electron microscopy were used to profile sev. quantitative mass spectrometry was used to characterize cq-induced changes in the sev proteome. antibody-conjugated magnetic beads were used in immunoprecipitation of sev. results: cq treatment did not substantially alter the physical properties of tnbc-derived sev. however, cq treatment altered the sev proteome and growth effects of sev on normal and endothelial recipient cells. cq treatment induced co-localization of mammalian atg proteins with endolysosomal markers in the cytoplasm, which coincided with an enrichment of atg s and their adaptor proteins in sev. cq-induced enrichment of atg s in sev required lipidation, and occured preferentially in one subset of sev. summary/conclusion: our study reveals changes in the content and function of cancer cell-derived sev in response to perturbation of intracellular trafficking pathways, demonstrates the flexibility and heterogeneity of sev composition, and has implications for cq efficacy in therapeutic settings. introduction: introduction: argonaute (ago ) is the essential component of the rna-induced silencing complex (risc) that binds mirnas and promotes mrna degradation. extracellular vesicle (ev)-carried mirnas have been shown to influence gene expression and functional phenotypes in recipient cells. many investigators have found ago in evs and it is postulated that ago is a major transporter of mirnas into small evs (sevs), such as exosomes. others have reported extracellular ago that is non-vesicular. we set out to evaluate the effect of growth factor signalling and serum contamination on the detection of ago in sevs. methods: methods: wildtype kras colorectal cancer cells, dks , were conditioned with different culture media (serum-free dmem, dmem supplemented with ev-depleted fbs, and opti-mem). evs were purified from conditioned media by cushion-density gradient ultracentrifugation. western blot analysis of dks total cell lysates, large evs and density gradient fractions was performed, probing for ago and ev marker proteins. the size and concentration of the evs were determined by particle metrix analysis. results: results: in all conditions, we found the highest abundance of sevs in fractions and , as assessed by western blot analysis. ago was detected in the same fractions as sevs in both the serum-free dmem and opti-mem conditions, although the levels of ago was higher in the serum-free dmem fractions compared to that of opti-mem. in contrast, ago was present in both vesicular and non-vesicular fractions in the dmem supplemented with ev-depleted fbs condition. no significant differences were observed in the size and number of evs collected in the three conditioning methods. summary/conclusion: summary/conclusion: the presence or absence of ago in evs has been controversial. multiple factors may affect the ability to detect vesicular ago , including serum and growth factors in the conditioned media that may provide sources of extravesicular ago and also regulate the trafficking of ago into vesicles. introduction: cancer-associated glycosphingolipids have been utilized as tumour markers and targets of cancer therapy. we have investigated roles of gangliosides in cancers, and clarified that cancerassociated gangliosides enhance malignant properties of cells by forming complexes with membrane molecules in lipid rafts. in this study, we analysed contents of gangliosides and membrane molecules on extracellular vesicles (ecvs) secreted from melanoma cell lines. methods: melanoma cell lines with various ganglioside patterns were used for isolation of ecvs. gangliosidemodified melanomas with genetic engineering were also used. genetic modification was done by cdnas of ganglioside synthase genes. ecvs were collected by ultra-centrifugation, or by tim -beads. contents in ecvs were analysed by immunoblotting or flow cytometry. roles of lipid rafts in the generation and secretion of ecvs were analysed by treating cells with mm methyl β-cyclodextrin. results: using melanoma cell lines, ecvs were isolated by ultra-centrifugation, and their sizes were analysed by nanosight. all samples showed uniform sizes between and nm. protein amounts in ecvs were measured, showing heterogeneous levels at ~ μg/ ml. then, gangliosides expressed on ecvs from these cell lines were analysed using tim beads and flow cytometry. gd and gd were detected on ecvs almost proportionally with expression levels of those gangliosides on the cell surface. then, immunoblotting was performed to analyse integrin levels in ecvs from transfectant cells expressing high levels of gd , showing increased levels of integrins in ecvs from gd + cells compared with those from gd -cell lines. integrin levels in cell lysates from these cells (gd + and gd cells) were almost equivalent. treatment of a gd -expressing melanoma cell line by mm methyl β-cyclodextrin resulted in marked reduction of secreted ecvs and amounts of tsg in them. summary/conclusion: ganglioside expression patterns on melanoma cells were well reflected in the expression of gangliosides on ecvs. these results as well as increased levels of integrins in ecvs from gd + cells suggest that gangliosides and lipid rafts are involved in the generation and secretion of ecvs. introduction: hypoxia, or low oxygen tension, is a common feature associated with tumour growth and is known to regulate tumour cell function, especially through rewiring of cell metabolism. however, how hypoxia influences tumour cell interactions with surrounding cells is not fully elucidated. we sought to evaluate how hypoxia alters metabolite and metabolism-associated mirna packaging in exosomes. methods: exosomes were isolated from t breast cancer cells cultured in normoxia ( % o ) and hypoxia ( % o ) via ultracentrifugation, optiprep gradients, and size exclusion chromatography. exosomes were further characterized by nanosight, qubit protein quantification, and flow cytometry analysis of exosome markers. metabolite and mirna profiling was performed on exosomes and exosome-producing cells in normoxia and hypoxia. results: secretion of exosomes was increased under hypoxic conditions. metabolite profiling revealed alterations in metabolites specific to exosomes derived from hypoxic cells. profiling of exosomal mirna showed packaging of metabolism-related mirna into exosomes derived from hypoxic cells. summary/conclusion: hypoxia alters the metabolite and mirna profiles of cancer cells, with selective packaging of these molecules into exosomes. we identified metabolites and mirna that are depleted and enriched in exosomes compared to cells. these studies identify hypoxia-associated shifts in exosome cargo, providing insight into exosome cargo packaging with potential implications for understanding how cancer cell-derived exosomes regulate recipient cell function. lysosomotropic agents prompts the release of extracellular vesicles carrying autophagy-associated markers: evidence of a general mechanism of secretion driven by lysosomal impairment introduction: drug-induced lysosomal storage disorders (lsds) are due to the transient intracellular accumulation, mostly of phospholipids, into multilamellar inclusion bodies within late endosomal/lysosomal compartment. they represent a major side-effect for many drugs of several pharmacological categories. most lsds inducers are cationic amphiphilic drug (cad), but the molecular mechanisms leading to accumulation of undigested substrates are unknown. extracellular vesicles (evs) have been implicated in cell waste disposal, but it is unclear whether they might be involved in extracellular release of undigested substrates. methods: to investigate this aspect, we developed hek cells stably expressing the fluorescent fusion proteins egfp-cd and mcherry-cd , separated evs by differential ultracentrifugation and quantified by evassociated fluorescence and nta particle count. results: evs released by these models upon treatment with drugs inducing the accumulation of phospholipids (amiodarone) or glycosaminoglycans (tilorone), showed the release of fluorescent medium/large evs ( k fraction) and small evs ( k fraction), whose size and distribution were similar to the same vesicles released by control cells, but enhanced the recovery of medium/large evs and to a lower extent of small evs, analysis of evs associated markers revealed a dosedependent increase of autophagy-associated markers in medium/large and small evs. similar results were obtained when autophagic flux was impaired by drugs raising lysosomal ph by different mechanisms, such as chloroquine and bafilomycin, but not when autophagic flux was stimulated by drugs such as curcumin or overexpression of the endosomal/lysosomal regulator tfeb. summary/conclusion: overall results show that impairment of autophagic flux, either by indigested substrates or higher lysosomal ph, is associated with an increased release evs enriched in autophagy markers, compatible with autophagomes and/or amphisomes, unravelling a connection with secretory autophagy. tomofumi yamamoto a , yusuke yamawaki b , yutaka hattori c and takahiro ochiya a a tokyo medical university, shinjuku-ku, japan; b national cancer center research institute, chuo-ku, japan; c keio university faculty of pharmacy, minato-ku, japan introduction: multiple myeloma (mm) is a haematological tumour. last decade, the prognosis of mm has improved by the development of therapeutic drugs; however, mm cells acquire drug resistance by longterm exposure of these therapeutic drugs. one of the possible explanations of drug resistance is that cells with drug resistance transmit information to other mm cells and their microenvironmental cells. although the elucidation of the mechanism of drug resistance in mm have been desired, it remains poorly understood. methods: in order to understand the mechanism of drug resistance in mm, lenalidomide resistant cell lines were established by long-term exposure of low concentration of lenalidomide. drug resistance was assessed by mts assay and caspase assay. the amount of ev was measured by exoscreen, which is ultra-sensitive detection method of evs by measuring surface protein of evs, such as, cd and cd (yoshioka et al., nat commun., ) . to identify the genes which involved in drug resistance, rna sequence among the drug-resistant cell lines and their parental cell lines was performed. results: firstly, characterization of these cells was confirmed. we found that all of the lenalidomide resistant cell lines secreted more evs than their parental cell lines. in addition to this, the size of ev derived from resistant cells are smaller than those of parental cells. next, we collected evs from resistant cells and parental cells by using ultracentrifugation, and added them to parental cells in the presence of lethal dose of lenalidomide. compared with ev derived from parental cell lines, the evs derived from lenalidomide resistant cell lines increased a number of living parental cells. these results suggested that the evs derived from lenalidomide resistant cells can affect the lenalidomide sensitive cells. as a result of rna sequence, several genes highly expressed in resistant cell line we found, which associated with lysosome pathway. among them, attenuating the sort and lamp genes could significantly reduce the ev secretion in mm cells, leading to enhance the lenalidomide sensitivity. summary/conclusion: our results showed that ev secretion via sort or lamp could induce the drug resistance in mm. study on biological stimulate mechanism of stem cell-derived exosome generation by nanoparticles introduction: mesenchymal stem cells (mscs) are pluripotent stromal cells known to release extracellular vesicles (evs) containing various growth factors and antioxidants that can positively affect surrounding cells. nanoscale msc-derived evs, such as exosomes, have been developed as bio-stable nano-type materials, but had low yield and were difficult to quantify. we hypothesized that the mechanism of nanoparticleenhanced exosome production would stimulate intracellular molecules. the aim of this study was to elucidate the molecular mechanisms of exosome generation by comparing the internalization of surface-modified positively charged nanoparticles and exosome generation from mscs. methods: mesenchymal stem cells (mscs) were cultured in mem-alpha with % fbs and × antibiotics. the positively charged nanoparticles were synthesized by poly-lactide-co-glicolide (plga) and polyethylenimine (pei) with cy . for tracking nanoparticles. all of the exosome image were identified using an electron microscope. additionally, it was confirmed the internalization of the nanoparticles by if. the primary antibodies used were anti-eea , anti-rab and anti-gm . in order to prove the development of exosomes, rt-pcr using autophagy-related mrna was performed. real-time rt-pcr was performed using the applied biosystems sequence detection system . lastly, mirna from msc-derived exosome analysed automatically in the affymetrix data extraction protocol using the provided affymetrix genechip® command console® software (agcc). all statistical testing and visualization of differentially expressed genes was conducted using r statistical language . . results: we determined that rab , located in the mvb and autolysosomal membrane, was increased upon exosome expression and was associated with autophagosome formation. these results suggested that nanoparticles migrated to lysosomes during treatment; however, intracellular exosome-forming factors were stimulated during endosomal maturation simultaneously. summary/conclusion: therefore, msc-derived exosome research using nanoparticles is useful for increasing exosome yield and the discovery of nanoparticleinduced genetic factors. theoretical description of formation of extracellular vesicles by budding of membrane introduction: understanding mechanisms of extracellular vesicles (evs) formation is of utmost importance for their effective use in science, medicine and technology. in particular, the discovery of universal mechanisms explaining the phenomena taking place in vesiculation appears to be crucial and highly warranted. mammalian erythrocytes and giant phospholipid vesicles have been largely used as model systems to study principles of membrane budding and vesiculation. the mechanisms conveniently studied in these simple systems are then generalized to other types of biological membranes. we present a theoretical description of membrane budding and compare the theoretically obtained shapes with the observed ones. methods: in accordance with the fluid crystal mosaic model, membrane is considered as composed of constituents (inclusions) subjected to the local curvature field created by surrounding constituents. constituents can attain different in-plane orientations in the membrane which correspond to different energies. the thermal motion oposes the complete orientational ordering. the single-constituent energy expresses a mismatch of the curvature of the membrane at the position of the constituent and the intrinsic principal curvatures of the constituent and inplane orientation of their principal axes. the free energy of the whole membrane is obtained by summing up (integration) the contributions of the constituents and using methods of statistical physics, and minimized by using numerical methods. results: to outline the principle of (outward and inward) budding, respective sequences of shapes corresponding to a formation of one (outward and inward) spherical bud were calculated by minimization of the free energy. also the corresponding shapes observed in evs (imaged by electron microscopy) and in erythrocytes and giant phospholipid vesicles (imaged by optical microscopy) are shown. it can be seen that theoretically calculated shapes and experimentally observed ones agree well over up to orders of magnitude (the order of the size of giant phospholipid vesicles is between and micro metres, in erythrocytes it is about micro metres and in evs it is about nanometres). summary/conclusion: budding of the membrane is an universal mechanism in formation of external and internal vesicles. introduction: the release of extracellular vesicles (evs) from cells is important for many cellular mechanisms both in normal physiology and in disease. arrdc (arrestin domain containing protein ) is an adaptor protein known to facilitate the ubiquitination of target substrates by nedd family ubiquitin ligases. it also traffics cargo to extracellular vesicles. previous studies show the involvement of arrdc in the trafficking of the divalent metal ion transporter dmt to evs in a ubiquitin-dependent manner, and we aimed to further understand this mechanism. methods: we performed mass spectrometry to identify ubiquitinated lysine residues in arrdc . we then generated arrdc wt and lysine mutant clones and expressed these in cells to determine the effect on ev biogenesis and protein trafficking. results: mass spectrometry data identified potential ubiquitinated lysine residues. out of these, lysine appeared to be the most important for arrdc function. arrdc k r mutation caused a decrease in the number of ev released by the cell compared to arrdc wt, and a reduction in trafficking of dmt to evs. furthermore, we also observed a decrease in dmt activity and an increase in its intracellular degradation in the presence of arrdc k r. k also appeared to be ubiquitinated with k polyubiquitin chains by the ubiquitin ligase smurf . summary/conclusion: our data suggests that k polyubiquitin chains are the signal for arrdc mediated ev biogenesis and protein trafficking, and loss of this signal causes cargo to be rerouted to intracellular degradation mechanisms. chair: tanina arab -department of molecular and comparative pathobiology, johns hopkins university school of medicine a d-printed model to represent the structure and nature of extracellular vesicles, for public engagement and education events. christian burton a , sara veiga a , jason webber a , kate milward a , muireann ni bhaoighill a , lauren evans a , andreia de almeida b , rachel j. errington a and aled clayton a a cardiff university, cardiff, uk; b cardiff university, research associate, uk introduction: explaining the field of extracellular vesicles to the lay public and young audiences can often be challenging. whilst diagrams and images of evs may be helpful, conveying clearly the shape and composition of an ev by these means is not always a success. whilst many members of the audience may be familiar with concepts of cells and related structures, others will find such discussions very abstract and challenging. in order to aid interactions with lay audiences we embarked on the design of a physical hand-held plastic model, representing a typical ev. incorporating flexibility in the design allowing the community to adapt it to showcase their own research. the second goal was to ensure manufacturability using widely available dprinting technologies. methods: the basic model design was conceived by dr c. burton, and iteratively developed using solidworks, , then exported for use in any cad environment (stl format). a model showing a halved ev hemisphere, with a visible lipid-bilayer was developed. attachable rings allow trans-membrane-molecules to be represented, current designs include mhc class-i, hspgs, integrins, tetraspanins and supported by handouts accompanying the models. intraluminal cargo is included via removeable "pegs", and examples representing rna or simple globular proteins, and a template has been created. results: the design is free and open source, and available to the community at: https://www.thingiverse. com/thing: . instructions for d printing are available from the uk extracellular vesicle society website; https://www.ukev.org.uk/public-engagementmaterials/. models have been produced using entrylevel d printers and trialled at engagement events with good early responses. summary/conclusion: the authors hope the community will use and develop this d-model design and that the approach provides an additional and helpful tool for educating audiences about the complexities and roles of evs in biology and disease. centrifugal filtration-sec is promising for extracellular vesicle isolation from d and d her + breast epithelial cell lines introduction: despite recent developments in breast cancer therapy, there is still need for a more targeted approach. extracellular vesicles (evs), endogenous nanovesicles released from human cells, are an attractive choice as nanodrug carriers due to their size, stability and their unique targeting specificity. the aim of this study was to determine if centrifugal filtration (cf) combined with size exclusion chromatography (cf-sec) would be useful for ev isolation from two epithelial breast cell lines d and d her +, representing the tissue of interest, and the amount of cell culture needed to get measurable ev concentrations. methods: cell culture media (without serum) from the immortalized breast epithelial cell lines d and d her + was concentrated with centrifugal filtration (cf) followed by isolation with size-exclusion chromatography (sec) using hiprep / sephacryl s- column run with Äkta start ( nm), min runs. each fraction ( - ml) was collected with fraction collector. dulbecco's particle free pbs was used as mobile phase. the resulting particles were analysed with nanoparticle tracking analysis (nta, nanosight ns , camera gain , static mode, capture time sec), western blotting (wb), microbca and transmission electron microscopy (tem, samples fixed with % formaldehyse and stained with % uranyl acetate, run at kv). results: although sec did not show any prevalent peaks from early eluting regions previously shown to contain extracellular vesicles, these fractions (f -f , - min) were collected from d her + cell culture medium. interestingly, both nta and tem suggest that f and f contained evs as the isolated particles measured and nm, respectively and tem revealed spherical particles - nm in diameter. wb was unable to detect the ev associated protein alix (but was present in the whole cell lysate). soluble proteins and protein aggregates eluted late in the sec chromatogram ( min), with protein analysis (microbca), tem and wb confirming their presence. summary/conclusion: cf-sec is a promising method for ev isolation for pharmaceutical applications, but further work is needed to optimize the isolation process using Äkta start for these cell lines. customer stories from the ev core of university of helsinki introduction: the ev core, world's first ev-dedicated technology platform established in , is a joint venture of two extracellular vesicle (ev) research laboratories at university of helsinki. as an academic research/service facility, the ev core provides infrastructure, state-of-the-art and emerging ev-technologies for research groups, hospitals, companies and authorities in the ev-field. the ev core provides ev isolation, purification and characterization services and offers contacts to downstream analyses in other core facilities based on optimized ev protocols. here, we present and discuss the customer experiences and prospects with the aim to further develop ev core services. methods: our most wanted services are nanoparticle tracking analysis, electron microscopy, ev isolation and rna isolation and consultation. currently, the key down-stream analysis methods are (mi)rna sequencing, metabolomics, flow cytometry and functional assays. results: we present the stories from our customers starting with their research questions and need for the ev expertise/consultation and equipment. next, we show how the projects advanced and what types of ev core -derived or other downstream services helped them to achieve their aims. in the end, we will acknowledge the customers experience and current status of their research. summary/conclusion: narratives of customer stories are an effective starting point for fruitful discussions about the current status and next developments in the young ev service field. recent isev workshops: open, reproducible and standardized ev research (ghent, ) and evs in immunology (buenos aires, ) introduction: since its founding in , isev has sought to further extracellular vesicle research in various ways including scientific meetings. these events encompass annual meetings as well as smaller, topically focused workshops, with the first isev workshop (on rna and evs) organized in new york city in october, . in december, , the workshop "open, reproducible, and standardized ev research" was held in ghent, belgium. in march, , the workshop, "evs in immunology" was held in buenos aires, argentina, with a preceding education day. methods: the international organizing committees of the and isev workshops prepared scientific programs around key themes of ev rigour and standardization (ghent, belgium, workshop) and evs in immunology (buenos aires, argentina, workshop). abstract and application submissions were invited. applications were reviewed and ranked by panels of ev experts for each event, and participants were invited. results: the and workshops assembled a total of more than individuals for talks and discussions around the themes of rigour and standardization and evs in immunology. the buenos aires workshop was preceded by an education day, coordinated by the isev executive committees for education and science and meetings. during these two workshops, poster presentations were permitted for the first time, affording additional presentation and interaction opportunities. the rigour and standardization workshop also featured real-time discussant polling to facilitate discussion. summary/conclusion: isev workshops such as those addressing rigour and standardization (ghent, ) and evs in immunology (buenos aires, ) continue to provide opportunities for focused discussion of small groups of experts on key topics in the field. often followed by published products, isev workshops help to lead and coordinate progress in ev science. for future isev workshops, educational activities may again expand the reach of each event, while poster sessions and app-driven real-time responses should be considered for enhanced interactions and participant canvassing. ev journal club: exchanging pizza for a worldwide audience during covid- kenneth w. witwer johns hopkins university school of medicine, baltimore, usa introduction: a monthly journal club focused on extracellular vesicle science was established at johns hopkins university in , featuring lunch and presentations by academic and industry participants. when covid- prevented in-person meetings beginning in march, , the journal club was converted to a virtual, weekly format on the popular online meeting app zoom. the journal club has persisted despite initial problems with online vandalism. most sessions are also made public on a youtube channel, https:// www.youtube.com/c/extracellularvesicleclub. methods: weekly ev club sessions are arranged by the host. most focus on a specific manuscript related to evs, but some weeks feature presentations of published or soon-to-be-published research by the presenting authors. sessions are advertised one week to several days in advance on social media platforms such as linkedin, twitter, and facebook, asking interested parties to sign up to join a mailing list via surveymonkey. the log-in information is then sent to the mailing list. upon clicking the link, participants are placed in a virtual waiting room for vetting by the host and volunteers. after admission, all parties but the host and presenter are muted to avoid distractions. questions and comments may be placed in a chat box. contributions are monitored and compiled by the host and volunteers to build a question-and-answer session at the end of the presentation. recorded sessions-with or without editing as needed-are placed on the youtube channel for additional access. results: despite initial problems with online vandalism known as "zoombombing," the journal club has continued weekly during the covid- shutdown in the host country (us). an audience of between and individuals is typical. participants typically ask more questions than can be answered in a one-hour time frame. the online format also allows for debate-style events and polling of the audience. summary/conclusion: this ev journal club is an example of how online tools can be used to facilitate international scientific interactions. further development of such formats could provide alternative approaches for isev activities in the science, education, and communication areas. the study aim is to assess whether the exposure to pm and pm , , chosen as paradigmatic environmental stressors, could modify the composition of nasal microbiota (nm) and extracellular vesicle (ev signalling network, showing a role in allergic ar exacerbation). methods: nm analysis were performed on v -v s rrna gene regions amplified from upper-airway tracts of ar cases and healthy individual controls to perform nm analyses. ev size, concentration and cellular origin for each subject were assessed by nanoparticle tracking analysis (nta) and flow-cytometry (fc). information on daily pm and pm , concentrations at the municipality of residence in the days preceding nasal sampling (i.e. day − to day − ) was assigned to each subject by arcgis software. multivariable and logistic analyses were applied on nm, nta and fc outcomes. results: when taxonomy composition was considered, in controls actinobacteria ( . %) was the most represented, followed by firmicutes ( . %) and proteobacteria ( . %) while in cases proteobacteria were . %, actinobacteria were . % and firmicutes were . %. cases showed a higher concentration of all the investigated ev types, derived from platelets (cd +), activated endothelium (cd e+), monocytes (cd +), eosinophils (cd +), neutrophils (cd +), mastocytes (cd c+), epithelial cells (epcam+), gram+ bacteria (lipoteichoic acid+), gram-bacteria (lps+). the effect was greatest in the case of mastocytes evs which were increased . fold in cases versus controls (p < . ). evs were modified by pm exposure at several time lags. in particular, a negative association between pm and eosinophil evs was observed (beta = − , ; pvalue = , ). as we clustered subjects according to their nm, we observed this variable was a strong effect modifier of the association between pm exposure and ev release. summary/conclusion: our findings start to provide an insight on the effect of air pollution on evs, taking into account the effect of nm, in patients with ar. further research is necessary to disentangle the mechanism exerted by inhaled pollutants in modulating evs and nm, and therefore ar exacerbation. funding: gsk investigator sponsered study aryl hydrocarbon receptor activation induces the expression of specific microrrnas in th cells that are release into extracellular vesicules and associated with arthritis introduction: in rheumatoid arthritis (ra), an autoimmune disorder characterized by a chronic sinovial inflammation, smoking is a major risk factor contributing to disease progression, and poor response to therapy. th cell is actively involved in worsening smooking-associates inflammation mediated by aryl hydrocarbon receptor (ahr), a cytoplasmic transcription factor involved in xenobiotic metabolism. both, ahr and th cells, has important implications during ra development. considering that cigarette smoke is a potent epigenetic modifier, we hypothesized that ahr activation, by cigarette components, would transcribe specific micrornas in th cells as a molecular mechanism to exacerbate inflammation in arthritis. methods: microrna expression was evaluated by largescale approach or real-time pcr. c /bl and ahr null mice were submitted to arthritis experimental models and exposed or not to cigarrete smoke (ethical committee approved / ). extracellular vesicles (evs) were isolated by ultracentrifugation, and characterized by western blot and nanosight. rankl-induced osteoclasts (ocs) differentiation in vitro was stained for trap. inhibition of mirnas were performed using anti-mirs transfection. results: we identified a specific group of mirnas induced in th cells after ahr activation. during arthritis progression, the micrornas are expressed and increases after exposure to cigarette smoke. in the absence of ahr their levels were drastically reduced. interestingly, we found that these micrornas are released by th cells into evs, and are able to promote osteoclastogenesis. ocs differentiation in vitro increases in the presence of th -derived evs, and this process is reduced in the absence of micrornas. summary/conclusion: microrna-mediated gene regulation plays crucial roles in the immune system functions, and their abnormal expression is highly correlated with the pathogenesis of ra. evs are known to function in cell-to-cell communication and are able to transmit their contents and cause changes in the target cell. our findings demonstrate a new molecular mechanism by which cigarette smoke could aggravate inflammation in arthritis; through the activation of ahr receptor in th cells, inducing the transcription of specific micrornas that are released into evs, and act as pro-inflammatory mediators. introduction: chagas disease (cd) is caused by the flagellated protozoan t. cruzi. trypomastigote forms are capable of releasing extracellular vesicles (evs) that contain the major surface molecules of the parasite. the parasite has a complex life cycle that leads to it a rapid adaptation in the environmental changes in the hosts. however, the effects of stress on on evs release are not completely understood. objetive: we evaluated the release of evs by trypomastigotes incubated under different stress conditions and the immunomodulatory role of these evs in pre-activated bone marrow-derived macrophages (bmdm). methods: nanoparticle tracking analysis (nta) and scanning electron microscopy (sem) showed an increase in evs releasing by trypomastigotes at °c under acidic conditions, evs released was affected and triggered amastigogenesis process. results: treatment with sodium azide (nan ) also caused changes in the release of evs regarding size and concentration. nitrosative stress caused by sodium nitrite (in culture medium mildly acidic, ph . ; in this condition nano releases nitric oxide) stimulated an increase in production of evs by t. cruzi. when the parasites were treated with nm s-nitrosoglutathione (snog), we observed a reduction in size and concentration of vesiculate material by trypomastigotes. at a higher snog concentration ( µm), the concentration of the vesiculate material increased. t. cruzi-derived evs exposed to stress conditions increased the expression of inos, arg , il- and il- genes in ifn-γ and lps pre-activated bmms. summary/conclusion: results suggest that the viability and/or integrity of the parasite are necessary for the evs releasing. in those in vitro conditions they triggered a proinflammatory response in host cells. this may be a strategy developed by the parasite to favour its establishment in the host. funding: fapesp, cnpq, capes and fapemig ppm-x / . immuno-toxicological evaluation of human mesenchymal stem cell- introduction: mesenchymal stem cells (mscs) have been widely used to the field of autoimmune diseases or tissue regeneration therapy. recently, many research groups have reported that mscs showed their ability via secreted paracrine mediators including extracellular vesicles (evs) rather than cell-to-cell contact. mscs mainly exist on bone marrow, peripheral blood, umbilical cord and adipose and can mostly secrete evs. it has emerged that evs alone are responsible for the therapeutic effect of mscs in plenty of animal diseases models. hence, msc-derived evs may be used as an alternative msc-based therapy in regenerative medicine. methods: as part of safety programme for human therapeutics, we performed immunotoxicological assessment of evs obtained from human mscs (hevs) in mice and human peripheral blood mononuclear cells (hpbmcs). firstly, mice were treated intravenously with a negative control, a positive control (lps; . mg/kg), or low-dose ( x e paticles/head) and high-dose ( x e paticles/ head) of hevs every other day for days and then analysed lymphocyte subsets from collected spleen by facs. next, we treated the evs on hpbmcs for days with low conc. ( x e particles/ml), high conc. ( x e particles/ml), pma/ionomycin as a cell activator or cpt ( μm) as an apoptotic inducer. annexin v/pi and csfe were analysed by facs. results: as a result, splenic nk cells and b cells were slightly increased about ~ % in hevs-treated mice, without biological significance, compared with a positive control (lps) as an immunogenicity inducer. and there were no effects on serum levels of inflammatory cytokines in mice. in addition, hevs had no cytotoxic effect on hpbmcs at both low and high conc. under the culture medium with evs-depleted fbs, the hevs appeared minimal anti-apoptotic effect on hpbmcs. for the cfse assay, the hevs showed slight proliferation on hpbmcs and pbmc activation induced by pma/ionomycin. summary/conclusion: in conclusion, the hevs have little immuno-toxicological effects in mice and hpbmcs. further detailed studies to elucidate immunological response of hevs for development of human therapeutics are needed. funding: this research was supported by a grant ( mfds ) from ministry of food and drug safety. investigation of immune response to mesenchymal stem cell-derived extracellular vesicles in the cancer setting introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) are thought to be a fingerprint of the secreting cell and therefore may retain the cancer targeting and immune privilege of mscs. thus msc-evs hold immense potential as tumour-targeted therapeutics for breast cancer. the aim of this study was to determine whether msc-ev administration in tumour bearing immunocompetent animals would initiate an immune response. methods: evs were isolated from conditioned media of both human and murine bone marrow-derived mscs through sequential differential centrifugation, microfiltration and ultracentrifugation. evs were characterized by nanoparticle tracking analysis (nta), western blot and transmission electron microscopy (tem). x ( ) human or murine msc-evs were administered intravenously into t breast tumour bearing balb/c mice (n = ) and healthy controls (n = ). tumour tissue, draining lymph node and spleen were then harvested, dissociated and flow cytometry performed targeting markers associated with a range of immune cells including t-cells, macrophages and natural killer (nk) cells. results: evs were successfully isolated from murine and human mscs with the appropriate size of small evs (sevs: - nm) and morphology including a lipid bilayer observed by tem. evs expressed tetraspanins cd , cd , cd ; cytosolic protein tsg and were negative for calnexin. ev concentrations ranged from . x ( ) - . x ( )/ml. in order to study a range of immune cell populations two antibody panels were created using complimentary fluorescent dyes. the proportion of t-cells (cd +, cd +, cd +), neutrophils (gr- +, ly- c+), dendritic cells (cd c+), macrophages (cd b+, mhci+, mhcii+), nk cells (cd +) and b cells (cd +) remained stable in the tumour, draining lymph node and spleen of all tumour-bearing animals that received either human or murine msc-evs, with no significant change observed in any category. summary/conclusion: the data presented supports the hypothesis that msc-evs retain the immune privilege of the secretory cell, with human cell-derived evs illiciting no immune response in mice. this is encouraging and reinforces the potential for use of msc-evs in the therapeutic setting. introduction: mycobacterium avium (m. avium) is a slow growth rate non-tuberculous mycobacterium (ntm). m. avium infection is a severe global health problem. but the mechanisms of pathogenicity of m. avium are poorly understood. outer membrane vesicles (omvs) that traverse the cell wall and contain a varied bioactive components inculding dna, rna, protein and toxins. previous studies have suggested that these omvs are produced in vitro and during animal infection, but the role of omvs secretion during the interaction of m. avium with host cells remains unknown. methods: in this study, m. avium were grown in middlebrook h medium (m h ) supplemented with % (v/v) oadc enrichment and . % (v/v) glycero. m. avium omvs were isolated by ultracentrifugation method. characterization of omvs by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). the raw . murine macrophages were incubated with the m. avium omvs to analyse inflammatory response and production of nitric oxide (no) and reactive oxygen species (ros) of macrophage. results: in this study, we demonstrate by fluorescence microscopy that murine macrophages can phagocytosis omvs produced by m. avium. incubation of m. avium omvs with murine macrophages resulted in increased levels of extracellular tumour necrosis factor alpha (tnf-α), interleukin- β (il- β), terleukin- (il- ) and interleukin- (il- ). meanwhile omvs stimulated macrophages produce no and ros. introduction: hospital associated venous thromboembolism (ha-vte) in paediatric patients is the second most common contributor to harm in hospitalized children. platelet-endothelial interactions are integral to the formation of vte, especially in inflammatory conditions such as sepsis. small extracellular vesicles (sevs) have the ability to reprogramme target cell phenotypes via their microrna contents and are known to contribute to vte formation. we hypothesize that sepsis alters platelet-derived sev micrornas capable of net upregulation of vascular endothelial procoagulant and downregulation of anticoagulant pathways. methods: using a precipitation solution and size exclusion chromatography, we isolated sevs from platelet poor plasma of children admitted to the paediatric intensive care unit for sepsis and from healthy controls. we positively selected platelet-derived sevs using immunomagnetic isolation for cd b platelet antigen and confirmed selection using flow cytometry. microrna was profiled using affymetrix genechip mirna . array. results: microrna from sepsis patients (median age . years; iqr: . - and % female) with a median psofa score of (iqr: . - ) and from healthy controls (median age years; iqr: . - . and % female) was isolated and compared. in septic vs. healthy patients mirnas were differentially expressed (false discovery rate (fdr)< . ; fold change ≥| . |) affecting mrna pathways. in septic children, pathways affecting chemotaxis and cell movement of leukocytes were predicted to be activated with z-scores ≥ . summary/conclusion: we developed a method to successfully isolate platelet-derived sevs. sepsis alters the platelet-derived sev microrna profile in paediatric patients with sepsis. these micrornas are predicted to target chemotaxis and cell movement pathways, important contributors in the formation of ha-vte. further analysis into specifically targeted pathways should be conducted as a potential target for the prevention of ha-vte in sepsis. introduction: sjögren´s syndrome (ss) is a systemic autoimmune disease that mainly affects salivary and lacrimal glands. mechanisms of ss pathogenesis are poorly understood. it is thought that inflammation leads to destruction of exocrine glands, however the triggers of autoimmunity and the mechanisms by which inflammation drives immunopathology are not characterized. our work identifies t cell-exosomederived mir- - p as a pathogenic driver of immunopathology in ss. micrornas (mirnas) are endogenous small noncoding rna molecules that regulate the expression of target genes through translational repression of mrnas. through transcriptomic profiling studies our group had previously documented a significant upregulation of mir- - p in patient ss tissues and in serum exosomes. methods: structured search for target genes of mir- - p involved in salivary gland (sg) physiology was performed with mirdip . serca b, ryr and ac were selected for further validation and functional analysis. binding of the mirna was confirmed by luciferase reporter assays in hsg cell lines and human-derived primary epithelial cells. the mrna and protein levels of serca b, ryr and ac were determined by qpcr and western blot, respectively. to investigate the cell-specific distribution of mir- - p in relation to the expression levels of serca b, ryr , and ac , a double fluorescent in situ hybridization was performed. ca + signalling and camp levels were measured using fluorescent sensor. to isolate exosomes, the t cell medium and serum of ss-patients and healthy volunteers (hv) were collected. results: we show that mir- - p is over-expression in the sgs of ss-patients. next, we demonstrated that mir- - p is contained in exosomes in serum of sspatients significantly more than serum of hv. we also show that activated t cells secrete exosomes containing mir- - p which transfer into glandular cells and affecting intracellular ca + signalling, camp production and protein production by mir- - p targets (serca b, ryr and ac ). summary/conclusion: this study provides evidence for a functional role of the mir- - p in ss pathogenesis and promotes the concept that t cell-activation directly may impair epithelial cell function through secretion of mi-rna containing exosomes. treg-derived il -coated extracellular vesicles promote infectious tolerance (p ) subunits, yet the forms that il assumes and its role in peripheral tolerance, remain elusive. methods: we induce cba-specific, il -producing t regulatory (treg) cells in tregebi wt c bl/ reporter mice, and identify il producers by expression of ebi tdtom gene reporter, plus ebi and p proteins. results: curiously, both subunits of il were displayed on the surface of tolerogen-specific foxp + and foxp neg (itr ) t cells. furthermore, il producers, although rare, secrete ebi and p on extracellular vesicles (ev) targeting a -to -fold higher number of t and b lymphocytes, causing them to acquire surface il . this surface il is absent when ev/exosome production was inhibited, or if ebi is genetically deleted in treg cells. summary/conclusion: the unique ability of ev to coat bystander lymphocytes with il , promoting exhaustion in, and secondary suppression by, non-treg cells, identifies a novel mechanism of infectious tolerance. funding: nih grants r -ai - (to w.j.b.), r ca and p ca (to d.a.a.v.) and the university of wisconsin carbone cancer center support grant p ca . unique formulated dual targeting antigen specific and delivered mirna- gene regulating exosomes acting at the immune synapse to induce apc-derived secondary suppressive exosomes introduction: an exosome-apc circuit we uncovered may be applicable beyond skin immunity we study in mice. methods: high antigen dose tolerized cd + t cells make suppressive antigen-specific exosomes due to chosen surface antibody light chains that enable targeting antigen presenting cells (apc) antigen-specifically for delivery of also chosen inhibitory mirna- to mediate specific functional gene alterations. results: both antigen and gene specificity aspects are lent to naïve but activated exosomes by simple in vitro incubations alone. for mechanism, these primary exosomes bind antigen peptides in mhc on apc that in turn make secondary suppressive exosomes that act peptide/mhc-specifically on the effector t cells at the immune synapse. they transfer another mirna for strong prolonged inhibition of active delayed-type hypersensitivity (dth) for days even, when the primary mirna- -pos exosomes are administered orally at the height of the in vivo response, in a physiological dose. summary/conclusion: it is shown possible to induce therapeutic exosomes with ag targeting of choice due to placed ab on the surface and that also target specific gene functions of acceptor cells due to carriage of a selected mirna. this dual ag and gene-specific therapy has applications in treatment of cancer, autoimmunity and allergies. introduction: previously, our group characterized distinct populations of extracellular vesicle (ev) released from neutrophilic granulocytes: ev formed spontaneously (sev) and upon activation with opsonized particles (aev). the aev differs in protein cargo and its ability to inhibit bacterial growth. we described that mac- integrin (cr receptor) plays key role in the aev production and extracellular calcium supply is crucial in this signalization. in the present work, our aim was to investigate whether mac- activation or casignalling on their own are sufficient for the initiation of the aev biogeneis. methods: we isolated neutrophil derived evs from peripheral human blood and murine bone marrow by two-step centrifugation and filtration. we tested the effect of ca-ionophore and examined the ev production on c bi coated surface and in soluble form. we quantified the vesicles by flow cytometry and determined their protein content by bradford assay. we examined their antibacterial effect in parallel with optical density-based measurement and our flow cytometry based method. results: on c bi coated surface, we observed an increased ev production, and these evs possessed antibacterial capacity. however, in soluble condition, c bi did not induce further ev production, and these evs did not show any antibacterial property. we found that ca-ionophore initiated ev formation, but these ev did not show antibacterial effect. we observed ev production increase after ca-ionofore treatment both in the presence and in the absence of extracellular ca. the ca-ionophore slightly increased the opsonized particle induced ev production, but did not potentiate their antibacterial capacity. summary/conclusion: mac- activation is not just crucial, but sufficient in initiation of the aev biogenesis. clustering of this receptor is required. while the ca-signal is crucial, it is not sufficient in the generation of aevs. extracellular vesicles and their microrna cargo in retinal health and degeneration: mediators of homoeostasis, and immune modulation yvette s. m. wooff, adrian cioanca, riemke aggio-bruce, joshua chu-tan, ulrike schumann and riccardo natoli the australian national university, canberra, australia introduction: photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases such as age-related macular degeneration (amd). however, the molecular mechanisms regulating these biological processes are largely unknown. extracellular vesicles (ev) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. evs, including exosomes, encapsulate and transfer microrna (mirna) to recipient cells and in this way can modulate the environment of recipient cells. dysregulation of evs however is correlated to a loss of cellular homoeostasis and increased inflammation. in this work we investigated the role of isolated retinal small-medium sized ev (s-mev) in the regulation of homoeostasis and immune modulation in both the healthy and degenerating retina. methods: isolated s-mev from healthy and degenerative (photo-oxidative damaged) mouse retinas were characterized using dynamic light scattering, transmission electron microscopy and western blot, and quantified using nanotracking analysis. small rna-seq was used to characterize the mirna cargo of retinal s-mev isolated from healthy and degenerating retinas. finally, the effect of exosome inhibition on s-mev-mediated immune modulation was investigated using systemic daily administration of exosome inhibitor gw and analysed by in situ hybridization of s-mev-abundant mirna. electroretinography and immunohistochemistry were performed to assess functional and morphological changes to the retina as a result of exosome depletion. results: our results demonstrated an inverse correlation between s-mev concentration and photoreceptor survival, with decreased s-mev numbers following retinal degeneration. small rna-seq revealed that s-mevs contained uniquely enriched mirnas, however no differential composition in s-mev mirna cargo following photo-oxidative damage was observed. exosome inhibition using gw exacerbated photoreceptor degeneration, with reduced retinal function and increased levels of inflammation and cell death seen following photo-oxidative damage. further, reduced translocation of the photoreceptor-derived s-mev was demonstrated following exosome-inhibition in photo-oxidative damaged mice. summary/conclusion: taken together, we propose that retinal s-mev and their mirna cargo play an essential role in maintaining retinal homoeostasis through immune-modulation, and have the potential to be targeted using gene therapy for retinal degenerative diseases. impacts of agricultural dust exposure on human lung-resident mesenchymal stromal/stem cells and their extracellular vesicles introduction: agricultural dust is considered a high-risk occupational hazard by the cdc, with impacts reaching throughout the communities surrounding these industries, leading to increased incidence of respiratory illness and disease among individuals within this occupation and these communities. lung-resident mesenchymal stromal/stem cells (lr-msc) have an important role in maintaining homoeostasis in the lung, and mediating pro-and anti-inflammatory effects, particularly during exposure to inhaled irritants, like agricultural dust. one way in which these lr-msc promote lung homoeostasis is through the release of extracellular vesicles (ev), with a variety of cargo that elicit changes among target cells. we hypothesize that exposure to agricultural dust modifies the quantity and cargo of ev released by lr-msc to promote lung tissue homoeostasis. methods: primary human lung-resident mesenchymal stromal cells were exposed to extracts of dusts collected from swine confinement facilities (de) for or hrs and the media from these exposures were collected and enriched for ev by opti-prep density gradient ultracentrifugation. the quantity of these ev were assessed by nanoparticle tracking analysis. additionally, cytokine and chemokine release by lr-msc were analysed by enzyme-linked immuno assays. results: as assessed at hr following treatment, deexposed lr-msc released pro-inflammatory cytokines, il- and il- , with il- release reaching statistical significance at . %, . %, and % de concentrations (p = . , < . , and < . respectively) and il- trending a similar dose response but only statistically significant at % de (p = < . ). de exposure of lr-msc also induced changes in the lr-msc-derived ev populations when compared to vehicle control, where lr-msc released significantly more ev in the and % iodixanol fractions (p = < . and . , respectively) at hr following de treatment. alternatively, there were significantly less ev in the and % density fractions in the media of deexposed lr-msc versus vehicle control. summary/conclusion: following exposure to agricultural dusts, lr-msc-derived ev populations more likely consist of exosomes and ectosomes, which play an important role in promoting lung tissue homoeostasis during exposure-related pulmonary inflammation. introduction: during analyses of single extracellular vesicles (evs) by flow cytometry (fcm), particles below the detection limit may exceed the trigger threshold, which is called swarm detection and generates false-positive counts. serial dilutions are recommended to find the minimal dilution for which swarm detection is absent. however, because particle concentrations in plasma vary, the optimal dilution differs > -fold between donors, but it is unfeasible to do serial dilutions for each clinical sample. therefore, our aims are to ( ) develop a faster method to avoid swarm detection, and ( ) increase the number of detected evs per second. methods: we measured serial dilutions of cd stained evs in platelet free plasma (pfp), with and without spiking of fitc beads, by fcm (apogee a -micro). we triggered either on side scatter or fluorescence. results: for scatter triggering with our fcm, swarm detection consistently occurred for plasma samples exceeding a (total particle) count rate of , - , events/s. the cd + evs concentration scaled linearly over . orders of magnitude of the dilution and most donors required > -fold dilution to avoid swarm detection, thereby reducing cd + ev counts. for fluorescence triggering, the cd + evs concentration scaled linearly over > orders of magnitude of the dilution. for all donors, swarm detection was absent after -fold dilution (relative to pure plasma). the count rates of cd + evs were - -fold higher compared to scatter triggering. the spiked fitc beads confirmed that the median signals remained constant. summary/conclusion: we have developed two clinically applicable ways to avoid swarm detection. for scatter triggering, the count rate provides direct feedback on the presence of swarm detection in plasma samples. for fluorescence triggering, swarm detection was absent for all plasma samples diluted ≥ -fold and compared to scatter triggering, count rates of cd + evs were - fold higher, thereby improving statistical significance. funding: edwin van der pol is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . benchmarking flow cytometric analysis of nanoparticles: a cross-platform study for single extracellular vesicle detection introduction: despite flow cytometry being widely used to analyse cells in suspension, most commercial instruments lack sensitivity when measuring nanoparticles (nps) and extracellular vesicles (evs). furthermore, the use of appropriate reference materials (rms) for calibration and quality control are essential to compare results acquired with different instruments. to work towards successful clinical applications for ev biomarker profiling, benchmarking studies including state-of-the-art flow cytometers are required. we here investigated the ability of three different flow cytometers to detect nps and evs. methods: the instrument sensitivity of light scattering detection was evaluated by using synthetic nps of different sizes and refractive indices. fluorescent calibration was investigated by using molecules of equivalent soluble fluorophores (mesf) beads. biological recombinant evs (revs) were used to validate the detection and quantification of fluorescent evs in a side-by-side cross-platform study using an n nanoflow analyser (nanofcm), an optimized bd influx and a cytoflex lx. results: we found that when light scatter based detection was used, the nanofcm detected the smallest non-fluorescent nps, the bd influx was able to provide reliable fsc information from the smallest detected nps and the cytoflex performance was greatly improved by the use of violet-ssc. biological revs showed that the nanofcm could clearly resolve fluorescent evs while the bd influx and cytoflex were unable to fully resolve revs from background, although fluorescence threshold improved detection. in addition, our findings revealed that different concentrations are required to ensure single ev detection in these platforms. summary/conclusion: we identified several strengths and limitations for each platform with respect to single ev analysis. furthermore, our results showed that proper calibration and rms are of utmost importance to ensure reliable interpretation of ev flow cytometric data. caution when using membrane dyes for sequential extracellular vesicle analysis diana pham, michael wong, desmond pink and john lewis nanostics, edmonton, canada introduction: confirmation that particles detected by microflow cytometry are actually extracellular vesicles (evs), or at least membranous in composition, can be achieved through a variety of methods. positively staining particles with a membrane dye strongly suggest that the particle contains a membrane; loss of stain (or detection) after detergent solubilization of the membrane-dyed particles provides even stronger evidence that the particles were evs. it is important to recognize that the labelling protocol provided by the membrane dye manufacturer may not be ideal for all types of evcontaining biological samples, such as blood, urine, semen etc.). removal of excess dye from stained evs is very difficult and can be impractical depending on the nature of the experiment. however, this means that the potential for excess dye to contaminate subsequent sampling is high. therefore, it is important to determine optimal working concentrations and labelling conditions when using membrane dyes for ev detection to understand properties that may impact your analyses. methods: to assess the utility of membrane dyes, titration curves were generated to determine the optimal working concentrations of membrane dyes for ev detection in conditioned media and human serum samples. once the optimal concentration was determined the potential of dye carry-over from sample to sample during microflow cytometry detection was evaluated by tracking dye positive (dye+) particles in phosphate buffered saline (pbs) blanks and matched, unlabelled, sample replicates. results: we found that optimal concentration of any membrane dye is dependent on sample type. even with the inclusion of system washes to prevent sample carryover, there was carryover of low amounts of dye+ particles into sequentially analysed pbs blanks. if unstained samples were analysed following a stained sample, excess dye (or at least dye+ events) appeared in the data. a sample concentration effect was also seen; samples of lower concentrations were more susceptible to dye carryover. summary/conclusion: when using membrane dyes to stain evs in biological samples, especially if an autosampler is employed to run a series of tests, it is critical to determine the optimal concentration of dye for each type of sample, as excess dye can carry over to the next sample in the queue. in addition, determining the necessary steps to clean any excess dye following each sample run will improve the accuracy of ev detection and analyses. funding: nanostics alberta innovates alberta cancer foundation correlation between size and protein expression of single exosomes by combined atomic force and fluorescence microscopy introduction: there are no universal markers of extracellular vesicles, but often they are identified by the presence of tetraspanins in their membrane. based on this, products have been developed to precipitate or quantify evs by acting upon cd , cd , and cd . however, evs also carry proteins from their parent cells, and capturing evs based their presence allows for a more complete understanding of vesicle heterogeneity from a single cell type, and for evs derived from specific tissues to be enriched from other biofluids in support of biomarker assessment. for example, evs derived from the brain could be captured from the general population of serum evs for better assessment of cargo associated with proteinopathy. the goal of this study was to identify specific antibodies to capture and label evs bearing the neural markers cd , snap , α-synuclein, tau, and ncam. methods: the targets were overexpressed in hek t cells through transient transfection of plasmids (origene). media was conditioned for - hours, and then centrifuged to remove cell debris. cell lysates and concentrated conditioned media (cm) were analysed by western blot. unpurified cm, or cm after performing size exclusion chromatography (sec, izon), were analysed in the exoview r system. diluted cm was incubated on custom antibody microarray chips overnight. then the chips were labelled with a cocktail of labelled antibodies, washed and imaged. vesicles were counted, sized, and phenotyped. next, commercially available pooled human csf was analysed in a similar fashion to determine their abundance in a relevant biofluid. results: multiple antibody clones were tested in different combinations for capture and labelling for the five different neuronal enriched proteins of interest, and optimal combinations were identified. some markers were identified on particles > nm in size that were negative for tetraspanins, while others colocalized with tetraspanins. through comparing permeabilized and intact evs with and without sec to remove non-vesicular proteins, we found that tau could be on the vesicle surface, within the vesicle, and free in solution. summary/conclusion: the exoview platform can be customized to enable the detection of proteins of interest and to determine whether they are on the ev surface, intravesicular, or non-ev associated. methods: forty non-smoking male and female subjects ( - y) at moderate risk for cvd were recruited for the study. evs from platelet-free plasma (pfp) were isolated using size exclusion chromatography (sec). the concentration and size distribution of evs were measured by nanoparticle tracking analysis (nta) and flow cytometry (fcm). three ev markers, including annexin v for the circulating phosphatidylserinepositive (ps+) evs, cd for platelet-derived evs and cd for endothelial-derived evs were used for phenotyping. in addition, coagulation and fibrinolysis were assessed using a thrombodynamics analyser (hemacore). platelet aggregation to determine platelet function was assessed by a high-throughput platelet function assay with a wide range of concentrations of agonists, including adenine diphosphate (adp), collagen-related peptides (crp-xl), epinephrine, thrombin receptor activating peptide (trap- ) and u . the association between thrombogenic risk markers for cvd and ev numbers was tested by pearson's correlation coefficient and linear regression model using the statistical program, spss. results: circulating ev concentration with threshold of nm, measured by nta, were positively associated with coagulation-related risk markers, including rate of clot growth (r = . ; p = . ) and clot size at min (r = . ; p = . ). ps+ evs derived from endothelial cells, determined by fcm, were negatively associated with lysis onset time (r = − . ; p = . ), whereas they were found positively correlated with lysis progression (r = . ; p = . ). both mean and mode size of cevs, detected by nta, were significantly correlated with u -induced platelet aggregation (r = − . ; p = . , r = − . ; p = . , respectively). summary/conclusion: in subjects at moderate risk for cvd, cev numbers were positively related to rate of clot growth and clot size and size of cevs was negatively related to platelet activity. higher numbers of endothelial cell-derived ps+ cevs were associated with lower rates of fibrinolysis. this suggests that cevs promote clot growth and reduce fibrinolysis, and may therefore be an indicator for greater risk of cvd. beyond stem cells: extracellular vesicles from human induced pluripotent stem cells (hipsc) and hipsc-cardiomyocytes as therapeutic approaches for heart failure introduction: heart failure is caused by a variety of underlying diseases, the most common being myocardial infarction. initially regarded as an alternative to pharmacological approaches, stem cell transplantation has failed to demonstrate clinically meaningful results. instead, it has become increasingly apparent that the therapeutic effects of transplanted cells are largely mediated by their secretome, while mounting evidence suggests extracellular vesicles (evs) play a major role in cardiac repair. within this framework, evs from human induced pluripotent stem cells (hipsc) and hipsc-derived cardiomyocytes (hipsc-cm), hold a tremendous potential to treat cardiovascular disease. we isolated evs from conditioned culture media at key stages of the hipsc-cm differentiation and maturation processes, i.e. from hipsc (hipsc-ev), cardiac progenitors (cpc-ev), immature (cmi-ev) and mature (cmm-ev) cardiomyocytes, with the aim of studying their potential role as therapeutics, and whether their effectiveness was influenced by the state of their parent cell. methods: hipsc were differentiated into cardiomyocytes in a d culture approach, using the protocols developed by our group. ev isolation was performed on an iodixanol density gradient, and the evs were characterized in terms of particle size and particle size distribution, presence of ev-specific markers, and imaging through transmission electron microscopy. functional studies were performed using human umbilical vein endothelial cells (huvecs) to evaluate evuptake, cell migration and angiogenesis. results: evs from all hipsc and cardiac derivatives presented a typical cup-shaped morphology and expressed cd and cd . ev yield varied along differentiation, with a minimum for cpc and a maximum for cmi. pkh -labelled evs were uptake by huvecs, and colocalized with calnexin, a protein from the endoplasmic reticulum. wound healing assays showed an increased cell migration in huvecs treated with cardiomyocyte-derived evs, in comparison with control evs isolated from foetal bovine serum. summary/conclusion: our findings suggest a different ev secretion profile along cm differentiation and maturation, with preliminary assays showing ev functionality. ongoing work aims at elucidating the possible differences in function and cargo amongst these types of evs. endothelial cells differentially load and secrete extracellular vesiclederived micrornas into apical and basolateral compartments this may play a role in microcalcification in calcific aortic valve disease (cavd), but this is poorly understood. annexin a is thought to be a marker of membrane-derived evs, but because it can be found on the cytoplasmic or extracellular side of the plasma membrane, its localization within or on the surface of evs is unclear. the goal of this study was to determine whether annexin a is found on the surface of evs in two cell lines relevant to cavd, and develop an assay that can be used to determine whether this changes under pathogenic conditions. methods: evs were isolated by differential ultracentrifugation from the conditioned medium (cm) of smooth muscle cells (smc) and valvular interstitial cells (vic). total protein in the cell lysates and ev pellets was analysed by western blot. evs from cells treated with control sirna or anxa -sirna were enumerated and phenotyped using the exoview r platform. evs with surface expression of cd , cd , cd , and annexin a were captured using a customized antibody microarray chip. then evs were labelled with fluorescent antibodies to assess ev number, size, and colocalization of ev proteins. the knockdown of annexin a allowed us to assess the specificity of the selected annexin a antibody. results: the ev fraction was positive for cd , and lacked markers of other vesicle types. western blot on the ev pellet and supernatant in ± edta indicated that there is annexin a both on the surface of and within the evs. using the antibody microarray chips, numerous annexin a + evs were captured on the annexin a spots from the control cm, and there was a marked decrease in capture and labelling from anxa -sirna treated cells. under both conditions, vesicles were also captured on tetraspanin probes, with the greatest number captured on cd , then cd and cd . there was a significant population of annexin a + evs that was negative for tetraspanins. summary/conclusion: annexin a is found on the surface of evs. the assay developed in collaboration with nanoview biosciences is well suited for assessing the number and phenotype of annexin a + evs derived from smc and vic cell lines, which could provide a useful method for understanding ev populations in cavd patient cell lines. funding: this work was supported by hl and hl . possibility of exosomal micrornas associated with chronic limb-threatening ischaemia, the end stage of atherosclerosis, as a promising biomarker introduction: chronic limb-threatening ischaemia (clti), the end stage of peripheral artery disease (pad), has poor prognosis and is attributed to lifestyle disease. with increasing of atherosclerotic disease all over the world, establishment of biomarker for should play a pivotal role for early detection and preventing aggravation of the disease. the aim of this study is to explore the possibility of liquid biopsy for atherosclerotic disease by analysis of clti-associated exosomal micrornas. methods: clti due to pad was diagnosed by anklebrachial blood pressure index, skin perfusion pressure (< mmhg) and angiography. ten preoperative clti patients and control patients without pad were analysed (all patients with diabetes and % of patients had end-stage renal failure [esrd] ). to identify biomarkers associated with clti, exosomes were extracted from patient's serum after ultracentrifugation and total rna including small rna was isolated from the exosomes. the expression profile of exosomal micrornas associated with clti were evaluated using a next generation sequencing. results: forty-three exosomal mirnas associated with clti were identified. intriguingly, these mirnas were clearly categorized with esrd, which was well known as end-stage of life-style disease: these were stratified into micrornas for esrd patients and micrornas for non-esrd patients. since esrd is the most important factor significantly related to patient's prognosis in clti, exosomal micrornas reflected patient's comorbidity onto the expression profile. summary/conclusion: a portion of the expression profile of exosomal micrornas associated with clti was identified. exosomal microrna could be a biomarker to stratify patient's condition along with their comorbidities and is very promising for individualized diagnosis in atherosclerotic diseases with risk diversity. postoperative plasma exosomal mir- and mir- a signature in patients with left ventricular reverse remodelling after surgical mitral valve repair underwent implantation of a prosthetic mitral ring. lv remodelling was assessed by cardiac magnetic resonance imaging and pexos were isolated by optimized ultracentrifugation before surgery (t ) and six months after surgery (t ). isolated pexos were quantified by nanoparticle tracking analysis and mir- , mir- , mir- a, and mir- a were measured by rt-qpcr. the same analysis was performed on healthy subjects with normal cardiac function (n = ). local ethical committee approved the study (emigrate study, approval n° ) and informed consent was obtained from all patients. results: pexos levels at t were lower (− %, p = . ) in patients with worst postoperative lv function, while they were higher at t (+ %, p = . ) in patients with reversed lv remodelling after surgery. at t , the increase in pexos levels was associated to decreased heart mass index (− %, p = . ) and higher levels of exosomal mir- (+ %, p = . ) and mir- a (+ %, p = . ) were detected in patients with improved lv function. summary/conclusion: higher postoperative levels of pexos delivering mir- and a depict lv reverse remodelling after surgical mitral valve repair. monitoring of exosomal micrornas cargo might predict postoperative outcome in patients with mr. expression of lipocalin- (lcn ) in circulating extracellular vesicles (evs) and femoral plaque-derived evs of peripheral arterial disease patients. introduction: clinically, the drug resistance situation of acinetobacter baumannii is becoming increasingly serious, and its drug resistance has become a difficult problem for nosocomial infection and clinical treatment. in view of the relatively slow development of antibacterial drugs, exploring the resistance mechanism of acinetobacter baumannii is of great significance to improve bacterial resistance and help clinical treatment. studies have shown that outer membrane vesicles (omvs) can transmit resistance genes to mediate the spread of drug resistance, and recent studies have confirmed that high expression of efflux pumps play an important role in the multidrug resistance of a. baumannii. in this study, we want to explore whether the outer membrane vesicles of acinetobacter baumannii can transfer the efflux pump related substances. methods: first, ultracentrifugation and density gradient centrifugation were used to extract the omvs of acinetobacter baumannii antimicrobial-sensitive strains (atcc ) and antimicrobial-resistant strains. then, nanoparticle tracking analysis (nta) technology was used to analyse the particle size and distribution range of omvs. transmission electron microscopy (tem) was used to identify their morphology and structure. bradford method was used to determine the protein concentration of omvs. next, the omvs of antimicrobial-resistant strains were incubated with the antimicrobial-sensitive strains and then the drug susceptibility test was done to determine whether omvs of antimicrobial-resistant strains could transmit antimicrobial-resistance information to the antimicrobial-sensitive strains. finally, pcr, qpcr and mass spectrometry were used to determine whether the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. results: nanoparticle tracking analysis (nta) detected the concentration and size distribution of omvs of acinetobacter baumannii strains. it showed that the extracted omvs have a relatively uniform particle size and a size between - nm. tem showed that omvs had a typical vesicle structure. omvs coculture experiments showed that omvs of the antimicrobial-resistant strains can indeed pass resistance to the antimicrobial-sensitive strains. and the efflux pump related genes were higher expression in omvs of antimicrobial-resistant strains than those in antimicrobial-sensitive strains. summary/conclusion: omvs of the antimicrobialresistant strains can indeed pass resistance to the antimicrobial-sensitive strains. the cause of acquiring antimicrobial resistance in sensitive strains may be caused by resistant strains passing efflux pump-related genes or proteins to sensitive strains. characterization of melanocytic extracellular vesicles during ageing of the choroid kelly coutant a , léo piquet a , nathan schoonjans b , philippe gros-louis a , julie bérubé c , stéphanie proulx a , alain r. brisson d and solange landreville a a université laval, quebec city, canada; b université de lille, lille, france; c centre de recherche du chu de québec-université laval, quebec city, canada; d université de bordeaux, bordeaux, france introduction: the choroid is located at the backside of the light-sensitive retina and is highly vascularized. it contains pigmented melanocytes, and their melanin protects them against oxidative stress. since ageing reduces the number of melanosomes in melanocytes and generates a stiffer extracellular environment, our hypothesis is that surrounding choroidal cells and the retinal pigment epithelium (rpe) are subject to more oxidative stress-related damages. this study aimed to characterize evs released by human choroidal melanocytes in the context of intercellular cooperation during ocular ageing. methods: melanocytic evs were recovered from the conditioned culture medium of young/old melanocytes grown on hydrogels of varying stiffness ( . - kpa) by differential centrifugation. the concentration and size distribution of melanocytic evs were determined by high-sensitivity flow cytometry. cryo-transmission electron microscopy combined with receptor-specific gold labelling were used to reveal their morphology, size and phenotype. the relative abundance of surface markers was evaluated with the exo-check exosome antibody array. the uptake of fluorescent melanocytic evs by the rpe and choroidal endothelial cells was assessed by confocal microscopy. results: choroidal melanocytes released evs positive for annexin- and the tetraspanin cd . young melanocytes produced more annexin- positive evs and evs larger than nm compared to older donors. the stromal stiffness impacted the concentration and size of melanocytic evs. we confirmed the uptake of melanocytic evs by endothelial and rpe cells. summary/conclusion: evs from choroidal melanocytes are internalized by surrounding endothelial cells and rpe. age-related stressors modify the phenotype of melanocytic evs. the identification of melanocytic factors that can protect retina/choroid cells from oxidative stress-induced cell death could lead to more efficient therapy for patients suffering from dry agerelated macular degeneration. introduction: owing to their proposed biocompatibility and ability to cross biological barriers, evs represent an attractive therapeutic delivery platform. however, evs are eminently heterogeneous. a better understanding of ev heterogeneity and its origins will allow for improved design of ev-based therapeutics. ev heterogeneity is mainly studied by focusing on distinct ev subpopulations. other sources of heterogeneity, such as heterogeneity within ev secreting cells themselves, have been investigated in lesser detail. in this study, we assessed the phenotypic drift of cell derived evs to explore the origins of ev heterogeneity and its potential impact. methods: three independent samples of two mda-mb- breast cancer cell sub-clones were cultured for six weeks. evs were harvested weekly and analysed using the macsplex exosome flow cytometry kit. at two time points the proteome of evs was analysed by lc-ms/ms mass spectrometry with subsequent gene ontology and reactome pathway analysis. results: the expression of over proteins was deregulated in evs derived from the two different cell clones. many de-regulated proteins were associated with biological processes predicted to affect potential ev toxicity (platelet activation, neutrophil degranulation, blood coagulation) and ev biological activity (antigen presentation, inflammation, tgf-beta/ mtor/wnt signalling). more surprisingly, within only two weeks, over ev proteins, many associated with immune modulation, apoptosis, interleukins, cytokines and cell signalling pathways (including those affecting t-cell/b-cell receptors) were de-regulated between the two ev isolation time points. summary/conclusion: results suggest that temporal changes can be observed in the ev proteome (potentially by clonal drift, epigenetic changes or cellular genomic instability) over short time periods. these changes could cause significant differences in biological effects and delivery capabilities between evs harvested from the same cells at different time points and conditions. in vivo tracking and biodistribution analysis of mesenchymal stem cellderived extracellular vesicles in a radiation injury murine model introduction: recent studies indicated that extracellular vesicles (evs) play key roles in intercellular communication and have great potential for clinical application. understanding the biodistribution of evs is therefore essential. our previous works have shown the ability of mesenchymal stem cell (msc)derived evs to protect haematopoietic cells from radiation damage. in this study, we evaluated the biodistribution of msc-evs in a radiated mouse model. methods: human msc-evs were harvested by ultracentrifugation and labelled with did lipid dye. the reliability of the labelling evs was confirmed by sucrose gradient fractionation analysis. the distribution of evs in radiation-exposed mice after ev intravenous administration were evaluated by fluorescence molecular tomography and further confirmed by flow cytometry and confocal microscopy analysis. results: we observed that did labelled msc-evs appeared highest in liver and spleen, lower in bone marrow in tibias, femurs, and spine, and were undetectable in heart, kidney and lung. we found the significantly increased msc-ev accumulation in spleen and bone marrow post-radiation appeared with an increase of uptake of msc-ev by cd b+ and f / + cells, but not b + cells, compared to those organs from non-irradiated mice. however, there was a predominant ev accumulation in lung and less accumulation in spleen and liver; in mice infused with human lung fibroblast cell derived evs (lfc-evs) and there was no significant lfc-evs accumulation change in the spleen or liver after radiation. we further found that increasing levels of irradiation caused a selective increase in vesicle homing to marrow and spleen. this accumulation of msc-evs at the site of injured bone marrow could be detected as early as hour after msc-ev injection and was not significantly different between and hrs. post-msc-ev injection. summary/conclusion: this study indicated the specific accumulation of ms-evs at the site of injury of haematopoietic tissue in radiation injury mice. funding: this work was supported by the nih grants uh tr , uh tr - s , p gm , and t hl . linking fat to colorectal cancer: extracellular vesicle crosstalk sheffield hallam university, sheffield, uk introduction: colorectal cancer is the third most common cancer worldwide, and fourth leading cause of malignancy related mortality. understanding the mechanisms of its growth and metastasis is key to elucidating new therapeutic targets and developing treatments in the clinical setting. epidemiological evidence indicates an increased risk of cancer in obese patients, pointing to bidirectional communication between colon and adipose cells. extracellular vesicles (evs) are small membrane enclosed packages released by cells, capable of transporting bioactive cargo from donor to recipient cells and inducing phenotypic changes. adipocytes are a key component of the tumour microenvironment and interactions between adipose tissue and tumour cells may be important in the growth and metastasis of cancer. in this study, we investigate the effects of colorectal cancer evs on adipocytes in vitro, and potential induction of dedifferentiation to a more fibroblastic, pro-inflammatory phenotype. methods: evs were isolated from sw and ht human colorectal cancer cell lines by differential ultracentrifugation and mature adipocytes generated by differentiation of the sgbs human pre-adipocyte cell line. adipocytes were treated with evs and their lipid content measured by oil red o to determine loss of lipids. inflammatory cytokine profile was measured by elisa to assess any increase in pro-inflammatory behaviour, and expression of late adipogenesis markers were determined by western blot. results: ev treatment was shown to reduce lipid accumulation in adipocytes, with up to % reduction in lipids observed at the µg/ml dose. treatment was also shown to reduce the expression of late adipogenesis markers, and increase secreted levels of proinflammatory cytokines il- and il- by over fold and fold respectively. these results provide evidence for colorectal cancer derived ev involvement in the dedifferentiation observed in cancer associated adipocytes in vivo, displaying an altered phenotype, releasing lipid energy stores to fuel tumour growth and increasing pro-inflammatory signalling. summary/conclusion: studies have shown colorectal cancer evs may be involved in signalling which induces functional changes in cells within the tumour microenvironment. our work indicates that ev mediated dedifferentiation of resident adipocytes may potentially contribute to a microenvironment favouring cancer cell growth and metastasis. further work aims to elucidate the specific ev cargo which mediates these effects. introduction: ageing is a major risk factor for many human diseases. it is a complex process that progressively compromises most of the biological functions of the organisms, resulting in an increased susceptibility to disease and death. senescence is a cellular phenotype characterized by a stable cell cycle arrest. senescent cells are accumulated in the body during ageing. it contributes to develop age-related diseases and cancer. the alteration in intercellular communication with age has been demonstrated to be due to senescent cells developing a phenomenon denominated senescenceassociated secretory phenotype (sasp). exosomes are small extracellular vesicles (sev) ( - nm) of endocytic origin whereas microvesicles are formed by shedding of the plasma membrane. they contain nucleic acids, proteins and lipid that generally reflect the status of the parental cell and can influence the behaviour of neighbouring cells. methods: in this study, we demonstrated that the small extracellular vesicles (sev) contribute for transmitting paracrine senescence to proliferative cells firstly, we evaluated the presence of exosome-like particles in the sev from senescent cells by detection of exosome markers (alix, tsg and cd ), transmission electronic microscopy (tem) and nanoparticle tracking analysis (nta). to determine that sev from senescent cells are mediators of the paracrine senescence, we performed functional assays using cre-loxp reporter system and high-throughput results: besides, we confirmed at a single-cell level that the proliferative cells internalizing sev from senescent cells activate senescence process using the cre-reporter system. sev protein analysis from senescent cells by mass spectrometry (ms) and validation of top candidates using a functional sirna screen identify interferon induced transmembrane protein (ifitm ), a component of non-canonical interferon (ifn) pathway, as partially responsible for transmitting senescence to proliferative cells. summary/conclusion: in conclusion, we found that sev are regulators of paracrine senescence and ifitm contained in senescent sev has an important role in the intercellular communication mediated through sev during cellular senescence . bin wu a , lei guan a , ye xu a , likang chin a , ting li a , youhai chen a , gordon mills b , jinqi ren a , ravi radhakrishnan a , rebecca wells a and wei guo a a university of pennsylvania, philadelphia, usa; b oregon health & science university, portland, usa introduction: extracellular matrix (ecm) remodelling and stiffening are associated with solid tumour progression. stiff ecm promotes cell proliferation, epithelial-to-mesenchymal transition (emt), metastasis and chemoresistance. hepatocellular carcinoma (hcc) appears frequently in patients with liver cirrhosis or fibrosis while the mechanism remains unclear. exosomes have been determined to serve as messengers to mediate intercellular communication and influence the extracellular. tumour-derived exosomes have been shown to influence tumour progression, metastasis, drug resistance, angiogenesis and immune regulation. thus, determining whether exosomes provide a mechanism by which stiff matrix modulates tumour microenvironment for tumour progression opens a new way to understand cirrhosis and oncogenesis. here we identified the molecular mechanism of matrix stiffening induced exosome secretion and showed the different effect of exosomes induced by soft or stiff matrix on tumorigenesis. methods: huh cells were cultured on acrylamide gels with the stiffness was modulated to pa (soft) or k pa (stiff). the exosomes in conditioned media were collected and analysed by nanoparticle trafficking analysis (nta) and immunoblotting. protein expression level in cells was screened by reverse phase protein array (rppa). inhibitor or shrna were used to inhibit target proteins function. in vitro phosphorylation and gef assay were used to verify rabin phosphorylation and activation. exosomes from cells on soft or stiff matrix were injected into mice to study their effect on tumour growth. results: ( ) stiff matrix promoted exosomes secretion. ( ) akt was activated by stiff matrix and was required for exosome secretion. summary/conclusion: matrix stiffening promotes exosome secretion via akt-rabin -rab pathway, contributing to tumorigenesis. tridimensional fibroblast culture revealed a novel exososome-dependent extracellular matrix secretion mechanism vincent clément a , bastien paré b , cassandra goulet a , thiéry de serres-bérard a , stéphane bolduc a , françois berthod a and françois gros-louis a a université laval, québec, canada; b norgen biotek corp., thorold, canada introduction: the extracellular matrix (ecm) is constituted of a variety of proteins and polysaccharides that are secreted locally and assembled into a thick d meshwork to provide biophysical and biochemical support to the surrounding cells, and regulate numerous cellular functions such as adhesion, migration and proliferation. dysregulation of ecm components or aberrant ecm remodelling can lead to various pathologies, as well as to play important roles in wound healing. although ecm secretion pathways are still largely unknown, the current paradigm is that ecmassociated proteins are synthesized in the endoplasmic reticulum and transported via the endosomes to the golgi apparatus en route to the cell surface and released by exocytosis. methods: to study ecm secretion pathway, we used dimensional ( d) cultured fibroblasts. this culture method technique has been used widely to generate tissue-engineered self-assembled stromal tissues, free of exogenous materials, and rely on long-term supplementation of sodium ascorbate into the culture medium. non-cancerous fibroblasts, grown in conventional two-dimensional ( d) cellular cultures, are known to be a poor source of secreted exosomes when compared to cancerous fibroblasts. results: here, we provide evidence that non-cancerous dermal fibroblasts can secrete high amounts of exosomes, containing different ecm proteins, when cultivated in a d fashion. we also demonstrated that dermal fibroblast-derived exosomes had the capacity to travel from one cell to another, induce cellular migration and promote wound healing. summary/conclusion: altogether, these findings reveal a novel exosome-dependent ecm deposition mechanism and suggest that the use of d-fibroblast cellular culture may emerge as an innovative approach in precision medicine to better study the role of patient-derived exosomes and ecm proteins in the establishment of cellular microenvironment in health and disease. anthony yan-tang. wu a , charles lai, yun-chieh sung b , steven t. chou c , vanessa guo c , jasper c. chien c , john j. ko c , alan l. yang c , ju-chen chuang c , hsi-chien huang b , syuan wu c , meng-ru ho d , maria ericsson e , wan-wan lin f , koji ueda g , yunching chen h , chantal hoi yin cheung i and hsueh-fen juan j introduction: bionanoparticles including extracellular vesicles and exomeres (collectively termed evs), have been shown to play significant roles in diseases and therapeutic applications. however, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a limited suitable method. methods: we developed a bioluminescence resonance energy transfer (bret)-based reporter, palmgret, to enable pan-bionanoparticle labelling ranging from exomeres (< nm) to small (< nm) and medium and large (> nm) evs and larger evs (> nm). results: palmgret emits robust, sustained signals and allows the visualization, tracking and quantification of bionanoparticles from whole-animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, bret, and fluorescence. using palmgret, we show that evs released by lung metastatic hepatocellular carcinoma (hcc) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. ev proteomics identified hcc-ev lung tropic protein candidates associated with cancer progression, in which slco a and clic expression on non-tropic evs conferred lung-tropism, while cd gave spleen tropism. our results further demonstrate that redirected lung tropism decreases ev distribution to the liver, whereas the spleen tropism significantly reduces over time delivery to most major organs distribution including the liver and kidney. summary/conclusion: we established a multimodal and multi-resolution palmbret method to enable pan-bionanoparticle labelling and imaging and therefore quantification in live cells, whole animals, and preserved tissues. the method can resolve the intricate spatiotemporal dynamics of evs. palmgret revealed that evs derived from lung metastatic hcc are lung tropic, and the tropism can be conferred to non-lungtropic ev- t by decorating evs with identified hcc-ev membrane proteins. importantly, the enhanced ev delivery to tropic organs also significantly alters its distribution to other major organs. our findings suggest that the dynamics of ev biodistribution and targeted design should be investigated at the organ systems level in ev biology and therapeutic developments, respectively. tracking mesenchymal stem cell-derived extracellular vesicles (evs) in a in vivo cancer model introduction: small extracellular vesicles (sevs) are nanoparticles ( - mn) encircled by a phospholipid bilayer, derived from the endocytic pathway and released by all cells. sevs have an inherent role in cell communication and deliver cargo to target cells. mesenchymal stem cells (mscs) and have a natural ability to home to tumours and metastases while avoiding the host immune response. it is hypothesised that msc derived sevs (msc-sevs) also possess tumourhoming and immune-evading capacities therefore could provide a novel targeted delivery vehicle for treatment of cancer. it is imperative to elucidate msc-sevs migratory itinerary in vivo to support translation to the clinical setting. methods: this study aimed to image the interaction of labelled msc-sevs with cancer cells in real time in vivo. sevs were isolated from wildtype mscs and mscs with stably expressing red fluorescent protein (rfp) (via lentivirus) by the combined techniques of differential centrifugation, microfiltration and ultracentrifugation. isolated sevs were extensively characterised by transmission electron microscopy (tem), nanoparticle tracking analysis and western blot. nod scid gamma (nsg) mice with dorsal skinfold window chamber (dsfwc) were injected with either mda-mb- luciferase (luc) expressing cells or ht- -luc cells. bioluminescence imaging was performed to confirm tumour formation. a dose of x ^ msc-rfp-sevs was directly added to the window chamber and rfp expression detected using a microscope with rfp filter attachments. x ^ evs were incubated with the radionuclide, technetium- m tagged duramycin ( mtc-dur) for minutes at room temperature. excess radiolabel was removed using exosome spin column (invitrogen™). the mtc-dur-sevs were then added directly to the window chamber and charged particle imaging carried out. results: hours post-administration; the rfp signal was localised at the tumour site. radiolabelled sev signal could be detected minutes and hours after administration. msc-sevs were successfully detected at the tumour site following direct administration using two different tagging and imaging approaches. summary/conclusion: this promising preliminary data supports the potential of this approach for tracking msc-sev migration in vivo. future studies will investigate systemic tracking of msc-sev migration. vaughn garcia ; aejez sayeed ; rachel derita ; shiv ram krishn ; peter a. introduction: tumor-derived small extracellular vesicles (sevs) have emerged recently as mediators of tumorigenesis. however, the role of sevs in response to irradiation, a widely used therapy in prostate cancer, is not fully understood. methods: our study involved the tramp mouse model of prostate cancer. we used plasma sevs isolated using differential ultra-centrifugation and further isolated using iodixanol gradient fractionation. we also used nanoparticle tracking analysis (nta) to analyze sevs. mouse pelvises were irradiated using gy, for consecutive days. results: we first observed that upon pelvic irradiation of tramp mice, the levels of the signaling oncogene c-src are reduced in plasma-derived sevs, while the average size of sevs is increased from - nms to - nms. furthermore, we show that the sevs from irradiated cells lose the ability to stimulate anchorage independent growth and migration of recipient cancer cells. additionally, sevs from irradiated mice increase the amount of dna damage in recipient cancer cells. summary/conclusion: overall, our data show that irradiation of tramp mice (and prostate cancer cells) significantly reduces the pro-metastatic and pro-anchorage-independent growth potential of sevs when tested on human cells. changes to the composition and behavior of a cancer cell sev population via radiation therapy offers promise for future therapeutic approaches for prostate cancer. introduction: there are emerging physiological and pathological functions of extracellular vesicles (evs) in neurodegenerative diseases including alzheimer's disease (ad). brain derived-evs contain pathogenic proteins, such as tau, amyloid beta (aβ), which have been reported to contribute to cell-to-cell propagation in those diseases. investigation of the brain-derived ev cargo, therefore, is important to further understand the mechanisms of progression in neurodegenerative diseases. we developed the ev separation method from unfixed frozen mouse and human brain tissues and assessed the protein composition. methods: to establish the ev separation method, we separated evs from frozen mouse brain tissue using sucrose density gradient ultracentrifugation (sg-uc) or size exclusion chromatography to compare the results from the particle number, morphology and protein profiling by nta, tem and mass spectrometry. evs were then separated from cortical grey matter of ad (n = ) and control (n = ) by sg-uc. tau and aβ in the evs were measured by immunoassay. differentially expressed ev proteins were observed by quantitative proteomics employing machine learning. results: the separated evs were enriched in ev molecules and devoid of contaminant proteins by sg-uc, showing our method was successful. the levels of ps tau and aβ - were significantly increased in ad evs. annexin a (anxa ), neurosecretory protein vgf, neuronal membrane glycoprotein m -a (gpm a), and alpha-centractin (actz) were differentially expressed in ad evs. a combination of these proteins were confirmed to predict ad with the % accuracy by machine learning. summary/conclusion: these data suggest our method were suitable for the separation of brain-derived evs and ev anxa , vgf, gpm a and actz can be potential biomarkers for monitoring the progression of ad. edta stabilizes the concentrations of extracellular vesicles during blood collection introduction: to establish reliable biorepositories for research on extracellular vesicles (evs) as disease biomarkers, the release of evs during blood collection and handling must be avoided. currently, citrate is recommended as the anticoagulant for blood ev research, but citrate does not inhibit the release of evs from activated platelets. the release of platelet-derived evs excludes pneumatic tube transport and makes assays time dependent, thereby limiting clinical compatibility. therefore, we aim to stabilize the release of platelet ev concentrations. methods: blood samples were collected from healthy individuals and subjected to common circumstances known to induce platelet activation. blood was (i) incubated with or without thrombin receptor-activating peptide (trap; n = ), a potent platelet activator, (ii) send to the lab by a routine blood transport (pneumatic tube system; n = ), and (iii) stored at room temperature or at °c for hours (n = ). the concentrations of evs from platelets (cd +), activated platelets (p-selectin+), erythrocytes (cd a+), and leukocytes (cd +) were determined by flow cytometry (apogee a -micro). results: following activation by trap, concentrations of platelet-derived and activated platelet-derived evs increased . -fold and . -fold in citrate-anticoagulated blood, compared to . -fold and . -fold in edta-anticoagulated blood (edta vs citrate: p = . and p = . , respectively). preliminary data show that during pneumatic tube transport and routine sample handling, both platelet-and activated platelet-derived evs were more stable in edta compared to citrate. the concentrations of evs from erythrocytes and leukocytes were unaffected under all studied conditions. summary/conclusion: to conclude, edta stabilizes platelet ev concentrations during and after blood collection, which would facilitate pneumatic tube transport, enhance reliability and thereby improves the establishment of reliable biorepositories for ev research. introduction: cancer-cell secreted extracellular vesicles, called exosomes, are an emerging biomarker for cancer liquid biopsy. profiling of cancer-associated exosomes usually required lengthy, and multi-step procedures; therefore simple and easy-setup sensing methods are urgently needed for diagnosing cancer in a timely manner. chirality, the foundational property of all biomolecules, including exosomal proteins, can be utilized for exosome detection and differentiation using recent advances in chiral nanostructures. we found that microfluidic sensors can be successfully implemented for successful detection of cancer-associated exosomes taking advantage of unusually high circular dichroism (cd) of chiral gold nanoparticles (aunps). circular dichroism-based exosome (cdexo) detection utilizes chiroplasmonic enhancement of cd signatures of cancer-associated exosomes. we first synthesized donut-shaped aunps conjugated with l-cysteine and immobilized the aunps on a glass slide using a layer-by-layer assembly. the aunps on slide glass were surface functionalized by the standard biotin-avidin reaction after mua treatment. biotinylated annexin v marker, targeting phosphatidylserine (ps) expression on cancer-associated exosomes, was conjugated to the aunp surface. μl of exosome samples from cancer cells (a and h ) or normal cells (mrc ) were injected into the pdms microfluidic device and incubated for minutes. the cd signal before and after exosome exposure was monitored, compared, and systematically analysed as a rapid technique for the detection of exosomes with high sensitivity. results: we showed that the cdexo signals from cancer exosomes showed . folds absolute cd peak value change and . folds shift, respectively, compared to that of healthy exosomes. importantly, the cdexo sensing method takes less than mins in terms of total scanning time and requires minimal sample volumes. from the preclinical studies using blood samples from cancer patients and healthy donors, we found that cancer patients show stronger band shift and signal change comparing to that of healthy donors, implying our platform could be used for cancer diagnosis. summary/conclusion: this new versatile and sensitive method based on chiroplasmonic exosome detection paves the way to profiling disease-associated exosomes in a timely manner for minimal volumes of liquid biopsies. ev classification and fractionation strategy using surface charge labelling takanori ichiki a , hiroaki takehara a , hirofumi shiono b and hiromi kuramochi a a the university of tokyo, bunkyo, japan; b innovation center of nanomedicine, bunkyo, japan introduction: the development of new classification technology is required based on the evaluation of physicochemical properties of exosome surfaces and the diversity of constituent molecules. in this presentation, we present the electric charge activated exosome sorting platform comprising microfluidic device technology and electric charge labelling technique. methods: the single nanoparticle analysis platform, which has been developed by our research group, images rayleigh scattered light (elastically scattered light) obtained by irradiating nanoparticles with convergent laser light and provides information of individual particles by image processing. the method that utilizes electrokinetic phenomena, unlike the method using fluorescent labels, measures the properties of the particle surface without serious difficulty in principle even if the particle size is on the order of tens nanometres, and further enables to perform fractionation. since the number of particles usually handled in exosome research or its envisioned application is enormous, it is not realistic to take an approach such as a cell sorter in which particles are sequentially manipulated one by one following the measurement results of individual particles. results: particles receive attraction or repulsion by an external field according to the charge density on the surface, so there is no need to control the external force, and it is possible to design a device that can autonomously fractionate particles according to the difference in zeta potential. summary/conclusion: in conclusion, we have proposed and demonstrated the new concept of electric charge activated ev sorter. funding: this research was partially supported by the center of innovation program (coi stream) from the japan science and technology agency. high throughput exosome analysis by using reversible microfluidic electrochemical sensor system introduction: exosome is one of the important extracellular vesicles (evs) released from parental cells and it contains various types of molecular cargos from its original cell including proteins, messenger rna (mrna), and micro rna (mirnas) [ ] . the exosomes have recently emerged as biomarkers for early stage cancer detection because the number of exosomes originated from cancerous cells are significantly higher than those from normal cells [ ] . since many different types of exosomes exist in the whole blood, it is necessary to isolate and detect disease-specific exosomes. for this reason, the isolation and the detection of exosomes is an important research issue and has been studied by many groups. however, limitations such as low throughput and low recovery still make it difficult to use exosomes in diagnostics and therapeutics. methods: in this study, we developed an integrated microfluidic electrochemical biosensor to extract plasma from whole blood and subsequently detect cancer related exosomes in a continuous manner. this consists of two parts. the first part is a channel for extracting plasma containing exosomes from whole blood, and the second part is a channel combined with an electrochemical sensor for multiple detection of various exosomes in the extracted plasma. previously, a multi-orifice flow fractionation (moff) channel that consists of a series of expansion and contraction structures has been developed in our group. in this channel, the blood cells are moved to sides of channels by hydrodynamic forces and then are eliminated to outlets. at this time, the plasma is moved to the electrochemical sensor part, the exosomes in the plasma are captured to the electrodes immobilized with the specific antibodies and are quantified the amount of cancer-related exosomes. results: using this chip, blood cells were eliminated from the whole blood with over % of separation efficiency at µl/min flow rate and exosomes were collected continuously with high recovery (~ %). in order to quantify various types of exosomes, a labelfree electrochemical biosensor with electrochemical impedance spectroscopy (eis) was used for the continuous detection of exosomes. the limit of detection was x ^ exosomes/ml. summary/conclusion: the developed device is an integrated device capable of separating exosomes from whole blood with high purity and quantitating exosomes through the electrochemical sensor in a continuous manner. , , ) . the development of highthroughput techniques capable of simultaneously monitoring physical and biochemical properties of evs would significantly simplify and accelerate the characterization process. in this context, microfluidic technology is emerging as an attractive platform. here, we present a microfluidic device based on the combination of diffusion sizing and multi-wavelength fluorescence detection to simultaneously provide information on ev size, concentration and composition. methods: the diffusion of evs in the microfluidic channel provides information on their size distribution, and four different staining protocols with high signalto-noise ratios track different ev native molecules. evs are separated from unbound fluorophores directly during the microfluidic analysis, therefore avoiding the need for sample pretreatments and allowing to operate the device as a single-step immunoassay. results: the microfluidic device coupled with complementary staining techniques allows to individually detect and size particle populations with different ev components such as lipids, primary amines and the ev marker cd . we demonstrate that this approach can probe the abundance of ev-specific markers and impurities such as lipoproteins with high throughput and low sample consumption. summary/conclusion: we present a microfluidic technique capable of characterizing and quantifying evs at low costs, in a time-scale of minutes and requiring only up to µl of non-pretreated sample. this method is an important complementary tool to the current array of biophysical methods for ev characterization, in particular for high-throughput screening applications. funding: h -eu. . . -fet open programme via the grant agreement . immunomagnetic isolation of specific subpopulations of exosomes for liquid biopsy via nano-architected porous materials introduction: exosomes offer the potential to reveal significant biological information in many areas of clinical importance by virtue of their rna contents and protein surface markers. this abstract reports the fabrication of a device for high throughput targeted immunomagnetic capture of exosomes via the use of highly-ordered nano-architected porous metal lattice materials. methods: we have invented a fabrication technique to precisely make millions of nanoscale exosome sorting devices that can operate on unprocessed plasma. each nanoscale device can precisely sort targeted exosomes from background vesicles but is too slow for practical use individually. however, the operation of millions of these devices in parallel preserves the precision of nanoscale sorting while also enabling high throughput and robust use on raw plasma samples. the metal lattice within which these devices are contained is assembled via metal electroplating onto a selfassembled polystyrene bead lattice with face-centred cubic (fcc) symmetry with nanometre pores. the devices feature a conformally-coated layer of nickel-iron with gold passivation atop a base layer of nickel, resulting in a lattice of millions of nanoscale pores capable of magnetic sorting of exosomes tagged via surface-marker-based immunomagnetic labelling with magnetic nanoparticles. results: compared to our previous work on immunomagnetic exosome capture via commercial track-etched membranes (tempo), this device offers superior capture due to increased surface pore density (> x) and three-dimensional pore density (> x) alongside lower required sample volume due to decreased noncapturing volume in the device. finite-element analysis simulations show that strong magnetophoretic traps emerge at the pore boundaries in this structure between higher-permeability metals such as nickeliron permalloy and the lower-permeability sample fluid in the device. preliminary experimental data shows that this device can isolate iron nanoparticles in solution with > x enrichment from input and x capture efficacy versus tempo. summary/conclusion: current methods of exosome isolation such as ultracentrifugation and column chromatography all suffer from low throughput and limited yield. the application of inverse opal materials towards exosome capture offers the potential for isolation of specific exosome populations from very low clinical sample volumes or sparse biological signals. micropatterned growth surface topography affects extracellular vesicle production colin l. hisey a , james hearn b , yohanes nursalim a , vanessa chang a , cherie blenkiron a and lawrence w. chamley a a the university of auckland, auckland, new zealand; b university of auckland, grafton, new zealand introduction: extracellular vesicles are micro and nanoscale packages released by all cells and play an important role in cell-to-cell communication by shuttling biomolecules to nearby and distant cells. however, producing enough evs for many in vitro studies using conventional tissue culture techniques can be challenging, and despite the success of some bioreactors in increasing ev-production, it is still unknown how many independent culture conditions like growth surface topography can alter the production and content of evs. methods: standard mm petri dishes were patterned with µm tall polystyrene microtracks spaced by , and µm across a mm area using standard microfabrication techniques including photolithography, soft lithography and microtransfer printing. the micropatterns were characterized with sem and profilometry, then activated with oxygen plasma and uv sterilized. mdamb cells were seeded onto patterned and smooth (control) dishes and grown in serum-free media for the final hours of culture. evs were isolated using sequential ultracentrifugation of conditioned media and characterized using nta, tem and western blot. cell morphology was imaged using immunocytochemistry and single cell migration was characterized using time-lapse microscopy and manual single cell tracking in fiji. results: we demonstrate the simple and repeatable fabrication of microtracks across a large surface area in order to culture cells on topographically patterned growth surfaces. furthermore, we show that the µm spacing produced significantly more evs than other patterns as well as the highest cell aspect ratio and average single cell migration speed (p < . ). summary/conclusion: these findings have implications in both biomanufacturing of evs and potentially in enhancing the biomimicry of evs produced in vitro. however, further experimentation to assess the differences in cargo on patterned growth surface topographies compared to conventional methods is still required. funding: this project was funded by the maurice and phyllis paykel trust. using miscroscale thermophoresis and surface plasmon resonance to measure the interactions of extracellular vesicles mst is a quick method, easy to handle, has a low sample consumption, has no limitation on molecule size, and enables measurements in solution, either in various buffers or complex biological liquids. these properties make mst an interesting tool for research of extracellular vesicles (evs); therefore, our aim is to apply this method to evs. methods: evs were isolated from jurkat cell line by differential centrifugation. microscale thermophoresis (mst) and surface plasmon resonance (spr) were used to analyse the interaction between antibody and evs. results: we have demonstrated that interactions of evs with antibodies could be analysed by mst. however, the tiny glass capillaries for sample mounting represent a challenge due to adhesion of evs to their surface. we have tested commercial capillaries as well as prepared capillaries in house coated by liposomes or bovine serum albumin. the interactions between evs and antibodies were confirmed by surface plasmon resonance (spr), which is an established method for studying the interactions of evs. introduction: the isolation of extracellular vesicles (evs) from cell culture supernatants and complex body fluids, such as blood and urine, is of high importance for ev research as well as for future medical applications in diagnostics and therapy. nevertheless, it is still challenging to reach the desired recovery, purity and specificity due to many manual and time intensive sample preparation steps. conventional centrifugation for ev isolation or sample preparation prior to affinity-based separation methods can damage evs and cells, leading to misinterpretation of results or inactive evs. alternative field flow fractionation methods employing acoustic fields are highly promising, but so far limited to laboratory usage, based on a complex (moulding) fabrication and/or hardly reproducible. here, we present an innovative surface acoustic wave (saw)-based acoustofluidic device for gentle sorting of cells and particles. methods: our device consists of interdigital transducers patterned on a piezoelectric substrate generating saw propagating on the substrate surface. upon interaction of saw with our on-chip structured, fluid-loaden microchannels, an acoustic pressure field is developed across the fluid wherein particles are suspended. this pressure field can be employed to simply manipulate cells and particles based on their intrinsic properties, such as size, density and compressibility in continuous flow. the device is manufactured using precise and low-cost microtechnological methods and is suitable for reproducible mass fabrication. results: we demonstrated the separation of blood components, i.e. the sorting of erythrocytes and thrombocytes. furthermore, we could also show results on thrombocyte activation indicating a gentle separation without damaging these shear-sensitive cells, as well as first results on plasma separation from whole blood samples and nanoparticle sorting. summary/conclusion: our unique acoustofluidic sorting technology for complex suspensions has the potential to overcome the need for time-effective, cheap and gentle separation of evs. funding: this work was supported by efre infrapro project "champ: chip-based acoustofluidic medtech platform". nanophotonic platform for cancer-associated exosomal microrna detection introduction: exosomes have an important role in intercellular communication at physiological and pathological processes. their cargo includes micrornas (mirs), single-stranded non-coding rnas, involved in alterations on recipient cells, such as development of tumourous phenotype and metastasis. more particularly, mir- excels due to its association with several cancers. determining exosomal mirs as cancer indicators demands selective and accurate methods, which are not currently available or entail high costs. colorimetric photonic-based assays are a promising label-free alternative, which dismisses complex apparatus for signal reading since biorecognition is detected by colour change. moreover, the clinical and economic systems have also been demanding a decrease on the green footprint of biosensors, requirement fulfilled with naturally derived biomaterials. methods: herein, the biosensor is constructed on a biopolymer matrix to meet the requirements of an eco-friendly disposable device, and it is based on a photonic structure obtained by imprinting a nanopattern on the polymer surface. then, the surface is functionalized with the complementary oligonucleotide sequence of mir- as sensing probe. a labelfree detection is thus envisioned and the sensor performance is evaluated by changes in the optical properties when the target is present. results: the combination of biological materials conducted to a biosensor support with great flexibility and low water permeability, allowing easy surface functionalization. the self-reporting ability of the photonicbased sensor enables high intensity colours detected by naked eye. summary/conclusion: the alliance with the high selectivity of oligonucleotide hybridization is expected to offer great exosomal mir- recognition ability and an optimistic perspective for utilization in clinical setups. funding: the authors acknowledge the financial support from the european commission/h , through mindgap/fet-open/ga project. introduction: urinary extracellular vesicles (uevs) are important intercellular communicators. by systems biology integration, uevs prove to be relevant in genitourinary disease detection. however, it has recently been shown that labelled evs administered to the circulation can be detected in the urinary system, as well. thus, this pilot study aimed at phenotyping haematopoietic surface markers on uevs to create enough plausibility for future non-invasive biomarker studies of circulation and immune disorders that may translate into urine but are not yet timely recognized. methods: urine was obtained from healthy men signing a written informed consent (n = ). sampling was approved by the local ethics committee and in compliance with the declaration of helsinki. cell-free urine was obtained by serial centrifugation and ml, each, were utilized for the macsplex exosome kit, human (miltenyi biotec). the manufacturer's recommendations were followed to examine distinct uev surface markers of cd +/cd +/cd + vesicles in a multiplexed bead-based manner including respective controls. the accuri c (bd) was utilized for data acquisition. for further misev -compliant characterization, cd +/cd +/cd + uevs were isolated by immunoaffinity and analysed by fluorescence nanoparticle tracking (f-nta), transmission electron microscopy (tem) and western blotting (wb). urinary creatinine (ucrea) was determined to control for variances in urinary dilutions and used for data normalization. results: except cd , all other surface markers could be identified. the most abundant markers were cd and cd , which were detected in % of samples, followed by cd / ( %), cd ( %), cd and cd ( %, each). cd ( %), cd , cd ( %), cd e ( %) and cd showed similar relative median fluorescent intensities (rmfi), while cd yielded significantly higher (p = . ) and all other markers significantly lower rmfi (p < . ). tem and f-nta revealed cup-shaped vesicles ( ± nm) with . ± . e + particles/g ucrea. wb indicated uev isolates that were positive for alix, syntenin, tsg , cd , cd and cd without any uromodulin or calnexin contamination. summary/conclusion: our results imply that considerable quantities of circulatory evs are, indeed, filtered into urine and could serve as valuable non-invasive biomarkers for systemic dysfunctions. cardiovascular risk markers are strongly related to numbers of circulating extracellular vesicles ruihan zhou a , esra bozbas a , plinio ferreira b and parveen yaqoob a a university of reading, reading, uk; b imperial college london, london, uk introduction: extracellular vesicles (evs) are small plasma membrane-derived vesicles released from various cells, which potentially affect many physiological and pathophysiological processes, and are emerging as a potential novel biomarker in cardiovascular diseases (cvds). however, there is little information about the association of circulating ev levels with traditional cardiovascular risk markers and cvd risk score. methods: • subjects (n = ) aged - yrs with moderate risk of cvds were recruited and assessed for body mass index (bmi), blood pressure (bp) and plasma lipid profile (triacylglycerol, total cholesterol and high-density lipoprotein). • evs were isolated from platelet-free plasma by size exclusion chromatography and analysed by both nanoparticle tracking analysis (nta) and flow cytometry (fcm). nta was used to measure the concentration and size distribution of evs population, and evs were phenotyped by fcm via a colour panel, which included annexin v (for the majority of circulating evs), cd (for plateletderived evs) and cd (for endothelialderived evs). • the association between risk markers and ev numbers was examined by pearson's correlation coefficient and stepwise multivariate regression model. analysis of covariance (ancova) was performed after adjustment for various variables to determine the correlation between the quartile range of ev numbers and -yr cvd risk detected by qrisk . results: ev numbers, as determined by nta, were strongly associated with bmi (r = . , p < . ), blood pressure (systolic bp: r = . , p = . ; diastolic bp: r = . , p < . ) and plasma triacylglycerol levels (r = . , p < . ). plasma total cholesterol level was positively associated with platelet-derived evs, determined by fcm (r = . , p = . ). a multivariate regression model demonstrated that plasma triacylglycerol and diastolic bp independently predicted total ev numbers, with plasma triacylglycerol concentrations explaining . % of the variance for total ev numbers. an additional . % of the variance in total ev numbers was predicted by diastolic bp. ancova of the -yr cvd risk score in the quartile range of total ev numbers were positively and independently associated. summary/conclusion: bmi, blood pressure, plasma triacylglycerol and total cholesterol levels are strongly associated with ev numbers. plasma triacylglycerol and diastolic bp independently predict circulating ev numbers. elevated numbers of evs are independently associated with -yr cvd risk. introduction: extracellular vesicles from cardiospherederived cells (cdc-evs) are known to be anti-inflammatory in various disease models. to further dissect the mechanism, we examined the effects of cdc-evs on t lymphocytes. methods: naïve cd + t cells were isolated from secondary lymphoid organs of foxp -rfp reporter mice, using magnetic-activated and fluorescence-activated cell sorting. cells were subsequently polarized into effector subtypes (th , th , and th ), as well as regulatory t cells (tregs), and the effects of exposure to human-derived cdc-evs on proliferation and cytokine production were assessed. cdc-evs were isolated from serum-free, -day conditioned medium, using ultrafiltration by centrifugation. results: after polarization and culture for days, cdc-evs resulted in dose-dependent and cell-specific proliferative responses. effector t cells (th , th , th ) showed either no change in proliferation (th ) or decrease in proliferation (th , th ), compared to the vehicle control. in contrast, tregs proliferated much more than control (p < . ). next, we sought to characterize the changes in cytokine production by each effector t cell and tregs. compared to the vehicle control, exposure of polarized effector t cells to cdc-evs had little effect on the expression of characteristic cytokine genes, including ifnγ and tnfα (th ), il and il (th ), or il a and il f (th ). in contrast, exposure of tregs to cdc-evs resulted in~ -fold increase in expression of il , a key paracrine agent utilized by tregs in suppression of inflammation. this response was specific to cdc-evs insofar as it was not recapitulated with dermal fibroblast exosomes. concentrations of il- in the culture media of cdc-ev-conditioned tregs mirrored the increases in gene expression. summary/conclusion: cdc-evs potentiate tregs by increasing their proliferation and enhancing production of il- . this offers an attractive therapeutic approach to inflammatory diseases that relies on harnessing an endogenous mechanism of immunosuppression. funding: nih t hl prostanoids impair platelet reactivity, thrombus formation and platelet extracellular vesicle release in patients with pulmonary arterial hypertension aleksandra gąsecka a , marta banaszkiewicz b , rienk nieuwland c , edwin van der pol d , najat hajji e , hubert mutwil f , sylwester rogula a , wiktoria rutkowska a , szymon darocha g , grzegorz opolski a , krzysztof j. filipiak f , adam torbicki g and marcin kurzyna g introduction: prostanoids (epoprostenol, treprostinil and iloprost) induce vasodilation in advanced pulmonary arterial hypertension (pah) but also inhibit platelet activation, thereby increasing the risk of bleeding. therefore, the platelet function and extracellular vesicle (ev) concentrations were measured in pah patients treated with prostanoids and compared to patients with pah not receiving prostanoids. methods: venous blood was collected from patients treated with prostanoids (study group; n = , ± years, % female) and patients not treated with prostanoids (control group; n = , ± years, % female). platelet reactivity was analysed in whole blood by impedance aggregometry using arachidonic acid (aa; . mm), adenosine diphosphate (adp; . µm) and thrombin receptor-activating peptide (trap; µm) as agonists. in a subset of patients, concentrations of evs from platelets (cd + and cd p+; pevs), leukocytes (cd +, levs) and endothelial cells (cd +, eevs) were measured in plateletdepleted plasma by flow cytometry (a -micro). platelet-rich thrombus formation was measured using a whole blood perfusion system. results: compared to the control group, patients treated with prostanoids had lower platelet reactivity in response to aa and adp (p = . ) and lower concentrations of pevs and levs (p ≤ . ). furthermore, thrombus formation was delayed (p ≤ . ) and thrombus size was decreased (p = . ) on prostanoids. epoprostenol did not affect platelet reactivity in vitro, but decreased the concentrations of cd + pevs (p = . ). in contrast, treprostinil and iloprost decreased both platelet reactivity in response to aa and adp (p ≤ . ) and the concentrations of pevs (p ≤ . ). all prostanoids delayed thrombus formation and decreased thrombus size (p ≤ . ). summary/conclusion: patients with pah treated with prostanoids have increased risk of bleeding both due to impaired platelet aggregation, ev release and thrombus formation, compared to patients not treated with prostanoids. antiplatelet effect of prostanoids varies: whereas epoprostenol decreases the release of pevs, treprostinil and iloprost impair platelet aggregation. funding: ag is supported by the national science centre, research project preludium / /n/ nz / . evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . nanoflow cytometry identifies an imbalance of epithelium-and neutrophil-derived extracellular vesicles in the airway environment of paediatric cystic fibrosis patients brian dobosh, vincent giacalone, milton brown, lucas silva, lokesh guglani and rabindra tirouvanziam emory university, atlanta, usa introduction: progressive lung disease is the leading cause of mortality in cystic fibrosis (cf), a chronic condition characterized by recruitment of polymorphonuclear neutrophils (pmns) into the airways. newly arrived pmns are exposed to extracellular vesicles (evs) from the airway epithelium and pmns recruited before them. in controlled experiments, these evs were necessary and sufficient to induce pathological changes including reduced bacterial killing and immunosuppressive activities towards macrophages and t-cells. however, children with cf do not always show a high pmn presence in their airways, which suggests that the balance between pmn recruitment and the activity of other cells is still in flux in early stage disease. methods: we utilized spectral nanoflow cytometry to profile the single ev content of the bronchoalveolar lavage fluid (balf) from cf children (< years of age). for nanoflow cytometry, evs were stained with di- -anepps, and with epcam, cd b and cd (to ascertain epithelial, pmn, and macrophage origins, respectively). violet side scatter and/or fluorescence threshold triggering were used for ev detection. the ratio of neutrophil-to epithelial-derived evs in cf balf correlated positively with the percentage of pmns that are present in the airways (p = . , spearman's rho = . ). this ratio also correlated with the pragma disease score, which quantifies airway damage by chest computed tomography (p = . , rho = . ). summary/conclusion: using a method to quantify evs from specific cell types in vivo, we demonstrated that the ratio of pmn-and epithelial cell-derived evs tracks with airway damage and neutrophil influx, suggesting a critical interplay between these cells in early cf disease. this ev-focused method can be applied to other diseases in which sampling cells is difficult. future experiments will use cf balf biobanks to strengthen data presented here. funding: cf foundation (tirouv a ), emory paediatrics flow core. the potential of crude extracellular vesicle micrornas for the diagnosis of community-acquired pneumonia and for the detection of pneumoniarelated sepsis as a severe secondary complication introduction: circulating cell-free micrornas (mirnas), often associated to extracellular vesicles (evs), are essential for cell-cell communication in the pathogenesis of infectious pulmonary disorders. as early pneumonia diagnosis is often clinically challenging, advances in disease detection could improve outcomes. we characterized crude ev mirnas as potential biomarkers for community-acquired pneumonia and sepsis as a severe secondary complication. methods: individuals were enrolled into our study, subdivided into a training (volunteer n = , pneumonia n = , sepsis n = ) and testing cohort (volunteer n = , pneumonia n = , sepsis n = ). after precipitating crude evs from sera (mircury exosome isolation kit-serum and plasma) and extracting total rna, small rna sequencing was performed. mirnas were selected as biomarker candidates by differential gene expression analysis (deseq ) and sparse partial-least-squares discriminant analysis (mixomics). technical and biological validation was performed by reverse transcription quantitative real-time pcr. group classification was predicted by partial-least-squares discriminant analysis. gene targets and causal networks were identified by ingenuity pathway analysis. results: differential gene expression analysis revealed significantly regulated mirnas in pneumonia compared to volunteers, and mirnas in pneumonia related to sepsis. based on sparse-partial least discriminant analysis, group separation was achieved by mirnas as discriminators with high sensitivity and specificity (area under the curve of the receiver operated curve: volunteer: . , pneumonia: . , sepsis: . ). mir- a- p (log fc = . , padj = . e- ) and mir- - p (log fc = . , padj = . e- ) differentiated between pneumonia and volunteers and mir- (log fc = − . , padj = . e- ) between pneumonia and sepsis. expression levels of mir- a- p and mir- were related to disease severity. mir- - p was higher expressed in pneumonia compared to volunteers and had equal expression in patient groups. prediction of group classification in the testing cohort was . %. signalling networks were constructed for "cellular and humoral immune response", "antimicrobial response" and "pathogen influenced signaling" involving the significantly regulated mirnas. summary/conclusion: crude ev mirnas are potentially novel biomarkers for community-acquired pneumonia and may help to identify patients at risk for progress to sepsis allowing early intervention and treatment. introduction: it remains unclear the specific mechanisms that lead to airways inflammation in asthma and the subsequent remodelling of the airways. exosomes, small extracellular vesicles, has become in an important mechanism of cell-to-cell communication and participate in diverse biological processes including inflammation. in this study, we hypothesize that exosomes and their mirna cargo play an important role in the proinflammatory status of the upper airway of asthma patients, especially in those patients with severe asthma. methods: in a pilot study, healthy subjects had induced sputum using standard methods. after several centrifugation steps, we were able to isolate exosomes from sputum supernatant by both precipitation and size exclusion cromatography (sec). exosome size was observed with transmission electron microscopy (tem) and the protein markers cd and cd were analysed by western blot (wb). then, total rnas were isolated from sputum exosomes and mirnas (mir- a-p, mir- - p, mir- a, mir- b- p, mir- - p, mir- - p, mir- - p, mir- - p, let- g- p), were evaluated by rt-qpcr. after the optimization of the methodology, healthy adults subjects and patients with persistent moderatesevere asthma, matched by age and sex were selected and induced sputum was collected. results: exosomes isolated with both methodologies (precipitation and sec) were observe under the tem with a correct range of size. furthermore, wb assay displayed a coherent protein profile for the exosome markers cd and cd . however, sec displayed low signal and the variability of between subjects was to higher. using the optimized method of precipitation, we observed that after normalization, mirna- a showed a significant increased (p = . ) in asthma patients compared to control. this mirna has been linked with an active proinflammatory status. summary/conclusion: our results confirm the presence of exosomes in induced sputum with promising applications in the field of asthma. the upregulation of exosomal mir- a, which is related with inflammation, suggest that exosomes could play a crucial role in the chronic inflammation of airway described in asthma patients. human nrf -active multipotent stromal cell exosomes reverse pathologic delay in the healing of cutaneous diabetic wounds introduction: multipotent stromal cells (mscs) have attracted much attention for their capacity to accelerate wound healing. exosomes, nanosized extracellular vesicles, may be key to translating msc therapy. we previously found that nuclear factor erythroid -related factor (nrf ) regulates msc promotion of diabetic tissue repair. here, we explore a novel role of nrf in exosome biogenesis and investigate whether exosome treatment recapitulates the effects mscs have on healing. methods: exosomes were harvested by differential ultracentrifugation of conditioned bone marrow derived msc media. for nrf -active exosomes, mscs were incubated with potent nrf activator, cddo-im. exosomes and mscs were vigorously characterized. full-thickness humanized-stented wounds were created on adult leprdb/db diabetic mice (db/db). exosomes were injected intradermally and circumferentially to the wound margin. results: mscs adopt an adherent fibroblast morphology, demonstrate robust osteogenic, chondrogenic, and adipogenic differentiation, express > % positive msc markers (cd , cd , cd , and cd ) and < % express negative markers (cd , cd , cd , cd , or hla-dr). immunoblotting of msc exosomes shows enrichment for positive exosomal markers cd , cd and tsg . nanoparticle tracking analysis (nta) shows a nanoparticle population with mean diameter of . ± . nm. transmission electron microscopy of exosomes reveals flattened cup-like spheres. nta demonstrates that nrf -active human mscs increase exosome secretion by %, compared to nrf -baseline mscs (p < . ). both nrf -baseline and nrf -active exosome treatment significantly reduced closure time to . and days respectively, compared to . days for vehicle-treated wounds (p < . ). this reduction eliminated the delay in closure time compared to wounds of c /b mice. nrf -active exosome treatment of db/db wounds reduced closure time by a further . days compared to untreated c /b wounds. at day , exosometreated db/db wounds have significant decreases in epithelial gap, expanded granulation tissue, and greater density of cd + vessels compared to vehicle-treated wounds. summary/conclusion: enhancing nrf function in mscs multiplies exosome yield. our results demonstrate exosome-based therapies hold tremendous promise and warrant further investigation for rapid translation. introduction: obesity increases prostate cancer aggressiveness and adipose tissue (at) is a rich source of extracellular vesicles (ev) that have been shown to contribute to pro-oncogenic effects in various malignancies. twist is a key mediator of tumour cell metastasis. the goal of this study was to determine molecular and phenotypic changes of prostate cancer cells in response to evs from obese human at and the role of different levels of endogenous twist . methods: ev were harvested from human at (atev) obtained from bariatric subjects or from at endothelial cells treated with proinflammatory cytokines (pic-ev) to mimic the obese at environment. evs were isolated by ultracentrifugation and characterized by electron microscopy, nta and protein markers. we determined the effect of atev and pic-ev on pc -ml prostate cancer cells proliferation and invasion. ev mirna cargo and transcriptome of pc -ml cells treated with atev or pic-ev were assessed using nanostring. to establish the contribution of twist to the ev-related phenotypic and molecular changes in recipient cells, we used pc -ml lines stably overexpressing or deficient in twist . results: atev from obese subjects and ev-pic from at endothelial cells both reduced invasion and increased proliferation in wild-type pc -ml cells. a molecular signature showing decreased expression of genes mediating invasion, adhesion and metabolism supported these functional effects. also atev and ev-pic shared a subset of mirna that target multiple mmps, inhibit glycolytic genes and target cell cycle inhibitory genes. pc -ml overexpressing twist showed an increase in both proliferation and invasiveness and this phenotype was supported by the transcriptomic analysis following ev treatment. summary/conclusion: ev produced by obese at or by at endothelial cells share a subset of mirna that in conjunction with increased twist expression contribute to tumorigenesis and metastasis of prostate cancer cells in vitro. introduction: exercise is associated with various health benefits, including the prevention and management of obesity and cardiometabolic risk factors. however, a strong heterogeneity in the adaptive response to exercise training exists. differential response to exercise training might be mediated by myokines (proteins, nucleic acids, metabolites) that can be released directly into the systemic circulation, or packaged within extracellular vesicles (evs). the objective of this study was to evaluate if changes in evs after acute aerobic exercise (ae) were associated with the responders phenotype following -week resistance exercise (re) training. methods: this is a secondary analysis of plasma samples from the exit trial (clinical trial # ). eleven sedentary obese youth ( . ± . years, bmi ≥ th percentile underwent an acute bout of ae ( % heart rate reserve, min). blood was collected before [time (at) − , min], during [at , , min], and after [ min (at ), min (at )] exercise. afterwards, youth participated in -week re programme, and were categorized into responders or non-responders (nr) based on changes in insulin sensitivity (above or below percentile). primary outcome: evs were isolated using size exclusion chromatography (izon®) at baseline (at ), immediately after ae (at ) and after recovery (at ). ev protein concentration, size, and zeta potential were analysed in a single-blind fashion. results: responders had larger evs (~ . nm) as opposed to nrs (~ . nm) at at (p < . ) and this pattern was maintained at at and at , though not significant (p = . ). nrs displayed differential ev size distribution (peaks at nm or nm), while ev distribution was highest at nm in responders. no difference in average zeta potential or total ev protein yield was observed between groups. an increase in ev yield with exercise time and recovery was observed in both groups. summary/conclusion: our preliminary data suggest that ev size is significantly increased after an acute bout of ae in obese youth responders. further research to delineate the role of evs as predictors of exercise adaptation is warranted. funding: funded by dream and research manitoba. using dual-fluorescent reporter mice to track tissue-specific extracellular vesicles andrea estrada, gabriella hehn, zackary valenti, christopher allen, nicole kruh-garcia and dan s. lark colorado state university, fort collins, usa introduction: extracellular vesicles (evs) from tissues like skeletal muscle (skm) and adipose tissue (at) have been implicated in human disease but are understudied. skm is likely a major player in ev biology as it accounts for~ % of total body mass. tools to define cellular ev origin are needed because tissues like skm are comprised of a variety of cell types. here, we describe our ongoing efforts using the dual fluorescent mg/mt mouse as a tool to analyse skm-myocyte derived evs. methods: wild-type (wt) and mg/mt mice were used for these studies. mg/mt mouse cells express membrane-tagged red (mt) or green (mg) fluorescent protein in the absence or presence of cre, respectively. we made skm myocyte mg expressing mice using a mouse expressing cre on the human skeletal actin promoter. blood was collected via cardiac puncture and platelet-free plasma was obtained via centrifugation. plasma evs were isolated using exoquick, exoquick-tc or size exclusion chromatography. skm and at were dissected into~ mm chunks, placed in serum-free dmem and incubated for hours. tissuederived evs were isolated using exoquick-tc. ev abundance was determined with a horiba viewsizer. individual evs were analysed with a cytek aurora spectral flow cytometer. settings were optimized using polystyrene beads and spectral unmixing was performed to allow detection of mg and mt. results: in wt mice, skm releases > times more evs than adipose tissue per unit of mass (p < . using paired student's t-test). since skm is also a major component of total body mass, these data further emphasize the importance of skm-derived evs. skmderived evs from wt mice were not fluorescent (< . % of events). evs from mg/mt mouse skm overwhelmingly expressed mg (> % of events) with negligible (< %) expression of mt. at-derived evs robustly expressed mt but lacked mg. summary/conclusion: these data provide "proof-ofprinciple" that mg and mt are readily incorporated into evs secreted ex vivo. surprisingly however, plasma evs from mg/mt mice expressed very little mg (~ %) or mt (~ %). this observation was confirmed with three separate isolation techniques. we are currently exploring possibilities to explain this finding, including: ) modification of evs post-secretion, ) clearance of fluorescent evs by the liver or ) that evs secreted from tissues remain predominantly in the interstitial space. funding: this work was supported by an innovative project award from the american heart association ( ipa ) to dsl. endothelial cd delivery of fa loaded extracellular vesicles is critical for thermogenesis. introduction: membrane cd facilitates tissue fatty acid (fa) uptake. we recently found that endothelial cell (ec) cd controls muscle and adipose tissue fa uptake, and influences the tissue's metabolic phenotype. the mechanism for cd -facilitated fa uptake is unknown. here we examined the role of ec cd in thermogenesis and in fa delivery to brown fat tissue. methods: adult male mice were housed individually, restricted from food during acute ( hr) cold exposure ( °c) with core temperature monitored every minutes. after hours, animals were sacrificed and samples collected for analysis. for cellular studies, human microvascular (lonza) or primary murine microvascular ec were used. for primary cells, crude cell pellets from lung homogenates were purified using mouse-cd magnetic beads (miltenyi). for microscopy studies, alkyne fa (cayman) was added to cells and to enable visualization of internalized fas, click chemistry (invitrogen) used to label alkyne-fa with alexa . for radioactive studies, primary lung ec were serum starved for hrs and incubated overnight with h-oleic acid bound to fa-free bsa ( : ratio). media was collected, clarified by centrifugation to remove microvesicles and debris. small extracellular vesicles (sevs) were isolated from clarified media using total exosome isolation reagent (invitrogen) and counted for radioactivity. results: basal core body temperatures are similar in mice lacking ec cd (eccd -/-) compared to controls (cd fl/fl). however, during cold exposure at °c , eccd -/-are unable to maintain body temperature (p < . ). plasma free fa are higher in cold exposed eccd -/-indicating fa clearance by brown fat is impaired. mitochondrial function and expression of thermogenic and mitochondrial genes in brown fat from eccd -/-and cd fl/fl mice were similar. these data suggested that endothelial delivery of fas is necessary for thermogenic maintenance of body temperature. to examine fa handling by ecs we used alkyne fas to visualize the process. we found that fas are transferred by microvascular ec through caveolae-mediated transcytosis involving src signalling and cav- phosphorylation. the internalized cav- and cd positive vesicles containing fas are released as sevs. to determine the dependence of cd on this process, we treated primary microvascular ec with radiolabeled fa and found that sevs secreted by cd -/-cells contain less labelled-fa (p = . ). summary/conclusion: endothelial delivery of fa is critical for thermogenesis. our working model for the mechanism of fa uptake by brown adipose tissue is the following: endothelial cells transfer the fa through caveolae-mediated transcytosis and secrete small extracellular vesicles (sevs) that help deliver fas to brown adipocytes. funding: this work is supported by nih grants dk and dk . introduction: diet-induced obesity modifies intestinal permeability leading to bacteria infiltration and to a decrease in the number of immune cells protecting mucosa. as orange consumption is beneficial for human health and used in preventive medicine, we determined whether orange juice-derived nanovesicles (onv) might be recommended as nutritional strategies for the treatment of intestinal complications associated with obesity. methods: onv isolated from fresh orange juices were characterized by lipidomic, metabolomic, microscopy, nta and for their stability during digestion. intestinal barrier (ib = caco- cells+ht- cells differentiated with oleic acid) were treated with onv and co-cultured with adipocytes to monitor ib fat absorption and release. obesity was induced in mice fed for weeks with a high-fat high-sucrose diet (hfhs mice vs standart chow diet mice). then half of the hfhs mice were gavaged with micrograms/day for weeks. results: onv did not modify high-fat high-sucrose diet-induced obesity and insulin resistance but reversed diet-induced gut modifications. six hours post-gavage, onv accumulated preferentially in jejunum involved in lipid absorption. in jejunum, and no other intestinal region, onv increased villi size, restored immune response and decreased barrier permeability in hfhsd mice. in addition, onv-treated mice had increased expressions of acat , angptl and dgat , but a decreased expression of fabp , fatp , mtp vs hfhsd animals, which indicated that fat absorption, tg synthesis and chylomicron release were strongly reduced. similarly to other plant-derived nanovesicles, these results were likely associated with onv lipid and metabolite compositions (strong enrichment in bioactive phospholipids: pe, pa, pc, pi and leucine) as onv did not resist to harsh digestive conditions in vitro and were poorly incorporated in enterocytes. as the effects of onv on the decrease in tg content and epithelial cell growth were also observed in vitro, gut microbiota unlikely participate to these effects. summary/conclusion: onv are important bioactive compounds of orange juice and for the first time we demonstrated that they can modulate lipid metabolism in the intestinal barrier associated with morphological changes. interestingly onv treatment targets mtp and angptl mrnas, therapeutic intestinal targets to reduce plasma lipids and for attenuating inflammation in gastrointestinal diseases. therefore, onv might be used to reduce the development of dyslipidemia-associated diseases and to restore intestinal functions in obese patients. funding: olga triballat institut; benjamin delessert institut, inrae institut. association, structure, and function of fibronectin in extracellular vesicles from hepatocytes xinlei li, ruju chen, sherri kemper and david brigstock nationwide children's hospital, columbus, usa introduction: we have shown that extracellular vesicles from normal hepatocytes have anti-fibrogenic activity and that they preferentially bind to hepatic stellate cells (hscs, the principal fibrosis-causing cell in the liver) and hepatocytes. in this study, our goal was to determine the molecular nature of the ev components involved in cell binding. fibronectin (fn ) is a key component of extracellular matrix, functioning in processes including cell adhesion, differentiation, and wound healing. two types of fn are present in vertebrates, of which the soluble plasma fn is derived principally from hepatocytes, while cell-associated fn is produced by numerous cell types. here we describe a novel function of plasma fn in facilitating binding of hepatocyte evs to target cells. methods: differential ultracentrifugation was used to collect evs released by parental mouse aml hepatocytes, fn ko aml cells in which fn was ablated using crispr-cas , primary human or mouse hepatocytes, or human hepg cells, or from human or mouse serum. evs were characterized by nanosight tracking analysis (nta), western blot, iodixanol gradient ultracentrifugation, and mass spectrometry. the binding efficiency of pkh -labelled evs from parental (ev-hep) or fn ko (ev-hepfn ko) aml cells was analysed in hepatocytes or hscs. swiss webster mice were injected with ccl for five weeks to induce liver fibrosis, with some mice also receiving i. p. administration of ev-hep or ev-hepfn ko over the last two weeks, followed by determination of hepatic fibrogenic genes by qrt-pcr. results: ev-hep or ev-hepfn ko were - nm in diameter and positive for common ev markers (cd , cd , flotillin- ). mass spectrometry showed that fn was the most abundant protein in ev-hep and comprised principally the plasma form. the abundant presence of ev fn was verified by western blot and co-immunoprecipitation with anti-cd or antiflotillin- . western blot showed that fn was also abundant in evs from primary human or mouse hepatocytes, hepg cells, and human or mouse serum. fn and ev-hep co-sedimented at a density of~ . g/ml. ev-hepfn ko yield and size-range were similar to those of ev-hep, suggesting that ev biogenesis is fn -independent. as compared to ev-hep, the binding of ev-hepfn ko to target cells was highly reduced whereas ev binding was independent of fn expression by the target cells themselves. both ev-hepfn ko and ev-hep were anti-fibrogenic in vivo but only ev-hep attenuated collagen ⍺ expression in mouse hscs in vitro. summary/conclusion: fn is abundantly associated with hepatocyte evs and facilitates ev binding to target hepatocytes or hscs. additional studies are needed to clarify the functional role of fn in mediating ev-hep anti-fibrogenic actions in vitro or in vivo. elevated glucose increases soluble and aggregated forms of human islet amyloid polypeptide in islet-derived extracellular vesicles -implications in type diabetes and islet transplantation introduction: type diabetes (t d) is characterized by reduced beta cell mass and function. islet amyloid, formed by aggregation of human islet amyloid polypeptide (hiapp), contributes to progressive beta cell loss in t d. amyloid also forms in human islets during pre-transplant culture and following transplantation in patients with type diabetes (t d) which is associated with graft failure. the cellular mechanisms underlying islet amyloid formation are still unclear. in this study, we examined the potential role of islet-derived extracellular vesicles (ev) in the clearance of soluble and aggregated (pro)iapp species from beta cells and amyloid formation. methods: human islets isolated from cadaveric pancreatic donors (n = donors) and wild-type or hiappexpressing (hiapp+) transgenic mouse islets (n = / group) were cultured in normal ( . mm) or elevated ( . mm) glucose to form amyloid. ev (exosomes) were isolated from culture medium using classical centrifugation and ultracentrifugation. purified ev were analysed by nanoparticle tracking analysis. western blot analysis and double immunogold transmission electron microscopy were performed to verify the presence of ev markers as well as (pro)hiapp species and oligomers (aggregates). results: human islets formed amyloid during culture with elevated glucose which was associated with progressive beta cell apoptosis. (pro)iapp species were detectable in ev released from human islets cultured in normal and elevated glucose. the latter markedly increased (pro)iapp content in islet-derived ev. interestingly, hiapp aggregates (oligomers) were present in the majority of ev released from human islets cultured in elevated glucose but were not detectable in islets cultured with normal glucose. similarly, ev released from hiapp+ mouse islets which formed amyloid during culture had higher (pro)iapp content compared to wild-type islet-derived ev. moreover, hiapp oligomers were present in ev derived from hiapp+ islets but not wt islets. summary/conclusion: in summary, our data show that (pro)iapp species are present in islet-derived ev and that elevated glucose increases (pro)hiapp and its aggregates in ev released from islets. islet-derived ev may play a key role in the process of amyloid formation in t d and human islet grafts. funding: university of manitoba research grants program (urgp). on. contraction, but not glycolysis, regulates the size of skeletal muscle evs secreted ex vivo. colorado state university, fort collins, usa introduction: skeletal muscle (skm) is a metabolically active tissue and accounts for~ % of total human body mass. acute exercise increases secretion of extracellular vesicles (evs), but the mechanisms responsible are unknown. muscle contraction increases the demand for atp which requires intercellular communication in order to adapt. we hypothesized that this "metabolic stress" during contraction increases skm ev secretion. methods: we tested our hypothesis using an ex vivo ev secretion assay. all studies were approved by the colorado state university institutional animal care and use committee. vastus medialis muscle (skm) from male c bl/ j mice (n = ) or female mt/mg mice (n = ) was cut into~ mg pieces and added to well plates (~ mg/well) filled with ml of serum-free dmem and placed in a cell culture incubator at c for hours. skm from male mice was treated with -deoxyglucose ( -dg) ( . nm - mm) to induce metabolic stress via inhibition of glycolysis. skm from female mice was treated with um of blebbistatin (bleb), a contraction inhibitor. after incubation, skm mass was measured and conditioned media was centrifuged ( , x g for min) to remove cell debris. evs were isolated using exoquick-tc. nta was performed on isolated evs using a horiba viewsizer . ev secretion was normalized to tissue mass and culture media volume then reported as ([particle]/ml/mg tissue). statistical comparisons for -dg experiments were made using a repeated measures -way anova. bleb experiments were analysed using a paired student's t-test. results: there was a trend towards greater ev abundance (p = . ) as a function of -dg treatment, but no effect on ev diameter (p = . ). bleb treatment did not alter ev abundance (p = . ), but significantly reduced ev mean diameter (p = . ; % decrease; dmso: . ± . vs. bleb: . ± . ). summary/conclusion: contrary to our hypothesis, inhibition of glycolysis with -dg did not stimulate skm ev secretion. however, bleb did appear to promote the release of small evs and/or inhibit secretion of larger evs. ongoing efforts are focused on testing other metabolic stressors and defining how blebbistatin promotes small ev secretion. funding: american heart association grant to dsl (ipa ). introduction: extracellular vesicles (evs, exosomes) are nanovesicles ( - nm) secreted from various types of cells. because of vesicular encapsulation of mirnas and enzymes, the evs play crucial roles in cell-to-cell communication by delivering these functional molecules to other cells [ ] . on the other hand, the evs are highly expected as next generation therapeutic tools due to pharmaceutical advantages such as controlled immunogenicity, effective usage of cell-tocell communication routes, artificial modification and encapsulation of functional molecules. however, cellular targeting and uptake efficacy of the evs are insufficient to be utilized as therapeutic tools [ , ] . in this study, we newly developed evs decorated with cellpenetrating sc or (sc ) peptides, which are derived from the c-terminal domain of the cationic antimicrobial protein, cap , because the peptides can be efficiently internalized by breast cancer cells. [ , ] . methods: all peptides were prepared by fmoc-solid phase synthesis. secreted evs from cd -gfp stably expressing hela cells were isolated by ultracentrifugation. cellular uptake of evs was analysed using a flow cytometer and a confocal laser microscope. encapsulation of saponin in the ev was conducted by electroporation. results: sc peptide is known as one of cell-penetrating peptides, and branched structure of sc peptides, (sc ) , further enhances the cellular uptake [ ] . in this research, we examined the effects of the peptide modification on cellular ev uptake, and modification of the sc or (sc ) peptides on ev membranes was conducted via stearyl moiety. as our results, increased macropinocytotic cellular uptake by modification of the peptides was successfully attained. especially, the modification of (sc ) peptides showed higher cellular uptake and macropinocytosis induction efficacy than that of sc peptides. in addition, anticancer protein, saporin toxin-encapsulated evs modified with the (sc ) peptides significantly enhanced their biological activity with dependency of glycosaminoglycan expression on targeted cells. summary/conclusion: the cell-penetrating (sc ) peptide-modified evs shows high abilities to be effectively internalized by cells and are applicable for intracellular delivery of therapeutic molecules. this study is expected to contribute to development of intracellular delivery techniques based on evs. [ introduction: rna therapeutics possess high potential which is yet to be realised, largely due to difficulties involved in delivery to the cytoplasm of target cells. extracellular vesicles (evs) possess numerous features that may help overcome this hurdle and have emerged as a promising rna delivery vehicle candidate. despite extensive research into the engineering of evs for rna delivery, little is known about how their intrinsic rna delivery efficiency compares to current synthetic rna delivery systems. using a novel crispr/cas based rna transfer fluorescent stoplight reporter system, we here compared the delivery efficiency of evs to state-of-the-art dlin-mc -dma lipid nanoparticles (lnps). methods: evs were isolated from mda-mb- cells expressing either a targeting or non-targeting control sgrna and applied to hek t stoplight+ reporter cells. lnps containing targeting sgrna were titrated onto hek t stoplight+ reporter cells to determine the minimum effective dose. lnp and ev particles were characterized using nanoparticle tracking analysis, dynamic light scattering and zeta potential analysis. sgrna copy number was determined using rt-qpcr. results: evs were ± nm in diameter as measured by dls and possessed a negative surface charge of − . ± . mv. rt-qpcr and nta analysis indicated that sgrna ev loading was low, with only in . e ± . e evs containing a single sgrna copy. nevertheless, evs containing targeting sgrna induced significant reporter activation while evs containing non-targeting sgrna did not. lnps were ± . nm in diameter and possessed a neutral charge. these particles also induced significant reporter activation when loaded with targeting sgrna. when delivered via evs, only between to sgrna copies per cell were required to induce statistically significant reporter activation. in contrast, the minimal effective sgrna dose when delivered by lnps was considerably higher at approximately e copies per cell. summary/conclusion: mda-mb- evs deliver rna in a highly efficient manner and are functional at sgrna concentrations several orders of magnitude lower than those required for lnp mediated delivery. this underlines the potential of evs as rna delivery vehicles and highlights the need to study the mechanisms by which evs achieve their efficiency in order for improved development of rna therapeutics. the role of circulating extracellular vesicles in patients with chronic chagas disease introduction: chagas disease is a neglected tropical disease (ntd) caused by the flagellated protozoan trypanosoma cruzi. it is a major public health problem in latin america, and it is now expanding over the globe through immigration of infected individuals. eukaryotic cells release extracellular vesicles (evs) that circulate in body fluids and have an important roles in intercellular communication, both in physiological and pathological conditions. objectives. our study proposes to characterize and to compare the circulating evs isolated from plasma of the chronic chagas disease (ccd) patients with healthy individuals (controls). methods: peripheral blood was collected from patients and controls in the presence of edta and evs enriched from plasma by differential ultracentrifugation. the obtained evs were characterized and quantified by nanoparticle tracking analysis (nta) and added to human thp- cells. after h, the cell supernatants were analysed by elisa for the presence of cytokines. results: lower amounts of evs were obtained from ccd patients in comparison with control individuals. however, the same amount of evs of ccd were more capable of inducing cytokines such as ifn-gamma and il- in relation to controls. summary/conclusion: although less evs are present in the blood of ccd, these evs induce high inflammatory reactions on macrophages suggesting a possible role of these evs in the establishment of chronic disease. funding: supported by fapesp, cnpq and capes. extracellular vesiclesa trojan horse for therapeutic agent delivery introduction: extracellular vesicles (evs) may prove to be one of the optimal payload carriers for therapeutic agents. while they travel through the extracellular space, the ev's lipid membrane layer shields their luminal cargo from deleterious external factors. when autologous evs are used to protect this therapeutic cargo, little immunogenic effects are expected compared to viral vectors and artificial structures, such as liposomes. their usage is potentially manifold, and they are ubiquitously present in all body tissues and fluids. the key is to develop a manageable ev loading agent for adoptive transfer therapies. methods: to exploit the unique properties of evs, highly positively charged proteins were used to load them with multiple biomolecules, such as a cas protein or dicer substrate dsrna as a functional payload and to improve their apparently inadequate natural ability to deposit cargo into the cytoplasm of recipient cells. results: highly positively charged proteins can associate with and/or diffuse through a phospholipid bilayer (thompson et al. ) . when these kinds of charged proteins are mixed with isolated evs in vitro, they are loaded into the evs. the positive charge of the protein has the advantage that it can associate with negatively charged agents, such as rna species, and aids the associated molecule to also incorporate into the ev. moreover, the positive charge of the protein helps with cargo delivery, and thus overcoming the bottleneck of the ev's cargo to escape the endosome post-uptake in a recipient cell. self-quenching fluorescent lipid dyes demonstrated that discharge of the highly positive ev cargo into the cytoplasm is concomitant with lipid mixing between the membrane of evs and the membrane of the recipient cell. when egfp-expressing microglia were exposed to evs loaded with a dicer substrate dsrna able to silence egfp via the positively charged protein, the uptake of dicer substrate dsrna was concomitant with a decrease in egfp expression in the microglia. a similar result was achieved when evs were loaded with cas protein conjugated to the highly positively charged protein. post-uptake of these cas -loaded evs, microglia expressing anti-egfp sgrna (single guide rna) lead to decreased egfp expression. summary/conclusion: our ev delivery technology has the capability of delivering multiple biomolecules, such as protein and rna cargo and demonstrates postuptake of the ev functionality of the ev delivered cargo in the recipient cell. hybrid extracellular vesiclesbiomimetic tool for drug delivery to repair endothelial cell dysfunction introduction: traditional drug delivery systems (dds) are usually based on liposomes, micelles or dendrimers. unfortunately, many dds cause side effects including organ toxicity and/or unexpected immune response. in living organisms, extracellular vesicles (evs) are responsible for delivering biologically active molecules to distant cells. in vitro loading of therapeutic compounds into evs is still not effective and needs developing new strategies. for these reasons we aimed to design hybrid extracellular vesicles (hevs) with high loading capacity for dds. methods: for hev synthesis, we used human endothelial derived evs. using freeze/thawing method we fused them with liposomes composed of cholesterol and one of the three lipids: dopc, sphingomyelin or phosphatidylserine. to confirm membrane fusion, we applied a spectroscopy ruler -fret (förster resonance energy transfer) and cryotem imaging technique. we characterized hevs using nta (for size distribution evaluation), dls (zeta potential) and western blot (for detection of evs markers). we evaluated loading efficiency using calcein as a model drug. additionally, we performed cytotoxicity tests. results: in the cryotem imaging, pure and homogenous hev population with a diameter of ± nm was detected. additionally, we observed changes in zeta potential and in size distribution after fusion. fret measurements showed increased fusion efficiency with the increasing number of freeze/thawing cycles and dependence on a lipid-to-protein ratio in evs. additionally, hev had higher loading efficiency than liposomes and sole evs and that their internalization by endothelial cells did not cause a cytotoxic effect. summary/conclusion: based on cryo-tem and fret, we confirmed that our fusion method of hybrid evs is effective and can be applied as a delivery platform for dds to endothelial cells. response to a range of stressors. the functional activity of these evs in recipient cells may, in part, be driven by changes to their biological cargoes. however, the molecular details of the underlying ev biogenesis and loading processes, and how this may vary in different conditions, is poorly understood. methods: we first studied the effect of oxidative stress on the functional activity of evs in recipient cells using cell viability and mitochondrial membrane potential assays in drosophila s r+ cells. we then carried out total rna sequencing of ev and cellular rna under three stress conditions and compared results to existing data in mouse cells. further to this we have used a bioinformatic pipeline to identify sequence motifs enriched in evs under stress. results: functional assays indicated changes to cell viability and mitochondrial membrane potential in recipient cells, which were donor cell-stress dependent. subsequent characterisation of rna showed an enrichment of ribosomal rna in evs relative to cells, but no significant changes to other biotypes. comparative analysis has also uncovered a set of genes enriched in evs under oxidative stress, and a further subset whose enrichment may be evolutionarily conserved in mouse. we also identified potential ev-loading motifs which may assist in rna loading specifically under stress. summary/conclusion: we have shown that evs derived from oxidatively stressed cells show dose-dependent differences in rna cargo and identified potential sequence motifs that may have a role in its loading. we are now validating the biological significance of these findings by combining different in vivo approaches in drosophila. this will enable us to gain insights into the basic mechanisms which govern ev loading in different contexts, and ultimately the molecular mechanisms underlying ev-mediated intercellular communication. ishai luz a , bibek bhatta a , kanaga sabapathy b and tomer cooks c a ben-gurion university of the negev, beer-sheva, israel; b national cancer centre, singapore, singapore, singapore; c ben-gurion university, beer sheva, israel introduction: mutations in tp are considered one of the most frequent genetic alterations in human cancer. besides the abrogation of the wild-type (wt) p -mediated tumour suppression, a distinct set of missense mutations was reported to endow mutant p proteins with novel activities termed gain-of-function (gof). even though mutations in tp are typically thought to arise in the tumour cells rather than in the stroma, the non-cell-autonomous effects of these mutants over the tumour microenvironment are poorly understood. in the presented studies, focusing on colon cancer as well as on lung cancer microbiome, we investigated intercellular interactions mediated by exosomes and outer membrane vesicles (omvs) in the context of cancers harbouring mutant p . methods: p results: in the colon, tumour cells harbouring mutp were found to exert a non-cell-autonomous effect over macrophages. when exposed to tumour cells harbouring mutp , monocytes became polarized towards a distinguished subset of macrophages characterized by tams-related markers. the mutant p affected tam were characterized as tnf-αlow/il- high, over expressing cd- and cd , with decreased phagocytic ability and increased invasion and matrix degradation potency. investigating the exosomal transfer from mutp tumour cells to macrophages, revealed a mutp -specific mirs signature led by mir- promoting the tam phenotype and creating an invasive front together with tumour cells. mir- was also found to be the top mutp -associated mir in a cohort of human colorectal resected tumours. separately, in two lung cancer cohorts, we identified a signature of microbiome members associated with p mutations. acidovorax temperans, a gram negative bacterium, was found to be abundant in tumours of patients with mutant p . we found a significant increase in tumour volume in animals inoculated with acidovorax temperans as compared to sham treated animals, and increased lung weight as a percent of total body weight. these preliminary data indicate that acidovorax temperans contributes to lung tumorigenesis in the presence of activated k-ras and mutant p . omvs shed by acidovorax temperans promoted inflammatory signalling in lung carcinoma cells and elevated cd expression on tumour cells and sirpα levels on macrophages. summary/conclusion: altogether, these findings are consistent with a microenvironmental role for specific "hot-spot" gof p mutants tightening the interaction between the tumour cell and the immune compartment in colon cancer. in both colon and lung cancer, mutant p facilitates cellular interactions within the tumour microenvironmets mediated by vesicles. funding: intramural funding from the national cancer institute, national institutes of health. lori zacharoff and mohamed el-naggar university of southern california, los angeles, usa introduction: the metal respiring bacterium s. oneidensis creates outer membrane extensions and outer membrane vesicles that are sculpted by the novel bar domain protein bdpa. these vesicles and extensions incorporate mutliheme cytochromes involved in extracellular electron transfer to metals and electrodes. however, the physiological relevance of incorporating these cytochromes into the higher order d architecture of a vesicle or extension is unknown. given that bar domains serve as a protein sorting mechanism in eukaryotes, we investigated the pathway crosstalk between bdpa and outer membrane multiheme cytochromes as means to understand the physiological significance of membrane architecture. methods: o this end, vesicle morphology and content was measured using dry weights, dynamic light scattering, fluorescence microscopy and comparative proteomics from wild type s. oneidensis and deletion strains. results: cells lacking bdpa make large amorphous vesicles that are dense with protein. in contrast, a strain lacking outer membrane cytochromes recruits less total protein into smaller vesicles. proteomics to show that both bdpa and multiheme cytochromes are involved in recruiting other proteins to outer membrane vesicles and have a reciprocal relationship. summary/conclusion: in the absence of bdpa, protein crowding has to become the main driving force of vesiculation and bdpa is essential for efficient incorporation of cytochromes. however, multiheme cytochromes are not only vesicular cargo, but are also important for shaping and loading vesicles. both of these situations make it clear that vesicles play a role in increasing the respiratory surface area of s. oneidensis cells. moving forward, we hope to be able to control bdpa and cytochrome levels for selective recruitment of technologically relevant payloads. introduction: fascioliasis caused by fasciola hepatica represents a major economic loss and clinical burden in cattle farming worldwide. extracellular vesicles (evs) contain pathogen-derived molecules that represent novel biomarkers of disease. in the present study, we have identified potential new biomarkers of f. hepatica infection in evs present in sera of infected cattle. methods: parasites and sera were obtained from local abattoirs (valencia, spain, and medellin, colombia, respectively). sera from infected and from healthy animals. parasites were cultured, and evs obtained by sizeexclusion chromatography (sec) and characterized by nta, tem and proteomic profiling. recombinant proteins from f. hepatica evs (enolase and fh . tegumentary protein) were produced, and coupled to magnetic beads. measurement of bovine igg antibodies was performed using luminex bead array technology. results: a total of proteins were identified associated with evs as shown by the presence of typical ev-markers (tsg , alix, cd ). two parasite proteins, enolase and the fh . tegumentary protein were produced as recombinant proteins and used for detection of cattle igg employing luminex bead array technology. interestingly, significant differences were found in the fluorescence values of both recombinant proteins allowing discrimination between sera from infected and non-infected cattle. the use of the fh . protein generated a highly significant difference between the two groups (p value = . ); as did enolase (p value was . ). summary/conclusion: this study demonstrates the usefulness of ev proteins as new biomarkers for early diagnosis of helminth infections using multiplex assays, a technology that may also be applied to other parasite ev molecules. life stage-specific glycosylation of schistosome-derived extracellular vesicles introduction: glycans play an essential role in pathogen-host interactions. larvae and adult worms from schistosoma mansoni release distinct subsets of glycoconjugates as excretory/secretory (es) products. extracellular vesicles (evs) are also among the es products. we recently found that schistosomuladerived evs are glycosylated and bind human dendritic cells via c-type lectin receptor (clr) dc-sign, leading to increased il- and il- release. here we investigated the glycosylation profile of evs released by s. mansoni adult worms, compared this to schistosomula evs, and addressed how this may affect parasite-host interactions via clrs. methods: evs from cultured s. mansoni parasites were obtained by ultracentrifugation and purified with iodixanol density gradients. isolated evs were analysed by nta and cryo em. n-glycan and lipid glycan content was determined by mass spectrometry. density gradient fractions with evs were loaded onto sds-page gels followed by western blot (wb) analysis using anti-glycan monoclonal antibodies (mabs). results: cryo em showed that adult worm evs lacked the long thin filaments that are characteristic for schistosomula evs. additionally, in contrast to schistosomula evs, glycolipids could not be detected in the adult worm evs. mass spectrometry analysis showed that the most abundant n-glycans in the adult worm evs contained galnacβ - glcnac (lacdinac, ldn) motifs, which correspond to previously published overall glycan profiles of this specific life stage. other differences in ev glycosylation between the two life stages were observed by wb using anti-glycan mabs: adult worm evs showed a paucimannosidic glycan motif whereas in the schistosomula evs galβ - (fucα - ) glcnac (lewis x) was detected in line with previous ms analysis. introduction: phloem plays a central role in plant function, as it is the responsible for the translocation of photoassimilates from source-to sink-organs, and a long-distance route for signals distribution. due to the sap high nutrient content, sieve elements are primary target for plant pathogens and pests. in this work we aimed to isolate and characterize extracellular vesicles (evs) from cucumis melo phloem sap, derived from plant either exposed or not to the melon aphid, aphis gossypii (hemiptera: aphididae). methods: phloem exudates from -week-old melon plants, either uninfested or infested with adults of a. gossypii (n = , replicates each), were collected by cutting the stem with a sterile razor blade between first and second expanded leaf from the top. evs were isolated by size exclusion chromatography, and analysed by nanoparticle tracking analysis (nta) and transmission electron microscopy. evs proteome was determined by quantitative mass spectrometry. results: evs from phloem sap were successfully isolated in every condition. no significant differences were detected among distinct samples, neither in particle concentration and size by nta, nor in protein concentration. most importantly, a total of different proteins were identified in phloem sap evs, including present in exosome databases (exocarta). on top of that, differentially expressed proteins were identified in evs derived from aphid infested or uninfested plants (p value < . ). summary/conclusion: understanding how plants trigger their defences against pests and pathogens is important to develop new control measures. the characterization of several proteins in evs from the phloem sap provide valuable information on long distance signalling in plants. moreover, as plants lack an immune system comparable to animals, the different protein content in phloem sap evs after exposure to aphids could indicate their important role in delivering inducible defences against invading pests and pathogens. extracellular vesicles from nematode species heligmosomoides bakeri and trichuris muris contain distinct small rnas that could enable niche specificity in the host introduction: gastrointestinal nematodes are extremely prevalent parasites that infect most animals and % of human population. their success as parasites is attributed to their ability to secrete diverse molecules that modulate the host immune system. extracellular vesicles (evs) are one of the immune modulatory compounds they release that directly modulate host cells. our goal is to understand how the small rna (srna) cargo underpins ev function, using a comparative analysis of ev cargo from diverse nematode species. methods: we first compared how different ev isolation methodologies (ultracentrifuge (uc), size fractionation, sucrose gradient floatation) effect the small rnas detected in h. bakeri evs using different library preparation kits (cleantag, truseq), with or without polyphosphatase treatment. we then compared this to small rna libraries from t. muris evs using comparable methods, uc ev purification, with or without polyphosphatase treatment and using the cleantag library preparation kit. results: evs from both species contained mirnas, however the mirna gene familes in h. bakeri and t. muris evs are distinct. the mirna content detected in ev samples collected by different purification protocols is robust. the largest difference in detected mirnas was found when comparing different library preparation kits. although both h. bakeri evs and t. muris evs were dominated by srnas derived from intergenic or repetitive elements in the parasite genomes, only in h. bakeri evs were these secondary sirnas. summary/conclusion: h. bakeri and t. muris evs contain distinct small rna cargos, which may underpin their ability to colonise different host niches, and/or modulate the host immune system differently. t. muris evs do not contain secondary sirnas, in contrast to h. bakeri, however they are dominated by srnas derived from intergenic or repetitive regions. comparative analysis of helminth evs could help pinpoint the srnas involved in cross-species communication. please provide any keywords if applicable.: nematode, cross-species communication, small rna introduction: extracellular vesicles (evs) are secreted from various cells including cancer cells and known to contain protein and small rnas including mirna isoforms (isomirs). therefore we also focused on isomirs including other small non-coding rnas for biomarker discovery. although liquid biopsies using small rnas are promising biomarkers for early detection of cancer, current approaches to detecting and analysing mirnas in the blood are still inadequate. artificial intelligence (ai) data analysis may provide better algorisms for diagnosing cancer. methods: small rnas were isolated from serum or purified evs using a mirneasy mini kit (qiagen) and quantified by using the ion s ™ next-generation sequencing system. (thermo fisher scientific). evs were purified using total exosome isolation reagent (invitrogen™). ai data analysis was performed using jmp® genomics and datarobot enterprise ai platform. results: three small rnas, isomir of mir- - p, mir- a- p, and trf-lys (ttt) were significantly upregulated in breast cancer patients compared with the healthy cancer-free individual. the combination algorithm using these three small rnas allows for a more accurate diagnosis of the area under the curve (auc) . . to test the possibilities that these small rnas are derived from cancer cells, we isolated evs from the serum and performed ngs analysis to profiled serum small rnas in evs. interestingly we found that two small rnas, mir- - p and mir- a- p, also high in breast cancer evs, indicating that these small rnas were expected to be derived from cancer cells. in oesophagus cancer, we also performed ngs analysis and identified twenty-four mir/isomirs candidates for diagnostic biomarkers. a multiple regression model selected mir- a- p and two isomirs (mir- - p and mir- - p) . the auc of the panel index was . . we also performed ai data analysis and discovered the novel algorisms that can diagnose breast and oesophagus cancer more accurately. summary/conclusion: we demonstrated combinations of circulating non-coding rnas containing evs potentially useful for the detection of early-stage breast and oesophagus cancers. in addition, the datarobot enterprise ai platform enables us to the more accurate diagnosis of cancers at the early stage. identification of novel ev-associated mirnas as toxic biomarkers in mouse introduction: recent findings reveal that extracellular vesicles (evs), secreted from cells, are circulating in the blood. evs are classified into exosomes ( - nm), microvesicles ( - , nm) and apoptotic bodies ( - , nm). evs contain mrnas, micrornas, and dnas and have the ability to transfer them from cell to cell. recently, especially in humans, the diagnostic accuracy of tumour cell type-specific evs as biomarkers is more than %. in addition, micrornas contained in the evs are being identified as specific biomarkers in blood for chemical-induced inflammation and organ damage. therefore, micrornas contained in the evs released into the blood from tissues and organs in response to adverse events such as chemical substances and medicine are expected to be useful as novel biomarkers for toxicity assessment. in this study, we aimed to identify target organs by comprehensive analysis of ev rnas in the blood of mice after chemical exposure to establish a highly sensitive "next generation type" toxicity test for chemical substances and medicine using ev rna in blood as a biomarker. methods: all animal studies were conducted in accordance with the helsinki declaration and the guidelines approved by the animal care committee of the national institute of health sciences. c bl/ j male mice ( weeks) were orally dosed with ccl (vehicle, , mg/kg). serum were separated from blood after , , and hours after ccl administration. the serum was centrifuged at , x g to remove cellular debris and subsequently ultracentrifuged , x g. the pellet is resuspended in pbs and ultracentrifuged , x g again. the comprehensive small rna-seq of collected evs were performed according to the manufacture's protocols. results: we succeeded in isolating more than novel small rnas, which could be used as novel highly sensitive biomarkers for hepatotoxicity due to carbon tetrachloride (ccl : mg/kg & mg/ kg). well known hepatotoxicity biomarkers, mirna- and mirna- were upregulated more than -fold in the administration of mg/kg ccl , but not responded in the administration of mg/kg ccl . summary/conclusion: these results suggest that mir- and mir- are mainly released from liver to blood directly only in the administration of mg/kg ccl , while novel more sensitive hepatotoxicity biomarkers which responded in the administration of both mg/kg and mg/kg ccl should be included in the ev. our novel biomarkers will accelerate a rapid evaluation of chemical substances and medicine in nonclinical safety evaluation. introduction: advancements in sequencing technologies have allowed analysis of the genomic landscape of cancer using circulating cell-free(cf) dna. however, cfdna does not originate only from tumour cells. we recently demonstrated that most of the dna circulating in plasma of cancer patients is associated with large evs (l-evs), and that l-ev-associated dna reflects genomic aberrations of the cells from which l-evs arise. since l-evs are specifically released by tumour cells, we explore their potential to report cancer-specific genomic alterations in patient plasma and compare it to cfdna. methods: differential ultracentrifugation, tunable resistive pulse sensing, qubit dsdna high sensitivity assay, capillary electrophoresis, whole exome sequencing ( - x), targeted sequencing (qiaseqtm), flow cytometry. results: we show here that l-evs in the size range of > micrometre are present exclusively in plasma obtained from cancer patients and absent in plasma from healthy donors. in agreement with this finding, double-stranded(ds) dna is detected only in l-ev fractions of patient plasma and not in those obtained from healthy donor plasma using the same protocol. we also demonstrate that the fragments of dsdna associated with circulating l-ev are larger in comparison with cfdna (> , bp versus~ bp). a large-scale analysis of l-ev dna obtained from plasma of patients with metastatic castration-resistant prostate cancer (mcrpc) as well as with non-small cell lung cancer (nsclc) demonstrates that dna associated with circulating l-evs reports cancer-specific genomic alterations in both types of cancer. we further investigate if l-evassociated dna is intra-or extravesicular and demonstrate that it is present in both forms. we finally compare the purity of the tumour signal in intravesicular l-ev dna, total l-ev dna, and cfdna obtained from patient plasma. summary/conclusion: our results demonstrate that circulating l-evs contain high quality, large molecular weight dna that contains cancer-specific genomic alterations, supporting the use of l-evs as a source of tumour-derived dna in plasma. introduction: epidermal growth factor receptor (egfr) mutation driven lung adenocarcinoma (ac) represents a unique subgroup that lends itself to treatment with oral egfr tyrosine kinase inhibitors. current methods that are used to detect these mutations (e.g. l r or the resistance mutation t m) involve invasive tumour biopsies or blood circulating tumour dna (ctdna) and cell free dna (cfdna). the sensitivity of blood ctdna and cfdna is limited by the frequency of genomic alterations in the egfr gene; additionally, ctdna does not reflect changes in the egfr protein, against which novel therapies are in development. there remains a need to develop bloodbased biomarkers that can circumvent these disadvantages and replace the more standard, invasive tumour biopsies. we propose the study of exosomes for treatment monitoring as well as to identify egfr resistance related genomic and proteomic changes. methods: we enrolled patients with metastatic lung ac: with egfr mutations and without (control). from the patients with egfr mutant lung ac, we processed blood samples through the patients' treatment course, using ultracentrifugation to isolate exosomes. we then used both droplet digital pcr (ddpcr) to test exosomal rna (exorna) for the mutation of interest and western blots to test protein resulting from exon deletion or l r mutations. results: from patients with egfr exon deletion mutations, we detected identical mutations in exorna from / samples. exorna based mutational load increased and mirrored clinical progression in patients. three patients whose cancer remained stable demonstrated a decrease in their exorna. one patient had blood drawn only at points and was therefore not plotted. exorna from patients with l r and t m mutations demonstrated the corresponding mutations; however, exorna did not mirror their disease course. we also demonstrated mutant egfr protein presence in exosomes from patients. finally, we tested cfdna for egfr mutations from four matched samples using ddpcr. we detected matched mutations in exosomes in all four, while cfdna mutations were only detected in / patients. summary/conclusion: in summary, we detected egfr mutations in / exosome samples isolated from metastatic lung ac. our results set the stage for optimization of exorna methods and inform future experiments relating to exosomal cargo in patients with egfr mutant lung ac. identification of plasma-derived, ev-based biomarkers for glioblastoma introduction: glioblastoma multiforme (gbm) is the most malignant and aggressive primary brain cancer in adults, with an incidence of . per , people. currently, diagnosis is only performed via histopathological investigation of a tissue sample from a gbm lesion, complemented with molecular diagnostics for identification of select biomarkers. mri is the standard of care for follow-up and monitoring of treatment response. therefore, development of a "liquid biopsy" to obtain disease-relevant information from patient's body fluids is highly desirable. methods: we present the results from a clinical study in which extracellular vesicle (ev)-derived mrnas and long non-coding rnas were profiled from the plasma of gbm patients and control individuals. we obtained plasma from patients at the time of initial diagnosis, and matched controls by sex and age. ev-associated rna was isolated from - ml plasma and rna-seq was performed using our proprietary pipeline. sequencing data was analysed for differential gene expression. results: we observed mrnas as differentially abundant between gbm and control samples, with mrnas enriched in gbm samples and mrnas enriched in control samples (p < . ). correlation based on differentially abundant mrnas separated gbm and control samples into two unique populations. eight differentially expressed mrnas were previously identified as part of the mesenchymal gbm subtype. these data, while preliminary, provide a potential basis for the further development of a noninvasive gbm gene panel test. summary/conclusion: we have identified a novel rna signature for gbm from plasma derived evs, which differs from previously identified biomarkers isolated from tissue. further work will refine this signature to enable detection, characterization, and patient monitoring for gbm with minimally invasive techniques. introduction: sjogren's syndrome (ss) is a systemic autoimmune disease in which inflammation progressively damages the moisture producing glands of the afflicted. million americans are estimated to be suffering from the disease, % of which are women with an average age of . overlapping symptoms with other health conditions and co-morbidities make ss particularly difficult to diagnose, with average time to diagnosis of years. saliva exosomal rna profiling has been primarily focused on small rnas and has been limited thus far due to the large contribution of sequencing reads from the oral microbiome. a noninvasive saliva exosomal rna (exorna) based test capable of diagnosis would be highly desirable. methods: we began by first developing a novel long rna-seq workflow to selectively enrich and profile human exosomal mrnas and long non-coding rnas (lncrnas) from saliva. we then profiled salivary exorna obtained from ss patients and healthy matched controls. finally, we performed differential gene expression analysis to obtain an exorna signature for ss. results: rna-seq data analysis demonstrated highly efficient enrichment of human transcriptome, with over % of reads mapping to the transcriptome. further rna biotype analysis showed over % of transcriptome reads mapped to protein coding genes and lncrnas. we detected over , mrnas and approx. lncrnas. differential expression analysis (dex) of ss vs. healthy control exorna identified upregulated genes, including mrnas and lncrnas (p < . ). genes were found to be downregulated in ss, including mrnas and lncrnas. gene ontology analysis of dex genes revealed enrichment of genes involved in various immune system related pathways. most importantly, principal component analysis (pca) resulted in clear separation of ss patients from healthy controls. summary/conclusion: our optimized rna-seq workflow enables saliva-based liquid biopsy for biomarker discovery. the gene signature identified in this ongoing study could potentially provide a non-invasive molecular means of diagnosing sjogren's syndrome. introduction: increasing embryo implantation rates has become one of the greatest challenges in assisted reproduction techniques. usually an endometrial biopsy is done to identify a receptive endometrium, which prevents embryo transfer in the same cycle, as it is detrimental for the implantation. the implantation is a complex process, which requires a synchrony between the development of the embryo and the endometrium, but also, an adequate embryo-endometrial cross talk. the presence of extracellular vesicles (evs) as mediators of this communication has been describe in the endometrial fluid. therefore, we hypothesize that the molecular analysis of the content of the evs and companion molecules from endometrial fluid could be a non-invasive method to recognize an implantative endometrium and consequently improve the implantation rates. methods: the objective is to define a simple, sensitive and reproducible non-invasive ev-based method that allow the quick identification of an implantative endometrium by means of mirna analysis. for the establishment of a robust methodology for analysing evs from endometrial fluid in clinical settings, where the sample is limited and no sophisticated equipment is available, five different methodologies were compared in triplicate. two of them consisted in the direct extraction of rna while in the other three, before the rna extraction an enrichment of evs was done. smallrnaseq was performed to determine the most efficient method. once the best method was selected, it was applied in a set of real samples with different implantation outcome. the content of mirnas (mainly associated with evs) of endometrial fluid samples from women in whom the implantation was successful (n = ) and unsuccessful (n = ) were analysed. results: our results show that the protocols with a previous enrichment step of evs obtained a higher mirna expression. the results obtained from the differential analysis of the set of samples with different implantation outcome are being analysed and it is expected that the results will be available by the time this communication is presented. summary/conclusion: this work demonstrates that it is possible to obtain and analyse evs and evs-associated mirnas from a small volume of endometrial fluid samples, which allows the use of ev-mirnas as a low-invasive biomarkers for the detection of an implantative endometrium. funding: jip is supported by a predoctoral grant from the basque government. small rna cargo of evs is affected by hormone treatment in prostate cancer introduction: small rnas are recently reported as a regulator for prostate cancer progression to castration-resistant disease. our previous work has shown that evs protein cargo is affected by male steroid hormone, dihydrotestosterone (dht). in this study, we assess the small rna cargo of evs in response to androgen manipulations. methods: androgen receptor-positive lncaps are grown in css medium to deplete the androgens. media were then replaced with vesicle-depleted css medium ± nm dihydrotestosterone (dht) ± µm enzalutamide (enz) for h. evs were isolated using sequential ultracentrifugation ( g for min, , g for min, , g for h), washed once in pbs. protein and rna were collected from both parent cells and conditioned medium to allow direct comparison between s-evs cargo and cells. small rna ngs libraries were prepared using the illumina's truseq small rna library prep kit and single-end sequenced at a read length of nucleotides (nt). fastq library files were processed using a custom-designed pipeline. adapters were removed using the cutadapt tool, trimmed reads were mapped with high stringency against ribosomal sequences using bowtie . snorna and trna fragments were identified using the flaimapper software. remaining reads were mapped against the human genome hg using bowtie . results: we found that the presence or absence of androgens does not significantly change the amount of total rna in small evs (s-evs). however, hormone stimulation altered the small rna content of s-evs, in parallel with our previous published data on ev protein cargo. dht increased the abundance of snorna in cells, while a reduction of snornas was observed in the s-evs fraction. interestingly, dht induced the formation of cell filopodia that are not inhibited by androgen inhibitor enzalutamide. pathway analysis indicates the p mediated regulation driven by mirnas found in s-evs upon exposure to dht. the expression profile of snorna and trna fragments in dht treated cells resembles results from clinical prostate cancer specimens. summary/conclusion: our findings show that androgen manipulation alters both s-ev derived protein and rna cargo. changes in the s-ev rna profile due to treatment with androgens are not identical to small rna profiles in parental cells, indicating a specific sorting mechanism of s-ev small rna upon androgen manipulation. further, dht induces the formation of cell filopodia irrespective of enzalutamide, suggesting cargo selection of s-evs. we conclude that small rna ev cargo can be utilised to as prostate cancer biomarkers in androgen targeted treatments. introduction: cancer immunotherapy, such as pd-l blockade, is a method to eliminate cancer cells. ectopic expression of pd-l , on the surface of tumour cells, has been associated with tumour persistence and as an important predictor of therapy response. a test that, specifically and accurately, detects pd-l is critically important in order to identify patients that would benefit from these treatments. emerging evidence has shown that extracellular vesicles (ev) can carry immune checkpoint molecules, such as pd-l , and whose expression have been correlated with tumour immunity response. with a multitude of commercially available antibodies identifying appropriate clones and associated assay is important in order to standardize the diagnostic modality used. methods: pd-l expressing cancer cell lines were used to generate evs. pd-l -myc vector was transfected to generate an overexpression system. exoview® sensors containing different anti-pd-l clones were generated. samples (cell derived and plasma) were incubated on chips to allow the antibody to bind the antigen on the ev. after incubation, chips were immunolabeled with fluorescently labelled antibodies against pdl- or ev associated markers. exoview r reader was used to enumerate the evs captured on the sensor surface and analyse the expression of pdl- on single vesicle through fluorescence imaging. immunoprecipitation and mass spectrometry (ip/ms) were employed as an orthogonal method to verify the specificity of the assay. results: to study the detection efficiency of the antibodies, engineered pd-l -myc evs were used. under these circumstances, all the tested antibodies were able to capture evs. when testing endogenous pd-l positive evs from different cancer cell lines, only . and clones consistently bound to evs. in addition, evs derived from plasma demonstrated to be positive for pd-l , however, only clone . was able to immobilize these evs. the results suggested that clone . could be a potential pd-l antibody to detect pd-l positive evs originating from various sources. to confirm these results, and assure the specificity of the antibody targeted ip/ms was employed. summary/conclusion: in combination with the exoview platform, anti-pd-l antibodies can be screened and potentially used to generate a non-invasive ev-specific assay that could detect this protein in patients. differences in extracellular vesicle protein cargo is dependent on head and neck squamous cell carcinoma cell of origin university of michigan, ann arbour, usa introduction: head and neck squamous cell carcinoma (hnscc) is the sixth most common, eighth most fatal cancer worldwide and includes cancers of the oropharynx, larynx, hypopharynx, and oral cavity. in , there were over , new cases and , deaths estimated in the usa alone. despite recent advances in treatment, including radiation, chemotherapy, surgery, concurrent chemoradiation, and immunotherapy, many tumours develop resistance and progress. patients develop metastases or tumours recur locally or regionally; the -year overall survival rate for hnscc is only - %. factors that contribute to poor survival for patients with hnscc include late stage diagnosis, lack of reliable markers for early stage detection, high level of biologic heterogeneity, and local recurrence and distant metastases after treatment. methods: this study used representative hpv-positive and hpv-negative hnscc cell lines, one hpvtransformed cell line. and two non-cancer oral keratinocyte cell lines. evs were isolated using differential ultracentrifugation and peg precipitation/ultracentrifugation. evs were characterized by tem, nta, and wes protein analysis for reported ev markers. ev and whole cell lysates were assessed by lc-ms/ms analysis using the tandem mass tag- plex kit. cluster analysis was performed on the fold-change peptide spectrum matches (psm) for the evs from the hnscc lines compared to the evs from the normal keratinocyte line (noksi). protein was measured using a capillarybased electrophoresis instrument. results: cd and annexinv were detected in all of the ev lysates tested, while calnexin was detected in all of the whole cell lysates and none of the ev samples tested. selected proteins stat , hla-a, tenascin, e-cadherin, β catenin, cytokeratin , epha , and cd , and hpv-related markers p , p , rb, cyclin d , and egfr were tested using the wes platform. evs from hpv-positive cell lines showed higher protein levels compared to evs from hpv-negative cell lines in stat , hla-a, and tenascin. only kert demonstrated lower protein levels in evs from hpvnegative cell lines. of the common hpv-associated hnscc markers: egfr, p , rb, cyclin d and p , only egfr was positive in any the evs tested. the remaining proteins queried, e-cadherin, β catenin, epha and cd showed varying protein levels in evs from both hpv positive and hpv-negative cell lines. summary/conclusion: our findings suggest that these proteins may be potential hnscc ev markers that may be ) selectively included in ev cargo for export from the cell as a strategy for metastasis, tumour cell survival, or modification of tumour microenvironment, or ) representative of originating cell composition, which may be developed for diagnostic or prognostic use in clinical liquid biopsy applications. validation of antibodies on western blot for extracellular vesicles from biological human samples and cancer cell conditioned media the brady urological institute, johns hopkins university school of medicine, baltimore, usa introduction: one of the major challenges in extracellular vesicles (evs) research is to prove the particles that are isolated are true evs, rather than other co-isolated contaminants, like lipoproteins. isev recommends using multiple assays to characterize evs. this study aims to validate the positive and negative protein markers for extracellular vesicles from plasma, urine and prostate cancer cell conditioned media (ccm). methods: membrane and cytosolic fractions of mcf cells served as positive and negative controls for all antibodies validated. evs were isolated from plasma of healthy volunteers, urine of healthy volunteers and ccm of pc- cells using differential ultracentrifugation. eight protein markers were assessed: positive markers cd , cd , cd , flotillin (flot ), alix and tumour susceptibility gene (tsg ), negative marker calnexin (canx), and contaminant markers apo-a for plasma and thp for urine. tetraspanins are small transmembrane proteins expressed in evs. flot is membrane protein that forms microdomains in the plasma. alix and tsg , an accessory protein of the endosomal sorting complex required for transport, are involved in the biogenesis of evs. they are positive markers for evs. canx is in the membrane of the endoplasmic reticulum. apolipoprotein-a (apo-a ) is the protein components of lipoproteins, therefore it is marker of contamination for plasma ev. tamm-horsfall protein (thp) is contamination marker for urine ev, because it is most abundant protein in human urine. results: all antibodies were validated in the correct positive and negative control, thus confirmed as usable and reliable antibodies for western blot. in plasma ev, cd , cd , cd and flot were positive and canx and apo-a were negative. in urine evs, cd , cd , flot- , alix and tsg were positive and canx and thp were negative. in ccm evs, cd , cd , flot , alix and tsg were positive and canx was negative. summary/conclusion: we confirmed a high degree of ev purity from sample types: urine, plasma, and ccm. of particular importance, we confirmed that evs isolated from biologic patient samples, plasma and urine, had low contamination. future work will use these methods to confirm purity of ev samples prior to addition analysis, such as examining ev cargo and biologic significance. proteomic study of mesenchymal stem cells derived exosomes modified using mir. introduction: the project we are working on is to modify the immunogenic profile of human cmms from the umbilical cord stroma through its stable transfection with anti-mir- - p, and therefore of the exosomes that these cells generate, for use in free-cell therapy to treat inflammatory process. methods: evs released from a primary culture of human umbilical cord mesenchymal stem cells and from primary culture of human umbilical cord mesenchymal stem cells mir -/-modified through stable lentiviral transfection were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (tem) and measured by nanoparticles tracking analysis (nta). protein extraction from evs was made using ripa buffer and after checking protein integrity the total ev proteins. we performed a shotgun proteomic study using a tmt ( -plex) label of the total mir -/-exosomes protein comparing it with normal exosomes. after labelling the ltq-orbitrap platform of proteored was needed for fraction injections and data acquisition. proteome discoverer . (thermo) was used for protein processing and quantification. results: a total of . proteins were identified at least with a unique peptide and we have able to establish the proteomic profile of mir -/-exosomes against normal exosomes. we found out several protein modulated by mir and related to inflammation. summary/conclusion: we have able to establish the proteomic profile of mir -/-exosomes against normal exosomes focusing on proteins involving inflammation process. all those results seem indicate that exosomes could be modified, which could be used as an anti-inflammatory free-cell therapy. funding: proteored concept test project grant. a novel extracellular vesicle isolation method used to discover urine liver disease biomarkers introduction: hepatocellular carcinoma (hcc) is the th most common cancer worldwide and the rd most common cause of cancer death; additionally, its incidence is increasing. while outcomes for early hcc are superior to those for late stage disease, early detection of hcc remains a challenge. current guidelines have suboptimal sensitivity and specificity. in this pilot study, we hypothesize that urine extracellular vesicles (evs) may identify candidate biomarkers towards the development of an inexpensive, widely accessible screening assay for the early detection of hcc. methods: urine samples from healthy subjects, subjects with cirrhosis, and subjects with cirrhosis plus hcc were collected and processed using ymatrix columns to isolate ev-associated protein and mirna. protein was analysed using a tandem mass tag method on a thermo scientific orbitrap fusion mass spectrometer with comet/paws and edger processing. mirna was analysed using a targeted firefly microarray from abcam. differential expression and predictive modelling for the presence of hcc and cirrhosis was performed to identify candidate mirna and protein biomarkers. results: for mirna, samples were eligible for analysis after low expression filtering. we used pair-wise ratios of cancer-associated mirnas by gradient boosting of decision trees to develop a predictive model for hcc. our best model had a sensitivity and specificity of . and . respectively using mirnas to distinguish hcc from cirrhosis. all samples were eligible for protein analysis. based on differential expression and biologic relevance, we identified protein candidate biomarkers. interestingly, we found liver-selective proteins and known hcc/cirrhosis plasma/tissue markers, demonstrating proof-of-concept for the method. summary/conclusion: urine extracellular vesicles contain liver-selective proteins and known liver disease serum biomarkers as well as novel mirna and protein biomarkers that are significantly up-regulated in disease samples. the described candidate biomolecules may be easily accessible biomarkers with which to develop a sensitive and specific universal screening diagnostic for the early detection of cirrhosis and hcc. introduction: the peptidergic g-protein coupled receptors (gpcrs) are cell-signalling transmembrane proteins, which in their native form comprise of seven segments embedded in the cell membrane. this structural advancement is believed to be maintained in extracellular vesicles (evs). in autoimmune diseases, the presence of autoantibodies towards gpcrs is not uncommon, and to detect plasma autoantibodies, evs carrying gpcr will be used as template in a novel microarray screening tool. methods: purified evs from hek cells were printed on different types of surfaces; polymer coated glass slides and hydrophilic and hydrophobic plastic well plates. five different print buffers were tested in a multiplex assays. spots containing evs were stained with biotinylated antibodies (cd , cd , cd , adrβ , hsp , epcam and flotilin- ) followed by binding of cy -labelled streptavidin and visualized microarray scanner. results: the outcome of these experiments was promising, as some of the chosen printing buffers showed increased tendencies to bind evs. the ev presence was verified with a panel of markers known to be present on small evs. in addition, the ev content of the adrenergic beta- receptor (adrβ -receptor), which is a gpcr of interest in autoimmune diseases, was verified in some of the experimental setups. summary/conclusion: the approach of using evs as template in a screening tool possesses the potential to easily screen for autoimmune illness markers in diagnostic purposes. using the microarray technology allows the screening to be multivariate, specific and highly sensitive. circadian variation of extracellular vesicles secreted in urine: analysis of time point collection and normalization strategy. introduction: urinary extracellular vesicles (uevs) are an ideal source of biomarkers for kidney and urogenital diseases. despite the great deal of interest generated by uevs, little is known about its collection time and normalization approach. the majority of the studies on uevs focus on spot urine collection based on the assumption that it accurately reflects the renal function, although time point of collection is not standardized. therefore the practice to collect spot urine does not allow for calculating and standardizing accurately the uev excretion rate which may vary during the day. in addition, no research has been carried out yet to show the quantitative and qualitative difference of uevs between spot urine and h collections.the aim of this study is to compare uevs excreted in all single voids during a hour collection period and compare it with hour collection performed. methods: uevs were enriched by differential centrifugation and electron microscopy, western blot, nanoparticle tracking analysis, tuneable resistive pulse sensing and imaging flow cytometry were used to quantify uevs and associated markers variation during the hour. creatinine, urine osmolality and particle concentration were used to normalize the assessed analytes. results: electron microscopy showed a heterogeneous population of evs and western blot confirmed the presence of ev markers (tsg , alix and cd ). rna was extracted by a column-based method (mirna extraction kit qiagen) and cel- mirna was spiked in each sample. a multiparametric detection of nephron markers podocalyxin, aquaporin- and uevs pan tetraspanins (cd + dc + cd ) was performed utilizing imaging flow cytometry. whereas the uev composition did not change across the hours analysis, the quantity of uevs and related markers fluctuated during the day depending on the hydration and excretion rate.the results of a hour urine collection reflected the average results of all single voids over a hr period. creatinine and particle count normalization failed to normalize "outliers". summary/conclusion: this study represents the very first report which compares single void urine versus hour uev analysis. we concluded that the hour collection is the preferred choice for a robust and rigorous assessment of uevs and its associated markers. porcine body fluids differ in small extracellular vesicle counts: comparison of blood plasma, seminal plasma and cerebrospinal fluid as vesicle sources for proteomic analyses helena kupcova skalnikova a , jakub cervenka b , jaromir novak a , karolina turnovcova c , bozena levinska a , jana juhasova a , stefan juhas a and petr vodicka a a institute of animal physiology and genetics, czech academy of sciences, libechov, czech republic; b institute of animal physiology and genetics cas, v. v. i. libechov, libechov, czech republic; c analysis tools were used to identify in silico biological pathways and functions governed by detected mirnas. expression of putative targets of selected mirnas was tested using qpcr after in vitro delivery of uterine evs to ptr cells. results: careful characterisation confirmed that uterine lumen is enriched with a diverse population of evs caring mirnas. interestingly, out of detected mirnas showed difference in abundance between tested days of pregnancy and half of them was exclusively detected on d . identified mirnas were characterized as potent regulators of cellular development, growth, proliferation, and movement, in addition to their involvement in organismal and embryonic development. the expression of genes identified as a possible mirna targets was tested after evs delivery to ptr cells in vitro. both down-(e.g., ptger ) and up-regulated (e.g., lifr) genes were found (p < . ); involved in the same molecular and cellular functions enriched by detected mirnas. methods: evs were harvested from wild type and arrdc -/-epididymal cells using differential ultracentrifugation, then characterised using nanoparticle tracking analysis and transmission electron microscopy. sperm motility was measured using computer assisted sperm analysis and imagej. fertilisation capacity was measured using the following assays: capacitation-associated tyrosine phosphorylation, calcium ionophore induced acrosome reaction, zona pellucida binding assay and in vitro fertilization with time-lapse imaging of embryo development. immunohistochemistry was also used to visualise two pronuclei formation and blastocyst morphology. arrdc -/-sperm was supplemented with wild type evs in the above assays to assess whether they could restore function. results: sperm from arrdc -/-mice develop normally through the testis but fail to acquire adequate motility and fertilization capabilities through the epididymis, as evidenced by reduced motility, premature acrosome reaction, reduction in zona pellucida binding and production of two-cell embryos. we observed a significant reduction in ev production by arrdc -/-epididymal epithelial cells, and addition of wild type evs to arrdc -/-sperm dampens the acrosome reaction and restores zona pellucida binding. introduction: gestational diabetes (gdm) is among the most common pregnancy complications. despite treatment, up to % of pregnancies complicated by gdm result in infants being born large-for-gestational-age (lga). this not only causes problems at birth but predisposes offspring to developing cardio-metabolic disease in adulthood. there are no treatments for lga as the cause is unclear, although it is associated with altered placental vascular development. micrornas (mirnas) regulate placental development; they are produced within cells but can be released into the circulation inside evs, which in turn can be transported into target cells and tissues to influence cellular processes. we aimed to characterise circulating evs in pregnancies complicated by gdm-lga and determine if ev-derived mirnas have the potential to influence placental development. methods: maternal serum and plasma samples were collected from women with pregnancies complicated by gdm at - weeks gestation; placental tissue was collected at delivery and birth outcomes recorded. serum and plasma evs were isolated and characterised by electron microscopy (shape), nanoparticle tracking analysis (nta; size/concentration), and western blotting (ev-enriched proteins). mirna qpcr arrays were performed on evs. mirnas were quantified in placental tissue via qpcr. results: em and western blotting confirmed isolation of evs and nta revealed no significant difference in size/ concentration in gdm-lga pregnancies (n = ) compared to gdm-aga (n = ; p > . ). several ev mirnas were altered in maternal circulation in gdm-lga compared to gdm-aga (n = /group; >twofoldchange; p < . ), including four skeletal muscle-specific "myomirs": mir- - p, mir- a- p, mir- b, and mir- a- p (all increased). all four myomirs were present in placenta but only mir- - p was significantly altered in gdm-lga compared to gdm-aga (n = - /group; p < . ). summary/conclusion: ev-bound myomirs could have predictive value for aberrant foetal growth in cases of gdm. mir- - p regulates vascular development in other systems, so we propose that mir- - p contributes to lga by influencing placental vascular development, however further work is required to establish this. introduction: seminal plasma is particularly rich in extra cellular vesicles. myelinosomes are membranous organelles described throughout the seminiferous epithelium of the testis but never reported in semen. the aim of this study was to look for the presence of myelinosome vesicles in human seminal plasma. methods: because of the viscosity of seminal gel and its water-holding capacity, classical transmission electron microscopy does not seem to be an optimal technique to reveal the presence of myelinosomes in this fluid. cryo-electron microscopy is a technique that allows visualization of nanosized structures without prior fixation or addition of heavy metals for contrast. the sample is therefore visualized as close to its native state as possible. using standard myelinosome preparation from tm sertoli cells, we first analysed the appearance of "standard" native myelinosomes by cryo em and then compared it with the vesicles from human seminal plasma samples. results: we have specified by cry-em the morphological aspect of "standard" myelinosomes isolated from the culture media of tm sertoli cells. the vesicles with the same morphological appearance were revealed in human seminal plasma specimens. summary/conclusion: myelinosomes are membranous organelles found in the seminiferous epithelium of the testis and secreted by the somatic sertoli cells in the lumen of the seminiferous tubules.the preparations from human seminal plasma contains a population of large ev (average diameter nm) whose morphological appearance resemble those of myelinosomes. defining the specific biomarkers and functionalities of myelinosomes in human seminal plasma are the concerns to be addressed in our further research. introduction: more than one million patients worldwide suffer from tuberous sclerosis complex (tsc) and have mutations in either tsc or tsc genes. together, the tsc proteins regulate mtorc activity. all tsc patient post-mortem samples exhibit renal disease and % of patients with tsc experience a premature loss of renal function. mouse and human studies are incongruity with the second somatic hit mechanism of disease, because of the low percentage of cystic cells exhibiting loss of tsc expression. we posited that the loss of a tsc protein expression may alter extracellular vesicle (ev) biology and contribute to disease. methods: we used crispr/cas to disrupt the tsc gene in mouse inner medullary collecting duct (mimcd) cells, and isolated evs using gel filtration from the isogenic cell lines. we characterized the evs using tunable resistive pulse sensing (trps), dynamic light scattering (dls), transition electron microscopy (tem), and wester blot analysis. we further performed mass spectroscopy on the ev proteins. results: loss of the tsc gene in mimcd cells induced a greater than three-fold increase in ev production compared to the same cells having an intact tsc axis. electron microscopy confirmed the purity and spherical shape of evs. both trps and dls demonstrated that the isolated evs possessed a heterogenous size distribution. approximately % of the evs were in the - nm size range. western blot analysis using proteins isolated from the evs revealed the cellular proteins alix and tsg , the transmembrane proteins cd , cd and cd , and the primary cilia-related hedgehog signalling-related proteins arl b. proteomic analysis of evs identified a significant difference between the tsc -intact and tsc -deleted cells that correlated well with the increased production. summary/conclusion: evs may be involved in tissue homoeostasis and cause disease by overproduction and altered protein content. the evs released by renal cyst epithelia in tsc complex may serve as a tool to discover the mechanism of tsc cystogenesis and in developing potential therapeutic strategies. introduction: we have shown that evs derived from amniotic fluid stem cells (afsc) of mouse origin present therapeutic effect in an animal model of chronic kidney disease, alport syndrome (as). in light of clinical translation, we isolated afsc-evs of human origin, characterized their cargo and evaluated thier therapeutic effect in vivo. methods: human clonal afsc were derived from amniotic fluid collected after volunteer donors provided consent. evs were obtained from afsc and identity and purity were assessed by rna-seq and proteomics. potency of hafsc-evs was evaluated by performing in vivo studies. ev biodistribution was evaluated by mri and therapeutic effect by measuring renal function and mice life-span. bulk rna-seq was performed on glomeruli obtained from injected and non-injected mice to identify potential ev regulating targets. results: proteomic profiling identified intact proteins and rna-seq data identified , mirs in hafsc-evs. hafsc-ev "fingerprint" was assessed by performing go analysis on the most highly expressed proteins and mirs. the results identified pathways involved in tissue homoeostasis such as mtor pathway, tgfβ and vegf pathways. when injected in vivo into as mice, biodistribution studies showed that hafsc-evs localized in the kidney, corrected proteinuria. no side effects (including teratoma) were noted in the treated mice. rna-seq of glomeruli obtained from treated as mice showed similar gene expression patterns to wilt type mice, by cluster analysis. our data indicated that hevs highly modulated pathways involved in collagen and matrix deposition remodelling, in addition to downstream targets of vegf, fgf, tnf, angiotensin and preserved glomerular cells structure and function. summary/conclusion: our protocol for hevs derivation is reproducible and allows derivation of ev lots with the same identity (specific cargo of proteins and mirs) and potency (present therapeutic effect in as). hafsc-evs modulated signalling pathways that are central to maintaining glomerular homoeostasis and preserved glomeruli structure with improved kidney function. this suggests the possibility of using hafsc-evs as a new therapeutic option for treating renal failure in humans. introduction: recent studies have shown that stem cell-derived extracellular vesicles (msc-ev) therapy improves renal outcomes in models of acute ad chronic renal disease. however, to better investigate the molecular mechanisms of ev-induced regeneration, and to define new ev sources, devices that mimic d organ architecture and flow conditions are needed. the aim of our work is to evaluate the regenerative potential of naïve and engineered ev in a millifluidic in vitro d model of glomerular damage in continuous perfusion. methods: methods: we set a millifluidic in vitro d model of glomerular filtration, a three-layers structure composed by human podocytes and glomerular endothelial cells, and, in between, of a basement membrane of collagen type iv. the barrier thus formed is set up inside a bioreactor, in a closed milli-fluidic circuit in which fluid flows continuously at a certain flow rate. we reproduced different pathological conditions and tested the localization and effect of evs in a dynamic system. : results: we obtained a standardized protocol and an adequate configuration of the milli-fluidic circuit subject to continuous reperfusion. renal damage was induced by doxorubicin or by hypoxia-reperfusion injury. we evaluated uptake, cargo transfer and effect of naïve and mirna engineered msc-evs or of klotho engineered ineffective evs administered into the dynamic co-culture system. evs were able to pass through the system and to deliver to podocytes proregenerative factors, promoting survival and limiting permeability. introduction: worldwide, renal cell carcinoma (rcc) is th most common cancer in men and th most common in women. new biomarkers are needed to aid rcc-diagnosis, provide prognostic information, and to predict response to modern targeted therapies. extracellular vesicles (evs) are an emerging source of cancer biomarkers because all cells, including cancer cells, secrete evs into biofluids as blood and urine. however, benign cells contribute to ev populations isolated from blood and urine reducing the diseasespecificity. we have developed a protocol for ev isolation directly from human rcc tissue that can increase tumour-specificity of biomarkers. methods: we obtained technical and biological replicates from normal kidney tissue and clear cell rcc tissue. serum-free media was incubated with the specimens. a combination of differential centrifugation, filtration, and ultracentrifugation was used for ev isolation. evs were quantitated using two methods, allowing for comparison between nanosight ns and nanofcm. tem was used to determine presence of intact vesicles in the ev samples. presence of ev introduction: urothelial carcinoma (uc) is a malignant cancer that affects the urothelial cells, representing % of all bladder tumours. at diagnosis % of bladder cancers are non-muscle invasive tumours. importantly, upon transurethral resection of the bladder tumour, nearly - % of these patients will experience disease relapse and - % will progress to muscle invasive tumour, requiring thereby, a rigorous and expensive follow-up. currently, this is performed through the frequent use of highly invasive cystoscopy and the low sensitivity urine cytology. thus, innovative liquid biopsy-based biomarkers that circumvent these drawbacks are highly desirable for improved uc clinical management. here, we aim to implement a protocol for the isolation and characterization of extracellular vesicles (evs) from uc patients' urine samples. methods: a two-step protocol involving ultracentrifugation (uct) and by size-exclusion chromatography (sec) was optimized for urine samples. the isolated urine-derived evs from uc patients were then characterized according to their size, concentration (nta), morphology (tem), protein amount (lowry method), presence of ev-associated and disease-associated protein markers (western blot). results: isolated urinary evs from uc patients had a size ranging from nm to nm with characteristic ev morphology, express ev-associated markers as cd and hsp and were negative for cell debris markers. the recovery yield and purity of isolated evs following each isolation technique was characterized. upon uct, sec was required to deplete most of the ev-associated thp and albumin protein contaminants. some disease-associated protein markers were highly enriched in isolated urinary evs compared to crude urine. summary/conclusion: taken together, these results indicate that a two-step ev isolation protocol was properly implemented and validated in uc patients' urine samples. notably, several ev-associated disease biomarkers were detected in the urine of uc patients. this ev-based liquid biopsy might provide the means for real-time monitoring of residual disease and relapse in uc patients. introduction: glioblastoma multiforme (gbm) is a very aggressive type of brain tumour. different gbm molecular subtypes (proneural, mesenchymal and classical) often co-coexist within the same tumour, with the mesenchymal subtype driving the tumour progression. recently, our lab demonstrated that the cargo of extracellular vesicles (evs) could mirror the molecular background of the gbm cells from which they were derived. altogether, we believe that gbm cell-derived evs can be directly involved in the expansion of the mesenchymal signature in tumours, thus supporting gbm aggressiveness. methods: non-mesenchymal (t & u ) gbm cells were "primed" using evs derived from mesenchymallike (u & ln ) gbm cells. ev-primed gbm cells were then co-cultured with their non-primed counterparts to determine whether the mesenchymal signature can "spread" from cell to cell via evs. effect on cell proliferation, migration and invasion (in hyaluronic acid hydrogels) was assessed following ev treatment and co-culture. the expression of mesenchymal gbm markers was measured by western blotting. further mass spectrometry analysis of cell and ev content was undertaken to describe potential underlying mechanisms. results: co-culture with ev-primed gbm cells significantly increased proliferation and hydrogel invasiveness of non-mesenchymal cells. interestingly, the stimulating effect of co-culture was even stronger on the proliferation of ev-primed gbm cells. moreover, further proteomic analysis revealed that expression of mesenchymal gbm markers such as cd was increased in non-mesenchymal cells following coculture. summary/conclusion: our data suggest that evs from mesenchymal gbm cells can be uptaken by gbm cells from different subtypes, thus stimulating tumour progression. overall, we think the present study provides with new insights for the understanding of gbm recurrence and the development of potential therapeutic strategies. introduction: triple-negative breast cancer (tnbc) is the most aggressive form of breast cancer. previously we reported that the heterogenous population of evs released from tnbc cells promotes the growth and aggression of recipient cells. here we investigated if, by using compounds proposed to inhibit ev release i.e. calpeptin and y (to block those budding at cell membrane) and gw and manumycin a (to block evs from mvbs), we could reduce the associated transmission of aggressive phenotype. methods: evs were separated from medium conditioned by tnbc cell line hs ts(i) , using a discontinuous optiprep density gradient, after the cells were treatment for hrs with the compounds listed above. evs (pooled fractions - with a density range of . - . g/ml) were characterised by nta, bca, lipid assay, immunoblot, tem and flow cytometry. to investigate the functional effects of the evs released, proliferation and migration assays were performed on hs t and mda-mb- cells using the ev to cell ratios of × evs/ x cells, × evs/ x cells, × evs/ x cells to evaluate doseresponse. ev-track id ev (score of %). results: gw significantly (p = . ) decreased ev release from hs ts(i) cells. manumycin a and a combination of calpeptin and y (combo) decreased ev release, but significance was not reached. conversely, calpeptin and y actually increased ev release; but not significantly. of the reduced numbers of evs released following gw treatment, hla-dr+ evs were significantly (p = . ) enriched. none of the evs analysed significantly changed hs t or mda-mb- growth rates. however, evs from cells treated with calpeptin (p = . ), gw (p = . ), manumycin a (p = . ) and combo (p = . ) caused significant reduction in mda-mb- migration compared to the effects of evs from untreated cells. similarly, ev from cells treated with gw (p = . ), and combo (p = . ) caused significant reduction in hs t migration. summary/conclusion: while gw was the only compound that caused a significant decrease in quantities of ev released, the evs that continued to be released following treatment with gw or calpeptin and y significantly reduced migration of both recipient cell lines. funding: phd funding: tcd scholarship and carrick therapeutics ltd extracellular vesicles from highly metastatic lung cancer cells induce barrier impairment, permeability, and epithelial-to-mesenchymal plasticity in a -day mature bronchial epithelium purdue university, west lafayette, usa introduction: epithelial-to-mesenchymal (emt) transition plays an integral role in cancer metastasis, which is responsible for as much as % of cancer mortality. cancer exosomes induce emt in bronchial epithelial cells, however, the epithelial cells inhibit emt when allowed to form a mature epithelial barrier with apicalbasal polarity. it is not known if cancer-derived extracellular vesicles (evs) can induce emt and more importantly, barrier disruption in a mature epithelium. here, we show that evs from a highly metastatic lung cancer cell line (calu ) are) are not only sufficient to induce emt in non-tumorigenic bronchail epithelial cells (beas- b), but are also capable of disrupting a -day mature bronchial epithelial barrier by significantly reducing teer, inducing sixfold increase in permeability and complete loss of e-cadherin at cellcell tight junctions. methods: beas- b and calu evs were characterized using electron microscopy, nanosight and western blotting for exosome-specific features. for permeability studies, beas- b cells were cultured in transwell for days to establish an intact epitheliumconfirmed by measuring teer (trans-epithelial electrical resistance). intact beas- b monolayers were treated with calu evs at , and μg/ml for hrs, and barrier intactness and permeability were evaluated by measuring teer, apical-basolateral translocation of dextran beads and confocal imaging of tight junctions (e-cadherin). for emt experiments, beas- b cells treated with calu evs at and μg/ml were evaluated for ecadherin and vimentin levels by qrt-pcr and western blot after hrs. results: beas- b and calu evs were enriched in - nm size range, and cd and cd were enriched in the ev fraction in contrast to the cell lysate and vice versa for gp . calu evs significantly impaired day mature beas- b monolayer's barrier properties, which at the highest dose caused % reduction in teer from . ± . to . ± . Ω.cm (n = ). this was further confirmed by~sixfold increase in dextran beads' apical-basolateral translocation in min ( . ± ng/ml in control vs . ± ng/ml in treated) (n = ) and complete loss of e-cadherin expression at cell-cell tight junctions (n = ). at the transcript level, calu evs induced significant downregulation of e-cadherin by % and upregulation of vimentin (mesenchymal marker) twofold (n = ) in beas- b cells, indicating transition into mesenchymal phenotype. summary/conclusion: we demonstrated the involvement of evs derived from highly metastatic lung cancer cells in inducing emt in bronchial epithelial cells and epithelial barrier disruptionthe initial stage of the intravasation process. grp plays a crucial role in the extracellular vesicle-promoted radioresistance of irradiated head and neck cancer cells introduction: small evs released from irradiated head and neck squamous cell carcinoma (hnscc) cells increase resistance of recipient hnscc cells to radiation in vitro. we have identified the glucose-regulated protein (grp ), a chaperone protein of the hsp family which is involved in cellular stress responses and associated with worse survival in head and neck cancer patients, as an essential component of the ev-mediated radioresistance. methods: small evs were isolated from conditioned medium from irradiated and non-irradiated bhy hnscc cells by combined microfiltration ( . µm) and differential ultracentrifugation. grp surface expression was measured by proteomic analysis, immunoblotting and bead-facs. radiation resistance of bhy cells was determined by a clonogenic survival assay. results: increased grp was identified on the surface of evs from irradiated cells. the increase in ev grp correlated with increased grp expression at the donor cell surface. the grp content of recipient cells also increased upon transfer of evs from irradiated, but not non-irradiated cells, ultimately leading to enhanced cell survival. to check a potential role of elevated grp in radiation resistance we overexpressed grp . here the modest ( x) overexpression of grp was sufficient to confer an enhanced radioresistant phenotype to the bhy cells. a correlation between grp -dependent increase of radioresistance and activation of the akt pathway is yet to be determined. summary/conclusion: our results suggest a pivotal role for ev-transferred grp in modulating the radiation response of recipient hnscc cells. radiation directly increases the cellular and vesicular grp levels, and subsequent ev-mediated transfer leads to enhanced grp levels and radioresistance in recipient cells. this study provides new mechanistic insights into the effects of evs in radiation response and elucidates an interesting target protein and novel strategies for the improvement of radiotherapy. d modelling of ev release in progressing prostate cancer introduction: the modelling of cancer progression should be capable to translate acquired knowledge of cell behaviour to the real human body conditions. however, the extracellular vesicles (evs) isolated from d cell models are commonly exploited in research. taking into account the specificity of the prostate cancer (pc) environment, and a strong need of early diagnosis of castrate-resistance by prostate cancer (crpc) patients, we suggest in-depth profiling of different ev subtypes isolated from d culture as a new tool to model the progressing pc. methods: cells from hormone-resistant prostate carcinoma -rv line were cultured in d and d conditions, using d coseedistm. acd plasma controlled for haemolysis and remaining platelets was taken from patients with pc and crpc. the fractions of ev subtypes from cell culture and plasma were obtained by differential centrifugation (dc) followed by iodixanol density gradient purification. each of the fractions was measured by nanoparticle tracking analysis (nta), tunable resistive pulse sensing (trps) followed by elisa. for that, cd and cd were used as ev markers, apob and apoa for lipoprotein contaminants control, and cd , cd and psma as tissue-specific biomarkers for determination of fractions containing evs of different origin. ev-contained fractions were subjected to next generation sequencing (ngs). results: in d conditions, the -rv cells produce up to -times higher ev number than in d. size and density distribution of evs derived from d cultures but not of d resembled plasma evs. size distribution and biomarker expression among different ev subtypes allowed distinguishing between pc and cprcderived samples, indicating a potential to translate these results into clinics for early cprc detection. summary/conclusion: this work demonstrates a new approach to study the secretome of a progressing pc under d conditions. the profiles of ev subtypes produced by cancer cells growing in a d spatial architecture resemble the profiles of plasma evs and can serve a useful tool for the establishment of new biomarkers. introduction: renal cell carcinoma (rcc) is the most common primary renal neoplasm, with over , cases in the us alone each year. early detection of rcc leads to consistently better patient outcomes, and extracellular vesicles (evs) isolated from patient samples may prove to be a valuable clinical tool in the future. evs are abundant in blood and urine and show a large amount of heterogeneity but are difficult to analyse due to their small size and difficulty in isolation. here, we employ a multiparametric analysis of ev surface markers to identify a set of markers that may prove clinically relevant in future studies. methods: rcc cell lines vok , vok , and vok were cultured in flasks containing ml of ev-depleted media ( % fbs, centrifuged hr x , g). when cells reached~ % confluency, the conditioned media was collected and spun at , g for mins two times to deplete any remaining debris, leaving~ ml of media. this media was concentrated to a final volume of~ ml using a pall jumbosep kda mwco filter. this concentrate was purified from protein by using an izon qev- column, collecting ml fractions. protein content of each fraction was analysed using a absorbance while concentration and diameter distribution were determined through nanoparticle tracking analysis (nta). pooled samples made of the three most concentrated fractions were concentrated to a final volume of~ µl using the pall microsep kda filter and then used for analysis in the miltenyi macsplex exosome kit. flow cytometric data were generated by the cytoflex s and analysed using flowjo and mpapass software. these positive signals were verified through bead-only controls and titrations. results: the mpapass software allowed for heatmap generation, data reduction, clustering and visualization of expression patterns. of the detection antibodies used across capture beads, cd , cd , cd , beta- microglobulin, and cd were found to be prevalent in these rcc evs. these markers were found to be co-expressed particularly with cd , cd , and cd . summary/conclusion: the use of multiplex analysis allowed for detection of five distinctive surface markers found to be prevalent in evs collected from rcc cell lines. these results demonstrate the utility of multiplex analysis and mpapass software for identifying potential markers of interest and provide proteins that are worth exploring further. the next steps to this work will be developing custom multiplex arrays that tailor capture and detection of evs specifically for rcc pathology. low molecular weight protein tyrosine phosphatase (lmwptp) carried by colorectal cancer cells-derived extracellular vesicles as a player in tumour-educated human fibroblast university of campinas -unicamp, campinas, brazil introduction: extracellular vesicles (evs) are doublemembrane-bound nanovesicles released by cells playing a key role as mediators of intercellular communication. low molecular weight protein tyrosine phosphatase (lmwptp) is upregulated in several cancers type, including colorectal cancer (crc), and it has been correlated with aggressiveness, chemoresistance and poor prognostic. methods: the aim of this study was to determine whether crc cells release lmwptp-enriched-evs and influence tumour microenvironment-associated cells as a representative tumour education. crc cells, hct and ht , were cultured in serum-free medium for hours. conditioned medium was concentrated by ultrafiltration (mwco kda) and evs were isolated by total exosome isolation reagent (invitrogen). evs were characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and western blotting (wb). lmwptp levels were analysed by wb and sandwich-elisa. to evaluate tumour education, hff- fibroblasts were used as recipient cells. the uptake of evs (pkh fluorescently labelled evs), proliferation (viability) and migration (wound healing assay) were analysed in a co-culture model of crc-derived evs and hff- . results: nta showed a higher concentration of evs released by ht . hct and ht evs displayed a mean diameter around nm and a cup-shaped morphology. isolated evs were positive for evs-markers cd and tsg and negative for gm a non-evs marker. ht lineage as well as derived-evs are lmwptp-enriched in comparison to hct cells and evs. upon incubation, fluorescently hct and ht derived evs were internalized into hff- cells in a perinuclear region. evs derived from both cells increased the viability and proliferation of hff- cells. intriguingly, evs derived from ht promoted cell migration. summary/conclusion: in conclusion, for the first time, we showed that lmwptp can be carried by evs derived from crc cells and lmwptp-enriched-evs can modulate biological aspects of hff- fibroblast. overall, our findings point lmwptp out as important player in tumour-educated fibroblast. exosomal mir- a inhibition by vincristine and prednisone in paediatric acute lymphoblastic leukaemia. introduction: vincristine and prednisone are standard agents in treatment of paediatric acute lymphocytic leukaemia (p-all). mechanistically, vincristine induces apoptosis by blocking microtubules formation, while prednisone binds to cytoplasmic receptors and inhibits dna synthesis, both of which lead to apoptosis. the effect of these agents on exosomal micro-rna expression and its functional regulation is not yet investigated. elevated levels of mir- a in circulating exosomes (nanoparticles) has been shown to lead to progression in several cancers, including all. we have previously shown that leukaemia-derived exosomes induce leukaemia cell proliferation via up-regulating of mir- a expression and silencing of exosomal mir- a reverses this exosomeinduced cell proliferating effect. the objective is to investigate the effect of vincristine and prednisone on exosomal mi-r a expression in all. methods: jm , sup-b , and nalm- leukaemic cell lines were treated in vitro with vincristine ( . to . µm) and prednisone ( . to . µm) in exo-free medium and apoptosis was measured by mts assay. total rna of exposed cell lines was isolated and cdna was prepared for mir- a analysis. expression of mir- a was analysed by q-pcr. exosomes from conditioned medium of exposed cell lines were isolated by ultracentrifugation method. purity and particle size of exosomes were confirmed by western blot and nanoparticle tracking analysis (nta) assay respectively. total exosomal rna was isolated from exosomes (exo-rna) by trizol method. synthesis of cdna was carried out with the miscript ii rt kit (qiagen). results: vincristine and prednisone promote apoptosis in leukaemia cell lines (jm and sup-b ) in a dosedependent manner. both cellular and exosomal mir- a expression was down-regulated by vincristine and prednisone exposure in all three leukaemia cell lines (jm , sup-b , and nalm- ). these observations demonstrate that cellular mir- a down regulation in the parental cells is stable and can be transferred to exosomes, confirming the concept that exosomes are the fingerprint of parent cells. summary/conclusion: our data suggest that the vincristine and prednisone anti-proliferative effect in p-all maybe induced by another yet unexplored pathway, that suppresses mir- a at a cellular and exosomal level in p-all, resulting in apoptosis. funding: this project is supported by the dimartino family foundation. secreted extracellular vesicles from renal cell carcinoma cells anatoliy samoylenko, artem zhyvolozhnyi, eslam abdelrady, naveed ahmad, genevieve bart and seppo vainio oulu university, oulu, finland introduction: clear cell renal cell carcinoma (ccrcc) represents the most common form of kidney cancer and is among the most lethal of all genitourinary cancers. despite surgery and medication therapy, most patients with metastatic ccrcc have a poor prognosis. intratumoural hypoxia is a key factor involved in renal cancer progression and it is known to promote secretion of evs by many types of tumour cells. methods: rcc-derived renca cells, embryonic kidney derived ub cells, and primary mouse hepatocytes were used in the study. evs were purified from cell culture media by gradient ultracentrifugation, sequential ultracentrifugation and exo-spin™ columns. before ev isolation cells were kept for h either under normoxia or hypoxia ( % oxygen). evs were analysed by transmission electron microscopy with negative staining and immunolabeling, by nanoparticle tracking analysis (nta) and western blotting. cells proliferation and viability were assayed by live cell imaging using incucyte zoom (essen bioscience), cell metabolic activity by seahorse xf analyser (agilent), rna expression by qpcr and ddpcr. proteins were identified by ultra-performance liquid chromatography-mass spectrometry (uplc-ms). rna libraries were made using nebnext small rna library prep kit, and sequenced on nextseq (illumina). results: we showed that hypoxia induced production of evs by rcc cells, and characterized differences in protein and rna content of evs generated by renca cells cultured under normoxic and hypoxic conditions. we also showed that rcc-produced vesicles modify key features of tumorigenesis (gene expression, metabolic activity, motility, and growth) of target cells. these data were obtained by using two target cell types: model mouse kidney cells and primary mouse hepatocytes, which represent typical site of rcc metastasis with an exceptionally poor prognosis. we proposed that a possible mechanism of ev action in rcc is related to changes in caveolin- function. we also tracked renca-derived evs in a chick embryo model and in a novel kidney organoid co-culture assay developed by our group (xu et al., ) . summary/conclusion: hypoxia may influence tumorigenic properties of rcc by changing rates of production and composition of evs. funding: the study was supported by finnish cancer foundation grants. exosomes synthesizing her mirna and engineered to adhere to her on tumour cells surface exhibit enhanced anti-tumour activity introduction: exosomes are small extracellular vesicles averaging - nm in diameter. they serve as a means of intercellular communication. typically they consist of structural proteins as well as selected proteins, mirnas, mrnas, and long noncoding rnas. thus in an earlier report this laboratory designed a mirna targeting a major herpes simplex virus regulatory protein. as predicted by the nucleotide packaging signal the mirnas were packed in exosomes and on exposure to infected cells significantly reduced virus yields. her (human epidermal growth factor receptor ) plays an important role in the neoplasia of some breast cancers. the protein is exhibited on the cell surface and is the target of therapeutic antibodies. methods: firstly, we report on the construction of a mirna targeting the synthesis of her both in cells constitutively expressing her and in cells transfected with a plasmid encoding her . secondly, we report that the mirna targeting the synthesis of her reduced the viability of her positive cancer cells both in cell culture and in implanted tumours. lastly, we enhanced the anti-tumour activity of the exosomes by binding to the exosome surface a ligand with affinity for the her on the surface of tumour cells. the -mir-her exosomes package with mirna designed to block her synthesis and deliver to cells. these exosomes kill cancer cells dependent on her for survival but have no effect on cells lacking her or which were engineered to have her but do not depend on it for survival. the -mir-xs-her exosomes carry in addition a peptide which enables the exosome to adhere her on the surface of the cancer cells. in consequence, these exosomes preferentially enter and kill cells exhibiting her on their surface. the exosomes with -mir-xs-her are significantly more effective in shrinking the size of her -positive tumours implanted in mice than the -mir-her exosomes. summary/conclusion: our studies indicate that exosomes carrying mirna against her have no effect on her negative cells it was nevertheless desirable to increase the uptake of exosomes carrying the her mirnas by her -positive tumour cells. to this end we modified the exosomes to exhibit on their surface a peptide that bound the exosomes to the her on the surface of cancer cells. in consequence, we significantly enhanced the uptake of exosomes carrying the mirnas directed against her by her positive cells. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd , shenzhen science and innovation commission project grants jcyj , jcyj to shenzhen international institute for biomedical research. systematic characterization of ovarian cancer-derived exosomes unveil mirnas interfering with cd + t cell activation introduction: cd + tumour-infiltrating lymphocytes (til) have been widely reported to correlate with cancer patient survival, including ovarian cancer. even with the presence of tils, immunotherapy has limited success in ovarian cancer. understanding the interaction between cd + til and tumour cells is thus important. our hypothesis is that tumour-derived exosomes are released and taken up by cd + til such that specific mirnas contained within modulate physiological processes that inhibit cd + t cell activation. we aim to identify mirnas carried in tumour-derived exosomes that inhibit cd + t cell activation in ovarian cancer. methods: we purified exosomes from nine ovarian cancer cell lines and stocked in high concentration. interferon-gamma (ifn-gamma expression screening was performed after days of co-incubation of tumour derived exosomes, cd + t cells, and activators in conditioned medium. cell counts and viability were tested by trypan blue staining at day and day . rna-seq for exosomes were generated to identify mirnas critical in differentiation effects on cd + t cell activations. microrna target matching uncovered target mrnas while enriched pathway analysis predicted potential signalling pathways involved. results: our ifn-gamma screening results indicated the exosomes exhibit different behaviours in interfering cd + t cell activation owing to different donors. exosomes derived from peo. and ovca cells have consistent polarized results in ifn-gamma expression. exosomes derived from peo. remained a low ifn-gamma expression and from ovca stayed at relatively high level. small rnas profiling analysis between the two cell lines identified mirnas (p < . ), and mirnas have been reported with validated targeting information, and out of have targets involved in immune signalling. mrna targets were uncovered by target matching. cmap search identified complex connections among mrnas with the top enriched pathways actively involved in cell cycle and immune related behaviours. summary/conclusion: our ifn-gamma screening identified crucial mirnas in ovarian cancer exosomes interfering cd + t cell activation. computational modelling on both experimental and public multiomics datasets predicted promising signalling pathways of tumour-immune crosstalk for functional validation. irradiation of breast cancer cells alters the quality of dna cargo in the exosomes that they produce sheila spada, paul zumbo, doron betel, tuo zhang, nils-petter rudqvist and sandra demaria weill cornell medicine, new york, usa introduction: irradiation of breast cancer cells with an immunogenic dose ( gyx ) leads to accumulation of cytosolic dna that is sensed by cgas leading to interferon type i (ifn-i) signalling via cgas/sting pathway [ ] [ ] [ ] . we previously showed that tumour-derived exosomes (tex) secreted by irradiated ( gyx ) (rt-tex) but not untreated (ut-tex) tsa carcinoma cells carry dna that stimulates the production of ifn-i in recipient dendritic cells (dc) via the cgas/ sting pathway [ ] . moreover, mice vaccination using rt-tex, but not ut-tex, elicited anti-tumour immune response inhibiting tumour growth [ ] . here, we hypothesized that the differential ability of rt-tex and ut-tex to activate ifn-i in recipient dcs is due to qualitative differences in dna cargo of rt-tex compare to ut-tex. methods: the length of dna purified from tex and from the cytosolic fraction of tsa cells was measured by agilent bioanalyzer. the dna cargo of tex was analysed by whole-genome sequencing (wgs) and whole-genome bisulphite sequencing. the percentage of methylation of total dna in tsa cells was quantified by -methyl cytosine dna elisa kit. results: dna fragments with size between and bp were enriched in rt-tex compared to ut-tex, as well as in the cytosolic fraction of irradiated compared to mock-treated tsa cells. wgs revealed that the entire genome was represented in tex dna cargo, regardless of rt. more than % of tex dna was of nuclear origin, but mitochondrial dna was increased in rt-tex. interestingly, we found that rt decreases the level of methylation in both exosomal and total dna in tsa cells compared to the controls. summary/conclusion: these data support the hypothesis that immunogenic rt alters some characteristics of the exosomal dna cargo, mirroring molecular changes occurring in parent irradiated breast cancer cells. the enrichment in dna fragments of - bp in rt-tex is intriguing considering that cgas is optimally activated by dna in this length range [ ] . we are currently investigating which features of the cargo dna that differ between ut-tex and rt-tex may explain the differential ability to induce ifn-i pathway activation in recipient dcs. the identification of a dna signature associated with the ability of tex to activate the cgas/sting pathway could provide a circulating biomarker of the rt-driven immunogenic tumour response. introduction: triple negative breast cancer (tnbc) is among the most difficult cancer subtypes to treat and continues to cause a high number of cancer-related deaths annually. extracellular vesicles (evs) transfer cell type-specific cargo and have important implications in disease initiation, therapy and outcome. upon treatment of cancer cells with low-dose chemotherapy, released evs are able to transfer phenotypic traits to other cancer cells. new treatment strategies for tnbc, like inhibitors of the er stress pathway (ire ) might impact on ev biogenesis, cargo delivery and response of cells in the cancer microenvironment. our aim is to identify immune modulatory alterations in breast cancer cells and cancer derived evs upon treatment with inhibitors of the er stress pathway. methods: human tnbc cell lines were treated with ire inhibitor mkc and cells were analysed for immune modulatory surface markers, like hla-i, b -h molecules and different integrins. mitochondrial and lysosomal activities were investigated by the use of a mito-and lysotracker and analysed by imagestream (isx) technology. extracellular vesicles were isolated from cell culture supernatants by sequential centrifugation, quantified by nanoparticle tracking (nta) and characterized by exosome bead array. single ev analysis of total cell free supernatants and of isolated evs was performed by isx and marker positive evs were quantified for absolute fluorescence signals and total amount by objectives/ml. ev uptake into t cells was investigated by the use of different ev labelling strategies. results: several immune relevant surface markers (hla-i and cd ) are downmodulated by ire inhibition across different cell lines. cell surface expressed cd and b -h show cell line specific downmodulation profiles upon ire inhibitor treatment. other immunomodulatory marker such as b -h and b -h , integrin cd , cell adhesion-promoting cd and stemness/metastasis marker (cd and ssea) are unaltered on ire treated breast cancer cells. cancer cell derived evs were tetraspanin positive (cd , cd , cd ), similar in number and showed differential expression of immune markers upon ire treatment. mitochondrial and lysosomal activities were unaltered under ire inhibition, whereas cell proliferation was diminished. no breast cancer-derived ev uptake of externally labelled evs into healthy t cells could be detected. summary/conclusion: ongoing analyses focus on the multicolour analysis of multiple markers on single evs by imaging flow cytometry and on the functional impact of cancer derived evs on t cells delivered by ev receptor binding. funding: dagmar quandt is supported by the sfi (cÚram research centre, /rc/ ), the european regional development fund and the dr. werner jackstädt-stiftung. chair: uta erdbrügger -university of virginia chair: larry harshyne -thomas jefferson university comparison of three isolation protocols to search extracellular vesicles signature in sickle cell disease patients introduction: sickle cell disease (scd) is an inherited disorder characterized by chronic haemolysis and continuous activation of different cell types. extracellular vesicles (evs) were described to be at increased levels in scd patient's plasma compared to healthy subjects and were associated with several clinical manifestations such as leg ulcers and stroke. scd patient's plasma has increased concentrations of haem, free-hb and other proteins and lipoproteins as chronic haemolysis consequence. here, we report the comparison of three mostly used isolation protocols to search ev signature in scd patient's plasma by flow cytometry. methods: blood samples were obtained from scd patients (n = ) following wisgrill et al., ( ) protocol. three different ev isolation protocols were used: differential centrifugation (dc), ultracentrifugation (uc) and size-exclusion chromatography (sec). lactadherin and calcein-am were used to detect phosphatidylserine (ps)+ vesicles and membrane integrity, respectively. platelet-derived evs (pevs), endothelialderived evs (eevs), leucocyte-derived evs (levs) and monocyte-derived evs (mevs) were quantified. silica beads were used to define evs gate and samples were acquired in the cytoflex cytometer platform. results: the quantification of pevs in uc, dc and sec samples was, respectively, x , , x and , x events/ml mean, eevs was , x , × and , x events/ml mean, levs was x , × and , x events/ml mean and mevs , x , , x and , x events/ml mean. uc samples demonstrated a higher concentration of evs, which could be more useful to functional studies than dc and sec, however, it took more time to separate than dc. dc was the fastest method to separate evs from plasma, being useful to study large patients cohorts, but showed the smallest overall number of evs. sec also demonstrated high capability to detect evs in plasma and the possibility of obtaining a purer sample, although it is the most expensive and time-consuming method among all tested. all evs populations were detected in the three protocols tested. summary/conclusion: in summary, all protocols tested were efficiently to detect evs in scd patient's plasma and the definition of the best protocol may vary based on the research aim and time and budget available. funding: fapesp / - . gabrielle lapping-carr, joanna gemel, yifan mao and eric beyer university of chicago, chicago, usa introduction: aberrant cell-cell interactions involving the endothelium are central to the pathophysiology of sickle cell disease (scd), including acute chest syndrome (acs), a deadly and unpredictable complication. we previously demonstrated that the plasma of scd patients contains increased circulating small extracellular vesicles (evs) compared to controls and that those vesicles can disrupt endothelial integrity in vitro by affecting adherens junctions and ve-cadherin. the current study was designed to examine the effects of those evs on other cellular junctions including tight (zonula occludens , zo- ) and gap junctions (con-nexin , cx ) and to test the hypothesis that the junctions would be more severely affected by evs isolated from patients during an episode of acs than by ones isolated from the same patient at baseline. methods: we identified subjects with scd in our biobank who had plasma isolated at baseline and at the beginning of an admission for acs. evs were isolated from platelet free plasma using established methodologies. to determine the effects on endothelium, cultures of human microvascular endothelial cells were treated with evs for h and studied by immunofluorescence, immunoblotting and rt-qpcr. gap junction-mediated intercellular communication was assessed following microinjection of lucifer yellow and neurobiotin. results: the distribution and abundance of zo- at the plasma membrane were minimally affected by scd evs. while baseline evs did not affect the distribution of cx , evs isolated during an episode of acs caused loss of cx from the plasma membrane. the integrated intensity of cx membrane staining was decreased bỹ % following treatment with acs evs. cx protein decreased on average by %, cx mrna levels by % and neurobiotin transfer by - % in cells treated with acs evs, compared to baseline evs. summary/conclusion: circulating evs in scd affect multiple components of endothelial junctions. gap junctions composed of cx are the most sensitive of the cellcell junctions, since their abundance and function are reduced by acs evs even when the endothelial monolayer appears intact. cx -mediated intercellular communication may be an early and sensitive event in the endothelial disturbance caused by evs in scd patients. funding: nih ul tr , comer hospital rbc race funds, ted mullin fund. the effects of platelet concentrate storage time on extracellular vesicle interactions associated with fibrin clot formation in-vitro jamie nash a , christine saunders b , amanda davies a and philip james a a cardiff metropolitan university, cardiff, uk; b welsh blood service, velindre university nhs trust, cardiff, uk introduction: platelet concentrates (pcs) have been utilised for decades to prevent bleeding in thrombocytopenic patients and to stop active bleeding. the storage of pcs however is a logistical challenge due to the limited day shelf life under standard conditions. during storage, platelets undergo a number of mechanical and biochemical changes contributing to the short shelf life of a pc. these changes are collectively known as the platelet storage lesion. platelet extracellular vesicles (pevs) are known to increase throughout pc storage, due to an increase in platelet activation. as pevs have previously been shown to be pro-coagulant and increase in annexin v binding over pc storage. the aim was to investigate the effect of pc storage time on extracellular vesicle interactions on fibrin clot formation. methods: pcs were sampled on alternate days up to days of storage and centrifuged to achieve acellular plasma. the plasma was subjected to ultracentrifugation ( , xg) to pellet evs. the size and concentration of evs was assessed using nanoparticle tracking analysis software, followed by a western blot to confirm evs were of platelet origin. the pevs were added at a fixed number to a control pooled plasma sample with added thrombin and tissue plasminogen activator. the time to clot and % lysis time were recorded by using the turbidometry of the plasma over time. results: evs isolated from the pc were confirmed to be of platelet origin by western blot using cd as a marker of platelet origin and cd as an ev marker. pevs caused a significant increase effect on the fibrin clot formation (p < . ) when compared to the control plasma. pevs also had a significant effect (p < . ) on the fibrinolysis time, extending the time taken to lyse the clot. characterization of mirna from serum derived exosomes in a mouse tibia fracture model of introduction: complex regional pain syndrome (crps) is a debilitating chronic disease that occurs after trauma to the periphery and is intimately associated with nerve injury. its presentation is often described as an injury that is disproportional to the inciting event and manifests neuropathic pain, systemic inflammation, and immune dysregulation. owing in part to our poor understanding of disease aetiology, current treatments for crps are insufficient and as a disease of exclusion there is a lack of quantitative diagnostic markers. exosomes are small extracellular vesicles (sevs) - nm in size which provide a means of cellular communication through their cargo molecules (protein, mirna, mrna, lipids) , and have demonstrated promise in uncovering mechanisms of disease manifestation and identifying potential diagnostic markers. we have shown previously that crps patients have differential expression of several mirnas in serum derived sevs as compared to healthy controls, but little is known on how this compares to the established mouse tibia fracture model of crps. methods: mice undergoing fracture were anesthetized and subjected to a unilateral tibia fracture followed by casting of the injured limb. after confirming the establishment of pain hypersensitivity, serum samples were collected from fracture model and control mice three weeks post-injury. sevs were isolated by differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna-seq analysis is being performed to identify differentially expressed mirnas. results: nanoparticle tracking analysis showed no significant difference in the number or size of sevs present in the serum from the fracture model and control mice. rna-seq is ongoing and differential mirna expression in sevs from fracture model will be compared to control samples. comparative studies identifying mirnas that are common between crps patients and the rodent model will facilitate the development of correlational outcomes between preclinical and human studies. summary/conclusion: identification of similarities and differences between crps patients and animal models will aid in directing future studies at clinically relevant aspects of crps aetiology and identifying potential diagnostic markers for crps patients. extracellular vesicle-based liquid biopsy in acute myeloid leukaemia: a reliable source of residual disease biomarkers? introduction: acute myeloid leukaemia (aml) is an haematopoietic stem cell disorder with a poor -year survival rate. monitoring of measurable residual disease (mrd) in aml patients receiving chemotherapeutic treatment is useful to assess therapy response and predict relapse. indeed, many different leukaemia associated immunophenotypic protein markers (laips) are presently useful to detect mrd. nevertheless, their analysis currently requires invasive bone marrow aspirates, thus severely hindering real-time monitoring of the disease. therefore, alternative peripheral blood-based methods are highly desirable for an easy, real-time and costeffective monitoring of aml progression. this work aims was to assess the feasibility of a peripheral blood ev-based liquid biopsy method for aml disease monitoring, based on the detection of laips with a known negative impact on the prognosis of aml. methods: the profile of evs isolated from paired samples from aml patients' blood plasma collected at diagnosis, complete remission (and some at relapse) was compared and correlated with clinical data. for that, a size-exclusion chromatography (sec) method was optimized to isolate the circulating evs from the blood plasma. the evs of the paired aml patients' blood samples were then characterized according to their size (dls/nta), morphology (tem), proteinto-lipid ratio (lowry/sulpho phosphovanillin assay), surface charge (zeta-sizer) and protein cargo (western blot). results: sec allowed the isolation of size-resolved plasmaderived evs from the peripheral blood of aml patients. isolated evs had a size ranging from nm to nm with an intact morphology, expressing ev-associated markers such as hsp , cd , cd and cd . size-resolved evs also had a differential expression of mitofilin, actinin- , syntenin- and annexin-xi proteins. several laips were detected in the isolated evs and their relative abundance changed throughout the stage of the disease. summary/conclusion: our preliminary data shows that aml patients' circulating evs carry relevant immunophenotypic protein markers, which might predict aml clinical outcome. introduction: cell plasticity regulated by the balance between the epithelial-to-mesenchymal transition (emt) and met is critical in the metastatic cascade. extracellular vesicles (evs) may play an important role in this balance by shuttling molecular cargos into recipient cells. this study aims to evaluate the feasibility of profiling mrnas of parental prostate cancer (pca) cells with different phenotypes and their daughter evs using the nanostring low rna input ncounter assay. methods: pc -epi and pc -emt cell lines representing epithelial and mesenchymal phenotype, respectively, were generated from original pc cell line. the cell culture supernatant was first pre-cleared for any dead cells and debris by centrifugation at × g for min. without disturbing the pellet, the supernatant was then transferred to a fresh ultracentrifuge tube and centrifuged at , × g for min at °c. the remaining supernatant was then centrifuged to isolate the evs at , × g for min at °c. the evs pellet was further washed in × pbs followed by a second centrifugation at , × g for min at °c. the final evs pellet was resuspended in × pbs for subsequent characterization (transmission electron microscopy, nanoparticle tracking analysis and western blot) and ncounter assays. the total rna of cells and their daughter evs were assayed by the ncounter pancancer progression panel to determine expression of selected mrnas. the nanostring ncounter low rna input kit with the multiplex gene primer pool was used for the pre-amplification of mrna and overnight hybridization with the pancancer progression panel. each sample type was submitted to the assay in biological triplicate. results: when comparing all samples, eisen cluster analysis separated all the cells and all evs into two groups, regardless of their phenotypes. in subgroup analysis, the expression patterns between pc -epi and pc -emt cells were significantly different. clec b, kdr, crip , il ra , cc d b were significantly upregulated in pc -emt cells, while cxcl , epcam, esrp , tgfb , cdh , s a , ovol were significantly downregulated in pc -emt cells. the expression patterns between pc -epi and pc -emt evs were also significantly different. tbx , cav , col a , slc a , myc, itgb , timp , camk b, ptgds, p h , itgb , vim, stat were all significantly downregulated in pc -emt cell derived evs. summary/conclusion: the nanostring low rna input ncounter assay can provide reliable mrna expression profiling of evs. the mrna expression patterns are very different between cells and their daughter evs. both cells and evs with different phenotypes have different gene expressions. cancer cell-derived evs containing alphav beta integrin regulate cd , il- and il- levels in peripheral blood mononuclear cells introduction: extracellular vesicles (evs) mediate communication in the tumour microenvironment and play an important role in cancer progression. previously, we have shown the enrichment of alphav beta integrin in small extracellular vesicles (sevs) isolated by differential ultracentrifugation and iodixanol density gradient from pc prostate cancer cells. we have also shown in the past that alphav beta -positive sevs induce peripheral blood mononuclear cell (pbmc) polarization by increasing the expression of pro-tumorigenic m markers, such as cd and cd . finally, we have demonstrated that down-regulation of alphav beta integrin up-regulates the stat -interferon stimulated genes (isgs) pathway in cancer cells and in sevs released by them. methods: in order to investigate whether prostate cancer cell-derived vesicular stat has a causal effect in pbmc polarization, we down-regulated alphav beta and stat in prostate cancer cells derived sevs using sirna as well as crispr-cas strategies. the sevs isolated from these cells were used to analyse m polarization by measuring the levels of cd in pbmc. the results show that sevs lacking alphav beta inhibit cd levels in pbmc in a stat -independent manner. analysis of cytokines released by pbmc upon incubation with sevs lacking alphav beta , show that pbmc selectively up-regulate the levels of il- and il- , which are predominantly anti-tumorigenic cytokines. in contrast, sevs lacking alphav beta do not upregulate pro-angiogenic cytokines, such as vegf. summary/conclusion: these findings suggest that cancer cell-derived sevs containing alphav beta integrin promote a pro-tumorigenic pbmc phenotype in the tumour microenvironment by regulating cd , il- and il- levels. introduction: the recognition of donor-mhc molecules by recipient t cells triggers the immune response leading to rejection of allografts. our recent studies have documented the presence of high numbers of recipient apcs displaying donor-mhc molecules (cross-dressed) on their surface in the lymphoid organs of mice after skin, heart or pancreatic islet transplantation. in addition, we have reported that acquisition of allogeneic mhc molecules by host apcs (mhc crossdressing) is mediated by donor-derived extracellular vesicles (evs) trafficking through blood and lymphatic vessels (marino et al. science immunology, ) . in the present study, we investigated the ability of allogeneic evs and allo-mhc-cross-dressed cells to initiate a t cell alloresponse in vitro and in vivo. methods: evs were isolated (using differential centrifugation) from balb/c bone marrow derived dendritic cells (bmdcs). these evs were used to cross-dress b splenocytes in vitro. the transfer of donor mhc class i and ii on b cells was analysed by imaging flow cytometry. next, t cells from b mice were cultured in vitro with either allogeneic bmdc-derived balb/c evs or b spleen cells crossdressed with allogeneic balb/c mhc. alternatively, × balb/c or b bm derived evs or × balb/c bm cells were injected iv to b mice. in both cases, the t cell response was assessed by activation markers detection, infg production and cell proliferation. results: apcs cross-dressed with allogeneic mhc molecules can trigger a pro-inflammatory direct alloresponse by t cells in vitro and in vivo. on the other hand, allogeneic evs alone were only able to induce early t cell activation but not proliferation in vitro. furthermore, injection of mice with allogeneic evs alone could induce some but suboptimal alloresponse in vivo and only when administered with complete freund's adjuvant. summary/conclusion: blocking donor evs release and subsequent recipient apc cross-dressing may represent a promising target to selectively inhibit anti-donor t cell inflammatory responses thus achieving long-term allograft survival. funding: r dk . antifungal antibiotic activity of outer membrane vesicles from adherent lysobacter enzymogenes c against therapeutic and biocontrol targets. rutgers university, new brunswick, usa introduction: lysobacter enzymogenes is a predatory gram negative bacterial species being studied for biocontrol activity against fungi. planktonic l. enzymogenes c produces outer membrane vesicles (omv) harbouring small molecule antifungal antibiotics (meers et al. ) . we show here that the more biologically relevant surface-associated c exerts remote antifungal activity via omv as well. the results have important consequences regarding the natural mechanism of biocontrol of fungal pathogens by c as well as isolation and delivery of therapeutically relevant antifungal compounds. methods: omv were isolated from scraped adherent c culture on agar by similar methods to meers et al . omv were stained in some cases with fluorogenic syto dna stain for microscopic observation. fungal growth was monitored via turbidity readings in liquid culture or photomicrographs on agar. c was also grown on polycarbonate filter membranes with defined pore sizes to monitor growth of fungal cells on the opposite side. vesicles were also labelled with an amine-reactive probe alexa- and washed x by sedimentation. binding of labelled omv to fungal cells was observed by epifluorescence microscopy. results: syto -stained vesicles from surface-adherent c were similar to previously observed~ nm vesicles (meers et al., ) . the isolated vesicles inhibited growth of saccharomyces cerevisiae or candida albicans in liquid cultures at similar potency and were active against the filamentous species fusarium subglutinans grown on agar or maize leaves. c cultures grown on filters with nm pore size but not nm were able to inhibit the hyphal growth of f. subglutinans on the opposite side. similarly c on filters with a nm pore size were able to inhibit growth of c. albicans. observation of fluorescently-labelled c omv after interaction with c. albicans showed binding specifically to hyphae or pseudohyphae and for f. subglutinans to the growing hyphal tips. summary/conclusion: the omv of c specifically bind and inhibit the growth of fungal hyphae of various species without direct c cell contact. these data elucidate mechanisms of biocontrol and suggest strategies for production of therapeutic antifungal antibiotics. meers et al. elucidating the cellular uptake and tissue distribution mechanism of cell derived vesicles, a novel therapeutic carrier hui-chong lau a , jae young kim a , jin-hee park a , jun-sik yoon a , min jung kang a and seung wook oh b a mdimune inc, seoul, republic of korea; b mdimune inc, seattle, usa introduction: cell derived vesicles (cdvs) are emerging as a novel therapeutic carrier. one of the crucial factors in the development and therapeutic applications of cdvs is to understand the precise mechanism by which vesicles find and enter the target cells. in this study, we aim to investigate the uptake mechanism of cdvs produced from natural killer (nk) cells using a manufacturing process established at mdimune inc. both in vitro uptake assay and in vivo distribution analysis were performed to provide precise insights into how cdv exert its effect at the cellular level. methods: nk cells were mainly used to produce cdvs. breast cancer cells, bt , and human and rodent endothelial cells, with a varying degree of icam- expression, were used to determine the effect of lfa- expressed on the surface of nk-cdvs in cellular uptake using facs and confocal imaging analysis. next, various inhibitors for uptake pathways, such as phagocytosis, dynamin dependent endocytosis, and receptor mediated endocytosis, were used to understand the underlying mechanism of cellular uptake of cdvs. biodistribution profile of cdvs were characterized using both normal and tumour xenograft models by ivis imaging. results: using a recently established manufacturing process, we demonstrate that nk-cdvs can efficiently enter the target cells. this study also shows that the cellular uptake depends on the molecular interaction between icam- and lfa- . in vivo distribution profile of nk-cdvs are also assessed using various tumour models. furthermore, we present a cellular uptake mechanism involved in the entrance of cdvs into the target cells. summary/conclusion: this study demonstrates that the cdvs produced at the manufacturing scale can be easily taken up by cells via specific cellular pathways. this finding will facilitate the development of more efficient therapeutics for cancer and other debilitating diseases. myofibroblasts-derived microvesicles increase dermal fibroblasts collagen production through plgf- syrine arif, sebastien larochelle and véronique j. moulin chu de québec -université laval, loex, québec, canada introduction: a proper wound healing of the skin involves angiogenesis, extracellular matrix (ecm) remodelling and re-epithelialization. these three mechanisms require well-organized interactions between different cell populations. a key role in this context is played by myofibroblasts (wmyo), a cell population mainly differentiated from dermal fibroblasts. these cells contract wound edges and synthesize new ecm. we previously showed that myofibroblasts predominantly produces microvesicles (mvs) and can favour angiogenesis. however, proteomic analysis of mvs from our previous studies indicated some molecules that can potentially be implicated in ecm remodelling. in this study, we evaluated whether myofibroblasts-derived mvs could affect dermal fibroblasts who are highly responsible for ecm regulation. methods: mvs were isolated by differential centrifugation of medium collected from wmyo cells. number and size of mvs were characterized by transmission electron microscopy and nanosizer. multiplex assays of cytokines were evaluated in mvs samples, wmyo and mvs-depleted medium. to examine the interaction of mvs with fibroblasts, we evaluated the uptake of mvs isolated from wmyo transduced with a fluorescent protein. we then treated fibroblasts cultures with mvs or a selected cytokine for days and evaluated collagen production. lastly, we neutralized the selected cytokine in mvs samples before evaluating collagen production. results: plgf- was the cytokine detected in mvs samples in large amount ( . ± . pg/µg proteins in mvs). fibroblasts treated with mvs or plgf- significantly stimulated pro-collagen i level production with a fold change of . ± . and . ± . . moreover, the neutralization of plgf- present in mvs significantly inhibited the production of pro-collagen i by dermal fibroblasts. summary/conclusion: our results indicated that mvs influence fibroblasts pro-collagen production through plgf- signalling. funding: this work was supported by natural sciences and engineering research council of canada (nserc) (rgpin - ); les fonds de recherche du québec-santé (frqs) via the research centre funding grant; the quebec cell and tissue and gene therapy network-thécell (a thematic network supported by frqs). structural insights on fusion mechanisms of extracellular vesicles with model plasma membranes introduction: extracellular vesicles (evs) represent a potent intercellular communication system. while their functional biological properties are more and more investigated, the biophysical aspects of their interaction with recipient cells are often overlooked. small size ( to a few hundred nanometres in diameter) of evs and their heterogeneous origin still pose a great challenge for their isolation, quantification and biophysical/biochemical characterization. in particular the complex network of interactions between differently classified evs and recipient cells remains to be further revealed. here we deeply investigate the fusion mechanism between evs and a model plasma membrane system by an interplay of different structural/morphological techniques to get a molecular description of the interaction helping to clarify the role of different membrane compartments on the evs uptake mechanism. standardized protocols and good manufacturing practice conditions were employed to derive highly stable vesicles of defined size and reproducible molecular profiles from umbilical cord multipotent mesenchymal stem (stromal) cells. after a thorough biophysical and biochemical characterization of evs non-contact liquid imaging atomic force microscopy (afm) and, in parallel, neutron reflectometry (nr), as well as small angle neutron scattering (sans) experiments were performed on evs to determine their interaction with model plasma membranes in the form both of supported lipid bilayers and suspended unilamellar vesicles of variably complex composition. results: we observed that evs tend to fuse with the model membranes with a preferential interaction with the external layer of the fluid membrane. moreover we revealed a stronger interaction with the liquid ordered domains, strengthening the hypothesis of a critical role of lipid rafts in fusion mechanisms. summary/conclusion: our results on the analysis of the interaction of evs with artificial lipid membranes could provide insights on the internalization mechanisms of evs. the approach shown here can be further extended to convey incremental complexity, adding glycolipid and membrane proteins to the model lipid bilayers. this approach combined with data on the specific biological function of each ev subpopulation as retrieved by standard functional assays, will turn useful to select the crucial molecular aspects of evs internalization by cells. introduction: platelet-derived extracellular vesicles (pev) are the most abundant circulating extracellular vesicle (ev) and exhibit platelet-like properties, hence the original term "platelet dust". direct phenotyping of ev surface markers within biofluids is challenging often requiring time-intensive purification steps that can significantly alter resultant ev population characteristics. the exoview™ (nanoview biosciences) specifically captures ev sub-populations and was used to characterise the ev content of platelet free plasma (pfp) and a potential novel haemostatic agent designed for the treatment of severe trauma and haemorrhage, platelet enhanced plasma (pep). methods: freeze-thaw cycling of platelet rich plasma/ expired platelet concentrates was followed by centrifugation to remove platelet remnants and yeilded pep. pfp controls were prepared by double centrifugation ( g for minuntes followed by , g for minutes). rotational thromboelastometry (rotem) and calibrated automated thrombography (cat) were used to assess ev driven haemostasis and thrombin generation. a dilutional and hypothermic model of coagulopathy was designed to assess pep. ev capture arrays comprised of anti-cd , anti-cd , anti-cd and anti-cd were used (exoview™, nanoview biosciences). captured vesicles underewent interferometric imaging and were quantified, sized and further probed with fluorescent tetraspanin markers, annexin-v and intravesicular markers. results: pep is highly procoagulant, exhibits enhanced thrombin generation and can restore haemostasis in a dilutional model of coagulopatic whole blood. pep can be generated from expired platelet concentrates, potentially allowing for upscalable production. the predominant vesicle population were pev with a large cd /cd population that contained a smaller subpopulation of phosphatidyserine positive procoagulant vesicles. pfp as expected has a much lower number of pev and a cd positive ev population. summary/conclusion: pep is a unique resuscitation fluid containing high pev levels for the potential treatment of severe trauma and haemorrhage. exoview measurements can be performed in unpurified plasma and may be useful for measuring circulating ev in health and disease. funding: defence and security accelerator, dstl therapeutic effect of exosomes in mice model of autism daniel offen a , reut horev a , nisim perets a , ehud marom b , uri danon b and yona gefen b a tel aviv university, tel aviv, israel; b stem cell medicine ltd., jerusalem, israel introduction: during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. we have previously shown that intranasal administration of msc-exo, cross the bbb and significantly ameliorate autistic-like behavioural phenotype in btbr and shank animal models of autism, representing a potential therapeutic strategy to reduce symptoms of autism spectrum disorder (asd). our objective is to study the mechanism of action and the cellular pathways in which the msc-exo activate their target, we performed rna sequencing analysis of primary neurons isolated from shank mice treated with msc-exo. methods: primary neuronal cell cultures were prepared from newborn shank homozygotes mice model of autism. cultures were treated with msc-exo ( ^ particles/ul), isolated from human adipocytes, followed by rna sequencing. the alterations in gene expression between the treated and intact neurons were analysed for gene ontology and pathways and were also compared to proteomics analysis of the msc-exo in order to find regulatory proteins that may lead to these differences. results: bioinformatic analysis revealed several up-regulators proteins that might be responsible for the increase in anti-inflammatory and protective factors seen in the mice neurons treated with msc-exo. one of them is bdnf which is known as an essential growth factor responsible for neuroprotection and neurogenesis. importantly, no difference in the genetic expression of cancer-related genes was identified following msc-exo treatment indicating for their safety. summary/conclusion: our data suggest that adipocytederived msc-exo carry therapeutic potential in asd via alternation in gene-expression related mainly to immuno-modulation, reduce neuroinflammation and increase neuroprotection and neurogenesis. the beneficial effects of the exosomes treatment in mice models is being translated into a novel, easy to administer, a therapeutic strategy to reduce the symptoms of asd. introduction: autologous blood-derived products gain increasing focus in regenerative medicine, especially in orthopaedics and osteoarthritis therapy. this disease is characterised by cartilage degradation and inflammation among other symptoms, which are targeted by conventional therapies, but genuine cartilage regeneration is rarely achieved. citrate-anticoagulated platelet rich plasma (cprp) is often clinically applied to stimulate soft and hard tissue healing. recently, cell-free alternatives to cprp including hyperacute serum (hypact™ serum) have been developed. cprp and hypact™ serum contain specific profiles of growth factors, however, they also contain extracellular vesicles (evs) that harbour signal molecules including mirna. methods: evs were enriched by ultracentrifugation (uc) followed by size exclusion chromatography (sec) to obtain purified evs. particle size and concentration of each fraction was measured by nanoparticle tracking analysis (nta). fractions with the highest amount of particles were pooled and concentrated via uc, before mirna expression was assessed via screening with a panel of mirna-specific primer pairs by rt-qpcr. presence of evs was confirmed by cryoelectron microscopy. results: the ev concentration tended to be lower in hypact™ serum than in cprp as determined via nta. similarly, lower diversity of mirna species was found in hypact™ serum than cprp evs. around % of detected mirnas were found in both blood products, whereas only % of mirnas were shared between evs from cprp and hypact™ serum. while mirnas such as mir- were consistently depleted in evs compared to the corresponding blood product, others like mir- a were in enriched in hypact™ evs, but not cprp evs, indicating release of specific mirnas via evs in response to clotting. summary/conclusion: although the purification resulted in high loss of evs, we identified specific mirnas enriched in evs from cprp and hypact™ serum. their functional spectrum with respect to osteoarthritis therapy focuses on inhibition of inflammation, inhibition of tissue remodelling via matrix degrading enzymes as well as preventing senescence. this renders blood product derived evs as interesting candidates for in vitro and in vivo testing with respect to cartilage regeneration. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst -f- / - . protective role of shiitake mushroom-derived exosome-like nanoparticles in d-galactosamine and lipopolysaccharide-induced acute liver injury in mice baolong liu, xingyi chen and jiujiu yu university of nebraska lincoln, lincoln, usa introduction: fulminant hepatic failure (fhf) is a rare, life-threatening liver disease with poor prognosis. new therapeutic interventions are urgently needed to treat this disease. administration of d-galactosamine (galn) and a low dose of lipopolysaccharide (lps) triggers acute liver damage in mice, which simulates many clinical features of fhf in humans and therefore is widely used to investigate the molecular mechanisms and potential therapeutic interventions of fhf. recently, suppression of the nucleotide binding domain and leucine rich repeat related (nlr) family, pyrin domain containing (nlrp ) inflammasome was shown to alleviate the severity of lps/galn-induced liver injury in animal models. therefore, the goal of this study was to identify food-derived exosome-like nanoparticles (elns) with anti-nlrp inflammasome function to potentially control fhf. methods: seven commonly consumed mushrooms were used to extract elns, which were examined for anti-nlrp inflammasome activities in primary macrophages. results: it was found that these mushrooms contained elns composed of biomolecules including rnas, proteins, and lipids. among these mushroom-derived elns, only shiitake mushroom-derived elns (s-elns) strongly inhibited nlrp inflammasome activation by blocking the inflammasome assembly. this inhibitory effect was specific for the nlrp inflammasome because s-elns had no impact on activation of the absent in melanoma (aim ) inflammasome. s-elns also inhibited the secretion of interleukin (il)- and both protein and mrna levels of the il b gene in macrophages. remarkably, pre-treatment of s-elns protected mice from lps/galn-induced acute liver injury. summary/conclusion: therefore, s-elns, identified as potent inhibitors of the nlrp inflammasome, represent a new class of agents with the potential to combat fhf. approaches to assess clinically available exosomes' quality and safety introduction: recent adverse events resultant from an exosome product use in a nebraska clinic, highlight the importance of assuring product quality and safety standards. an often-overlooked safety risk is ancillary reagents remaining within a finished product. when processes to obtain exosomes utilize cow proteins such as fbs or bovine sera albumin, failure to adequately remove these can result in significant adverse allergic reactions. we evaluated different exosome products to test the hypothesis that purity of some products may not be consistent with actual product quality and safety profiles claimed. methods: three different exosome products (manufacturer a, b, and c) were prepared per their instructions for use. sample source identity was blinded from assaying scientists. an independent cro service was used to conduct the experiments to ensure unbiased assay execution and data collection. exosome suspensions were sampled undiluted for bovine protein content using commercially available bovine secretome protein arrays from ray biotech. a total of different proteins found in bovine serum were quantified. results: six of proteins were not detected in any sample. of array antibodies were found to cross react with human antigens. of the bovine proteins that were acceptable for analysis, manufacturers a, b, and c exosomes contained of proteins, of proteins, and of proteins, respectively. concentrations of individual bovine proteins ranged from . to , . ng/ml. summary/conclusion: these results indicate manufactures a and b are selling potentially dangerous products. the successful implementation of exosome products into the clinic requires equivalent demonstrations of safety and quality. this requires adopting strict quality standards and safety testing during their production. physicians must require safety data prior to clinical use. engineering pro-healing ev cargo using a closed-system bioreactor. introduction: chronic wounds, including diabetic ulcers and pressure ulcers, are difficult and expensive to treat. while tissue engineering approaches have largely failed as a viable treatment for chronic wounds, we hypothesize that stem cell-derived extracellular vesicles (evs) may provide several unique advantages. zenbio, inc has developed a methodology to generate commercial-scale stem cell-derived exosomes using a closedsystem hollow fibre bioreactor capable of continuous ev production. additionally, we have shown that by manipulating the cellular environment, we can improve the pro-healing capacity of the evs.this technology leverages the complex healing capabilities of stem cells without the obstacles of replicating cells. methods: we have demonstrated that a mild heat shock resulted in evs enriched for stress-response proteins and increased pro-healing activities in vitro. we extended this innovative approach to include stimulating adipose stem cells with combinations of heat shock and growth factors to generate differential extracellular vesicle packaging that enhances pro-healing activity. to monitor reproducibility across lots and batches, we rigorously characterized tuned evs for particle size and number as well as surface marker and cargo composition. results: our results using tuned evs showed efficacy using cellular models of inflammation, motility, vascularization, collagen production and metalloprotease activity. we utilized an established murine model of pressure ulcers to assess the in vivo efficacy of the tuned evs. these studies showed a single injection into the wound site activated a more rapid wound closure, increased collagen deposition and reduced dermal thickness compared to saline control. summary/conclusion: these data strongly support our hypothesis that evs may be selectively modified to improve their wound healing activity by modulating the culture or tissue microenvironment. future studies will use chronic wound models to determine optimal dosing and routes of administration. introduction: mesenchymal stem cell-derived extracellular vesicles (msc-evs) can reduce inflammation, promote healing and improve organ function thereby providing a potential "cell-free" therapy. prior to clinical translation, there is a critical need to synthesize existing preclinical evidence supporting their efficacy. this systematic review provides the most comprehensive evidence map of methods, safety and efficacy for msc-ev research to date. methods: medline and embase were systematically searched for in vivo interventional studies using msc-evs. two reviewers extracted data for: ) methodology, ) study design, ) intervention details and ) efficacy/ adverse events. results: after screening articles, studies met our eligibility criteria. msc-evs were used to treat a variety of diseases including renal ( %), neurological ( %) and cardiac ( %) conditions. benefits were described in % of studies across all organ systems and adverse effects were seen in only three studies; two showing tumour growth. however, several key methodological concerns were evident. based on size criteria for ev subtypes (exosomes/small evs~ - nm, microvesicles~ - nm) only % of studies used appropriate nomenclature. ultracentrifugation ( %) and isolation kits ( %) were the most common isolation methods despite marked differences in yield and purity. evs were inconsistently dosed by protein ( %), particle number ( %) or cell count ( %), hindering inter-study comparisons. two-thirds of studies used xenogeneic evs suggesting immunocompatibility. techniques to determine size, protein markers and morphology was highly heterogeneous, and only and studies met the characterization standards recommended in the misev and guidelines, respectively. finally, % of studies did not incorporate randomization which represents a high risk for bias and only a quarter performed biodistribution studies. summary/conclusion: this systematic review reveals extensive heterogeneity in methods and intervention details for animal studies of msc-evs. nonetheless, nearly all studies showed significant benefits in a wide range of distinct conditions. the knowledge gaps we identified highlight important opportunities for improving preclinical design and the need for more standardized approaches in this growing field of ev therapeutics. msc-exosomes as next generation therapeutics for atopic dermatitis exocobio inc, seoul, republic of korea introduction: atopic dermatitis (ad) is a systemic inflammatory disease with unknown cause. recent approval of a targeted therapy, dupilumab, opens new era of ad management. however, current therapeutic options for ad are only targeting inflammation, a component of ad vicious cycle including itching and barrier disruption. human mesenchymal stem cells (mscs) have been highlighted as a novel therapy for suppressing allergic progress of ad in clinical studies. unfortunately, phase iii clinical study of human umbilical cord blood mscs for ad was failed with unknown reason. previously, our group reported that exosomes derived from human adipose tissue-derived mscs (asc-exosomes) alleviated the pathological symptoms in a murine ad model with concomitant reduction of inflammation. methods: our group has further investigated the therapeutic effects of human asc-exosomes in an alternative murine ad model with skin barrier defects. large scale isolation of asc-exosomes was performed by tangential flow filtration and isolated asc-exosomes were characterized according to the recommendation by the isev. the protein and lipid cargo were also analysed. results: we found that asc-exosomes induced restoration of skin barrier by inducing de novo lipid synthesis and reduced the levels of multiple inflammatory cytokines. in addition, asc-exosomes suppressed the expression of itching-causing cytokines. transcriptomic analysis of ad skin lesions revealed that asc-exosomes reversed the abnormal expression of genes functioning in skin barrier function, lipid metabolism, and cell cycle. summary/conclusion: taken together, asc-exosomes could be a promising cell-free therapeutic option for the treatment of ad, which affecting inflammation, skin barrier function, and itching. cell derived vesicles: unravelling the science of novel vesicles with therapeutic promises introduction: cell derived vesicles (cdvs) are nanosized vesicles produced by serially extruding cells through small pores. a growing number of studies have implicated their therapeutic potentials, with superior yield compared to other extracellular vesicles (evs). however, two key objectives remain to be accomplished to demonstrate the utility of cdvs in clinical applications. first, a manufacturing process has to be developed to allow a large-scale production of cdvs. next, these novel vesicles need to be thoroughly characterized at multiple levels. methods: manufacturing-scale extruders were developed to allow extrusion of large volume of cell suspension in a single process. cdvs with approximately - nm in diameter were obtained by a serial extrusion. crude samples were then purified using the tangential flow filtration method to further remove cellular impurities. finally, physical and biochemical characteristics of purified cdvs were analysed using dls, nta, cryo-em, and facs analysis. additionally, cdvs were subject to multi-omics profiling to comprehend our understanding in molecular contents of cdvs. both mesenchymal stem cells (mscs) and natural killer (nk) cells were used for this study. results: in this study, we first demonstrate that the large-scale extruder efficiently produce cdvs with consistent quality at the scale that are compatible for clinical applications. surface marker and membrane composition analyses show that the cdvs are primarily formed using plasma membrane of source cells, with characteristic cellular markers enriched on the surface. comprehensive profiling of molecular components reveals the unique properties of cdvs as well as the underlying mechanism of formation of cdvs. summary/conclusion: recently, we have established a manufacturing process to enable clinical applications of cdvs. this study also highlights key molecular features of cdvs that can be harnessed to offer a powerful tool for regenerative and anticancer medicine. antifibrotic properties of extracellular vesicles derived from human induced pluripotent stem cells introduction: fibrosis is a pathological condition resulting from abnormal healing of various tissues. it is triggered by activation of fibroblasts and their subsequent transition to myofibroblast. in consequence, excessive deposition of extracellular matrix proteins leads to impaired organ function. to revert this process, we employed extracellular vesicles (evs) derived from human induced pluripotent stem cells (hipscs). as a model system, we used human cardiac fibroblasts (hcfs), since heart fibrosis constitutes a serious socioeconomic problem worldwide. methods: we isolated evs from conditioned media from three hipsc lines using ultrafiltration combined with size exclusion chromatography methods. next, we analysed the evs by nanosight, transmission electron microscopy, mass spectrometry and western blot methods. finally, we treated tgf-b-stimulated hcfs with hipsc-evs and evaluated expression of fibrosisrelated genes using real-time qpcr, western blot and fluorescence microscopy. results: we detected anti-fibrotic properties of hipsc-evs exerted on hcfs pre-stimulated with tgf-b. the evs significantly decreased the expression levels of acta , fn, tnc, snai , col a and reduced the number of myofibroblasts. the canonical profibrotic tgf-b-dependent smad / pathway was significantly attenuated in response to ev-treatment. summary/conclusion: in this study we demonstrated strong anti-fibrotic function of hipsc-evs. our findings can further be exploited for future medical applications to treat fibrotic diseases, such as heart fibrosis. funding: this work was supported by the project sonata : umo- / /d/nz / from the national science centre of poland to sbw. induced pluripotent stem cells-derived extracellular vesicles ameliorates d-galactosamine and lipopolysaccharide induced acute liver failure tianjin third central hospital affiliated to nankai university, tianjin, china (people's republic) introduction: liver failure is among the most causes of death in patients with liver disease. promoting liver regeneration will help patients with liver failure recover on their own. extracellular vesicles (evs) can released by induced pluripotent stem cells (ipscs) through paracrine effects and play a pivotal role in inter-cellular communication in the treatment of disease. in this study, we investigated whether the ipscs-evs have therapeutic effects on acute liver failure. methods: the ipscs-evs were isolated by ultracentrifugation and identified using nanoparticle tracking analysis, transmission electron microscopy and western blotting. the isolated ipscs-evs were administrated d-galactosamine-injured heprg cells in vitro and tail intravenously injected into d-galactosamine and lipopolysaccharide induced acute liver failure model mice in vivo, respectively. the anti-apoptosis role and potential mechanism were evaluated using flow cytometry and immunofluorescence staining. and alanine transaminase (alt) and aspartate transaminase (ast) in serum, h&e staining and tunel staining were explored the effect of ipscs-evs on liver injured and liver function. finally, high throughput sequencing of small rnas was performed to investigate mirna expression profiles in ipscs-evs and ipscs. results: the ipscs-evs that were all - nm, doublelayered and oval or round cellular vesicles and expressed the marker proteins cd , tsg and hsp . in vitro, the ipscs-evs treatment inhibited heprg apoptosis induced with d-galactosamine in a time-and dosedependent manner and promote the proliferation of hepatic stem cells. in vivo results showed that ipscs-evs significantly alleviated liver failure, improved liver function and prolonged the survival period. tunel assay showed that ipscs-evs suppress apoptosis of hepatocytes. moreover, mirna expression profiles analysis found that mir - a cluster and mir - cluster were enriched in ipscs-evs and ipscs. summary/conclusion: these findings indicated that ipscs-evs could ameliorate d-galactosamine and lipopolysaccharide induced acute liver failure to attenuate hepatocyte apoptosis, which will be benefit for therapy of liver disease in the future. msc-derived extracellular vesicles promote human cartilage regeneration by control of autophagy introduction: osteoarthritis (oa) is a rheumatic disease leading to chronic pain and disability with no effective treatment available. recently, allogeneic human mesenchymal stromal/stem cells (msc) entered clinical trials as a novel therapy for oa. increasing evidence suggests that therapeutic efficacy of msc depends on paracrine signalling. here we investigated the role of bone marrow msc-derived extracellular vesicles (bmmsc-evs), an important component of msc secretome, in cartilage repair. methods: to test the effect of bmmsc-evs on oa cartilage inflammation the tnf-alpha-stimulated human oa chondrocytes were treated with bmmsc-evs and inflammatory gene expression was measured by qrt-pcr after h. to access the impact of bmmsc-evs on cartilage regeneration the bmmsc-evs were added to the regeneration cultures of oa chondrocytes, which were analysed after weeks for glycosaminoglycan content by dmmb and qrt-pcr. paraffin sections of the regenerated tissue were stained for proteoglycans (safranin-o) and type ii collagen (immunostaining). results: we show that bmmsc-evs promote cartilage regeneration in vitro. treatment of oa chondrocytes with bmmsc-evs induces production of proteoglycans and type ii collagen and promotes proliferation of these cells. msc-evs also inhibit the adverse effects of inflammatory mediators on cartilage homoeostasis. our data show that bmmsc-evs downregulate tnfalpha induced expression of pro-inflammatory cox- , pro-inflammatory interleukins and collagenase activity in oa chondrocytes. the anti-inflammatory effect of bmmsc-evs involves the inhibition of nfκb signalling, activation of which is an important component of oa pathology. autophagy, a cellular homoeostatic mechanism for the removal of dysfunctional cellular organelles and macromolecules, is essential to maintaining chondrocytes survival and differentiation. the expression of autophagy regulators is reduced in osteoarthritic joints, which is also accompanied by increased chondrocyte apoptosis. our preliminary data indicate that bmmsc-evs carry mrna of natural autophagy inducers and promote autophagy in oa chondrocytes. therefore, we hypothesize that msc-evs exert their beneficial effects on cartilage regeneration by restoring the expression of autophagy regulators. summary/conclusion: in summary, our findings indicate that bmmsc-evs have ability to promote oa cartilage repair by reducing the inflammatory response and stimulation of oa chondrocytes to produce extracellular matrix, the essential processes for restoring and maintaining cartilage homoeostasis. thus, msc-evs hold great promise as a novel therapeutic for cartilage regeneration and osteoarthritis. large-scale preparations of small extracellular vesicles from conditioned media of mesenchymal stromal cells modulate therapeutic impacts on a newly established graft-versus-host-disease model in batch dependent manners introduction: extracellular vesicles (evs) harvested from supernatants of humane adult bone marrow-derived mesenchymal stem/stromal cells (mscs) can suppress acute inflammatory cues in a variety of different diseases, including graft-versus-host disease (gvhd) and ischaemic stroke. furthermore, they can promote regeneration of affected tissues. following a successful clinical treatment attempt of a steroid refractory gvhd patient, we intend to optimize msc-ev production strategies for further clinical applications. as we observed functional differences of independent msc-ev preparations in vitro, we aimed to adopt an in vivo gvhd model for the more advanced functional testing of different msc-ev preparations. methods: to this end we set up a bone marrow transplantation mouse model in which endogenous bone marrow was myeloablated by ionizing irradiation (iir). gvhd was induced by the transplantation of major histocompatibility mismatched allogeneic spleen-derived murine t cells. if not treated otherwise, myeloablated mice developed severe gvhd symptoms. results: the gvhd symptoms were effectively suppressed, when msc-ev preparations were applied at consecutive days, which exerted immune modulatory effects in a mixed-lymphocyte reaction assay. msc-ev preparations lacking in vitro immune modulating activities, however, hardly improved the symptoms of the gvhd mice. thus, our results demonstrate that not all msc-ev preparations harvested from adult bone marrow-derived mscs contain the same therapeutic potential. summary/conclusion: thus, successful transplantation of msc-evs into the clinics requires a platform allowing identification of msc-ev preparations with sufficient therapeutic, most probably immune modulating activities. funding: this research was funded by sevrit leitmarkt lifescience.nrw ls- - - g. introduction: malnutrition impacts approximately million children worldwide and is linked to % of global mortality in children below the age of five. severe acute malnutrition (sam) is associated with intestinal barrier breakdown and epithelial atrophy. extracellular vesicles including exosomes (evs; - nm) can travel to distant target cells through biofluids including milk. since milk-derived evs are known to induce intestinal stem cell proliferation, this study aimed to examine their potential efficacy in improving malnutrition-induced atrophy of intestinal mucosa and barrier dysfunction. methods: mice were fed either a control ( %) or a low protein ( %) diet for days to induce malnutrition. from day to , they received either bovine milk evs enriched using differential ultracentrifugation and sucrose gradient purification or control gavage and were sacrificed on day , hours after a fluorescein isothiocyanate (fitc) dose. tissue and blood were collected for histological and epithelial barrier function analyses. results: mice fed low protein diet developed intestinal villus atrophy and barrier dysfunction. despite continued low protein diet feeding, milk ev administration improved intestinal permeability, intestinal architecture and cellular proliferation. summary/conclusion: our results suggest that evs enriched from milk should be further explored as a valuable adjuvant therapy to standard clinical management of malnourished children with high risk of morbidity and mortality. funding: cb was generously awarded a catalyst grant from the centre for global child health at the hospital for sick children to support this work. the impact of spheroids culture on mesenchymal stem cells and ev production introduction: mesenchymal stem/stromal cells (mscs) are now widely believed as bio-factories releasing bioactive products responsible for their therapeutic effect, i.e. cytokines, chemokines, and extracellular vesicles (evs). mscs are highly sensitive to physical stimuli from their surrounding microenvironment and can change their characteristics in response to their environment. the application of d spheroids cell culture allows mscs to adapt to their cellular niche environment which, in turn, influences their paracrine signalling activity. we aim to determine how d and d culture microenvironments can modulate the ev production and investigate their anti-fibrotic activity. methods: for d culture, bone marrow-derived mscs were cultured on standard tissue culture plastic. for d culture, mscs were aggregated into spheroids using non-adherent -well plates and cultured with addition of . % methylcellulose. to collect conditioned media, both d and d mscs were cultured using serum free medium for days. evs were isolated by serial ultracentrifugation and were characterised on exoview platform which allows simultaneous detection of particle size and expression of cd /cd /cd . cell lysates were collected for mirna isolation and qrt-pcr was performed to analyse expression of candidate mirnas. to model the progress of lung fibrosis, human lung fibroblasts (hlfs) were cultured with tgf-β to induce fibroblast activation, subsequently exposed to d and d evs, and collagen production was measured. further, d and d msc-evs were added into human lung mscs isolated from healthy and ipf patients and cell proliferation was assessed using mts assay. results: d and d msc-evs have similar ev characteristics in terms of particle size and ev tetraspanin markers expression. exoview analysis showed expressions of cd /cd /cd and average particle diameters of < nm. on a cellular level, we identified a panel of anti/pro-fibrotic mirnas which are differentially expressed in d and d mscs. d and d msc-evs have similar anti-fibrotic activity shown by their ability to reduce collagen deposition in hlf cultures. both d and d msc-evs could promote cell proliferation on ipf lung mscs but no overall effect on healthy lung mscs. summary/conclusion: this concept of engineering the cellular microenvironment to promote ev production is as yet untouched and we foresee that in d cultures, we can culture mscs for longer timeframe and therefore maximising the overall ev production process. the outcome presents future potential for d culture of msc to increase the efficiency and feasibility of scalable ev production. outer membrane vesicles from photobacterium damselae subsp. piscicida: characterization and antigenic potential introduction: photobacterium damselae piscicida (phdp) is a gram-negative bacterium that causes a septicaemia in > fish species worldwide. it represents a major drawback for aquaculture, whose importance has been sharply growing as a food supplier. given the phdp massive mortality and widespread antibiotic resistance, an effective vaccine is highly needed. extracellular products (ecps) have an essential role in phdp virulence, containing important antigens. however, the ecps' identity remain undisclosed. in our efforts to dissect their composition, we found that they contain high amounts of outer membrane vesicles (omvs). these particles are potent weapons for bacteria and are being explored in the field of vaccinology, since omvs present antigens in native conformations and are strongly immunogenic, without requiring adjuvants. this potential associated to the urgent need for an anti-phdp vaccine prompted us to isolate and characterize the omvs shed by phdp. methods: in order to harvest high amounts of pure phdp omvs, a reproducible optimized protocol was developed: the bacteria-free supernatant from a phdp overnight culture is concentrated, dialysed and ultracentrifuged to collect the omvs. results: analysis of the obtained omvs preparations by transmission electronic microscopy and dynamic light scattering indicate that the main population of vesicles has sizes around - nm. proteomic analysis of the vesicles revealed the presence of the apoptogenic ab toxin aip that is known to play a major role in phdp virulence, a putative pore-forming toxin, a putative adhesin/invasin and several outer membrane proteins (omps), including a kda omp, predicted to be involved in iron acquisition, and other omps ( - kda), with an ompa-like structure that may act as adhesins. moreover, preliminary in vivo studies suggest that some of those proteins may have important roles for virulence, since injection of knock-out strains in sea bass induced a decreased mortality comparing to the wt strains. summary/conclusion: our findings suggest that omvs are a promising vaccine candidate and we are currently studying their biological activities and determining the antigenic potential of the identified proteins. introduction: whole body exposure to high doses of ionizing radiation (ir) can potentially be lethal if radiation injury is not diagnosed and treated expeditiously. when considering a non-invasive approach for the identification of biomarkers of ir exposure, we and others have studied molecules in plasma, serum, saliva, and urine. however, these matrices can potentially have significant background noise, obscuring potential biomarkers of biological importance. extracellular vesicles (evs) are fast becoming a platform for biomarker discovery in radiation research as well as in other pathologies. however, no groups have investigated the use of metabolomics to analyse evs derived from urine in the context of ir exposure. furthermore, the dominant protocols for ev isolation from urine require a large (up to ml) amount of starting volume, which may not be available for many studies. the aim of this study was to optimize ev isolation from rat urine and assess radiation-induced alterations in urine ev number and metabolic content. methods: as a proof of concept, we compared and optimized several ev isolation methods on small volumes of urine from male wag/rijcmcr rats exposed to gy or gy x-rays to the whole body except the hind leg. starting with either µl or µl of urine, we isolated evs using ultracentrifugation (uc) with filtration, size exclusion chromatography (sec), and a proprietary bead-based isolation method developed by a rd party provider. ev samples were characterized using nanoparticle tracking analysis. metabolomics profiles were measured using lc-qtof-ms. results: we found that sec resulted in the highest yield of evs from as little as µl of urine, while uc was the poorest performing. lc-qtof-ms analysis revealed that sec and uc had the most consistent identification of features, whereas the bead-based method contained artefacts likely as a result of the extraction method. we next used sec to isolate evs from a larger cohort of rats exposed to ir and analysed with ms. ev metabolic content will eb related to differences in survival and organ function between sham and irradiated groups. summary/conclusion: we conclude that sec is the preferred method for isolating evs from small volumes of urine for broad-based mass spectrometric analysis, and that the ev metabolome may be a sensitive and specific early indicator of radiation injury. introduction: there is growing evidence that contents (including rna and proteins) of exosomes may serve as biomarkers for early diagnosis and prognostic prediction of cancers. here we aim to identify potential protein markers for oesophageal cancer. methods: using our newly developed label-free exosome automated preparation system (leaps), exosomes were isolated from ml culture medium of various oesophageal cancer cells with different differentiation profiles and different sources of metastasis. exosomes from µl plasma of cancer patients at different clinical stages or with/without relapse and healthy controls were also prepared by leaps. matrix-assisted laser desorption ionization time-of-flight mass spectrometry (maldi-tof ms) was employed to directly analyse exosomes. protein identities of exosomal fingerprint peaks were tentatively assigned by correlation with top-down and bottom-up proteomics. results: start from ml culture medium or µl plasma, high-quality exosomes rapidly isolated by leaps are sufficient for maldi-tof mass spectrometry. it seemed that poorly differentiated cells showed more exosome release. maldi-tof ms fingerprints of exosomes in cells is cell line specific. ms profiles from poorly differentiated cells showed more peaks than that from highly differentiated cells. fingerprints also allowed classification of cancer cell lines through software mathematical analysis. we identified different numbers of significantly differentially expressed peaks in exosomes of various cancer cells. fingerprints of exosomes derived from the poorly differentiated cells showed more elevated peaks. top four peaks ( , m/z, , m/z, , m/z, , m/z) were commonly down-regulated in exosomes of most cancer cells. top four protein peaks ( , m/z, , m/z, , m/z, , m/z) that might be correlated to the differentiation profile of cancer cells were also identified. maldi-tof ms detection of exosomes in the plasma and clarifying identities of potential biomarker peaks will be done in the future. summary/conclusion: the combination of leaps and maldi-tof mass spectrometry provides a fast and high-throughput tool for exosomal marker discovery. potential biomarker identified in exosomes derived from oesophageal cancer cells or from plasma of cancer patients by this tool might be useful in cancer diagnosis and prognosis. fraction-based proteomic profiling of serum extracellular vesicles derived from cervical cancer patients introduction: current evidence indicates that extracellular vesicles (evs) can release from most of cell types and affect adjacent or distant cells by circulating in all bodily fluids. proteomic analysis of evs from clinical samples is complicated by the low abundance of ev proteins relative to highly abundant circulating proteins. size exclusion chromatography (sec) has been overcome as a method to deplete protein contaminants and enrich evs. methods: we collected serum of healthy women and cervical cancer patients with stage i-iii and then counted concentration and size distribution of the evs using nanoparticle tracking analysis (nta). differential ultracentrifugation combined with sec was used to isolate and purify evs from contaminant proteins. isolated evs were investigated their characteristic based on morphology using transmission electron microscope (tem) and on expression of cd , cd , cd protein markers using western blot analysis. fraction no. - of isolated evs in among sample groups were profiled by nano-liquid chromatography tandem mass spectrometry (nanolc-ms/ms) analysis. results: nta shows that the concentration of evs is increased in patients compared with healthy women. proteome profiles of evs isolated by sec were compared in each fraction. moreover, we detected molecular evidence for fraction-specific molecular pathways in connection with cancer progression and complied a set of protein signatures that closely reflect the associated clinical pathophysiology. summary/conclusion: these unique features in each fraction among sample groups would be the informative considering in order to select for further analysis as in vitro. introduction: recently, diagnostic biomarkers from exosomes by proteomic analysis have been reported, but it is required to optimize the isolation protocol to screen out more effective biomarkers. for serum-originated exosomes, it has been also reported to isolate them selectively, however, it is observed that a different method resulted in different protein profiles in -d gel electrophoresis. methods: we isolated exosomes by two discrete methods, using ultracentrifugation and magnetic separation. before ultracentrifugation and magnetic separation, precipitation using polymer materials was perforemd. the isolation of exosomes by these two methods followed by comparison of their size, total vesicle number, morphology, and protein markers. to identify protein biomarkers, proteomic analyis using -d gel electrophoresis was performed. results: both methods induced enrichment of exosome-specific proteins, but protein profiles in each exosome fraction was totally different. the protein profiles showd that the magnetic seperation following a polymer-based precipitation step was more efficient to screen out candidate biomarkers, which showed nearly protein profiles originated from exosomes. summary/conclusion: in our study, magnetic separation of exosomes from serum fraction was optimized for -d gel electrophoresis to observe identifiable biomarkers. an extracellular small rna-seq data processing pipeline optimized for high-performance computing chenghao zhu and angela zivkovic department of nutrition, uc davis, davis, usa introduction: a variety of rna species is found in extracellular biofluids such as blood, bile, and urine, carried by extracellular particles including extracellular vesicles (evs) and lipoproteins (e.g high density lipoproteins (hdls)). the extracellular rna (exrna) carried by evs and hdls is of great interest for two reasons: ) the exrna within different carriers could be diagnostic of the state of the tissues from which the particles originate, and ) exrna has been shown to affect gene expression in target cells. although the origin and functions of exrnas remain largely unknown, there is growing interest in exrna research for the development of diagnostics and new therapeutic targets. small rna sequencing is widely used to estimate the abundance of exrnas in biofluid samples. here we present a data processing pipeline for extracellular small rna sequencing. sequencing data are pre-processed through quality control, and then aligned to the endogenous genome to obtain the gene counts for various rna biotypes, including microrna, trna, rrna, piwi-interacting rna, long non-coding rna (lncrna) and protein coding rna. it also aligns sequencing reads to exogenous databases, including the ribosomal rna sequence database silva, and all sequenced bacteria genomes available on ensembl, to estimate the abundance of exogenous genes. results: we analysed a publicly available small rna-seq dataset of hdl from three systemic lupus erythematosus (sle) patients and three healthy controls using this pipeline. the mirna hsd-mir- , lncrna al . and ac . were elevated in sle patients compared to controls. exogenous rna reads mapped to bacteroidetes were also elevated in sle patients. summary/conclusion: our pipeline is able to process exrna sequencing data and estimate the abundance of major exrna species, as well as exogenous rna taxonomy. the pipeline is optimized for the job scheduler slurm, and can therefore utilize the full computational power of high-performance computers. the pipeline is publicly available on github (www.github. com/zhuchcn/excernapipeline). introduction: ibd is a chronic hyperinflammatory disorder that severely compromises the intestines. the aetiology of ibd is poorly understood. however, it has been associated with a dysregulation of the immune system and gut microbiota and with genetic and environmental factors. cumulative evidence indicates that evs play an essential role in modulating immune responses. recent research suggests that evs derived from dendritic cells, saliva and intestinal epithelial cells may be involved in the progression of ibd inflammation. however, little is known about the contribution of immune cells-derived evs with this pathology. the goal of this study is to shed light on the contribution of pbmc-derived evs on ibd pathogenesis. here we characterized and compared the composition of evs derived from pbmcs of ibd patients and healthy control. evs were isolated by differential centrifugations from the supernatant of pbmc activated with cd -cd beads for days in serum-free media. size and concentration were analysed using a nano sight instrument, while the presence of known evs markers (cd , cd , hsp ) was analysed by immunoblotting. whole evs proteome was performed by ms/ms and functional-enrichment analysis was done using funrich with uniprot database. results: proteomics analyses identified a total of proteins in the four groups. of those, ( . %) were present in both the ibd patients and control. this group of protein was composed of several ras-related proteins, eukaryotic initiation factors, granzyme, cd , tubulin, and serpins among others. patients' evs shared proteins in common such as proteasome subunit beta type- , t cell receptor beta, and the amine oxidase containing copper . interestingly, each patient sample had a unique group of proteins. among these are myeloperoxidase, neutrophil elastase, proteasome subunit alpha type- , and signalling lymphocytic activation molecule (slamf ). summary/conclusion: these preliminary studies show that the ev composition from pbmcs of ibd patients is specific and differs from a healthy control. this exclusive composition has the potential to be used as a biomarker for diagnostics and progression of the disease, and it could also provide new insights into our understanding of the cellular pathways involved in the pathogenesis of ibd. the studies were performed with corresponding irb approvals. proteomic analysis of exosomes isolated using precipitation and columnbased approaches introduction: exosomes are a subtype of small extracellular vesicles (evs) involved in various physiological and pathological processes with huge potential as biomarker resources or as therapeutic tools. although several exosome isolation approaches are available, complementary studies focusing on optimizing the methods for human bloodderived exosomes isolation and method-specific comparative exosomal proteomic profiles will be of clinical value. methods: blood-derived evs were isolated through precipitation-and column-based methods and characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. serumderived exosomal proteomes were analysed by mass spectrometry (ms). the resulting proteomes were then overlapped with the proteomes obtained from exosome-related databases, to determine the % of similar content. in addition, bioinformatic analysis, including gene ontology (go) was carried out. results: both methodologies tested isolated particles with the expected morphology and size range, although the column-based method isolated a higher number of particles. about % of the exosomal proteins identified through ms overlapped with the proteomes extracted from the databases. go terms were similar for the proteomes isolated from the column-and precipitationbased methodologies. the top go terms identified for molecular function were ion binding, peptidase activity and enzyme regulator activity and for biological process were immune system process, transport and response to stress. further, partial least square analysis revealed a clear segregation of proteomes obtained by the distint methodologies and complementary statistical analysis revealed the proteins differently expressed. summary/conclusion: no major differences were found in the top biological processes and molecular function based on go analysis. nonetheless, the two approaches result in different evs yields and significant proteome differences were identified. characterization of distinct methods for blood-derived exosomes isolation can be useful in the context of evs potential in disease diagnostics/therapeutics. introduction: we and others are developing biomarkers for neurodegenerative diseases using neuronalenriched evs immunocaptured from a suspension of total plasma evs. here we assess how the isolation method for total evs affects the yield, purity and enrichment of neuronal evs. methods: for n = subjects, total evs were isolated by ev precipitation solution (exq), ev precipitation solution plus bipartite resin columns (exu) and size exclusion chromatography (qev) from . , . and . ml plasma, respectively. then, neuronal-enriched evs were immunoprecipitated using anti-l cam antibody. in total and l cam evs, we measured particle concentration by nanoparticle tracking analysis, protein concentration, and novel multiplex electrochemiluminescence immunoassays for tetraspanins cd , cd and cd on intact evs. results: for total evs, yield followed the order of exq > qev > exu, assessed by particle (p < . ) and protein concentrations (p < . ). l cam evs immunocaptured after exq showed -fold higher particle (p < . ) and fivefold higher protein (p < . ) concentrations compared to l cam evs after exu, and -fold higher particle (p < . ) and -fold higher protein (p < . ) concentration compared to l cam evs after qev. l cam evs after ev precipitation (exq) showed , and -fold higher cd , cd , and cd concentrations (p < . ) compared to l cam evs after exu, and , and -fold higher cd , cd , and cd concentrations (p < . ) compared to l cam evs after qev. l cam evs following different methods had equal purity assessed by ratios of particle/protein concentrations (p = ns), and tetraspanin/particle concentrations (p = ns). summary/conclusion: l cam ev immunocapture preceded by exq exceeded the yield of immunocapture preceded by exu or qev. recovered l cam evs showed equal purity by particle/protein and tetraspanin/particle metrics. neuronal enrichment results will be available by the time of isev. immunoprecipitation following exq, often considered impure, purifies final isolates as effectively as more onerous methods typically considered purer. balancing sensitivity, purity and scalability is essential for implementation of blood biomarkers in the clinical setting and may be achieved by combining techniques. funding: this research was supported in part by the intramural research program of the nih, national institute on aging. characterisation of breath exosomes: towards non-invasive diagnosis deanna ayupova a , renee goreham b and paul teesdale-spittle c a school of chemical and physcial sciences, victoria univeristy of wellington, wellington, new zealand; b university of newcastle, newcastle, australia; c school of biological sciences, victoria univeristy of wellington, wellington, new zealand introduction: breath-derived exosomes present new potential for non-invasive diagnosis of lung cancer. however, breath-derived exosomes have not been well characterized and methodology for their purification has not been optimised. in order to exploit their potential for diagnosis, it is first necessary to develop methods that reproducibly provide high quality pure exosomes from breath. in this study, we optimise methods for their isolation and characterise them in comparison to exosomes derived from cell culture models. methods: in order to characterize exosomes from exhaled breath condensate (ebc) it was first necessary to optimize methods for isolation of pure, intact, and high quality exosomes. to this end, isolation methods were optimised on cell-derived exosomes and then applied to ebc, yielding high quality exosomes from size exclusion chromatography (sec). ebc exosomes were compared with those from a and wi-cells using dls, tem, and cryo-sem. an immunoblotting-grid technique was used to validate the presence of exosome-specific markers cd and cd . protein content of exosomes were quantified and compared. results: sec-based isolation was more effective at isolation of pure and intact exosomes than ultracentrifugation, with the highest purity exosomes obtained in the middle fractions of the exosome-containing eluate. exosomes from ebc had a size range ( - nm), protein content ( - ug/ml) and molecular markers typical of cell-derived exosomes. summary/conclusion: breath-derived exosomes isolated through size exclusion chromatography are sufficiently pure for diagnostic purposes and are phenotypically similar to exosomes derived from other sources. we foresee their use in non-invasive diagnostics for lung cancer as an important future application. ligand-based exosome affinity purification (leap) is a rapid and reproducible method for the enrichment of functional evs introduction: platelet-derived extracellular vesicles (pevs) represent the next generation of therapeutic biologics as they enable a more refined and targeted approach when compared to crude blood derivatives currently used for treating diseases such as cancer, thrombocytopenia and chronic wounds. however, development of an ev-based therapeutic is hindered by the lack of a scalable, validated and reproducible purification process. in this study, pevs were isolated from activated platelet concentrates and purified using exopharm's ligand-based exosome affinity purification (leap) technology to produce a functionally active ev therapeutic. methods: platelet concentrates (n = ) were obtained from the australian red cross blood service and were activated by exopharm's proprietary process. activation was verified by measuring cd p using flow cytometry. the resulting platelet releasate ( ml) was subjected to leap purification to isolate pevs. for characterization, protein concentration was determined by a bicinchoninic acid assay, microfluidic resistive pulse sensing (mrps) was used to perform a particle count and transmission electron microscopy (tem) enabled visualization of ev morphology. key ev markers were detected using mass spectrometry (ms) and western blots. to confirm biological activity, human dermal fibroblasts were subjected to serum starvation for hours before treatment with pevs ( µg/ml). cell growth was recorded by the real-time xcelligence system and differences in proliferation were statistically analysed using a one-way anova. results: mrps and tem both revealed isolated pevs to be - nm in size. the final product was positive for platelet markers (cd , cd p) and key ev markers (tsg , alix, cd ). treatment with purified pevs significantly increased proliferation in serumstarved fibroblasts over hours. summary/conclusion: exopharm's leap technology is a rapid and reproducible purification process which produces pevs that adhere to misev guidelines and are functionally active. funding: all funding was through exopharm ltd (asx:ex ) a novel but simple method to obtain purified exosomes by one-step ultracentrifugation introduction: exosomes are extracellular vesicles (evs) that are derived from endosome membrane. they are usually - nm in diameter, actively secreted in most living cells. originally, exosomes were thought to act as cellular garbage disposals. recent studies showed that exosomes not only can serve as biomarkers for diagnosis, but also can be used as an ideal delivery vehicle for drugs in therapeutics. exosomes are natural carrier for mrna, mirna, sirna, protein, dna and peptide for long distance intercellular communication. isolation of exosomes is challenging due to their small size and heterogeneity. traditional differential ultracentrifugation method is still the gold standard for exosome purification. to further explore the potentials of exosomes being as the therapeutic delivery vehicle or diagnostic reagent, it is an essential step to purify them in high quality at high yield. methods: here, we report a novel method to obtain intact shape, high-quality and high purity exosomes with one-step ultracentrifugation by using "exojuice". results: data of nanoparticle tracking analysis (nta) and western blotting showed "exojuice" can yield exosomes with a simpler method to obtain higher purity exosomes in comparison to previous method of cushion ultracentrifugation using optiprep. summary/conclusion: our method can be used to purify exosomes from cell culture medium, serum, urine, saliva, and other biofluids. a straightforward device to extract apoplastic fluid from succulent fruits for higher purity of extracellular vesicles introduction: edible plants are emerging as a sustainable source for extracellular vesicle (ev)-based drug delivery vehicles. however, current isolation methods (e.g. grinding or squeezing) may cause destruction of plants' biostructures, and in turn leads to unwanted effects in downstream applications and complicates the study of nanovesiclescell. therefore, we designed a simple device that allows the extraction of apoplastic fluid (af) from succulent fruits, facilitating ev isolation as well as effective downstream applications. methods: an inner filter tube was designed to extract af with a determined membrane pore size. af was collected by low-speed centrifugation method and then filtered to eliminate the impurities from the cytoplasm and damaged cells. minced juice (mj) was homogenized by a blender and then centrifuged to remove large fragments. subsequently, the differential centrifugation method was employed to extract evs from af and mj. fourier-transform infrared spectroscopy (ftir), nanoparticle tracking analysis (nta), and transmission electron microscopy (tem) were performed to discriminate af, mj and their evs. results: the "spectroscopic" protein-to-lipid (p/l) ratio of af ( . ± . ) is significantly lower than that in mj ( . ± . ), showing the higher lipid contents in af, which may result from the loss of lipids in mj obtained from grinding or juicing methods. similarly, ftir showed the difference in p/l ratio between af and its evs ( . ± . and . ± . , respectively). nta showed the sharper peak and smaller vesicle size in the following order: mj ( . ± . nm), af ( ± . nm), af-derived evs collected at , × g and , × g ( . ± . nm and . ± . nm, respectively). furthermore, tem study indicated that the collected evs exhibited a typical lipid bilayer of extracellular nanovesicles. summary/conclusion: by using a reusable filter device, we successfully isolated af from succulent fruits, paving the way to collect plant evs without an interference of significant biodestruction or damaged cells, hence improving the purity of evs and facilitating downstream applications. moreover, this method is straightforward, reproductive, and can be potentially used in a large-scale production. method to simultaneously capture multiple classes of intact extracellular rna carriers including extracellular vesicles and lipoprotein particles introduction: extracellular particles including extracellular vesicles (evs), lipoproteins, and free proteins are carriers of extracellular rna (exrna), which has been shown to regulate cellular function. because these particles have different physiological origins, they have different rna signatures, so the first step to understanding the biology of exrna is to isolate individual particle fractions with high purity and efficiency. current methods for isolating evs are optimized for increased yields and purity of ev fractions but typically require multiple millilitres of starting plasma and do not capture the other exrna carrier particle types. methods that can capture evs from low starting plasma volumes and can also capture other exrna carriers simultaneously are needed for analysing samples from previously conducted large cohort studies, biorepositories, and in populations where sample volume is limiting. methods: we have developed a method adapted from lipoprotein isolation that requires only µl of starting plasma, and uses brief ultracentrifugation (uc) followed by fast protein liquid chromatography (fplc) to capture classes of purified exrna carriers including evs, ldl, hdl, lipidated albumin, proteins, and vldl/chylomicrons. we have validated successful capture of evs by microfluidic resistive pulse sensing (mrps, spectradyne), transmission electron microscopy (tem), and single particle interferometric reflectance imaging system (sp-iris; exoview) with optional fluorescence. results: we have observed . × particles per ml from a ml fplc fraction of evs measured from , events by mrps, confirming that evs are being captured by this method. there were also . × particles/ml and . × particles/ml in the two subsequent ml fractions that are known to contain lipoprotein particles, though these were measured from , events each. by tem we confirmed these observations that evs are eluting before lipoprotein particles with some evs eluting later in fractions containing lipoproteins. summary/conclusion: these results confirm the efficacy of the method in isolating multiple exrna carrier fractions simultaneously from a single ul plasma sample, making it amenable for the analysis of exrna in samples from large cohort studies, biorepositories, and vulnerable populations such as the elderly and young children. funding: nih/nia r ag ; nih ug ca - optimizing the isolation of placental mesenchymal stromal cell-derived extracellular vesicles in a d bioreactor system leora goldbloom-helzner a and aijun wang baaaaa a uc davis, davis, usa; b uc davis medical center, sacramento, usa introduction: extracellular vesicles (evs) derived from placental mesenchymal stromal cells (pmscs) have the potential to provide neuroprotection at sites of injury. however, a rate limiting step in ev research is the low yield, high technical time, and high cost of current isolation procedures. to address this inefficiency, we cultured pmscs in a unique bioreactor system to increase the absolute yield of evs per ml of media and per cell. future studies will determine if this system can improve pmsc ev yield without altering the demonstrated neuroprotective properties of pmsc-evs. methods: pmscs were cultured in the bioreactor for ten weeks. ev-conditioned media was collected weekly and evs were isolated through differential centrifugation. nanoparticle tracking analysis (nta) measured ev size and concentration. western blots tested for normal ev markers (cd , cd , and cd , calnexin(-)) and enzyme-linked immunosorbent assays (elisa) measured levels of characteristic growth factors in conditioned media including vascular endothelial growth factor (vegf), brain-derived neurotrophic factor (bdnf), and hepatocyte growth factor (hgf). results: evs remained consistent until week eight, after which a decrease in both ev size and concentration was seen. western blots revealed normal positive expressions of cd , cd , and cd and negative expressions of calnexin. levels of vegf, bndf, and hgf were comparable after weeks. cost analysis revealed an overall increase in ev yield for shorter labour time and lower material cost. summary/conclusion: this initial study uses a bioreactor system for a unique source of cells and has brought us closer to optimizing pmsc ev isolation protocols for increased yield, lower cost and time commitment, and maintained sample purity. preliminary data suggests the ev phenotype and cell secretome are consistent with those present in current culture settings. future experiments will assess the preserved neuroprotective properties of the pmsc evs. a novel method for isolating extracellular vesicles from cell culture media and plasma using polyethylenimine introduction: due to their ability to transport dna, rna, and protein cargoes between cells, extracellular vesicles (evs) are becoming popular for biomarker discovery as well as for therapeutic delivery. here we describe the development of a novel precipitation method for the isolation of evs from cell culture media and plasma that is based on polyethylenimine (pei), an inexpensive, water-soluble, and biocompatible cationic polymer. pei is a group of hydrophilic cationic polymers that are synthesized as either linear or branched forms of varying molecular masses ( , to , da) and are widely used in the biomedical field as a coating and transfection agent. methods: linear and branched pei of varying molecular weights (mw) were tested for their ability to precipitate evs from either conditioned culture media (ccm) or human plasma. isolated evs were characterized by western blotting and nanoparticle tracking analysis (nta). the small rna profile of evs isolated using pei from human plasma was analysed by ngs and ev-specific mirnas were confirmed by digital droplet pcr (ddpcr). mass spectrometry (ms) was used to analyse the proteome of pei-captured evs from plasma. hek cells producing gfp+ evs were used to optimize conditions for release of evs from both linear and branched pei by fluorescent spectrophotometry and flow cytometry measure-ments. results: linear and branched pei were both able to precipitate evs as determined by western blotting for ev protein markers; however, branched pei with mw > , da and linear pei with mw > , da were more efficient for ev precipitation than lower mw forms. despite its known ability to bind nucleic acids pei was unable to capture cell-free dna from plasma, although rna and in particular ev-associated mirnas such as mir- - p were recovered. ms revealed that pei enriches extracellular exosome proteins from plasma. evs captured from ccm by pei could be released from the complex using heparin or high salt conditions. summary/conclusion: pei has an unexpected preference for associating with evs compared to nucleic acids in complex biological samples and has a hitherto unrecognized application for ev precipitation. introduction: there is ongoing debate about which is the most appropriate method for isolation of evs, with most labs using some combination of differential ultracentrifugation (uc), size-exclusion chromatography (sec), and/or density gradient ultracentrifugation (dg). here we applied a surface-enhanced raman spectroscopy (sers) analysis platform to compare chemical composition of the isolate from each method against lipoprotein standards to assess the relative purity of the ev preps. methods: - ml of plasma was separated from whole blood collected from head and neck cancer patients. each sample was split into batches and evs were isolated by either uc, sec, or dg. following isolation, samples were incubated on commercial sers substrates and raman spectra were collected. lipoprotein standards were purchased and also measured for comparison. using principle component analysis (pca), spectra were analysed for chemical variability. results: sers analysis of sec, uc, and dg isolated evs were chemically distinguishable using simple pca. the chemical changes could in large part be attributed to fitting the differences in spectra to lipoprotein standards. we found that uc isolated populations clustered with the high-density lipoproteins (hdl), sec populations with the low-and very low-density lipoproteins (ldl, vldl), and dg populations were more variable, but mainly clustered together with the highdensity-lipoproteins (hdl). summary/conclusion: this set of experiments matches our expectation that various lipoprotein would contaminate ev preps according to their relative size and density distributions. no single isolation method could separate pure ev samples. this study also illustrates the utility of label-free sers analysis for rapid chemical characterization of evs. bioreactors: lessons to develop an extracellular vesicle factory vanessa chang, priscila dauros-singorenko, lawrence w. chamley, colin l. the university of auckland, auckland, new zealand introduction: high density mammalian cell culture systems (bioreactors) provide valuable advantage for large scale production of secreted products such as extracellular vesicles (ev). however, optimisation of design selection, handling and operational costs can be quite challenging. here we provide our experience with a celline bioreactor system. methods: cultures of adherent cell lines were established in celline ad bioreactors and propagated for up to weeks. media was changed twice weekly and cells shed into serum-free conditioned medium were counted and assessed for viability. nanoevs were isolated by sequential centrifugation ( g - , g - , g) and size exclusion chromatography (sec). nanoevs were characterised in their protein (bca) and particle (nanoparticle tracking analysis) amount, ev markers (western blotting) and morphology (transmission electron microscopy, tem). results: the viability of shed cells varied between cell lines and through time, suggesting a changing dynamic during reactor establishment and continuous growth phases, that was specific to each cell line. hdfa, bt and bt consistently shed mainly dead cells ( - %), as opposed to mcf and mda-mb- which predominantly shed live cells. sec fractionation of nanoevs identified a dominate ev-rich peak and significant quantities of smaller proteins, highlighting the need for further purification. nanoev yields from each - day culture averaged - × particles, representative of yields obtained from cells grown in to conventional t tissue culture flasks. ev markers and tem confirmed the protein profiles and morphology of evs obtained from bioreactors. summary/conclusion: high density bioreactor cultures offer a physiologically relevant, cost and space efficient approach to produce significant amounts of evs, providing sufficient material for numerous experimental uses. in our hands, with careful twice weekly management, they can be propagated for up to weeks without significant changes to the evs. introduction: extracellular vesicles (evs) have potential applications for clinical theranostics. ultracentri-fugation is most commonly adopted to the evs isolation, which is recommended as a gold standard method. however, ultracentrifugation is time-consuming and expensive equipment requirement, resulting in the coisolation of contaminants such as protein aggregates. therefore, our aim is to develop a rapid and efficient platform to isolate heterogonous evs based on the insertion of lipid molecules into the evs membrane to avoid co-isolation of non-membranous protein particles. methods: herein, a defected nanoscale functional metal organic framework (mof) was constructed as an efficient platform for evs isolation. typically, one single-stranded dna was designed and modified with a phosphate group at the ʹ-end and cholesterol at the ʹ-end to form a capture dna named phosphate−dna−cholesterol (pdc). the phosphate group forms a strong covalent bond with the designed defeated site of zr (iv) in mof uio- -nh and the cholesterol inserts into the phospholipid bilayer to capture evs without non-membranous particles contamination. the formed mof−phosphate−dna −cholesterol−evs (mof@pdc@evs) system was further treated with dnase i for dna hydrolysis to give high pure evs. results: a rapid and efficient isolation platform of evs based on a defected mof functionalized with phosphate-dna-cholesterol (mof@pdc) has been constructed successfully. compared with ultracentrifugation, mof@pdc platform promises to isolate size heterogeneous evs i) without non-membranous particles contamination, maintaining evs intact membrane structure, protein components, and biological functions; ii) with the ability to capture evs with % isolation efficiency; iii) makes evs isolation process simple and fast, which could be finished in minutes without requirement of the expensive equipment. summary/conclusion: in conclusion, this rapid and efficient platform is suitable for isolation evs from biological fluid for downstream protein analysis. this work opens a new perspective in mof-based separation researches and may shed light on further studies towards evs isolation. introduction: incorporation of pharmacologically active molecules on the surface or the lumen of extracellular vesicles (evs) is an important strategy for maximizing the therapeutic potential of evs. genetic engineering of producer cells by introducing dna through random or site-specific integration are promising strategies for creating engineered evs. longterm stability with consistent transgene expression in the ev producer cells and therapeutic potency of resulting engineered evs are crucial for biomanufacturing. we present a comprehensive study to investigate stability of transgene expression and potency of two potential therapeutic engineered evs derived from stably selected pools transfected by either random integration (ri) or site-specific integration (ssi). methods: producer cells were engineered to make evs displaying interleukin (il ) or interferon gamma (ifng) by ri or nuclease-mediated ssi into aavs locus. following puromycin (puro) selection, longterm cellular stability and transgene expression without selective pressure was investigated. evs were generated from stable cell pools at , , and months post-thaw and purified by density gradient ultracentrifugation. purified evs were biochemically characterized by nta, bca, western blot, and cholesterol quantitation. transgene expression and biological activity of evs displaying il and ifng were assessed by alphalisa and in vitro reporter assays. results: transfection by ssi resulted in faster recovery in puro selection compared to ri. all stable cell pools, regardless of integration method, resulted in comparable cell culture performance, ev yield, and lipid and protein content at all time points tested. the engineered evs also demonstrated long-term stability of il and ifng transgene expression and in vitro activity from both integration strategies. summary/conclusion: both methods for generating stable cell lines were comparable in terms of cell stability, transgene expression, ev titre and potency, with ssi having the advantage of speed, allowing for more rapid iteration cycle times. thus, both methods are suitable for the precision engineering of therapeutic evs. this work demonstrates feasibility to manufacture therapeutic engineered evs from stable cells from either integration strategy for clinical development. transport of outer membrane vesicles as a model therapeutic delivery system in pathogenic and commensal bacteria introduction: outer membrane vesicles (omvs) in gram-negative bacteria have been shown to be important carriers of biomolecules, including toxins and other virulence factors, peptidoglycan, and nucleic acids. it has been shown that omvs play an important role in the delivery of these biomolecules to host cells and bacterial cells. while many thorough studies have explored omv delivery to host cells, few studies have explored the mechanisms of delivery of omvs to bacterial cells. our goal was to study the delivery of omvs to other bacterial cells. specifically, we were studying the oral pathogen aggregatibacter actinomycetemcomitans (a.a.), a gram-negative organism associated with localized aggressive periodontitis, to study the process by which vesicles from this organism communicate with other bacterial cells. overall, we want to understand the roles specific surface components of omvs play in the transport of these omvs to other bacterial cells. methods: we studied omvs from two strains of a.a.: jp , a highly pathogenic strain, and , a natural commensal strain. af -labelled omvs were incubated with fresh bacterial cultures. association of the omvs with the bacterial cells was quantified using flow cytometry. to examine the role of surface-associated dna in this process, dna was digested with dnase, and the amount of surface-bound dna was quantified with the membrane impermeable dna stain, toto- . results: using flow cytometry, we observed jp omvs were delivered to , cells, and at a lesser amount to jp cells. alternatively, , omvs associated readily with jp cells, more than to , cells. this suggests that the delivery of omvs to bacterial cells may be a targeted delivery mechanism. furthermore, we hypothesized surface-associated dna may play a role in this interaction. we next digested the surface-associated dna on the omvs with dnase, and observed a decrease in association between the omvs and bacterial cells. this supports our hypothesis that dna on the surface of the omvs plays a role in association. current experiments are investigating this interaction in more detail. summary/conclusion: we have demonstrated that omvs are selectively delivered to bacterial cells, and surface-associated dna plays a role in this process. we propose to investigate this process to further understand omvs delivery to bacterial cells. funding: r de & r de . utilizing a gaucher's disease cell line for the evaluation of a novel exosome-based replacement therapy annie k. brown a , jiayi zhang b , brendan lawler b and biao lu b a santa clara university, san jose, usa; b santa clara university, santa clara, usa introduction: engineered nano-scale exosomes have great potential as new and targeted delivery vehicles for the treatment of gaucher's disease, the most common lysosomal storage disease. recently, we have reported the design, production, and isolation of exosomes loaded with lysosomal β-glucocerebrosidase (gba). people suffering from gaucher's disease do not have functional gba, which results in toxic build-up of undegraded substrates within the cell. methods: to evaluate the efficacy of this exosomebased therapy, a human gaucher's disease model is required. here, we have utilized near-haploid human cells (hap ) modified via crispr-cas to model gaucher's disease in vitro. these cells contain a bp insertion in the th exon of the gba gene, resulting in non-functional gba. pcr, enzyme activity assays, and flow cytometry have been employed to confirm the diseased genotype and phenotype. results: characterization of gba-knock out cells shows a total loss of gba enzyme activity. further characterization demonstrates a normal growth rate but an increased number of lysosomes, indicating a diseased phenotype. summary/conclusion: the utilization of a human gba-knock out cell line will enable the evaluation of the efficacy of our engineered exosomes. disease models will be an important resource for the evaluation of new biologic therapeutics, including exosomes. funding: we would like to acknowledge the santa clara university school of engineering for their support. thrxosomes: a novel exosomes based theranostic for lung cancer introduction: chemotherapy is the first-line of treatment for lung cancer. however, inefficient bio-distribution and reduced accumulation of drugs in the tumour results in treatment failure. therefore, improved drug delivery and diagnostic systems are warranted. herein, we propose a novel theranostic system "thrxosomes" where exosomes are loaded with super paramagnetic iron nanoparticles (spions) conjugated to an anticancer drug via a phresponsive linker for controlled release. we hypothesize that thrxosomes will exert profound anticancer tumour activity that can be concurrently be monitored by magnetic resonance imaging (mri). methods: thrxosomes were produced by combining normal human lung fibroblast (mrc ) cell-derived exosomes with spions conjugated to and anti-cancer drug (chemodrug or mirna) via a ph cleavable linker. the physical and biological properties of thrxosomes were determined using transmission electronic microscopy (tem), nanotracker-analysis (nta), inductively coupled plasma mass spectrometry (icpms), western blotting, cell viability, and mri. results: exosomes used in preparing thrxosomes were nm in size with a typical lipid bilayer structure, and were positive for cd , cd , flotillin and negative for annexin a confirming presence and purity of exosomes. charge analysis, tem, and icmps data showed successful loading of spion-drug conjugate. biological studies showed selective and enhanced drug release under acidic condition (ph . ) compared to drug release at ph . . cell uptake and viability studies demonstrated increased uptake and killing of thrxosome-treated human a lung cancer cells compared to mrc- cells. in vivo studies demonstrated accumulation and detection of spions by mri in in-situ tumours of a tumour-bearing mice. summary/conclusion: our study demonstrates thrxosomes will produce profound anticancer activity in lung cancer that is measurable by mri. exosome-modified nanoparticles as an alternative delivery system for small rnas in cancer therapy petro zhupanyn a , alexander ewe b , thomas büch c and achim aigner a a independent division for clinical pharmacology at rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany; b dr., leipzig, germany; c rudolf-boehm-institute for pharmacology and toxicology, faculty of medicine, university of leipzig, germany, leipzig, germany introduction: gene knockdown by rna interference (rnai) is an alternative, non-invasive method for inhibiting proliferation or promoting apoptosis in tumour cells. this technique allows the specific targeting of key signalling proteins or mutated genes. most of the available transfection compounds suffer from rather profound cytotoxicity in vitro. the aim of our study was to establish a novel targeted small nucleic acid delivery system to the cells, with good cellular biocompatibility and applicability for in vivo studies. for this aim, we used native, cell own vesicles-exosomes. since exosomes are known to transport peptides and different rnas between cells and tissues, these unique, small extracellular vesicles (ev) may also be useful as transport vehicles for therapeutic sirna. methods: as detected by multiple cell surface protein expression analysis, exosomes carry specific surface expression markers, allowing the cellular uptake by the most of tissues. we established an ev purification protocol from tumour cell culture supernatants and a strategy for the efficient ev loading with our test sirnas or antimirs. here we used the combination of polyethylenimine (pei)-complexation of the rnas with ultrasound treatment for their loading into the evs. our ev-modified, ultrasound-treated nanoparticles were tested in vitro by measuring knockdown efficacies in luciferase reporter cell lines or by rt-qpcr gene expression analysis. results: more efficient cellular sirna uptake was observed upon ev-modification of our pei/rna nanoparticles, accompanied by efficient inhibition of gene expression. biological efficacies were retained also after storage for several days at room temperature. the monitoring of the ev-based particles by facs revealed a different time resolution of cellular uptake and nucleic acid release compared to the classically formulated peinanoparticles. in an in vivo therapy study in tumour xenograft-bearing mice, high biocompatibility, significant biological knock-down and tumour inhibition were observed after injection of anti-survivin sirnas formulated in our ecv-modified pei nanoparticles. summary/conclusion: our data demonstrate the usability of ecv-modified nanoparticles as efficient delivery system for small rnas in cancer therapy. microglial extracellular vesicles as therapeutic vector for neuroinflammation giulia marostica a , annamaria finardi b and roberto furlan a a san raffaele scientific institute, milan, italy; b san raffaele scientific institute, milan, italy introduction: microglia is considered an eligible target against the progressive multiple sclerosis (ms), but currently available therapies do not allow its efficient targeting. as many cell types, microglia communicate with the neighbouring cells through a complex system of extracellular vesicles (evs) exchange. recently my group described that microglia derived-evs, engineered to encapsulate il , are taken up by microglia itself, mediating a phenotype switch to a protective phenotype. in vivo studies suggest that these evs can ameliorate established neuroinflammation, thus making them a promising drug-delivery tool to target cns in ms. my project focuses on understanding the mechanism of action and the signalling pathway of evs delivery and to exploit this knowledge to specifically deliver different potential therapeutic molecules. for this purpose, we decided to characterize the evs through trps technology. methods: a murine microglia cell line (bv ) was engineered to stably overproduce endogenous il . this cell line was cultured in exosome-depleted rpmi and stimulated with pma( mg/ml) for min. evs isolation was carried out by collecting supernatant and subjecting it to consequential centrifugation of g, min, rt and g, min, °c. the resulting supernatant was filtered ( µm) and ultracentrifuged at , g for h at °c. the evs pellet was re-suspended in ice-cold pbs. the evs analysis with trps shows two populations of evs, one with a mean diameter of - nm and a broad zeta potential ranging from − mv to − mv, while the second population has a mean diameter of - nm and a zeta potential of − /- mv. this difference can be consistent with the different pathway formation of exosomes and microvesicles. we demonstrated in vivo the strong phenotypic change induced by our evs to resting microglia in a dose-and time-dependent effect. then, impairing the physiological procedure of the endosome acidification, the effect of our evs on recipient cells is higher. thus, suggesting an endocytic pathway for the internalization of the vesicles. we further demonstrate with gradient ultracentrifugation the capability of our formulation to vehicle endogenous il inside the vesicles. even if some protein is co-purified in the procedure, we know that the half-life of this cytokine is too short to elicit a strong in vivo response. consequently, we assume that the anti-inflammatory effect of our evs in vivo is a result of the il internalized in our formulation. summary/conclusion: these data help us understand more in detail the process of internalization and phenotype change mediated by these evs. our next goals are to discriminate between different internalization pathways and further validate the efficacy of our therapy on the eae mouse model. targeting il- rα on tumour-derived endothelial cells blunts metastatic spread of triple negative breast cancer via extracellular vesicle reprogramming introduction: the lack of an approved targeted therapy and the early onset of metastasis highlight the need for new treatments for triple-negative breast cancer (tnbc) patients. interleukin- acts as an autocrine factor for tumour-endothelial-cells (tec), and exerts pro-angiogenic paracrine action via extracellular vesicles (nevs). il- rα blockade on tec changes tec-ev (anti-il- r-evs) microrna cargo and promotes the regression of established tumour vessels. as tec are the doorway for "drug" entry into tumours, we have aimed to assess whether il- r blockade on tec impacts tumour progression via their unique ev cargo. methods: human tnbc samples, mda-mb- , mda-mb- and mcf cell lines were evaluated for the expression of il- rα. nevs and anti-il- r-evs were characterized by electron-microscopy, macsplex-exosome-kit and western blot. proliferation, migration, apoptosis and sphere formation were evaluated. scid mice were used for in vivo experiments. results: we noticed that, besides tec and inflammatory cells, tumour cells from . % of the human tnbc samples expressed il- rα. mda-mb- and mda-mb- , but not mda cells, expressed il- rα. in vitro, nevs provide survival and migratory signals, while anti-il- r-evs promoted apoptosis as well as reduced cell viability and migration of human tnbc cell lines. in vivo anti-il- r-ev treatment induced vessel regression in established tumours formed of mda-mb- cells and almost abolished the spread of liver and lung metastasis. moreover, decreased β-catenin and twist were found in tumours from animals treated with anti-il- r-evs. in addition, anti-il- r-evs reduced lung metastasis that was generated via the intravenous injection of mda-mb- cells. nevs that were depleted of mir- - p (antago-mir- - p-evs) were effective as anti-il- r-evs in down-regulating twist and reducing lung-vessel density and metastatic lesions in vivo. summary/conclusion: overall, these data provide the first evidence that il- rα is highly expressed in tnbc cells, tec and inflammatory cells, and that il- rα blockade on tec impacts tumour progression. introduction: high-grade serous ovarian cancer (hgsoc) is the deadliest gynaecologic cancer. its lethality is explained for late diagnosis at advanced stages and frequent recurrences despite achieving complete response with standard therapy. most of recurrences occurs at abdominal cavity with multiple metastasis. therefore, identifying key determinants of metastatic process remains as priority to find better therapies. current evidence assigns a central role of the exosomes in conditioning the metastatic niche in epithelial cancers. recently, we demonstrated that statins reduce metastasis in hgsoc in preclinical models. here, we decided to study the effects of statins on hgsoc-derived exosomes and its capability to condition the metastatic niche. methods: exosomes were isolated from heya ovarian cancer cell line and primary tissue cultures established from advanced-stage hgsocs (with signed informed consent and irb approval) by differential ultracentrifugation and quantified by nanoparticle tracking analysis (nta). enriched-cancer initiating cells (cic) spheroids were established from heya cells by using stem-selecting conditions. the paracrine effect of exosomes on cic migration/invasion was studied using either d migration or boyden chamber invasion assays. previous to exosome isolation, heya cells were treated with simvastatin ( um, h) or solvent and proteins involved in exosome biogenesis/uptake (alix, tsg ), its trafficking (rab a, rab a) and in conditioning the metastatic niche (emmprin) were measured by immunoblotting. results: exosomes isolated from heya cells or hgsocs enhance the metastatic potential of heya established spheroids in d migration or boyden chamber invasion assays. upon simvastatin treatment, we observed a significant reduction in migration/invasion induced by equivalent number of exosomes in heya -derived cics. under same treatment, we observed a significant decrease in protein levels of alix and tsg and an increase in the inactive forms of rab a and rab a in heya cells. we also observed a decrease in emmprin levels in heya -derived exosomes. summary/conclusion: here, we demonstrated a paracrine effect of hgsoc-derived exosomes that favour the metastasis process. in addition, we demonstrated that simvastatin reduces metastasis induced by cancerderived exosomes. such an effect is partially explained by changes in the expression of proteins involved in exosome biogenesis/uptake, its endocytic trafficking and in the content of proteins conditioning the metastatic niche. thus, simvastatin arises as potential therapeutic target to improve outcomes in this disease. funding: this research was supported by fondecyt granted to mauricio a. cuello label-free optical imaging and characterization of cancer-associated extracellular vesicles in tissues introduction: cancer-associated extracellular vesicles (evs) visualized in the tumour microenvironment have been identified as a potential biomarker for cancer-related tissue changes. analyses of evs have traditionally been performed in cells or isolated evs, with no temporal or spatial information that could be critically important for elucidating their roles in carcinogenesis. since the unperturbed distribution and organization of evs in the tumour microenvironment is associated with their cellular function and can potentially serve as a diagnostic and prognostic biomarker, there is a strong need for visualizing evs in freshly isolated tissue specimens. currently, only fluorescent labelling methods enable visualization and tracking of evs. we used a custom label-free multimodal multiphoton optical imaging system to detect and characterize evs and classify them using their optical signatures both in isolated tissues and in situ tumours. methods: label-free multimodal multiphoton imaging was used to provide simultaneous, co-registered structural and functional images of evs in untreated samples. heterogeneous populations of evs could be identified from their unique optical signatures. results: the intrinsic metabolic and structural properties of evs enabled reliable visualization and optical characterization of evs from cell cultures and in situ imaging of tumour-bearing rats. unique optical signatures were then used for identification of cancer-related evs in tissues from human breast cancer patients, and their density was found to be highly correlated with clinical diagnosis. in the current study, evs were isolated from urine of tumour-bearing dogs, and urine and serum from breast cancer patients. analysis of ev content showed higher concentration of nad(p)h in evs isolated from cancer subjects, than from healthy subjects, which reflects the reprogramming of cellular metabolism in carcinogenesis. summary/conclusion: these results suggest a potential label-free optical methodology to detect and characterize evs by their optical signatures, which can be utilized as possible diagnostic and prognostic biomarkers for cancer. funding: this research was conducted under protocols approved by the iacuc and irb at the university of illinois and carle foundation hospital, and supported by funding from nih. novel potential anticancer therapies based on interference with nuclear entry of cancer cell-derived extracellular vesicle components in recipient cells introduction: the intercellular communication mediated by extracellular vesicles (evs) in the tumour microenvironment plays an important role in tumour progression. experimental evidence indicates that evs derived from highly metastatic cells influence the behaviour of less aggressive cancer cells. we have previously described a novel intracellular pathway where a fraction of endocytosed ev-associated proteins and nucleic acids is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into nucleoplasmic reticulum (nr). here, we better characterize this pathway and report that it is required for the induction of an aggressive behaviour induced by evs released from highly metastatic sw colon cancer cells in isogenic primary cancer cells. methods: super resolution-structured illumination microscopy and magnetic-based co-immunoisolation studies were employed to identify the protein components of the nuclear pathway and to monitor the entry of ev-containing late endosomes into the nucleoplasmic reticulum. human sw carcinoma cells expressing er-gfp and rab -rfp were exposed to evs from sw cells and then live imaged. results: we have previously reported that the tripartite protein complex, containing vap-a, orp and rab orchestrates the localization of ev-carrying late endosomes into nr. we now report that silencing of orp or vap-a, but not its homologue vap-b, reverses the pro-metastatic changes induced by evs isolated from metastatic cells on their non-metastatic counterpart, including transition to an ameboid phenotype, cell rounding and blebbing. moreover, we found that certain nuclear pore complex proteins and importin-beta are co-immunoisolated with orp , vap-a and rab suggesting the formation of a large protein complex at the entry of nuclear pores. summary/conclusion: interfering with the mechanisms regulating this novel intracellular pathway may find therapeutic applications particularly in ev field and oncology. educated osteoblasts regulate breast cancer proliferation via small extracellular vesicles thomas jefferson university, philadelphia, usa introduction: breast cancer commonly traffics to bone, where breast cancer cells (bccs) can survive undetected for years before metastatic outgrowth. in bone, bccs interact with surrounding stromal cells, including osteoblasts (obs), to shape the metastatic niche. our lab discovered there are at least two subpopulations of obs in the bone-tumour niche, based on protein marker expression. one group, "educated osteoblasts" (eos) have engaged in crosstalk with bccs whereas another group, naïve obs, have not. we have novel evidence that eos regulate bcc proliferation. the purpose of this study was to determine if extracellular vesicles (evs) produced by eos play a role in regulating bcc proliferation. we hypothesized evs produced by eos would decrease bcc proliferation. methods: eo-derived small evs from culture media were isolated via ultracentrifugation and characterized evs for size, protein marker expression, and density floatation to validate the purity of ev samples. the functionality of eo-derived evs on bcc proliferation was examined using edu and checkpoint proteins p and p . bcc protection from chemotherapy induced cell death was also examined. results: we found that evs produced by eos, but not naïve obs, decreased both triple negative and erpositive bcc proliferation in a concentration dependent manner. furthermore, using an edu assay, we found that exposure to eo-derived evs induces bccs to undergo cell cycle arrest. interestingly, the cell cycle arrest was reversible and bcc proliferation was restored upon removal of eo-derived evs. in addition, exposure to eo-derived evs leads to increases in bcc expression of the g checkpoint proteins, p and p . we next wanted to investigate proliferative signalling pathways that may be deregulated in bccs following exposure to eo-derived evs. we found that eoderived evs reduce bcc levels of erk / . because our data indicate eo-derived evs induce sustained cell cycle arrest in bccs, we desired to know if eo-derived evs protected bccs from chemotherapy-induced cell death. we found that bccs exposed to eo-derived evs and the chemotherapy drug, doxorubicin, have decreased cell death compared to bccs exposed only to doxorubicin. summary/conclusion: altogether, our data suggest eos play a crucial role in bone-tumour microenvironment by regulating bcc proliferation. funding: supported by nih r ca and commonwealth of pennsylvania -department of health sap for kmb. phosphorylation of tyrosine in annexin a is essential for its association with exosomes and for imparting invasive and proliferative capacity to other cells priyanka prakash desai a , pankaj chaudhary b , xiangle sun b and jamboor vishwanatha a a unt health science center at fort worth, fort worth, usa; b unt health science center, fort worth, usa introduction: triple negative breast cancer (tnbc) accounts for %- % of all breast cancer cases. the lack of targeted-based therapies highlights the importance of studying tnbc. elevated levels of annexin a (anxa ), a ca+ -dependent phospholipid binding protein, has been correlated with worse overall survival in tnbc patients. our previous data implicate that exosomal anxa is involved in creating a pre-metastatic niche and facilitating metastasis in tnbc. moreover, n-terminal phosphorylation of tyrosine (tyr) in anxa has been implicated in regulating several anxa activities in cancer progression. here, we demonstrated that n-terminal phosphorylation of anxa at tyr is important for its association with exosomes which imparts invasive and proliferative phenotype to other cells. hence, dissecting the regulatory pathway will be critical for verifying the value of anxa as a therapeutic, diagnostic or prognostic marker in tnbc. methods: pn -egfp plasmids expressing the constitutive phosphomimetic (anxa -y e) and non-phosphomimetic mutant (anxa -y f) gene expressing mutation at tyr site were overexpressed in mda-mb- tnbc cells. mutant cells were experimentally validated for anxa specific functions like migration, invasion and proliferation. exosomes were isolated from the mutant phosphomimetic (exo-anxa -y e-gfp) and non-phosphomimetic (exo-anxa -y f-gfp) cells and were analysed for exosomal surface expression of anxa by immunoprecipitation and flowcytometry. cal- breast adenocarcinoma epithelial cells were treated with exo-anxa -y e-gfp and exo-anxa -y f-gfp to analyse the rate of invasion and proliferation by transwell invasion and proliferation assay, respectively. transfer of exosomal anxa in cal- was studied using immunofluorescence and its implications on signalling pathways were studied by western blot. results: mda-mb- phosphomimetic tnbc mutant cells showed increased migratory, invasive and proliferative capacity compared to non-phosphomimetic tnbc mutant cells. exo-anxa -y e-gfp had elevated surface anxa expression compared to exo-anxa -y f-gfp. cal- cells treated with exo-anxa -y e-gfp showed high migratory, invasive and proliferative characteristics, with a higher expression of p-anxa (tyr ), p-src(tyr ) and p-paxillin(tyr ) compared to exo-anxa -y f-gfp treated cells. summary/conclusion: n-terminal phosphorylation of tyr in anxa in mda-mb- tnbc cells (phosphomimetic mutant cells) is essential for its association with exosomes and for conferring increased invasive and proliferative capacity to other breast cancer cells. funding: the above study is funded by national institute of health ro ca and nimhd's u md to dr.j.k.vishwanatha. a novel method for epithelial-derived extracellular vesicle isolation and enrichment in patients with advanced prostate cancer arpit rao, helene barcelo and bharat thyagarajan university of minnesota, minneapolis, usa introduction: evaluation of changes in prostate cancer biology is difficult due to presence of lymph nodal or bony metastatic disease in a majority of patients. a number of liquid biopsy assays have shown clinical utility in prostate cancer, but are limited by low sensitivity (e.g. circulating tumour cells-based assays) or inability to perform transcriptome sequencing (cellfree dna-based assays). epithelial-derived extracellular vesicles (epi-ev)-based assays are uniquely positioned overcome both these limitations as evs are abundantly secreted into the blood and have rnacargo that mirrors the cell of origin. however, a reliable method to enrich for epi-evs is currently lacking. methods: plasma was isolated from the peripheral blood collected from patients with metastatic prostate cancer enrolled in an institutional biobanking study before initiation of systemic antineoplastic therapy. evs were isolated from µl of plasma using a commercially available qev size exclusion column (izon inc.). without subjecting the evs to any physical stressors such as centrifugation, cd magnetic beads were used to fractionate the evs into cd + (platelet derived) and cd -(non-platelet derived) fractions. multiparameter flow cytometry was used to evaluate evs that expressed cd and epcam and were negative for calnexin. nanotracking analysis (nta) was used to quantify both total ev and cd + and cd fractions in all patient samples. the average ± standard deviation of total evs obtained from the patients was . x ^ ± . x ^ evs/ml of plasma (coefficient of variation [cv] : %) while the average and standard deviation of cd -evs was . x ^ ± . x ^ (cv: %). the cd -ev fraction represented a variable amount of the total evs in prostate cancer patients ranging from . % to . %. multiparameter flow cytometry showed that over % of total evs were cd + and calnexin-, suggesting an endosomal origin for a vast majority of the evs in these plasma samples. however, the proportion of evs expressing epcam (marker of epi-evs) was higher among the cd -fraction ( % - %) as compared to the cd + fraction ( . % - %). summary/conclusion: our novel method was able to isolate and enrich the epi-ev from the plasma of advanced prostate cancer patients. correlation between clinical characteristics and ev quantity is being evaluated to identify the reason(s) for large variations in cd -ev fraction. future studies are planned to use our method in improving the sensitivity of ev-based assays and increase the rna yield to facilitate transcriptome sequencing. funding: this work was funded by grants from randy shaver community and research fund, minnetonka, mn. exosomes drive medulloblastoma metastasis in a mmp and emmprin dependent manner introduction: recurrent/metastatic medulloblastoma (mb) is a devastating disease with an abysmal prognosis of less than % -year survival. the secretion of extracellular vesicles (evs) has emerged as a pivotal mediator for communication in the tumour microenvironment during metastasis. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the blood-brain-barrier. matrix metalloproteinases (mmps) are enzymes secreted by tumour cells that can potentiate their dissemination by modification of the extracellular matrix. we hypothesise that exosomal mmp and its inducer emmprin could enhance metastasis of mb. methods: proliferation, invasion and migration assays were used to evaluate the phenotypic behaviour of primary cell lines pre-treated with metastatic tumour cell-derived exosomes. gelatin zymography and western blotting were performed to confirm mmp functional activity in cell lines and exosomes. nanoscale flow cytometry was used to measure surface exosomal emmprin levels. exosomal mmp and emmprin were modulated at the rna level. results: number of exosomes is directly related to the migratory behaviour of parental mb cell lines (p < . ). notably, functional exosomal mmp and emmprin levels also correlate with this. furthermore, exosomes from metastatic cell lines conferred enhanced migration and invasion on their matched isogenic primary (non-metastatic) cell line pair bỹ . -fold (p < . ). exosomes from metastatic cell lines also conferred increased migration on poorly migratory foetal neuronal stem cells. summary/conclusion: together this data suggests that exosomal mmp and emmprin may promote medulloblastoma metastasis and supports analysis of exosomal mmp and emmprin levels in patient cerebral spinal fluid samples. introduction: exosomes secreted from cancer cells harbour the potential to regulate intracellular signalling and promote metastasis. wherein, metastasis suppressor genes (msgs) play a pivotal role in regulating such signalling cascades. however, the regulation gets hampered due to low expression of msgs under metastatic conditions. nm -h , product of first identified metastasis suppressor gene nme , is significantly downregulated under metastatic conditions. nm -h serves as a regulator of small gtpases. several evidences have highlighted an involvement of small gtpases (such as rab , rab and rab ) in the biogenesis of exosomes. in addition, bacterial homolog of nm has been shown to interact with rab and rab . however, experimental evidence supporting a relationship between exosomes and nm -h is lacking. our current focus is to deduce the relationship between exosomes and msgs. methods: breast cancer cell lines were used to assess the effect of exosomes isolated from highly metastatic cells (mda-mb- cells) on lower/non metastatic cells (mcf- cells). nme was overexpressed in mda-mb- cells and subsequently used to isolate exosome fractions. equivalent amount of isolated exosome fractions from mda-mb- cells and mda-mb- /nme cells were utilized to access their effect on migration and difference in exosome markers. results: we observed an enrichment of nm -h in the exosomes isolated from mda-mb- cells upon overexpression of nme . proteinase k protection assay confirmed the packaging of nm -h inside the exosomes isolated from mda-mb- /nme cells and excluded the possibility of membrane association of nm -h . additionally, overexpression of nm -h led to a significant reduction in the ability of mda-mb- exosomes to stimulate movement of mcf- cells as confirmed by wound healing assays. our data also highlights a clear reduction in the protein levels of exosome markers such as cd , cd and alix in the exosome fraction isolated from mda-mb- /nme cells as compared to mda-mb- cells. interestingly, rab a, a protein involved in the endosome-lysosome fusion was also present in lower amount in the exosomes isolated from nm -h overexpressing cells. summary/conclusion: our data highlights an antimigratory effect of nm -h via exosomes. these findings support a regulatory role of nm -h in the packaging or release of exosomes in highly metastatic breast cancer cells, and further suggest that metastasis suppressor proteins may be involved in the regulation or packaging of exosomes. additional studies will be required to decipher the downstream signalling of nm -h which affects the biogenesis of exosomes as well as to assess the effect of nm -h overexpression on the content of exosomes. these insights could help us delineate the complex exosome biogenesis pathway and provide new potential drug targets for exosome regulation. introduction: exosomes (exs) are emerging as novel players in the beneficial effects induced by exercise on vascular diseases. our recent study has revealed that moderate exercise enhances the function of circulating endothelial progenitor cell-derived exosomes (cepc-exs) on protecting endothelial cells against hypoxia injury. in this study, we aimed to investigate whether exercise-regulated cepc-exs contribute to the beneficial effects of exercise on ischaemic stroke (is). methods: c bl/ mice performed moderate treadmill exercise ( m/min, -wks) before is induced by middle cerebral artery occlusion surgery. acute injury was evaluated at day by determining neurologic deficit, infarct volume, cell apoptosis in the penumbra and neurologic recovery was assessed by determining angiogenesis/neurogenesis, sensorimotor functions at day . the correlations of cepc-exs and their carried mir- with neurological parameters were analysed. the underlying mechanism of the effects of cepc-exs isolated from exercised mice was explored in a hypoxia neuron model. cellular mir- level, apoptosis, axon growth ability and gene expressions (cas- , bdnf and akt) were measured. results: ) exercised mice had a smaller infarct volume on day , which was associated with decreased cell apoptosis and cleaved cas- level, and a higher microvessel density than those in control; ) the elevated cepc-ex level positively correlated with tepc-exs in ischaemic brain of exercised mice on day . the upregulated mir- level positively correlated with the numbers of tepc-exs in ischaemic brain; ) the numbers of cepc-exs and their carried mir- level negatively correlated with the infarct volume, cell apoptosis and positively correlated with the microvessel density in the peri-infarct area on day ; ) exercised mice had decreased infarct volume, increased microvessel density, promoted angiogenesis/neurogenesis and improved sensorimotor functions on day , accompanying with upregulated levels of bdnf, p-trkb/trkb and p-akt/akt; ) cepc-exs of exercised mice protected neurons against hypoxia-induced apoptosis and compromised axon growth ability which were blocked by mir- and pi k inhibitors. summary/conclusion: our data suggest that the protective effects of moderate exercise intervention on the brain against mcao-induced ischaemic injury are ascribed to cepc-exs and their carried mir- . funding: this work was supported by american heart association ( post ) and nih ( r ns ). syndecan- regulates alveolar type epithelial cell senescence mediating through extracellular vesicles during lung fibroproliferation tanyalak parimon a , changfu yao a , adam aziz a , stephanie bora a , marilia zuttion zuttion a , dianhu jiang a , melanie koenigshoff b , cory hogaboam a , paul nobel a , barry stripp a and peter chen a a cedars-sinai medical center, los angeles, usa; b university of colorado, denver, usa introduction: alveolar type epithelial cell (at ) senescence is implicated in the pathogenesis of lung fibrosis, a progressive fatal condition. syndecan- , a heparan sulphate proteoglycan, is overexpressed by at cells of human idiopathic pulmonary fibrosis (ipf) and bleomycin-injured wt mice and the overexpression of syndecan- is profibrotic. moreover, syndecan- deficient (sdc -/-) mice had less lung fibrosis after bleomycin injury. we reported that extracellular vesicles (evs) in bronchoalveolar lavage (bal) of bleomycin-injured wt mice augmented lung fibrosis whereas the sdc -/--bal-evs attenuated the process. moreover, wt-bal-evs expressed lower level of anti-fibrotic mirnas (mir- b- p, − - p, − - p, and − - p) compared to the sdc -/-bal-evs. these mirnas targeted genes in the cellular senescence pathway indicating that syndecan- altered microrna profiles in the bal-evs to promote cellular senescence during lung fibrogenesis. we investigate how syndecan- regulates at senescence through evs. methods: bleomycin was intratracheally given into wt and sdc -/-mice. at day , lungs were processed for single-cell rna sequencing (scrnaseq) and western blot (wb). evs were isolated using ultrafiltration centrifugation method. human (a ) and mouse (mle- ) lung epithelial cell lines were used for in vitro experiments. results: scrnaseq analysis indicated while bleomycin stimulated an overexpression of cellular senescencespecific genes on at cells of wt mice, these genes were significantly downregulated on sdc -/-at cells. senescence proteins, p and p , were also less expressed in the lungs of sdc -/-than of the wt mice by wb. to determine the functional effects of evs in bal, a cells were treated with human ipf or control lung wash-evs and evaluated for beta-galactosidase activity. we found that ipf-evs markedly increased beta-galactosidase enzymatic activity. corroborating with these data, bleomycin-injured bal-wt-evs also significantly upregulated senescence marker, p , by wb on mle cells whereas sdc -/--bal-evs inhibited p expression. summary/conclusion: our data indicate that syndecan- regulates lung fibrosis through the senescence signalling pathway on at cells. furthermore, syndecan- controls at senescence mediating through extracellular vesicles in the bal. lastly, the most likely cargo molecules mediating this process are micrornas. immortalized cardiosphere-derived cell ev-associated pirna, imev-pi, protects against ischaemic injury in the heart alessandra ciullo, ahmed ibrahim, liang li, chang li, weixin liu and eduardo marbán smidt heart institute, cedars sinai medical center, los angeles, usa introduction: cardiosphere-derived cells (cdcs) are a population of heart-derived progenitors with demonstrated therapeutic efficacy in preclinical and clinical settings. cdcs function by secreting extracellular vesicles (evs), lipid-bilayer nanoparticles laden with bioactive molecules. recently our group developed a strategy for immortalizing cdcs (imcdc) that retains their therapeutic potential and enhances cdc function indirectly through their secreted evs. imcdc show a different rna content(mirna, mrna, rrna, trna and pirna) compared to primary cdc. in particular, we focus on piwi rnas (pirnas), small rnas bound by piwi proteins, important regulators of both the epigenome and transcriptome. we seek to explore the role of a pirna highly enriched in imcdc-evs (imev-pi). methods: evs are prepared by conditioning cells for hrs in serum-free basal media, in hypoxic culture. after hrs conditioned medium is cleared of cellular debris and evs isolated using ultrafiltration by centrifugation (ufc). fractions were analysed in terms of particle size, number, and concentration and pirna content. in vitro, bone marrow derived-macrophages (bmdm) were exposed to imcdc-ev, imev-pi and control and transcriptomic profile and potentially activated pathways were assessed. in vivo, - week-old wistar-kyoto female rats received ^ imcdc-ev, imev-pi, scramble or vehicle intracoronary minutes after ischaemiareperfusion(i/r). cardiac troponin i levels, scar size and monocytes were assessed at and hrs. results: by small-rna sequencing analysis we found that pirnas are enriched in both cdc-ev and imcdc-ev. imcdc show a different pirna composition compared to primary cdc. imev-pi was identified as one of the most highly-expressed non-coding rnas (the number of reads were x higher in imcdc-ev compared to cdc-ev). in vitro, imexo-pi-conditioned bmdm exhibit a different transcriptomic profile compared with control, with upregulation of pathways involved in the inflammatory response, cell death, and cell-to cell signalling. in vivo, imev-pi is cardioprotective, as shown by reduced scar size and lower cardiac troponin levels compared to vehicleand scramble-injected animals at hrs post i/r. imev-pi only minimally alters neutrophil counts profile in blood but it alters monocytes profile with a decreased number at hrs and an increase at hrs. summary/conclusion: we posit that imev-pi is a key determinant of imcdc-ev therapeutic efficacy. our results indicate that target cells may be macrophages/ monocytes, given that imev-pi exposure modifies their composition and mrna profile both in vitro and in vivo. introduction: extracellular vesicles (exosomes, evs) are cell membrane particles ( - nm) secreted by virtually cells. during intercellular communication in the body, secreted evs play crucial roles by carrying functional biomolecules (e.g., micrornas and enzymes) into other cells to affect cellular function, including disease progression and tissue regenerations. literature previously reported that the macropinocytosis pathway contributes greatly to the efficient cellular uptake of evs. the activation of growth factor receptors, such as epidermal growth factor receptor (egfr), induces macropinocytosis. in this study, we demonstrated the effects of evs on demal papilla and hair follicle regeneration. methods: identification of distinct nanoparticles and subsets of extracellular vesicles from umbilical cord blood stem cell by asymmetric flow field-flow fractionation. results: the effects of evs from umbilical cord blood stem cell on the propagation of demal papilla and hair follicle regeneration were observed. summary/conclusion: the enhancement of extracellular vesicles from umbilicalcord blood stem cell the propagation of demal papilla and hair follicle regeneration were observed and confirmed. mechanisms of host resistance to plasma membrane damage induced by pneumolysin attack introduction: bacterial pore-forming toxins (pfts) are major virulence factors produced by pathogens. pfts target host plasma membrane (pm) and create transmembrane pores, which allow uncontrolled flux of ions and small molecules across the pm disrupting cellular homoeostasis. to survive, cells display poorly understood repair mechanisms to recover the cell homoeostasis. several mechanisms were proposed to participate in cell recovery: exocytosis of cortical lysosomes; endocytosis of pfts pores; pm blebbing and shedding. methods: we used increasing concentrations of purified ply to intoxicate cells. pm permeability was assessed by flow cytometry using propidium iodide dye. cytoskeleton rearrangements were investigated by confocal immunofluorescence microscopy. extracellular vesicles released during pm repair were isolated by high-speed centrifugation and characterized by nanoparticle tracking analysis (nta), transmission electron microscopy (tem) and mass spectrometry/ liquid chromatography analysis. results: ply triggers a complete reorganization of the actomyosin cytoskeleton inducing the formation of cortical actomyosin bundles at sites of pm remodelling. these structures assemble upon loss of pm integrity and disassemble as pm recovers. we detected the release of microvesicles during the recovery of pm integrity. vesicle population is heterogeneous with sizes ranging from to nm, with the majority of them measuring - nm. vesicle proteomic analysis revealed that they contain ply, suggesting they participate in pore removal, proteins involved in vesicle trafficking, pm repair and exosome biogenesis. summary/conclusion: our data demonstrate that cells are able to recover from the damage induced by sublytic concentrations of ply. actomyosin cytoskeleton undergo massive changes with the assembly of cortical bundles possibly at sites of pm damage. we showed that cells produced extracellular vesicles during the process of repair. we are now focusing on understanding the biogenesis of those vesicles and its importance during the process of repair. introduction: despite of high expectations, mesenchymal stromal cell (msc)-based therapies still lack efficacy, partially due to loss of cell viability and function upon administration. msc-derived extracellular vesicles (msc-ev) emulate the regenerative potential of msc, shifting the field towards cell-free therapies. clinical applications require the establishment of a scalable and gmp-compliant processes for the production and isolation of msc-ev, combined with robust characterization platforms. methods: to develop a well-established process for the production of therapeutic msc-ev, we compared different msc sources (bone marrow, adipose tissue, umbilical cord matrix), culture media compositions (dmem supplemented with foetal bovine serum (thermo fisher scientific), dmem supplemented with human platelet lysate (aventacell biomedical) and stempro msc sfm xeno free medium (thermo fisher scientific)) and culture parameters (oxygen tension and shear stress) in two different culture platforms ( d static tissue culture flask vs d dynamic spinner vessels). subsequently, msc-ev were isolated by ultracentrifugation or a commercially available isolation kit and characterized according to isev guidelines. results: msc derived from different sources/donors were able to grow under normoxia and hypoxia in d t-flasks and d spinner vessel culture systems, while maintaining their immunophenotype and differentiation potential, according to the minimal criteria defined by the isct. the time point for pre-conditioning and collection of conditioned medium for msc-ev isolation was also optimized for both d and d culture systems. msc-ev were characterized according to misev guidelines, using techniques as nta, protein and lipid quantification, western blot, imaging and fourier-transform infrared spectroscopy (ftir). the results indicate that msc-ev derived from different sources/donors have similar size distribution, however, ev yields tend to be higher for the d culture system. of notice, several spectral regions were identified by ftir, enabling the detection of differences in the biomolecules present in msc-ev, msc-conditioned media and cells produced under different conditions. summary/conclusion: in summary, this study contributes to the establishment of a scalable process for msc-ev production. evaluation of three different isolation methods for small extracellular vesicles from human plasma in prostate cancer diagnosis introduction: extracellular vesicles (evs) have great potential in prostate cancer (pca) diagnosis and progression monitoring to complement the inaccurate prostate specific antigen (psa) screening and invasiveness of tissue biopsy. however, current methods cannot isolate pure evs and therefor evs characteristics remain largely unknown. in order to develop an accurate approach for ev isolation, we aimed to compare three emerging methods with different characteristics of small evs (sevs) from human pca plasma samples and to choose the best one for diagnostic and functional studies. methods: pca patients and age-matched healthy controls (hc) plasma (n = in each group) were used to isolate sevs with different isolation methods including commercial exoquick ultra kit, qev and qev size exclusion chromatography (sec). isolated sev were characterized by nanoparticle tracking analysis, immunoblotting, cyrogenic electron microscopy, flow cytometry (fc) and proteomics analysis. for fc characterizing surface marker expression, the sevs were further purified by cd and cd commercial immunoaffinity magnetic beads . lipoprotein was captured by streptavidin biotinylated apob magnetic beads to measuring the lipoprotein contamination. results: the sev size, morphology, surface protein and protein cargo with proteomics were analysed between the three isolation methods. sevs isolated from sec methods had a lower particle size, protein amount, protein/sev marker ratio and apob+/sev marker ratio than those from exoquick ultra method. in addition, sevs isolated from qev demonstrated a significantly higher sev content, more up-regulated and down-regulated pca proteins from proteomics but lower sev marker/protein ratio and a higher protein contamination than those from qev . furthermore, sev marker signal also showed a good correlation with particle numbers instead of protein content in all the methods. summary/conclusion: qev method demonstrated better performance in isolating relatively pure sevs from human plasma; qev has the better performance in isolating samples with higher sev content; exoquick ultra isolated samples with closely sev content to the qev but with the highest non-sev protein contaminations. people can choose higher sev content or higher sev purity according to the downstream analysis. moreover, sevs may also be used for treatment monitoring, as recent studies suggested that the expression levels of certain markers may change during therapy, reflecting tumour response. for cancer diagnostics and therapeutic purposes in clinical settings, it is important to have a device which allows multiplexed measurements, in order to scan a large number of markers simultaneously and compare the expression levels of different patients, or same patients at different treatment stages, in a time efficient manner. methods: herein, we propose a multiplexed platform for label-free detection and surface protein profiling of sevs. the technique is based on the electrokinetic phenomena of streaming current and zeta potential (\zeta*) and measures the\zeta* change upon sev binding on functionalized microcapillary surfaces. for the purpose, we used sevs derived from lung cancer cells. in its current form, the platform can measure up to channels simultaneously, however, it can be further expanded. results: having demonstrated that our electrokinetic sensor successfully detects sevs in a specific way, we tested its ability to measure the expression level of membrane proteins. the analysis showed that it could detect differences in the expressions of egfr on sevs, with a sensitivity of %. we then extended the platform for multiplexed analysis, by connecting and measuring four capillaries, functionalized with different capture probes, simultaneously. for the purpose, we targeted specific tumour markers, i.e. egfr, and exosomal tetraspanin family proteins, such as cd and cd . the results showed successful multiplexed ev detection. summary/conclusion: being the sensor suitable for multiplexed sev detection, we shall present our investigation on a set of pleural effusion samples collected from a cohort of lung-cancer patients with different genetic makeup. introduction: extracellular vesicles (evs) are released to biological fluids from different tissues and organs and they contain molecules proposed as biomarkers for multiple pathological conditions. however, most ev biomarkers have not been validated due to the lack of sensitive techniques compatible with high-throughput analysis required for routine screenings. using immunocapture techniques, combining antibodies against tetraspanins and candidate tumour-specific markers we have recently optimized several assays that greatly facilitate ev characterization. methods: we have improved flow cytometry and elisa assays, increasing substantially the sensitivity for ev detection. using dls, em and analytical ultracentrifugation, we have characterised the biophysical basis of this enhancement. the final methodology can be performed in any laboratory with access to conventional flow cytometry or elisa reader. results: using combinations of antibodies specific for the tetraspanins cd , cd and cd , it is possible to detect evs in minimal volumes of urine and plasma samples without previous enrichment. additionally antibodies against other less abundant markers, like the epithelial marker epcam, have been used to capture and identify evs directly in minimal volumes of urine or plasma with sensitivity higher than western blot analysis of isolated evs. furthermore, we demonstrate that additives altering the biophysical properties of an ev suspension, increased detection of tumour antigens in these immune-assays. summary/conclusion: the development of sensitive, high-throughput methods, easily translatable to clinical settings, as elisa and flow cytometry described here, opens a new avenue for the systematic identification of any surface marker on evs, even scarce proteins, using very small volumes of minimally processed biological samples. these methods will allow the validation of ev biomarkers in routine liquid biopsy tests. introduction: when ev subpopulations are enriched on antibody microarrays and probed for their surface proteins, the detection signal is biased towards abundant subpopulations as it is dependent on both the protein expression level and the number of evs captured. to address this challenge, we developed a novel normalization approach allowing: ) the estimation of a target signal independent of ev subpopulation size through dye-based ev quantification, and ) the assessment of subpopulation target enrichment relative to the population average by leveraging tim as an unbiased, lipid-based ev capture. here, we investigated the expression of cancer-associated proteins, particularly metastasis-associated integrins (itgs), in breast cancer evs with varying metastatic potential and organotropism. methods: the relative protein enrichment profiles for various ev subpopulations were established from evs of skbr (her +), t d and mcf- (er+pr+), bt and mda-mb- (triple negative) breast cancer cell lines, as well as five mda-mb- -derived cell lines of four different organotropisms (brain, bone, lung, liver) using our custom antibody microarrays with our normalization approach. results: as expected, her was broadly detected in her + skbr evs. interestingly, her -t d and mcf- evs also expressed her where it was highly enriched in its epcam+ subpopulations. itg α , β and β were only found in triple negative and organotropic evs with itg β and β differentially enriched based on the organotropism. the population average of mda-mb- and lung-tropic evs had high expression of itg β , where subpopulations of cd + evs showed positive enrichment while cd + and cd + evs showed negative enrichment. itg α , β and β were absent in the bone-tropic cd + ev subpopulation, a profile atypical in other organotropisms. lastly, egfr was negatively enriched in tetraspanin+ subpopulations in mda-mb- evs, but positively enriched in these subpopulations in organotropic evs, especially for brain-tropism. summary/conclusion: following normalization, we were able to quantify specific protein associations, uncovering a multitude of co-enrichment profiles that characterize specific metastatic and organotropic cell lines. notably, we found enrichment signatures that distinguish between different organotropisms derived from the same parental cancer line. introduction: the tissue microenvironment surrounding tumours is complex and the cross-talk between cancer and non-cancer cells is essential for tumour growth and progression. we have previously shown that heparan sulphate proteoglycans (hspgs), on the surface of prostate cancer evs, are required for delivery of tgfβ and initiation of a disease-supporting fibroblast phenotype. however, hspgs are known to bind numerous growth factors, so here we have explored the repertoire of such proteins tethered to evs by hspgs. methods: evs were isolated from du prostate cancer cell conditioned media by ultra-centrifugation onto a sucrose cushion. vesicular hspgs were modified either by removal of heparan sulphate (hs) glycosaminoglycan (gag) chains using the enzyme heparinase iii (hepiii), or attenuation of hspg core protein expression using shrnas to knockdown specific hspgs within the parent cell. differences in proteins present in control vs modified evs were identified by a sensitive protein array, based on proximity-ligation technology, and selected targets validated by elisa. functional delivery of growth factors by ev-associated hspgs to recipient fibroblasts is being explored using a variety of in vitro techniques. results: proteome analysis identified targets that bind to hs-gag chains, and also different proteins that showed altered expression following the loss of one or more hspgs from evs. using elisa, we have been able to quantify selected candidates on wild type vesicles, some of these are lost following hsdigestion. we were also able to validate proteins on hspg-deficient vesicles. gene ontology analysis suggests that ev hspg-mediated delivery of growth factors is important for control of processes such as angiogenesis, tumour invasion and immune regulation. functional validation of proteins identified is ongoing. summary/conclusion: here we demonstrate that hspgs play a key role in loading of evs with a complex assortment of growth factors, and therefore subsequent ev-mediated growth factor delivery. we anticipate that loss or damage of ev-associated hspgs will result in attenuation of ev induction of a tumour-supporting fibroblast phenotype. introduction: ovarian cancer (oc) is the fifth leading cause of cancer-related death in women, partly due to difficulty in early diagnosis. extracellular vesicles (evs) show promise for use in early diagnostics of oc. here, evs from cervical mucus (cm) of ovarian cancer patients were used for discovery of oc biomarkers for diagnostics. machine learning was used to mine ev mirna data to develop an oc biomarker panel (validation via the cancer genome atlas). examination of the mirna targets reveal that the panel is a sufficiently accurate predictor of oc. methods: evs from the cm of patients ( highgrade serous, low-grade, benign) were isolated for small rna-sequencing. the top differentially expressed mirnas were used in a random forest and "voom" (variance modelling at the observational level) model. unsupervised approaches were used and then vetted against patient symptomology data. a tcga ovarian cancer dataset (n = ) was used for validation. results: an oc biomarker panel of micrornas (voom: . % accuracy; random forest: % accuracy) was generated. the panel consists of members from the mir- family and the mir- family, among others. the mirna targets are associated with molecular functions and pathways specific in oc progression. summary/conclusion: our method has identified ev mirna biomarkers that may be crucial for early, noninvasive detection of oc. data science has been used to develop a feedback system integrating biochemical experiments, smaller datasets, and previously available data to identify and verify a biomarker panel for oc diagnostics. introduction: liver disease has become a significant cause of morbidity and mortality among hiv patients. alcohol exposure can further exacerbate liver damage by activating hepatic stellate cells (hscs), leading to hepatic fibrosis or cirrhosis, often seen at all levels of alcohol exposure among people with hiv. due to the potentiating effects of alcohol on hiv-induced hepatocytes (hep) damage, as well as the effect of ethanol in hsc-mediated extracellular remodelling, it is imperative to understand the interplay of hep and hscs. here, we focus on the exosomes released by hiv-and ethanol exposed hep and how these exosomes modulate the functional behaviour of hscs. methods: human hepatocyte huh . cyp e [hepatoma cells stably transfected with cyp e designated as rlw cells] were infected with hiv in the presence or absence of alcohol metabolite, acetaldehyde using the acetaldehyde-generating system (ags). the conditioned medium was collected from groups of cells: untreated, hiv-, ags-and hiv+ags. quantification of exosomes number and size were evaluated with zetaview or nanosight and further characterized for exosome markers following the guideline from minimal information for studies of evs (misev ). the human hepatic stellate lx- cell line was exposed to hepatocyte-derived exosomes and assessed for the activation using pro-inflammatory markers il- β, il- , tnfα, and fibrotic markers acta , and timp using quantitative pcr. we also analysed exosome mirna content in primary human hepatocytes (phh), which potentially regulates the function of recipient cells by "programming" their inflammation/fibrosis status. the network analysis for mrna and mirna were carried out using gene ontology consortium, and mirror . and david bioinformatics resources . . results: ags treatment further enhanced the release of hiv-induced exosome from hepatocytes. size distribution assessed by zeta view or nanosight revealed that approximately - % of particles distributed in the range of to nm, with a peak at~ nm. enriched expression of hiv protein p was observed in fractions f -f . western blotting of hepatocytederived exosome demonstrated positivity for exosome-enriched proteins alix, tsg and cd specifically in f -f fractions and negative for endoplasmic reticulum protein calnexin. the uptake of hepatocytederived exosomes by hscs was apparent as demonstrated by immunofluorescence. the internalization of hepatocyte-exosome induced activation of hscs as evidenced by increased expression of pro-inflammatory il- β, il- , tnfα markers in the latter cells. summary/conclusion: we conclude that ags treatment in hiv-infected hepatocytes potentiates the release of exosomes, which, following uptake by the hscs, leads to their activation. funding: this work is supported by nih- r aa - a . antimicrobial peptide ll- induces neutrophil-derived extracellular vesicles with antibacterial potential and protects murine sepsis yumi kumagai, taisuke murakami, kyoko kuwahara and isao nagaoka juntendo university, bunkyo-ku, japan introduction: extracellular vesicles (evs) released from immune cells or other host cells upon microbial infection modulate the immune response and thereby regulate the infection. sepsis is a life-threatening multiple organ dysfunction caused by systemic dysregulated inflammatory response to infection. nevertheless, numerous therapeutic trails concerning immune dysfunction have still been disappointing outcomes. we have previously shown that ll- , a human cathelicidin antimicrobial peptide, improves the survival of caecal ligation and puncture (clp) septic mice. here, we investigated the induction of ev release by ll- and functions of ll- -induced evs in murine sepsis. methods: evs were isolated from peritoneal exudates of clp mice and the supernatant of ll- -stimulated mouse bone marrow neutrophils by differential centrifugation or size exclusion chromatography. isolated evs were analysed by flow cytometry, western blotting, and nano particle analysis. neutrophil-derived evs were injected into clp mice to assess the protective function of evs against septic mice. the antibacterial activity of evs was evaluated by incubating with escherichia coli. results: in clp mice, ll- augmented the level of evs. evs from ll- -injected clp mice contained higher amounts of neutrophil-derived antibacterial proteins (lactoferrin and cramp, cathelicidin-related antimicrobial peptide) and exhibited higher antibacterial activity compared to evs from pbs-injected clp mice. furthermore, ll- stimulated mouse bone marrow neutrophils to release evs with antibacterial potential, and administration of the ll- -induced evs reduced the bacterial load and improved the survival of clp mice. summary/conclusion: ll- induces the release of antimicrobial evs from neutrophils in clp mice, thereby reducing the bacterial load and protecting mice from lethal septic condition. identification of mirna profiles of serum exosomes in active tuberculosis introduction: tuberculosis (tb) has exceeded hiv as the most lethal infectious disease globally for two consecutive years, mainly due to difficulties in achieving early and definitive diagnosis, and timely treatment. exosomes carrying rna, particularly mirna, have demonstrated their functional and diagnostic potential in diseases including tb. however, few published studies have explored whether exosomal mirnas could be used for diagnosis of tb. thus, more systematic and comprehensive study of exosomal mirnas with regard to their potential as non-invasive tb biomarkers is still urgently needed. methods: we searched the gene expression omnibus database for datasets published before december , and performed meta-analysis on available exosomal mirna profile data for healthy control (hc) and active tb clinical specimens . reprocessing next generation sequencing data under uniform parameters and utilizing state-of-the-art bioinformatics analysis. results: we identified many distinct up-regulated and down-regulated differentially expressed exosomal mirna across multiple studies, and further screened the top , which might provide a potential panel for differentiation of hc and tb. we classified all differentially expressed mirnas into six expression patterns and identified two persistently up-regulated mirna (hsa-mir- - p, and hsa-mir- - p) as potential markers during tb progression. moreover, the differential expressed exosomal genes that we screened from the datasets were consistent with the genes overlapped with predicted mrna targets of differentially expressed mirna. pathway and function analysis further demonstrated down-regulated signalling pathways/immune response and up-regulated metabolism and apoptosis/necrosis. introduction: trypanosoma cruzi is a protozoan parasite that causes chagas disease, a relevant source of morbidity in latin america, which has spread to many countries as result of immigration of the people from endemic areas. many studies have been showed that trypomastigote forms of t. cruzi release extracellular vesicles (ev) that increase parasite infection. objectives. here, we aim to test if previous immunization with evs in adjuvant can generate a protective immune response by decreasing the effects of evs in experimental chagas disease. methods: female balb/c mice were immunized by intra peritoneal (ip) administration with × or evs isolated from trypomastigotes forms, with aluminium hydroxide adjuvant (aloh). injections were administered intravenous in doses during days ( days interval). after immunization, mice were infected intra-peritoneally with trypomastigotes forms. parasitaemia was quantified by counting motile parasites in fresh blood sample drawn from lateral tail veins. mortality and weight were analysed during the infection. in control group, the mice were immunized with aioh. results: the immunization with evs with aloh decreased the blood parasitaemia and the animals survived, while all animals died in the group aloh alone. the animals immunized with evs had an increase of f / + cd b+ and cd /cd expression in cells isolated from the peritoneum. summary/conclusion: these results indicate that t. cruzi ev antigens can induce an immune response that controls the development and establishment of the experimental chagas disease. introduction: acinetobacter baumannii (ab) is a nosocomial pathogen, of major concern due to its multidrug resistance (mdr) and the recent appearance of hyper-virulent strains in the clinical setting. the world health organization included ab as a critical priority pathogen for the development of novel antibiotics. ab pathogenesis is associated with a multitude of potential virulence factors (vf) that remain poorly characterized. there is growing evidence that outer membrane vesicles (omv) are used as vehicles to transport bacterial proteins that contribute to set up the conditions for the infections. in the present work we studied the physiopathology of mdr ab. we focused on the contribution of non-characterized outer membrane proteins (omps) associated to omvs, with special focus on lipoproteins (lp). methods: we conducted a bioinformatic prediction using available datasets to construct a list of omv-associated omps putatively acting as vf in ab . seven genes were selected and the corresponding mutants were obtained from manoil lab collection. physiological analyses of the mutants were performed, and the involvement of the selected proteins in ab pathogenesis was evaluated by adherence, invasion, and cytotoxicity assays on human lung cells a . results: biochemical analysis indicated similar growth rates in rich media, as well as similar levels of omv production for all the mutants as compared to wt. also, no differences in susceptibility to chaotropic agents were observed, indicating no alteration of the om function as a general permeability barrier. all mutants similarly reduced a cell viability, but to a lesser extent than the wt. moreover, three of them exhibited less adhesion and invasion compared to the wt, and omv isolated from these mutants displayed variable levels of cytotoxicity. summary/conclusion: these results suggest roles for the mutant gene products in ab pathogenesis and contribute to the better understanding of ab virulence mechanisms, revealing novel possible targets for therapeutic development. funding: agencia nacional de promoción científica y tecnológica (anpcyt, pict - ) medicine, nanfang hospital, southern medical university, guangzhou, , china, guangzhou, china (people's republic); d zhujiang hospital, southern medical university, guangzhou, china, guangzhou, china (people's republic) introduction: talaromyces marneffei (t. marneffei) grows as a mycelial form in the environment but multiplies rapidly as a yeast form in the host and within macrophages. the yeast can cause disseminated and progressive infections or lethal talaromycosis. but the mechanisms of pathogenicity of t. marneffei are poorly understood. fungal extracellular vesicles (evs) have previously been shown to transmit a proinflammatory message to macrophages. however, the characteristics and effects of t. marneffei evs on the progress of infection have not yet been investigated. methods: in this study, evs of t. marneffei yeasts were isolated by ultracentrifugation method. evs were detected and confirmed by electron microscopy and nanoparticle tracking analysis (nta). the raw . murine macrophages were incubated with the t. marneffei vesicles to observe the changes of macrophage morphology and function, especially in inflammatory response. the proteins, dnas, rnas of t. marneffei vesicles were respectively removed with protease, dnase and rnase. all treated evs were used to incubate with murine macrophages observe the effect on macrophages in inflammatory response. results: we observed that evs secreted by t. marneffei have a typical spherical shape with a diameter of to nm. t. marneffei evs were internalized by raw . murine macrophages and promoted the production of no and proinflammatory cytokine by macrophages in a dose-dependent manner. t. marneffei evs stimulate macrophages to generate reactive oxygen species (ros). addition of t. marneffei evs to macrophages also promoted transcription of the m -polarization marker cd and diminish that of the m markers cd . incubation of t. marneffei vesicles with murine macrophages resulted in increased levels of extracellular interleukin- β(il- β), interleukin- (il- ) and interleukin- (il- ). the proinflammatory effect of vesicles was weakened when the proteins of the vesicles were destroyed. in contrast, no similar changes were observed in degraded dna and rna. summary/conclusion: our results indicate that the extracellular vesicles of t. marneffei can stimulate macrophage towards to m polarization phenotype and promote proinflammatory function. plasma-derived extracellular vesicles as potential biomarkers in chronic chagas disease patients introduction: chagas disease (cd), caused by the parasite trypanosoma cruzi (t. cruzi), is a neglected tropical disease affecting about million people worldwide. currently, one of the main clinical problems is the lack of effective biomarkers for therapeutic response and disease prognosis during chronic infections. in that context, extracellular vesicles (evs) are raising attention as novel, minimally invasive, and inexpensive method for diagnostic and screening of diseases, as well as a new source to identify new biomarkers. the main objective of this study is to use evs derived from biological fluids of chronic cd patients for identifying novel biomarkers, specifically in the context of therapeutic response and disease prognosis. methods: plasma, saliva and urine from a cohort of chronic cd patients are being collected before and at the end of benznidazole treatment. as negative controls, healthy donors have been also included. the purification and characterization of the evs was performed by size exclusion chromatography, followed by nanoparticle tracking analysis, bead-based flow cytometry assay and transmission electron microscopy. a proteomic analysis of the evs was also performed. results: proteins associated with evs secreted by infective t. cruzi have been previously identified in cell culture, but never in human samples. our results, based on the analysis of a single heart-transplanted patient with chronic cd, showed the presence of t. cruzi and human proteins specifically associated with plasma-derived evs. noticeably, several human and parasite proteins identified in evs obtained from plasma samples, were present or upregulated before chemotherapy and were absent or downregulated following treatment. currently, proteomics analyses are being performed with higher numbers of cd plasma samples. summary/conclusion: to the best of our knowledge, this is the first proteomic profiling of plasma-derived evs from a heart-transplanted patient with chronic cd. these results thus open the possibility of using evs from biological fluids as a tool for the identification of new biomarker candidates in chronic cd. these biomarkers are essential for assessing disease introduction: eukaryotic cells communicate with one another through multiple pathways. an established route of communication between eukaryotic cells is via the production of a range of different membrane bound signalling "packages", called extracellular vesicles (evs). evs are produced by all domains of life and carry proteins, nucleic acid (rna and dna), and other biological material, travelling between cells and around the body to deliver a range of chemical messages. bacteria can also produce evs that communicate with each other to coordinate population behaviour, as well as with eukaryotic cells to stimulate host defence or induce tolerance. here i investigate the poorly explored axis where evs are the vehicle for communication between eukaryotic cells and bacteria. methods: as a first step, i have isolated evs from tissue cultured eukaryotic cells grown in advanced rpmi media with minimal ev-depleted fbs. nanoevs were isolated from spent culture media using sequential centrifugation ( , × g, , × g) and concentration ( kda filter) before purifying using size exclusion chromatography columns. nanoev-rich fractions were pooled based on particle (nanoparticle tracking analysis) and protein quantity data. nanoevs were characterised by electron microscopy and expression of exosomal markers. eukaryotic nanoevs were then characterised in their effect upon the growth of escherichia coli as a model bacterium, also grown in tissue culture media to mimic relevant in vivo conditions. results: further experiments with increased dosages are required to determine the effect of human evs on bacteria. summary/conclusion: our work will investigate whether human evs communicate with the resident and pathogenic microbiota, while examining the mechanisms behind this communication. escherichia coli pathogenic bacteria commensal bacteria hydrogen sulphide (h s) derived extracellular vesicles: a potential protective role in response to respiratory syncytial virus (rsv) infection methods: evs were isolated from untreated (control evs) and gyy treated (gyy-evs) a cells, a human alveolar type ii-like epithelial cell line. evs were purified using a two-step enrichment procedure. evs were characterized using particle sizing (size and concentration) and western blot for the ev markers. electron microscopy and immunofluorescence staining were used to investigate presence of multivesicular bodies (mvbs), evs precursors, in both groups. recipient a cells were cultured for hours in the presence or absence of control-or gyy-evs, then infected with rsv for hours. viral titres by plaque assay were measured in recipient infected a cells. results: we confirmed the presence and purity of our evs. we found that gyy reduced the particles number of evs, but did not change ev size. a cells treated with gyy showed an accumulation of mvbs/lysosomes-like structures, as well as an increase in cd expression, a mvbs marker, compared to untreated cells. recipient a cells treated with gyy-evs showed lower viral replication than control ev-treated cells in response to rsv infection. we are currently investigating the potential mechanism for this observation and characterizing the rna cargo composition of gyy-evs. summary/conclusion: no vaccine or effective treatment is currently available for rsv. cellular pretreatment with gyy-evs reduced the rsv replication in airway epithelial recipient cells, suggesting that h s could exert its antiviral activity in the context of rsv infection potentially through modulation of ev composition. therefore, gyy-evs could represent a future novel pharmacological approach for ameliorating virus-induced lung disease. effects of extracellular vesicle-mediated transmission on reoviridae infection results: taken together, these data suggest that multiple particles of reovirus and rotavirus egress in large, virus-modulated evs, and that transmission in evs increases segment complementation compared to transmission as free particles. summary/conclusion: these discoveries may be broadly applicable to viruses that travel in evs and will contribute to general principles of virus transmission and diversification. continued studies will illuminate the specific cellular pathways reovirus and rotavirus utilize for successful egress. these pathways may prove to be critical targets for the improvement of vaccines and oncolytic therapy. multiparameter flow cytometry analysis of the human spleen and its interaction with plasma-derived evs from plasmodium vivax patients introduction: the spleen is a secondary lymph organ that filters blood and elicits immune responses against blood-borne pathogens, such as malaria parasites. extracellular vesicles (evs) are membrane-bound particles involved in intercellular communication. evs play several roles in malaria ranging from modulation of immune responses to induction of vascular alterations. here, we report the first integrated characterization of human spleen cells using multiparameter flow cytometry (mfc) describing subpopulations of splenic leukocytes and red blood cells (rbcs), and studied their interaction with plasma-derived evs from p. vivax patients (pvevs). methods: human spleens were obtained from organ transplantation donors. myeloid, lymphoid, erythroid and haematopoietic stem cells (hscs) were immunophenotyped by mfc. t cells, dendritic cells (dcs) and rbcs were enriched by density centrifugation and immunomagnetic isolation. pvevs and healthy donors evs (hevs) were purified by size-exclusion chromatography (sec) and characterized by bead-based flow cytometry. enriched evs were labelled with fluorescent lipophilic dyes and incubated with total splenocytes or enriched populations. evs-cells interaction was assessed by flow cytometry. results: human spleen immunophenotyping showed that cd + cells included b ( %), cd + t ( %), cd + t ( %), nk ( %) and nkt ( %) lymphocytes. myeloid cells comprised neutrophils ( %), monocytes ( %) and dcs ( . %). erythrocytes represented % whereas, unexpectedly, reticulocytes were . % of total cells. in addition, we also detected hscs, which accounted for . %. sec separated evs from the bulk of soluble plasma proteins as shown by the enrichment of cd , cd l and cd markers. interaction studies showed an increased proportion of t cells (cd + -fold and cd + -fold), monocytes ( . -fold) , b cells ( . -fold) and erythrocytes (threefold) interacting with pvevs as compared to hevs. summary/conclusion: the integrated cellular analysis of the human spleen and the methodology employed here allowed in vitro interaction studies of human spleen cells and evs. a larger proportion of monocytes, t and b lymphocytes as well as erythrocytes was found to interact with pvevs compared to hevs. future functional studies of these interactions can unveil pathophysiological processes involving the spleen in vivax malaria. neuroblastoma-secreted exosomes carrying mir- promote osteogenic differentiation of bone marrow mesenchymal stromal cells introduction: bone marrow (bm) is the major target organ for neuroblastoma (nb) metastasis and its involvement is associated with poor outcome. yet, the mechanism by which nb cells invade bm is largely unknown. tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (mscs) have been recognized as a fundamental part of the associated tumour stroma. here, we explore the potential role of nb-derived exosomes in induction of a pro-osteogenic phenotype on bm-mscs. introduction: extracellular vesicles (evs) are nanosized particles delimited by a lipid bilayer which transfer functional molecular cargos from the cells of origin to target cells. this intercellular crosstalk controls both physiological and pathological conditions. given their presence in body fluids and their characteristics, these nanocarriers might be potentially used in diagnostics and/or therapy. breast cancer is the most frequently diagnosed malignancy and ranks as the leading cause of cancer mortality in women worldwide; the triple negative breast cancer, in particular, is the most aggressive subtype with a poor prognosis. since it is recognized that cell stiffness of cancer cells play a crucial role during the metastatic spreading, we set ourselves the goal of clarify the effects and the activity of small-evs (i.e. with a diameter below nm) in metastatic breast cancer, with a special attention on their correlation with the biomechanical properties of cells. methods: functional assays were performed on the non-invasive mcf breast cancer cell line, before and after the cellular uptake of small-evs originating from the invasive mda-mb- triple negative breast cancer cell line. the mechanical properties (cell stiffness, cytoskeleton organization and focal adhesions) of mcf cells were investigated before and after the vesicle uptake. results: the uptake of small-evs derived from mda-mb- significantly reduces the young's modulus values of mcf cell line making them more invasive. moreover actin and focal adhesion variations were observed in mcf cells before and after small-ev's uptake, suggesting a molecular rearrangement inside mcf cells upon uptake. summary/conclusion: our results evidence that small-evs play a key role in altering biomechanical properties of target cells and underline their relevance in cell-cell crosstalk. our approach is very promising to identify new molecular mechanisms through which evs perform their oncogenic function. stratification of angiogenic or non-angiogenic lesions in colorectal cancer liver metastases patients using extracellular vesicle mirna introduction: colorectal carcinoma (crc) is the second leading cause of cancer death in the western world. over % of the crc patients develop liver metastasis (lm) and % will die from metastatic disease. in the current clinical setting, liver resection provides the only possible cure, but only % of crclm patients are resectable. the combination of angiogenic inhibitors with chemotherapy is used to downsize crclm with the goal of converting unresectable patients to resectable ones. however, only - % of these patients can be successfully converted to a resectable state. we have no way of identifying those crclm patients that would respond/benefit to the addition of anti-angiogenic therapies (e.g. bevacizumab: bev)). proper stratification of patients into angiogenic inhibitor responders and non-responders will permit a proper assessment of the efficacy of angiogenic inhibitors. crclm forms distinct histopathological growth patterns (hgp): angiogenic (desmoplastic) and nonangiogenic (replacement) hgp. we demonstrated that crclm patients with predominant angiogenic lesions receiving bev plus chemotherapy have a more than double -year overall survival compared to patients with non-angiogenic lesions. therefore, nonangiogenic lesions do not respond to angiogenic inhibitors. our study focuses on stratifying angiogenic vs non-angiogenic lesions of crclm through extracellular vesicle mirnas. we are using two approaches in the selection of mirnas to target: . text mining of published ev mirna from crclm patients; and . differentially expressed mirnas present in tumour tissue from both lesion types, we have obtained by sequencing - patients. these two strategies will generate a list of mirnas that we will target using qpcr on plasma-derived ev mirna from the patients used in approach , where we have classified the lesions in the patients. preliminary data on patients will be presented. methods: ev isolation was performed using the gold standard centrifugation method. rnaseq and qpcr are used to generate the expression profile for angiogenic vs non-angiogenic type of crclm. results: the research is under progress. summary/conclusion: the research is under progress. the introduction: it is known that bone metastasis causes a reduction in the quality of life of cancer patients due to fractures and nerve compression. therefore, it is important to elucidate the mechanism of bone metastasis and develop new treatments. metastatic bone tumours occur at particularly high rates in cancers of the prostate, breast, and lung. in this study, we focused on extracellular vesicles (evs) in bone metastasis, and investigated that the role of evs derived from cancer cells in osteolysis. methods: the prostate, breast, and lung cancer cellderived evs were added to osteoclast precursors with rankls. the osteoclast differentiation was evaluated by tartrate-resistant acid phosphatase (trap) stain and by measuring the expression level of osteoclast markers using by qrt-pcr. a proteome analysis (lc-ms/ms) and sirna approaches were used to identify molecules which are responsible for promotion of osteoclast differentiation in the prostate cancer cellderived evs. to investigate whether the molecules are suitable for the detection of bone metastasis in serum evs, we isolated evs from serum of prostate cancer patients, and analysed the protein level of the molecules by western blot analysis. results: we found that the prostate cancer and lung cancer-derived evs significantly promoted the rankl-stimulated osteoclast differentiation. our analysis revealed that cub domain-containing protein (cdcp ), which is a membrane protein on the prostate cancer cell-derived evs, was responsible for promotion of osteoclast differentiation. moreover, cdcp was markedly detected in the evs-derived from serum of prostate cancer patients who had bone metastasis than that of normal subjects. we also found that cdcp exits on the breast and lung cancer cell-derived evs. summary/conclusion: we showed that the evsderived from bone metastatic tumours have a role in activation of osteoclastogenesis. moreover, we revealed that cdcp in the evs is responsible for promoting of osteoclast differentiation. these evs could be the novel diagnostic and therapeutic target for bone metastasis. increased expression of chemokine receptor cxcr in non-invasive colorectal cancer cells after incorporation of platelet-derived extracellular vesicles. introduction: blood platelets and platelet-derived extracellular vesicles (p-evs) play a crucial role in tumour growth and metastasis. p-evs, also referred to as platelet microparticles, are recognized as a carrier for proteins and nucleic acids that control cell-to-cell communication, mediate the formation of metastatic niches and affect tumour invasion and metastasis. among the other factors, p-evs contain the chemokine receptor cxcr , known as a co-receptor for hiv entry but also regarded as important in cancer development due to the importance of cxcr /cxcl signalling. overexpression of cxcr was reported in various, especially in invasive cancers, including colorectal cancer (crc). crc, the third most commonly diagnosed cancer, is usually diagnosed at the late stage and patient's death is mainly related to metastasis. increased levels of cxcr has been reported as a poor prognostic factor for survival of crc patients and its blocking has been suggested as therapeutic approach. the aim of this study was to analyse the effect of p-evs on the levels of cxcr in crc cells on various epithelial-to-mesenchymal transition stage. methods: we used crc cell lines ht and sw , which represent distant invasive potential and different phenotypes, epithelial and strongly mesenchymal, respectively. p-evs were isolated from outdated concentrates of human blood platelets after activation by thrombin in the presence of calcium ions, by subsequent centrifugation and ultracentrifugation. the p-evs were labelled using pkh fluorescent dye to visualize their uptake into cell lines by confocal microscopy. we also quantified the levels of cxcr in ht and sw by western blot analysis. the effect of p-evs uptake on the migration of crc cells was studied by "wound healing" method. results: we found that the levels of cxcr in crc lines used in the study were correlated with their emt stage. we show here that p-evs released by activated platelets were incorporated into both ht and sw cell lines. the expression of cxcr in ht was increased after the uptake of p-evs. additionally we observed that migration rate of ht cells with incorporated p-evs was elevated as compared to control cells. summary/conclusion: we posit that circulating p-evs can be incorporated into yet not invasive crc cells to significantly increase the level of cxcr receptors and that may lead to the their more invasive characteristics. introduction: for cancer therapy it is important to identify markers and key processes induced during cancer progression. one of them is epithelialmesenchymal transition (emt) which is associated with cell acquisition of invasiveness, stem cell characteristics and resistance to apoptosis and therapy. also the extracellular vesicles (evs) released from tumour cells, which can be taken up by cells constituting pre-metastatic niches, can alter cancer progression by promoting cells' reprogramming. our group has recently reported that snail transcription factor, a key factor of emt, when overexpressed in crc ht cells, drives their early emt and alters the expression of microrna (mirs). in the present study we analysed the mirs profile of evs released from those cells. methods: evs from three ht clones stably overexpressing snail and from control ht -pcdna were isolated by differential centrifugation and ultracentrifugation of conditioned media after h of culturing in serum-free medium. total rna was isolated and nextgeneration sequencing (ngs) analysis of the mirnas was performed followed by gene ontology ( introduction: prostate cancer (pca) is the most common malignant tumour in male urinary system and osteoblastic bone metastasis is the most observed metastasis in prostate cancer patients. it has been demonstrated that circulating micrornas contained in extracellular vesicles are potential early biomarkers and therapy targets for many diseases. however, the potential role of micrornas in prostate cancer bone metastasis, is not yet to be fully explored. methods: after isolation and purification evs using ultracentrifugation from conditioned media of bone metastatic co-opting prostate cancer cells and normal cells, total rna was extracted. subsequent to library preparation and small rna-seq, differential gene expression analysis was performed. data were filtered by mean mirna expression of ≥ reads, two fold up or down regulation between . − . and adjusted pvalue ≤ . . the uptake of pca-sevs was performed. three candidate mirnas (has-mir- c- p; has-mir- ; has-mir- - p) were internalized and osteoblast differentiation were detected by qpcr, histochemical staining and protein activity detection. results: total reads of mirnas in bone metastatic co-opting pca-evs exceeded significantly than that in normal evs (p < . ), indicating that mirnas delivered by pca cells play critical role in pca bone metastasis. pca-cm enhanced osteoblast differentiation and can be reversed by gw . the uptake of pca-evs by mc t -e was efficient. the high expression of the three candidate mirnas in pca-evs was verified by qpcr. all the three candidate mirnas promoted osteogenesis, verified by mrna expression of osteoblastic markers (alp, ocn, runx , osx), alp activity, alp staining and aliza red s staining. summary/conclusion: these findings suggest that mirna cargos in pca-evs play a pivotal role in the development of osteoblastic bone metastasis of pca, which can be potential early biomarkers and therapy targets for prostate cancer bone metastasis. funding: this work was supported by grants from the national natural science foundation of china ( ); xijing hospital science and technology foundation project (xjzt ptk ). introduction: retinoblastoma (rb) is the most common intraocular cancer of childhood. despite recent advances in conservative treatment have greatly improved the visual outcome, local tumour control remain difficult in presence of massive vitreous seeding. thus, the identification of new biomarkers is crucial to design more effective therapeutic approaches. traditional biopsy has long been considered unsafe in rb, due to the risk of extraocular spread. exosomes, nano-sized vesicles containing nucleic acids and proteins, represent an interesting alternative to detect tumour-associated biomarkers. the aim of this study was to determine the protein signature of exosomes derived from rb tumours (rbt) and vitreous seeding (rbvs) primary cell lines. methods: exosomes from rbt (hsjd-rbt , hsjd-rbt , hsjd-rbt , hsjd-rbt ) and rbvs (hsjd-rbvs , hsjd-rbvs , hsjd-rbvs ) cell lines were isolated by high speed ultracentrifugation. vesicles number and size were confirmed by nanosight and scanning electron microscopy. protein content was analysed by bicinchonic-acid assay and high resolution mass spectrometry. results: a total of proteins were identified. among these, and were expressed in exosomes rbt and one rbvs group respectively. gene enrichment analysis of exclusively and differentially expressed proteins and network analysis identified identified in rbvs exosomes upregulated proteins specifically related to invasion and metastasis such as proteins involved in extracellular matrix (ecm) remodelling and interaction, resistance to anoikis and metabolism/catabolism of glucose and aminoacids. summary/conclusion: in conclusion, in this study, we isolated exosomes from rb primary tumour and vitreous seeding cell lines and characterized their content with a proteomic approach. this is the first evidence describing a proteomic exosome signature specifically associated with vitreous seeding in rb. this characterization may represent a starting point for future analyses that allow defining exosomal markers as promising diagnostic and potential prognostic markers in rb as well as therapeutic targets. activation of hepatic stellate cells by extracellular vesicles released by uveal melanoma cells introduction: uveal melanoma (um) is the main intraocular tumour in adults, and is particularly resistant to treatments when disseminated to the liver. our hypothesis is that extracellular vesicles (evs) released by the primary tumour are priming the liver stroma for metastatic cell colonization by activating hepatic stellate cells (hstecs). this study aimed to characterize evs from um cells, and to determine their interactions with liver cells. methods: evs were isolated from cell lines derived from ocular tumours and liver metastases by differential centrifugation. their concentration/diameter range were determined by high-sensitivity flow cytometry. cryo-tem combined with receptor-specific gold labelling was used to reveal the morphology/size of melanomic evs. the presence of melanoma and ev markers was assessed by western blotting. the internalization of fluorescent melanomic evs in hstecs and their subsequent activation were assessed by confocal imaging using alpha-smooth muscle actin (alpha-sma) and phalloidin stainings. ev impact on invasion was measured with a tumour spheroid model embedded in extracellular matrix. melanomic evs were inoculated into the retro-orbital sinus of immunodeficient mice to study their selective organ distribution. results: melanomic evs were positive for annexin- , tetraspanins, as well as some melanoma markers. stellate cells with internalized melanomic evs expressed more alpha-sma, reflecting their activation. adding evs on tumour spheroids increased the invasion process. melanomic evs were localized into different murine organs, but mainly into the liver, as observed by in vivo fluorescent imaging. introduction: exosomes are being tested for their use as therapeutic agents in degenerative and chronic diseases. however, the optimal source of exosomes is currently under investigation. amniotic fluid (af) is a naturally-rich source of exosomes that is easily obtained for use in regenerative medicine. organicell flow™ is a minimally-manipulated, acellular product derived from human af and consist of over cytokines/chemokines as well as exosomes derived from the amniotic membrane and surrounding tissues. we characterized the exosome fraction of our product to elucidate the protein cargo of af exosomes and demonstrate the therapeutic potential as a novel regenerative therapy. methods: the exosome fraction of our product was analysed using nanosight nanoparticle imaging and macsplex exosome surface marker array analysis. exosomes were precipitated using size-exclusion filtration followed by ultracentrifugation from independent products (in triplicate) and subjected to protein lysis and preparation for mass spectrometry analysis using the easy nlc and q exactive instruments. tune (version . ) and xcalibur (version . ) was used to collect data while proteome discoverer (version . ) was used to analyse data. protein expression lists were created by merging the sample replicates together and commonly expressed proteins were determined using vinny . vin diagram analysis. webgestalt tool kit classification system was used to identify top protein function and pathway hits. results: organicell flow™ contain a mean concentration of . x ^ particles/ml (n = ) with a mean mode size of . nm (n = ). surface marker analysis confirms the presence of exosome associated proteins cd , cd , and cd in addition to a high expression of cd (n = ). the completed analysis revealed commonly detected proteins across products. the top molecular functions of identified proteins included protein-binding, ion-binding, and nucleic acid-binding with enzymes, transcription regulators, and transporter proteins representing the most abundant protein groups. pathway enrichment analysis revealed top hits for integrin, pdgf, and p pathways. a deeper dive into the enzyme category of the protein cargo further demonstrates the presence of proteins that promote dna repair such as dna polymerase (beta and lambda), telomerase reverse transcriptase, and brca . summary/conclusion: organicell flow™ characterization demonstrates the therapeutic potential of afderived exosomes. proteomic analysis revealed protein cargo that may regulate various growth factor and cellcycle associated pathways. furthermore, the presence of dna damage response proteins suggests a possible mechanism for induction of cellular repair. generation of car-t and γδt cell-derived exosomes for future cell free immunotherapies γδt cells are a subset of t cells with dual innate and adaptive qualities. this duality provides various advantages over their more studied and used counterpart, αβt cells. in the present study, we sought to compare the immunotherapeutic potential of car-t cell and γδt cell-derived exosomes as novel cell-free based alternatives. methods: cd -targeting car-t cells were obtained following the isolation, expansion and transduction of αβt cells using a lentiviral vector bearing the car construct. γδt cells were isolated and expanded from peripheral blood mononuclear cells (pbmcs) following innate or adaptive stimulation. exosomes from both cell sources were isolated after a -day culture in serum-free media using ultracentrifugation-based methods. exosomes were characterized by nanoparticle tracking analysis (determination of size) and western blot assays (detection of the appropriate surface markers). nalm- (b cell precursor leukaemia) cells were used as target cells for assessment of exosome cytotoxic/ killing function. car-t cell and γδt cell-derived exosomes were incubated at particles/target cell for -hours. total viable cell counts were assessed via imaging-based cytometry (nc- ) utilizing acridine orange and dapi staining. results: exosomes derived from γδt cells activated via innate mechanisms showed significant killing of nalm- as compared to exosomes from non-activated or adaptively activated γδt cells. in comparison, car-t cell-derived exosomes showed minor killing capabilities of the target cells. summary/conclusion: here, we report for the first time that exosomes derived from cd car-t cells and innately activated-γδt cells show/exert inhibitory action on nalm- cells. further studies are currently underway to identify the underlying mechanism(s) responsible. introduction: age-related cognitive dysfunction is associated with increased oxidative stress, low-level chronic neuroinflammation, and waned hippocampal neurogenesis in the brain. from this perspective, biologics capable of modulating oxidative stress and neuroinflammation, and stimulating neural stem cell activity in the brain might be useful as anti-ageing interventions. methods: we investigated the efficacy of intranasal administration of extracellular vesicles (evs) generated from cultures of rat subventricular zone neural stem cells (svz-nscs) in the middle-aged mice to alleviate cognitive and mood dysfunction, increased oxidative stress, neuroinflammation, and neurogenesis decline in old age. mice were treated intranasally with nsc-evs once weekly for three weeks ( billion per administration) starting from . months of age. a month later, the animals were examined for cognitive, memory, and mood function using multiple behavioural tests, and brain tissues were examined for oxidative stress, neuroinflammation, and neurogenesis. results: object-based tests revealed that aged animals receiving vehicle displayed cognitive impairments for discerning minor changes in the environment as well as for distinguishing similar but not identical experiences. these animals also exhibited spatial memory dysfunction and anhedonia. in contrast, aged animals receiving nsc-evs showed improved cognitive and mood function. biochemical analyses of brain tissues revealed that nsc-ev treatment normalized elevated concentrations of oxidative stress markers malondialdehyde and protein carbonyls and the proinflammatory cytokine interleukin- beta. moreover, nsc-ev treatment stimulated increased production of antiinflammatory protein interleukin- and the antioxidant superoxide dismutase. immunohistochemical analysis revealed modulation of neuroinflammation typified by reduced activity of reactive astrocytes and activated microglia and improved hippocampal neurogenesis. summary/conclusion: the results suggest that the intranasal administration of nsc-evs is a promising approach for maintaining better cognitive and mood function in ageing through modulation of oxidative stress, neuroinflammation, and neurogenesis. funding: supported by a grant from the national institute of neurological disorders and stroke ( r ns - to a.k.s.) chemically modified myocytes-derived evs for the treatment of cardiac fibrosis. marta prieto-vila a , asao muranaka a and takahiro ochiya b a tokyo medical university, tokyo, japan; b tokyo medical university, shinjuku-ku, japan introduction: myocardial fibrosis is a disorder that may occur after cardiac injure due to a malfunction of the cardiac remodelling. fibroblasts resident in myocardium are erroneously activated causing an excessive accumulation of extracellular matrix, which decreases cardiac function and eventually, leads to death. it is known that cardiomyocytes communicate with the surrounding cells such as fibroblast and endothelial cells by extracellular vesicles (evs). the loss of this communication is thought to play a central role in cardiac fibrosis. therefore, cardiomyocytes-derived evs may be a promising a cell-free system for the treatment of fibrosis inhibition. methods: a novel culture medium was stablished to improve the expansion of primary cardiac myocytes. this was tested using two commercially available primary myocytes cell lines. evs were collected by serial ultracentrifuges, and their effect on fibrosis was tested. for that, prior to any treatment, and to mimic fibrosis, primary cardiac fibroblast were activated overnight with tgfβ. results: by the use of a defined conjunct of chemicals, mature cardiomyocytes culture was highly improved to ensure a high collection of evs. terminal differentiation markers, as well as senesce apparition was delayed in comparison to predetermined culture medium. interestingly, those primary cells secreted a rather large amount of evs, which expressed common evs membrane marker. tgfβ-treated cardiac fibroblasts were co-cultured with myocytes showing a decrease of fibroblast activation markers both at mrna and protein levels. similar results were found when activated fibroblast were treated with evs. summary/conclusion: our findings indicate that the use of evs derived from chemically modified myocytes is a promising treatment for ischaemic myocardial fibrosis. however, further molecular experiments have to be done to identify the molecules within evs responsible for the inactivation of fibroblast. evaluation of osteoinductive and anti-inflammatory properties of spinederived exosomes renaud sicard a , tania del rivero b , jonathan messer c , shabnam namin c and timothy ganey c a vivex, biologics, inc., miami, usa; b vivex, biologics, inc., miami, usa; c vivex, biologics, inc., miami, usa introduction: over the last decades, mesenchymal stem cell-derived exosomes have been shown to play a crucial role in a myriad of cell function such as extracellular matrix synthesis, proliferation, differentiation or cell migration. biological sources of exosome (heterogeneous or homogeneous cell population, serum, urine etc.) have a direct influence on the content of their cargo and their therapeutic application and potential. in this study, we evaluated exosomes excreted from cadaveric spine-derived cells. we hypothesized that exosomes derived from a bone source such as the spine, will drive the osteogenic differentiation of progenitor cells. we also investigated their effects on inflammation in nucleus pulposus cells using an in-vitro assay. methods: after their isolation and characterization, exosomes derived from cadaveric human spines were assayed for osteoinductive properties. a c c myoblast cell line was treated with different concentrations of exosomes and expression of alkaline phosphatase was measured after days incubation. treatment with bmp- was used as positive control. anti-inflammatory properties were assessed by incubating tnf-treated nucleus pulposus cells with exosomes for days. qpcr analysis of mrna expression of inflammatory cytokines (il- , il -beta, il- ) metalloproteinases (mmp and adamts ), and apoptotic genes (bax, bcl ) was used to determine the effects of exosomes on inflammation. results: spine-derived exosomes positively expressed the exosome flow cytometry markers tested (cd , cd and cd ). the mean number of exosomes per microgram of protein was . ± . x indicating a relatively high purity. osteoinductive (oi) testing was performed using different concentrations of exosomes. the oi index of treatment of c c cells with bmp- , x , x , x , × or × exosomes alone was . , . , . , . , . and . respectively. anti-inflammatory properties of exosome are currently being assessed and will be presented at the time of the poster presentation. summary/conclusion: administering exosomes alone or in combination with an exogenous scaffold has the potential to repair injured tissue and to restore bone function. the clinical significance of this application is aimed to promote the patients' bone healing process and provide a cell-free therapeutic platform that is safe and effective. administration of human mesenchymal stem cell derived extracellular vesicles modulates the abnormal plasticity of newly born neurons and neuroinflammation in a rat model of status epilepticus maheedhar kodali a , daniel gitai b , dong ki kim a , mariam atobiloye a , bing shuai c , sahithi attaluri c , raghavendra upadhya c , leelavathi n madhu a , olagide w. castro a , darwin j. prockop a and ashok k. shetty c decline in the percentage of newly born neurons displaying basal dendrites. besides, ev treated animals displayed higher percentages of resting microglia (ramified microglia), reduced percentages of activated microglia (microglia expressing iba- and cd ), in comparison to animals receiving vehicle after se. interestingly, diminished abnormal plasticity of newly born neurons was accompanied by the preservation of interneurons positive for reelin; a protein believed to guide newly born neurons to their correct locations. summary/conclusion: the results suggest that even a low dose in administration of msc-derived evs after se can limit neurons loss, dampen the abnormal plasticity of newly born neurons, and modulate the activation of microglia. introduction: autism spectrum disorders (asd) are neurodevelopmental disorders characterized by three core symptoms that include social interaction deficits, cognitive inflexibility, and communication disorders. they have been steadily increasing in children over the past several years, with no effective treatment. two percent of all asd patients are suffering from a disorder caused by a mutation in the shank gene. shank is an important synaptic protein, disruption of this gene directly leads to cognitive and motor impairments. during the recent decade, exosomes that derived from mesenchymal stem cells (msc-exo) have been spotlighted as a promising therapeutic target for various clinical indications, including neurological disorders. here we test three different autistic mice models. btbr as a multifactorial mice model of autism and two different shank mutated mice. the first is a complete deletion of exon ( q . ) and the second is a specific insertion mutation of guanine to position in the gene (insg ) that leads to stop codon. methods: exosomes were isolated using differential centrifugation protocol and characterized using the misev guideline recommendations. each animal received an intranasal administration of ul containing exosomes/µl. for intravenous administration, the same number of exosomes, were used, injected in µl. results: all three animal models showed significant improvement in their autistic behavioural phenotypes following intranasal administration. the improvement seems to be dose-dependent and was better achieved via intranasal vs intravenous administration. biodistribution of msc-exo showed accumulation in the brain within hours, yet the reduction of the signal was observed in the kidneys, heart and lungs. summary/conclusion: our data suggest that exosomes derived from adipose msc, carry a therapeutic potential in asd, via non-invasive intranasal administration in three different mice models. these data further emphasize our potential therapeutic strategy to reduce symptoms of autism in clinical trials. funding: stem cell medicine ltd. israel. equine tendon injury treatment by evs: an in vitro study introduction: current treatment options for tendinopathies (chronic, painful tendon disorders), are not able to restore the functional properties of native tendons. hence, new treatment options are sought. the efficacy of mesenchymal stem cells (mscs) therapies, which combined with a rehabilitation programme including controlled exercise is the current gold standard in equine tendon treatment, has been shown to be largely due to the cells´paracrine activity. the aim of this study was therefore to evaluate the effect of bone marrow msc derived autologous and allogeneic conditioned medium (cm, full secretome) and their extracellular vesicles (evs) on "tendon healing" in vitro. methods: to compare the "therapeutic" effect of msc derived evs and cm, a standardized scratch assay (wound healing assay) was performed. cm from equine tenocytes, ev depleted medium and medium with or without fcs served as controls. tendons and bone marrow aspirates were obtained from three horses ( , and years) which were euthanized for reasons unrelated to this study. mscs were isolated by ficoll density gradient centrifugation and tenocytes were obtained by migration from tendon explants. for cm and ev production, cells were cultured in ev depleted medium. evs were harvested by a stepwise ultracentrifugation approach and characterized by nanoparticle tracking analysis (nta), western blot (cd , cd ) and transmission-electron microscopy (tem). results: western blot, nta and tem confirmed successful isolation of evs from equine mscs. the strongest positive effect on wound healing (fastest gap closure) was achieved by msc-cm (p < . ). the gap closure achieved with msc-evs was slower than with msc-cm (p < . ) but faster than with cm of tenocytes (p < . ). donor specific differences in wound healing capability were shown for both autologous and allogeneic application. summary/conclusion: treatment with msc-cm resulted in significantly faster wound healing of adult tenocytes in vitro than msc-evs or tenocyte-cm. mscs donor age shows a significant effect on gap closure following autologous but not allogeneic administration. ev-enriched secretome fraction from gmp-compatible, scalable, human ipsc-derived cardiac progenitors improve heart function in chronic heart failure mice introduction: we have shown that research-use-only grade (res) human ipsc-derived cardiac progenitors (cpcres) can produce a secretome whose small-evenriched fraction (svf) can treat chronic heart failure (chf) in mice. gmp-compatible, scalable processes for a cpc-derived svf suitable for human therapeutic use is needed. methods: ipsc-derived cpc were produced and cultured using gmp-compatible, scalable processes (cpctx). media without cells were "cultured" in parallel for "virgin media" controls (mv). cpcres were cultured as previously described. as a proof of concept, svfs were isolated from conditioned media by ultracentrifugation: cpctx-ev, cpcres-ev and mv. particle size distributions/concentrations (nanoparticle tracking analysis), protein levels (bsa), and the presence of cd- (elisa) were determined. in vitro activity was assessed by huvec scratch wound healing assay, and by rat and human cardiomyocyte (cm) survival assays. c bl/ mice in chf received echoguided myocardial injection of pbs vehicle control ( ul, n = ), cpctx-ev ( ul, n = ), or cpcres-ev ( ul, n = ). change in cardiac function was assessed by echocardiography. results: cpctx-ev particle sizes were polydisperse (mode~ nm) at a concentration of~ . e particles/ml (~ , particles/cell) and~ . mu cd /ug protein. cpctx-ev increased wound healing, human cm survival, and rat cm survival in vitro by . x, . x, and x, respectively over mv controls. in chf mice, significantly less cpctx-ev mice, and less cpcres-ev mice had severely progressive heart failure (left ventricular end systolic volume, lvesv, increased > %) than pbs control mice (pbs vs cpctx-ev, p < . ; pbs vs cpcres-ev, p < . ), and the average ejection fraction of the pbs group deteriorated . x more than the cpctx-ev group (− % vs − . %, respectively; ns). summary/conclusion: we have a process for cpc differentiation and production of conditioned media suitable for use in human clinical trials from which can be made an svf with the potential to treat chf, possibly through re-vascularization or preservation of cm viability. introduction: exosomes are nanoscale vesicles that mediate cell-to-cell communication via exchanging molecular cargo. mesenchymal stem cell (mscs) modification towards an osteogenic path can occur by uptake of exosomes from other cells. it is less clear whether vesicle placement in the absence of cells will facilitate site-specific delivery through acellular transfer of osteogenic activity. an electrospun fleece was combined with bone marrow-derived exosomes in the absence of cells to evaluate osteoinductive potential that might be thermo-stable and be used in a biologically neutral collagen carrier. comparisons were made of standard laboratory assay of osteoinductivity (oi), and in vivo expression in a mouse calvarial defect model. methods: electrospun type-i collagen was prepared with and without hydroxyapatite (ha) (spinplant gmbh, leipzig) as a foundation base for application of the bone marrow-derived exosomes. individual discs of the collagen enhanced scaffolds ( -mm) were prepared and placed in a mouse calvarial skull defect. animals were followed for and weeks. exosomes were isolated from qualified cadaveric human spines by differential ultracentrifugation. microscopic observation, quantitative assessment of oi with an alkaline phosphatase assay, and flow cytometry were used to evaluate the composition, the hybrid nature of the addition to the nano-collagen fibres. a fluorescent protein reporter transgenic mouse model expressing osteocalcin, type-i collagen, phex, and sp (osterix) was evaluated at and weeks to determine bone formation across the defect. results: alp activity on the scaffold with ha demonstrated an approximate tenfold increase to that of the collagen scaffold alone. while a dose-dependent effect, with higher doses of exosomes resulting in a greater amount of alkaline phosphatase expression, expression that exceeded that of the ng bmp- control. dose escalation from . , , and e resulted in similar increases in expression that was statistically greater with the combination of the fleece with the exosome component. bone formation in the mouse calvaium did not demonstrate gap closure at or at weeks, but did demonstrate enhanced osteoclastivity and robust bone remodelling at the margins of the defect. summary/conclusion: bone marrow-derived exosomes dried into an electrospun fibrillar collagen demonstrated in vitro osteoinductive potential that might provide site-specific placement that could enhance biologic potential. with the capacity for ambient temperature storage, the provision of site-specific placement becomes a technical consideration. placement of the human tissue derived exosomes in a transgenic mouse calvarial defect model did not demonstrate bridging bone across the defect. exosomes loaded with pten-interfering rna enables functional recovery in rats after complete spinal cord transection daniel offen a , nisim perets a , shaowei guo b , oshra betzer c , rachela popovtzer c and shulamit levenberg b a tel aviv university, tel aviv, israel; b technion, haifa, israel, haifa, israel; c bar ilan university, israel, ramt gan, israel introduction: complete spinal cord transection is a debilitating disease that usually leads to permanent functional impairments, with various complications and limited spontaneous recovery. the current investigation of molecular mechanisms controlling axon regeneration, (e.g., signalling networks and environmental cues), led to new strategies to enhance axonal regeneration. we have previously shown that intranasal administration of mesenchymal stem cells derived exosomes (msc-exo), cross the blood-brain barrier and significantly ameliorate motor and behavioural phenotype in several animal models of neurotrauma and neuropsychiatric disorders. methods: msc-exo were isolated from human bone marrow and were loaded with phosphatase and tensin homolog small interfering rna (pten-sirna). the exosomes were given intranasally to rats two hours after complete spinal cord transaction. eight weeks later we followed the motor function and histology and electrophysiology study was performed in order to reveal the connectivity and the biochemical changes in the treated rats. results: we demonstrate that intranasal (in) administrations of msc-derived exosomes could penetrate the blood-brain barrier, home selectively to spinal cord lesion via chemotaxis, and integrated in neurons within the lesion. furthermore, in rats with complete spinal cord transection, msc-exo loaded with pten-sirna silenced pten protein expression in the lesion and promoted robust axonal regeneration and angiogenesis, companied with decreased astrogliosis and microgliosis. moreover, the intranasal treatment partially restored electrophysiological and structural integrity, and most importantly, enabled the remarkable functional recovery and significant improvement in their movements. summary/conclusion: this rapid, non-invasive, approach, using cell-free nano-swimmers carrying molecules to target pathophysiological mechanisms suggest novel strategy for clinical translation to spinal cord injury and beyond. a novel umbilical cord derived wharton's jelly formulation for regenerative medicine applications introduction: musculoskeletal injuries have traditionally been treated with activity-modification, physical therapy, pharmacological agents and surgical procedures. these modalities have limitations, as well as potential side-effects. over the last decade, there has been an increased interest in the use of biologics for regenerative medicine applications (rma), including umbilical cord (uc) derived wharton's jelly (wj). despite this increase, there is insufficient literature assessing the amount of growth factors, cytokines, hyaluronic acid (ha) and extracellular vesicles (ev) including exosomes in these products. the purpose of this study was to develop a novel wj formulation and evaluate the presence of growth factors, cytokines, ha and ev including exosomes. methods: wj was isolated from human-uc obtained from consenting c-section donors and formulated into an injectable form. randomly selected samples from different batches were analysed for sterility testing and quantified for presence of growth factors, cytokines, ha and particles in ev size range. the results showed all samples passed the sterility test. growth factors including igfbp , , , and , tgfα, pdgf-aa were detected. expression of several immunomodulatory cytokines, rantes, il- r, il- , were also detected. expression of pro-inflammatory cytokines mcsfr, mip- a; anti-inflammatory cytokines tnf-ri, tnf-rii, il- ra; and homoeostatic cytokines timp- and timp- were observed. cytokines associated with wound-healing, icam- , g-csf, gdf- , and regenerative properties, gh were also expressed. high concentrations of ha were observed. particles in the ev size range ( - nm) were detected and were enclosed by the membrane, indicative of true ev. summary/conclusion: our results confirmed the presence of numerous growth factors, cytokines, ha and ev in the wj formulation. more studies are underway to confirm the presence of exosomes in detected ev using exosome-specific markers. we believe the presence of multiple factors within one wj formulation may play a role in reducing inflammation, pain and augment healing of musculoskeletal injuries. this offers a potential expanded use for rma. funding: this study was funded by biointegrate llc, new york, ny, usa. collagen sponge loaded with mesenchymal stem cell-derived small extracellular vesicles promote robust bone regeneration shang jiunn chuah a , chee weng yong a , jacob ren jie chew a , ruenn chai lai b , yi ann cheow a , raymond chung wen wong a , asher ah tong lim a , sai kiang lim c and wei seong toh d introduction: mesenchymal stem cell (msc) therapy has demonstrated effective bone regeneration in clinical studies. however, the therapeutic efficacy of mscs have been attributed to the secretion of extracellular vesicles (evs), particularly - nm small evs (sevs). here, we investigate the efficacy of msc-sevs loaded in collagen sponge in the regeneration of critical-sized calvarial defects in immunocompetent rats. methods: sevs were isolated from conditioned medium of human mscs and stored at − c. calvarial defects of -mm diameter were surgically created on thirty-two -week-old male sprague-dawley rats. these rats were then randomly assigned to groups (n = rats/group): defects treated with collagen sponge containing μg of sevs in μl saline (cs/sevs) and defects treated with control collagen sponge containing an equivalent volume of saline (cs/control). at and -week post-surgery, the calvarial bone samples was harvested for analyses by micro-computed tomography (micro-ct), histology, immunohistochemistry and histomorphometry. results: at -week post-surgery, micro-ct analysis showed little bone formation at the defect site in both cs/sevs and cs/control groups. no statistical differences were observed in micro-ct and histology scores in both groups. interestingly, cs/sevs group showed significantly higher osteocalcin (ocn)+ area of . ± . % than that of cs/control group ( . ± . %; p = . ). cd + microvessels at sizes ≤ µm and > µm in cs/sevs group ( . ± . and . ± . microvessels/hpf) were also significantly higher than that of cs/control ( . ± . and . ± . microvessels/hpf; p = . and p = . respectively). by weeks, cs/sevs group displayed enhanced new bone formation that completely bridged the calvaria defect. in contrast, rats in cs/control showed limited bone formation. consequently, cs/ sevs group displayed a micro-ct score of . ± . which was significantly better than that of cs/control group ( . ± . ; p = . ). cs/sevs group also exhibited >twofold increase in bone volume, and improved bone quality with higher trabecular thickness and number, and smaller separation (p < . ), compared to cs/control group. consistently, cs/sevs group displayed a significantly better histology score of . ± . than that of cs/control ( . ± . ; p = . ). moreover, cs/sevs group showed significantly higher ocn+ area of . ± . % than that of cs/control group ( . ± . %; p = . ). summary/conclusion: this study demonstrates that single-stage implantation of collagen sponge loaded with ready-to-use msc sevs can promote robust bone regeneration in a rat calvarial defect model. funding: national university of singapore, r , national medical research council singapore, r . immunomodulatory potential of extracellular vesicles derived from mesenchymal stromal cells introduction: extracellular vesicles (evs) derived from mesenchymal stem/stromal cells (mscs) are promising new agents in regenerative medicine and immunotherapy. considering that independent msc-ev preparations might differ in their therapeutic function, we have set up a functional assay allowing testing for the potential immunomodulatory properties of independent msc-ev preparations. methods: human peripheral blood-derived mononuclear cells (pbmcs) were pooled from up to different healthy donors warranting high allogeneic cross-reactivity, even following an optimized freezing and thawing procedure. after thawing, mixed pbmcs were cultured for days in the absence or presence of msc-evs. thereafter, cell morphologies were documented, supernatants were harvested for cytokines quantification and cells were phenotypically characterized by flow cytometry. by analysing the expression of a collection of different lineage and activation markers, we selected a panel of antigens apparently being regulated by msc-ev preparations considered to be therapeutically active. results: we observed that in the presence of active msc-ev preparations more cd + (monocytes) are recovered from the mlr assay than in corresponding control samples. focusing on t cells, we learned that active msc-ev preparations reduced the content of cd and cd t cells expressing activation markers like cd and cd . summary/conclusion: the mlr assay allows elaborated functional testing of immunomodulatory activities of given msc-ev preparations. currently, we are comparing the immune modulatory capabilities of evs derived from distinct sources and optimize the marker panel to distinguish discrete immune cell subtypes such as different cd cell types, i.e. th , th , th and tregs. extracellular vesicles in platelet-rich plasma: dependency on sample processing zala jan a , saba battelino b , darja božič c , matej hočevar d , ales iglič e , marko jeran c , manca pajnič a , ljubiša pađen a , domen vozel f and veronika kralj-iglič a introduction: platelet-rich plasma (prp) proved effective in regenerative medicine. numerous protocols for its preparation and application are available in the published literature. prp possesses important immune, haemostasis and regenerative factors, however, the mechanisms of their action are yet poorly understood. extracellular vesicles (evs) could be one of the important factors that would contribute to the beneficial effects of preparations. this study was performed as a part of a registered randomised controlled clinical trial (nr: nct ). prp was used to treat chronic middle ear inflammations. here we present the results of prp analyses from blood samples of volunteers with no record of disease. methods: plasma obtained from ml of blood was depleted of erythrocytes and enriched with other particles by repetitive centrifugation of samples. flow cytometry (fcm) was employed to monitor particle contents (cells and smaller particles) throughout the sample processing. the platelet gate was divided into two parts: intact platelets and smaller particles. identity and morphology of particles in the preparations were examined by scanning electron microscopy (sem). standard laboratory tests of blood were performed. results: sem images revealed the presence of heterogeneous population of particles in the preparation of prp, most of which were activated and partially fragmented platelets. the population of smaller particles measured with fcm, was identified as evs. the erythrocyte sedimentation rate was statistically significantly correlated to the volume of plasma obtained in the initial centrifugation step (r = , , p < , ) and to the concentration of evs (r = , ; p < , ). time from sample collection to the preparation of prp was negatively correlated with the concentration of platelets in prp and positively with the concentration of evs (r = , , p < , ). platelet concentration in preparation samples was found to depend on the concentration of platelets in the blood and parameters of sample processing connected with larger centrifugal and shear forces on the samples during centrifugation. these include: sample volume, the size and shape of the centrifuge tube and the distance of the sample from the rotor axis. summary/conclusion: evs are gradually forming upon activation and degradation of cells in the sample throughout the sample processing. optimal processing may importantly contribute to the healing properties of preparation. funding: authors acknowledge support from the european union's horizon research and innovation program under grant agreement no. (ves us project) and slovenian research agency (arrs, grants p - , p - , j - ). satellite cell-derived extracellular vesicles as a therapeutic for mitochondrial dysfunction in duchenne muscular dystrophy duchenne muscular dystrophy (dmd). sc-derived extracellular vesicles (sc-evs) may unlock the therapeutic potential of scs by overcoming these limitations. to investigate their therapeutic potential, we assessed the ability of sc-evs to reverse mitochondrial dysfunction, a key pathological feature of dmd, in oxidatively-damaged c c and primary dmd myotubes. methods: scs from c mice were isolated and cultured. evs were isolated from the supernatant of scs via polyethylene glycol precipitation and characterized using nanoparticle tracking analysis. the ability of sc-evs to deliver protein cargo to c c myotubes, and the localization of the cargo once delivered, were analysed using fluorescence microscopy. to examine sc-ev potential to restore the function of damaged mitochondria, c c myotubes were treated with µm h o for h followed by treatment with . x sc-evs for h. separately, cultured dystrophic myotubes were treated with . × evs every h for h. in both sets of experiments, maximal oxygen consumption rate (max ocr) was measured via seahorse xf cell mito stress test. where appropriate, a t-test was performed to test for statistical significance (p < . ). results: based on estimated cell number and ev quantification, each sc released approximately . × ± . x evs/day. evs delivered protein cargo into myotubes within h. fluorescent labelling of intracellular mitochondria showed co-localization of delivered protein and mitochondria. incubation of myotubes with h o resulted in a % decline in max ocr relative to untreated myotubes. subsequent treatment with sc-evs resulted in a % increase in max ocr. treatment of undamaged myotubes with sc-evs had no effect on max ocr. primary dmd myotubes treated with sc-evs showed a % increase in max ocr relative to untreated dmd myotubes. summary/conclusion: sc-evs rapidly deliver proteins into myotubes, much of which co-localizes with mitochondria, and reverses mitochondria dysfunction in oxidatively-damaged and dystrophic myotubes. introduction: flow cytometry has been used extensively for analysis of ev particles stained with fluorescent antibodies directed to the known cell surface markers. quantitation of the surface markers in terms of the number of molecules or the number of antibodies bound per specific marker has remained one of the largest challenges in the ev research field. changes in instrument setup as well as changes in fluorescent antibodies from different vendors, all impact the relative mfi values for the same ev sample. in this work we report a standardization method of quantitating extra-cellular vesicle surface markers with mesf liposomes. methods: liposomes labelled with fitc fluorescent dye were prepared with a bd proprietary technology. dynamic light scattering analysis was used for size determination of the liposomes. bd facsaria™ fusion system, modified with a small particle side scatter module (sp ssc), was used for analysis of the labelled liposomes by flow cytometry. results: we created a set of nm fitc-modified liposomes of various fluorescent intensities with a known number of fitc molecules incorporated in each liposome intensity. the mfi values of each liposome population (intensity) had a linear relationship to the amount of fitc used for labelling the liposome nanoparticles, suggesting that no self-quenching of fitc fluorescence had occurred. the number for the fitc fluorophores for each liposome intensity was expressed in the units of molecules of equivalents soluble fluorochrome (mesf). a plot of mesf vs. the fluorescent intensity of the liposomes (mfi values) obtained from flow cytometry analysis provided a calibration curve, from which the fluorescent intensity (mfi value) of a stained ev sample can be converted to the number of fluorophores bound (mesf value) to the surface of the ev particles. summary/conclusion: by this approach, the mfi values of stained ev particles are converted to standardized mesf values that are independent of instrument variation, resulting in further improvement of inter-laboratory standardization. furthermore, utilization of liposomes with similar size and refractive index to ev particles simplifies the data evaluation and improves the accuracy of ev surface marker quantitation by flow cytometry. currently, other fluorescent dyes are being explored to expand the utility of mesf liposomes with other fluorescent colours. measuring cholesterol as a high-throughput method for quantifying extracellular vesicles introduction: the extracellular vesicle (ev) field currently lacks a high-throughput method for accurately quantifying evs in solution. ev quantification has traditionally relied on nanoparticle tracking analysis (nta), which is time intensive and indiscriminately counts non-ev particles, such as membrane fragments and protein aggregates. we have rigorously assessed two commercially available methods for measuring cholesterol, a major lipid component of the ev lipid bilayer, and evaluated the utility of these assays to quantify evs in minimally processed samples. methods: the amplex® red cholesterol assay and cedex bio ht were used to quantify cholesterol in ev samples via enzymatic oxidation, with dynamic ranges of - , ng/ml and - µl/ml, respectively. samples throughout various stages of purification were analysed, from clarified cell culture medium to highly purified evs separated on an iodixanol gradient. we evaluated several pre-processing methods, to remove non-ev cholesterol content prior to analysis. results: the amplex® and cedex bio ht assays were found to perform comparably for quantifying cholesterol in purified evs (r = . ). importantly, cholesterol quantification on purified ev samples, ranging from e to e particles/ml, correlated well with nta measurements (r = . ). both µm filtration or an additional , rcf centrifugation step following clarification removed cholesterol associated with cellular debris or other non-ev sources, allowing for accurate quantification of conditioned medium samples or ultracentrifugation pellets (ucp) instead of needing to rigorously purify samples with an iodixanol density gradient. summary/conclusion: cholesterol quantitation can be used to accurately estimate ev concentration, allowing for rapid characterization of samples from clarified cell culture supernatant to highly purified evs. this highthroughput analytical capability may enable more comprehensive assessment of methods to boost ev yield through mass screening of cell culture conditions. optimization of nanoparticle tracking analysis of extracellular vesicles isolated from plasma and bronchopulmonary lavage fluid of patients with non-small cell lung cancer introduction: recent studies show that tumourderived extracellular vesicles (evs) greatly influence the tumour microenvironment and impact the therapy. in non-small cell lung cancer (nsclc), bronchopulmonary lavage fluid (balf) appears to be a good source of tumour-derived evs, providing more accurate information about the tumour microenvironment than evs from plasma. so far there is a lack of accurate and standardized methods for ev quantification. fluorescence nanoparticle tracking analysis (fl-nta) is an emerging method of ev-analysis, allowing discrimination of evs and exosomes from impurities. here we perform an optimization of the fl-nta method to compare evs from plasma and balf of nsclc patients and healthy controls (nc). methods: evs were isolated using homemade sizeexclusion chromatography (sec) columns (plasma) and ultrafiltration or differential ultracentrifugation (balf). nta was performed using zetaview pmx (particle metrix) after ev-staining with membrane dyes or fluorescence-labelled antibodies against typical ev-marker (cd , cd , cd ). results: nta scatter measurements showed a higher total particle concentration in plasma than in balf. however, membrane-specific staining showed a much greater purity of ev-preparations from balf, where nearly % of the particles detected in scatter mode showed positive membrane-staining. in contrast, only around - % of particles in the plasma ev-preparations were positive for the membrane dyes. fluorescence-staining for ev surface marker requires further optimization to obtain reproducible results. summary/conclusion: classical nta using only the scatter mode fails to discriminate between evs, lipoproteins and protein aggregates. for ev-analysis from complex biofluids like plasma, fla-nta and staining for specific ev marker is necessary to receive reliable data. balf seems to be a better source of tumourderived evs than plasma, since the obtained ev-preparations show a higher purity. improving conditions for fluorescence-staining and nta measurement of evs from plasma and balf of nsclc patients will provide an additional method for quantifying and phenotyping of evs. introduction: the exoviewer platform currently enables the user to capture extracellular vesicles (ev) by means of surface antigen-specific antibodies (e.g. targeting tetraspanins), making possible the enumeration of individual particles using single-particle interferometric reflectance imaging sensor (sp-iris, interferometric) imaging as well as fluorescence. currently, through interferometric imaging particles smaller than nm cannot be detected, while fluorescently stained ev smaller than nm can be well resolved. further, it is conceivable that small ev contain antigen numbers in the single digits, making antigen-specific immunostaining a challenge. to further characterize ev populations of different sizes and surface marker composition, it would be highly advantageous to target the vesicular nature of the detected particles linked to a fluorescence readout. methods: the goal of this project is to detect ev with a probe that is ubiquitously distributed across the surface (or lumen) of the vesicle. small ( - nm) ev present fairly distinctive lipid membrane features in the extracellular environment, turning the ev membrane into a "universal" marker, and as such may serve as an alternative marker that is complementary to canonical ev surface markers. results: here we present data on successfully staining ev with the membrane dye di- -anepps (di- ) and the luminal dye calcein-am. we demonstrate that ev from different sources can be efficiently stained with either dye, allowing the quantitative characterization of ev in an unbiased manner using exoviewer's fluorescence mode. while both dyes certainly have their own unique strengths, they exhibit the wanted linear correlation of ev staining versus concentration. further, both dyes are compatible with subsequent immunostaining applications, allowing the user to target specific surface or luminal markers (di- ). summary/conclusion: while a large-panel screening featuring other powerful dyes is continuously ongoing, the current data support the notion of providing the experimenter with a reference for total particle count and at the same time fully exploring the larger dynamic range of the fluorescence mode. moreover, the universal probe will enable the user to correlate intensity and particle size measurements, thereby significantly improving the exoviewer platform and its applications. membrane labelling is essential for the identification and quantification of extracellular vesicles via facs introduction: extracellular vesicle (ev) research is challenged by the lack of standard protocols to identify and distinguish between exosomes and ectosomes being released via exocytosis or plasma membrane shedding, respectively. analysis of small ev populations requires high-resolution technology and can be further improved using fluorescent labels such as carboxyfluorescein diacetate succinimidyl ester (cfse). at the inner leaflet of the plasma membrane, cfse is cleaved enzymatically resulting in covalent binding of the dye. in this study we optimized the conditions for membrane labelling of evs and their subsequent detection by flow cytometry to obtain a maximum yield of intact evs. methods: using sequential centrifugation, we separated ev subpopulations from supernatants of colo pancreas carcinoma cells based on size and mass. after , x g centrifugation, we reconstituted evs from the pellet. we used cfse for ev detection and analysed the expression of tetraspanins by facs to confirm the lipid bilayer structure. furthermore, we determined size distribution of evs by nanoparticle tracking analysis (nta) and electron microscopy. detecting evs as cfse+ events, we quantified our samples and investigated the impact of threshold adjustment on ev quantification. results: after high speed centrifugation of cell free supernatants, we identified cfse+ events as evs, which appeared as round structures under the microscope, and ranged from to nm in size. interestingly, tetraspanin markers cd and cd were detectable only on a subpopulation of purified evs, suggesting heterogeneity of our preparations. for sufficient labelling of evs, minimal temperature variations and short incubation times correlated with ev stability. of note, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of labelled evs and hence, is central for data comparability. summary/conclusion: protocol standardization is of major importance for the use of evs as diagnostic markers in liquid biopsies. funding: this project has been supported in part by annelise-asmussen foundation, luebeck (grant ), leo pharma germany (grant ). surface plasmon field-enhanced fluorescence spectroscopy (spfs) system for quantitative and qualitative extracellular vesicles total evaluation without any sample pretreatment introduction: the function of extracellular vesicle (ev) is interested in the immunology and oncology fields as a key transmitter for cellular communication. however, the conventional ev evaluation methods are required complicated evs preconcentration from the sample, its leads ev analysis uncertainty. in this study, we applied the spfs highly sensitive automated system for quantitative and qualitative ev evaluation without any sample pre-concentration and preparation step. methods: spfs automated system and plastic disposable sensor had been developed by konica minolta corporation in house. anti-membrane protein (cd , cd , cd ) antibody was chemically bonded on hydrophilic polymer which was immobilized through the gold thin film on the spfs sensor. the concentration of standard ev materials was evaluated by the qnano system before using. ev detection without preconcentrating was achieved by sandwich immunoassay step in microchannel round-trip flow reaction (tat min) with the spfs system, and elisa was adapted as a conventional standard method. after spfs highly sensitive fluorescent measurements step, extracted and detected ev were effectively recovered by using the recovery buffer reaction. results: the ev sensitivity performance between spfs and elisa clearly showed a significant difference, and the lod of spfs ( . particles/μl) method was estimated times superior to the lod of conventional elisa ( , particles/μl). the spfs calibration curve showed a wide dynamic range at least over logs as an additional specificity. spfs method also showed fine results in the dilution linearity test with high reproducibility under the serum/plasma sample condition. the data for recovery test of ev expected us that highly accurate measurement can be guaranteed under the condition of dilution about times or less even in the whole blood sample. after the spfs measurement, extracted ev on the spfs sensor chip could be effectively recovered and could be analysed nucleic acid which contains micro rna. summary/conclusion: spfs system might have great potential for quantitative and qualitative ev evaluation. our strategy with spfs system for ev proteomic and genomic profiling will be possible for applying to ev quality control as well as a novel biomarker development. identification of a novel compound that inhibits small ev secretion and tumour progression by a sensitive elisa screening. yunfei ma a , takeshi yoshida a , duc tuan nguyen a , kazutaka matoba b , katsuhiko kida b , taito nishino b and rikinari hanayama c a kanazawa university, kanazawa, japan; b nissan chemical corporation, tokyo, japan; c wpi nano life science institute, kanazawa university, kanazawa, japan introduction: small evs from tumour cells are known to promote tumour progression, therefore, it is expected to develop drugs that regulate small ev secretion, which can be used in clinical applications. methods: to identify such regulators, we first developed a sensitive elisa system for the quantification of small ev secretion using a high-affinity ev binding protein tim . by using this elisa system, we screened for small compounds that promote or inhibit small ev secretion using a drug-repositioning compound library (about , compounds). results: as a result, we identified eight promoters and two inhibitors, including compound a, which significantly reduced small ev secretion from various cell types without affecting cell growth. we further investigated the effects of compound a on a mouse model of osteosarcoma and found that compound a suppressed tumour progression efficiently. summary/conclusion: these data suggest that compound a would be useful not only for the characterization of small ev function but also for the clinical therapy against tumour progression, by inhibiting small ev secretion. introduction: for many years it was believed that several proteins such as cd , cd and flotillin- were unique for exosomes, however recent studies have shown that several of these markers also can be present in other subpopulations of evs (kowal et al pnas ) . furthermore, few markers have been identified as uniquely present in microvesicles. the aim of this study was to in depth compare the proteome of microvesicles and exosomes. methods: mda-mb- -luc-d h , -d h ln and -bmd a were cultured in ev-depleted media. microvesicles ( , x g, min) and exosomes ( , x g . h) were isolated using a combination of differential ultracentrifugation and a density cushion (~ . g/ml). purity and yield of evs were determined by nanoparticle tracking analysis (nta), western blot, and electron microscopy (em). quantitative mass spectrometry (tmt-lc-ms/ms) was used to identify differently enriched proteins in microvesicles and exosomes (n = x cell lines). results: in total proteins were quantified, with being quantified in all samples. in total and proteins were significantly upregulated in exosomes and microvesicles, respectively. go terms associated with the proteins significantly upregulated in exosomes were "extracellular exosome" and "plasma membrane", while the microvesicle proteome was associated with "membrane" and mitochondrion". in exosomes tetraspanins, annexins, escrt and rab proteins were significantly upregulated. in contrast, proteins that were upregulated in microvesicles were involved in protein translocation into the mitochondrial membrane (timm and tomm proteins), in cytokinesis, and in micos complex. however, flotillin- was not differently expressed in the ev subtypes. summary/conclusion: this study identifies several proteins to be differently enriched in exosomes and microvesicles. several of the proteins suggest recently by kowal and colleagues, such as adam and mitofilin could be validated. additionally several novel proteins could be identified. identifying markers separating microvesicles and exosomes is of high importance for the ev field and future studies will have to validate them also in other cells to determine if they are generic. introduction: the cellular elements composing the lining of brain ventricles have drawn much attention from neuroscientists, especially the role of subependymal cells in neurogenesis, but the role of ependymal cells in brain function and disease is still neglected. our objective is to study the morphological aspects of rat brain ventricles and the ependymal cells as analysed by transmission and field emission scanning microscopy in normal or ischaemic rats. methods: for this purpose, male wistar rats were submitted to minutes of global brain ischaemia and divided into two groups: a) sham-operated animals and b) saline-treated ischaemic animals. all animals were allowed to survive for seven days. all procedures were approved by the ethics committee of the federal university of são paulo ( / ). transmission and scanning electron microscopic analysis of lateral brain ventricles were done in buffered , % glutaraldehyde/ %formaldehyde perfused brains. cerebrospinal fluid was collected for nta analysis. results: the morphological characterization of brain ventricle revealed a slight rarefaction of ciliary tufts of animals submitted to ischaemia when compared to normal animals. field emission electron microscopy revealed the secretion of vesicles by the ependymal cilia in the lateral ventricle. size and concentration of particles in the cerebrospinal fluid was confirmed by nta and transmission electron microscopy. summary/conclusion: our results are unprecedented and bring innovative potential regarding the role of extracellular vesicles in both the physiology and pathogenesis of the nervous system. these data may also contribute to the development of new technologies for diagnosis and therapy of chronic degenerative diseases. introduction: the function of mitochondria relies on precise and effective quality controls. neurons have high metabolic demands and employ multiple mechanisms to ensure functional mitochondria. we investigated mitochondrial vesiclesa less understood quality control mechanism for mitochondriaand assessed the effect of cellular stress. methods: we surveyed mitochondrial vesicles in rat and planaria brains with electron microscopy. we quantified these vesicles with serial-section electron microscopy (fib-sem). we also conducted confocal microscopy with airyscan analysis of cultured neurons expressing fluorescently tagged mitochondrial markers. results: electron microscopy showed the ultrastructure of various types of mitochondrial vesicles. serial-section electron microscopy revealed the d ultrastructure of mitochondrial vesicles and their prevalence in neurons. confocal microscopic analysis showed increased numbers of mitochondrial vesicles in neurons under mild stress. summary/conclusion: our findings provide direct structural evidence for mitochondrial vesicles in neurons and their abundance in response to neuronal stress. their detection in the extracellular compartment (evidence for which is expected to be presented by the time of isev) may allow for development of biomarkers for mitochondrial health, with relevance to numerous pathologic conditions. from endosomes, might be involved in the impairment of rna, specific feature of als disease. combining high-resolution flow cytometry and surface marker analysis using an automated platform to study extracellular vesicle in cerebrospinal fluid unity health toronto, toronto, canada introduction: there is growing enthusiasm that extracellular vesicles (evs) carry the potential for a variety of applications in medicine. as biomarkers, evs may aid clinicians in the evaluation of diagnoses, disease progression, or even response to therapy. however, proper characterization of the amount, size, and phenotype of evs in a given sample remains challenging due to their sub-micrometre size and heterogeneity. over the last years, technologies, including high-sensitivity flow cytometry and automated platforms that simultaneously assess ev amount, size, and phenotype, have matured, providing new opportunities to study evs for future clinical applications. using such technologies to analyse cerebrospinal fluid (csf), which is in direct contact with the brain and spinal cord, may yield valuable insights into neurological disease processes. while there is often uncertainty about the exact source of evs in a biological sample, cd has emerged as a surface marker that suggests a neuronal origin. methods: csf samples that had been stored at - degrees celsius for advanced biomarker studies were analysed using two distinct approaches. a becton, dickinson and company (bd) aria iii flow cytometer was converted into using violet side scatter (ssc) for improved detection of evs with instead of nm ssc. for the combined analysis of amount, size, and phenotype, samples were analysed with the nanoview bio r platform. phenotype analysis included probing for the classic tetraspanins associated with exosomes (cd , cd , cd ) and the neural cell adhesion molecule l (cd ). results: flow of csf samples showed similar vesicle counts in control vs. disease and an increase of counts in later disease stages when neurodegeneration is thought to be more prominent. all csf samples showed some binding to classic exosomal markers (cd , cd , cd ). the sample taken at the latest time point showed relatively high vesicle counts, overall larger vesicle size, and abundant cd binding. interestingly, the cd positive evs were not positive for any of the classic exosomal markers (cd , cd , and cd ). summary/conclusion: this data supports the notion that analysing the amount, size, and surface markers of evs in csf can reveal intriguing dynamics in such basic ev characteristics over time and suggests important differences between ev populations in different disease stages. while previous studies indicated that cd could identify an ev to be of neuronal origin, it remains to be determined whether such specific surface markers will emerge as clinically relevant tools to support the evaluation of people affected by neurological diseases. a distinct microrna signature in plasma derived small extracellular vesicles of different neurodegenerative diseases introduction: exploring identifying robust biomarkers is essential for early diagnosis of neurodegenerative diseases. blood stream transports large (levs) and small extracellular vesicles (sevs), which are extracellular vesicles of different sizes and biological functions that are transported in blood. aim of our study was to investigate mrna/mirna signatures in plasma derived levs and sevs of amyotrophic lateral sclerosis (als), alzheimer's disease (ad), parkinson's disease (pdpd), fronto-temporal dementia (ftd) and alzheimer's disease (ad) patients. methods: levs and sevs were isolated from plasma of patients and healthy volunteers (ctr) by ultracentrifugation and rna was extracted. whole transcriptome and mirna libraries were prepared with truseq stranded total rna kit and truseq small rna library kit (illumina). results: our data suggested that the rna cargo in levs and sevs varies among different diseases. mirna analysis in sevs provided the most informative disease specific signatures, while whole transcriptome analysis did not show any specific signature. als was characterized by a small but specific group of circulating mirnas. mirnas profiling revealed that pd and ftd can be subgrouped in two classes while ad appears to be a homogeneous disease population. furthermore, mirnas profiling show the presence of overlaps in the signatures between the analysed diseases. mirna profiling in levs is similar to that observed in sevs, although in levs the overall differences between diseases are less marked. summary/conclusion: in this study we have demonstrated that mirnas are the most interesting subpopulation of transcripts transported by plasma derived sevs since they discriminate a disease from the other and they can provide a signature for each neurodegenerative diseases. may be linked with apoe genotype, we investigated the possible effect of apoe genotype on brain-derived evs (bdevs) and their protein and rna molecular cargo. methods: cortical brain tissues of ad patients with different apoe genotypes [ε /ε (n = ), ε /ε ( ), ε /ε ( ), ε /ε ( )] and non-ad controls (n = ) were obtained. bdevs were separated by size exclusion chromatography plus ultracentrifugation (uc) and characterized per misev . proteins were analysed by mass spectrometry. after protein identification, data were normalized using the cyclicloess method and analysed by principal component analysis (pca). nested factorial design highlighted differentially expressed proteins. rna from bdevs was extracted by mirneasy mini kit. small rna libraries were constructed using the ion total rna-seq kit and sequenced on the ion torrent s ™ using ion™ chips. reads were aligned to human reference transcriptomes using bowtie. differential gene expression was quantified by edger and limma. results: among proteins dysregulated in ad bd-sevs, several have reported roles in ad, e.g., microtubule-associated protein tau and peroxiredoxin- . regarding apoe genotypes, proteins were differentially expressed between ε carriers (ε /ε and ε /ε ) with non ε carriers (ε /ε and ε /ε ). however, ev markers did not differ by apoe genotype. in contrast to protein cargo of bdevs, the overall small rna expression pattern was similar among ad patients with different apoe alleles and non-ad patients. only a few mirnas showed different abundance level between ε /ε and ε /ε groups, or between ad and non-ad groups. summary/conclusion: bdevs carry proteins and mirnas related to ad development and apoe genotypes. further verification of protein and rna expression in brain and plasma derived evs may reveal mechanisms of ev function in neuroinflammation and develop biomarkers for ad disease. funding: this project was funded by mh . efficient pathology spread by extracellular vesicles from human brain tissues in mouse brain and tissue cultured neurons: transmission and propagation to gabaergic neurons however, whether human brain-derived evs induce tau pathology has not yet been characterized in the mouse brain. here, we assess the mechanisms of disease spread after intrahippocampal injection of human brainderived evs into the aged mouse model. methods: ev-enriched fractions were isolated from unfixed frozen human brain samples from ad, prodromal ad (pad), control (ctrl) cases, and tau knockout (tko) mouse brains. isolated evs containing pg of human total tau were sterotaxically injected into the right outer molecular layer of the dentate gyrus of months-old c bl/ female mice. . months after the injection, hippocampal slices were prepared for whole-cell patch clamp recordings of ca pyramidal neurons were undertakent. hippocampi were analysed with immunohistochemistry using phosphorylated-tau (p-tau) epitopes including at . evs were examined for protein composition by protein mass-spectroscopy, the neuronal uptake in vitro, and structural analysis by the atomic force microscopy (afm). results: semiquantitative brain-wide immunohistochemistry of p-tau revealed that inoculation of ad or pad-evs induced tau propagation throughout the hippocampus, including the dentate gyrus, ca and ca subregions. at was localized primarily in gad + gabaergic neurons in pad and ad evs groups, accompanied with reduced amplitude of inhibitory postsynaptic currents and excitatory-inhibitory ratio in amplitube of postsynaptic currents in ca pyramidal neurons in pad evs. afm analysis showed higher density of tau oligomers in both ad and pad evs while only ad evs showed significantly higher neuronal uptake compared to ctrl evs. finally, proteomic analysis showed that ad evs are enriched in disease and glia-related molecules compared to ctrl evs, which may contribute to their enhanced neuronal uptake. summary/conclusion: intracranial injection of ad or pad evs induced p-tau accumulation primarily in gabaergic neurons throughout the hippocampus, resulted in higher uptake by neurons, and tau oligomer conformation, indicating of their pathogenic potency as seeding factors. gabaergic neuronal dysfunction in the hippocampal neuronal circuitry reported in early ad brains could be attributed to specific ev mediated tau propagation in this cell type, a phenomenon meriting further investigation and validation. funding: nih rf ag , nih r ag , nih r ag , cure alzheimer's fund, brightfocus foundation, curepsp, coins for alzheimer's research trust introduction: extracellular vesicles (evs) are released by cells of the central nervous system as a result of injury, including mild traumatic brain injury (mtbi). since mtbi may alter circulating levels of evs, this study aimed to investigate differences in circulating ev numbers between contact sport athletes with and without acute mtbi. methods: circulating evs containing cd (cd + ev), cd (cd + ev), and neural cell adhesion molecule (l cam+ev) were analysed in young, male athletes with or without mtbi ( - yo, n = per group). sodium citrate-treated blood samples were obtained from athletes with mtbi within -hours of injury and from control athletes free of mtbi for one year. athletes were best matched for age and history of prior mtbi. samples were double-centrifuged to obtain platelet-poor plasma and stored at − °c until analysed. quantification of evs was performed using a spectral flow cytometer. the study was approved by temple university's irb, and all athletes provided written informed consent. results: mann-whitney u tests showed that population percentages of small size ( - nm) cd + ev, cd + ev and l cam+evs were significantly higher in mtbi athletes (mean rank: . , . , . ) than controls (mean rank: . , . , . ) (u = . , p = . ; u = . , p > . ; u = . , p > . , respectively). population percentages of large size ( - nm) cd + ev, cd + ev and l cam+evs were also significantly higher in mtbi athletes (mean rank: . , . , . ) than controls (mean rank: . , . , . ) (u = . , p = . ; u = . , p > . ; u = . , p > . , respectively). there were no significant differences between percentages of evs associated with blood brain barrier function (cd + ev) or platelets (cd a+ev) among mtbi athletes or controls. introduction: parkinson's disease (pd) is characterized by clinical heterogeneity, different rates of progression and absence of definitive biomarkers. extracellular vesicles (evs) are easily isolated from plasma and play a central role in intercellular communication which is highly relevant for inflammatory processes implicated in protein misfolding-related neurodegenerative disorders. thus, we characterized distinctive plasmatic ev subpopulations of pd and atypical parkinsonisms (ap) patients, with the aim to identify candidate biomarkers among evs surface membraneproteins. methods: plasmatic evs were collected from pd, matched healthy controls (hc), ap with multiple system atrophy (msa) and ap with tauopathies (ap-tau). evs were quantified by nanoparticle tracking analysis. the expression of ev-surface markers, related to inflammatory and immune cells, were measured by macsplex and correlated to clinical scales. a diagnostic model based on ev markers expression was built via supervised machine learning algorithms and validated in an external cohort ( pd, hc, msa, ap-tau). the cantonal ethics committee approved the study protocol. all enrolled subjects gave written informed consent. results: pd showed the highest ev concentration compared to others groups. pd and msa displayed a greater pool of overexpressed immune markers compared to ap-tau. ev antigens correlate to cognitive impairment and disease gravity in pd and msa. the roc curve analysis of a compound ev marker showed optimal diagnostic performance for pd (auc . ; sensitivity . %, specificity . %) and msa (auc . ; sensi-tivity %,specificity . %)andgoodaccuracyforap-tau (auc . ; sensitivity . %, specificity . %). a diagnostic model based on ev markers expression, cor-rectlyclassified . %ofpatientswithreliablediagnostic performance after validation in an external cohort ( % of accuracy). summary/conclusion: this analysis of multiple immune surface markers of circulating evs in pd and ap well captured the clinical heterogeneity of pd and showed optimal diagnostic performance. furtherly it suggests a different immune dysregulation in pd and msa vs. ap-tau, to be confirmed by functional analysis in experimental models of disease. funding: supported by abreoc. separation and characterization of extracellular vesicles from human cerebrospinal fluid introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off , ), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions ( + ) after the sec void volume as evaluated by detection of cd and cd markers (immunoblotting) and annexin a (peptide mapping by nanolc-ms/ms). there, nanoparticles around nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between and nm, with average diameter ± nm. cd was visualized by immunocytochemistry at the surface of ev around nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin- binding protein (lgals bp or k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between and nm; some of those nanoparticles had ring-like appearance. occasionally k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from k nanoparticles to investigate their individual physiological relevance and biomarker potential. introduction: extracellular vesicles (ev) are released from cells to the surroundings and are found in human biofluids, where they constitute promising targets for novel biomarker identification. ev have been found in cerebrospinal fluid (csf) where they may provide with markers for neurological diseases. here, we aimed at purifying and characterizing ev from human csf. methods: csf was collected by lumbar puncture from patients with amyotrophic lateral sclerosis. patients gave written consent and studies were agreed by the local ethics committee. csf was fractionated by ultrafiltration (vivaspin, cut-off , ), and size-exclusion chromatography (sec; qevsingle izon science). eluted fractions were analysed by dynamic light scattering (dls) and electron microscopy. proteins were analysed by immunoblotting and nano-liquid chromatographytandem mass spectrometry. results: ev eluted in early fractions ( + ) after the sec void volume as evaluated by detection of cd and cd markers (immunoblotting) and annexin a (peptide mapping by nanolc-ms/ms). there, nanoparticles around nm were identified by dls. in agreement, electron microscopy showed ev with characteristic shape and sizes typically between and nm, with average diameter ± nm. cd was visualized by immunocytochemistry at the surface of ev around nm. on the other hand soluble proteins igg and albumin eluted in later fractions. curiously, galectin- binding protein (lgals bp or k) was also partially detected in early-eluting fractions as nanoparticles of irregular shapes and heterogeneous sizes typically between and nm; some of those nanoparticles had ring-like appearance. occasionally k also appeared on ev of variable dimensions. summary/conclusion: in conclusion, ev from the csf may be separated from soluble proteins and small molecules by a combination of ultrafiltration with sec fractionation. however, using this strategy a population of k-containing nanoparticles co-eluted with ev from the csf. further separation techniques need to be applied to separate ev from k nanoparticles to investigate their individual physiological relevance and biomarker potential. release of extracellular vesicles from platelets requires platelet-platelet interaction aleksandra gąsecka a , naomi c. buntsma b , sytske talsma c , krzysztof j. filipiak d , rienk nieuwland e and edwin van der pol f introduction: arterial thrombosis is a major and global cause of human death and disability, but a biomarker for early-diagnosis of thrombosis is absent. platelet activation and aggregation are the first steps of plateletrich thrombus formation, but their relative contribution to platelet extracellular vesicles (pevs) release is unknown. methods: to study the relation between pev release and platelet interaction (aggregation), citrate-anticoagulated whole blood (wb) from healthy donors was diluted , , , and -fold and activated by μm thrombin-receptor activating peptide (trap). in addition, undiluted wb and -fold diluted wb, which totally blocked pev release, were activated with various trap concentrations. concentrations of pevs (cd + and cd +, cd p + > nm) and activated platelets (cd +, cd p+ > nm) were measured by flow cytometry (apogee a -micro). platelet aggregation was assessed using impedance aggregometry. results: a -fold dilution of wb blocked both aggregation and the release of pevs. compared to baseline, activation of undiluted wb with trap increased the concentrations of cd + . -fold and cd +-cd p + pevs . -fold. the concentration of cd + (r = . ) and cd +-cd p+ (r = . ) pevs as well as platelet aggregation (r = . ) scaled inversely (reciprocal) with the dilution of wb. further, we found a linear correlation between the % of activated platelets and the concentration of cd + (r = . ) and cd +, cd p+ (r = . ) pevs in undiluted wb, which was absent in -fold diluted blood (r < . ). summary/conclusion: the absence of aggregation and pev release upon platelet activation in -fold diluted blood shows that aggregation directly depends on the distance between platelets, which is confirmed by the reciprocal relationship between pev release and blood dilution. because pevs are only released when platelet activation is followed by aggregation, pevs are a potential early biomarker of thrombosis. funding: ag is supported by the national science centre, research programme preludium / / n/nz / . evdp is supported by the netherlands organisation for scientific research -domain applied and engineering sciences (nwo-ttw), research programmes veni . age-dependent alteration in concentration and size distribution of extracellular vesicles in plasma of normotensive and hypertensive rats kosuke otani, muneyoshi okada and hideyuki yamawaki laboratory of veterinary pharmacology, school of veterinary medicine, kitasato university, towada, japan introduction: spontaneously hypertensive rats (shr) are the most widely used animal model of human essential hypertension. we previously reported that plasma small extracellular vesicles (sevs) in shr regulate systolic blood pressure, however, the mechanism has not been clarified. in the present study, we compared the concentration and size distribution of plasma evs (sevs and large evs) from young and aged normotensive wistar kyoto rats (wky) and shr. methods: heparin-anticoagulated plasma was collected from male wky and shr at ~ -(young) and -(aged) week-old. large evs were isolated from the plasma by centrifugation ( x g). sevs were isolated by ultracentrifugation ( , x g) following precipitation with polyethylene-glycol. the concentration and size distribution of sevs and large evs were measured by a tunable resistive pulse sensing analysis. results: there was no significant difference in the total concentration of plasma sevs between wky and shr or between young and aged rats. the mean diameter of plasma sevs from aged rats was larger than that from young rats in both wky and shr. also, the number of particles with a diameter of smaller than nm in plasma sevs from aged rats was lower than that from young rats. the concentration of plasma large evs from aged rats was higher than that from young rats in both wky and shr. there was no significant difference in the size distribution of plasma large evs between wky and shr or between young and aged rats. summary/conclusion: the present results for the first time demonstrate that the concentration of plasma large-sized evs is increased by ageing, while there is no difference in the concertation and size distribution of evs between wky and shr. further research is required to clarify the cause of age-dependent alternation in plasma ev size distribution and its physiological meaning. microrna profiling of circulating extracellular vesicles is involved with susceptibility to age-related diseases: relevance to cardiovascular signalling in ageing process ionara rodrigues siqueira a , laura cechinel b , rachael batabyal c and robert freishtat c a universidade federal do rio grande do sul (ufrgs), porto alegre, brazil; b universidade federal do rio grande do sul, porto alegre, brazil; c children's national hospital, washington, usa introduction: ageing represents a central risk factor for several diseases, such as cardiovascular diseases. our hypothesis is that extracellular vesicles (evs) can be potential mechanism of spreading molecules, such as micrornas, involved with susceptibility to chronic age-related diseases and geriatric syndromes. in this context, the role of micrornas in age-induced detrimental changes in the cardiovascular system has been suggested. although evs can protect micrornas from endogenous rnases and internalization of these vesicles into cells is involved with cell communication, delivering micrornas even to distant tissues, the relationships between evs micrornas profile and chronic age-related diseases has not been evaluated. our aim was to investigate the microrna profile of circulating evs during ageing process and their downstream signalling pathways. methods: the ethics committee (ceua -comissão de Ética no uso de animais -ufrgs; nr. , ) approved all animal procedures and experimental conditions. male wistar rats of -and -month-old were used, and plasma was obtained from the trunk blood. evs were isolated with exoquick following the manufacturer's instructions. microrna was isolated from evs and then amplified. microrna was labelled using the flashtag biotin hsr rna labelling kit and profiled on affymetrix genechip microrna . arrays. ingenuity pathway analysis (ipa) was used to identify pathways regulated by significantly altered micrornas. results: microarray analysis revealed micrornas. of these micrornas, were differentially expressed between aged and young-adult animals, micrornas were significantly upregulated and were downregulated in aged animals compared to young adult (p < . ; fold change of | . |). a conservative filter was applied on ipa and only experimentally validated and highly conserved predicted mrna targets for each microrna was used. ipa analysis showed that cardiac hypertrophic signalling is ranked as highly predicted targets for these differentially expressed micrornas (p < . ). moreover, ipa demonstrated that this canonical pathway is upregulated in aged animals when compared to young adult. in addition to cardiac hypertrophic signalling, other relevant cardiovascular canonical pathways, such as endothelin- signalling and intrinsic prothrombin activation pathway have predicted targets. summary/conclusion: our results showed for the first time that micrornas profile in circulating evs has a potential role to drive heart senescence and consequent cardiac diseases which represents the leading cause of death. introduction: introduction: the vascular endothelium and smooth muscle form adjacent cellular layers that comprise part of the vascular wall. here, we examined the extent to which extracellular vesicles (evs) vesicles participate in endothelial-vascular smooth muscle cell communication. methods: methods: evs were collected from rat aortic endothelial and smooth muscle cell serumfree media by ultracentrifugation. vesicle morphology, size and concentration were evaluated by transmission electron microscopy and nanoparticle tracking analysis. endothelial cell and vascular smooth muscle cell cultures were subjected to various concentrations of evs for various times. functional assays were performed. results: results: western blot as well as shot gun proteomic analyses revealed sets of proteins common to both endothelial-and smooth muscle-derived ev as well as proteins unique to each vascular cell type. functionally, endothelial-derived evs stimulated vascular cell adhesion molecule- (vcam- ) expression and enhanced leukocyte adhesion in vascular smooth muscle cells while smooth muscle evs did not elicit similar effects in endothelial cells. evs from endothelial cells also induced protein synthesis and senescenceassociated β galactosidase activity in vascular smooth muscle cells. proteomic analysis of vascular smooth muscle cells following exposure to endothelial cellderived evs revealed upregulation of several proteins including pro-inflammatory molecules, high-mobility group box (hmgb) and hmgb . pharmacological blockade of hmgb and hmgb and sirna depletion of hmgb in smooth muscle cells attenuated nfkb (p ) phosphorylation and nuclear translocation, vcam- expression and leukocyte adhesion induced by endothelial cell evs. summary/conclusion: conclusions: these data suggest that endothelial cell-derived evs can enhance signalling pathways that induce a pro inflammatory in vascular smooth muscle cells. introduction: graft patency is one of the major determinants of long-term outcome following coronary artery bypass graft surgery (cabg). biomarkers, if indicative of the underlying pathophysiological mechanisms, would suggest strategies to limit graft failure. many studies have generated compelling data on the sensitivity of mvs as biomarkers of cardiovascular disease progression and events. the mv usefulness in cabg has been tested only in a study that highlighted their importance in surgical haemostasis. no information is so far available on the association between the amount or pattern of circulating mvs and cabg outcome. we aimed to evaluate whether mv pre-operative signature could predict mid-term graft failure. methods: this was a nested case-control substudy of the coronary bypass grafting: factors related to late events and graft patency (cage) study that enrolled patients undergoing elective cabg. of these, underwent coronary computed tomography angiography months post-surgery showing % graft occlusion. flow cytometry mv analysis was performed in patients ( /group with occluded [cases] and patent [controls] grafts) on plasma samples collected the day before surgery and at follow-up. results: before surgery, cases had two-fold (p = . ) and four-fold (p = . ) more activated platelet-derived and tf+ mvs, respectively than controls. the mv thrombin generation capacity was also significantly greater (p < . ). this mv signature predicted graft occlusion (auc of . [ %ci: , - , ], p = . ). by using a mv-score ( - ), the or for re-occlusion for a score above was . ( % ci . - . , p < . ). summary/conclusion: the pre-operative signature of mvs is an independent predictor of mid-term graft occlusion in cabg patients and a cumulative mvscore stratifies patient's risk. since the mv signature mirrors platelet activation, patients with a high mvscore would benefit from a personalized antiplatelet therapy. exosomes from engineered immortalized human heart cells improve ventricular function and attenuate fibrosis in mice with arrhythmogenic cardiomyopathy yen-nien lin, lizbeth sanchez, rui zhang, thassio ricardo ribeiro mesquita, chang li, ahmed ibrahim, eduardo marbán and eugenio cingolani heart institude, cedars sinai medical center, los angeles, usa introduction: arrhythmogenic cardiomyopathy (ac) is characterized by progressive loss of cardiomyocytes and fibrofatty tissue replacement. currently, there is no effective treatment for this disease. exosomes (imexos) secreted by heart stromal cells, engineered to be immortal and overexpressing β-catenin, exert anti-inflammatory and anti-fibrotic effects and improve ventricular function in models of ischaemic injury (ibrahim et al., nature bme ). methods: to investigate the effectiveness of imexos in a murine model of ac, four-week old homozygous dsg knockout (dsgko) mice and wild type (wt, age-and strain-matched) mice were compared. dsgko mice were randomized to receive weekly imexos or vehicle via intravenous injection for weeks. neonatal rat ventricular myocyte (nrvm) proliferation and apoptotic assays were performed to explore potential effects of exosomes. results: biodistribution studies of dir-labelled imexos revealed some cardiac uptake, along with strong signals in spleen. at weeks, dsgko mice which had received intravenous imexos showed improved cardiac function (echocardiographic ejection fraction ± vs ± % in vehicle mice, p = . ), with an underlying attenuation in myocardial fibrosis by histology. electrophysiology test showed shorter qrs duration ( . ± . ms imexo vs . ± . ms vehicle, p = . ) and effective refractory period. programmed ventricular stimulation showed dsgko mice which had received imexos were remarkably less prone to ventricular tachycardia induction ( . ± % vs . ± % in vehicle, p = . ). in vitro study showed nrvm exposed to imexos for days exhibited higher brdu expression relative to vehicle group, and less annexin-v expression after oxidative stress induced by -minute illumination with nm uv. summary/conclusion: intravenous administration of imexos improved cardiac function, reduced cardiac fibrosis, and suppressed arrhythmogenesis in ac. our findings motivate clinical testing of imexos in ac, an orphan disease with great unmet medical need. funding: nih r hl (to em) cardiac-derived extracellular vesicles contribute to communication between heart and brain in chronic heart failure (chf) and target nrf / are signalling changhai tian a , lie gao b and irving zucker b a department of cellular and integrative physiology, university of nebraska medical center, omaha, usa; b department of cellular and integrative physiology, university of nebraska medical center, omaha, usa introduction: mirnas regulate the translation of proteins that are involved in redox homoeostasis in the heart and brain. intra-and/or inter-organ communication takes place by multiple mechanisms including extracellular vesicular (ev) transport. our previous studies suggested that cardiac derived mirna-enriched evs contribute to the dysregulation of nrf /antioxidant enzyme (are) signalling in the myocardium via intercellular cross-talk, and result in the decreased nrf /are signalling in the sympatho-regulatory areas of the brain in chf. however, it is unclear if cardiac derived evs circulate to the central nervous system evoking sympatho-excitation by disrupting central redox homoeostasis. methods: cardiac-specific membrane gfp+ mice were generated to track the brain distribution of cardiac evs in rats with chf (coronary ligation). the isolation and characterization of evs were carried out by differential ultracentrifugation, tem, nanosight, western blotting, and qrt-pcr. transfection, labelling, and microinjection of evs into the rostral ventrolateral medulla (rvlm) were performed. results: nrf protein was reduced in the rvlm of chf rats consistent with an upregulation of nrf -targeting mirnas. nrf -targeting mirnas were enriched in cardiac and circulating evs of chf rats. nrf -targeting and cardiac-specific mirnas were abundant in brain-derived evs. circulating evs were taken up by neurons in sympatho-regulatory areas of the brain. mirna-enriched evs from chf animals increased sympathetic tone which was prevented by a cocktail of nrf -targeting mirna inhibitors. summary/conclusion: myocardial infarction-induced mirna-enriched evs mediate the inter-organ crosstalk between heart and brain in the oxidative regulation of sympathetic outflow through targeting the nrf / are signalling pathway. these findings suggest that cardiac-derived ev mirnas targeting nrf /are signalling may act as an endocrine signalling mediator of chf that has potential as a novel therapeutic target. introduction: a fine-tuned communication between cardiac cells is vital to maintain myocardial integrity and contractility. not only an impairment of gap junction (gj)-mediated intercellular communication, but also defects in ev-mediated communication have been associated with ischaemic heart disease, a major causative factor of heart failure. we have previously shown that cx , the main ventricular gj protein, assembles into channels at the evs surface, mediating the release of vesicle content into target cells.the main objective of this work was to characterize the signals underlying protein sorting into extracellular vesicles (evs) in a human pathophysiological context, using connexin (cx ) as a model substrate. methods: animal models of ischaemia/reperfusion (i/ r) injury by ligation of the left anterior descending coronary artery, ex vivo and in vitro ischaemia models and human patients were used to investigate the secretion of ev-cx . results: release of cx was downregulated in circulating vesicles from i/r-injured mice and patients with st-segment elevation myocardial infarction, as well as in intracardiac and cardiomyocyte-derived evs. additionally, we show that ubiquitin signalled the release of cx in basal conditions but appeared to be dispensable during ischaemia. depletion of the autophagy adaptor p partially restored the secretion of cx , suggesting an interplay between ischaemiainduced cx degradation and secretion. summary/conclusion: overall, we demonstrated that ischaemia impairs the sorting of cx into evs, which may ultimately affect long-distance communication. through the identification of the underlying molecular mechanisms and players, these results pave the way towards the development of innovative diagnostic and therapeutic strategies for cardiovascular disorders. introduction: remote ischaemic conditioning is a cardioprotective intervention which protects the heart against ischaemia/reperfusion injury. transient activation of toll-like receptor (tlr ) and its downstream regulators (tnfα and il- ) have been implicated in cardioprotective interventions. extracellular vesicles (evs) play a role in cardioprotection through the activation of the tlrs. however, since isolation of evs in high amounts with suitable purity from blood is a challenge, our aim was to develop a cellular model system from which tlr-inducing, cardioprotective evs can be isolated in a reproducible manner. methods: ev release from hek cells was induced by calcium-ionophore a . evs were characterized, cytoprotection by evs against simulated ischaemia/ reperfusion injury and its mechanism were investigated in h c and ac cell lines. results: a induction of hek cell induced ev release and the isolates contained mostly large evs. evs decreased cytotoxicity and apoptosis due to h ischaemia followed by h reperfusion in h c and ac cells in a dose-dependent manner. evs activated tlr and its downstream signalling pathway in h c and ac cells as well as the expression of cytoprotective haem oxigenase (ho- ) in h c cells. summary/conclusion: a -induced evs exert cytoprotection in h c and ac cells by inducing tlr signalling and ho expression. therefore, evs released via calcium-ionophore treatment may serve as a basis of an efficient carpdioprotective therapy. introduction: biliary strictures may be benign or malignant. the major malignant causes of biliary stricture are a primary cholangiocarcinoma (cca) or pancreatic ductal adenocarcinoma (pdac). there is ongoing debate about adequate diagnostics in biliary strictures of unknown aetiology. micrornas (mirnas) are small non-coding rnas important in tumourigenesis. mirna have been found to be enriched in exosomes, small membrane-bound extracellular vesicles (ev) of endocytic origin, which is a novel pathway for intercellular signalling within the tumour microenvironment and have been implicated in loco-regional pre-metastatic niche formation. this project aims to investigate circulating-free and ev mirnas as biomarkers that can aid diagnosis in patients with a biliary stricture. we will ( ) isolate and characterise evs in plasma and bile from patients with benign and malignant biliary strictures (i.e. pancreaticobiliary cancers); and ( ) identify differentially expressed circulating-free and ev mirnas in plasma and bile suitable for detecting malignancy. methods: sample size (n = ) was calculated for a study power of % and α error of % for the ability of extracellular mirnas to discriminate benign from malignant biliary strictures. prospective matched plasma and bile samples will be collected from patients with benign (n = ) and malignant (n = ) biliary strictures undergoing endoscopic retrograde cholangiopancreatography (ercp). evs will be isolated from the biofluids by ultracentrifugation and/or size exclusion chromatography and then characterised (tem, nta and immunoblotting). circulating-free and ev-associated mirnas will be profiled using small rna sequencing. extracellular mirna "signatures" will then be validated by rt-qpcr, and diagnostic accuracy confirmed (sensitivity, specificity, auc). results: evs derived from patient samples have been characterised using nta, western blotting and tem. sec derived evs appear to be more well-defined than uc evs with marker positivity for cd , cd and cd . ongoing work will be focused on rna profiles of evs from both malignant and benign cohorts. summary/conclusion: there is currently no effective method to differentiate benign from malignant biliary strictures. novel plasma and bile circulating-free and ev-associated mirna biomarkers may improve the speed and accuracy of diagnosis, resulting in considerable patient benefits. furthermore, as little is known about the ev-associated function of these tumours, candidate ev-mirnas could be taken from "bedside to bench" and their function further investigated using in vivo, vitro and silico models. introduction: urine is a source of extracellular rna (exrna) biomarkers that can be obtained non-invasively throughout pregnancy. several studies have profiled extracellular mirnas in biofluids during pregnancy, but few have profiled extracellular mrnas (ex-mrnas) in urine. objective: to optimize methods for ex-mrna isolation and rna-seq library preparation from urine of healthy pregnant and non-pregnant females. methods: rna was isolated from pooled non-pregnant urine using kits based on ev precipitation (mircury exosome kit for csf/urine, seramir), ev affinity purification (exorneasy), and protein precipitation (mirneasy serum/plasma advanced). next, long (> nt) and short rnas were isolated from ev enriched urine of pregnant (n = ) and non-pregnant (n = ) individuals using the mircury kit followed by the mirneasy micro kit. rna-seq libraries were prepared using the smart-seq v ultra low input rna (oligo(dt) priming) and the smarter stranded total rna-seq kit v -pico input (random priming) methods (takara). preliminary data were obtained using the illumina miseq, and aligned using star v. . . .a. results: overall, rna isolation using mircury followed by the smart-seq v library preparation kit yielded the highest % of mapped reads: % in pooled non-pregnant, % in individual non-pregnant, and % in individual pregnant urine. for rna extracted using the mircury kit, the smart-seq v libraries had higher % of mapped mrna reads compared to pico libraries (p < . , t-test). in contrast for mirneasy advanced it was reversed ( % vs %). summary/conclusion: early results from low-depth sequencing show the highest mrna mapping rates for mircury followed by the smart-seq v kit. high-depth sequencing data are now being generated, which will enable us to perform detailed comparisons of different rna species from the rna profiles obtained using different library preparations and rna isolation methods from urine of pregnant and non-pregnant subjects. funding: this study was funded by nih k hd - , nih u hl , and a ucsd igm-illumina mini-grant. il- mutein-induced changes of exosomal mirna cargo in a humanized mouse model emily lurier, erik sampson, patrick halvey, mike cianci and katalin kis-toth pandion therapeutics, cambridge, usa introduction: regulatory t cells (tregs) are key contributors to immune homoeostasis. decreased number and/or function of these cells are frequent features of many autoimmune diseases linked to the development of tissue inflammation. while interleukin- (il- ) is essential for pan t cell proliferation and performance, low dose il- treatment has been shown to preferentially affect tregs and is being evaluated as an intervention in autoimmune diseases. pt is a novel il- mutein fc fusion molecule (il- m) designed to selectively engage with tregs. using a humanized nod-scid il rn-null (nsg) mouse model we have shown that pt expanded tregs without significant effects on other immune cells. we have also shown that tregs from pt -dosed humanized mice exhibit increased expression of foxp and cd , and demethylation of foxp and ctla- genes, suggesting enhanced function and stability. in the current study we investigated the mirna content of plasma exosomes isolated from pt -or vehicle-treated mice in order to identify treg specific mirnas from the il- m treated animals. methods: cd + haematopoietic stem cell humanized nsg mice were dosed once subcutaneously with pt or vehicle. plasma samples from mice were collected at day and exosome isolation was conducted using the exoquick method. small rna was extracted and quantified using the bioanalyzer small rna assay. an illumina nextseq instrument was used for library preparation and sequencing with bp single end reads at an approximate depth of - million reads per sample. raw sequences were mapped to human genome grch and analysed via a pipeline provided by the university of california santa cruz. results: rna within the exosomes from vehicle and il- m-treated groups was mostly comprised of mirna and trna. plasma was pooled from animals per treatment group and differential expression was determined using a twofold change cut-off. we found that pt treatment actively altered the mirna content of plasma exosomes, compared to exosomes from vehicle-treated mice. many of the differentially expressed mirnas are involved in immunoregulation. summary/conclusion: plasma exosomes from pt treated humanized mice encapsulated treatment-specific mirnas which can potentially be used as systemic biomarkers of treg expansion and function. identification of potential biomarkers in microglial specific exosomes isolated from prion-infected serum introduction: transmissible spongiform encephalopathies (tse) are neurodegenerative disorders caused by the misfolding of the cellular prion protein (prpc) to the beta-sheet rich abnormal prion protein (prpsc). prpsc aggregates in the brain and causes amyloid plaques, neuronal loss, spongiform degeneration and microglial activation. currently, definitive diagnosis of tse diseases is only confirmed post-mortem thus a diagnostic test in accessible body fluid is of interest. exosomes are a good resource for biomarker discovery since they cross the blood-brain barrier easily and contain protein, lipids and nucleic acids from the cells of origin. the goal of this study was to look at biomarkers from brain-originating exosomes (specifically microglia) isolated in the serum of prion-infected animals. methods: westerns and nanoparticle tracking analysis (nta) were used to look at the composition of microglial-specific exosomes. as proof of principle, exosomes were isolated from a microglial cell line (bv cells). a cd antibody was labelled with a fluorophore and binding to exosomes was visualized via nta. exosomes were isolated from serum of both prioninfected and mock-infected mice throughout disease course. a macrophage specific antibody (f / ) was bound to beads which were used to isolate exosomes which includes those of microglial origin. microrna was extracted from these exosomes and next-generation sequencing (ngs) was performed using the illumina platform. clc genomics workbench was used for bioinformatics analysis. results: microglial and macrophage proteins (tmem and iba ) were identified in exosomes isolated from bv cells and prion-infected mouse serum. macrophage exosomes were isolated via a novel antibody-bead based system. results of the ngs analysis of the microrna isolated from these exosomes indicated a series of mirna that could differentiate between control and infected samples as well as age-specific markers. summary/conclusion: to our knowledge, this is the first time microglial-specific exosomes have been isolated from prion-infected serum from early and end stage disease. the results of this analysis could facilitate the diagnosis of prion disease in easily-accessible biofluids pre-mortem. comparison of urinary extracellular vesicle isolation methods for transcriptomic biomarker research in diabetic kidney disease introduction: urinary extracellular vesicles (uevs) are emerging as a source for early biomarkers of kidney damage, holding the potential to replace the conventional invasive techniques including kidney biopsy. several methods are available for uev isolation. our aim was to compare different workflows and isolation by hydrostatic filtration dialysis (hfd), ultracentrifugation (uc) and a kit based isolation method for their subsequent use in mirna-seq and rna-seq for biomarker discovery in diabetic kidney disease. methods: type diabetic patients (t d) with macroalbuminuria and normoalbuminuric healthy controls were included in the study. sample collection and all experiments were performed in accordance with the declaration of helsinki. evs were isolated from - ml of h urine collections by uc, hfd, or a commercially available kit (purification based on spin column chromatography, urine exosome purification and rna isolation midi kit, norgen biotech, canada) each with different established urine clarification steps. quality control of the evs was performed with negative staining em, nta and western blotting. isolated rnas were profiled with bioanalyzer pico kit and subjected to mirna and mrna sequencing. for rna-seq, cdna library was prepared using smart-seq v ultra low input rna kit for sequencing (takara bio, japan). rna-seq was performed using hiseq (illumina). mirna-seq library was prepared using qiaseq mirna library kit (qiagen, germany). mirna-seq was performed on the illumina hiseq platform (illumina). results: our data showed that uev yield, morphology and size distribution were closely similar in hfd and uc preparations, while lower yields were obtained using the kit. by western blot, ev markers were detectable in samples isolated by hfd and uc but not readily in samples isolated with the kit. tamm-horsfall protein was detected in all the samples and albumin levels appeared higher in hfd and kit isolated samples relative to uc samples. the number of paired-end reads for rna-seq in hfd and uc samples (in both > m) were closely similar. instead, rna reads were lower than m for the kit samples. for mirna-seq, the number of reads as well as the molecular biotype distribution were similar for the three methods. by principal component analysis of the rna-seq data, we observed that hfd and uc grouped together showing similarities. however, for mirna-seq data such similarities were not obvious. this suggests that the three different workflows and isolation principles may enrich different mirna-rich uev preparation components. summary/conclusion: our transcriptomics data shows that hfd and uc are suitable methods to isolate uevs for mirna-seq and rna-seq. the kit based method appears better suited for mirna-seq. introduction: exosomes contain a variety of biomolecules including dna. knowledge of cfdna distribution and localization in bioliquid is important for understanding both biological function of cfdna and exosomes. some publications state that a large proportion of plasma cfdna is localized in exosomes. to quantify cfdna content in free vs. exosomal form in human plasma, urine, and saliva, we employed subx technology, which allows affinity capture dna via phosphates groups of the polynucleotide chain and exosomes via membrane surface phosphate moiety clusters. subx is a proprietary compound that can simultaneously bind to both cfdna and exosomes in bioliquids, thus allowing precipitation of the [subx-dna/ subx-exosomes] complexes without ultracentrifugation. methods: detection of subx-dna and exosomes binding was done by measurement of particle sizes using zetasizer nano zs and nanosight ns . the samples were processed with the subx exo-dna isolation kit following the standard protocols. dna, protein and lipid concentrations were measured by fluorescent assays using qubit fluorometer. results: subx efficiently and selectively captures and co-precipitates cfdna and exosomes directly from bioliquids. exosomes are easily extracted from the pellet in exosome reconstitution buffer (erb), followed by subsequent isolation of tightly bound cfdna from the subx pellet. erb does not extract dna form the [subx -dna] pellet and thus does not contaminate reconstituted exosomes with cfdna. thus, we separate two distinct types of extracellular materialintact exosomes and purified cfdna in a single protocol from the same sample. over % of dna in plasma and urine exist as a free circulating pool, while in saliva up to % is associated with exosomes. thus, cfdna distribution is probably bioliquid-specific and must be evaluated by methods that eliminate cfdna-outer exosomal membrane aggregation. summary/conclusion: subx technology is suitable for simultaneous isolation of both cfdna and exosomes from the same bioliquid sample. subx separates cfdna fragments non-specifically attached to the outer lipid layers of the exosome membrane from the true intra-exosomal cfdna. in contrast, salting-out peg technique is associated with aggregation of macromolecules and vesicles and thus leads to overestimation of exosome-associated polymers content, including cfdna. tracing extrachromosomal dna inheritance patterns in glioblastoma using crispr eunhee yi, amit gujar, hoon kim, albert cheng and roel verhaak jackson laboratory for genomic medicine, farmington, usa introduction: glioblastoma multiforme (gbm) is the most lethal brain tumour; it is characterized by poor response to standard post-resection radiation and cytotoxic therapy, resulting in a dismal prognosis with a five-year survival rate of %. recurrence after therapy for gbm is unavoidable. there are substantial differences among the cells of gbm tumours in the abundance and types of genetic material. this heterogeneity likely is the major cause of therapy failure, the development of treatment resistance, and ultimately recurrence. a recent study has suggested that the amount of a particular type of dnaextrachromosomal dna (ecdna)differs substantially among different gbm tumours, and differs within a given gbm tumour over time. despite the speculation that ecdna is a key factor of tumour heterogeneity, how ecdna is propagated and distributed amongand how it behaves withincancer cells is completely unknown. methods: to address this gap in knowledge, this study focused on developing a novel cytogenetic crisprbased tool that enables visualization and tracking ecdna behaviour in live gbm cells. results: we found breakpoint sequences resulting from genome rearrangements during ecdna formation by performing computational analysis from whole genome sequencing data. and each breakpoint was regarded as a unique target sequence for ecdnaspecific labelling. the uniqueness of each breakpoint was validated by breakpoint-pcr (bp-pcr). furthermore, the location and the amount of each breakpoint were observed by breakpoint-fish (bp-fish) analysis in gbm cells. summary/conclusion: this results will be strong evidence to make ecdna-specific crispr system in further research. tracing ecdna dynamics will provide new insight into the impact of ecdna on cancer evolution. introduction: small extracellular vesicles (sevs) are - nm vesicles that mediate intercellular communication by transferring rna and proteins to the recipient cells. these cargo molecules are selectively sorted into sevs and mirror the physiological state of the donor cells. given that sevs can cross the bloodbrain barrier and their composition can change in neurological disorders, there is an increasing interest in elucidating the molecular signatures of sevs in circulation as disease biomarkers. however, circulating sevs are derived from multiple cellular sources and determining their source is challenging. information on sev composition can be beneficial in predicting whether these sevs are released predominantly from central nervous system cells. we hypothesized that differentially expressed mirnas between neuronal sevs and astrocytic sevs could be used as cell-typespecific signatures. methods: small extracellular vesicles were isolated from cell culture media of postnatal mouse primary neurons and astrocytes using differential centrifugation and characterized using nanoparticle tracking analysis, transmission electron microscopy and western blotting. rna from neurons, astrocytes, and their respective sevs were used for transcriptome and small rna sequencing. results: we observed that only a subset of cellular mirnas was packaged into sevs; differential expression of specific mirnas between sevs and their corresponding cells suggest that cells employ special mechanisms to sort mirnas into sevs. these mechanisms could be celltype specific since neuronal sevs showed a different mirna profile compared to astrocytic sevs. exomotifs, the short sequence motifs that control the loading of rna into sevs, were present in differentially expressed mirnas. we also observed that five rnabinding proteins, which are associated with passive or active rna sorting into sevs, were differentially expressed between neuronal and astrocytic cells. summary/conclusion: mirna signatures of sevs from neurons and astrocytes could be beneficial in determining if these cell types contribute to the alterations of sev composition in circulation in neurological disorders. cell-type-specific selectivity in rna loading might be attributed to the differential expression of rna-binding proteins. introduction: analytes present in the extracellular fraction of bodily fluids (ex. blood, urine) have utility as a tool for uncovering the molecular landscape of tumours and hold great potential for discovery of individualized cancer medicine. urine, being noninvasive as a sample type, has an obvious advantage over blood when used for liquid biopsy purposes. however, potential for microbial proliferation and the labile nature of host cells and extracellular vesicles (evs) at the point of sample collection/transport to the lab drives the need for stabilization of urine samples. development of such sample stabilization opens up capability for the detection of various biomarkers present in the extracellular fraction to be used in liquid biopsy. this is of particular concern as studies around urinary analytes for cancer diagnosis, progression and therapeutic effect are rapidly expanding in cohort sizes. multi-site collections and at-clinic collections are increasingly prohibitive for large scale recruitment and also lead to variability in the time between collection and processing. methods: in this study, we have analysed two commercially available ev extraction kits and compared them with ultracentrifugation technique for size, concentration and specificity of the isolated evs from human urine samples with and without our proprietary preservation solution using nanoparticle tracking analysis and western blot analysis for exosomal membrane markers. ev rna contents in various urine fractions (first morning first void, random first void and midstream) were compared using rt-qpcr assay to provide better understanding of the collection techniques and fractionations that are ideal for ev research work. results: in our current work, we have bench-marked human urine collection and ev extraction in order to provide recommendations in standardization of sample acquisition and processing for urinary ev studies. we have utilized these standardization in order to develop a novel and efficient sample stabilization principle for preservation of evs and ev rna in urine samples during an ambient temperature hold. summary/conclusion: taken together, we have established a framework for evaluating technologies and techniques in the ev sample processing space, which can be utilized by other research groups. vn -isolated plasma extracellular vesicles improve tumour mutation detection by next-generation sequencing compared to cell-free dna and correlate with tissue biopsy of nsclc patients introduction: liquid biopsy is a minimally-invasive diagnostic method that detects circulating biomarkers and has the potential to improve access to molecular profiling for nsclc patients when tissue biopsy material is unavailable or insufficient. although isolation of cell-free dna (cfdna) from plasma is the standard liquid biopsy method for detecting dna mutations in cancer patients, the sensitivity can be highly variable. vn is a amino acid peptide with an affinity for heat shock proteins that are exposed on the surface of extracellular vesicles (evs); peptide-ev aggregates readily sediment using a benchtop centrifuge and therefore the vn peptide provides a rapid, clinically-amenable procedure for ev isolation. in this study, we determine whether isolation of evs from nsclc patient plasma improves the sensitivity of single nucleotide variants (snvs) detection compared to cfdna and correlate genetic changes observed by liquid biopsy with tumour ffpe tissue biopsy. methods: blood was collected from stage iii/iv nsclc patients with informed consent in either edta or cell-free dna bct® collection tubes and plasma was harvested within minutes. total nucleic acid (tna) was extracted from either vn -isolated evs from edta plasma or directly from plasma collected in edta or cell-free dna bct® tubes (cfdna). snvs were detected by next-generation sequencing (ngs) results: vn isolation of evs from plasma resulted in higher recovery of dna than cfdna isolation. the snvs detected in both ev-dna and cfdna correlated well with those reported in matched ffpe tumour tissue using ngs, including % specificity for egfr mutations. no improvement in snv detection was observed using cell-free dna bct® collection tubes compared to edta tubes. isolation of evs with the vn peptide prior to sequencing improved a number of ngs parameters including library yield, total reads, median read coverage and molecular coverage, resulting in improved sensitivity of snv detection. summary/conclusion: in summary, our research demonstrates that vn -based ev isolation is useful for molecular profiling of nsclc patients for whom tissue biopsy is not an option, thereby improving access to molecular profiling and targeted therapies. funding: atlantic canada opportunities agency novel markers for neuroendocrine prostate cancer divya bhagirath a , michael liston b , theresa akoto a and sharanjot saini a a augusta university, augusta, usa; b veteran affairs, san francisco, usa introduction: prostate cancer (pca) is fuelled by androgens and androgen receptor (ar) signalling. therefore, ablation of ar signalling by androgen deprivation therapy (adt) is the goal of first-line therapy that results in cancer regression initially. however, two to three years post-adt, the disease develops into castration-resistant prostate cancer (crpc). as a second-line of therapy, next generation of ar pathway inhibitors (api) such as enzalutamide (enz) are used that are effective initially followed by emergence of drug resistance. a subset of api-resistant tumours emerges to an ar independent state via undergoing a trans-differentiation to neuroendocrine lineage, a process referred to as neuroendocrine differentiation (ned). due to lack of ar signalling, these pca variants, referred to as neuroendocrine prostate cancer (nepc), are impervious to anti-androgen therapy and constitute an aggressive variant of advanced crpc with poor prognosis. currently, there is a lack of effective molecular biomarkers for predicting api therapy resistance and emergence of therapy-induced ned. methods: exosomes/evs were isolated from sera of a patient cohort with/without ned. the study was conducted in accordance with ethical guidelines of us common rule and was approved by the institutional committee on human research. written informed consent was obtained from all patients. following extensive characterization of evs by electron microscopy, nanosight tracking analyses and western blotting of exosomal markers, small rna sequencing was carried out on illumina hiseq platform to identify differentially expressed transcripts. machine learning algorithms were applied to clinical sequencing data to train a "mirna classifier". further, we probed the proteomic profile of exosomes isolated from nepc cellular model nci-h and enzalutamide resistant crpc cell lines by mass spectrometry. results: we identified that transition from crpc-adenocarcinomas to neuroendocrine states is associated with significant ev-mirna dysregulation, with a specific dysregulation in certain mirna families. with the application of machine learning algorithm, we identified an ev-based "molecular classifier" that can robustly stratify crpc-ne tumours from crpc-adenocarcinomas. proteomic analyses identified novel nepc-specific, glycosylated proteins that can be exploited for nepc diagnosis. summary/conclusion: our data suggest that ev mirna and protein profile can predict neuroendocrine differentiation in advanced castration-resistant prostate cancer patients. exosomal mrna in diagnosis strategy for hepatocellular carcinoma aleksandr abramov, alisa petkevich, vadim pospelov and pavel ogurtsov peoples' friendship university of russia (rudn university), moscow, russia introduction: exosomal cargo is informative source illustrating the genetic events happening in cells, what can be especially advantageous in case of cancer development for disease progression or treatment effectiveness monitoring. methods: plasma samples of hepatocellular carcinoma (hcc) patients, plasma samples of patients with liver cirrhosis - on the hepatitis c virus (hcv) background, healthy donors' plasma samples. exosomes were isolated with ultracentrifugation, western blot (cd , cd ) was performed. total mrna was isolated with exosomal rna isolation kit, norgen biotec corp. sequencing was carried out on a minion sequencer. housekeeping genes (gapdh, b m, actb, tuba a). detected mutations were confirmed by real-time pcr with specific highly sensitive lna probes. results: significant changes in expression levels were identified for genes in hcc and liver cirrhosis groups (increasing up to x compared to control samples and decreasing up to no detected expression). in out of patients with hcc mutant burden was significant increased compared to mutant burden in groups with cirrhotic samples. in out of patients with hcc increased expression for mrna line- was identified compared to cirrhotic patients. summary/conclusion: exosomal mrna expression levels may serve as a prognostic and diagnostic marker for patients with liver cirrhosis caused by hcv for hcc risk development. funding: research is supported with federal funds " - " circulating extracellular vesicle signatures in small cell lung cancer michela saviana a , giulia romano a , giovanni nigita b , robin toft a , patricia le a , kai wang c , mario acunzo a and patrick nana-sinkam a a virginia commonwealth university, richmond, usa; b the ohio state university, columbus, usa; c institute for systems biology, seattle, usa introduction: lung cancer is the leading cause of cancer deaths worldwide and classified primarily as either non-small cell lung cancer (nsclc) or small cell lung cancer (sclc). compared to nsclc, sclc has a faster growth rate, earlier widespread metastasis, and shorter overall survival. the early diagnosis of sclc and the development of novel therapeutics have proven challenging. thus, progression and recurrence rates remain high. non-invasive methods for cancer detection are increasingly being used to inform clinical decision making. extracellular vesicles (evs) have recently emerged as potential carriers of genetic contents such as micrornas (mirs) to induce reprogramming of components of the microenvironment in cancer initiation and progression. moreover, extracellular mirs expression profiles have been shown to have signatures related to tumour classification, diagnosis, and progression. methods: we selected a cohort of patients divided into groups: high-risk smokers, adenocarcinomas, squamous carcinomas, and sclc. we extracted total circulating ev and plasma rna from plasma ( patients in total) and rna from plasma in a separate group ( patients in total). utilizing both next-generation sequencing (ngs) and nanostring platforms, we analysed for global microrna (mirs) expression patterns. candidate mirs were then validated by qrt-pcr. results: we identified several deregulated mirs in both evs and plasma of sclc patients compared to the other groups. for evs, we validated mir- - p as a significant biomarker for the late stage of sclc compared to controls. in the case of plasma, we validated the upregulation of mir- in sclc compared to controls. summary/conclusion: our results indicate that a potential combination of plasma (mir- ) and ev-based (mir- - p) mirs be valuable biomarkers for sclc detection and serve as a basis for a non-invasive sclc classifier. funding: virginia commonwealth university, doim -nih/nci introduction: the isolation of evs from milk is technically challenging due to the complexity of milk. currently used separation procedures allow for the removal of milk fat globules and cells (by low speed centrifugation of fresh milk), removal (by acidification), or disruption (by addition of edta) of casein micelles. using these protocols the integrity, composition and targeting of bovine milk evs has been evaluated and has led to believe that milk evs might withstand these conditions. however, the effects on functionality of milk evs (i.e. immunomodulatory properties) after processing and isolation have not been studied. therefore, we have set up an in vitro culture system using a human t cell line that allows for the rapid screening of milk ev functionality. methods: fresh bovine milk was defatted and cells were removed after x , g centrifugation, followed by differential centrifugation at , g and , g. this milk was either subjected to acidification with hcl, or edta was added, or the milk supernatant remained untouched. top down optiprep density gradient separation followed by sec was used to further purify evs. these highly purified milk evs were added to human jurkat t cells, which were simultaneously stimulated using anti-cd and anti-cd antibodies. after h t cell activation was measured by il- cytokine production. results: precipitation or disruption of casein micelles allowed for the substantial removal of proteins during isolation compared to directly isolated evs, which aids in the purification of milk evs. in vitro analysis revealed that in the presence of directly isolated, or edta isolated milk evs, jurkat cells were suppressed in their activation as measured by il- production. remarkably, evs isolated from hcl-acidified milk were impaired in their suppressive capacity to inhibit il- production. summary/conclusion: although casein removal from bovine milk greatly improves purity of isolated milk evs, the detrimental effects on ev functionality should be considered. interestingly, evs exposed to acidic conditions lost their ability to modulate t cell activation, which is in contrast with the general believe that milk evs could withstand the gastro-intestinal tract. funding: this work is funded by the european union's horizon framework programme under the grant fetopen- evfoundry. optimising methods for separation and characterisation of extracellular vesicles from skim milk and infant milk formula introduction: infant milk formula (imf) is intended to impart nutrition to infants, similar to breast milk. however, although industrial imf production involves harsh treatment, potential consequences on extracellular vesicles (ev) in imf are not yet established. this study aimed to optimise methods for separating evs from imf and skim milk (sm) and to characterise the evs in accordance with misev . methods: sm and imf were either not treated (nt) or treated with acetic (aa) or hcl acid (isoelectric precipitation, ip), to remove caseins. samples were then subjected to differential ultracentrifugation (duc) or gradient ultracentrifugation using iodixanol solution (guc). for duc, ml samples were centrifuged at k g, k g, k g, k g and k g sequentially for min each and pellets re-suspended in ml pbs. preparation of agarose microspheres for high-efficient separation of extracellular vesicles cheng-tai chen, chien-an chen, carolyn yen and nien-tzu chou industrial technology research institute, chutung, taiwan (republic of china) introduction: size exclusion chromatography (sec) is becoming a widely used technique for separating of extracellular vesicles. various commercially available products were launched on the market, however, their separation efficiencies were not fully disclosed. herein, novel porous agarose microspheres with the tunable diameter and pore size were synthesized by emulsion reaction. the performance was evaluated and compared with commercial products. the modified sec column packing materials were shown to exhibit advantages for rapid, high-recovery and high-purity separation of extracellular vesicles from cell culture-conditioned medium and human plasma. methods: the homemade sec column was packed by gravity flow. μl of the sample was loaded and the pbs buffer was used as eluent. factions were collected and analysed by cd /cd sandwich elisa assay and by micro bca assay for determining respectively extracellular vesicles and total protein content. results: agarose microspheres were prepared by emulsification. the particle size can be controlled by the types and concentrations of surfactants. the product was collected by desired screen meshes and used as packing materials of the sec column. our results showed that the extracellular vesicles were clearly separated from proteins. more than . % of proteins were removed while the recovery of extracellular vesicles was close to %, which is much higher than % of the commercial product. the total separation time was less than min. summary/conclusion: we have established an approach for generating spherical agarose microspheres as packing materials of homemade sec columns, which are capable of separating extracellular vesicles from complex samples with high efficiency. further validations with additional samples are currently ongoing. immunomagnetic sequential ultrafiltration (isuf) platform for enrichment and purification of extracellular vesicles from large and small volumes of biofluid eduardo reategui, jingjing zhang, luong t. h. nguyen, richard hickey, nicole walters and andre f. palmer the ohio state university, columbus, usa introduction: evs derived from tumour cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. however, compromises have to be made when using a particular technology/methodology for the isolation of evs. currently, there is a trade-off between sample volume and specificity in ev isolation technologies that limits quantitative molecular analysis of ev contents, ultimately impacting the utility of evs in cancer diagnostics. here, we present an approach called immunomagnetic sequential ultrafiltration (isuf). our platform combines ultrafiltration and immunoaffinity separation. using isuf, we demonstrate that small or large volumes of biofluid can be processed (~ µl or > ml) while concomitantly removing . % contaminating proteins. we also processed serum from breast cancer patients enabling the characterization of different tumour and immune biomarkers on the isolated evs. methods: human samples were collected under an approved irb. size distribution and concentration of evs were measured using a tunable resistive pulse sensing (trps) method. ev proteins and rnas were extracted and quantified using a bca protein assay and uv spectroscopy. isuf and other ev isolation methods were compared for ev concentration, protein, and rna quantity. results: ml of cell culture media (ccm), . ml serum, and ml urine samples were processed with the isuf platform and recovered in µl. for all cases, evs were enriched with recovery efficiency greater than %. the processing time for a ml sample was min with over % of purity. we compared ev concentration and purity isolate from . ml serum using isuf and other commercially available methods, isuf demonstrated superior performance on isolating evs at high concentrations and purities. analysis of total rna amounts in the isolated evs using different methods was corresponding to higher ev recovery efficiency of isuf. we also compared protein and rna levels of evs enriched with isuf present in urine and serum samples from the same donors (n = ), and we found that for the same number of evs, the ev rna concentration from both biofluids showed no significant difference. finally, we have processed serum samples from metastatic breast cancer patients and demonstrated that their isolated evs have expression levels of her , cd and mir biomarkers at significantly higher levels than healthy controls. summary/conclusion: the isuf platform can be scale-down or -up to work with small or large volumes of biofluids for the isolation of evs. using the isuf platform with clinical samples shows the potential of our platform to be used for cancer diagnosis or monitoring treatment response. funding: national institutes of health (nih) grants ug tr (e.r.); r hl , r hl , and r eb (afp). challenges in exosomes isolation from primary biological samples derived from multiple myeloma patients introduction: multiple myeloma (mm) remains incurable despite advances in its treatment and research progress on the crosstalk between mm and surrounding host cells. exosomes are important regulators of the cellular niche. their importance for diagnostic and therapeutic applications has been proven in many cancers. in this context we hypothesized that a better understanding of the molecular role and features of mm-derived exosomes would provide a basis for their use for both risk stratification and as predictive biomarkers of response to anti-mm drugs already in use in clinical settings, given the optimization and validity of their isolation/purification method. methods: exosomes were isolated from human mm cell lines (hmcls) supernatants and peripheral blood plasma (pbpl) isolated from healthy donors, mm and mgus (monoclonal gammopathy of undetermined significance) patients. both fresh and frozen samples were tested. we evaluated commercially-available kits, density-based separation and ultracentrifugation. results: higher purity and recovery, evaluated by western blotting, nanoparticle tracking analysis and electron microscopy, were observed for supernatant density-based purification and for pbpl resin-based isolation. exploring the function of mm-derived exosomes, we observed an increase in proliferation of the immortalized stromal cell (sc) line hs treated with exosomes when compared to untreated cells, and a higher increase in proliferation of scs treated with mm-exosomes when compared to exosomes derived from normal and mgus pbpl samples. summary/conclusion: the method of isolation represents a critical step in the study of exosomes as many factors can affect the purity, yield and downstream application. our data demonstrated that density and resin-based isolation methods provided functional mm-derived exosomes with proliferative effects on scs. altogether our findings may serve as a guide to choose exosome isolation methods for mm studies. further optimization steps, including albumin-depletion from plasma samples and use/type of serum in cell cultures, should be taken into consideration when planning proteomics and genomics as downstream applications. funding: australian government rtp and monash departmental scholarship. a rigorous method for exosome isolation from post-mortem eyes introduction: in order to determine and validate the tissue-specific content of extracellular vesicles (evs) in biofluids, robust ev isolation methods from tissues must be developed. however, to date very few rigorous methods to isolate or enrich for intact evs from tissues have been reported. we present a comprehensive exosome isolation method with a sufficient level of characterization to unequivocally demonstrate true ev identity from ex vivo eyes. methods: iodixanol (optiprep) buoyant density gradient ultracentrifugation (dguc), cushioned dguc (c-dguc), and our newly developed c-dguc immunocapture (c-dguc-ip) method were used to compare yield and enrichment of exosomes isolated from porcine eyes between to hours post-mortem. yield was assessed by nanoparticle tracking analysis (nta) and immunoblotting for exosomal markers along with total protein quantitation. enrichment was assessed by comparison of exosomal markers, ocular-specific markers and known contaminant markers, plus in-depth proteomic mass spectrometry analyses. results: high enrichment of posterior eyecup small evs (sev) were achieved by dguc and c-dguc, with c-dguc resulting in an eightfold increase in yield by nta and two to fivefold increases of exosomal protein markers such as syntenin- and cd by immuno-blotting compared to dguc. interestingly, in-depth proteomic analyses revealed that a majority of these sevs with densities of . - . g/ml isolated by dguc and c-dguc were likely of endoplasmic reticulum (er) and golgi origin, suggesting er-to-golgi transport vesicles resulting from post-mortem tissue cell rupture. in order to enrich further for sevs (including exosomes) we subjected sevs isolated by c-dguc to anti-cd immunocapture. the resulting sev proteome was enriched . -to -fold for bona fide sev and exosome markers compared to c-dguc. summary/conclusion: the c-dguc method provides an enhanced yield and purity of sevs and exosomes from ex vivo eye tissue. however, to avoid significant contamination with er and golgi-derived vesicles from postmortem eyes, a final ev-specific immunocapture step is required to achieve sufficient purity for subsequent analyses. our highly rigorous method paves the way for identification and validation of ocular-derived exosomes in blood and their potential use as eye disease biomarkers. characterization of the extraction of extracellular vesicles using a lab-ona-disc filtration system introduction: personalized treatment for cancer is a promising way to face the multiplicity of the disease, to increase the efficacy of drugs and to decrease their toxicity. as part of this strategy, liquid biopsy explores a new non-invasive approach to diagnose cancer, guide treatment and monitor its efficacy. extracellular vesicles (evs) are nanometric lipid bilayers micelles with high potential as biomarkers. they are involved in the transfer of information (proteins, rna and dna) between cells. evs include a broad spectrum of particle sizes, from the tens to thousands of nanometres. the isolation of evs from complex matrices is the first step of any protocol and is particularly important for the reproducibility and fidelity of the results presented, as it could bring bias in further analysis. in order to explore the heterogeneity of evs, a full characterization (physical and biological) of the extracted evs is needed. we evaluate and compare evs purification methods, including ultracentrifugation, sizeexclusion chromatography (sec) column and an emerging microfluidic technology: labspinner filtration labon-a-disc device isolating evs between two filters of and nm. methods: a cell supernatant was used as a model matrix. we compared three methods of extraction of evs: ultracentrifugation with two cycles of h at , g at degrees celsius (rotor type ti, beckman floor ultracentrifuge optima l k), qev size exclusion chromatography columns from izon (qevoriginal/ nm) and lab-on-a-disc filtration system (labspinner, exodisc c). evs characterization was conducted with nta (nanosightns ), trps (izon), nanodrop (spectrometernd ), tem (fei tecnai kv) and custom micro-immuno-assay. results: in this study, we characterize a filtration system made of two serial filters of nm and nm pores for isolation of evs. compared to ultracentrifugation and chromatography columns, yield of extraction is up to times higher and the size of the extracted particles is smaller. tem imaging was used for assessment of the quality of the extracted evs. however, albumin concentration measurement tends to show that the purity of the solution is decreased. the immuno-labelling analysis shows that the proteomic signature of the extracted evs differs according to the extraction methods. the new filtration technology seems to give us access to a broader range of evs compared to standard methods. summary/conclusion: in this study, we characterized purification methods including lab-on-a-disc filtration, and were able to demonstrate an increase of the concentration of evs by a factor of , a decrease of the size of the accessible extracted particles and access to new proteomic signatures. funding: we acknowledge the support of génome québec and action marie skłodowska-curie. effects of sample processing on isolation of extracellular vesicles from blood plasma by centrifugation darja božič a , matej hočevar b , veno kononenko c , marko jeran a , urška Štibler d , immacolata fiume e , manca pajnič f , ljubiša pađen f , ksenija kogej g , damjana drobne c , ales iglič h , gabriella pocsfalvi i , veronika kralj-iglič f and darja bozic j introduction: the isolation of extracellular vesicles (ev) from body fluids is still controversial and the poor understanding of vesicle stability and effects of sample processing is probably one of the core issues preventing the breakthrough of this field into applicative practices. methods: we performed an in-depth study of sample changes in blood, blood plasma and samples throughout the increasing speed of centrifugation, considering the number, size, contents and shape of particles in the isolates. flow cytometry, light scattering, mass spectrometry and scanning electron microscopy were employed to reveal the properties of material in the samples. results: the particles of size about - nm with characteristic topology of membrane vesicles without internal structure were observed by the scanning electron microscope only in ev isolates prepared from fresh blood sample. inspection of the tube surface in which the isolation took place suggests that those particles are likely formed from activated platelets tearing at the tube wall due to the centrifugal pull. the isolates prepared from frozen blood plasma prepared by centrifugation with different forces contained different amounts of particles with similar protein contents, predominated by highly abundant human plasma proteins, including albumins and immunoglobulins. some lipoprotein clearance and fibronectin precipitation were however observed through increased speed and time of centrifugation. summary/conclusion: the results of this study [ ] contribute to the understanding of stability and dynamics of membrane particles. the reported evidence provides the support for viewing ev isolates as a product, shaped by uniqueness of the starting samples and the thermal and mechanical stress applied upon processing. we believe this kind of insights strengthen our ability of reading the story of evs. introduction: apoptosis is a form of programmed cell death with diverse roles in the tumour microenvironment and emerging data show that, besides its role in tumour suppression, it can also promote oncogenic proliferation. highly aggressive tumours such as burkitt lymphoma (bl) show high levels of apoptosis, which has a diagnostic and prognostic value for classifying and staging the disease. we hypothesize that amongst other elements, extracellular vesicles (ev) are key mediators of apoptotic cell-derived tumour microenvironment signals. here, we report on ev released in vitro by apoptotic bl cells (apo-ev) in relation to their potential use as cancer biomarkers. methods: basic physical properties of apo-ev such as structure, size distribution, surface charge and membrane fluidity are discussed using cryo electron microscopy (em) and tomography, nanoparticle tracking analysis, dynamic light scattering and fluorescence anisotropy respectively. for phenotypic analysis we apply immunocapture and flow cytometry, immunogold labelling on transmission em, fluorescence microscopy and quantitative pcr. in addition, we study the interaction of apo-ev with blood components such as platelets, leucocytes and red cells, in order to understand their effects in the circulation and therefore their potential for analysis in blood samples. results: looking at the differences between apo-and non-apo-ev, apo-ev have larger diameter, while structurally are not different. however, we have identified distinct apo-ev markers such as active caspase and histones, or dna and small non-coding rna-y. there is also strong interaction of ev with platelets and leucocytes but not with red cells, indicating potential routes of transfer of ev cargo in the circulation. summary/conclusion: it is concluded that for the characterization of the heterogenous ev populations, combination of multiple techniques is often required, and also, understanding the strengths and limitations of each method is essential for choosing the appropriate set of analytical tools. finally, we consider that monitoring free circulating apo-ev or blood cells with which they have interacted is a promising approach to improve cancer diagnosis, prognosis and evaluation of therapeutic response. casting a small netrin: functional roles of a novel surface factor on stroma-derived extracellular vesicles in pancreatic cancer kristopher s. raghavan a , ralph francescone b , janusz franco-barraza b and edna cuckierman b a drexel university; fox chase cancer center, philadelphia, usa; b fox chase cancer center, philadelphia, usa introduction: pancreatic ductal adenocarcinoma (pdac) is a devastating disease driven and supported by changes in its microenvironment, or stroma. here we dissect the intercellular communication that exists between the primary stromal component, cancer-associated fibroblasts (cafs) and pdac. pdac communicates with its microenvironment, in part, through the exchange of specific types of extracellular vesicles (evs). specifically, we focus on the mechanism by which caf-secreted evs support pdac survival, with an additional goal to identify biomarkers suitable to generate a future "liquid biopsy" test for early pdac detection and prognosis. methods: evs are isolated from patient-derived pdac-associated fibroblasts via differential ultracentrifugation and validated by isev standards. human pdac cell lines used as recipient cells are treated with caf-evs to assess their role in supporting pdac survival. recombinant proteins, neutralizing peptides, and non-functional mutant proteins are used to block ev interaction with target cells. results: we observe sub-types of caf-evs containing unique surface receptors. one ev sub-population of interest contains a novel surface protein (nsp) expressed on the plasma membrane of pancreatic cafs, but not their healthy counterparts. further, pdac cells up-regulate nsp's lone binding partner, suggesting a role for these factors in pdac-selective ev uptake. functional assays designed to test pdac viability suggest these nsp(+)-evs protect pdac cells from programmed cell death as a result of physiological stress. this ev-mediated survival benefit can also be inhibited by blocking the interaction of nsp and its binding partner, suggesting the engagement of these two factors is necessary for cafs to support pdac via evs. pursuing our biomarker goal we confirm stromal nsp expression increases during early panin stages prior to tumour development, and we are currently seeking to validate nsp(+)-evs in blood of pdac patients. summary/conclusion: this research shines light on a novel mechanism of tumour-stroma communication that may be crucial for cancer progression during early disease stages and a potential target for disrupting the supportive role of the tumour microenvironment. additionally, we describe a sub-population of nsp (+)-evs that have the potential to serve as biomarkers for identifying pdac development. exosomes carry distinct mirnas that drive medulloblastoma progression introduction: extracellular vesicles (evs) represent an ideal source of functional biomarkers due to their role in intercellular communication and their ability to protect cargo, including rna, from degradation. the most investigated ev's are exosomes, nanovesicles secreted by all cell types and able to cross the bloodbrain-barrier. here we characterised the rna of exosomes isolated from medulloblastoma cell lines, with the aim of investigating exosomal rna cargo as potential functional biomarkers for medulloblastoma. methods: exosomes derived from a panel of matched (original tumour and metastasis) medulloblastoma cell lines were isolated and characterised by nanosight, electron microscopy, western blotting and nanoscale flow cytometry. exosomal mirna and mrna from our matched cell lines and foetal neuronal stem cells, which were used as a normal control, were analysed by rna-sequencing technology. results: based on hierarchical clustering, malignant derived exosomes were distinctly separated from normal control exosomes. mirna profiling revealed several established oncomirs identified in our malignant derived exosomes compared to control samples. using interaction pathway analysis, we identified that our malignant exosomes carry numerous mirnas implicated in migration, proliferation, cellular adhesion and tumour growth. several previously identified oncomirs were also identified to be present at higher levels in metastatic exosomes compared to primary and normal, including hsa-mir- - p and hsa-mir- a- p. summary/conclusion: this study shows that exosomes from mb cells carry a distinct mirna cargo which could enhance medulloblastoma progression. the use of circulating exosomes as markers of metastatic disease could be an innovative and powerful noninvasive tool. introduction: inflammatory changes in the bone marrow (bm) and suppression of haematopoietic stem and progenitor cell (hspc) function during acute myeloid leukaemia (aml) significantly contribute to patient morbidity and mortality. our laboratory has previously shown that aml-derived extracellular vesicle (ev-aml) trafficking confers a state of enforced quiescence and leads to lasting dna damage in hspcs. here we explore the underlying cause. specifically, we hypothesize that ev-aml incite inflammatory regulators as potential mediators of dna damage. methods: as a validated model of aml, we utilized the murine tib cell line as a source of ev-aml. ev-previous work has indicated that mirnas, notably mir- a and mir- , play a critical role in scc tumour development. evs are membrane-bound vesicles involved in cell-cell communication carrying actively sorted cargo, protected from degradation. the potential pathways these vesicular mirnas modulate and the implication they have on cancer biology is under active investigation. we have previously shown that the cadherin dsg , a stem cell marker, modulates ev release. dsg is upregulated in a number of cancers, including scc, and correlates with poor prognosis. here we aim to elucidate the impact of ev-associated mirnas in sccs by bioinformatic analysis. methods: scc cells stably expressing dsg were generated and evs isolated by sequential ultracentrifugation. total cellular and ev rna was isolated by mirneasy, analysed using rnaseq and identified by grch alignment. results were confirmed by qpcr. altered pathways based on targets were identified using mirnet and kegg pathway analysis. potential cancerassociated cytokine targets were confirmed by antibody array. results: rnaseq revealed cellular and ev mirnas that were differentially expressed in response to dsg with overlapping. the highest altered mirnas were validated by qpcr. kegg pathway analysis determined that these mirnas have the highest number of shared targets in cancer, cell cycle, and p signalling pathways. interestingly, mir- was upregulated while mir- a was dramatically downregulated in evs. targets of mir- a, icam- , il- , and il- , cytokines critical for cancer progression were upregulated. summary/conclusion: these results suggest that the mirna content of evs is tightly regulated. by altering the mirna profile, dsg contributes to the pathogenicity of these evs by increasing levels of cytokines important for cancer stem cell renewal and metastasis. in addition, these mirnas may serve as non-invasive diagnostic markers for sccs. funding: nih r cancer cells grown in d release distinct extracellular vesicles during tumour growth and invasion jens c. luoto, sara bengs, leila coelho rato, lea sistonen and eva henriksson Åbo akademi university, turku, finland introduction: cancer cells secrete extracellular vesicles (evs) that affect tumour progression. the characteristics of evs produced during tumour growth and invasion are however poorly understood. in this study, we identify the composition and characteristics of evs produced by noninvasive and invasive tumours and correlate these characteristics with the invasive status of the tumour. for that purpose, we established a protocol for isolating evs from extracellular matrix (ecm)-based three-dimensional ( d) cancer cell cultures. methods: human prostate cancer pc cells were grown in d cultures using ecm-based hydrogel, in standard d culture conditions and in bioreactor. evs were isolated from these cultures with differential and density gradient centrifugation. the isolated evs were characterized with nanoparticle tracking analyses, electron microscopy, immunoblotting and mass spectrometry (ms). results: our results demonstrate that d ecm-based hydrogel cell cultures secrete evs that can be isolated from both the conditioned media and the hydrogel. the invasive d cultivated pc organoids were found to secrete large amounts of evs compared to the non-invasive organoids. interestingly, our ms results revealed that non-invasive and invasive organoids secrete evs with partially distinct protein cargo. summary/conclusion: we have established a novel protocol for ev production in a d cell culture system utilizing ecm-based hydrogel, in which invasive tumour growth can be mimicked. our method allows the specific isolation and characterization of evs derived from different stages of d culture, such as non-invasive and invasive organoids. importantly, we found that tumour-derived evs change in composition during the tumour progression. taken together, our method can be used to define the distinct ev characteristics involved in cancer invasion. we previously showed extracellular vesicles (evs) to be causally involved in transmitting drug resistance. this study aimed to evaluate compounds proposed to reduce/block ev release. specifically, we selected calpeptin and y (proposed to inhibit evs budding from the cell membrane) and manumycin a and gw (proposed to inhibit evs deriving from mvbs). associated effects on -and consequences of-ev release were then investigated. methods: suitable compounds concentrations that were non-toxic to cells were first selected by performing cytotoxicity assay and flow cytometry (fc). conditioned medium (cm) was collected from docetaxel-resistant pc (pc rd) cells after h incubation in dfbs-medium with or without the compounds. evs were separated from tangential flow filtration concentrated cm using optiprep density gradient. . - . g/ml fractions were then pooled and washed. evs were characterised using nta, immunoblot, tem and lipid assay and fc. influences on growth and migration, of evs continuing to be released (at x evs/ x cells, x evs/ x cells), were evaluated on recipient du and rv cells. evtrack id ev , score % results: calpeptin and y , alone and in combination, did not significantly affect quantities of evs released. however, gw significantly (p < . ) increased quantities of released evs, of a larger size; very high protein to lipid ratio; and carrying grp compared to control evs (p < . ). this effect was reverted when gw was combined with manumycin a (p < . ). following all compounds treatments, x evs/ x cells inhibited rv proliferation (p < . ), while at x evs/ x cells only evs from manumycin a (p < . ) and y (p < . ) treated cells reduce rv proliferation. evs following gw treatment significantly (p < . ) inhibited du migration compared to bulk non-treated control and compared to the effect obtained using the entire pool of evs (p < . ). summary/conclusion: while none of the proposed inhibitors significantly reduced ev release, the resulting evs were less potent in transmitting aggressive behaviour, such as proliferation and migration, to receiving cell lines. patient-derived organoids represent a novel tool to study the effect of intra-tumoral heterogeneity on ev release in non-small cell lung cancer introduction: lung adenocarcinoma (luad) is the leading cause of cancer-related death with a low -year survival. although the importance of intra-tumoral cellular heterogeneity of solid tumors in the clinical outcome and treatment is emerging, proper models to study its effects on ev release and cargo in human tissues still lack. the d organoid technology maintains the cellular and genetic heterogeneity of in vivo tissues and has proved to be so far the best ex vivo model of human cancers. by using patient-derived and mouse organoids we set out i) to compare the ev release from normal and tumor tissues and ii) to follow changes in ev secretion when the relative ratio of tumor cell subpopulations is shifted. methods: we used mouse and luad patient-derived normal and tumor organoids. the medical research council of hungary approved our experiments with human samples and informed consent was obtained from patients. evs were detected by antibody-coated beads, nta and tem. intra-organoid heterogeneity was proved by immunostaining and rt-qpcr. results: we provide evidence that both mouse and human normal organoids contain all the bronchiolar cell types. interestingly, luad organoids selected for tp mutation contained not only ki + proliferating cells, but differentiated cell types as well. furthermore, all the lung organoid cultures produced evs and this was shifted to the smaller size range. interestingly however, when modifying the proportion of organoid cell types, we observed an increased ev release when more ki + proliferating cells were present both in normal and in luad samples. summary/conclusion: our data show that patientderived lung organoids represent a novel model to study the role of intra-tissue heterogeneity in ev functions in the humans, leading to improved diagnosis. funding: this work was funded by the national competitiveness and excellence program nvkp_ - (national research, development and innovation office, hungary) and by the national excellence program in higher education (ministry of human resources, hungary). exosome mediate heart-adipocyte communication after myocardial ischaemia/reperfusion and impairs adipocyte endocrine function yajing wang, lu gan, dina xie, wayne lau, theodore christopher, bernard lopez and xinliang ma thomas jefferson university, philadelphia, usa introduction: by incompletely understood mechanisms, mi patients sustain systemic metabolic disorder. adipocytes are an important cellular type regulating energy homoeostasis. the impact of mi upon adipocyte function remains unknown. exosomes (exo) are critical vehicles mediating organ-organ communication. however, whether and how exo may mediate post-mi cardiomyocyte/ adipocyte communication have not been previously investigated. methods: adult male mice were subjected to mi/r. serum exo were isolated hours after r and incubated with t l cells for hours. the effects of exo upon adipocyte function were determined. results: compared to control, mi/r exo significantly altered the expression of genes known to be important in adipocyte function. go analysis revealed that genes associated with endoplasmic reticulum (er) function and adipocyte endocrine function are the primary two pathways altered by mi/r exo. venn analysis identified mi-rnas as cardiac-enriched, adipocyte-poor, and er function-related mirnas. rt-qpcr confirmed the mir- a/ a/ - family members are the most markedly increased mi-rnas in mi/r exo. incubation of t l cells with mi-r a mimic significantly downregulated edem , dsba-l, and pparn, and upregulated perk and chop. conversely, mi-r a inhibitor significantly decreased the impact of mi/r exo upon er function genes. additional studies demonstrated edem and pparγ (two critical molecules maintaining er function and adipocyte endocrine function) to be direct targets of mi-r a. one of the most significant endocrine molecules of adipocyte origin, adiponectin is regulated by pparn at the transcriptional level and by dsba-l at the post-translational level. we next determined whether mi/r exo may affect adiponectin expression/ assembly. incubation of t l cells with mi/r exo significantly inhibited total and high molecular weight adiponectin expression, an effect blocked by mir a mimic. finally, in vivo administration of gw (exo biogenesis inhibitor) or mir a inhibitor attenuated adipocyte er dysfunction and restored plasma adiponectin level in mi/r animals. summary/conclusion: we demonstrate for the first time that mi/r causes significant adipocyte er and endocrine dysfunction by exo mediated cardiomyocyte/adipocyte communication via mir- a/ a/ - . funding: nih and american diabetes association pancreatic cancer cell extracellular vesicles drastically alter the behaviour of recipient normal pancreas cells charles p. hinzman a , yaoxiang li a , meth jayatilake a , jose trevino b , partha banerjee a and amrita cheema a a georgetown university medical center, washington, usa; b university of florida health science center, gainesville, usa introduction: pancreatic cancer (paca) is predicted to become the rd leading cause of cancer-related deaths by . patients diagnosed with pancreatic ductal adenocarcinoma (pdac) have a -year survival ratẽ %. detection of pre-neoplastic lesions can potentially improve survival. however, there is currently no screening test for early stage detection. importantly, paca tumours are % non-tumorigenic cells. a better understanding of early paca oncogenesis is needed. cancer cells shed extracellular vesicles (evs) that are internalized by neighbouring and distant cells to induce a myriad of cancer progression events. we hypothesize that in early paca oncogenesis, evs mediate a behavioural change in surrounding normal cells, leading to the formation of this unique stroma. the purpose of this study was to develop a model to examine the phenotypic changes undergone by normal human pancreas cells when they are exposed to paca cell evs. methods: evs were isolated using differential ultracentrifugation with filtration from established (panc- , sw- , capan- and miapaca- ) and patientderived xenograft (ppcl- and ppcl- ) paca cell lines. cells were grown using ev-depleted fbs. ev isolations were validated and quantified using transmission electron microscopy, quantitative elisa, immunoblot and nanoparticle tracking analysis. normal pancreas cells (htert-hpne and hpde-h c ) were co-cultured with cancer cell evs for - hours. metabolic activity was measured using a mito stress test on a seahorse xfe extracellular flux analyser. results: we discovered that normal cells undergo vast behavioural transformations, including significant morphological changes, increased proliferation and an uncharacteristic invasive capability, when co-cultured with paca cell evs. these responses were ev dose dependent. further, paca cell evs metabolically reprogrammed normal cells, causing a bioenergetic switch, from a quiescent, aerobic profile to a highly energetic and glycolytic profile. summary/conclusion: our results indicate that paca cell evs confer enormous transformational properties to normal human pancreas cells in vitro. we hypothesize that evs impart distinct transformational properties to normal cells in vivo and this influence could unveil novel mechanisms regulating cancer onset and progression. these signals may be detectable before progression of early-stage paca to pdac, leading to the development of assays for earlier diagnosis in patients. further studies are underway to identify the biochemical mediators of these changes. plasma extracellular vesicles-mirnas released by hypoxic cells are associated to pro-tumorigenic and immunosuppressive microenvironment in lung cancer introduction: extracellular vesicles (ev) containing specific subset of functional biomolecules, such as micrornas (mirnas) are released by all cell types. it has been widely demonstrated that ev-mirnas from cancer cells can manipulate the tumour microenvironment modulating the gene expression of recipient cells. we previously identified a three levels risk classifier (msc) based on plasma-mirnas associated with lung cancer development and prognosis. the aim of this study was to investigate the potential role of ev- mirnas as mediators of pro-tumorigenic features. methods: evs were isolated from plasma of heavysmoker individuals with high (mscpos) or low (mscneg) risk of lung cancer by differential centrifugation method. purity of evs isolated was confirmed by sizing using nta and tem analysis and expression of ev-enriched proteins. mirna levels were analysed by dpcr. in vitro and in vivo analyses were used to assess the biological effect of plasma evs on different recipient cells. results: levels of mirnas in evs correlated with determination of whole plasma ( % of correlation with msc classifier). mscpos-evs stimulated d and d proliferation of non-tumorigenic epithelial cells through c-myc transfer into recipient cells. furthermore, mscpos-evs increased the ability of huvec to form tubular structures compared to mscneg-evs. in vivo co-injection of mscpos-evstreated huvec with a lung cancer cells resulted in an increase of tumour growth compared to mscneg-evs-treated huvec. mir- modulation in evs with mirna mimics or in recipient cells using mirna inhibitors demonstrated that this mirna is implicated in the autocrine proangiogenic modulation of huvec phenotype. mscpos-evs induced m polarization of macrophages both in vitro and in vivo. we demonstrated using synthetic oligonucleotides that mir- is responsible for the immunosuppressive modulation of these cells. regarding the potential origin of evs in mscpos individuals, we observed that hypoxia stimulated the secretion of evs containing c-myc from fibroblasts, mir- -evs from endothelial cells and mir- -evs from granulocytes. summary/conclusion: these data show that plasma evs of high-risk individuals display pro-tumorigenic features, as documented by their ability to induce a pro-angiogenic and immunosuppressive microenvironment suggesting an involvement of evs in lung cancer development. exploration of ev-associated metastatic targets on melanoma cells introduction: cancer cells secrete evs that may harbour metastatic proteins. previous studies have demonstrated the decrease of cd tetraspanin expression in melanoma cells are correlated with enhancing its metastatic potential. however, other proteins, such as cd , cd , met and nrp which are overexpressed in melanoma cells, are also associated with the spread of cancer. in this study, we sought to investigate the expression of metastatic proteins in evs derived from murine melanoma b f lineage. methods: b f cells were cultured in dmem supplemented with % fbs and antibiotics. the cells supernatant were harvested each hours, filtered through . µm and ultracentrifuged at x g for min at ºc to pellet evs. next, size and concentration was determined using nanoparticle tracking analyses technique, and the morphology of evs were analysed by negative-staining transmission electron microscopy (tem). the ev's surface protein were characterized by flow cytometry and protein content was profiled by mass spectrometry. results: our flow cytometry results have shown the presence of tetraspanins markers cd , cd and cd on vesicle´s surface. in addition, our assay demonstrated a diminished expression of cd molecule in comparison to cd and cd . we have profiled melanoma-evs by mass spectrometry, identifying the presence of proteins that may be associated to metastasis, such as cd , cd , met and nrp . summary/conclusion: these preliminary results are consistent with the literature and suggest that melanoma-derived evs harbour proteins, which may contribute to promote tumour metastasis. in our next step, we plan to generate b f lineages harbouring shrna vectors, in order to knockdown gene expression of cd , cd , cd , met and nrp to investigate the metastatic potential in vitro, in comparison to parental cells. we also may use syngeneic mice models to explore metastatic potential of genetically modified b f -derived cells. introduction: overexpression of her occurs in % of breast cancers and confers aggressive behaviour and poorer prognosis. thankfully, a number of drugs such as neratinib have been developed to target her , potentially providing substantial benefit for many patients. nevertheless, it is estimated that up to % of patients with her -overexpressing tumours do not gain benefit, as a result of innate or acquired drug-resistance. this study aimed to investigate if nano-sized membrane-surrounded extracellular vesicles (evs) released from drug-sensitive and drug-resistant cells reflect the her status of their cells of origin and thus have potential as minimallyinvasive biomarkers. methods: evs were isolated from conditioned media (cm) of her -positive cell lines (hcc and skbr ) and their neratinib-resistant counterparts (hcc -nr and skbr -nr) that we developed in our laboratory. evs from cm of a triple-negative breast cancer (tnbc) cell line variant, hs ts(i) , were evaluated as negative control for her . in brief, cm was centrifuged at g, for min x to get rid of any cells. the supernatant was then centrifuged at , g for h at ºc to collect evs. the resulting evs were washed in pbs, centrifuged as before, and resuspended in μl pbs. ev quantities were estimated by nanoparticle tracking analysis (nts). ev lysates were characterised by immunoblots, for established positive and negative ev markers. particle concentration as well as size distribution of evs were measured using the zetaview (particle metrix, germany). surface proteins on single evs were analysed by highresolution flow cytometry (fc), using an amnis imagestreamx mark ii. all data was submitted to ev-track (ev-track id: ev ). results: neratinib-resistant cell line variants were found to have reduced her protein expression compared to their respective drug-sensitive counterparts. neratinib-resistant cell line variants released fewer evs, when normalised per number of secreting cells, compared to their-drug sensitive counterparts. furthermore, evs from drug-sensitive cells carried her , while those from drug-resistant cells lacked her (similar to the evs from the tnbc cells); reflecting the her status of their cells of origin. summary/conclusion: this study indicates that a reduction in her protein expression is a mechanism by which cancer cells manifest resistance to her -targeted drugs (i.e. by making fewer her receptors available on the cells surface to accommodate the drugs activity). furthermore, ev-carried her seems to reflect the her status of their cells of origin. this suggest that analysis of her on evs in the peripheral circulation may help predict response to her -targeted drugs. thus, analysis of evs in appropriate cohorts of patients' specimens is warranted. introduction: rab a, a small gtpase involved in exosome biogenesis by regulating mve docking at the plasma membrane, and its expression level highly correlated with ohsv replication ability in vitro. oncolytic viruses is a newly promising therapeutic agent for cancer treatment. however, more than % of tumours naturally showed highly resistant to oncolytic viruses for unknown reasons. uncovering the underlying mechanisms of resistance to ohsv can offer potential therapeutic targets to enhance ohsv activity to kill tumour cells. in addition, it will give new insights into the identification of therapeutic biomarkers, which can be used to predict patient response to ohsv in clinical. methods: deep-sequencing data showed that lower expression level of rab a is present in ohsv resistant tumour cells compared to that in sensitive tumour cells. then an ohsv resistant mc tumour cell line was established by repeated injections with ohsv in mc tumour-bear mouse model. lastly, it was verified that ohsv resistance is associated with a downregulation of rab a and overexpression of rab a can rescue ohsv replication. results: ) the lower expression level of rab a is shown in ohsv resistant tumour cells compared to that in sensitive tumour cells shows in deep-sequencing data. ) furthermore, we established an ohsv resistant mc tumour cell line by repeated injections with ohsv in mc tumour-bear mouse model. similarly, in ohsv resistant mc cell line, rab a expression was lower than parental mc cells. and the release of exosomes and virus was decreased in ohsv resistant mc cell line. these results were confirmed by rab a sirna knockdown. ) we verified that in ohsv naturally resistant human cancer cell lines, ohsv resistance is associated with a downregulation of rab a and overexpression of rab a can rescue ohsv replication. summary/conclusion: downregulation of rab a expression restricts the efficiency of ohsv replication and the spreading ability of the released progeny virus which also provide rab a as a potential target and biomarker for ohsv cancer therapy. funding: these studies were supported by grants from shenzhen overseas high-calibre peacock foundation kqtd , shenzhen science and innovation commission project grants jcyj , jcyj to shenzhen international institute for biomedical research. inactivation of emilin- by proteolysis and secretion in extracellular vesicles favours melanoma progression and metastasis introduction: studies have demonstrated that melanoma-derived extracellular vesicles (evs) home in distal organs and sentinel lymph nodes favouring metastasis. although lymph node metastases are themselves rarely life threatening, they could be considered as one of the first step of metastasis in many cancer types. therefore, defining the mechanisms involved in lymph node metastasis and pre-metastatic niche formation in lymph nodes could bring the clue to block the metastatic process from the beginning. methods: we have characterized secreted exosomes from a panel of mouse melanoma models representative of low metastatic potential (b -f ), high metastatic potential (b -f ) and lymph node metastasis (b -f r ). we analysed the rna expression in cells and protein content of exosomes derived from mouse melanoma lymph node metastatic models by rna sequencing and mass spectrometry respectively. we validated expression by western-blot, qpcr and immunofluorescence. to define the mechanism of emilin- secretion cells were treated with the ev secretion inhibitor (non-competitive inhibitor of sphingomyelinase), gw . cell viability and cell cycle assays with an overexpression model (b -f -emilin- ) were also performed. in vivo experiments based on subcutaneous and intrafootpad injection for studying the role of this protein during melanoma progression were performed to define the relevance of our findings in vivo. human paraffin samples were analysed for emilin- expression by immunohistochemistry. results: we found a signature of over-expressed genes and hyper-secreted proteins in exosomes related to lymph node metastasis in the b mouse melanoma model. among them, we found that emilin- , a protein with an important function in lymph node physiology, was hyper-secreted in exosomes. interestingly, we found that emilin- is degraded and secreted in exosomes as a mechanism favouring metastasis. further, we found that emilin- has a tumour suppressive-like role regulating negatively cell migration. importantly, our in vivo studies demonstrate that emilin- overexpression reduced primary tumour growth and metastasis in mouse melanoma models. analysis in human melanoma samples showed that cells expressing high levels of emilin- are reduced in metastatic lesions. summary/conclusion: overall, our analysis suggests that the inactivation of emilin- by proteolysis and secretion in exosomes reduce its intrinsic tumour suppressive activities in melanoma favouring tumour progression and metastasis. funding: this work was supported by grants from mineco (saf r), asociación española contra el cáncer, fundación de investigación oncológica fero and mineco-severo ochoa predoctoral program. introduction: the amplification of erbb gene and the consequent overexpression of the encoded protein her have an important role in breast cancer classification at diagnosis and subsequent treatment decision with the anti-her monoclonal antibody trastuzumab. fish and ihc have been used so far to detect erbb gene amplification and her protein overexpression respectively in tissue biopsies. in this context, a major goal for liquid biopsies is to take advantage of the information carried by circulating tumourderived materials (such as extracellular vesicles (evs) and cell free dna (cfdna)) to noninvasively detect erbb gene status in the blood. however, the isolation of diverse tumour-derived materials from a single aliquot of patients' plasma and the accurate detection of cancer biomarkers is still challenging. methods: by adopting a recently published nickelbased evs isolation (nbi) protocol that allows for recovery of cfdna after evs isolation, we generated a high-sensitivity molecular assay to accurately detect erbb amplification and consequent her overexpression on a limited volume of plasma ( . ml) collected from breast cancer patients (stage i, ii and iii) at diagnosis. results: ) we detected erbb amplifications by droplet digital pcr (ddpcr) on the cfdna isolated from the plasma of erbb positive patients; ) we confirmed her overexpression on a subset of patients by setting up an antibody-based affinity reaction designed to detect her protein on the surface of the isolated evs; ) we succeeded in the quantification of her transcripts enclosed within evs by performing ddpcr in samples of patients showing a range of circulating tumourderived material. the specificity and sensitivity of these novel methodological assays were tested on a cohort of healthy individuals (n = ) and on a cohort of her positive (n = ) or her negative breast cancer patients (tnbc; n = ). summary/conclusion: here we report a pilot study on a novel multimodal method for erbb detection from a minimal amount of plasma. this approach integrates information from cfdna with evs-derived rna and proteins analysis. this proof of concept may ultimately translate into relevant clinical applications for disease diagnosis as well as for therapy selection and monitoring of disease progression. introduction: the biological and medical importance of exosomes recognized over the last decade has given rise to a crucial need for the discrimination between true evs and co-purified nanoparticles, such as lipoproteins, protein aggregates or debris. additionally, it is imperative to develop methods to identify and characterize ev sub-populations. considering ev biology and the reliability of labelled biomolecules, we developed both exogenous and endogenous labelling protocols, taking advantage of different dye properties which can target a multitude of compartments. this approach reveals key aspects of ev structure and integrity. methods: nanosized evs/exosomes were purified by size exclusion chromatography (sec) from model cell lines and human plasma. diverse dyes were orthogonally evaluated through different single particle and bulk analysis technologies, such as high-resolution cytometry, nanoparticle tracking analysis and plate fluorimeter. concomitant profiling of specific ev subpopulations was optimized using antibodies (abs) against tetraspanins and cell type specific targets and assessed by single particle analysis and elisa. to assess specificity of labelling protocols we used specific controls such as recombinant rfp expressing vesicles and purified lipoproteins. results: our ev staining protocols allowed for high labelling efficiency and unprecedent ev discrimination, quantification and characterization by combining single particle analysis and bulk measurements in simple matrices (saline buffers) and in complex biofluids (i.e. plasma). different approaches have diverse and complementary advantages (costs, capacity, sensitivity, informative readout) for implementation in research and diagnostic development flows, directly feeding in-house r&d and qc pipeline. summary/conclusion: overall, fluorescent evs are versatile and valuable tracers that can be applied in the optimization of pre-analytical and purification protocols, selection of target biomarkers and diagnostic assay calibration and validation. funding: endevor (por-fse - ) region tuscany and exosomics r&d program. subpopulations of tissue-derived extracellular vesiclesmethodological evaluation for vesicle size measurement introduction: introduction: subpopulations of extracellular vesicle (evs) are commonly classified by their different size, however, the ev size cut off is still under discussion. the aim of the study was to evaluate size range of six ev subpopulations using three methods: electron microscopy (em), nanoparticle tracking analysis (nta) and exoview™. methods: methods: ev subpopulations were isolated from melanoma tissues by a centrifugation based protocol recently established in our lab. large and small evs (levs and sevs) were isolated with differential ultracentrifugation and these were further separated into low and high-density fractions (ld and hd). size of ev subpopulations was then evaluated by: em introduction: small-extracellular vesicles have an important role in cell metabolism and cell-to-cell communication. moreover, sevs when secreted from cells are capable to act as mediators of various neurological diseases. however, sevs show heterogeneity and this may impact their functions. therefore, to characterize sevs at a single-particle level is important to better detail the associated biological activity. in this scenario, we innovatively propose the structured illumination microscopy (sim) as a technique able to complement the non-optical methods (transmission electron microscopy, tem) to analyse single sevs and their markers. methods: human plasma sevs were separated from healthy cognitive control (ctrl), mild cognitive impairment (mci) and demented subjects. the sevscontaining pellet was resuspended in % paraformaldehyde or % glutaraldehyde solutions. for sim, sevsenriched preparations were washed with the blocking solution and incubated with the primary antibody (l cam). the secondary fluolabelled antibody was then added. between the steps with the blocking solution, the primary and secondary antibodies, two ultracentrifuge steps were performed. the image acquisition was done on a nikon sim system with a x oil immersion objective. sevs were imaged with a d-sim acquisition protocol. tem was performed on mesh formvar copper-coated grids. sevs-enriched preparations were incubated first with the blocking solution and then, immunogold-labelled for cd . samples were counterstained with uranyl acetate and observed under a jeol ex electron microscope. data were recorded with a morada digital camera system. participation to the present study was approved by relevant local ethics committee of mendrisio and lugano hospital and written informed consents were obtained from subjects. results: for sim methodology, only vesicles in the range from to nm were detected, as the final resolution achieved was nm. instead, for tem, sevs under nm were identified. none of these methods provided relevant information about possible difference in morphology of the ctrl-, mci or demented subjects-derived sevs. summary/conclusion: even if both methods identified cd or l cam-positive vesicles, sim resolution and the complexity of the protocol represent some disadvantages respect to tem, that may be the first choice screening technique for evs analysis to be then completed by sim for particular tasks. fabrication of nanopore structures via conformal metal-film deposition for ev sensing kwanjung kim a , seung-min han b , soo-hyun kim c and sung-wook nam d luminex corporation, seattle, usa introduction: extracellular vesicles (evs) are membrane-derived structures that include exosomes, microvesicles, and apoptotic bodies. these evsreleased under normal physiological conditions as well as in the pathogenesis of neurological, vascular, haematological, and autoimmune diseaseshave been shown to transfer biological molecules such as protein and rna between cells, potentially transmitting signals. to understand more about these signalling mechanisms, there is a need for detecting and quantifying evs with cargo protein and rna in a reproducible and reliable manner. however, this has been challenging due to the small size of evs (ranging from to nm in diameter), and the lack of specific staining reagents. methods: here, we utilize the amnis® cellstream® flow cytometer, which enables high-throughput flow cytometry with increased sensitivity for detecting small particles. we demonstrate that a charge-coupled device (ccd)-based, time-delay-integration image capturing system can be used to detect and quantify evs and their cargo labelled with exoglow™-protein or exoglow™-rna. results: in this study, we show flow cytometry data quantifying ev samples that have been labelled with cargo markers for proteins and rna. the ev cargo contents along with the appropriate control samples will be shown. summary/conclusion: the ccd based detection of the cellstream flow cytometer has the sensitivity to quantify evs and their cargo. single ev imaging reveals novel ev biomarkers and dna cargo siobhan m. king a , ricardo bastos a and andras miklosi b a oni, oxford, uk; b oni (oxford nanoimaging ltd), oxford, uk introduction: extracellular vesicles (evs) are cellderived membrane-bound particles that range in size from - nm and carry active molecules such as dna, rna and proteins. upon secretion, evs can execute many biological functions such as initiating intracellular communication or regulating immune responses. depending on their origin evs have different characteristics and cargo, making them attractive candidates for early diagnostic and therapeutic applications. however due to their small size and heterogeneity, direct visualization and characterization of the surface markers expressed remains a challenge since these vesicles are below the resolution limit of standard light microscopy. methods: here, we describe a method that provides size analysis of single-evs, which falls below the diffraction limit of light. this was done with purified evs, immunostained using fluorescently labelled primary antibodies raised against ev surface markers (cd , cd , cd ), specific cargo such as dna and probed for tissue specific cargo. characterization of the molecular content and structural properties of surfaceimmobilized evs was performed using single-molecule localisation microscopy (smlm) on the nanoimager platform. results: multicolour smlm was used to detect up to three ev biomarkers showing successful characterization of the molecular signature for different ev subpopulations. the distribution of novel components on urinary evs were visualized for the first time using this approach. in addition, smlm revealed the presence of dna on both the surface and also as a cargo inside evs isolated from tumour cell culture media, which was validated using complementary biochemical characterization. summary/conclusion: smlm is a powerful technique for single-ev analysis and characterization. visualization of single-urinary evs enabled accurate sizing and further insights into novel components expressed on the subpopulation's membrane surface. together, the data demonstrates that the quantitative abilities of smlm can significantly enhance our understanding of evs, as structure, phenotypes, and cargoes can now be successfully resolved. introduction: working skeletal muscle is a common site for injury due to unaccustomed exercise with or without underlying pathology. direct analysis of skm injury requires invasive tissue biopsies. circulating extracellular vesicles (evs) are abundant in blood and have been shown to be enriched in microrna; profiles of which may reflect the state of tissues. evs may therefore serve as a non-invasive indicator of muscle injury and regenerative processes in vivo. methods: two consecutive bouts of muscle-damaging exercise (plyometric jumping and downhill running) were performed by healthy male volunteers. serum creatine kinase (ck) and plasma evs were analysed at baseline, and hr post-exercise. perceived muscle pain (pmp) was assessed at and hr post-exercise. large evs were isolated using a g centrifugation step, and small evs were isolated using qev columns. ev-enriched isolates were visualized using tem, and size and numbers were quantified using nta. based on nta results the highest particle fractions ( - ) were pooled for rna analysis. qpcr was done on plasma, large evs and small evs. a group of muscle and immune cell-important mirs were analysed by means of normalization to an exogenous control. results: ck and pmp increased post-exercise, providing evidence for muscle damage. tem revealed an abundant and heterogeneous pool of evs. a concomitant abundance of evs was seen with nta (mean = . x particles/ml plasma). mean ev diameters were ± nm across all timepoints. no change in ev size nor number was seen over time, however, mir- decreased at hr when compared to hr in the small ev isolate only. plasma displayed an immediate increase in myomirs- and − at hr, which returned to baseline at hr. in contrast, myomirs- b and remained elevated over the hr period. myomir- b and , as well as immune-mirs, did not change in evs or plasma as a result of the intervention. summary/conclusion: the decrease in mir- in small evs at hr is consistent with previous data. no decrease in mir- in large evs suggests specific packaging and hence a specific response to the muscle damage in small evs. more changes occurred in plasma myomirs suggesting less specific passive leakage into circulation from damaged cell membranes. funding: south african national research foundation pulsed electromagnetic fields potentiate the paracrine function of mesenchymal stem cells for cartilage regeneration yingnan wu a , dinesh parate a , eng hin lee a , zheng yang a and alfredo franco-obregón b a national university of singpaore tissue engineering program, yll school of medicine., national university of singapore, singapore, singapore; b biolonic currents electromagnetic pulsing systems laboratory, biceps, national university of singapore, singapore, singapore introduction: the mesenchymal stem cell (msc) secretome, via the combined actions of its plethora of biologically active factors, is capable of orchestrating the regenerative responses of numerous tissues by both eliciting and amplifying biological responses within recipient cells. mscs are "environmentally-responsive" to local microenvironmental cues and biophysical perturbations, influencing their differentiation as well as secretion of bioactive factors. we have previously shown that exposures of mscs to pulsed electromagnetic fields (pemfs) enhanced msc chondrogenesis. here, we investigate the influence of pemf exposure over the paracrine activity of mscs and its significance to cartilage regeneration. also, the subsequent extracellular vesicles analysis and isolation are processed for the understanding of how the pemfs affect stem cell evs and consequent differentiation induction. methods: conditioned medium (cm) was generated from mscs subjected to either d or d culturing platforms, with or without pemf exposure. the paracrine effects of cm over chondrocytes and msc chondrogenesis, migration and proliferation, as well as the inflammatory status and induced apoptosis in chondrocytes and mscs was assessed. the cms which have significant effects during chondrogenesis will be analysed by protein and mirna studies. results: we show that the benefits of magnetic field stimulation over msc-derived chondrogenesis can be partly ascribed to its ability to modulate the msc secretome. mscs cultured on either d or d platforms displayed distinct magnetic sensitivities, whereby mscs grown in d or d platforms responded most favourably to pemf exposure at mt and mt amplitudes, respectively. ten minutes of pemf exposure was sufficient to substantially augment the chondrogenic potential of msc-derived cm generated from either platform. furthermore, pemf-induced cm was capable of enhancing the migration of chondrocytes and mscs as well as mitigating cellular inflammation and apoptosis. the cms protein results in the significant promotion chondrogenesis condition showed an increase in proliferation and anti-inflammatory cytokines. summary/conclusion: the findings reported here demonstrate that pemf-stimulation is capable of modulating the paracrine function of mscs for the enhancement and re-establishment of cartilage regeneration in states of cellular stress. the pemf-induced modulation of the msc-derived paracrine function for directed biological responses in recipient cells or tissues has broad clinical and practical ramifications with high translational value across numerous clinical application. effects of extracellular vesicles from blood derivatives on osteoarthritic chondrocytes within an inflammation model introduction: the degenerative disease osteoarthritis (oa) is one of the leading causes of disability especially of elderly people. besides various treatment options depending on the severity of the cartilage degradation, the application of blood derived products such as platelet rich plasma (prp) are getting more and more popular in clinical practice due to its high concentration of platelets and the perceived high growth factor levels. drawbacks of using prp include high donor variability, discrepancies among preparation protocols and the presence of cells (platelets, leukocytes) which can evoke cellular processes, especially inflammation, when injected into the diseased tissue. one possibility is to isolate only extracellular vesicles (evs) from blood derivatives to overcome these problems. in the current study the effects of evs isolated from blood derivatives on oa chondrocytes within an inflammation model was investigated. methods: cd positive primary monocytes were isolated from citrate anticoagulated whole blood by magnetic bead sorting. monocytes were differentiated into resting m macrophages and activated into m macrophages according to published protocols. elisa measurements verified successful differentiation and activation as il β and tnfα levels increased. as control, thp monocytes were used. patient-derived oa chondrocytes were grown in well plates and co-cultivated with activated m macrophages which were seeded into thincerts and added to the wells representing the inflammation model. furthermore, cells were treated for hours with media containing fcs, ev depleted fcs or evs isolated from prp or hypact serum. results: successful differentiation and activation of monocytes (thp and primary monocytes) into m macrophages was demonstrated by elevated levels of the inflammatory cytokines il β and tnfα. within the inflammation model (co-culture of oa chondrocytes with m macrophages), addition of evs isolated from prp or hypact serum resulted in decreased secretion levels of il β and tnfα compared to media supplemented with either fcs or ev depleted fcs. summary/conclusion: taken together, evs from blood derived products might be chondroprotective and anti-inflammatory mediators which protect cartilage from being degraded during oa. funding: the work was jointly supported by the european fund for regional development (efre) and the fund for economy and tourism of lower austria, grant number wst -f- / - . α, (oh) d regulates growth cartilage matrix vesicle micrornas niels asmussen, michael mcclure, zhao lin, zvi schwartz and barbara boyan virginia commonwealth university, richmond, usa introduction: matrix vesicles (mvs) are small ( - nm in diameter) lipid bound extracellular organelles isolated from calcifying tissues including the growth zone (gc) of growth plate cartilage. α, (oh) vitamin d ( α, ) is a regulator of gc chondrocytes and the mvs they produce. these mvs are key players in the mineralization process and are selectively enriched with enzymes and growth factors. we found that mvs are also selectively enriched with micrornas (mir), including mir- , mir- and mir- . the aim of this study was to determine the regulatory role of α, in the packaging of mirna in mvs by gc cells. methods: gc cells were isolated by enzymatic digestion from costochondral gc cartilage harvested from wk-old male sprague dawley rats (iacuc approved). confluent fourth passage gc cell cultures were treated with - m α, or vehicle for h. media were removed, cell monolayers digested with trypsin and cells and mvs isolated by differential ultracentrifugation. rna was precipitated from cells and mvs. small rnaseq data were trimmed, aligned and counted before undergoing differential expression analysis. experimental groups had an n = per variable. significant differences (p < . ) were determined using r v . . . results: α, treatment altered expression of mv mirs compared to control mvs, whereas cell mirnas were differentially expressed. . % of significantly up or down regulated mir found in mvs overlapped between α, and vehicle groups with the remaining being uniquely differentially expressed. α, increased mv mir- and decreased mir- - p two mirs known to regulate osteoblast proliferation ( increases, decreases). summary/conclusion: α, regulates gc chondrocyte and mv behaviour and this study demonstrates that it also impacts the mir packaging within mvs. mir discovered in mvs have been demonstrated to impact chondrocyte behaviour and the present study indicates that α, regulates the growth plate through mir delivered by mvs. introduction: increasing evidence has proposed extracellular vesicles (evs) as mediators of many of the therapeutic features of mesenchymal stromal cells (msc) that have been widely studied in clinical trials over the last years. these evs have been recognized as nanocarriers of important biological information, which play a central role in cell-to-cell communication. in this context, evs can be used as an alternative to a cell-based therapy, with reduced risks. the present work aimed to evaluate the impact of different culture conditions on the msc-derived evs molecular composition through fourier-transform infrared (ftir) spectroscopy. methods: evs derived from msc from different sources, expanded in two different culture media ((xenogeneic -free (xf) vs serum-containing medium (fbs)) were characterized by ftir spectroscopy, a highly sensitive, fast and high throughput technique. moreover, principal component analysis (pca) of preprocessed ftir spectra of purified evs was conducted, enabling the evaluation of the replica variance of the evs chemical fingerprint in a reduced dimensionality space. for that, different pre-processing methods were studied as baseline correction, standard normal variation and first and second derivative. results: evs secreted by mscs cultured with serumcontaining medium presented a more homogenous chemical fingerprint than evs obtained with xf medium. the regression vector of the pca enabled to identified relevant spectral bands that enabled the separation of samples in the score-plot of the previous analysis. ratios between these spectral bands were determined, since these attenuate artefacts due to cell quantity and baseline distortions underneath each band. statistically inference analysis of the ratios of spectral bands were conducted, by comparing the equality of the means of the populations using appropriate hypothesis tests and considering the significance level of %. it was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of evs chemical fingerprint in function of the culture medium used for msc expansion and the msc donor. summary/conclusion: this work is a step forward into understanding how different culture conditions affect msc-derived evs characteristics. funding: fundação para a ciência e tecnologia (ptdc/equ-equ/ / , uidb/ / ). performance qualification for microflow cytometers: understanding technical limitations to improve your research desmond pink a , michael wong a , diana pham a , renjith pillai a , leanne stifanyk b , sylvia koch b , rebecca hiebert a , oliver kenyon c and john lewis a a nanostics, edmonton, canada; b dynalife, edmonton, canada; c apogeeflow systems, hemel hempstead, uk introduction: as microflow cytometry and other techniques mature as validated modalities for analysing extracellular vesicles (ev), there has been a concerted effort to improve reproducibility . in order for this reproducibility to occur there has to be a critical understanding of advantages and limitations for each technology. for microflow cytometry, several instruments are available to analyse evs. each platform has different limitations as well as advantages over other platforms. to provide the optimal data for your specific research, it is critical to understand the limitations of your platform. to accurately define these limitations, a performance qualification (pq) of your instrument should be undertaken. methods: an apogee a platform was used in these experiments. experiments were designed with expected ranges and cut-offs for acceptance criteria.initial tests included autosampling of a well plate with either single or double aspiration, single sample reproducibility and linearity proportional to flow rate. other experiments designed to show machine performance included minimal time to achieve valid data, sample volume required for double aspiration, determination of coincidence; detection sensitivity using a spiked sample; flow rate stability for extended periods ( - minutes) . tests should also be performed to determine carryover at a range of sample concentrations. if present, the means to remove contaminating samples should be determined. any performance tests should be applicable to any instrument in the field. results: auto-sampling helped demonstrate consistent data; reproducibility of total events and biomarkers was - % c.v. detected bead concentrations were linear with flow rates between . and . ul/min. double well aspirations provided similar data with aspirations between - ul. valid data was achieved for a low abundant target (~ - events/ul) after only s, < %c.v. detection sensitivity was determined to be~ / , . carryover ranges were determined in the presence of nominal unstained serum. an optimal number of machine washes was determined. some membrane stains, such as cell mask and cfse require much more rigorous cleaning to remove stain carryover. summary/conclusion: to improve data reproducibility, performance qualification of any instrument is key. operational limitations help define optimal performance parameters of any technology. understanding the types of experiments to perform for your particular type of characterization technology depends on the requirements you set for your research. a good performance test should be applicable to any related instrument in the field. funding: funding provided by nanostics, the alberta cancer foundation, and alberta innovates. introduction: cancer cells release more evs than normal cells and evs secreted from tumour cells can promote tumour progression, survival, invasion and angiogenesis. the ev cargo may mirror the altered molecular state of the cell of origin. therefore, evs have potential for the development of non-invasive markers for early detection of cancers. evs and their cargo also have the potential to be multiplexed with other molecular markers or screening modalities (e.g., imaging) to develop integrated molecular-based computational tools for the early detection of cancer. one challenge with using evs as a biomarker is the lack of robust and reproducible methods for the isolation of a pure vesicular population. there is a lack of clear consensus for an optimal method of isolation of a pure ev population that is devoid of contamination with similar-sized vesicles of different origins. there is also a lack of standards to ensure rigour reproducibility. methods: the current funding opportunity announcement (foa), par - , is promoting research on the isolation and characterization of extracellular vesicles (evs) and their cargo for the discovery of biomarkers to predict cancer and cancer risk. results: the previous cycle of this foa, par - / , successfully funded r and r grants. these awards are focused on proteomics profiling of evs, effect of methodological and biological variability, asymmetric-flow field-flow technology, therapeutic monitoring, lss and sers lab on a chip optical spectroscopic, evs in obesity-driven hepatocellular carcinoma, nanoscale structure and bio-molecular heterogeneity, urinary ev dna, and ev markers in paediatric cancers. progress from these awardees have shown separation of two discernible exosome subpopulations and identified a distinct nanoparticle, the exomere (nature cell biology, ); and have shown that large-evs contain the entire genome of the cell of origin, including cancer-specific genomic alterations (journal of extracellular vesicles, ). protocols that critically evaluate and refine the existing methodologies to improve utilization of evs in clinical use have been shared (nature protocols, ). summary/conclusion: drs. sudhir srivastava and matthew young are the programme directors for the par which began accepting applications on january . this and other ev funding opportunities will be discussed. funding: this is a funding opportunity announcement offered by the national cancer institute. introduction: traumatic brain injury (tbi) is characterized by diverse primary mechanisms of injury that lead to the development of secondary pathological cascades that drive neurological deficit post-tbi. inability to separate patients based on the presence of these different endophenotypes represents a major challenge for diagnosis and treatment of tbi. extracellular vesicles including exosomes isolated from patient plasma have emerged as promising potential biomarkers for tbi due to their ability to cross the bbb into systemic circulation with molecular cargo intact for analysis. we have developed a novel microfluidic platform for rapid isolation of brain-derived evs providing a tool with which the biochemical state of neurons and glia can be directly assessed post-tbi. we used the ultra-sensitive, single molecule array (simoa) to quantify concentrations of protein biomarkers from the plasma and brain derived evs from mild tbi patients and controls. by combining multiple protein biomarkers, we could discriminate mtbi patients from controls in both the training and the blinded test set. building on this work, we are also characterizing single ev heterogeneity of neuron derived evs by developing novel droplet based digital assay for single ev quantification at ultra-low concentration. droplet based assay for single ev analysis would potentially be very informative for early disease diagnosis and therapy decision. methods: our microfluidic platform for ev isolation consists of tracked-etched membranes with millions of nanopores ( nm), coated with a magnetic film (nife) to precisely capture immunomagnetically labelled brain-specific evs from plasma. single molecule array (simoa) was used to quantify concentrations of the protein biomarkers (tau, uchl- , nfl, gfap, il , il , and tnf) in the plasma and brainderived exosomes of mild tbi (mtbi) patients and controls. to identify single ev, we applied droplet based enzyme-linked immunosorbent assay and encoded the fluorescent signal for single ev quantification within parallelized microfluidic platform. results: we report that concentrations of plasma and exosome gfap, nfl, and uchl were elevated in mtbi patients compared to controls (p < . ), and that each of these biomarkers are uncorrelated with one another. discrimination of mtbi patients from controls was most accurate when machine learning algorithms on the panel of biomarkers. specifically, combining plasma nfl, gfap, il and tnf-with tau from glur + evs showed % accuracy with % sensitivity and % specificity. summary/conclusion: this data suggests that neuronderived exosomes contain information that characterizes the injured and recovering brain. it also suggests that analysis of a panel of biomarkers from a combination of both blood and exosomal compartments could lead to more accurate diagnosis of mtbis. ps . = op . l cam is not associated with extracellular vesicles in cerebrospinal fluid or plasma maia norman, dmitry ter-ovanesyan, wendy trieu and david walt wyss institute, boston, usa introduction: neurons in living psychiatric and neurological patients are inaccessible for cell type specific analysis of rna and protein. our understanding of these diseases instead relies upon imperfect sources of biochemical information such as post-mortem brain tissue analysis and animal models. furthermore, there is a paucity of biochemical assays available to diagnose and manage brain diseases. extracellular vesicles (evs) present an opportunity to noninvasively sample the contents of neurons in cerebrospinal fluid (csf) and plasma. in order to isolate neuron-derived evs (ndevs), a cell type specific transmembrane protein is necessary for immunocapture. l cam, a protein abundant on the surface of neurons, has been used extensively in the literature for ndev isolation. however, l cam exists in humans in several isoforms without a transmembrane domain, and as such it can be secreted as a free protein. additionally, the ectodomain of l cam can be cleaved off of the cell surface in physiological processes. it remains to be demonstrated whether the l cam found in csf and plasma is ev associated, or if it is instead a spliced or cleaved isoform behaving as a free protein. methods: using single molecule arrays (simoa), a digital form of elisa, as well as western blotting, we quantify ev markers (cd , cd and cd ) as well as l cam and albumin. we use these assays to determine in which fractions of size exclusion chromatography (sec) and density gradient the l cam appears. we also immunocapture l cam from csf and plasma and perform western blots for the internal and external domains of l cam. results: simoa and western blot analysis of sec and density gradient fractions demonstrated that while the ev markers peaked all together, l cam eluted in the free protein fractions along with albumin in both csf and plasma. when immunoprecipitation was performed, western blotting revealed different isoforms of l cam in csf and plasma. summary/conclusion: our data utilize a multitude of distinct techniques that converge to demonstrate that l cam is not associated with evs in csf or plasma. furthermore, our data suggest that the isoforms present in csf and plasma are distinct, which indicates that the l cam in plasma is likely not coming from the brain. this data call into question the utility of l cam as a ndev marker and point to the need to find novel candidates for immunoprecipitation of ndevs. introduction: in parkinson's disease (pd), α-synuclein (α-syn) aggregates known as lewy bodies (lb) are present in both the central and peripheral nervous system. furthermore, data showing that α-syn can spread from pd patients to transplanted tissue has led to a new theory postulating that pathological forms of α-syn can drive disease by "infecting" healthy cells and corrupting normal proteins. the exact routes and mechanisms involved in such spreading are yet to be fully understood but it is known that α-syn can be secreted from cells and transported via extracellular vesicles (ev). ev derived from erythrocytes (eev) are of particular interest in this regard as they have been shown to contain α-syn. methods: we first optimized a protocol for the isolation of fluorescently labelled human eev. the capacity of these eev to cross the blood-brain barrier (bbb) was then evaluated in vitro using a boyden chamber composed of primary human brain endothelial cells. next, eev were added to a more complex and physiologically relevant d human bbb model including ipsc-derived brain microvascular endothelial cells. in both in vitro protocols, flow cytometry was performed on media collect from each compartment to determine the number of eev. immunofluorescence was performed to assess the localization of fluorophore tagged eev. we are also using an in vivo paradigm for the extraction and testing of eev spread and an in situ cerebral perfusion (isbp) model in wt mice to investigate if and how eev cross the bbb using confocal microscopy. results: in both in vitro models, flow cytometry analyses showed that fluorescently tagged eev added to the luminal side traversed the endothelial cell barrier. confocal analysis revealed that some eev could also be found within endothelial cells themselves. ongoing experiments are being conducted in our newly developed d bbb to further confirm these results. our preliminary in vivo experiments showed that fluorescently labelled beads, similar in size to eev, used in the isbp experiments are detectable in the brain parenchyma of injected wt mice using confocal microscopy. preliminary work also includes isbp injections of eev in -month-old wt mice, (n = /groups) derived from pd patients (at different stage of the disease) and a healthy individual as a control. summary/conclusion: our preliminary data suggests that eev can indeed move across the bbb in both in vitro and in vivo experimental setups. ongoing experiments will determine the dynamics and processes involved in this transport and whether eev can precipitate and/or exacerbate disease-related features. introduction: neuroblastoma accounts for % of childhood cancer mortality. amplification of the oncogene n-myc is a well-established poor prognostic marker for neuroblastoma. whilst n-myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of n-myc in the aggressiveness of the disease is poorly understood. exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. hence, characterising the exosomal protein components from n-myc amplified and nonamplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. methods: in this study, comparative proteomic analysis, nanoparticle tracking analysis, transmission electron microscopy, rnai-based knockdown, migration and cellular survivability assays were performed to understand the role of exosomes isolated from cells with varying n-myc amplification status. results: label-free quantitative proteomic profiling revealed proteins that are differentially abundant in exosomes released by the n-myc amplified and nonamplified neuroblastoma cells. gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in n-myc amplified exosomes. treatment of less aggressive sh-sy y cells with n-myc amplified sk-n-be cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. incubation of exosomes from n-myc knocked down sk-n-be cells abolished the transfer of resistance to doxorubicin induced apoptosis. summary/conclusion: these findings suggest that exosomes could play a pivotal role in n-myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. ps . = op . results: murine ctl evs were broadly divided into two populations that were eluted at low salt (l-s: . m- . m nacl) and high salt (h-s: . m- . m nacl) concentrations. l-s ctl evs were abundant in late endosome-related proteins, integrins, rabs, and effective mirnas, indicating exosome characteristics, and had biological activity for preventing tumour metastasis after depletion of tumoural mesenchymal cell populations by intratumoral administration (see seo et al., nat. commun. : , ) . contrary, h-s ctl evs were rich in dna, core histones, ribosomal proteins, cytoskeleton proteins, and housekeeping proteins, considering microvesicles and apoptotic bodies, and easily phagocytosed by a kupffer cell line (kup : kitani et al., results immunol. : - . ). in addition, there were noticeable differences between ls and h-s ctl evs in the negative zeta potential width and membrane glycan structure. summary/conclusion: thus, ion exchange can be an optimal mass fractionation method for discriminating bioactive exosomes from cargos for nucleic acids in evs. funding: cryotem was conducted in nara institute of science and technology (naist), supported by nanotechnology platform program (synthesis of molecules and materials: # ) of the ministry of education, culture, sports, science and technology (mext). this work was supported by grants from the japan agency for medical research and development (translational research network program (nagoya univ. seeds a )) and the japan science and technology agency (crest [jpmjcr h ]). clic is essential for breast cancer metastatic competence and predicts disease outcome introduction: metastatic breast cancer is a consequence of complex interactions between cancer cells and the host. clic , a member of a conserved gene family in the glutathione-s-transferase superfamily, mediates crosstalk between tumour and host in breast cancer. tcga and metabric data indicated that elevated clic expression was associated with breast cancers from young women, those with poor prognosis, and those with early stage metastatic disease. methods: since bulk tumour analysis does not distinguish between cancer and host stromal cells, we used genetic modifications of established syngeneic breast cancer mouse models to evaluate the contributions of clic in the host or tumour cells to develop metastases. results: experimentally, the essential clic host contributions for metastatic competence were related to circulating levels of pro-metastatic soluble factors, neoangiogenesis, tumour cell attachment to lung tissue, myofibroblast differentiation, and leukocyte migration. clic was detected as cargo in circulating extracellular vesicles (evs) from breast cancer patients. similarly, circulating evs from tumour-bearing mice have abundant clic in comparison to those from mice bearing tumours that lack clic . tumour cells released evs that induced myofibroblast conversion of wildtype but not clic ablated lung fibroblasts. summary/conclusion: these results illuminate clic expression as a prognostic marker for breast cancer patients, and experimentally, clic is a critical host factor for metastatic competence and potential target within host tissues for anti-metastatic therapy. funding: this work was supported by the intramural program of the national cancer institute under project zia bc . the application of flow cytometry in an ev-based liquid biopsy for the detection of cancer multidrug resistance in myeloma gabriele de rubis a , krishna sunkara a , sabna rajeev krishnan and mary bebawy b a laboratory of cancer cell biology and therapeutics, discipline of pharmacy, graduate school of health, the university of technology sydney, australia, sydney, australia; b the university of technology sydney, sydney, australia introduction: multiple myeloma (mm) is an incurable cancer of bone-marrow plasma cells. it is characterized by unpredictable and highly variable therapeutic response and poor survival, attributed to the development of multidrug resistance (mdr) to chemotherapy. presently, no clinical procedures allow for a continuous, minimally invasive monitoring of mdr. we identified unique extracellular vesicle (ev) populations in the blood of myeloma patients, which serve as biomarkers of disease evolution and mdr to combination chemotherapy. we describe approaches used to optimise the use of flow cytometry (fcm) for ev summary/conclusion: although further investigation is required, our results potentially promise an effective and inexpensive priming agent (i.e., ethanol) for the production of anti-inflammatory msc-evs. this, combined with the significant increase in yield via d dynamic culture, presents practical solutions to both ev manufacturing scalability and potency issues. donor source affects potency of mesenchymal stem cell-derived extracellular vesicles introduction: mesenchymal stem cell (msc) therapies have been heavily investigated for their utility in applications such as wound healing and regenerative medicine due to their angiogenic, immunomodulatory and anti-apoptotic effects. recently, msc-derived extracellular vesicles (evs) have been implicated as primary effectors in msc-based therapies via protein and nucleic acid cargo transfer to patient cells. msc evs represent a superior alternative to msc-based therapies, as they lack the ability to replicate and are much smaller in size, circumventing related safety concerns such as immunogenicity, teratoma formation and blood vessel occlusion. however, a key drawback with msc therapies in general is their variable therapeutic potency, which is dependent on donor source. as a cell derived therapeutic, this crucial limitation is hypothesized to exist in msc evs as well. here, we demonstrate the varying bioactivities of isolated msc evs from differing donors and tissue sources. methods: six separate msc lines were obtained from different donors, with three msc lines derived from donor adipose tissue, and the other three from the bone marrow of separate individuals. evs were isolated from each msc line at passage via differential centrifugation and ultrafiltration. these isolated msc evs were then characterized for size/concentration via nanoparticle tracking analysis, and ev markers (tsg , alix, cd ) via western blot. pro-vascularization capacities of msc evs were determined by a gap closure assay using human umbilical cord vein endothelial cells (huvecs). results: characterization of msc evs revealed similar sizes and ev marker expression across donor groups, frontiers in chemistry, submitted funding: this work was funded by the momentum programme (lp - ), by the national competitiveness and excellence program catalan institution for research and advanced studies (icrea) proteomic profiling of retinoblastoma-derived exosomes reveals potential biomarkers of vitreous seeding angel montero carcaboso g , andrea petretto b , franco locatelli a and angela di giannatale a a department of paediatric haematology/oncology and cell and gene therapy, irccs, ospedale pediatrico bambino gesù ps : separation and concentration a laboratory of clinical biophysics, faculty of health sciences ps : diverse ev biomarkers chair: pia siljander -faculty of biological and environmental sciences urinary evs were isolated using low vacuum filtration method followed by ultracentrifugation. raman spectra of urinary evs were recorded using a renishaw invia raman spectrometer. data analysis was performed using principal component analysis (pca) and hierarchical cluster analysis (hca). the size distribution and morphology of evs were analysed by transmission electron microscopy and nanoparticle tracking analysis methods. results: average raman spectra obtained for urinary evs from studied groups showed differences in intensities of specific bands in the region of - cm- . we found significant correlations between mean area under curve (auc) calculated for raman bands (phenylalanine, dna, proteins, lipids and amide i) and selected clinical parameters such as: egfr, serum creatinine, glucose, urine creatinine. chemometric methods showed spectral pattern responsible for separation between studied groups. nta measurements visualized evs with size of . ± . nm. summary/conclusion: our results showed that characteristic raman spectra of urinary evs are promising candidates for new, non-invasive biomarkers for dkd isolation of circulating extracellular vesicles and cfdna allows for erbb detection in a single aliquot of breast cancer patients plasma michela notarangelo a , mattia barbareschi b , antonella ferro c , orazio caffo c , vito d'agostino a and francesca demichelis d a department of cellular results: results: tissue-derived large and small evs showed difference in size (mean nm vs nm) when examined by em, whereas nta and exoview™ were unable to show a clear difference between the populations (nta: mean . nm vs nm nta can only detected vesicles above nm and exoview™ only measures vesicles between - nm. of the three different methods, em analysis of single vesicles visualized in a significant number of micrographs was the only one able to distinguish ev subpopulations by size. funding: funding: swedish research council knut och alice wallenberg foundation imaging of human plasma-derived small-extracellular vesicles using transmission electron microscopy and structured illumination microscopy mitovesicles: a new extracellular vesicle of mitochondrial origin altered in ageing and neurodegeneration alldred b , chris goulbourne b , hediye erdjument-bromage d , monika pawlik b , mitsuo saito e , mariko saito f , stephen d. ginsberg b an in vitro and in vivo perspective on the role of erythrocyte-derived extracellular vesicles in parkinson's disease pathology frédéric calon c , Éric boilard f and francesca cicchetti b a centre de recherche du chu de québec and faculté de médecine, département de psychiatrie & neurosciences département de microbiologie-infectiologie et d'immunologie evidences on microalgal extracellular vesicles: a morphological assessment antonella bongiovanni i , ales iglič j and veronika kralj-iglič j a laboratory of clinical biophysics, faculty of health sciences a faculty of dentistry, national university of singapore, singapore, singapore, singapore; b institute of medical biology, agency for science, technology and research, singapore, singapore, singapore; c exosome of cancer-associated fibroblast induce anti-cancer drug-resistance of nsclc so-young kim a and yeon-ju lee b a chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea; b chonnam national university hwasun hospital biomedical research institute, gwangju, republic of korea introduction: the understanding of interaction mechanisms between cancer cells and the tumour microenvironment (tme) is crucial for developing therapies that can arrest tumour progression and metastasis. cafs are the major constituent of the tme in many cancers. recent studies indicate that exosomes harbour the potential to regulate proliferation, survival and immune status in recipient cells. most of the current studies are focused on cancer cell secreted exosomes; and little is known about cafderived exosomes and their influence on cancer cells. methods: nsclc cell lines (pc gr) and mrc (normal fibroblast cells) were grown in culture with exosome-free fbs. cutured media was filtrated by tangential flow filtration systems. exosomes in supernatant were isolated with the exoquick-tc™ system. considering the important role of cell extrinsic factors on cell growth and survival, we assessed whether factors contained in the mpa exosome could affect proliferation and survival of recipient cancer cells. cells were then treated with μm osimertinib or pbs for days prior to cell quantification of live cells.to investigate mechanisms of resistance to osimertinib mediated by ma or mpa-exosome in nsclc cell lines, we test cell viability by crystal violet assay in trametinib or osimertinib treated after pretreated ma or mpa-exosome, pc gr during days. we will investigate how mpa-exsomes activate erk signalling pathway in pc gr cells to induce antitumor effects by western blot. results: mpa exosome increased proliferation of pc gr cells by more than % compared to control pbs. pc gr cells grown in mpa-exosome and subsequently treated with osimertinib showed a significant increase in cell survival compared to pc gr cells grown in ma-exosome. osimertinib is used to treat egfr-mutant non-small cell lung cancer (nsclc) with tyrosine kinase inhibitor resistance mediated by the egfr t m mutation.these data show that "mrc -pc gr-crosstalk factors" affect proliferation and adaptive drug resistance of cancer cells. mpa-exosome mediates erk signalling activation and attenuated after treatment of um osimertinib. summary/conclusion: cafs support cancer growth and invasion. co-cultured nsclc with mrc lung fibroblast increased cell viability and exosomal mir- through the tgf-ß pathway in treatment osimertinib. exosomal mir- up-regulation in cocultured nsclc with mrc- induced drug resistance to drug-induced apoptosis. thus, exosomal mir- expression in co-cultured nsclc with mrc may support drug tolerance persister cells. introduction: neural stem cell (nsc) therapy has shown promise for brain repair after injury or disease mostly through bystander effects. nevertheless, the translation of nscs derived from human induced pluripotent stem cells (hipscs) to the clinic remain constrained due to safety issues, which include immunogenic risks, tumorigenesis potential, and incomplete differentiation. a way to avoid these issues is by using extracellular vesicles (evs) generated from nscs, as nsc-evs likely have similar neuroprotective properties as nscs and are amenable for non-invasive administration as an autologous or allogeneic off-the-shelf product. however, this would require reliable purification and characterization processes, and testing of evs for composition and biological properties. methods: we generated evs from hipsc-derived nscs using a combination of ion-exchange chromatography (iex) and size-exclusion chromatography (sec) and investigated their composition through small rna sequencing and proteomics. we also performed in vitro and in vivo experiments to determine their biological and functional properties. results: iex and sec facilitated purification of hipsc-nsc evs nearly to homogeneity, which expressed ev markers such as cd , cd , cd , and alix with a mean size of nm. small rna sequencing revealed enrichment of mirnas related to different neuroprotective signalling pathways and diverse metabolic functions consistent with their role in cell-cell communication. the proteomic analysis identified > , proteins, including ev markers and many other proteins involved in central nervous system function and cellular processes. the evs also displayed antiinflammatory activity in an in vitro mouse macrophage assay. intranasal (in) administration of nsc-evs resulted in their rapid incorporation by neurons, microglia, and astrocytes in virtually all regions of the brain. functionally, in administration of nsc-evs reduced inflammatory activity in the brain in a model of status epilepticus, and increased hippocampal neurogenesis in the adult brain. summary/conclusion: biologically active evs with antiinflammatory and neurogenic properties could be purified and harvested from hipsc-nscs. such evs also contain many mirnas and proteins that are of interest for brain repair after injury or disease. funding: supported by a grant from the national institute of neurological disorders and stroke ( r ns - to a.k.s.) introduction: extracellular vesicles (evs) generated from human bone marrow-derived mesenchymal stem cells (hmscs) display anti-inflammatory and neuroprotective properties. our recent study has shown that intranasally (in) administered hmsc-evs incorporate into significant percentages of neurons and microglia in virtually all regions of the intact as well as the injured forebrain within hours (kodali et al., int j mol sci, ) . in this study, using a rat model, we investigated the efficacy of a low dose of hmsc-evs administered intranasally for alleviating the abnormal plasticity of newly born neurons and the activation of microglia after se. methods: approximately billion evs were dispensed bilaterally into both nostrils of young f rats that experienced two hours of kainate-induced se. animals were euthanized seven days after se, and brain tissue sections were processed for immunohistochemical staining of neun (a neuronal marker), dcx (a marker of newly born neurons), iba- (a microglial marker), and parvalbumin (pv) and reelin (markers of subclasses of interneurons). in addition, activated microglia were quantified using iba- and cd dual immunofluorescence. results: in administration of evs reduced the seinduced loss of pyramidal neurons in the hippocampal ca subfield. also, ev administration after se maintained higher levels of pv+ interneurons in the dentate gyrus. furthermore, ev treatment after se modulated abnormal neurogenesis, which was evidenced by a the role of small extracellular vesicles in chronic neuropathic pain zhucheng lin a , renee jean-toussaint b , yuzhen tian b , ahmet sacan a and seena ajit b a drexel university, philadelphia, usa; b drexel university college of medicine, philadelphia, usa introduction: chronic pain is the most prevalent, disabling, and expensive public health condition in the usa. exosomes are - nm extracellular vesicles that can transport rnas, proteins, and lipid mediators to recipient cells via circulation. exosomes can be beneficial or harmful depending on their source and contents. we hypothesized that the composition of small extracellular vesicles (sevs) can be altered following nerve injury and these alterations can provide insight into how the body responds to neuropathic pain. methods: to characterize changes following nerve injury, small extracellular vesicles (sevs) were purified by ultracentrifugation from mouse serum four weeks after spared nerve injury (sni) or sham surgery. mirna profiling and proteomics analysis using tandem mass spectrometry were performed to determine differential expression of mirnas and protein cargo respectively. for in vivo studies, sevs were administrated intrathecally into the mouse lumbar region. animals were evaluated for mechanical and thermal hypersensitivity over days after injection. results: our mirna profiling showed a distinct mirna signature in sni model compared to sham control. proteomics analysis detected gene products. of these, were unique to sni model. neuropathic pain can induce the activation of the complement cascade and we observed significant upregulation of complement component a (c a) in sevs from sni model. intercellular adhesion molecule (icam- ), required for the leukocyte recruitment, adhesion and homing of exosomes was also upregulated in sevs from sni model compared to sham control. administration of sevs from sni model increased paw withdraw threshold in naïve recipient mice and inflammatory pain model, indicating a protective role for sevs in attenuating chronic pain. summary/conclusion: our preliminary studies suggest a critical role for sevs cargo in regulating pain. additional studies are ongoing to determine the functional significance of alterations in sevs composition using mouse models of pain. introduction: amyotrophic lateral sclerosis (als) is a progressive adult-onset neurodegenerative disease caused by selective motor neurons (mns) death. the rapid disease progression strongly suggests that cell-tocell spreading of noxious factors could take place in als pathogenesis. extracellular vesicles could potentially spread the disease. in this study, we characterized large (levs) and small extracellular vesicles (sevs) isolated from plasma of sporadic als patients and healthy controls and determined their different composition in order to understand their neuroprotective or neurotoxic role in als pathogenesis. methods: levs and sevs were isolated from plasma of als patients and healthy volunteers by differential centrifugation and characterized by nanosight ns . cd , cd , cd , cd a and annexin v were used for flow cytometry. sod , tdp , fus protein level was investigated by western blot. for raman spectroscopy, evs were dried on top of a caf slide and raman spectra were acquired using a nm laser line. mirna libraries were prepared by truseq small rna library kit (illumina). results: the mean size both for levs and for sevs resulted increased in als patients compared to controls. levs derived from als patients were enriched in sod- , tdp- and fus proteins compared to ctrls. sevs showed a distinct spectral pattern from levs. in addition, levs of als patients were richer in lipids and had less intense bands relative to aromatic aminoacids compared to healthy controls. we also found a great presence of leukocyte derived levs (lmvs) in als patients compared to ad patients and healthy donors and significant correlation with the progression rate of the disease. on the other hand, mirna and rna whole transcriptome sequencing identified a specific signature of mirnas in plasma derived sevs of als patients compared to a group of healthy controls and three neurological groups of control. summary/conclusion: these data may suggest that levs derived from als patients, enriched in lipids and toxic proteins, might play a role in prion-like propagation and immunity of als disease, while sevs, deriving ps . introduction: dendritic spines are actin-rich structures at the postsynaptic sites of most excitatory synapses in the central nervous system. they are highly important structures for higher brain functions such as learning and memory. several live imaging studies have shown that long, thin, actinrich protrusions called dendritic filopodia are precursors of dendritic spines in hippocampal and cortical neurons. so far, many intracellular factors that regulate filopodia formation have been identified. however, extracellular mechanisms of filopodia formation are largely unknown. also, detailed molecular mechanisms by which astrocyte secreted factors regulate synaptogenesis are not well understood. small extracellular vesicles (sevs)/exosomes have potential to regulate filopodia, spine and synapse formation in autocrine or paracrine manner due to their unique cargo composition. here, we examine role of exosomes in filopodia, spine and synapse formation. methods: primary rat hippocampal and cortical neurons were transiently transfected with the multivesicular body (mvb) docking regulator gfp-rab b or with shrnas against the exosome secretion and biogenesis regulators rab b and hrs. transfected neurons were immunostained for synaptic proteins and analysed for filopodia at day in vitro (div) or spines at div . for rescue experiments, exosomes were isolated using differential ultracentrifugation method from conditioned media of div cortical neurons or primary astrocytes and characterized for their size, common protein markers and morphology. results: here, we find that mvb docking factor gfp-rab b localizes to both the tips and bases of actin-rich filopodia and spines in primary neurons. furthermore, genetic regulation of exosome secretion by overexpression or knockdown of rab b or hrs leads to respective increases or decreases in the number of filopodia, spines and synapses. the defects of exosome-inhibited neurons in filopodia density are rescued by add-back of neuronal exosomes. additionally, treatment of primary neurons with exosomes isolated from primary astrocyte cultures leads to enhanced spine and synapse formation. summary/conclusion: these results indicate that autocrine and paracrine communication via exosomes are a key part of the process of neuronal filopodia, spine and synapse formation. effects of apolipoprotein e genotype on protein and small rna profiles of brain tissue-derived extracellular vesicles of alzheimer's disease patients introduction: multiple sclerosis (ms) is the most frequent chronic inflammatory disease of the young adult central nervous system. nevertheless, the pathogenesis remains largely unknown. it is therefore relevant to better characterise in cerebrospinal fluid (csf), which irrigates the brain, novel bioactive compounds whose dysregulation could be involved in ms pathology. the concentration of extracellular vesicles (evs) has been already found affected in ms patient fluids but the content in bioactive molecules, particularly the micrornas (mirnas), remains barely investigated. the mirna are short oligonucleotides that are major posttranscriptional regulators and we previously showed the dysregulation of specific mirnas in csf of ms patients. evs can potentiate mirna effects by allowing remote action through the shuttling within biological fluids such as csf while providing a protection from circulating rnase. nevertheless, csf remains a challenging fluid to analyse due to limited access, low volume and presence of lipoproteins (other putative mirna carrier) that can be co-isolated with evs. methods: we performed a comparative analysis of ev isolation from csf by size-exclusion chromatography (sec), density-gradient ultracentrifugation, ultrafiltration or chemical precipitation (chemp) to determine the optimal technique(s) to enrich ev. results: sec applied on csf of control patients showed optimal ev purification with sufficient evs from . ml of csf for downstream ev characterization. furthermore, we were able to isolate mirnas from csf and determined their enrichment in evs by rnase-sensitivity treatments. finally, we have combined chemp and sec to enable a fast and largescale isolation of evs from > ml of csf, which successfully provided an increase in particles detected by nanoparticle tracking analysis. we are currently characterising the particles to confirm that they are purified evs, cleared from contaminants. summary/conclusion: this work opens perspective to analyse evs from ms patients and to determine whether mirnas participates in ms pathogenesis through their transit in evs. funding: fondation louvain, charcot foundation. differences in circulating number of extracellular vesicles between contact sport athletes with and without acute mtbi: a pilot study meghan rath a , jacqueline sayoc a , soo-young choi a , karlee burns b , aja corchado c , jane mcdevitt b , jingwie wu d , ryan tierney b , michael selzer e , xiaoxuan fan f and joon-young park a for bottom-up guc, increasing iodixanol gradients with . ml of samples were centrifuged at k g for h. fractions were then pooled based on densities ( . - . g/ml). bca and sds-page were used to analyse total protein; nanoparticle tracking (nta) and transmission electron microscopy (tem) for ev presence; and immunoblotting and imaging flow cytometry (ifcm) to evaluate ev specific markers. (ev-track id: ev ). results: immunoblotting showed absence of actinin from all samples, while cd and tsg were detected for all samples; apart from imf_ip. nt_samples were not analysed reliably by nta and ifcm, due to the high concentration of casein micelles present (~ ^ /ml in milk) that otherwise would be co-counted with evs. as expected, following ip, which most efficiently removed casein micelles, bca showed that samples had lowest total protein. this was confirmed by sds-page. thus, most effects were then focused on the ip casein-depleted samples. ifcm indicated that, post-guc, sm_ip evs had significantly (p < . ) more cd -positive particles/ml of milk vs all other guc and kduc samples. while there were no significant differences in sizes of ev separated from sm or imf, directly comparing the ip pre-treated samples, sm had significantly (p < . ) higher quantities of evs when compared to imf. additionally, tem indicated that evs separated from sm by guc were intact with limited background debris, whereas those separated from sm by duc and all imf evs were not. summary/conclusion: in conclusion, regardless of the method used, imf has fewer intact evs compared to sm. also, to obtain purest sm evs, ip followed by guc separation is optimal. introduction: extracellular vesicles (evs) exist as subpopulations with heterogeneous content. the surface heterogeneity of evs may reflect differences in functionality between ev subpopulations, as interactions with recipient cells may differ between ev subpopulations with different surface profiles. however, it is currently challenging to study functional differences between ev subpopulations due to the lack of suitable techniques to purify intact evs based on their surface signature. here, we showcase a novel capture-and-release platform to enrich intact ev subpopulations by their surface profile and compare their characteristics. methods: mda-mb- and skov- cell-derived evs were isolated using size exclusion chromatography. ev subpopulations were enriched based on surface markers cd , cd , cd or phosphatidylserine (ps) using a novel magnetic bead-based capture-and-release platform. obtained evs were characterized by transmission electron microscopy (tem), nanoparticle tracking analysis (nta) and western blotting. evs were fluorescently labelled using pkh and celltracker deep red (ctdr) and their uptake by recipient cells was examined using flow cytometry. results: western blot analysis showed that ev subpopulations enriched for the selected tetraspanins and ps were successfully isolated using a novel capture-andrelease platform. interestingly, evs isolated based on ps exposure (ps+) lacked most canonical ev markers. all ev subpopulations showed intact, cup-shaped morphology when analysed by tem, but contained less protein contaminants compared to the initial ev isolate. ps+ evs were slightly larger than other ev subpopulations when analysed by tem and nta. to test the capacity of ev subpopulations to interact with recipient cells, evs were labelled with pkh and ctdr prior to subpopulation fractionation. after fractionation, ps+ evs showed a significantly higher ctdr/pkh ratio than other ev subpopulations as determined by fluorescence spectroscopy, suggesting higher esterase activity of ps+ evs compared to other tested subpopulations. furthermore, mda-mb- derived evs isolated based on cd and cd expression were taken up more efficiently by hmec- and mda-mb- cells than evs isolated based on presence of cd or ps. summary/conclusion: using a novel technology to isolate ev subpopulations based on their surface profile, we here show that composition and cellular uptake efficiency differs between ev subpopulations. theoretically, this technology is applicable to any surface marker of interest, allowing its use to further establish ev surface-functionality relationships and enrich evs with desirable characteristics for therapeutic purposes. funding: this work was supported by a veni grant (no. ) of the dutch research council (nwo). aml were harvested from tib cells cultured in evfree medium using serial ultracentrifugation. hspc (ksl; lin-sca + ckit+) clonogenicity and inflammatory responses were assessed using colony-forming unit (cfu) assay and real-time polymerase-chain reaction, respectively. ifn-alpha receptor (ifnar ) expression and intracellular reactive oxygen species (ros) levels were assessed by flow cytometry. dna damage were assessed by quantifying nuclear γ-h ax using immunofluorescent microscopy. results: similar to evs derived from aml patients, tib ev-aml elicited double-stranded breaks in hspcs, and actively suppressed hspc clonogenicity. transcriptional profiling revealed that exposure to ev-aml induced the upregulation of several inflammatory mediators in hspcs, including isg , il- , ifnα, ch h. inflammatory signalling triggered by ev-aml did not depend on ifnα signalling as evident from suppression of clonogenicity in ifnar -null hspcs as well as the lack of evs-induced stat phosphorylation or ifnar downregulation. instead, we found increased levels of ros following ev-aml exposure. summary/conclusion: our findings support a model whereby ev-aml inflammatory signalling and oxidative stress lead to dna damage in hspcs. introduction: basic leucine zipper atf-like transcription factor (batf ) is implicated in inflammatory response and anti-tumour effects. although the tumour suppressive function of batf has been reported, its extracellular role in maintaining a non-supportive cancer microenvironment has not been explored. methods: in this study, we established gbm orthotopic and subcutaneous tumour models in nude and balb/c mice and flow cytometry analysis determined the batf inhibitory effects of mdscs recruiting. we used transwell assay to determine batf -positive evs (evs-batf ) inhibitory of the chemotaxis of myeloid-derived suppressor cells (mdscs) in vitro. in addition, exo-counter detection during the development of the gbm-batf model to demonstrate evs-batf crosstalk with distant tissues. amd blocking in tumour model confirms that evs-batf dominated by the sdf- a/cxcr signalling pathway. in addition, exo-counter detection of evs in pairs of gliomas in different stages proposes plasma-evsbatf (plevs-batf ) as a prognostic marker. results: we found that tumour-derived evs-batf regulate crosstalk between glioma cells and tumour microenvironment by inhibiting mdscs recruitment. evs-batf can be detected in plasma and bone marrow of glioma-bearing mice, this provides direct evidence that glioma-derived evs can communicate with distant site by crossing blood-brain barrier. besides, evs-batf injection significantly reduced sdf- α expression in the tumour tissues. after blocking sdf- α signalling by amd , the inhibitory effects of batf overexpression on mdscs recruitment were rescued. evs-batf inhibit mdscs recruiting and secreting mmp , mmp , and vegfa which promote gbm progression. strikingly, exo-counter detection of evs in pairs of gliomas in different stages reveals that the number of plevs-batf can distinguish stage iii-iv glioma from stage i-ii glioma and healthy donors. summary/conclusion: our results suggest that evs-batf may be an effective circulating biomarker associated with glioma progression. of note, we are the first to determine the regulatory role of evs-batf in regulating tumour microenvironment and propose plevs-batf as a prognostic marker predicting glioma progression and candidate target for gbm therapy. introduction: electrofluidics is an emerging technology of combining electronics and nanofluidics. one important device in electrofluidics is an ion transistor in which the ionic current through a nanopore is regulated by gate voltage bias. here, we suggest a fabrication method of nanopore by introducing focused ion beam (fib) and atomic layer deposition (ald) to sense extracellular vesicle (ev) via metal electrode structures. methods: we deposited nm-thick silicon-nitrite layers on both sides of silicon wafer by low-pressure chemical vapour deposition (lpcvd). we fabricated rectangular patterns by photolithography followed by reactive ion etching (rie) on the backside of the wafer. anisotropic silicon etching by koh was performed. the front side of the chip was patterned by photolithography followed by ti/au deposition for the fabrication of electrode structures. we drilled ~ nm pores in the si n membrane by fib. by the ald process, we deposited highly-conformal metal film, either platinum (pt) or ruthenium (ru) to shrink nanopores by a self limiting process. results: we expect that the ion current through the nanopore is efficiently controlled by the gate-surrounding structures. the nanopore ion transistor can be used to count the number of evs. summary/conclusion: we suggest a fabrication method of nanopore ion transistors by introducing focused ion beam (fib) and atomic layer deposition (ald). this device will be applicable for single ev sensing. introduction: extracellular vesicles (evs) are key players in cell-cell communication and increasing evidence has shown that evs function in cancer by promoting cancer cell motility and metastasis. analysing tumour-derived evs in biofluids is attractive because it would be a novel approach to a non-invasive liquid biopsy. unfortunately, evs are highly heterogeneous. they vary greatly in size, lipid composition, and cargo and are difficult to distinguish from other small particles in complex biofluids. we have developed a novel flow cytometry method to generate a distinct ev fingerprint to profile biological specimens. methods: evs from cell culture media (purified and unpurified) and biological fluids (plasma and urine) were detected by flow cytometry using features on individual evs produced by intrinsic (cd -phluorin) and extrinsic (lipophilic dye, di- -anepps, and antibodies) fluorescent labels. ev subpopulations were visualized with dimensional reduction (t-sne and umap) of - features that defined the vesicle size, shape, and fluorescent emission spectra associated with the fluorescent marker. unsupervised density based clustering (hdbscan) in conjunction with supervised machine learning (xgboost) was subsequently used to define subpopulations. we refer to this method as "ev fingerprinting". results: ev fingerprinting was successfully used to detect evs in complex biological specimens and trace their differential enrichment through conventional purification methods. evs were readily distinguished from protein complexes, lipoproteins and non-lipid particles. calibration with externally validated purified ev, as well as size, lipid, and fluorescence standards enabled ev fingerprinting as a rigorous and reproducible method for resolving heterogenous ev samples. ev fingerprinting applied to conditioned medium from tumour cells and biological fluids from cancer patients reveals unique ev profiles generated by cancer, further supporting the potential of ev fingerprinting as a liquid biopsy. summary/conclusion: our single-ev analysis approach characterizes whole ev populations in complex biological fluids without the need for purification, reducing time intensive purification protocols and subsequent sample loss, permitting efficient analysis of liquid biopsy samples. detection and quantification of extracellular vesicles with cargo protein and rna using the amnis® cellstream® flow cytometer introduction: the particle size distribution (psd) of extracellular vesicles (evs) is commonly measured by tunable resistive pulse sensing (trps) and nanoparticle tracking analysis (nta). both trps and nta have limitations that hamper the accurate measurement of the psd of evs, specifically in the size range from to nm. an alternative technique for measuring the psd of evs is micro-fluidic resistive pulse sensing (mrps). because a standard operating procedure (sop) for characterizing evs by mrps is absent, we aim to establish a reliable sop to ensure reproducible psd measurements of evs by mrps. methods: measurements (n = ) of red-blood cell, prostate cancer cell line supernatant, and human urine and plasma evs were acquired in × s acquisitions. two microfluidic cartridges were used to study a dynamic range of - nm. samples were diluted into phosphate buffered saline with different concentrations of tween or bsa. because the excess of particles affects the detection limit, serial dilutions were performed to find the optimal dilution for each sample. data were evaluated using data viewer software. results: the optimal dilution was determined for each sample by maximizing the particle rate and minimizing the measurement time while preserving a robust detection limit of or nm. moreover, we developed a procedure to optimize the peak filter settings of data viewer by fitting data to normal distributions and identifying threshold values for signal-to-noise ratio, symmetry, and transit time within % confidence. summary/conclusion: we recommend to use . % w/ v bsa in dpbs as sample diluent, because tween affects evs as confirmed by flow cytometry. by using orthogonal techniques and well-characterized biological test samples, we developed and validated a sop for ev detection by mrps, thereby making mrps a valuable tool for ev researchers. real-time measurements of extracellular vesicles binding kinetics achieved through interferometric imaging in a multiplexed microarray modality introduction: extracellular vesicles are very promising diagnostic biomarkers. as a matter of fact, the properties of these biological nanoparticles depend on the health conditions of each individual. however, experiments that involve evs phenotyping are time consuming, due to h-or overnight incubations. in order to get accurate results, maximizing binding efficiency is a necessity; that normally involves ensuring the saturation of the capture reaction, which can result in an unnecessarily long incubation time. with the ability of labelfree kinetic binding measurements using interferometric reflectance sensing in a microfluidic chamber, we perform an optimization of the incubation time in different flow conditions, while demonstrating a new way of multiplexing for real-time evs specific capture and detection.methods: all the real-time binding measurements were performed with the interferometric reflectance imaging sensor (iris). iris chips were first coated with an organic polymer (mcp- ), which provides an active surface for probe immobilization. then, antibodies against cd , cd , cd markers were spotted at different densities in a microarray modality. the chips were then encapsulated with a glass window to form a microfluidic chamber that allows for imaging the sensor surface. samples of hek-derived extracellular vesicles were flowed across the sensor surface in the iris system and real-time images were acquired. incubation was performed at different flow rates, and in static and stopflow modalities. results: in this work, we focus on the specific capture of evs under different flow conditions to achieve an optimization of the incubation time. indeed, through the acquisition of real time binding data, we are able to precisely monitor the equilibrium point of the capture reaction. in this configuration of iris, low magnification optics allow for simultaneous detection of binding on hundreds of capture ligand spots. therefore, surface probes (surface density and specificity) as well as assay conditions can be optimized. we report on the optimization of antibodies against cd , cd , and cd markers. since the sensor chips are identical to the single-particle detection assays developed by nanoview biosciences, the optimization of binding assays will directly impact the phenotyping of individual exosomes. summary/conclusion: our method proved to be very efficient in optimizing the most crucial aspects concerning evs captureflow conditions, incubation time, surface density and sample concentration. introduction: diabetes is a life treating diseases extending its impairing influence on more than billion of people around the world within upcoming years. the most harmful complication generating high treatment and social costs is diabetic nephropathy, which develops in about % of patients suffering diabetes. still we do not have an effective and direct prognostic biomarker to diagnose renal complications in the primary stage of renal disease. methods: extracellular vesicles were concentrated from diabetic patients' urine and washed to perform spectral analysis: fourier transform infrared spectroscopy (ftir), based on the molecular absorption of electromagnetic radiation in the infrared region of the spectrum in a range from cm- to cm- and raman spectroscopy (rs) as a technique based on inelastic scattering of monochromatic light. both techniques provide information on the chemical structure of compounds by identifying functional groups with high molecular specificity. results: average spectral signature obtained for evs from urine samples of patients in the different stage of kidney damage allowed distinguishing specific bands, representative for amide (i/ii), lipids, cholesterol and nucleic acids. spectral parameters correlated with a clinical stage and a commonly used indicator of renal function (creatinine) in diabetic patients. summary/conclusion: infrared and raman spectroscopy are promising tools to diagnose and monitor renal function in diabetes. introduction: several existing bioanalytical strategies for purifying and characterizing exosomes have allowed for fundamental progress to be made. mixtures of evs can be enriched for exosomes by techniques such as ultracentrifugation and size-exclusion chromatography. but, these processes require large amounts of material that are often difficult to obtain and many different types of particles have similar sizes and densities. it is likely that unique subfractions within enriched samples exist, particularly in complex biological matrices such as blood, urine or milk which remain difficult to characterize and isolate with existing analytical technologies. methods: bovine milk exosomes were isolated via differential ultracentrifugation and resolubilized in mm ammonium acetate. these data were recorded using charge detection mass spectrometry (cdms). in cdms, individual particles are reflected back and forth through an electrostatic ion trap where they pass through a sensitive charge detector. each time a trapped particle enters and exits the detector, its charge (z) and mass-to-charge (m/z) ratio is measured. mass distributions are generated by multiplying the m/z values by the charge measured for each ion and binning the resulting masses.results: the masses of particles in a bovine milk extracellular vesicle (ev) preparation enriched for exosomes were directly determined for the first time by cdms. particle masses and charges span a wide range from m~ to~ mda and z~ to~ e and are highly dependent upon the conditions used to extract and isolate the evs. in total, , particles were detected from eight cdms measurements. a simple two-dimensional gaussian model suggests that eight unique subpopulations of particles may be resolvable based on charge and mass. complementary em and proteomics analyses confirm that samples are enriched for exosomes. particles associated with the s , s , and s families that are centred at~ . ,~ . , and~ . mda, respectively, appear too small to be ascribed to exosomes. the remaining , ( %) particles detected by cdms are within the mass range expected for exosomes. while cdms measurements are at an early stage of development, this approach appears to provide a new physical basis for separating and characterizing ev particles. summary/conclusion: this work describes a novel biophysical approach for measuring and characterizing the masses and charges of the extremely heterogenous population of exosomes and other extracellular particles enriched in bovine milk. as new sample preparation methods, aimed at purifying specific types of exosomes from different cell lines, tissues, and other body fluids continue to evolve, rapid and sensitive cdms measurements of the physical properties of mass and charge may become an important means of assessing the efficacy of different protocols. funding: nih (r gm - ). bab is supported by indiana university quantitative chemical biology fellowship (t gm ). in situ detection of exosomal microrna- b by fusion with liposomeencapsulated nanomotor introduction: breast cancer is the most common cause of cancer-associated death in women and has raised global health concerns. early diagnosis and treatment are crucial to improve the prognosis and survival rate of breast cancer patients. liquid biopsy is expected to provide a strategy for early diagnosis of breast cancer. exosomes have been regarded as novel liquid biopsy biomarkers due to their stable cargo of rnas, lipids, and proteins from their origin cells. exosomal micro (mi)rnas have recently been recognized as promising indicators of cancer occurrence and progression. however, most of the reported exosomal mirna detection methods require the lysis or extraction process, which increases the possibility of sample loss. in situ detection strategies avoid interference from body fluid. in this study, we developed a gold nanomotor fluorescence platform based on liposome fusion for breast cancer exosomal mirna in situ detection. the exosomal mirna detection platform was constructed using a gold nanomotor (detector) and liposomes (carrier). the dnazyme amplification sequences which could be especially triggered by mirna- b were identified by sds-page before modified on gold nano-motor and the capacity of the nanomotor was assessed using synthetic target sequence, breast cancer cell mda-mb- , mirna- b-encapsulated anionic liposomes, and mirna- b-expressing exosomes. three kinds of liposomes were synthesized, characterized, and assessed for loading ability. membrane fusion effect was evaluated by confocal laser scanning microscopy (clsm) and nanoflow cytometry. the performance of this method to discriminate between breast cancer patients and healthy individuals was investigated. results: the chosen dnazyme amplification sequences transformed "locked" status to "cleavable" status on target addition, releasing a fluorescence signal. the modified gold nanomotor showed a ten times higher fluorescence signal in the presence of mirna- b than the background and no noticeable fluorescence changes from a single-base-mismatch sequence. moreover, among the three different liposomes, cationic liposomes exhibited great stability, high loading efficiency, and excellent membrane fusion effect. furthermore, the fluorescent experiments confirmed that cationic liposomes could load and transfer the nanomotors into exosomes for mirna- b detection. finally, we were able to distinguish breast cancer patients and healthy individuals by sensing exosomal mirna- b directly from plasma samples without exosome isolation. summary/conclusion: a separation-free and sensitive assay based on dnazyme amplification technique and membrane fusion effect was established for breast cancer-derived exosomal mirna- b detection, which could be a promising tool for the liquid biopsy of breast cancer. isolation of exosomes by membrane affinity column increases non-exosomal rna recovery in comparison to differential ultracentrifugation introduction: exosome-based liquid biopsy is a potential aid in the diagnosis and prognosis of cancer patients. however, in order to incorporate exosomes into clinical routine, there is a need to compare different isolation methods. here we analysed the impact, in exosomal rna yield, of two intermediate recovery/ intermediate specificity methods: differential ultracentrifugation (ucd) and a membrane-affinity column (mac) kit. although mac has a faster performance which is more suitable to the clinic, we found that ucd results in a higher recovery of exosomes and less contaminating non-exosomal rna.methods: exosomes were enriched by mac and ucd from identical volumes of human plasma ( , xg, min/ . ) m filtration/ , xg, h)(n = ) and lymphoma conditioned medium( xg, min/ xg, min/ xg, min/ , xg . h/ , xg, h) (n = ). all exosomes were characterized by nanoparticle tracking analysis (nta), immunoblotting of cd /cd /flotilin/alix and electron microscopy (tem). exosome pellets were pre-treated with proteinase k ( mg/ml/ °c/ min) and rnase a ( mg/ ml/ °c/ min) before phenol-chloroform/glycogen rna extraction. rna yield was measured by both fluorometer and bioanalyzer.results: isolation of exosomes by ucd, in both plasma and medium, resulted in a higher yield in comparison to mac. this was shown by an augmented intensity of marker bands in the ucd samples (p = . , n = ) as well as by an increased number of exosomes in tem.in contrast, mac final exosomal fraction (from both plasma and medium), resulted in a -fold and fold increase in rna, respectively, in comparison to ucd when measured by fluorometer. this was confirmed by bioanalyzer. introduction: there is a need for better techniques for characterizing ev populations. we developed a sensitive multiplexed electrochemiluminescence (ecl)based assay format to characterize evs in cell-conditioned medium (ccm) and human biofluids. here we use the format to analyse ev samples for the presence of ev surface proteins, and to identify changes in ev phenotype associated with different cell lines, purification methods and growth conditions. methods: multiplex plates were prepared on msd's u-plex® platform with antibodies for putative evsurface proteins. each well displayed an array of nine specific capture antibodies and a negative control antibody. evs from samples were captured on the arrays and then detected with a cocktail of anti-tetraspanin antibodies (cd , cd and cd ) conjugated to an ecl label. three distinct cell types were grown at two sites, msd and atcc. resulting ccm were each purified by four common methods: tangential flow filtration, peg-based precipitation, size-exclusion chromatography and centrifugal ultrafiltration. all samples were also assayed without purification.results: fifty-five of the surface markers were detected on intact evs from at least one evaluated cell type. datasets were analysed using correlation matrices, hierarchical clustering, and machine learning. for each cell type, when comparing unpurified ccm grown at different sites or evs prepared by different purification methods, we typically observed correlations above . , indicating that the purification methods did not introduce bias to ev phenotypes, and that the assay format can provide robust phenotypic information without any purification of evs. two unsupervised clustering analyseshierarchical clustering and t-distributed stochastic neighbour embeddingboth generated wellseparated clusters for each of the cell types, regardless of purification method or source. summary/conclusion: we developed multiplex ev surface marker assays and demonstrated their use for multimarker ev phenotyping. this flexible format enables rapid assay development for new ev subpopulations with or without sample purification. these results also demonstrate ev surface marker phenotyping via multiplex ecl assays may be used to distinguish ev populations from various cell types, and characterize bias introduced by purification. detection of misev recommended ev protein-markers using automated western blotting method for isolation of evs and a simple western blotting platform for automated protein separation and immunodetection of misev-recommended proteins.methods: total evs were isolated by affinity-membrane spin columns from pre-filtered . - ml plasma or - ml urine, respectively. intact vesicles were eluted and the ev-depleted biofluid fraction was collected from the flow-through. a small fraction ( μl) was analysed by a simple western blot workflow providing automated capillary electrophoresis-based protein separation and immunodetection, characterizing each fraction for presence or absence of misevrecommended proteins.results: a range of specific antibodies were identified and the ev fractions were shown to be enriched in evproteins, whereas contaminating non-ev proteins were significantly reduced. isolation of evs was necessary to allow detection of the low abundant ev protein markers, whereas non-ev proteins were readily detectable both in the neat biofluids and in the ev-depleted flowthrough. we characterized the effect of washing on the purity of ev isolates and defined the dynamic range of the workflow using titrations of input volume of both plasma and urine ev isolations. summary/conclusion: simple western blotting protocols were established for quality control of isolated evs in accordance with misev-guidelines. evs isolated using affinity-membrane spin columns were shown to be enriched in ev markers and depleted for non-ev proteins. al-pha beads: a library of extracellular vesicle-associated metalloproteinase biosensors (adams) and a disintegrin and metalloproteinase with thrombospondin motifs (adamtss) are highly promising cancer biomarker candidates that have complex roles in cancer pathogenesis and metastasis. importantly, within the context of lung cancer, the detection of adam proteolytic activity might be more informative than the level of adam protein.therefore, the development of low-cost metalloproteinase biosensors could serve as useful biomarker research tools. methods: to this end, we developed advanced proteolytic detector polyhydroxyalkanoates (al-pha) beadsa library of biodegradable, biopolymer-based protease biosensors. broadly, these biosensors utilise phac-reporter fusion proteins that are bound to microbially manufactured bioplastic beads. these phac-fusions also incorporate specific protease cleavage sites. in the presence of a specific protease, reporter proteins are cleaved off of the al-pha beadsresulting in a loss of bead fluorescence that can be measured using flow cytometry. these biosensors were assayed using either metalloproteinases, conditioned media or evs from in vitro cancer models.results: human metalloproteinase recognition motifs were identified in the literature and a total of different al-pha bead biosensors were designed. a control, tev-specific biosensor detected . introduction: brain extracellular vesicles (evs) are heterogenous and include previously described microvesicles and exosomes. herein we characterized a formerly unappreciated population of mitochondriaderived evs that we term "mitovesicles". mitochondrial dysfunction is a well-established hallmark of ageing and neurodegenerative disorders as down syndrome (ds). hence, we examined mitovesicle levels and cargo under these conditions to characterize in vivo mitovesicle biology and responsiveness to mitochondrial stressors. methods: employing a high-resolution density gradient, distinct and novel populations of evs were isolated from murine and human ds and diploid control postmortem brains or from cell media. morphometric ev features were analysed by nanoparticle tracking analysis and cryogenic electron microscopy, while ev constituents were characterized by western blotting, mass spectrometry, lipid profiling and mitochondrial rna qpcr.results: we identified a population of double-membrane, electron-dense brain evs containing multiple mitochondrial markers ("mitovesicles") that are highly distinct from microvesicles and exosomes. proteomic data show that mitovesicles contain a unique subset of mitochondrial proteins while lacking others, such as tom . mitovesicles have a lipid composition that is unlike that of previously described evs and is consistent with mitochondrial origin. functionally, the complex-iii inhibitor antimycin-a stimulated in vitro mitovesicle release into the cell media, suggesting an interrelationship between mitochondrial dysfunction and mitovesicle biology. in mouse brains, mitovesicle levels increased with age and were found to be higher in ds compared to diploid controls. mitochondrial rna and protein levels were also altered in ds compared to diploid controls. summary/conclusion: we describe a previously unidentified type of metabolically competent evs of mitochondrial origin that we designate mitovesicles. our data demonstrate that brain mitovesicle levels and cargo are tightly regulated in normal conditions and are modified during pathophysiological processes in which mitochondrial dysfunction occurs, suggesting that mitovesicles are a previously unrecognized player in mitochondria quality control and may have a role in the trans-cellular tissue response to oxidative stress. introduction: alzheimer's disease (ad) is a devastating neurodegenerative disease leading to progressive memory loss and ultimately death with limited therapeutic options. growing evidence supports the theory that toxic proteins, like tau and amyloid, may propagate from diseased cells by packaging toxic proteins into extracellular vesicles (evs) and releasing them to infect other cells. one enzyme involved in the isev abstract book biogenesis of evs is neutral sphingomyelinase (nsmase ), which catalyzes the hydrolysis of sphingomyelin to produce phosphorylcholine and ceramide. several groups have reported improved cognition and reduced tau propagation when nsmase is pharmacologically inhibited or genetically knocked down in ad mouse models. unfortunately, current nsmase inhibitors are not suitable for clinical development due to poor solubility and inadequate pharmacokinetic profiles.methods: our group carried out a high-throughput screening campaign followed by extensive medicinal chemistry efforts leading to the discovery of phenyl (r)-( -( -( , -dimethoxyphenyl)- , -dimethylimidazo [ , -b] pyridazin- -yl) pyrrolidin- -yl) carbamate (pddc), an orally active, nm potent inhibitor with excellent selectivity and brain penetration. we tested pddc's ability to inhibit exosome release in cultured primary glial cells as well as an in vivo model of acute ev release. we then treated xfad mice with mg/ kg of pddc daily for six months and monitored their behaviour in the fear conditioning assay.results: pddc dose dependently reduced ev release from cultured primary glial cells and significantly reduced plasma ev numbers in an in vivo model. following chronic treatment with pddc, xfad mice demonstrated significantly improved cognitive function in the fear conditioning assay. summary/conclusion: these promising findings are currently being expanded using mouse models of tau propagation. if successful, these data would support pddc as a novel compound for targeting the pathological spread of tau as a therapeutic for ad. profiling evs in the anterior cingulate cortex of individuals with major depressive disorder introduction: major depressive disorder (mdd) is one of the leading causes of disability worldwide, affecting % of the population. the environment has been thought to play a role in the disease development, resulting in biological changes mediated by epigenetic mechanisms. microrna's (mirna) are well known epigenetic regulators that are disrupted in the depressed brain, and they are packaged into extracellular vesicles (evs). evs have emerged as means of intercellular communication, a process that is also disrupted in mdd. they are thought to transfer mirna between cells, which can alter gene expression in recipient cells. therefore, we hypothesize that ev cargo is altered in mdd subjects compared to healthy controls (hc). the aim is to extract evs from human postmortem anterior cingulate cortex, a region previously associated with depression, and profile the mirna cargo and compare it between mdd subjects and hc. methods: post-mortem human brain tissue from the anterior cingulate cortex of mdd subjects and hc was mildly dissociated in the presence of collagenase type iii. residual tissue, cells, and large vesicles were eliminated, and evs were isolated using size exclusion chromatography. the quality was assessed by western blots and transmission electron microscopy (tem). rna was extracted and a small-rna library was constructed and sequenced using the illumina platform. differential expression analysis was then performed.results: western blots showed little to no endoplasmic reticulum (calnexin), golgi (bip), or mitochondrial (vdac) contamination, along with enrichment of the exosomal marker cd . tem images showed the typical cup-shaped morphology with sizes mostly between and nm. preliminary sequencing results revealed that mir- a- p, which is predicted to target glutamate receptors, is downregulated in evs from mdd subjects. summary/conclusion: high quality ev extractions can be obtained from post-mortem brain tissue using our method. this will be the first study to profile brainderived ev mirna in the context of depression. future studies will be needed to determine the effect of the different levels of mir- a- p. this could provide novel mechanistic insights into the pathophysiology of mdd and will serve as a starting point to examine the potential role of evs in mdd pathology. methods: we use ifc to characterize evs released by glioma using -ala, fluorescently labelled ev (cfda-se, cd ) and glioma specific (tenascin c and epidermal growth factor receptor viii, egfrviii) markers. furthermore, we characterized evs released by egfrviii positive glioma cells treated with dexamethasone, a steroid commonly used in glioma patients, to determine the effect of steroids on ev release. evs were quantified by ifc and results were confirmed by qpcr for the levels of egfrviii mrna. results: firstly, we optimized protocols to label glioma sevs using fluorescently labelled ev markers (cfda-se, cd ) and tumour specific markers (tenascin c and egfrviii). of the total evs (cfda-se), we demonstrate that % are tenascin c positive, . % are egfrviii positive and . % are -ala positive. there was only a minor overlap (< %) between the sub-populations. finally, we show that dexamethasone treated glioma cells release lower total evs ( . -fold), tumour specific evs ( . -fold; egfrviii), egfrviii mrna compared to mock treated cells. summary/conclusion: we demonstrate the potential of ifc to monitor sevs released by glioma cells exposed to different stimuli. this allows the characterization of ev sub-populations providing a working model to understand the dynamics of tumour evs at a single vesicle level. introduction: extracellular vesicles (ev) released by infective forms of trypanosoma cruzi, the agent of chagas' disease, modulate inflammatory response of macrophages through the activation of toll receptor (tlr ) via mitogen-activated protein kinase pathway. this induces the production of nitric oxide (no) and expression of the cytokines tnf-α, il- and il- , which could explain the inflammation observed in experimental chagas' disease, and eventually in the progression of human disease. evs released by the parasite are heterogeneous and it is unknown which factor, or factors present in the different vesicle populations act during the interaction with host cells.objectives. the goal of the present work was to characterize and isolate the different populations of evs released by t. cruzi and test their effects on macrophages. methods: ev released by trypomastigotes forms of t. cruzi (y strain) were purified by asymmetric flow field-flow fractionation (af ) and characterized by nanoparticles tracking analysis (nta). the different populations of evs were incubated with host human monocytes cells (thp- ) and cytokines production determined by elisa and qpcr. the different ev populations were also incubated with llcmk- epithelial cells and the infection by t. cruzi determined. results: we found two distinct populations of evs. a population with to nm (ev ) and another with to nm (ev ). ev induced more tnf-alpha, il- , ip- and ccl than ev . it was also more effective in promoting t. cruzi infection in epithelial cells. due to unknown reasons, making these systems insufficient for use in drug development and infectivity assays.noroviruses are known to attach to gram-negative enteric bacteria and this facilitates infection in vitro. however, the microbiome-norovirus-host communication link is missing. noroviruses infect immune cells present in lamina propria during acute infection, but bacteria themselves are large enough to cross the mucosal and the tight epithelial barrier which separates gut lumen from lamina propria. we hypothesized that binding of noroviruses to bacteria enhances extracellular vesicles (ev) production. because commensal bacterial evs by themselves do not have any detrimental effects on host cells, we believe using evs in in vitro culture will enhance norovirus infection, thus producing higher titre of viruses for vaccine and anti-viral drug development. methods: attachment assay: purified norovirus was incubated with enterobacter cloacae, lactobacillus acidophilus and bacteroides thethiotaomicron, and grown to produce evs. the attachment was confirmed via qpcr.isolation of evs: clarified media supernatants were subjected to ultracentrifugation at varying speeds and . um filtration. co-purification of norovirus with the evs was checked.ev quantification and characterization: ev total protein content was measured by microbca. the number of vesicles were quantified by nanoparticle tracking analysis. scanning and transmission electron microscopy was performed to check quality of ev preparation and determine if virus was attached to the vesicles. internal ev protein content was evaluated using ms-hplc. the evs were also check for infectivity via tcid assay. results: incubation of noroviruses with commensal bacteria resulted in significant increases in production of evs compared to uninfected controls. murine norovirus (mnv), used as a surrogate, was found to be associated with evs. em analysis determine association of viruses with the bacteria as well as the mvs, while also showing certain surface structural changes in virus attached bacteria compared to mock bacteria. the evs were found to cause infection in naive macrophages. summary/conclusion: changes in ev production and content by bacteria exposed to noroviruses will provide insight into its pathogenesis and possible solutions to the low viral output from hunov culture systems.ps . = op . kylie krohmaly a , claire hoptay b , andrea hahn a and robert freishtat a a children's national hospital, washington, usa; b childrens national hospital, washington, usa introduction: bacteria constitutively produce biologically active extracellular vesicles (evs), which contain rna, dna, and/or proteins. bacteria use these evs for communication with other bacteria and recent research suggests bacterial evs can also affect host cells. given these findings, it is necessary to examine the role of bacterial evs in human disease. current methods of bacterial ev isolation from human specimens cannot distinguish between bacterial species. however, there is utility in examining evs from specific species, as bacterial species and their evs may have unique contributions to human disease. our objective was to isolate circulating evs specifically from escherichia coli (eevs) and haemophilus influenzae (hevs), two known colonizers and pathogens in the gut and airway, respectively. methods: total evs were isolated from the blood of six healthy volunteers via precipitation and size exclusion chromatography. evs were then selected via a novel latex bead-based fluorescent antibody construct targeting species-specific outer membrane proteins. we used flow cytometry to evaluate the isolated evs. results: the constructs were saturated with eevs at an antibody concentration of . µg/ml of plasma, as geometric means ≥ . µg/ml were nearly equal. hevs were detected at µg/ml of plasma, but saturation is yet to be determined. eevs were imaged by a fei talos f x electron microscope and measured between - nm, and hevs were between - nm. both types of evs were spherical. summary/conclusion: using this novel technique, we were able to isolate, detect, and visualize eevs and hevs. this technique enables the study of specific bacterial evs. in the future, ev contents will be assayed. furthermore, this technique will be modified so that specific bacterial evs from body fluids can be used for downstream functional applications. this is the first time that bacterial evs from targeted bacterial species have been detected in blood from healthy humans. introduction: nasopharyngeal carcinoma (npc) is characterized by a large presence of regulatory t cells (tregs) and the production of tumour-derived exosomes with immunosuppressive properties. our team showed that npc-derived exosomes favour the suppressive activity and recruitment of human tregs via ccl chemokine, thus contributing to npc immune escape (mrizak et al., jnci, ) . more recently, our team has shown that npc-exosomes could induce tregs by altering the maturation of dendritic cells (dcs) and promoting tolerogenic dendritic cells (tdcs) (renaud et al., herpas congress ). our main objectives in this study are (i) to define and compare the metabolic status of mature dendritic cells (mdcs), control tdcs and tdcs generated in the presence of npc-exosomes (exocnptdc) and (ii) to evaluate the chemoattractive potential of npc-exosomes on exocnptdcs, and notably to investigate the involvement of ccl in this recruitment. methods: dcs are generated from human monocytes in the presence or absence of npc-exosomes. the maturation status of dcs was evaluated at a phenotypic level by studying the expression of maturation markers using flow cytometry and at a functional level by analysing cytokines secretion using elisa. this cytokine analyse has been performed in both conditions, on treated dcs and during co-culture assays of autologous cd t lymphocytes with treated dcs. in a second step, a mitochondrial metabolic and glycolytic study was performed using the seahorse technology (ocr and ecar measurement). finally, the chemoattractive potential of npc-derived exosomes on the different induced dcs was analysed (i) using boyden chamber chemoattraction assays or real-time videomicroscopy (chemotaxis µslide ibidi) and (ii) using rt-qpcr analysis of the receptor expression of ccl (ccr ).results: npc-exosomes alter dc maturation, which gives rise to tolerogenic dcs that favour the induction of tregs. in addition, the metabolic analysis of dcs seems to put foward a specific metabolic signature of the tdcs induced by npc-exosomes. and finally, chemoattraction assay suggests that npc-exosomes preferentially attract tdcs and exocnptdcs in a ccl dependant manner. summary/conclusion: taken together our results should allow us to characterize the major role of npc tumour exosomes on the maturation and the recruitment of dc and so identify them as anti-tumoural therapeutic targets. cytotoxic t lymphocyte ev that prevents tumour metastasis by collapse of tumoural mesenchymal stroma is classified into exosome, but not microvesicle or apoptotic body.naohiro seo a , junko nakamura a , tsuguhiro kaneda a , takanori ichiki b , asako shimoda c , kazunari akiyoshi c and hiroshi shiku a a mie university graduate school of medicine, mie, japan; b the university of tokyo, bunkyo, japan; c kyoto university, kyoto, japanintroduction: recently, instead of ultracentrifugation, development of new preparation protocol is demanded for research of reliable bioactivity and drug discovery of extracellular vesicles (evs). in this study, we propose a novel method for large scale preparation of highperformance extracellular vesicles focusing on membrane negative charge. methods: murine cytotoxic t lymphocyte (ctl) evs in supernatant were concentrated more than times at over % purity without leaking by kda mwco ultrafiltration, and subjected to ion exchange deae column chromatography after replacing with pbs. after ion exchange, evs were characterized by bca assay, nta assay, cryotem observation, proteome analysis, dna content measurement, mirna microarray analysis, zeta potential measurement, lectin array analysis, and target cell analysis.biomarker detection and analysis and detail strategies for cross-platform analytical validation. methods: we conducted a cross-platform analysis using two commercially available flow cytometers designed for ev detection. scatter resolution, enumeration accuracy and precision were determined across both platforms by analysing submicron silica beads (apogeemix, - nm) of known concentration.we detected large evs, as established by reference size beads, electron microscopy, expression of phosphatidylserine and the presence of integral membrane proteins of cell of origin. we analysed evs isolated from plasma by high-speed centrifugation ( , g) as well performing analysis by direct plasma labelling followed by validation by detergent lysis of vesicular constituents. a clinical operating range was defined which ensures linearity and avoids swarm detection. we observed comparable scatter resolution, enumeration accuracy (error ≤ %) and precision (cv ≤ %) across both platforms used. we defined two ev size gates: a "latex" gate ( to nm polystyrene latex beads), and a "silica" gate ( to nm silica beads) for evs at the lower end of our size range of interest. to improve detection sensitivity, we identified common contributors to signal noise and applied workflow strategies to minimize these. finally, we identified linear ranges which avoid swarm detection, and which ensures reproducible ev counts (cv < %) across both instruments. summary/conclusion: we present an optimised, standardised and cross-platform reproducible working protocol which supports the use of fcm in an ev-based liquid biopsy application. funding: the project is funded by spark oceania and uts innovation commercialisation seed fund scheme to mb. metabolomic profiling of serum and exosomes isolated from head and neck cancer patients after radiotherapy introduction: cancer radiotherapy (rt) induces the response of the whole body that could be detected at the blood level. searching for new molecular signatures which could correlate with treatment response in cancer patients is of particular importance. radiation-induced changes in proteome and transcriptome of serum have been widely described. however, metabolomic changes in serum, exosomes and other classes of small extracellular vesicles (ev) of cancer patients after rt have not been given as much attention. metabolomics of serum and ev of cancer patients could provide a valuable insight into the response of both tumour and whole organism to the treatment. the aim of the study was to compare serum and ev metabolomic profiles in head and neck cancer (hnc) patients before and after rt. methods: serum samples from hnc patients were taken before (a) and after (b) rt. healthy volunteers were used as a control group (c). ev were isolated from ml of serum using size-exclusion chromatography (sec). selected sec fractions were subjected to extraction of metabolites. a mixture of meoh/h o was used for extraction of metabolites from serum and ev samples. samples were analysed by gas chromatography-mass spectrometry (gc-ms).the study protocol adhered to the tenets of the declaration of helsinki and was approved by the bioethical committee of the maria skłodowska-curie national research institute of oncology, branch gliwice, poland (permit nr. do/dgp/ / / / / /g). results: an untargeted gc-ms-based approach allowed the detection of metabolites in serum samples and exosomal small molecules, of which joint. the identified compounds included amino acids, fatty acids, carboxylic acids, sugars, and others. there were metabolites which levels discriminated compared groups (a,b,c) of serum samples and compounds that discriminate the ev isolated from hnc serum before and after rt from hc. summary/conclusion: rt caused significant changes in levels of serum and ev metabolites witch are involved in amino acid metabolism, lipids metabolism, energy metabolism and oxidative stress response. capable of contributing to intercellular communication and metastasis. numerous studies have focused on elucidating their role in cancer progression. we recently showed that sevs isolated from pancreatic cancer cells can function as an initiator in malignant cell transformation. here, using a mass spectrometry (ms)-based proteomics approach, we analysed the differences in the protein cargo of sevs secreted from normal pancreatic and cancer cells to better understand their biological characteristics. methods: sevs were isolated from human pancreatic cancer cell lines (capan- , mia paca- , and panc- ) and normal pancreatic epithelial cells (hpde) using a combined ultrafiltration-ultracentrifugation method coupled with a sucrose density gradient purification. proteomic profiling of sevs was carried out using an lc-ms/ms method. protein identification from resulting ms/ms spectra was conducted using proteome database search software followed by gene ontology (go) enrichment and reactome pathway analysis.results: a total of , unique proteins were identified confidently across the combined samples. the proteins present in all four sev types ( , proteins) consist of general housekeeping proteins. proteins were uniquely found in all cancer sevs but not in the normal hpde sevs. this group contains an enrichment of proteins that function in the endosomal compartment of cells responsible for vesicle formation and secretion and suggest their important role in driving the increased production of sevs from cancer cells relative to normal cells. moreover, this group includes a set of proteins that have been implicated in malignant cell transformation, consistent with our previous work showing that each of the cancer sevs analysed here could initiate malignant transformation of nih/ t cells. conversely, there were proteins uniquely found in normal hpde sevs. this group includes a number of immune response proteins that are not found in any of the pancreatic cancer cell sevs. summary/conclusion: the differences in the proteomes of cancer and normal sevs may be indicative of their varying roles in cell transformation and helpful in delineating the types of evs that are being produced. in addition, these differences point towards their potential value as cancer biomarkers. proteomic profile of tumour-derived exosomes in plasma of melanoma patients introduction: in the past years, extracellular vesicles (evs) have attracted considerable interest due to their ability to provide valuable diagnostic information from liquid biopsies. the high abundance in all bodily fluids and their cargo stability confers evs the potential as a powerful tool to not only obtain novel biomarkers from inaccessible tissues, therapy response and monitoring, but also to reduce infection risks of conventional highly invasive biopsies. virtually all cells continuously release vesicles into the extracellular environment, diverse in size, content and features depending on the biogenesis, origin and function. this heterogeneity adds a layer of complexity when attempting to isolate and characterize tissue-specific vesicles. methods: hence, we aimed to use a immunomagnetic capture approach for prostate-derived evs from cell culture supernatants, with further investigation into human plasma and urine samples. analysis was performed by nanoparticle tracking analysis, western blotting and electron microscopy. additionally, an in-house spotted antibody microarray is in development. here, we intend to detect different ev sub-populations based on their surface markers. results: isolated immunocaptured ev populations based on the classical ev marker cd show an increased signal for the luminal protein tsg . ev populations targeting the tissue-specific marker prostate specific membrane antigen (psma), were found positive for tsg in a lower extent indicating a subpopulation of evs. the microarray uses less than µl of sample (concentrated cell culture supernatant, human plasma, urine) and leads to a faster characterization within h for ev surface marker as compared to western blot. summary/conclusion: immunomagnetic isolation might be a promising approach for liquid biopsy and thereby the microarray could be valuable to identify potential capture targets. the current design for different surface marker from samples simultaneously could be easily extended for sample size and surface profiling allowing for a more economical way to multiplex samples. paving the way for implementing a feasible and reliable technique for assessing urinary extracellular vesicles as biomarkers for bladder cancer in clinical practice introduction: extracellular vesicles (ev) in urine have been proposed as biomarkers for bladder cancer (bc). however, at present there are no standardized methods for ev isolation or urine sampling. our goal was to evaluate the ev isolation performance between different methods, the effect of the sampling time and the importance of urinary creatinine (ucr) normalization. methods: two urine samples of ml were collected from patients with non muscle-invasive bc: one from the first micturition and another from any time of the day. twenty ml were used for ucr measurement and ml were used for ev isolation by either precipitation with polyethylene glycol (peg), concentration by filtration (uf, centricon plus- , k, millipore), sepharose size exclusion column (sec), or combinations of these methods. additionally, the effect of protease inhibitors (pi) and dtt treatment after collection or during processing was analysed. size and number of particles were evaluated by nanosight and the presence of exosomal markers was evaluated by western blot. results: among the methods evaluated, uf + sec showed the best performance retrieving the highest number of particles in the range of - nm, and the highest protein expression of exosomal proteins. uf alone showed the highest concentration of ev, but with a tendency to isolate larger particles. particle concentration was positively correlated with ucr, reflecting the importance of ucr normalization before journal of extracellular vesicles comparing between patients. finally, no differences in the performance according to the time of collection, nor in the use of pi or dtt were observed. summary/conclusion: uf + sec gave the highest ev yield and was not affected by the time of urine collection. the use of pi and dtt can be avoided, and normalization to ucr should be considered when implementing this technique for assessing evs as biomarkers for bc in clinical practice. funding: pida . the introduction: human tumours, including pancreatic ductal adenocarcinoma (pdac), often harbour a subpopulation of cancer cells with extra centrosomes. we found, that these cells secrete an increased number of small extracellular vesicles (sevs), within the - nm size range. sevs play a role in cancer signalling and progression and are widely studied for their diagnostic potential. we aim to understand the role of sevs secreted by cells with extra centrosomes in shaping pdac-associated stroma, particularly fibrosis. methods: to study the sev mediated changes in the pdac microenvironment, we purified sevs through serial ultracentrifugation and size exclusion chromatography, characterised the content through silac-based proteomics, and assessed phenotypic changes in pancreatic stellate cells (pscs) and extracellular matrix (ecm) production through immunofluorescence staining. results: our data indicates, that the sevs secreted by cells with extra centrosomes are exosomes due to their endocytic origin, and we found, that they can activate pscs, key mediators of fibrosis in pdac. indeed, we observed an increased level of collagen i produced by pscs activated by sevs from cells with extra centrosomes as compared to cells without extra centrosomes. interestingly, we found, that psc activation through sevs is not mediated by tgf-β, assessed by the level of nuclear smad accumulation downstream of tgfβ activation, suggesting a novel mechanism of pscs activation. summary/conclusion: pdac cells with extra centrosomes contribute to a novel type of psc reprogramming, which could alter their ecm deposition and contribute to the extensive fibrosis observed in pdac. we are currently characterising the signalling pathways associated with sev mediated psc activation and how it impacts padc progression to better understand the role of centrosome amplification in the cancer-stromal crosstalk. exosomal carboxypeptidase e confers and cpe-shrna loaded exosomes inhibit growth and invasion of hepatocellular carcinoma cells. methods: exosomes were isolated from the culture media of high metastic hcc h cells and incubated with low metastatic hcc l cells. in other experiments, cpe-shna loaded exosomes from hek cells were incubated with hcc h cells. the recipient cells were analysed for proliferation using mtt assay, colony formation, and matrigel invasion. results: analysis of exosomes derived from hcc h cells revealed cpe-wt mrna and protein. exosomes released from hcc h cells were able to enhance proliferation and invasion of hcc l cells. when cpe expression was suppressed in the hcc h cells before exosome isolation, the exosomes had no effect on proliferation and invasion. these data demonstrate the ability of exosomes to confer growth and invasion in hcc cells and the role of exosomal cpe in driving the process.previously it was shown that down-regulation of cpe expression by shrna can reverse tumour growth and metastasis in an hcc mouse model. we therefore loaded cpe-shrna into exosomes by infecting hek (human embryonic kidney) cells with adenovirus carrying cpe-shrna-gfp. these modified isev abstract book exosomes were used to transfer cpe-shrna to hcc h cells, resulting in significant reduction in proliferation and colony-forming ability of these cells. cpe-shrna loaded exosomes were found to down-regulate the expression of cyclin d and c-myc, two genes with high relavance to tumour growth and metastasis. summary/conclusion: our results demonstrate the ability of exosomal cpe to enhance proliferation and invasion in low metastatic hcc cells and the potential to use shrna loaded exosomes to target cpe as a therapeutic strategy to treat liver cancer.funding: intramural program of the eunice kennedy shriver national institute of child health and human development, and national cancer institute, national institutes of health, bethesda, md. . stress hormones promote prostate cancer aggressiveness through modulation of mir- - p expression and exosome release north carolina central university, durham, usa introduction: despite proactive screening and steady declines in mortality, prostate cancer (pca) remains one of the most prevalent cancers among men. evidence suggests that chronic activation of stress signalling pathways can result in an altered mirnas transcriptome and affect exosomal content and release. here, we study the interaction between leptin and mir- - p expression, previously shown to be downregulated in pca patients. in addition, explored the effect of stress hormones cortisol and leptin on exosomal release and content from pca cells.methods: we utilized normal prostate cell line rwpe- , and pca cells pc , lncap and mda-pca- b. proliferation of cells treated with leptin in the presence or absence of mir- - p mimic or negative control was assessed by mtt, colony formation, wound healing, and expression of targets affected by mir- - p was assessed by western blotting. moreover, exosomes were isolated via differential centrifugation from pca cells treated with leptin or cortisol and exosome number was determined by nanotracking analysis. exosome content was determined by western blotting and proteomic analysis by mass spectrometry.results: we observed that leptin significantly decreased expression of mir- - p in rwpe- cells.co-treatment with mir- - p mimic and leptin abrogated these effects in a cell dependent manner. we also observed that co-treatment with leptin affected mir- - p target jag and other molecules involved in epithelial to mesenchymal transition. in parallel, we demonstrated that cortisol increases exosome secretion particularly in pc cell exosomes with a . -fold increase at nm cortisol compared to untreated. western blotting revealed the presence of gr in exosomes particularly at nm cortisol. summary/conclusion: understanding epigenetic regulation through mirnas and exosomes may be the key to understand stress hormone influence in pca progression. these findings suggest that stress hormones effectively affect mir- - p expression and exosomal release and signalling.introduction: extracellular vesicles (evs) are promising drug delivery vehicles for therapeutic microrna (mirna). for the loading of exogenous cargo, researchers broadly seek to either manipulate the evs directly or the cell that produce them. electroporation, sonication, and direct ev transfection are common methods that work by physical disruption or irreversible chemical addition, which may irreparably damage the molecules intended for therapy. on the other hand, transfection into the producer cells is a simple option that does not imperil ev integrity.methods: there are multiple factors that contribute to ev loading efficiency, including transfection reagent used, timing, and dosage. thus, we sought to establish a basic protocol and improve understanding of the underlying dynamics involved in a basic system consisting of hek t cells and mir- a- p mimic.results: in this work, we examined how different reagents lead to variable ev loading. then we looked at variable dosages, specifically the relationship between rna amount added to reagent, amount present in cell, and amount exported to evs. summary/conclusion: these results will help future studies produce evs with exogenously loaded small rna, and suggest future optimizations. funding: national institutes of health. r and t (host pathogen interactions at university of maryland). we report a single ev trapping method via aptamermediated assembly between au nanoparticle (aunp) and au superlattice template. we propose a chip-based ev trapping technique based on semiconductor processes. methods: we introduce aptamer coated au nanoparticle (aunp) and au superlattices as a template to capture evs. first, we fabricated poly(methyl methacrylate) (pmma) hole pattern on au-coated si substrates by using electron beam lithography (ebl). we designed nm-diameter hole patterns to capture one ev in each hole. to connect the aunp and the au superlattice template, we used an aptamer molecule as a linker strand. also, to capture individual evs, the aptamer molecule is designed to have a hairpin structure to specifically bind to cd , a protein marker of ev. we modified ʹ-terminal and ʹ-terminal of the cd aptamer with thiol group for the formation of self-assembly monolayer (sam) on both aunp and au superlattice surface. results: first, we coat the cd aptamer on the surface of aunp. afterwards, we load the aptamer-coated aunp into au superlattice template. ev solution is specifically bound to cd aptamer. after washing step, each ev is expected to locate within a single hole due to the size confinement of the hole. to separate the evs from the aptamer, we use restriction enzyme, bamhi, to recognize specific dna sequence and cleave them. summary/conclusion: in this report, we propose a aunp -linked au superlattice chip by aptamer molecules for trapping evs. we selected cd aptamer for specifically binding with cd in evs. in addition, we designed cd aptamer as a linker strand to connect introduction: a hallmark of platelet activation is the release of internal granules as extracellular vesicles/ microparticles. thrombolux is a dynamic-light-scattering-based (dls) instrument that was developed for use in clinical setting to check for platelet activation before transfusion. compared to traditional dls, the thrombolux requires no cleaning (single-use capillary) and requires very little sample ( µl). hence the thrombolux may be a useful instrument beyond platelet pack test in blood transfusion laboratory. we have evaluated its use as an in-process monitoring tool for industrial ev manufacturing, for both quantifying cells (input) and evs (output). methods: the thrombolux was used to test the activation status of expired platelet packs (donated by arcbs for research purpose). the readout was compared with platelet swirling test and flow cytometry data (surface marker). furthermore, the thrombolux was also tested for process development and ev manufacturing monitoring purposes at different stages of the process for its ability to rapidly obtain particle presence and size information on evs. time to result was also compared between different particle analysis methods. results: the thrombolux was a better predictor of platelet packs variability compared to the traditional platelet swirling method. however, we did not observe a strong correlation between the activation status and the flow cytometry-based activation marker data. the thrombolux was able to provide a useful estimation of particle presence and sizing of evs in-process.results are obtained rapidly, within minutes, with minimal sample prep. summary/conclusion: although we did not observe a significant direct correlation between flow cytometry activation data and the % microparticles (within a small sample size), the thrombolux has shown potential to become a useful tool for in-process monitoring for ev manufacturing and other ev research, in particular through its speed and ease of use. funding: all funding was through exopharm ltd (asx:ex ). secreted introduction: a major manufacturing challenge related to exosome bioprocessing is that of robust and scalable purification. as efforts to translate exosomes into clinics grows, the more important the design of quality systems which can reproducibly purify the product becomes. the current gold-standard, ultracentrifugation, was adopted from the viral vaccine industry, but remains imperfect in terms of scale up and manufacturing due to labour and time intensive process requirements. in order to follow the preferential adoption of more standard bioprocesses, as previously achieved by the viral vaccine industry, we show the development of two monolith chromatography steps which can be used to purify exosomes from a clinically relevant, allogeneic stem cell product (ctx e ). methods: t-flask expansion of ctx e cells was performed to yield batches of - l of conditioned medium. the medium was subsequently clarified by benchtop centrifugation, and concentrated into a crude concentrate by tangential flow filtration [tff], using a combination of . µm dead-end filtration prior to concentration in a kda hollow-fibre tff system. tff retentate was loaded onto ml hic or aex monoliths, for further purification. potency was assessed by a fibroblast wound healing assay in vitro. results: exosome presence was verified in the tff material by detection of cd and cd . exosomes recovered in this manner could achieve full wound closure in vitro over hours, when dosed at µg. further purification by monolith chromatography showed high levels of reduction of albumin, detected by western blot, as well as heightened ratios of particles to both total protein, and total dna. the results indicate that neither aex nor hic steps cause detrimental loss to product function, either alone or in combination with one another. introduction: custom-made platelet pellet lysate (ppl) and heat-treated ppl (hppl) exert strong neuroprotective effects of neurotoxin-exposed dopaminergic luhmes neuronal cell culture. this effect is significantly enhanced using hppl, which was also highly protective of th-expressing neurons in mice parkinson's disease (pd) model. introduction: there is a critical unmet medical need for new therapies to treat age-related diseases including cardiovascular diseases such as stroke. exosome derived from stem cells have shown intrinsic therapeutic potential in a variety of animal models of ischaemic diseases. we have identified scalable exosome production cell lines (purestem) as a source of angiogenic exosomes and are aiming to generate good manufacturing practice (gmp) grade therapeutic exosomes that can effectively mediate angiogenesis and tissue regeneration. we are developing exosome production and purification protocols that combine methods of tangential filtration flow (tff) and size exclusion chromatography (sec). the particle number and size were measured by both tunable resistive pulse sensing (trps) as well as nanoparticle tracking analysis (nta) for comparison. exosomes were characterized by detection of exosome surface markers and absence of cellular markers. purity was assessed by measuring particles per ug of total protein content. the angiogenic activity of purestem-exosomes was assessed using live-cell imaging to measure endothelial wound-healing and tube formation assays. we further investigated the molecular cargo of purestem-exosomes by screening mirnas targets, rna-seq analysis, and mass spectrometry analysis.results: the isolated purestem-exosomes using our developed protocols were highly purified, resulting purity in the range of e - e particles/ug. we selected angiogenic exosome-producing cell lines from our purestem library by screening for functional activity and characterizing their molecular cargo. we found that purestem progenitor-derived exosomes showed higher angiogenic potency than primary mesenchymal stem cell (msc)-derived exosomes. furthermore, angiogenic micrornas such as mir- were enriched in purestem-exosomes from certain producer cell lines. summary/conclusion: these data demonstrate the potential for using purestem lines as a highly scalable source of therapeutic exosomes. we were able to obtain highly pure exosomes that retain their angiogenic activity. we anticipate that purestem-exosomes will be a valuable resource for developing ev therapies for stroke and other ischaemic diseases. we have developed purification methodologies aimed at achieving a robust and scalable exosome production compatible with gmp for clinical grade purestem-exosomes. these developments have great potential as therapeutic agents for future preclinical in animal model of stroke and clinical trials. neuronal introduction: the hallmark of parkinson's disease (pd) is a-synuclein accumulation, predominantly in dopaminergic neurons, causing neurodegeneration. pd is also associated with insulin resistance, a condition characterized by phosphorylated insulin receptor substrate- (irs- ). besides motor symptoms, some pd patients develop mild cognitive impairment (pd-mci) or dementia (pd-d). given the importance for prognosis, there is an urgent need to develop biomarkers for distinguishing pd with normal cognition (pd-n) from pd-mci/d. neuronal-origin extracellular vesicles (nevs) contain cell signalling and pathogenic proteins (including a-synuclein), which may serve as biomarkers for alzheimer's disease, pd and other dementias.methods: from . ml of plasma from pd-n, pd-mci, and pd-d patients, we immunocaptured nevs using anti-l cam antibody. then, irs- pser and irs- ptyr and a-synuclein were measured in nevs using electrochemiluminescence immunoassays.results: a-synuclein was lower in pd-mci and pd-d compared to pd-n (p < . ) and significantly decreased with increasing motor symptom severity measured by mds-updrs iii score (p = . ). irs- pser was lower in pd-d than in pd-n. irs- ptyr significantly decreased with increasing mds-updrs iii score (p < . ). no biomarker was associated with disease duration. summary/conclusion: pd patients with cognitive impairment exhibited lower nev levels of a-synuclein than cognitively intact pd patients, whereas a-synuclein and irs- ptyr were inversely associated with pd motor symptom severity. additional biomarkers and measurements will be available by the time of isev. plasma nevs is a valuable tool for discovering biomarkers in pd and investigating aspects of disease progression. introduction: despite decades-long advancement in transplant medicine, there is a necessity for personalized approach regarding early kidney allograft injury recognition and immunosuppression therapy towards improved transplant outcomes. biopsy, a gold standard for assessment of kidney allograft injury, cannot be serially used for the diagnosis of subclinical injury due to it's invasiveness and possible sampling errors. instead, urine is easily obtainable and bearing extracellular vesicles (evs), potential carriers of pathological signals related to kidney injury. our aim was to set up a urinary ev (uev) isolation protocol that would allow consistent and reliable identification of their characteristics and cargo. methods: second morning urine sample ( ml) was collected from patients and processed within hours. oxalate precipitation, ph and dilution variability, uromodulin polymerization and high protein content were taken into account. isolated evs were defined by transmission electron microscopy (tem) and nanoparticle tracking analysis (nta). uev specific proteins and mirnas were analysed by western blot and qpcr, respectively. results: the optimal protocol relied on low speed urine centrifugation ( . x g, rt) for cell removal and storage at − °c prior to further analyses. after urine thawing at rt, added edta averted cryoprecipitate and uromodulin polymer formation, while concentrated pbs neutralized the ph. filtration through . µm pores was used for large particle removal, while centrifugal kda membrane units (amicon®, milipore) served for sample concentration followed by particle separation on sizeexclusion chromatography (sec; qevoriginal, izon q). protein vacant sec fractions (as rated at a ) were pooled and concentrated to a volume of µl. tem micrographs revealed high sample purity and cup-shaped morphology of uevs. as per nta results, the average mean size of evs was , nm with concentration range of × particles/ml of starting urine. uevs were positive for the tested marker proteins hsc , flotillin, tubulin, gadph and cd . qpcr verified mirna presence in uevs, with ct for mir let- i at . summary/conclusion: we successfully isolated pure uevs. the set up protocol will be used to assess uevs as non-invasive biomarkers of allograft injury in kidney transplant recipients. astrocyte-derived extracellular vesicles regulate dendritic spine formation and neuronal network connectivity introduction: recent advancements in the biology of extracellular vesicles have begun to implicate glial released microvesicles as mediators of glia to neuron communication, suggesting that alterations in the release and/or composition of astrocyte microvesicles could impact neuronal function. methods: astrocytes were allowed to constitutively release extracellular vesicles (adev-cr), or stimulated with atp (adev-atp). adevs were isolated by ultracentrifugation followed by proteomic analysis. we developed a normative whole transcriptome database using primary neurons exposed to adev-cr, and identified changes in neuronal gene expression produced by exposure of neurons to adev-atp. we identified a number of pathways associated with the biological response of synapse, spine and neurite outgrowth that were regulated by adev-atp. the molecular cargo of adev-atp responsible for regulating synaptic functions in neurons were characterized by biochemical, molecular, and functional assays. results: adev-atp enhanced the maturation of dendritic spines and produced functional enhancements in neuronal activity and network connectivity. the mechanism for this effect involved the delivery of integrin- and epha that were enriched in adev-atp. integrin- facilitated binding of adevs to the neuronal surface, and epha -receptor signalled through ephrin to the tyrosine kinase erbb / that regulated the phosphorylation and activation of trkb without increasing expression of the natural ligands bdnf or ntf . this direct activation of trkb increased the expression of the synaptic scaffolding proteins disc , arc, and cplx to promote the maturation of dendritic spines. this increase in mature dendritic spines was associated with increased neuronal activity and network connectivity demonstrating a functional strengthening of synapses. summary/conclusion: these data identify a molecular mechanism whereby modifications in adev protein cargo produced by the stimulation of astrocytes with atp regulates synaptic maturation through activation of trkb in a manner independent of growth factors. stephanie kronstadt and steven m. jay university of maryland, college park, college park, usa introduction: mesenchymal stem cell extracellular vesicles (msc-evs) have been shown to have an immunosuppressive effect in both autoimmune and inflammatory disorders. despite this, clinical translation of ev therapies is hindered by potentially low potency in vivo and the lack of a scalable biomanufacturing process. cell culture parameters are critical in modulating both yield and bioactivity of evs. thus, we hypothesized that the combination of chemical priming and d dynamic culture would enhance the yield and potency of immunosuppressive msc-evs. methods: bone marrow-derived mscs cultured in flasks were chemically primed using ethanol or curcumin. mscs were also cultured using a d-printed scaffold-perfusion bioreactor using a flow rate of ml/min. anti-inflammatory effects were assessed following application of msc-evs to lipopolysaccharide (lps)-stimulated murine macrophages. subsequent inhibition of the production of the pro-inflammatory cytokine il- , quantified using an elisa, was used to characterize evs as anti-inflammatory. in addition, both chemical priming and the bioreactor will be simultaneously utilized to potentially uncover any synergistic effects on ev immunomodulation abilities. nanoparticle tracking analysis (nta) was used to assess ev size and concentration while protein mass was measured via a bca assay. results: preliminary data suggests that priming mscs with µm ethanol for hours prior to ev collection results in a strong inhibition of il- production in stimulated murine macrophages. nta revealed that msc-ev yield increased by about two orders of magnitude in the bioreactor ( . e ± . e ) when compared with flasks ( . e ± . e ). protein measurements also indicated that ev production in the bioreactor (~ µg) was much greater compared with production in the flasks (~ µg). additionally, average protein content per ev was reduced in the bioreactor when compared with flask evs. regardless of tissue source. furthermore, comparison of adipose tissue-derived (ad) msc evs from three donors indicates varying pro-vascularization bioactivity between those donors evaluated in vitro via gap closure assay. similar results were observed for the bone marrow-derived (bm) msc ev donor groups. summary/conclusion: this work highlights the need for screening of donor derived-mscs before use for therapeutic ev production. additionally, standardized criteria for msc donor selection are needed before isolated msc evs can be used as a large-scale, repeatable therapeutic treatment. analysis of extracellular vesicle populations from malaria-infected erythrocytes by field-flow fractionation reveal distinct sub-sets alicia rojas a , paula abou-karam a , anna rivkin a , yael fridmann-sirkis b , yifat ofir-birin c and neta regev-rudzi c a department of biochemical sciences, weizmann institute of sciences, rehovot, israel, rehovot, israel; b wis, rehovot, israel; c weizmann institute of science, rehovot, israel introduction: malaria is one the most devastating infectious disease in the world and plasmodium falciparum (pf) represents the deadliest species. this parasite invades human red blood cells (rbcs) and releases extracellular vesicles (evs) carrying dna, rna and protein cargo components which are involved in the pathogenesis of the disease. recently, it has been shown in mammalian systems that evs are subdivided into different subpopulations, each with a distinct biological function. however, it is still unknown whether pfinfected rbcs (pf-evs) release different ev subpopulations with distinct cargo. methods: we isolated evs from pf-infected and uninfected rbcs, pf-evs or ui-evs, respectively, using differential centrifugation. the ev pellet was subjected to field flow fractionation (fff). the different subpopulations were collected, concentrated with size-exclusion filters and evaluated by nanoparticle tracking analysis. additionally, the presence of ev markers (sr and hsp ) were examined by western blot analysis. results: the fff analysis showed four particle subpopulations derived from the pf-evs and five in the ui-evs. the first three subpopulations were similar in their detection signals in both samples, but the fourth subpopulation was consistently higher in ui-evs than in pf-evs. moreover, hsp was detected in subpopulations and of both pf-evs and ui-evs, whereas sr only in subpopulation . isev abstract book summary/conclusion: pf-ev and ui-ev have similar separation profiles and proteins markers in their subpopulations, consistent with the fact that both samples are derived from host rbcs. additional data regarding the dna and rna cargo, as well as microscopic observations of the pf-ev and ui-ev subpopulations is necessary. this will clarify how malaria parasites sort their components into evs and which fractions are associated to immune evasion and pathogenesis. we have established a small size laboratory production of the microalgae culture in order to harvest the extracellular vesicles (evs) for pharmaceutical and medical uses. in this work we report on globular particles in the isolates from media of microalgae of two types, that we recognize as evs. we observed changes in their production at different temperatures and conditions. methods: samples were fixed by various combinations of aldehyde fixatives and/or osmium tetroxide. they were dehydrated in a graded series of ethanol, hexamethyldisilazane, and air dried. they were au/pd coated for inspection with scanning electron microscopes (sem) crossbeam fib-sem gemini ii (zeiss, germany) and jsm- f field emission scanning electron microscope (jeol ltd., tokyo, japan). results: microalgae were incubated overnight at °c and °c in growth medium and in growth medium supplemented with detergent. the samples obtained from the microalgae culture contained particles that we recognized as extracellular vesicles, however, these particles do not correspond to characteristic shapes of membrane enclosed entities without internal structure. increased temperature and/or presence of surfactant (triton x- and sodium dodecyl sulphate) stimulated formation of evs of different shapes and sizes. the isolates of these samples were rich with evs. in the presence of surfactant, the cell-walls detached from the cell and collapsed upon dehydration. this was documented by sem. summary/conclusion: focused ion beam technique revealed complex internal structure of the algae. it seems from the shapes of the observed structures that the particles deposited on the surface of the microalgae do not derive from budding of the membrane surface, but are instead shed by the cells from the cell interior upon the rupture of the cell wall. key: cord- -uftc inx authors: nan title: abstract of th regional congress of the isbt date: - - journal: vox sang doi: . /vox. sha: doc_id: cord_uid: uftc inx nan in the fin de siecle was heavily concentrated in vienna. freud, boltzmann, schr€ odinger and mach might be the first names to find, whenever one cites austrian scientists. but more related to transfusion are the noble prize winners max perutz and karl landsteiner. landsteiner s fate illustrates the brain drain beginning in the early s escalating in with the "anschluss", which lead to the forced emigration of many scientists. a loss which was not regenerated in the post war years and was further aggravated by dubious and often undisclosed relations and scandals in the nazi-era. all together this leads to a severe loss of credibility and productivity of universities across decades. opening university access in the early s and intensive historical work-up of scandals transformed the austrian universities to open and effective scientific institutions driving innovation in the country. austria has achieved a great economic deal in recent decades, which was accelerated by the eu membership in . as a result of strong long-term economic performance, the country's gross domestic product (gdp) per capita is the eighth highest among oecd countries and fourth in the eu . levels of poverty and income inequality are both below the oecd average. investment in research and development (r&d) increased since the eu accession, when austria's r&d intensity (aggregate r&d expenditure as a percentage of gdp) was well below the oecd average and significantly far lower than switzerland -a country to which austria prefers comparison. the eu target of % r&d intensity was first met in and is the sixth highest among oecd countries and the second highest in the eu . austria showed the second highest increase in r&d intensity of all oecd countries, exceeded only by korea. the rapid expansion was matched by a similar increase in human resources and scientific output of universities. austrian science in quantum mechanics, quantum communication and information is world renown. vienna is a major biotech hub, as is linz in mathematics and mechatronics and graz in automotive and production technologies. austria has been a net resource recipient in the horizon and the preceding th framework programme. small and mediumsized enterprises show a high propensity to co-operate with universities and other research organisations and more and more included in scientific grant schemes. vienna is the largest student city in the german-speaking world and consistently ranks among the top cities in the world on quality-of-life indices. as austria possesses globally recognised cultural attractions ranging from famed salzburg festival to the vienna new year concert its inhabitants are not aware of the progress made in r&d and how thriving innovation is going on in their country. they still love to show their cultural heritage and events and impress the world with some kind of eternal sound of music. patients with refractory b-cell malignancies as non-hodgkin lymphomas (nhl) resistant to standard therapies have a dismal prognosis. the outcome is even poorer in patients relapsing after autologous stem cell transplantation. most of these patients do not qualify for an allogeneic hematopoietic cell transplantation (hct) due to refractory disease, lack of a suitable allogeneic donor, higher age or cumulative toxicity of previous chemotherapy. despite patients undergoing allogeneic hct normally profit from a graft-versus -lymphoma effect, overall survival in patients with nhl after hct remains short. a similar situation can be observed for patients with acute lymphoblastic leukemia (all). therefore novel treatment modalities are urgently needed. chimeric antigen receptor (car)-t cells, a new class of cellular immunotherapy involving ex vivo genetic modification of t cells to incorporate an engineered car have been used in clinical trials. in the majority of studies b-cell malignancies treated with cd targeting car-t cells have been analyzed. austria had the advantage to participate in two international trials in the past and is currently involved in further car-t studies. recently, results from cd directed car-t cell trials with an increased follow-up of patients led to fda (food and drug administration) and ema (european medicines agency) approval of tisagenlecleucel and axicabtagene ciloleucel. common adverse events (aes) include cytokine release syndrome and neurological toxicity, which may require admission to an intensive care unit, b cell aplasia and hemophagocytic lymphohistiocytosis. these aes are manageable when treated by an appropriately trained team following established algorithm. in this presentation, results of four large phase ii cd car-t cell trials for patients with nhl and all and focus on aes is summarized. preoperative anemia is a known risk factor for increased perioperative morbidity and mortality in patients undergoing major surgery. previous studies have not only shown higher in-hospital mortality, but also an increased hospital length of stay, greater postoperative admission rates to intensive care and prolonged use of mechanical ventilation and intensive care resources in patients with anemia compared to those with normal preoperative hemoglobin concentrations. about % of patients scheduled for major surgery suffer from preoperative anemia. this figure is even higher in patients requiring orthotopic liver transplantation, where up to % of all patients are diagnosed with anemia prior to surgery. transfusion of packed red blood cells (prbcs) is commonly used to correct anemic hemoglobin values. however, transfusion of prbcs has been associated with increased morbidity and mortality in patients undergoing cardiac, orthopedic, and abdominal surgery. additionally, transfusion of prbcs is associated with a greater incidence of postoperative acute kidney injury in patients undergoing orthotopic liver transplantation. as preoperative anemia might increase the perioperative use of prbcs, negative effects observed after prbc transfusions might even be augmented. data on the influence of preoperative anemia on morbidity and mortality after orthotopic liver transplantation are limited. thus, we retrospectively analyzed the association of preoperative anemia and mortality in adult patients undergoing orthotopic liver transplantation at our institution. in addition, we examined the influence of anemia on perioperative parameters such as transfusion requirements, surgical complications, early allograft dysfunction, acute kidney injury, and the need for renal replacement therapy. based on the results obtained in the retrospective analysis, an ongoing prospective randomized clinical trial was initiated. the two suspensive treatments in sickle cell disease (scd) are hydroxycarbamide, inducing the production of the functional hbf, normally repressed at birth, and red blood cell (rbc) transfusion, a critical component of scd management. however, rbc transfusion is not without risk. repeat exposure to allogeneic rbcs can result in the development of rbc alloantibodies which can make it difficult to find compatible rbcs for future transfusions. however, the main concern of alloimmunization is the development of hemolytic transfusion reaction, with in the most severe cases, hyperhemolysis, leading to multi organ failure and death in % of the cases. the prevention of this life threatening condition must be based on risk factors. however, although some risk factors, such as alloimmunization, have been identified, much of the mechanism underlying dhtr remains a mystery, particularly in severe cases presenting hyperhemolysis. here we will describe the current and future development to prevent and treat this severe syndrome in order to decrease exposure to transfusion in scd but also improve red blood cell quality, some new products are developed. oxidative damage is one of the parameter that could be diminished. some work is also ongoing to prevent filter blockage during leucodepletion of precious rbcs units from afro-caribbean donors carrying the sickle cell trait. finally, in countries with higher risks of transmission of infectious disease, treatment of red blood cell units against infectious agents can be discussed. the only current curative treatment of scd is hematopoietic stem cell transplantation (hcs). however, the occurrence, frequency, and effects of immune hematologic complications in hcs remain and will be discussed. finally, gene therapy is a real hope as a definitive curative treatment. clinical trials are ongoing in france and will be discussed as well as the remaining place of transfusion in this therapeutic. in the context of the chronic myeloid leukemia (cml), we have hypothesized that quiescent leukemic hematopoietic stem cells (hsc) compartment, escaping to the current tyrosine kinase inhibitors (tkis) treatment, in part associated in the molecular relapse, may be targeted by cart-cells immunotherapy. gene expression profiling studies have established that a cell surface biomarker il- rap is expressed by the leukemic but not by the normal cd + /cd -hsc. this talk will focus of the whole process of development of a cart-cells starting from recombinant il- rap protein mice immunization to produce a specific monoclonal antibody (mab), to the proof of concept demonstration, before moving into the clinic. we produced and selected a specific anti-il- rap mab (#a c clone, diaclone sa, besanc ßon, france). after molecular characterization of antigen-binding domain, nucleotide sequences were fused with rd generation t cell activation coding sequences and cloned as a single chain into a lentiviral backbone comprising a safety switch suicide gene icasp (inducible caspase ) and a monitoring/selection cell surface marker Δcd . we demonstrated in-vitro and in an in-vivo xenograft murine model that il- rap car t cells can be activated in the presence of il- rap+ cell lines or primary cml cells, secrete pro-inflammatory cytokines, degranulate and specifically killing them. we also demonstrated that multi-tkis treatment over a -year period does not affect transduction efficiency of cml patient t-cells by il- rap car vector and that autologous cart-cells are able to target il- rap+ leukemic primary hsc. "off-tumor-on target" toxicity prediction, by studying il- rap expression on a tissue macroarray comprising normal human tissues ( donors) , with #a c , detected various il- rap intensity staining in only few tissues. regarding the healthy hematopoietic system, #a c flow cytometry staining did not detect hematopoietic cells, except monocytes that express poorly il- rap. as expected, monocyte subpopulation is targeted by autologous il- rap cart cells (ratio e: t = : ), but at a lower level that il- rap cml cell line. in-vivo investigation of specific toxicities of autologous il- rap cart-cells against hsc and/or immune cells on a human-cd + cord blood cell engrafted/nog murine model, but also by an in-vitro cd + colony forming unit assay didn't reveal any significant toxicities in immunocompetent cell subpopulations, suggesting that healthy cd + hsc are not affected. finally, to overcome potential toxicity, functionality of the icasp / rimiducid â safety switch was demonstrated in-vitro but also in-vivo in a nsg tumor xenograft model, showing that, when activate, the system is able to eliminate more than % of cart-cells, after exposure to ap . in conclusion, based on cml model, we demonstrated that il- rap is an interesting target for cart-cell immunotherapy, with a limited "on target, off tumor" predictable toxicity. next step will be the up-scaling of the process in order to match with the use in human regarding also the regulatory requirements. this strategy may be applied, in the future, in other hematological malignancies. mortality ranges from to % for trauma victims with severe bleeding and is largely dependent on the transfusion therapy from which they can benefit. the nature of this therapy has an impact on prognosis with a halving of mortality when the plasma/prbc ratio is greater than ½ and a decrease of about % when the proportion of platelets transfused is close to that of whole blood. the speed with which such therapy is actually administered has a major impact as well with an increase in mortality of % for each minute of delay in making the entire therapy available. this can be explained mainly by the fact that the probability of death of these patients is greater within minutes of their admission to hospital with a median time to death of h after admission. to allow plasma, platelets and prbc to be made available in a timely manner, north american trauma centers have mandated that trauma centers have massive transfusion packs at the patient's bedside within min. to further simplify and speed up logistics from distribution to transfusion, several trauma centers now use whole blood stored at °c. this return of an "old" product is largely inspired by military experience where whole blood is mainly used "warm" immediately after collection with compelling evidence of its effectiveness. its return to civilian practice requires the ability to deleukocyte it while preserving platelets and to store it while maintaining their hemostatic functions. good quality data shows this is achievable and several clinical studies are planned to begin in the coming months. in france, the french blood establishment and the french army are cooperating to initiate the prospective randomized non-inferiority storhm trial (sang total pour la r eanimation des h emorragies massives) which will be comparing whole blood to separate blood components in an / / ratio in severely bleeding trauma patients. the primary endpoint will be a thromboelastographic parameter (maximum amplitude) assessed at the th hour after admission. secondary endpoints will include early and overall mortality, lactate clearance (reflection of the effectiveness of resuscitation) and h organ failure. this trial will be recruiting patients in french trauma centers and is planned to be initiated second half of . local/neighbours day: innovation in germany c- - mesenchymal stromal cells for regenerative therapy bm-msc / asc obtained from these protocols have been characterized in detail in preclinical evaluations. manufacturing licenses for msc and asc and a platelet-derived growth factor concentrate have been obtained and they have been explored in several clinical trials for treatment of bone defects (ortho-ct : eudract number: - - ; ortho-ct : eudract number: - - ; maxillo : eudract number: - - ) . we will summarize results of completed clinical trials which confirmed feasibility and safety of autologous msc /asc treatment and provided evidence for efficacy (gjerde et al, stem cell res. introduction: in vitro produced megakaryocytes (mks) may serve as source to produce platelets (plts) ex vivo or in vivo. we have established a strategy to differentiate mks from induced pluripotent stem cells (ipscs) in bioreactors. this study aimed at the large-scale production of mks using microcarriers to increase the mk yield and to characterize their phenotype and functionality after irradiation as a method to decrease possible safety concerns associated to the ipsc origin. methods: ipscs were cultured in an aggregate form in presence or absence of microcarriers using ml stirred flasks. cells were differentiated into mks using tpo, scf and il- in apel medium for a period of days. non-irradiated or irradiated ipsc-derived mks were analysed for polyploidy, phenotype and proplt production using flow cytometry and fluorescence microscopy. also, plt-production was investigated in vivo. non-irradiated or irradiated mks were transfused to nod/ scid/il- rcc-/-mice and blood was analyzed for human plts. results: differentiation of mks in presence of microcarriers resulted in an -fold increase of mks per ipsc in comparison to only aggregates. this resulted in mean of total mk harvest of . ae . in microcarrier-assisted bioreactors in comparison to . ae . mks collected from bioreactors containing only aggregates. interestingly, mks produced in microcarrier-assisted bioreactors showed higher proplt formation capacity than mks derived from only aggregates bioreactors. mk phenotype and dna content was comparable between mks derived from both types of bioreactors. irradiation of mks did not affect their phenotype and capability to form proplts or plts after transfusion into nod/scid/il- rcc -/mice. conclusion: microcarriers showed to significantly increase the yield of ipsc-derived mks in stirred bioreactors to clinically relevant numbers. this may facilitate the use of ipsc-derived mk for ex vivo production of plts, direct transfusion or for innovative mk-based regenerative therapies. although the rosetta stone, found by the troops of napoleon in egypt near the city of rosetta (rashid) contains only a small amount of text in three languages it was key in deciphering hieroglyphs. the rosetta mission tried to achieve something similar: by looking at a tiny body its goal was to decipher the origin of the solar system, planets including earth and life. after more than years the rosetta spacecraft softly crashlanded on comet churyumov-gerasimenko on september , . it has travelled billions of kilometers, just to study a small ( km diameter), black boulder named p/ churyumov-gerasimenko. the results of this mission now seem to fully justify the time and money spent in the last decades on this endeavor. where are we from? where are we going? are we alone in the universe? these are some of the big questions. in this talk i will show which answers we got from rosetta and comet chury. we follow the pathway of the material which makes up our solar system from a dark cloud to the solar nebula and finally to planets and life. i will show that indeed we are the result of stardust and that what happened here may happen elsewhere in the universe. cells, tissues and entire organs can collectively be seen as "living drugs". genetically unaltered cells are routinely used in clinical practice to treat diseases as diverse as anemia, bleeding disorders, leukemia and organ failure. ground-breaking advances in genetic and genome engineering technologies are propelling cell therapies to the frontline of medical research and practice. the hematopoietic system is particularly amenable to genetic engineering because specific cell types can be purified based on the expression of specific surface proteins and the ability to culture and expand cells ex vivo. the recent unprecedented clinical success of killer t cells reprogrammed by chimeric antigen receptors (cars) to attack cd expressing tumor cells demonstrates the power of immunotherapy with genetically engineered immune cells. however, given the rapid development of novel genome engineering and synthetic biology tools we are likely only at the beginning of a new era of engineered cellular therapies. i will present recent progress in immune cell reprogramming, gene correction, safety aspects and remaining challenges such as manufacturing. d- - cell free nucleic acids (cfna) circulate in the plasma of all individuals and are thought to be released by host and foreign cells into the circulation. after fractionation by centrifugation, cfnas can be extracted from the supernatant of whole blood samples or manufactured blood products. these dna or rna sequences can be of human, bacterial, viral or fungal origin. most of them are human double stranded dnas. research on cfnas is increasing, thanks to technological advancements in molecular biology. some of their results are already implemented in clinical practice in the areas of prenatal diagnosis, oncology and infectious diseases. the latter investigation focuses on the exploration of non-human cfnas, the field of metagenomics. high throughput sequencing associated with bioinformatics, the so-called new generation sequencing (ngs), has sped up the investigations of non-human cfnas. this tool provides the opportunity to classify cfnas into a human or non-human category, and then to identify them. it is thus possible to explore simultaneously the whole landscape of bacterial, viral and fungal populations. presently, ngs of human blood has already proven its feasibility and its value in identifying emerging viruses or investigating clinical cases of fever of unknown etiology. ngs of cfnas is also particularly effective in analyzing the different genotypes of a virus in case of a co-infection (e.g. hepatitis c virus). studying cfnas with the new molecular technologies is therefore of great importance in transfusion medicine, especially regarding security and clinical transfusion reactions. first, transfusion transmitted infections are the most feared adverse complications. second, febrile non-hemolytic transfusion reactions are also the most frequently reported adverse events in hemovigilance systems and their physiological mechanism -if only one-remains not clearly elucidated. investigating cfnas could thus improve our understanding and strategy aiming at reducing those two clinical adverse events. surveying comprehensively the composition of circulating infectious agents in a blood product by ngs technology could be very interesting for investigating a severe febrile transfusion reaction. moreover, when the costs of analysis will be reduced, it might be possible to screen prospectively and regularly the whole metagenomics of asymptomatic blood donors, in addition to the classical epidemiological surveillance. for instance, in a study testing a ngs method on manufactured fresh frozen plasmas, an astrovirus (mlb ) has been identified. finally, it is the responsibility of transfusion physicians implicated in the manufacturing of blood products to ensure that cfnas within a blood product do not have a clinical impact on the innate immunity of the recipients. according to recent research in vitro, cfnas purified from blood products can induce the transcription of inflammatory cytokines by mononuclear cells. as nonhuman cfna have an effect on toll-like receptors (tlr-linked inflammatory pathways), it would be also relevant to insure that donor's cfnas have no significant effect on the immune system of the recipient. in conclusion, cfnas are very diverse molecules contaminating blood products. technological progress makes now their investigation more available. besides being useful markers of infection in asymptomatic donors, their impact on the recipients' immunity should be further investigated. an active life and regular training is part of a healthy life style and for many this includes participation in endurance exercise competition at different levels. thus, it is highly relevant to know how a blood donation affects exercise performance and how close this can done to an endurance competition. endurance exercise performance is determined by many factors, but three of the primary are maximal oxygen uptake, the relative load that an individual can sustain over time and finally the efficiency of movement in the given discipline. over the years, a number of studies have sampled blood volumes ranging from - ml and applied different methodological approaches to measure maximal oxygen uptake over a recovery period ranging between - days. overall, the general finding is a reduction in blood haemoglobin and an attenuated maximal oxygen uptake as well endurance performance after blood donation. in normal to well-trained men maximal oxygen uptake and performance was normalized after two weeks in one study after a normal blood donation ( / ml), but remained attenuated after four weeks in another study, despite the change in blood haemoglobin concentration was similar and the design and methodology also similar in the two studies. in addition to maximal oxygen uptake the relative load that can be tolerated during exercise is probably also attenuated, through a decreased arterio-venous oxygen extraction, but the available data is very limited. the first part of this talk will highlight the major findings and discuss some of the methodological issues that complicate interpretation and conclusions. there are sex differences in circulating blood volume, haemoglobin concentration, haematocrit and hormone levels and thus it is entirely possible that there is a sex difference in the effect of blood donation on physical performance and the recovery after blood donation. in addition to the basic physiological sex differences, there is also a higher prevalence of iron deficiency in premenopausal women, physically active women and women donating blood. therefore, we studied the influence of a standard ml blood donation on maximal oxygen uptake and endurance performance and the subsequent recovery in physically active women. we observed that in iron sufficient women blood haemoglobin concentration and maximal oxygen uptake were back to baseline days after blood donation, but endurance performance was normalized already after days. the second part of this talk will discuss the sex differences in the effect of blood donation on maximal oxygen uptake and endurance performance. overall, the available data suggest that, with a careful conservative approach, - weeks are needed after a normal blood donation to be fully recovered to participate in endurance exercise competitions. more than one in ten attempts to donate blood result in a temporary deferral, due to concerns about the impact of the donation on the donor or recipient. there is well established evidence that temporary deferrals impact negatively on donors, with a large proportion of those deferred failing to return at the end of the deferral period. this presentation provides an overview of deferrals from the donor perspective, describing the likelihood of receiving a deferral for different donor subgroups. the impact of temporary deferrals on the future donation of donors, considering both short-term and longer-term donation patterns, will also be reviewed, outlining which donors are at highest risk of non-return following a deferral, and what is known about the accumulative impact of multiple deferrals on donors. several hypotheses have been proposed to account for the strong negative impact of deferrals on donor behaviour, and there is preliminary evidence of psychological factors, such as emotional reactions, predicting intention to return. research is also beginning to emerge on the effectiveness of tailored interventions to mitigate the impact of deferrals on donor behaviour. the evidence for these preventative interventions, and for strategies to reactive donors once they have lapsed post-deferral, will be reviewed. recommendations for blood centres will be made, as well as suggestions for future research to address continuing gaps in knowledge. in his influential study "the gift relationship" ( ) , richard titmuss coined the idea of voluntary, non-paid, blood donation being the gift of life for a fellow citizen. this metaphor has been powerful in mobilizing donors (busby ) . it conveys a direct relationship between blood donation and patients' vitality, as well as a difference between gains and costs. as the gift of life, blood donation is seen to symbolize pure altruism and promoting solidarity between strangers. but can we apply the metaphor as successfully into donating blood for research? we asked a group of finnish blood donors what they would think if the frc blood service invited them to give a blood sample and personal information for research. the blood donors were usually willing to contribute to research for the public benefit, because they saw great potential in science to create solutions to help patients in the future. however, based on our interview data and previous research, we suggest that the analogy between gift of life and donation for research did not work all the way. the metaphor fails to address donors' questions on new types of relationships, interests and risks related to the use of personal data for research. left unanswered these could discourage donating for research. hence, we argue that the gift of life metaphor is not applicable to donor recruitment at the research context. in this presentation we wish to look for a better metaphor for donation for research that blood services collecting research data could apply. academy day: transfusion challenges in patients with sickle cell disease a- - immunohaematological features of patients with sickle cell disease (lfa) should be considered as polymorphic antigens in the african population and these lfa are not present in most commercial panels. the situation is even more complicated when recipients lack high-frequency antigens, the most common ones being hr-, hr b -, sec-, u neg , u+ var , js(b-), (hy-), and jo(a-). finally, there is a high rh diversity among people of african descent. because they harbor variant alleles and/or partial rh antigens, they are at risk of developing alloantibodies. in this setting, screening for partial rh antigens makes sense. the figures illustrating this diversity vary with the approach used. one of them is to take into consideration rhd or rhce*ce variant alleles. in several american studies, their prevalence was estimated to be - % and - %, respectively. other teams take into consideration d, c and e partial antigens. their prevalence was estimated to be . - %, . - . %, . - . %, respectively, and the alloimmunization rates were . %, . - %, . %, respectively. as a result of these phenotype discrepancies, scd patients are more likely to be alloimmunized. an overall immunization rate of - % is commonly admitted in the general population. depending on the unit selection policy and/or the study design, the immunization rate in scd patients varies from % to %, the highest figures being established when an abo/rh -only matching policy is implemented. in a meta-analysis of publications, the overall alloimmunization rates were around %. alloimmunization is thought to be enhanced by an inflammatory state, which is often present in scd patients. they are more prone to develop a new alloantibody. using a stochastic modeling of alloimmunization, they have a % increased risk of producing additional antibodies versus % in the general population. autoantibodies have been identified as a risk factor of alloimmunization. as a result, scd patients often have complex mixtures of allo and autoantibodies. rh antibodies and those considered as irregular natural antibodies are present in a significant proportion. another characteristic of the antibodies in scd patients is their evanescence; up to % of alloantibodies become undetectable within a few years of their initial development. relatedly, about a third of dhtrs are reported to happen in patients with no previous history of immunization. in addition, a third of patients will not develop an antibody after a dhtr. identifying patients at risk of developing a dhtr is key to managing them properly. alloimmunization is a serious risk of red blood cell transfusion in patients with sickle cell disease (scd) and can result in severe (delayed) haemolytic transfusion reactions, exacerbation of clinical symptoms and life-threatening hyperhaemolysis. once alloimmunized, the presence of alloantibodies in the patients' blood further complicates pretransfusion testing and hampers the selection of compatible blood products. numerous studies have shown that scd patients have a relatively high risk of alloimmunization as compared to the 'general' population. this is not only explained by the large number of transfusions given but also by the increased exposure to foreign antigens as a result of differences in the antigen make-up of the scd recipients and the blood donor population. other factors involved in the immune response such as age at first transfusion, inflammatory state, hla typing are under investigation and are starting to unravel. because blood transfusion is still one of the main treatment modalities for scd and some patients have a life-long transfusion dependency it is important to minimize the alloimmunization rate. theoretically, complete matching for all relevant blood group antigens would prevent alloimmunization. this however, is only possible when all donors are comprehensively. matching strategies should be developed to minimize alloimmunization while balancing patients' need and donor availability and is cost effective. to develop a (preventive) matching strategy some factors need to be established; ) which antibody specificities are clinically relevant ) which antigens are most immunogenic ) what is the availability of specific antigen typings in the donor population ) how should recipients (and donors) be typed, phenotypically and/or genotypically and to what extent. the latter is especially important in scd patients since they are of african descent and the prevalence of genetic variations in this population is relatively high. rhd and rhce variants are common and can remain undetected when serological typing is used but can be discovered with high resolution molecular typing. patients with partial rh phenotypes are at risk for alloimmunization. apart from special rh phenotypes in individuals from african descent, the fy(a-b-) phenotype related to the gata-box mutation in the fyb allele and the u-or u var phenotype resulting from genetic variations in the mns alleles are also common. several studies have shown that in scd patients antibodies directed against rhd, rhe, rhc and k are most frequently found when unmatched transfusions are given. preventive matching for these antigens has proven successful in reducing alloimmunisation. extended matching for all rh antigens fy(a), jk(a) and jk(b) can further decrease the alloimmunisation rate. currently, different countries have preventive matching strategies in place for this vulnerable patient group. as genotyping is more and more available and within reach, optimal antigen typing approaches for patients and donors, combining serology and genetics are being developed. in this lecture several aspects of antigen typing approaches and preventive matching strategies that will most benefit scd patients of will be discussed. artificial intelligence has become a buzzword that will appear about anywhere in the media. we can forget that ai, or the subfield in this computer science field machine learning, has been around for over years. improvements in computing power, abundance of data, progress in computer science, and the arrival of affordable cloud solutions have now brought it to our daily lives. also in health care news about ai has become omnipresent. and some landmark papers have come out on algorithms outperforming (teams of) physicians in the diagnosis of all kinds of skin disease, eye disease from retina scans, and detect cancer in ct scans. however, little of these solutions have actually shown up in our clinical practice yet. in anaesthesia, we worked with the first algorithm to come to anaesthesia practice; hypotension during surgery is associated strongly with poor outcome like myocardial ischemia, surgical complications, renal failure and even mortality. we worked on a machine-learning trained algorithm that predicts hypotensive events using the arterial blood pressure curve up to - min before the actual event. to get fda and ce approval, however, mere mathematic validation is required. this can be achieved on retrospective datasets. in reality, we need more before we can use these algorithms to support our decision-making; after internal (retrospective) and external (prospective but passive use) validation steps, clinical (i.e. rct validation is needed. moreover, we will need to assess the economic impact too. ultimately this tool has now reached clinical practice and is starting to help us go from reactive to more proactive hemodynamic management. like this, we have started to work on machine-learning tools to predict the incidence of specific types of patients coming into a&e and predicting infections after surgery. we will discuss our approach, essentials to start with machine learning, practical learnings. we will also discuss a first project design to use machine learning in managing bleeding patients to get the best therapy advice for blood product use like plasma, fibrinogen et cetera. how can we start using this tool in unison with our existing tools to improve science and clinical practice in our respective (bio)medical fields? thrombocytopenia is a very common hematological abnormality found in newborns, especially in preterm neonates. two subgroups can be distinguished: early thrombocytopenia, occurring within the first h of life, and late thrombocytopenia, occurring after the first h of life. early thrombocytopenia is associated with intrauterine growth restriction, whereas late thrombocytopenia is caused mainly by sepsis and necrotizing enterocolitis. platelet transfusions are the hallmark of the treatment of neonatal thrombocytopenia. most of these transfusions are prophylactic, which means they are given in the absence of bleeding. however, the efficacy of these transfusions in preventing bleeding has never been proven. in addition, risks of platelet transfusion seem to be more pronounced in preterm neonates. because of lack of data, platelet transfusion guidelines differ widely between countries. in a recent randomized controlled trial (planet- /matisse study) among preterm infants with severe thrombocytopenia, we found that those randomly assigned to receive platelet transfusions at a platelet-count threshold of /l had a significantly higher rate of death or major bleeding within days after randomization than those who received platelet transfusions at a platelet-count threshold of /l. this presentation summarizes the current understanding of etiology and management of neonatal thrombocytopenia. transfusion-associated circulatory overload (taco) is a severe transfusion adverse reaction that is associated with increased mortality and morbidity. the incidence of taco in adults varies from % to %, but is probably underdiagnosed and underreported. the incidence in the pediatric population is undetermined. taco usually occurs in patients who receive a large volume of blood product over a short period of time. it is more common in patients with known risk factors such as cardiovascular disease, renal failure, and older or younger age (> years or < years). hospitalised patients and intensive care patients are also more at risk. the typical presentation of taco is respiratory distress (dyspnea, tachypnea) occurring within h of a blood transfusion. associated signs and symptoms are hypoxia, hypertension, tachycardia, positive fluid balance, high central venous pressure, and acute or worsening pulmonary edema on chest x-ray. echocardiography and measurement of brain natriuretic peptide (bnp) or its n-terminal prohormone (nt-probnp) is helpful for diagnosis. several definition criteria have been proposed for taco, but none are adapted for children, particularly critically-ill children who are more at risk. this is probably the main reason why taco is even more underdiagnosed and underreported in the pediatric population. in a recent study, we compared the incidence of taco in a pediatric intensive care unit using the international society of blood transfusion (isbt) criteria, with two different ways of defining abnormal values: ) using normal pediatric values published in the nelson textbook of pediatrics; and ) using patients as their own controls and comparing pre-and post-transfusion values with either a % or % difference threshold. we monitored for taco up to h post-transfusion. a total of patients were included. taco incidence varied from . % to %, depending on the definition used. with such wide variability, we conclude that a more operational definition of taco is needed in pediatrics, particularly for critically-ill children. differential diagnosis from other dyspnea-associated transfusion adverse reactions (e.g. transfusion-associated lung injury, anaphylaxis) is important because treatment differs, as do guidelines to the blood bank. treatment for taco is similar to that of any other cardiogenic pulmonary edema: oxygen, diuresis, ventilatory support. prevention is possible by avoiding unnecessary transfusions, transfusing only the necessary amount of blood product, avoiding rapid transfusions, and using diuretics. background: the risk and importance of transfusion-transmitted hepatitis e virus (tt-hev) infections by contaminated blood is currently a controversial discussed topic in transfusion medicine. in particular, the infectious dose is a not finally determined quantity. the different countries have chosen different approaches to deal with this pathogen. one central question is the need of individual nat screening (id) versus minipool nat screening (mp) approaches to identify all relevant viremias in blood donors. aims: comparison and evaluation of the available screening strategies in relation to the infectious dose to minimize the risk of tt-hev infections. methods: we systematically reviewed the presently known cases of tt-hev infections and available routine nat-screening assays. furthermore, blood donation screening strategies for hev ehev in effect in the european union were compared. we also describe our own experiences of hev screening utilising an id-nat-based donor screening algorithm compared to mp-nat in pools of samples. from november to january , a total of , blood donations were screened for the presence of hev rna using a mp-nat (in house, realstar hev rt-pcr kit) and an id-nat (cobas platform). results: the review of the literature revealed a significant variation regarding the infectious dose causing hepatitis e. in the systematic case review, all components with a viral load (vl) greater than . e+ iu caused infection (definitive infectious dose (difd) . the lowest infectious dose resulting in tt-hev infection observed in general was . e+ iu (minimal infectious dose (mifd). the infectious dose of the different blood products is mainly influenced by the remaining plasma content. our data comparing the two different hev screening algorithms revealed eight hev rna positive donations using a mp-nat (incidence : , ) , whereas hev rna positive donations were identified by id-nat (incidence : ); all id-nat only positive donations had vl < iu/ml. summary/conclusions: taken into account the current knowledge on the required mifd, the difd, and the analytical sensitivities of the screening methods, we extrapolated the detection probability of hev-rna positive blood donors using different test strategies (nat assay, id vs. minipool with different pool sizes). we also considered the amount of plasma in the different blood products and calculated the infectious doses needed to be detected. only id testing would be sufficient to detect the minimum vl in the donor to avoid tt-hev infections based on the currently known mifd, but a highly sensitive mp-nat should be adequate as a routine screening assay to identify high viremic donors and avoid tt-hev infections based on the difd. we have also determined that the incidence of hev infection was approximately % higher if id-nat was used. however, vl were below iu/ml and will most likely not result in tt-hev infection taken into account the currently known mifd or difd. the clinical relevance of and need of identification of these low level hev positive donors still require further investigation. in the last years several pathogen inactivation (pi) technologies have been developed to be applied to blood components. technologies for inactivating pathogens in plasma and platelets are available in the european union, and some others are currently under development. the first pi technology introduced in the market was for plasma, and was based on the addition of methylene blue and the illumination with light (theraflex mb-plasma, macopharma and gr ıfols). for platelets and plasma two technologies are licensed, one is based on the addition of amotosalen and the illumination with ultraviolet light (uv) (intercept â , cerus) and the other one combines the addition of riboflavin (vitamin b ) and the illumination with uv light (mirasol â , terumo bct). currently another technology for platelet inactivation, based on the illumination with uvc light and strong agitation is under development (theraflex, macopharma). for red blood cells one technology based on the addition of one molecule (amustaline, cerus) is being developed. the mechanism of action, and the spectrum and level of inactivation of pathogens varies among the different technologies. in addition, the number of studies with clinically relevant endpoints and the number of patients included in the studies is not homogeneous. there is published evidence for most of them that show that the treated blood components are safe and efficacious for the patients although, for treated platelet concentrates some decrease in the posttransfusion recovery and survival of the transfused platelets occur, with differences between the different technologies. however, cumulative experience on the use in routine, for some of the technologies for almost years, support the concept of the safety and efficacy of the blood components treated with pathogen inactivation technologies without a significant increase in utilization. the use of pathogen inactivation for blood components is not widespread. differences in epidemiology between countries, infectious risk perception, concerns about potential adverse effects associated with its use and economical considerations might explain the differences observed in its implementation. the history of the p and p k antigens is complicated and sometimes confusing because of several changes to the nomenclature. the association between the antigens and their genetic home has raised many questions as well as the longstanding enigma regarding the molecular mechanism underlying the common p and p phenotypes. the system (isbt no. ) currently includes three different antigens, p , p k and nor. the p antigen was discovered already in by landsteiner and levine while p k and nor were described in and , respectively. as for the abo system, naturally-occurring antibodies of igm and/or igg classes can be formed against the missing p /p k carbohydrate structures. anti-p is usually a weak and cold-reactive antibody very rarely implicated in hemolytic transfusion reaction (htr) or hemolytic disease of the fetus and newborn (hdfn). however, some antibodies against p have been reported to react at °c, bind complement and cause both immediate and delayed htrs. the p k antibodies can cause htr and anti-nor is regarded as a polyagglutinin with unknown clinical significance. a higher frequency of miscarriage is seen in women with the rare phenotypes p and p k /p k . the rbc of the fetus as well as of the newborn express low amounts of p , p and p k antigens but the placenta shows high expression and is consequently a possible target of the antibodies and the cause of the miscarriages. the p k and p antigens have wide tissue distributions and can act as host receptors for various pathogens and toxins. furthermore, altered expression of p k antigen has been described in several cancer forms. a longstanding question has been why individuals with p phenotype not only lack p k and p expression but also p . recently it was clarified that the same a galtencoded galactosyltransferase synthesizes both the p , p k and nor antigens and in addition the p and p phenotypes was confirmed to be caused by transcriptional regulation. transcription factors bind selectively to the p allele in the '-regulatory region of a galt, which enhance transcription of the gene. it has been debated whether the p k and p antigens exist on glycoproteins in the human rbc membrane or if glycolipids are the only membrane components carrying these epitopes. a recent publication shows that the p antigen can be detected on human rbc glycoproteins and thus glycosphingolipids can no longer be considered as the sole carriers of the antigens. the blood group system which started out with one antigen, p , has now gained two more members namely p k and nor. step by step the biochemical and genetic basis underlying the antigens expressed in this system has been revealed but still many questions remain to be solved. neither gata nor klf represent a blood group system but mutations in the genes encoding these transcription factors (tfs) have been shown to result in simultaneous altered expression of blood group antigens in certain rare blood group phenotypes. in particular, mutations in the klf gene are responsible for the dominantly inherited in(lu) phenotype, commonly referred to as lu(a-b-) because of the gross reduction in lutheran antigens expression. red cells from in(lu) individuals, though, have also weakened expression of other blood group antigens, like the high-incidence antigen anwj, the antigens of the indian blood group system (cd ) and p , among others. since the first description of klf variants associated with the in(lu) phenotype, many other variants of this gene have been reported with an impact on blood group antigen expression and they are listed on the klf table of blood group alleles. other than klf , a mutated gata gene has also been found associated with the x-linked form of the lutheran-mod phenotype and has likewise been registered in the gata allele table. besides the effect of tf variants on blood group antigen expression, there are transcription factor-binding site polymorphisms in regulatory regions of blood group genes, which also have an impact on the expression of the encoded antigens in red cells. the first example of such type of polymorphisms was described in , when the disruption of a gata motif in the ackr gene promoter was found to abolish erythroid gene expression in fy(a-b-) individuals of african descent. the impact of mutations affecting gata binding sites has also been described in some abo subgroups, like the am and bm phenotypes. a regulatory element with gata binding sites in the first intron of the abo gene has been found to be altered in individuals with these phenotypes, either by deletion or by a point mutation disrupting the gata motif. recent findings have also revealed that xga expression on red cells is dependent on gata binding to a control element located . kb upstream of the xg gene. a single nucleotide polymorphism (snp) within this region was shown to correlate very well with the expected distribution of the xga negative phenotype in different populations. further work has demonstrated that this g>c snp disrupts a gata binding site and consequently abolishes erythrocyte xg expression. overall, these investigations have allowed to elucidate the underlying genetic basis for xga expression and have made xga genotyping possible. similar to xga, the p antigen has been known for a long time to be determined by the a galt gene but the molecular basis underlying the common p /p phenotypes has remained elusive till recently. several cis-regulatory snps had been identified in non-coding sequences around exon a, which showed a very good correlation with p antigen expression. interestingly, potential binding sites for several hematopoietic tfs were identified in the same region. finally, recent investigations have demonstrated the role of the runx tf in the expression of p antigen, by selective binding to a regulatory site present in p but not in p alleles. to summarize, variation in blood group antigen expression may result from mutations or polymorphisms in the regulatory region of blood group genes. recent reports have unravelled the molecular mechanisms underlying the expression of p and xga blood group antigens, which involves tf binding to allele-specific regulatory elements. similar mechanisms may also regulate antigen expression in other blood group systems. c- - clinical immunology, copenhagen university hospital, copenhagen, denmark since the discovery of cell-free fetal dna (cffdna) in pregnant women's blood, the development of noninvasive prenatal testing (nipt) has provided new diagnostic applications in prenatal care. in transfusion medicine and clinical immunology, cffdna is extracted from maternal plasma to predict fetal blood groups with the purpose of ) guiding targeted rh prophylaxis in non-immunized rhd negative women and ) assessing the risk of hemolytic disease of the fetus and newborn (hdfn) in immunized women. i will give an overview of noninvasive prenatal testing of fetal blood groups. based on the literature, i will summarize the current experience with noninvasive prenatal testing of fetal rhd and other blood groups. for rhd negative pregnant women, routine clinical testing is available in several countries world-wide to assess the risk of hdfn in d immunized women, and routine testing to guide rh prophylaxis is now implemented as nationwide service in - european countries. noninvasive prenatal testing for fetal rhd is highly accurate with sensitivities of . %, as reported from clinical programs. in general, the sensitivity is challenged be low quantities of cffdna, especially in early pregnancy. the specificity is challenged by the polymorphic rh blood group system, where careful attention is needed to navigate among the many rhd variants. rhd variants may complicate cffdna analysis and interpretation of results, especially in populations with mixed ethnicities. despite these challenges, fetal rhd testing is very feasible when implemented with careful attention to these issues. for blood groups that are determined by snps, such as kel or rhc, the main challenge has been interference from the maternal dna when analyzing the fetal dna which has resulted in low accuracy and lower sensitivity, when using qpcr. with the application of more novel techniques such as next generation sequencing and droplet digital pcr, accurate noninvasive prediction of these fetal blood groups has been demonstrated. the success of predicting fetal rhd and its successful clinical implementation into national programs should encourage wide-spread use of cell-free dna based analysis. future work on noninvasive prenatal testing of fetal blood groups determined by snps may consolidate the application for cell-free dna testing for such targets, including human platelet antigens. at isbt, the newly formed cfdna subgroup of the red cell immunogenetics and blood group terminology working party will work to facilitate clinical applications, implementation and evaluation of cell-free dna testing. blood banks in most of the nordic countries all share a vein-to-vein approach which in short means that the collection of blood, the preparation of blood components, testing/release and storing is served by a single actor. on top of that recipient and donor blood grouping, crossmatching, delivery, registration of transfusion and of any complications is usually handled in a single blood banking information system (bbis). this means that blood banks in the nordics are traditionally operated by a single vendor. the needs for process control in a single vendor bbis, the present solutions, unsolved challenges and untapped possibilities of streamlining processes have been scrutinized with the intention to describe separate processes and to acquire best of breed or best of suite it-systems. the aim for integrations, rather than building an integrated it-system, to support the need for a vein-to-vein process is a precondition in the nordic countries. with multiple it-systems supporting isolated processes, we intend to facilitate development in these and furthermore increase the flexibility in the whole process. we set out to reveal any existing knowledge in the literature on it vendor strategies for blood banks, but we didn't succeed in identifying any relevant literature. however, a systematic literature study on vendor strategies when choosing health it was based on the prisma method, and identified studies, but only was eligible for full text review and met the inclusion criteria. even this broader literature study reveals very little evidence. two studies find single vendor strategies poor and conclude "best of suite" solutions to be optimal. one study was not able to correlate vendor strategies to the investigated productivity, but concludes that best of suite and best of breed strategies requires larger organizational changes than a single vendor strategy. in summary, the existing research is contradictory. this paper adds basic knowledge for breaking down the process control of blood banking in smaller processes. this adds the possibility for identifying best of breed or best of suite vendors, instead of relying on single vendor it solutions. furthermore it is a call for more research in the field of vendor selection strategies which this study didn't succeed in identifying. d- - applying drones to supply blood to remote areas: rwanda's experience biomedical services, rwanda-ministry of health-national blood services, kigali, rwanda background: in rwanda, blood transfusion services started in . during the genocide against the tutsis almost all the socioeconomic fabric of rwanda was destroyed as well as its health infrastructure. the healthcare system was suffering in its aftermath, and there were health inequalities between urban and rural areas, including access to blood for transfusion. from , the government started to rebuild all courses of life including the health system and the blood service in particular. the national center for blood transfusion (ncbt) was then mandated to provide safe, effective and adequate blood and blood components to all patients in need. this was pivotal in achieving health related mdgs , and . today, rwanda has an ambitious vision to put all million citizens within minutes of any essential medical product. while every second matters in emergency management, the use of drones was the perfect solution to many of the last mile challenges that have been traditionally difficult to overcome. it is impossible to forecast accurately down to the need of a single patient. the government has provided an easy solution by centralizing supply and providing on-demand, emergency medical deliveries by drone. doctors are now empowered to provide the quality care with all the supplies on hand, patients can now be treated close to home, and we eliminate waste from potential overstocking when health workers know that they have a quick and reliable source of supply. description: in , the government of rwanda started to operate the world's first national drone delivery program for blood and other lifesaving medical products. these drones can carry two to six units of blood at a time and deliver in - minutes depending on a hospital's location. the average duration was between - hours round trip with the vehicle system, before the use of drones. drones currently deliver blood to health facilities throughout the country and are set to reach % of transfusing health facilities outside kigali by the end of the year. within the first year, healthcare workers saved an average of . hours per delivery and a total of , hours of lost time on road pick up they could instead dedicate to patient care. by march , over , deliveries have been made, with % of those being emergency deliveries. a total of more than , blood units have been delivered. in february , zipline obtained the highest rating from the health facilities being served in a performance evaluation conducted by the national center for blood transfusion. when a doctor or medical staffer needs blood, they place an order through the hemovigilance order portal. they are then sent a confirmation message saying a drone is on its way. the drone flies to the health facility at up to km/h. when it is within five minutes of the destination, the medical staffer receives a notification. the drone then drops the package, attached to a parachute, into a special drop zone. conclusion: supply is not a developing country problem, it is a global issue. rwanda was just the first one to recognize the potential of this technology and decided to do something about it first and fast, to ensure access to universal access to all blood products. d- - scottish national blood transfusion service, edinburgh, united kingdom supporting the provision of viable transfusion services in remote/rural areas is more than just a geographical challenge. limited qualified blood bank resource; small throughput volumes; increased regulation are only three of the additional factors combining to threaten safe and sustainable transfusion service delivery. inventory management and out of hours service provision were identified as essential areas where it was thought that technology, in the form of a remotely controlled blood fridge, could provide a key element of the overall solution. a radio frequency identification (rfid) fridge racking system was installed in a standard blood bank fridge and connected to the laboratory information management system (lims) common to both the remote and central blood bank. the central blood bank was enabled to test patient samples from the remote laboratory, identify components located in the remote fridge suitable for the patient and allow correct component issue, even when qualified staff were unavailable in the remote laboratory. testing has concluded that installation of remote fridge management can play a major role in helping to maintain a remote inventory permitting patient compatible components to be issued. by sustaining transfusion services in remote communities we can avoid transportation of patients who require transfusion support to locations miles from home. antibodies to hna- b, fcgriiib and hna- have been reported, too. neonatal alloimmune neutropenia results from maternal antibodies transferred transplacentally to the fetus and is caused by all known hna-antibody specificities, i.e. hna- a, - b, - c, - d, hna- , hna- a, - b, hna- a, - b and hna- a specificity. hna and hla antibodies can induce mild febrile transfusion reactions and trali. since the introduction of the male only plasma strategy, in many countries the trali incidence decreased but it is still one of the most common causes of severe transfusion reactions. especially hna- a antibody containing plasma from female donors is responsible for severe or even fatal reactions. but also hna- a, - b, hna- and hla class i and class ii antibodies were reported. the latter activate monocytes to secrete soluble factors that act on the primed neutrophils in the narrow lung capillaries. laboratory testing: laboratory work-up requires the knowledge of the patient's clinical condition and the methods that are appropriate to detect the relevant antibodies. the classical granulocyte agglutination test (gat) in combination with the granulocyte indirect immunofluorescence test (gift) can detect nearly all relevant antibodies. hna- , - , - , - and hla class i antibodies are clearly detectable in the gift while hna- antibodies strongly agglutinate neutrophils in the gat. the monoclonal antibody-specific immobilization of granulocyte antigens (maiga) test detects all hnaantibodies except for hna- with high glycoprotein specificity and sensitivity but is time consuming and requires highly skilled personnel. for trali diagnostics laboratory testing is completed by methods like the indirect lymphocyte immunofluorescence test (lift) or elisa for hla class i antibodies and hla class ii specific elisas. since several years fluorescent bead based assays (luminex) enable faster and more automated hna antibody detection but to date not all specificities, especially hna- , can be reliably detected so that still the classical gift and gat have to complete the methodological spectrum. serological typing today is mostly reduced to the determination of hna- in the gift because the molecular reason for the hna- -null phenotype is not completely understood. establishing only one pcr-asp reaction for the main cd * a>t polymorphism would comprise the risk to miss other molecular causes. however, for all other hna allelotyping by pcr methods is the first choice. summary/conclusions: granulocyte serology still today is widely based on a variety of manual methods and will be reserved to specialized laboratories as it requires experienced laboratory staff and profound knowledge of granulocyte immunobiology. d- - norwegian national unit of platelet immunology, laboratory medicine, university hospital of north norway, tromso, norway maternal alloantibodies against antigens on human platelets can cause severe thrombocytopenia and bleeding in fetus or newborn, identified as fetal/neonatal alloimmune thrombocytopenia (fnait) . although most cases the thrombocytopenia is selfresolving within the two first weeks of life, some infants present bleeding symptoms and thus require platelet transfusion. a set of laboratory analyses are required to confirm the fnait diagnosis. in addition to guiding compatible platelets to the affected newborn, the correct diagnosis will be valuable to assess the risk of fnait in subsequent pregnancies. in addition, platelet alloantibodies may also complicate platelet transfusions by immune-mediated platelet refractoriness, and require proper identification of the patient's antibody specificities prior to selection of donor platelets. the algorithm for laboratory investigations include both serological and molecular assays, and depend on the objective and timing: whether there is an urgent need for platelet transfusion, follow-up of a pregnancy with known risk, or to do full-scale laboratory testing to confirm diagnosis. molecular genotyping should include all hpa systems relevant for the local population (in caucasians hpa- , - , - , - and - ), preferably with optional extended panels for systems for low frequency populations due to immigration/mobility and for less frequently seen alloantibodies (hpa- , - to - are most commonly included). serological testing of antibody binding to platelets is often initially tested by flow cytometry analysis (direct test and/or cross-match). however, the detection of platelet-specific antibodies is often complicated by the presence of anti-hla class i antibodies and thus require sensitive platelet glycoprotein-specific assays. serological testing for platelet-specific antibodies includes as a minimum panels of antigens on gpiib/iiia, gpib/ix, gpia/iia and cd and preferably additional targets for populations with asian/african origin. several methods are available; i.e. bead-based assays and elisa based methods. however, most reference laboratories perform variants of the monoclonal antibody immobilization of platelet antigens assay (maipa), as reported by the th international platelet immunology workshop of isbt ( ) . the investigations also include measurement of the anti-hpa- a by quantitative maipa if present, as this is reported to potentially predict the severity of fnait. for pregnancies with known risk of fnait, there are methods available to perform non-invasive prenatal typing from maternal plasma. the most feasible and so far appropriate for routine testing is fetal hpa- typing with quantitative pcr or by melting curve analysis. other sophisticated, yet resource-demanding techniques have also recently been reported -importantly also for typing of other hpa-systems. d- - molecular basis of hna- expression justus liebig university, institute for clinical immunology and transfusion medicine, giessen, germany human neutrophil antigen (hna- ) is a neutrophil-specific antigen located on gpi-anchored glycoprotein cd (also known as nb ). hna- is absent on the neutrophil surface of - % of the healthy individuals that divided the population to hna- positive and hna- null individuals. exposure of hna- null individuals to hna- positive neutrophils during pregnancy, after transfusion or transplantation, induces immunization against hna- and consequently the production of iso-antibodies. the hna- iso-antibodies are involved in the mechanism of neonatal alloimmune neutropenia (nain), transfusion-related acute lung injury (trali) and graft failure following bone marrow transplantation. presence of cd on a neutrophil surface of hna- positive individuals follows a bimodal expression that categorizes the circulating neutrophils to hna- positive and negative subsets. the cd gene contains exons encoding a protein of amino acids. the lack of hna- (in hna- null individuals) is associated with the presence of a missense mutation, cd *c. a>t in exon of cd gene inducing a premature stop codon in codon . this mutation alone or in combination with cd *c. delg has been introduced as the main reason for the absence of cd in hna- null individuals. a pseudogene (cd p ) highly homologous to exons - of cd gene is located downstream of the cd gene. conversion of exon of cd p into cd gene is responsible for the generation of cd *c. a>t missense mutation. in addition, the heterozygosity or homozygosity of cd *c. a>t is accounted for regulation of hna- negative and positive neutrophils subpopulations. genotyping has revealed the hna- null individuals, heterozygous for cd *c. a>t mutation without cd *c. delg, indicating the presence of a complementary mechanism regulating cd expression. newly in hna- null individuals and individuals with atypical cd expression a cd * g>a polymorphism in combination with cd * a>t is described. altogether these data indicate a complex compound mechanism(s) for regulation of cd expression on the neutrophil surface. this presentation will summarize recent findings on cd expression and highlights the potential genotyping methods for genetic assessment cd expression of donors and patients. blood products ordered, transfusion start and end times, whether patients experienced a reaction to the transfusion as well as vitals measurements taken before and during the transfusion, including temperature, oxygen level, blood pressure and heart rate. for the validation process, transfusion nursing notes were sampled and reviewers assessed the accuracy of the information regarding ) blood product ordered, ) whether the patient experienced a reaction, and ) the start and end times of the transfusion. for each of these fields across all sampled notes, the claritynlp tool reproduced these data points with percent accuracy. in addition, the tool supplied transfusion end times for numerous structured records that were missing this key data point. summary/conclusions: claritynlp can very efficiently digest a large number of transfusion nursing notes simultaneously and also does an excellent job of extracting the main characteristics of a transfusion, which can be used in partnership with structured data to produce a more accurate and more complete picture of patient transfusions. immunohematology and genomics, new york blood center, new york, united states antibody-based typing, with a positive result reflected in agglutination of the red cells (rbcs), has served the profession for nearly a century enabling safe and effective transfusion therapy. the power of rbc typing by serologic methods lies in the availability of standardized antibody reagents which target many of the specificities of significance for transfusion, and the ability to directly detect antigen expression on the rbcs. hemagglutination has historically been relatively inexpensive, particularly for abo and rhd as the most important blood groups in most populations. serologic rbc typing is reliable, requires no sophisticated equipment, is generally straightforward to perform, and is fast requiring less than h to results. hence, antibody-based testing has been considered the "gold standard" for blood group typing. with the age of genomics, dna-based genotyping is increasingly being used as an alternative to antibody-based methods. most antigens are associated with single nucleotide changes (snps) in the respective genes. genotyping has been validated by comparison with antibody-based typing and has been shown to be highly correlated. the power of genotyping of rbcs lies in the ability to test for antigens for which there are no serologic reagents, and to type numerous antigens in a single assay using automated dna-arrays. this increases accuracy and weak antigen expression can be revealed. fresh rbc samples are not required for dna extraction, and there is no interference from transfused rbcs or igg bound to the patient's rbcs. dnabased typing is economical in that it provides much more information, but testing requires special equipment, training, and -h turn around. what then is the best approach to use? will serologic typing be replaced by dnabased typing? indeed, genotyping will increasingly be used in the practice of transfusion medicine, especially with the growth of whole genome sequencing (wgs). however, because serologic typing for abo and rhd is fast and accurate and often relied on for sample identification, genotyping will not be the sole means for routine typing. genomic sequencing approaches will certainly reveal unrecognized changes and genetic variability in rbc membrane proteins, but not all variation will be immunogenic. a genetic polymorphism must be associated with antibody production to be considered a blood group antigen. the importance of an antibody, and antibody reactivity, then will continue to be the central defining principal in transfusion. as two sides of a coin, both are key to safe and effective transfusion therapy. since the mid- s, research in the molecular basis of structural and functional aspects of proteins carrying and producing the antigens has led to an upgraded and modern understanding of blood group variation. most commonly, single nucleotide polymorphism (snp) based basic molecular typing techniques were utilized to test new findings on a smaller subset of samples, and resulted in concordant sero-and genotyping results in general. however, quite commonly a small number of all samples delivered discrepant results, triggering consecutive rounds of analysis and resolution, finally resulting in a better knowledge with respect to the underlying blood group variation. such rounds of repetitions represented the synergistic incremental process of learning, learning for serologists and molecular biologists. inheritance of public, presence of high frequency, or low frequency, or partial antigens and notice of weakly expressed, or almost undetectable antigens marked the path of incremental learning and may best be exemplified by discoveries within the blood group systems abo, rhd and kell. naming for pheno-and genotypes coevolved alongside the permanent discovery of new antigens. at present, antigens and their antithetic counterparts (if tested and if existent), are more commonly reported independently, as exemplarily shown for the following kell phenotype consisting of three antithetic antigen-couples: kk, kp (a+b+), js(a+b+). alternatively, the same phenotype could be stated as: kel:- , , , , ( ), , . genotypes, on the other hand, rather mirror the actual biological background, e.g. display the two parental alleles (or "haplotypes") present in an individuals' sample. genotype of the above mentioned example would read: kel* . / . (italicized). in an idealized diagnostic environment and for most blood group systems encoded by proteins, every single blood group allele would be defined by its full genomic sequence, derived from one parental chromosome (including "some" 'and '-untranslated regions). thereby, every such "ideal allele" would fully declare presence or absence of all its public, low-and high-frequency antigens and possess its "ideal name". by trend, biallelic snps and their immediate relation to antithetic antigen couples might have distracted from the originally intended meaning of "blood group alleles", more recently. finally, genotypes only dependent on (ideal) allele names, and considering mendelian inheritance patterns (dominant, recessive), would allow for fully comprehensive phenotype predictions. more recently, blood group serology, e.g. the "second side of the coin", seems to gain momentum. since the advent of whole genome sequencing and access to many more than human genomes, it seems that dozens of new blood group alleles are discovered, almost on a daily basis. beside the challenge of naming this multitude of alleles, respective discoveries are frequently made in samples lacking any phenotypic blood group pre-values. clear procedures will be needed to address naming and analyzing the phenotypes resulting from previously unknown alleles. as a consequence, questions asked years ago have changed: today, molecular biologists looking at hundreds of newly discovered blood group alleles find themselves not being asked by serologists any more: "can you confirm my serology?", but instead, pose their question to the experts for the blood group phenotype: "can you confirm my genotype?" a-s - background: the screening of blood donors and returning travelers from active transmission areas have highlighted the importance of diagnosis of acute arboviral infections. in the context of co-infections and similar clinical signs in endemic zones, the differential diagnosis of arboviruses is essential to discriminate the causative agent. the detection of viral nucleic acids in serum or plasma provides a definitive diagnosis, however, in most instances, viremia is transient within less than two weeks after the onset of clinical illness. in addition, the cross reactivity due to the high degree of structural and sequence homology between zikv and other flaviviruses is a significant concern. the combination of molecular (identification of viral genomes) and immunological assays (detection of the immune response) is a key challenge to follow the natural history of these infections and to improve the patient management and the epidemiological surveillance. aims: in this context, we have developed an innovative platform based on agglutination of superparamagnetic nanoparticles (npmag) covalently grafted either with nucleic or proteic probes to face the continuing emergence of arboviruses. methods: dengue (denv) and zika (zikv) viruses are selected as models in this study. a pan-flavivirus rt-pcr is used for the molecular assay to amplify the viral genomes. then, biotinylated viral amplicons are captured specifically on complementary original polythiolated probes coated on npmag. for the immunological assay, npmag are grafted with viral ns proteins to capture anti-denv or anti-zikv antibodies potentially present in the plasma samples. both tests are performed in disposable cuvettes in a homogeneous format. a magnetic field generated by an electromagnet is applied to the reaction medium to align the npmag into chains to enhance the capture of the targets between two npmag. aggregates formed are detected when the field is turned off. the optical density is measured in real-time at nm during several cycles of magnetization / relaxation. results: in this study, molecular analytical performances were evaluated on human samples from blood donors with no history of infections as negative controls, on viral standards and on clinical samples. using viral references standards, we have observed sensitivities of - tcid /ml for zikv and denv (serotypes / / / ) after a detection phase of around min. the first results obtained on zikv (+) clinical samples previously tested by commercial real-time pcr (ct < , altona) showed an % correlation between the two detection methods. no false positive results or cross reactions were observed. concerning immunological assays, commercial human plasma from donors tested positive for denv or zikv antibodies were detected positive with our innovative approach in less than min (sampling + detection) instead of h with classical elisas. further assays on clinical samples are planned to confirm these preliminary results. summary/conclusions: this innovative strategy combining molecular and immunoassays on the same analytical platform offers new opportunities for rapid blood testing to improve the surveillance and the prevention of arboviral infections. background: zika virus (zikv) caused a dramatic epidemic in puerto rico (pr) during , with up to~ % of blood donors reactive for zikv rna in id-nat testing at the peak in june . aims: perform a serosurvey for anti-zikv igg using six panels of donor specimens each collected in march , at the beginning, peak and end of the epidemic, and from march and april . methods: we employed a commercially available zikv igg elisa antibody (ab) assay based on the zikv ns antigen from bio-techne to characterize zikv seroprevalence in the cross-sectional sample sets (anonymized with selected demographic information). results: pr donor samples collected in april were initially evaluated using the manufacturer supplied cut-off to confirm that the zikv ab results were largely negative ( positive, equivocal) despite the high dengue virus seroprevalence (> %) in pr that could potentially lead to false positive zikv ab results. we then used this dataset, together with known positives collected - months postdetection from zikv nat yield donors, to set a population-specific cut-off based on receiver operating characteristic (roc) curve analysis. this cut-off yielded sensitivity and specificity values of > %, and an area under the curve (auc) of . , demonstrating a highly accurate assay. we used this new cut-off to calculate final rates of seroreactivity in the additional sample sets ( samples) and estimate seasonal incidence. rates of reactivity, together with mean net od for only the reactives (shown in parentheses), were calculated for each sample set: background: sex hormone intake in blood donors occurs in three demographic groups: premenopausal women who take contraceptive drugs (progestins with or without estrogens), postmenopausal women who receive estrogen replacement therapies that may be combined with progestins, and testosterone therapies in men. we hypothesized that sex hormone therapies may modulate the quality of red blood cell (rbc) products via alterations of rbc function and predisposition to hemolysis during cold storage. aims: the objectives of this study were to evaluate the association between sex hormone intake and rbc measurements of hemolysis, and to examine possible mechanisms by which sex hormones interact with rbcs. methods: self-reported sex hormone intake and menstrual status were evaluated in , female blood donors from the national heart, lung and blood institute's rbc-omics study. the associations between hormone intake and donor scores of spontaneous storage, osmotic, or oxidative hemolysis were determined in all women and by menstrual state. the interactions between sex hormones and rbcs were determined by sex hormone (progesterone, b-estradiol, or testosterone) potency to inhibit calcium influx or hemolysis during incubations or cold storage. the calcium fluorophore, fluo- am, was used to define rbc calcium influx in response to treatments with sex hormones or drugs that modulate transient receptor potential cation (trpc) channel activity including hyp (a selective trpc activator). results: sex hormone intake by menstrual status was higher in premenopausal women ( . %) than in postmenopausal women ( . %). female hormone intake was significantly (all p < . ) associated with reduced storage hemolysis in all females ( . ae . % versus . ae . % in controls), enhanced susceptibility to oxidative hemolysis ( . ae . % versus . ae . % in controls), and reduced osmotic hemolysis in postmenopausal women ( . ae . % versus . ae . % in controls). in vitro, supraphysiological levels of progesterone ( or lmol/l), but not b-estradiol or testosterone, inhibited spontaneous or hyp -induced calcium influx into rbcs, and were associated with lower spontaneous hemolysis after day cold storage ( . ae . % versus . ae . %, progesterone lmol/l versus solvent control (dimethyl sulfoxide, . %), p < . ). co-incubations ( . h, °c) of rbcs in the presence of progesterone and a trpc activator (hyp , lmol/l) suggested that progesterone protected against hyp -induced hemolysis ( . ae . % and . ae . % versus . ae . %; hyp + progesterone at or lmol/l versus hyp alone, p < . by one-way anova). summary/conclusions: this study revealed that sex hormone intake in blood donors is capable of modulating rbc predisposition to hemolysis and led us to propose new mechanistic pathways by which progesterone regulates calcium influx and hemolysis in human rbcs. pre-and postmenopausal women respond differently to hormone intake and its effects on rbc responses to osmotic or oxidative stress. progesterone modulates calcium influx into rbcs via a mechanism that may involve interactions with membrane trpc channels, activation of which is associated with pre-hemolytic events such as senescence and eryptosis. a-s - international cooperation, swiss red cross, wabern, switzerland background: red cross and red crescent societies were playing an important role in setting up blood transfusion establishments in many low resource countries. by the mid- s, the red cross was active in the national blood programs in approximately % of countriesmostly in blood donor recruitment and education. today, major organizational developments in blood transfusion services were made in high income settings, where nearly % of all worldwide donations take place (home to only % of the population). who data shows that the median annual blood donation rate in high-income countries is . % of the population compared to . % in low-income countries. the factors for this low turnout are multilayered, but it is well-known that most resource poor settings suffer from a low rate of regular donors and challenges to set-up and financially sustain a national blood donor program. the red cross and red crescent (rc) societies assume a key role by reaching and retaining donors from the communities and contribute significantly to better safety and availability of blood. partnerships and international collaboration, such as the swiss red cross (src) program, are aiming to strengthen national structures to improve blood safety and to face today's epidemiological, demographical, and technological challenges. aims: the present work aims to review the role, the mandate and the impact of rc societies in improving blood safety through systematic "voluntary non-remunerated regular blood donor" (vnrbd) programming and international partnerships. methods: data and evidence is drawn from the src international cooperation projects over the last years, more specifically partnering with three rc societies, and the data from the global advisory panel (gap) of the ifrc including their global mapping. results: the promotion of vnrbd has been a specific objective in all src supported programs. through the engagement of the rc society, the training of volunteers and partnering with the health authorities, the projects significantly increased the blood donor rate by recruiting and retaining donors from the communities. for example, the rc societies increased total donations by % in lebanon; vnrbds by % annually in kirgizstan, and from practically zero to ' in south sudan. the importance of rc societies was also underlined in the published global mapping of gap, which showed that ( %) of them provide level a (full blood service), ( %) are level b (systematic blood donor recruitment) and ( %) are level c (vnrbd blood promotion) blood services. gap has also commenced a new three year vnrbd support program aimed at establishing tools and materials for national societies. summary/conclusions: the red cross / red crescent movement has a unique mandate and position in improving global blood safety at all levels; with its huge network of volunteers, even blood donors in remote communities can be reached and retained. rc societies in low resource settings with a level b or level c role should further capitalize on partnerships with local and international actors to leverage technical assistance and funding for their activities. background: it is essential to motivate and encourage the public to donate blood and be eager to help saving lives, in order to maintain safe and adequate national blood supply. aims: "technical assistance for recruitment of future blood donors (europeaid/ /d/ser/tr)" project aimed to avoid problems in supplying the safest blood to contribute to the improvement of public health by (i) increasing the knowledge of primary and secondary school students regarding blood donation, (ii) creating sensitivity in school principals and teachers regarding voluntary non-remunerated blood donation (vnrd), (iii) motivating family members of the students for blood donation and (iv) creating public awareness through media. methods: an effective coordination is established between ministry of health (moh), ministry of national education (mone) and turkish red crescent (trc). the existing curricula and textbooks of primary and secondary schools were reviewed and revised, and corresponding materials on the importance of blood donation were created. the human resources capacity of moh, mone and trc to support raising awareness on blood donation were developed. to raise public awareness on blood donation nationwide, education and recruitment campaigns on were organized in pilot schools. additionally, media and public relation campaigns on blood donation were organized throughout the country. results: ( ) existing curricula and textbooks relevant to promoting blood donation were reviewed, revised and reported to the board of education of mone. ( ) corresponding educational materials for students and teachers were developed and distributed. ( ) blood donation clubs were established in pilot schools. ( ) trainings were conducted for personnel of moh and trc on blood donation regarding their responsibilities. ( ) cascade trainings were conducted for personnel of transfusion centers and school principals in provinces. ( ) information seminars were delivered to . students and . teachers and family members of students during school campaigns. ( ) four animation films on blood donation were produced and broadcasted on the national tv channel (trt). ( ) three different computer games targeting different age groups were developed and uploaded to the web portal of the project and distributed to the pilot schools. ( ) media spots were produced and broadcasted . times in different tv and radio channels. ( ) billboard posters and brochures were prepared and distributed to provinces for raising public awareness. ( ) advertisements about the project and the importance of vnrd were displayed times on national and local newspapers, . times on online news, and broadcasted on national tv channels. ( ) during the campaigns, . units of whole blood were collected in pilot schools. ( ) visibility kits to recruit future blood donors are prepared and distributed throughout the project activities. ( ) awareness and knowledge level of students and their teachers/parents on the importance of blood donation are increased to . % and . % respectively, assessed through pretest and posttest. voluntary non-remunerated donation rate of national demand increased from . % to . in two years. summary/conclusions: training and campaign programmes successfully increased the knowledge on blood donation. to achieve national self-sufficient safe blood supply, efforts for recruitment should be continued. background: despite % of pakistan's population being under years, only % of blood supplies come from voluntary donors while remaining blood is collected from 'family replacement donors'. in pakistan the system has outsourced the mobilization of blood donors to the patient families. as a result many people reach out to their networks including on facebook to locate blood donors. there are thousands of posts each month in pakistan seeking blood donors on facebook. to facilitate needy families, the global social media giant facebook launched a special blood donation feature for pakistan in collaboration with sbtp, pakistan. the feature makes it easier for people to sign up to become blood donors and helps connect these voluntary donors with people and organizations in need of blood. similar features have been launched by facebook in india, bangladesh and brazil to address the problem of blood shortages in those countries. however, among these four countries pakistan has unique position because of the existence of a national counterpart, sbtp which can facilitate facebook in promoting its feature and provide the feedback on the impact of this innovative effort for continuous improvement of the feature. aims: to promote voluntary blood donations and blood safety in pakistan through facebook. methods: the facebook and sbtp teams launched a pilot to study the impact and effectiveness of the facebook blood donation feature as a tool of community engagement. a six months plan has been chalked out to measure the impact of this tool in five selected blood centres. a checklist called "p checklist" was shared with these blood centres to fulfill some basic requirements for an official blood bank page including a display picture, cover photo, contact information, directions, etc. regular skype meetings are held between the teams of sbtp, facebook (san francisco and singapore) and the blood centres to monitor the progress of the pilot and generate feedback. results: the facebook blood donation feature has recorded remarkable success with over one million signups within few months in pakistan. the blood centres participating in the pakistan study have experienced enhancement in the voluntary blood donations trend with - walk-in donors and an average of more than telephonic queries regarding voluntary blood donation per month in each center. the trend is gradually surging as the feature is being refined on the basis of feedback received. the pilot will end in june . the statistics generated since january are very encouraging and underscore the importance of social media in reaching out to the untapped potential blood donors. the study will be used to plan an effective nationwide strategy to increase donor mobilization, recruitment and retention. background: in our region, an increasing number of patients of african or asian origin with sickle cell disease (scd) or transfusion dependent thalassemia (tdt) require red blood cell (rbc) transfusions, and many have rbc alloantibodies. selecting optimally matched rbc units for these patients is essential for preventing not only acute hemolysis but also further alloimmunisation. beside antigen-matching for abo, rh d, c, c, e, e and k, patients with scd and tdt should ideally receive rbc units matched also for m, s, s, fya, fyb, jka and jkb (extended phenotype). this is the policy at our center, which currently provides rbc products to patients with hemoglobinopathies. because the vast majority of our blood donors are caucasians, the selection of matched rbc units for patients of different ethnic origin can be difficult. therefore, expanding the number of available african and asian blood donors is becoming increasingly necessary. aims: hereby the recruitment strategy of non-caucasian blood donors introduced at our center is described and the results obtained during six years are reported. methods: since . . , whenever a first-time blood donor of non-caucasian origin is registered, an alert is entered in the donor file to trigger the determination of the extended rbc phenotype along with routine testing. rbc antigen determination is performed in our laboratory with serologic methods. in selected cases (i.e. suspected rhd or rhce variant), samples are sent for molecular analysis (ssp pcr). rare rbc phenotypes relative to ethnicity are, among others, fy(a-b-), s-s-, lu(b-) and those with uncommon rh phenotypes. if a rare rbc phenotype is detected, a coded comment is entered in the donor data and the donor is listed in the national rare donor file. results: from . . until . . , an extended determination of rbc antigens was performed in subjects presenting for blood donation. twenty-nine rare donors ( %) were identified and included in the rare donor file: fy(a-b-), lu (a-b-), lu(b-), fy(a-b-) and s-, ccddee (r'r'). overall, these donors provided rbc units (range . to date, all donors are still active and are reserved for dedicated donations. the internal price of rbc antigen testing per donor is approximately . -chf, resulting in a total financial effort of around , .-chf in the time since the project was started. summary/conclusions: in our experience, a "passive" recruitment of non-caucasian blood donors based on ethnicity has an overall low efficiency from a logistic and financial point of view. moreover, african and asian blood donors may require investigations for hemoglobin variants and serology for malaria in addition to routine testing. nevertheless, a targeted determination of extended rbc antigen phenotype does allow the identification of persons with rare phenotypes. currently, measures for the active recruitment of potentially rare blood donors are being implemented at our center. after a pilot phase, a project for a nationwide recruitment strategy will be elaborated. a further goal is to build a national registry of patients with hemoglobinopathies requiring transfusions. blood transfusion is an essential treatment. transfusion safety consists of several components. although all are important, ion richer countries the order of priority is typically: .) avoidance of transfusion transmittable infections; .) quality of the blood product with a strong focus on component therapy; .) prevention of severe transfusion reactions; .) avoidance of clerical errors; .) sufficient availability of blood. the keynote of this lecture will be that the order of priorities on transfusion safety should probably be different in resource limited environments. .) sufficient availability of blood and proper utilisation; .) avoidance of transfusion transmittable infections; .) avoidance of clerical errors; .) prevention of severe transfusion reactions; .) quality of the blood product. most important, in regions with limited resources patients suffer from under-transfusion because not enough blood is available. all efforts should be made to reduce wastage of the available blood either by inappropriate storage, handling, or nonindicated transfusions. in addition, prevalence and number of pathogens transmittable by transfusion is much higher than in the richer parts of the world. this is aggravated by the fact that rejection of blood donors based on their history is problematic when the blood bank is empty. the aim to develop centralized national blood services with few sites manufacturing cost effective (low personnel costs) high-quality blood products, which are distributed to regional hospitals is not matching the reality of infrastructure, governmental support, and functionality. in healthcare systems with limited resources usually available personnel (hands) is not a problem, while reagents, equipment and even electricity are precious resources. the lecture will propose to focus on staff training and education, establishing local hospital-based transfusion services, which provide the blood products for the region, based on donor recruitment campaigns adjusted to the available technology, local culture, including replacement donor programs, with retention of safe donors as highest priority. fractionation of blood into components had been standard in transfusion medicine, but recently whole blood has experienced revival for patients with acute blood loss. given main transfusion indications such as postpartum hemorrhage, or severe trauma, in regions with limited infrastructure, whole blood might be the more appropriate product. most patients requiring transfusion in these regions are younger and volume overload by whole blood is not a major issue. in addition, frequent electricity failures do not allow prolonged storage of plasma at - °c (this is therefore mostly wasted), although this issue can be overcome by solar powered freezers. ideally whole blood should be pathogen inactivated for which two methods are currently available. to reduce frequencies of acute hemolytic transfusion reactions again education and training to minimize clerical errors in the transfusion process are most important. extended testing for other rhesus antigens and k beside abo and rh-d in transfusion dependent hemoglobinopathy patients may help to reduce delayed hemolytic transfusion reactions. currently a leukodepleted pathogen-inactivated whole blood product might mostly serve the needs for blood transfusion in regions with limited infrastructure . the developed world should invest research efforts to develop such a product available at affordable costs. background: in modern transfusion medicine, serological investigations for blood cell antigens are complemented by genotyping arrays and pcr assays. whilst these platforms are informative in the majority of reference investigations, they are limited in their ability to define nucleotide variants associated with rare antigens and unable to detect novel variants potentially affecting antigenicity. next-generation sequencing is increasingly being employed in reference settings, providing information that cannot be obtained through these methods. whole genome and whole exome sequencing have been successfully employed in many investigations of novel and rare antigens, however concerns remain regarding the collection of genomic data unrelated to reference investigations, and reporting clinically significant incidental findings. these concerns can be addressed through the use of targeted sequencing panels. we report on the design, testing and efficacy of a panel providing a comprehensive genotyping profile for red cell, platelet and neutrophil antigens in a single test. aims: design a customised targeted exome sequencing panel for red cell, platelet and neutrophil antigen genes, and benchmark the efficacy against a commercial medical sequencing panel (illumina trusight one -tso). -test the panel and in-house genotype prediction script on sequence outputs from samples with known red cell, platelet and neutrophil genotypes and phenotypes and determine whether predictions are concordant. methods: the panel was designed with probes covering exons of genes associated with red cell, platelet and neutrophil antigens. using illumina nextera rapid capture technology, samples were tested over five sequencing runs, on standard and micro chemistries, to determine optimal sample plexity per standard run, and the efficacy of smaller flow cells for lower throughput applications. an in-house python script was used to predict star-allele genotypes based on variants listed in isbt and embl databases. these predictions were compared to results from serology, snp array and previous tso data. results: coverage consistently averaged > , with % of target at a quality of q . optimal sample plexity for a standard run was determined to be samples, allowing for sufficient coverage of all clinically significant variants. for red cell samples with previous typing data (excluding rh structural variants), the script correctly predicted . % of snp based red cell genotypes. script predictions were % concordant for platelet genotypes, and four of five neutrophil antigen genotypes. hna genotypes defined by cd could not be reliably determined. the increased target coverage of the panel allowed for detection of a clinically significant heterozygous variant in scianna system, previously undetected by the tso panel due to extremely low coverage. additionally, a variant defining a potentially novel null allele was detected in the p pk system. summary/conclusions: the panel demonstrates considerably higher coverage, quality and throughput compared to the tso and allows for detection of variants previously overlooked due to low sequencing coverage. up to samples can be reliably sequenced in a single run. our script correctly predicts over % of snp based alleles; however, rh structural variants require further manual analysis. background: to ensure the safety of a transfusion it is critical to identify the blood type of both donor and recipient. serological methods for typing abo, rh and kel use monoclonal antibodies, however, typing reagents for rare blood groups are expensive, unavailable or unreliable. dna-based identification of human blood groups has been used to overcome these limitations and its application has reduced rates of alloimmunisation in chronically transfused patients. however, to date, the cost per sample has prevented the universal application of dna-based donor typing aims: to achieve universal adoption of dna based donor typing, the blood transfusion genomics consortium (bgc) set out to develop an affordable dna based platform, capable of typing all red cell antigens, hla class i and ii and human platelet antigens. methods: the uk biobank axiom array, previously used to type , uk citizens, was redesigned for donor typing using three approaches: i) mining transfusion medicine knowledge, e.g. isbt allele tables; ii) inclusion of loci associated with donor health; iii) extraction of all coding variants in relevant genes with a frequency > : , identified in large-scale sequencing data. samples from nhsbt and sanquin blood donors (n = , ) were used for performance assessment. red cell and platelet antigens for each donor were inferred from genotypes using the bloodtyper algorithm and concordance with clinical serological typing results assessed. results: concordance between genotypic and serological typing results was . % for , comparisons; of the discrepancies were serologically negative and genotypically positive for a given antigen (k/k, fy[a/b], lu[a/b]). in all cases dna variants known to modify or weaken antigen expression were detected, displaying the power of genotyping to detect variant 'weak-antigen' expression. across antigens for which serology was available, genotyping provided a . -fold increase in the number of typing results available per donor ( . vs . ). furthermore, genotyping provided data on an additional clinically relevant antigens, allowing identification of antigen-negative donors and blood group identification for which antibodies are not commercially available. the power of a genotyped panel of donors to support patient management was demonstrated by a retrospective analysis of clinical cases referred to nhsbt. from , patient referrals with > alloantibodies between and , unique alloantibody profiles were identified. we found that there was a . -fold greater likelihood of finding o negative compatible donors for these patients when using genotyping data from the , nhsbt donors. importantly, the number of alloantibody combinations for which no compatible antigen-negative donor could be identified fell from to , representing an additional patients that could be provided with directly compatible blood using the same donors. summary/conclusions: through the bgc efforts an affordable fully automated genotyping platform, including the processes for quality assurance and data analysis, has been developed. furthermore, we have demonstrated the real-world benefits more extensive donor characterisation can provide when selecting blood for patients with multiple antibodies. the results of this international collaboration provide opportunities to introduce fully-automated genotype-based donor typing in a safe and cost-efficient manner in blood supply organisations. c-s - biobank performs whole-genome sequencing (wgs) from individuals nation-wide. these data are suitable for allele frequency analysis to demonstrate gene expression and genetic profile in our population, also to estimate the significance of each antigen in transfusion practice. aims: we aim to provide and verify population-based blood group antigen profile using wgs and dna samples from taiwan biobank. methods: a near wgs and demographic data were analyzed. annotations of blood group antigen were performed according to variants from isbt allele tables, including transcription factors; variants for the lewis system were obtained from previous studies. annotations of blood group variants were verified by dna samples with targeted sequencing on illumina miseq, and specific variants were verified by dna samples with the commercial genotyping kit or sanger sequencing. allele frequencies from wgs analysis were compared with population serology data using two-proportion z test. results: population-wide blood group antigens were analyzed, revealed in-depth antigen expression profiles in all systems (except ch/rg). the antigen frequencies from wgs were similar compared with published serology data, except for the antigens and possible explanations listed as follow, ) m, n: insufficient sequencing reads, ) c, c: identical rhce exon with rhd exon for c allele, ) mur: insufficient read length/depth for gypa/gypb hybridization calling and individuals from high prevalence of mur antigen in aboriginal tribes were not enrolled. blood group antigen predictions and variants from wgs were accord to dna verification. furthermore, systems shown no genetic variations, predicting uniform antigen expression in our population, and we can manage transfusion with minimum concerns for antigen mismatch in these systems. moreover, we found weak and null alleles in our population for blood group systems that we had previously no knowledge of, such as lan, jr, and vel. these variants were helped to identify a patient with anti-jr a carrying homozygous jr a null alleles. summary/conclusions: taiwan biobank wgs is suitable for full blood group antigen profile determination with few adjustments required for specific antigens. the population antigen allele frequency provides valuable insight to antigen significance in transfusion practice, matching strategy for our patients, and estimation of the likelihood to obtain for specific antigen negative blood from mass population. also, the genetic variants revealed in this study can help us to locate rare donors, and to integrate variations into routine donor blood group screening to provide suitable blood at a low cost efficiently. background: providing adequate amounts of safe, appropriately matched blood to meet the demands of an expanding and aging patient population presents a challenging global problem. for these reasons, sustainable in vitro sources of red cells may offer a desirable alternative to reliance on donor blood. the first stable immortalized early adult erythroblast cell line, bel-a , has been shown to differentiate efficiently into mature, functional reticulocytes (trakarnsanga et al., nat commun. : , ) and consequently could provide a readily available tool for diagnostic use and proof of principle for future therapeutic use. aims: at ibgrl, next generation whole exome sequencing (wes) has been used previously to accurately predict blood group phenotype in a number of blood group systems, including abo, rh and mns (tilley & thornton, transfusion medicine (suppl. ) : , ). here we have used it to analyse and document bel-a blood group-related genotypes and predict blood group phenotypes. additional genes involved in cell-growth and enucleation were also analysed in order to further elucidate the characteristics of the bel-a cell-line. methods: bel-a cells (day ) were cultured in expansion medium and genomic dna (gdna) was isolated from cells on day . for wes, gdna libraries were prepared using nextera â rapid capture exome enrichment and sequenced on illumina â miseq. sequence alignments for genes encoding all known blood group systems and further genes encoding transcription factors and cell enucleation-associated proteins were visualised using integrative genomics viewer, whilst illumina â variant studio was used to identify observed mutations. mutations in coding regions were used to determine bel-a genotype and predicted phenotype. results: good coverage of most of the selected genes was achieved. alignment of homologous blood group genes including rhd/rhce, gypa/gypb and c a/c b was problematic and additional analysis of coverage of these genes was required for accurate interpretation. despite a number of polymorphisms observed across the tested genes, bel-a did not express any novel or rare blood group antigens. genotyping results predicted a common antigenic profile, in agreement with previous serological and genotyping results where available. although a number of missense single nucleotide variations were detected in analysed genes, including cr , cdan and tmx , these were common polymorphic variants and unlikely to be of any functional significance. summary/conclusions: wes was used to determine bel-a genotype in relation to blood group genes and selected genes encoding transcription factors and proteins associated with cell enucleation. wes allowed accurate prediction of blood group phenotypes, showing full concordance with available serological data (trakarnsanga et al, ) . a small number of mutations were identified which are of unknown significance and require further work to determine any potential phenotypic effects. this complete record of the bel-a blood group-related exome will enable reliable gene editing strategies for future diagnostic and therapeutic purposes. additionally, knowledge of the full cell line exome will allow analysis of any emerging genes of interest and provide better insight into the mechanisms of erythroid differentiation and enucleation. background: emerging evidence, especially in neonates, has shown potential harm associated with liberal platelet transfusion strategies. very little evidence exists regarding optimal platelet transfusion thresholds in critically ill children. randomized controlled trials may be difficult due to lack of equipoise from providers. if regional variation in practice exists, comparative effectiveness studies may be an alternative approach. aims: to describe regional variation in platelet transfusion practices in critically ill children. methods: secondary analysis of a prospective, observational study. subjects were grouped according to region (north america, europe, middle east, asia and oceania) and nation. transfusions were analyzed as prophylactic (given to prevent bleeding) or therapeutic (given to treat bleeding). the primary outcome was the total platelet count (tpc) prior to transfusion. sub-groups analyses were performed in children with an underlying oncologic diagnosis and those supported by extracorporeal life support (ecls). the dosing and processing of the platelet transfusions were analyzed as secondary outcomes. results: five hundred and forty-nine children from countries were enrolled ( % in north america, % in europe, % in oceania, % in asia, and % in the middle east). overall, the median (iqr) tpc prior to prophylactic transfusions (n = ) differed significantly on a regional basis (p = . ) and ranged from ( - ) x cells/l in the middle east to ( - ) x cells/l in asia. the median tpc prior to prophylactic transfusions did not significantly differ between countries (p = . ), nor did the tpc prior to therapeutic transfusions (n = ) differ on either a regional (p = . ) or national (p = . ) basis. for children supported by ecls (n = ), there were no regional (p = . ) or national (p = . ) differences for prophylactic transfusions. however, significant differences in the tpc prior to therapeutic transfusions were observed on both a regional (p = . ) and national ( . ) basis with the middle east, in particular israel, transfusing at the lowest median (iqr) tpc [ ( - ) x cells/l]. for children with an underlying oncologic diagnosis (n = ), no differences were seen in the tpc for prophylactic transfusions (n = ) on a regional (p = . ) or national (p = . ) basis. nor were differences seen in the tpc prior to therapeutic transfusions on a regional ( . ) or national (p = . ) basis. there was significant variability in the dosing of platelet transfusions on both a regional (p < . ) and national basis (p < . ). the median (iqr) dose based on volume ranged from . ( . - . ) ml/kg in north america to . ( . - . ) ml/kg in europe. the vast majority of transfusions were leukoreduced and irradiated but significant variation exists in storage duration on both a regional (p < . ) and national (p < . ) basis. summary/conclusions: regional and national variation exists in platelet transfusion practices among critically ill children, especially in those given therapeutic transfusions while supported by ecls. considering this variation, comparative effectiveness studies may be an appropriate approach to gain evidence to optimize platelet transfusion thresholds. background: the optimal threshold for prophylactic platelet (plts) transfusion in pediatric patients with cancer is still controversial and current clinical practice comes from studies on adults and on inpatient setting. the international guidelines (icmtg, ) recommend, for all age patients, a prophylactic platelet transfusion when plts count is ≤ /l and a platelet dose of . per square meter (sm) of body-surface area (bsa) in inpatient and . /sm in outpatient setting. aims: in january we started in our children's hospital a prospective protocol in order to evaluate the impact on bleeding risk of current clinical practice of prophylactic platelet transfusion in inpatients and outpatients onco-haematological patients. methods: bsa was calculated from age-standardized weight. inpatients received a dose per transfusion of . /sm and outpatients a dose per transfusion of . /sm. platelets were transfused when the count was ≤ /l or in presence of bleeding signs; pediatric aliquots were obtained from buffy coat derived pooled platelet concentrates or apheresis platelet concentrates, according disponibility. results: from january to december a total of platelet pediatric aliquots were transfused: ( . %) were obtained from apheresis platelet concentrates and ( . %) from buffy-coat-derived pooled platelet concentrates. the majority of platelets pediatric aliquots ( - . %) were transfused to onco-hematological patients undergoing hematopoietic stem cells transplant (hsct) or conventional chemotherapy. among them, aliquots were transfused in inpatient setting: ( %) in the hematology unit, ( . %) in the oncology unit and ( . %) in hsct unit. a total of ( . %) aliquots were transfused in outpatient setting: ( . %) to patients affected by hematological malignancies and ( %) to patients with solid tumors. five major bleeding events (who grade ≥ ) were observed during the study period and all of them occurred in hospitalized patients. two patients with solid neoplasm developed a who grade bleeding event. two patients with hematologic malignancies and a patient with neuroblastoma (n = , . %) developed intracranial bleeding (who grade ). the platelet count at the time of the event was /l, /l and /l, respectively. summary/conclusions: our results showed the efficacy, in onco-hematological pediatric patients, of a prophylactic platelet transfusion protocol based on international guidelines: a very low incidence of who grade bleeding has been observed in inpatients setting only ( . % vs % of plado trial, sj slichter, nejm, ) , while in outpatients setting the double platelet dose prevents the major bleeding event (who grade ≥ ) occurrence. background: the problem of blood-borne infections remains relevant in transfusion medicine. pathogen reduction technologies (prt) provide a preventive approach to a wide range of transfusion-transmitted infectious diseases. to date, prt widely used for platelet concentrates and blood plasma, however, the use of these technologies for the treatment of red blood cell-containing blood products undergo research. aims: the aim of our study was to evaluate the safety and efficacy of transfusions of pathogen-reduced (test group) red blood cell suspensions (rbcs) and compare these data with gamma-irradiated rbcs (control group). methods: the technology based on the combined action of riboflavin and ultraviolet (mirasol prt, terumo bct, belgium) was used to reduce pathogens in whole blood. subsequently, the rbcs of the test group were derived from pathogenreduced whole blood. the control rbcs were irradiated at the gammacell elite (best theratronics, canada) at a dose of gray. all rbcs were used for transfusion for days from the harvest day. pediatric patients with various oncological and hematological diseases were randomized to groups of members in each group. the test group of patients received transfusions of a pathogen-reduced rbcs; the control group received transfusions of a gamma-irradiated rbcs. the next day after transfusion were assessed hemoglobin and hematocrit increment, the level of potassium and haptoglobin in the patients' serum, the frequency and severity of transfusion reactions. - days after the transfusion, the direct antiglobulin test (dat) was performed and after - days the indirect antiglobulin test (iat) was performed. the interval to the next need for transfusion was also evaluated. results: the increase in hemoglobin and hematocrit (p = . ), as well as the concentration of potassium (p = . ) and haptoglobin (p = . ) in the patients' serum after the transfusion did not differ between groups. none of the patients in both groups had hyperkalemia after transfusion. in each group, two patients had febrile non-hemolytic transfusion reactions of comparable severity (p = ). all dat and iat tests were negative in both groups. the interval between transfusions were not significantly different between groups (p = . ). only in the test group was found the correlation between the increase in the hemoglobin and hematocrit values with the volume of transfusion, with the dose and the adjusted dose of hemoglobin obtained for the transfusion on body weight. and in this group was found inverse correlation between the hemoglobin and hematocrit increment with the level of hemolysis in the rbcs. summary/conclusions: we found that the clinical efficacy and safety of rbcs of the compared groups did not differ. there was no evidence of immune elimination and allo-sensitization caused by pathogen-reduced rbcs. according to our data, the spectrum of efficiency and safety indicators of pathogen-reduced rbcs is no worse than that of gamma-irradiated rbcs, provided that rbcs is used for days of storage. the founded correlation suggests that the efficiency of pathogen-reduced rbcs transfusions is more dependent on the characteristics of the rbcs. background: patient blood management (pbm) programs are expanding at an international level. a recent nationally representative study from united states observed pediatric age group as the only age group showing lack of objective evidence of pbm initiatives (goel et al, jama ) . aims: this study aims to identify trends in peri-operative blood utilization in children undergoing elective and non-elective surgeries over years duration from to . methods: using years data ( ) ( ) ( ) ( ) ( ) perioperative transfusions decreased steadily per year from . % in to . % ( % cumulative decline) in for children of all ages (or . ; % ci . - . ; p trend < . ). the cumulative change in elective procedures was . % versus . % decrease in urgent/emergent procedures (p trend < . ). summary/conclusions: in this large prospective registry study of > , children undergoing elective/non-elective surgeries, a statistically significant decrease in utilization of peri-operative rbc transfusions was seen across years from through with more significant decrease in urgent/emergent procedures than elective procedures while these findings need evaluation for non-surgical indications of transfusion, these results may provide first evidence of peri-operative pediatric patient blood management strategies being implemented to optimize transfusions in pediatric population. adverse events -tti, immune interactions and risk c-s - transfusion-transmitted infections (tti) are a long-standing and well recognized concern in medicine, which is tackled on the highest level to guarantee the safety of the transfusion procedure for all stake holders. these include the recipient patients, the donating volunteers, the health care workers involved, and their respective contact persons. accordingly, current national and international guidelines including expert societies and the who provide medical, technical, and legal frameworks, which are the basis for the standard operating procedures. nevertheless, there are important challenges, which render tti a "moving target", and reflect the dynamics in three main areas. first, a change in the type and number of recipient patients with past or ongoing immunomodulatory / immunodeficiency component (examples being hiv/aids, sot, allogenic hct, monoclonal antibody therapies, small molecule inhibitors). second, changing exposure to known agents in donors due to global travel, migration and displacement, as well as environmental/climate change. third, discovery and diagnostics of old and new agents with their known or presumed impact as tti. these aspects will require careful review of data and studies, and judicious discussion of the potential action such as selection versus close monitoring to keep tti rates as low as possible, to deliver maximal safety of patients and stakeholders. background: the implementation of nucleic acid testing (nat) and the development of sensitive and specific serologic assays to detect hbsag and anti-hbc antibodies significantly reduced the risk of hbv transfusion-transmission. the apparent redundant testing for two direct viral markers prompted debates on maintaining hbsag screening, particularly in low endemic countries where blood donations are screened for anti-hbc. however, frequencies of - % of hbsag-confirmed positive/nat negative donations have been reported depending on the sensitivity limit of the molecular assays used. the nature of this discrepancy between hbsag and dna remains largely unknown and it is essential to evaluate any potential negative impact on blood safety before considering removing hbsag testing. aims: the prevalence in blood donors and the molecular mechanisms responsible for a persistent undetectable or barely detectable level of viral replication in the presence of a sustained hbsag production were investigated in a collaborative study including five laboratories/blood centers in europe and south africa. discrepancy between viral dna and hbsag levels suggested the presence of mutations that may negatively affect hbv replication and/or infectious viral particle production. methods: donor samples from france, south africa, poland, and croatia were selected for having hbsag levels ≥ iu/ml and being id-nat (procleix-ultrio plus tm [ % lod: iu/ml]) non-reactive/non-repeatable reactive (nr/nrr) with undetectable viral load (vl) or < iu/ml (n = ) or nat repeat reactive (rr) with vl < iu/ml (n = ). french samples initially tested nat nr/nrr with procleix-ultrio (lod %: iu/ml) were retested with ultrio plus prior inclusion in the study. hbv dna load was quantified (cobas taqman hbv [loq: iu/ml]). hbv dna was purified from to ml of plasma after ultracentrifugation. the whole hbv genome, pre-s/s, precore/core and bcp regions were amplified and sequenced. results: following viral concentration, hbv dna presence was confirmed in % of all samples with undetectable or vl < iu/ml. hbv genotypes were a ( . %), a ( . %), a ( . %), b ( . %), c ( . %), d ( %), and e ( %). all samples were anti-hbc positive and % of ultrio-negative samples tested positive with ultrio plus. unusual - nt insertions/deletions identified in bcp regulatory elements (tata boxes, pginr, epsilon domain) suggest altered viral replication. amino acid substitutions (n = ) or deletions (n = ) at positions reported involved in nucleocapsid formation, particle envelopment and virion formation were observed in the core protein of samples. the replicative properties of the bcp and core variants are currently evaluated in vitro as a surrogate model for direct infectivity testing. preliminary results indicate that the variants tested so far have replicative capabilities similar to those of control viruses. analysis of pol, s, and hbx proteins is ongoing. summary/conclusions: these data confirmed the presence of extremely low level of circulating dna-containing viral particles in id-nat non-reactive or nonrepeated reactive blood donations with concomitant high hbsag levels and anti-hbc reactivity. despite the presence of mutations in the viral genomes potentially affecting virion production, preliminary data indicate that some of the viruses in plasma retain the ability to replicate in vitro and to constitute a potential infectious risk. c-s - background: in switzerland highly sensitive nucleic acid screening in an individual donation format for hepatitis b virus (hbv id-nat) and hepatitis b surface antigen (hbsag) detection is mandatorily performed (guidelines of swiss transfusion src, switzerland). since , hbv (hb) vaccination is recommended in switzerland for children and adolescents until the age of and for adults belonging to known risk groups. aims: to highlight that low anti-hbs titers several years following hbv vaccination still confer protection and enable the host immune system to clear hbv dna without development of serologic markers of disease. methods: a retrospective donor interview was conducted to complete information not covered by the questions included in the standard donor questionnaire. routine hbv serological donor screening was performed on a quadriga system (diasorin, former siemens) with the enzygnost hbsag assay (diasorin, former siemens). further hbv tests were performed on the abbott architect i analyser (hbsag neutralisation, hbeag, anti-hbc igg/igm, anti-hbc igm, anti-hbe and anti-hbs). routine id-nat screening for hiv/hcv/hbv was performed with the roche cobas mpx test on a roche cobas platform. hbv id-nat positive samples were confirmed with a quantitative hbv nat assay (abbott). background: hepatitis b core-related antigen (hbcrag) is a structural antigen of hbv, consisting in hbcag, hbeag and the p cr precore protein. quantitative hbcrag measurement is a sensitive marker of viral replication reflecting the cccdna content and persistence of disease. hbcrag positivity was found to be a significant risk factor of hbv reactivation in hbsag-, anti-hbc+, hbv dna-patients (occult hbv infection, obi) undergoing immunosuppressive therapy. aims: no data about hbcrag status in apparently healthy subject with obi are available. the aim of this study was to analyse this marker in our cohort of obi blood donors. methods: hbcrag was measured in blood donors confirmed to be carriers of obi (hbsag-, hbv dna+). of them, / ( . %) donors were anti-hbc positive, and ( . %) negative. donors had both anti-hbc and anti-hbe reactivities. a group of young blood donors vaccinated for hbv infection (hbsag-, hbv dna-, anti-hbc-), and patients with chronic hbv infection (hbsag+, hbv dna+) were used as negative and positive controls group, respectively. serum hbcrag was measured using a chemiluminescent enzyme immunoassay on the lumipulse g automated analyzer (fujirebio, tokyo, japan). the lower limit of detection (lod) of the quantitative assay is logu/ml and the lower limit of quantification (loq) is > logu/ml, due to nonlinearity results between and logu/ml. levels of hbcrag were tested in the three groups and analysed in comparison to the presence of anti-hbc and anti-hbe. statistical analysis was performed by the ibm statistics spss . . . results: all donors in the negative control group had undetectable hbcrag levels, whereas all patients in the positive control group have detectable hbcrag (mean value: . logu/ml, range . - . ), confirming that individuals without prior exposure to hbv would not have detectable hbcrag. hbcrag was detectable in / obi donors ( . %), with a mean value of . logu/ml (range . - . ). hbcrag could be measured only in obi donors ( . and . logu/ml), being below the loq of the test in the majority of obi ( / ). considering the presence of anti-hbc, hbcrag was detected in / ( . %) anti-hbc+ and in / ( %) anti-hbc-obi, with no significant difference in their mean levels ( . ae . vs . ae . ; p = . ). interestingly, the presence of anti-hbe ( / ) was independently associated with higher hbcrag levels ( . ae . vs . ae . ; p = . ). summary/conclusions: identification of donors with obi is critical to prevent the risk of hbv transfusion-transmission. being hbcrag associated with the cccdna content and replication, our results suggest that the presence of hbcrag, even if not quantifiable, could be useful marker to confirm the occult infection status, even in anti-hbc negative donors. the association between hbcrag, anti-hbc and anti-hbe could also be a useful marker to identify obi donors with a higher risk of hbv reactivation. c-s - hc group. human peripheral blood mononuclear cells (pbmcs) from blood donors were stimulated with hbv polypeptides pool in vitro. t cell proliferation assays (cfse) was used to detecting t cell proliferation, enzyme-linked immunospot assay (elispot) was used to detecting the frequency of hbv-specific ifn-c secreted t cells. spss . statistical analysis software was used for statistical analysis. the measurement data of normal distribution were tested by two independent samples t test; and the comparison between multiple groups was analyzed by one-way anova. mann-whitney u test was used for comparison between non-normal data sets. p < . was considered statistically significant. results: . proliferation characteristics of t cells. the proliferation of cd + t lymphocytes was mainly stimulated by specific hbv polypeptide pool, and the proliferation rates of obi group and chb group were significantly higher than those of hc group ( . %, . % vs. . %), with significant difference ( . % vs. . %, p = . , . % vs . %, p < . ). . the frequency of specific ifn-c secreted t cells. the response intensity of the obi group ( sfc/ pbmcs) and chb group ( sfc/ pbmcs) was higher than that of the hc group ( sfc/ pbmcs) under the stimulation of hbv polypeptide pool, and the positive rate of t cell response to the stimulation of hbv polypeptide pool was the highest in the obi group ( . %). summary/conclusions: both obi and chb had higher rates of hbv-specific t effector cell proliferation and ifn-c secretion than the healthy control group. compared with the chb group, obi group had a higher positive rate of t cell response, which may be one of the causes of host immunity resulting in obi. further studies on other immune factors are required. background: western blood transfusion practices are currently changing due to various drivers such as blood management policies, ongoing technological developments, and new therapeutic options. in the netherlands, as in many high-income countries, these have resulted in a diminishing trend of red blood cells. therefore, it is important for blood bank management to anticipate the future demand of blood products for the sake of medium and long term decision making. to support this decision making, we have employed scenario development, which is used in many other sectors (such as finance and transportation) and can also be applied to blood transfusion. building upon a prior literature review and semi-structured interviews of international experts, we gathered experts together for scenario sessions to assess the opportunities and threats for sanquin's medium-term ( - years) strategy using an online platform and face-to-face discussions. aims: to assess for opportunities, threats, and the organizational implications thereof for the medium-term future of sanquin, the dutch national blood bank. methods: twenty-one multidisciplinary experts in blood transfusion agreed to participate and were separated into two groups for half-day interactive sessions. using an iterative process through an online platform, experts brainstormed opportunities and threats for sanquin, which were categorized into themes. these themes were ranked according to importance and certainty, and through consensus, experts chose two themes with high impact and high uncertainty. for these chosen themes, specific actions for the blood bank were listed to mitigate and/or enhance the threat or opportunity. discussions were ample throughout. results: with regards to opportunities and threats for sanquin's medium term strategy, experts brainstormed many ideas and categorized them under themes: political context/ changing legislation, novel products and alternative applications, donors, international markets, commercialization, digitalization, change in perceptions, research, demand, and organizational structure. after ranking for importance and certainty, six themes were chosen: change in perceptions, international markets, political context (opportunities), demand (opportunities), research (vulnerabilities), and donor (vulnerabilities). for each of these themes experts provided specific actions for the organizations to mitigate threats or stimulate opportunities accordingly. these actions included increased transparency and improved communication with the (donor) public, lobbying in political spheres, increased activities in educational institutes and large funding organizations, and creating and collaborating on novel blood products on an international level, to name a few. summary/conclusions: these results show that mapping and assessing a blood bank's future using a multi-disciplinary group of experts is conducive as an effective means of collection a diverse range of opportunities and threats. this provides an opportunity for blood bank management to become proactive towards these potential opportunities and threats and possibly evolve future strategies for the organization. showed that iron-deficient female blood donors were more likely to have depressive symptoms than non-iron deficient female blood donors. among participants with depressive symptoms, females with low plasma ferritin levels had significantly increased odds for reporting a "feeling of lacking energy and strength" (or = . ; % ci: . - . ). as it is known that blood donors are at an increased risk of iron deficiency, it is important to determine whether those genetically predisposed to lower plasma ferritin levels have a higher risk of experiencing the tiredness/lack of energy symptom. aims: to investigate whether there is an association between polygenic risk scores (prss) based on plasma ferritin levels and the tiredness/lack of energy symptom in blood donors. methods: the dbds is an ongoing nationwide blood donor cohort, of which genome-wide genotype data are available for , participants. genotyping was performed using the infinium global screening array (illumina â ) and imputation was achieved based on a scandinavian reference genome. ferritin prss, based on an icelandic ferritin gwas (n = , ), were calculated for all dbds participants. , female donors were available for the analysis. data on depressive symptoms were obtained using the validated major depression inventory scale (mdi), a selfreport mood questionnaire, which assesses the presence of depressive symptoms. a donor was classified as "tired" if they responded "all the time" or "most of the time" to the question "how often do you feel that you lacked energy and strength?". logistic regression analysis was performed, adjusting for age. for generating the quantile plots, the participants were distributed evenly into six quantiles based on their prs, whereby quantile contained the donors with the lowest prss (genetically predisposed to lower ferritin levels) and was set as the reference quantile with or = from the age-adjusted regression analysis (tiredness~quantile). results: prss in females ranged between - . and . (mean . ). a total of , female donors were classified as "not tired" and ( . %) were classified as "tired". no significant difference in ferritin prs was found between "tired" and "not tired" female donors (tired mean prs: . ; not tired mean prs: . ). an age-adjusted logistic regression model found this to be insignificant (or: . , % ci: . - . ), p = . ). to visualise the lack of association, a quantile plot was created, separating the female donors into six equal quantiles based on their prs. no clear trend was observed; donors with the highest prss (in quantile ) had or = . (p = . ) of being tired when compared to those in quantile (or set as ). summary/conclusions: no significant association was found between the ferritin prss of female blood donors and the tiredness/lack of energy symptom. further studies are needed to understand the effect of blood donation versus genetic constitution on tiredness among female iron-deficient blood donors. background: antiretroviral therapy (art) is critical for the control of clinical progression of human immunodeficiency virus (hiv) infections. however, the outcome of art could be limited by drug resistance-associated mutations (drms), even lead to the transmission of drug-resistant hiv to treatment na€ ıve patients such as blood donors, which is a huge concern to art. drms surveillance in hiv infected groups is strongly recommended by world health organization. characteristics of genetic diversity and drms of hiv among blood donors may provide comprehensive data to monitor viral evolution and optimize art, play important roles in blood safety. aims: limited data concerning the epidemic of hiv- subtypes and drms of blood donors is available in china. this study is to investigate genetic characteristics and drms of hiv- infected blood donors. methods: from - , blood donations collected from blood centers, covering almost the whole of china, were confirmed as hiv- positive by national centers for clinical laboratories using abbott realtime hiv- assay or cobas taq-man hiv- test, version . . then hiv- gag ( bp, hxb : - ), pol genes ( bp, hxb : - ) (encoding the whole protease (pr) and a part of reverse transcriptase (rt)) was sequenced after viral rna extraction and amplification. hiv- subtype based on gag and pr-rt regions was determined by comprehensive analyses of los alamos hiv blast tool, rega hiv- subtyping tool, phylogenetic trees and online jphmm program. drms analysis was performed in the stanford hiv drug resistance database. results: among donations, gag and pr-rt regions of samples were sequenced successfully. the distribution of hiv- genotype was as follows: crf_ bc = ( . %), crf_ ae = ( . %), b = ( . %), crf_ bc = ( . %), crf _ b = ( . %), crf _ b = ( . %), crf _cpx = ( . %), crf _ b = ( . %), crf _ = ( . %), crf _bc = ( . %), urf_ = ( . %) and urf = ( . %). of hiv- isolates were identified to have drms. there were ( . %, / ) protease inhibitors (pi) accessory drms, pi major drms and ( . %, / ) non-nucleoside reverse transcriptase inhibitors (nnrti) drms. most of blood donors with drms were crf _ae and crf _bc ( . %, / ). of pi accessory drms were q e. the pi major drms included m l, m i and n s. n s could result in hlr to atazanavir (atv) and nfv, llr to indinavir (idv) and saquinavir (sqv). v d/e is main nnrti drm ( . %, / ). a combination of v d and k r among two samples acted synergistically to reduce efavirenz (efv) and nevirapine (nvp) susceptibility. furthermore, two blood donors with k n mutation in reverse transcriptase gene had high level-resistance to efv and nvp. summary/conclusions: overall, the most prevalent subtypes among blood donors in the study were crf _bc ( . %), crf _ae ( . %). besides, other rare crfs and several urf_ and urfs were also found in these hiv- isolates, which suggested the epidemic of hiv has been shifted from high risk populations into general populations, including blood donors in china. drms were observed in . % donors in the study, which may result in resistance to pis and nnrtis, especially the hiv- variants with n s mutation in pr gene and k n mutation in rt gene. in summary, our findings indicate that increasing diversity of hiv- in blood donors and remind us the necessity of timely genotypic drug resistance monitoring and molecular epidemiology surveillance of hiv- among blood donors. background: labeling of platelets is required to measure the recovery and survival of transfused platelets in vivo. currently a radioactive method is used to label platelets. however, its' application is limited, due to safety issues and the inability to isolate transfused platelets out of the circulation. biotin-labeling of platelets is an attractive non-radioactive option, however, no validated protocol to biotinylate platelets is currently available for clinical purposes. aims: the aim of this study is to develop a simple, standardized, reproducible method to label platelets with biotin as a non-radioactive alternative to trace transfused platelets in vivo. methods: six pooled buffy coats derived platelet concentrates (pcs) stored in % plasma were biotinylated at day and day of storage. to distinguish the effect of the processing steps from the effects of biotin incubation, 'sham' samples were processed. for the biotinylation procedure, ml of pcs was washed twice and incubated with mg/l biotin, dissolved in phosphate buffered saline-pas-e ( : ), for min. stability of the biotin labeled platelets after irradiation was tested. annexin v and cd p expression were assessed as measures of platelet activation. applicability of this method to other platelet products was assessed in three pooled pcs stored in % pas-e and three single donor apheresis pcs. results: the method was reproducible performed in a closed system. after biotinylation, . % ae . % of platelets were labeled. platelet counts, ph and 'swirling' were within the range accepted by the dutch blood bank for standard platelet products. the number of annexin v positive cells was not significantly altered by the biotinylation procedure in both fresh and stored platelets. in contrast, cd p expression was increased in biotinylated platelets . % iqr( . - . %) compared to the control samples . % iqr( . - . %) on day of storage. however, biotinylated platelets were not more activated compared to sham samples % iqr( . - . %). thus only the procedural steps led to increased cd p expression and not the biotin label itself. all samples showed maximal response to thrombin receptor-activating peptide. for platelets labeled at day , a similar pattern was observed. irradiation of biotin labeled platelets did not alter the stability of the biotin label nor cell quality. furthermore this method is also applicable to pooled pcs stored in pas-e and apheresis pcs, with similar patterns in annexin v and cd p expression. summary/conclusions: we developed a standardized and reproducible protocol according to good practice guidelines (gpg) standards, for biotin-labeling of platelets for clinical purposes. the procedural steps, which are similar to the steps used for production of hyperconcentrated platelet products, led to an increased cd p expression, but did not alter the annexin v expression. this method can be applied as non-radioactive alternative to trace and recover transfused platelets in vivo. blocking activity over the prototypic chs insulator in cell lines and substantially reducing genotoxicity in a c-retroviral vector-mediated carcinogenesis mouse model. in contrast to chs , these insulators are small-sized ( - bp vs . kb) and can be easily accommodated in gt vectors without detrimentally affecting vector titers. aims: we aimed to test whether a , one of the newly discovered cis, could reduce vector-mediated genotoxicity in the challenging context of sin-lvs, by insulating a therapeutic globin-vector. methods: we tested the genotoxicity effect in the il- -dependent d cells, which upon transduction with oncogenic vectors become il- -independent, leading to transformation. d cells were transduced with sin-lvs: the b-globin-ΤΝs . . -, the insulated b-globin-a -tns . . and the oncogenic sffv-gfp-vector. transduced cells were expanded in % il- and transduction efficiency was determined by vector copy number (vcn). transduced d cells were seeded in methylcellulose with % or - % il- to detect the il- -independent and potentially transformed clones. the il- -independent clones were further expanded in % il- and infused in partially myeloablated and il- -treated c h/hej mice. wbc analysis, blood smears and bone marrow(bm) cytospins were performed. results: the a insulator did not negatively affect vector titers (ΤΝs . . , a -tns . . , sffv-gfp: . , . , . x ^ iu/ml, respectively). d cells were successfully transduced with all vectors (%vcn positive colonies: - %) and expanded up to -fold. the a -insulator decreased the number of il- -independent colonies by - % over the uninsulated vectors. the uninsulated vector-transduced, il- -independent colonies, were greatly expanded in culture with % il- over the a -transduced colonies (sffv, ΤΝs . . , a -tns . . : , , fold change, respectively). il- independence as a transformation event was confirmed in vivo by the development of overt leukemia (hyperleukocytosis, splenomegaly, bm-and extramedullary site-infiltration) in mice transplanted with the il- -independent and expanded colonies. summary/conclusions: under forced oncogenic conditions, the a insulator effectively protected a therapeutic vector from vector-mediated genotoxicity. a may serve as a safety feature in the construction of globin-sin-lvs. background: novel rare nucleotide substitutions are frequently identified in rhd, the gene encoding the immunogenic d antigen of the clinically-relevant rh blood group system, resulting in d variant phenotype. so far, it has been commonly accepted that substitutions of amino acids located either in a transmembrane or intracellular domain of the rhd protein induce weak d phenotype, i.e. reduced d antigen density at the surface of red blood cells. recently we showed by functional analysis using a "minigene splicing assay" (msa) that a decrease in d antigen expression may be due also to alteration of cellular splicing. aims: here we pay attention to the general disruption of this mechanism and the related phenotypic consequences in novel and previously reported single-nucleotide variations in rhd. we then sought to characterize functionally by msa novel candidate splicing variants in rhd. then we extended the project by studying prospectively all single-nucleotide variations reported in rhd exons, in order to assess globally the correlation between in silico prediction and functional analysis and to gain insights into the reliability of bioinformatics tools in line with the available phenotypic and/or clinical data. methods: seventeen novel or uncharacterized rhd variations, including missense, synonymous and intronic substitutions, were selected for functional analysis by msa in human cell models. a second set, including missense variants reported in rhd exons and , was further analyzed. functional data were compared with an algorithm derived from the quepasa method and tools available in the alamut suite. a published d protein model was used to visualize the location of missense amino acid substitutions and to assess potentially their respective phenotypic consequence. results: a novel "universal" minigene was validated and used successfully to characterize eleven novel splicing variants. those variants include six intronic and four missense substitutions close to the consensus dinucleotide splice sites, as well as the c. c>t synonymous variation associated with a weak d phenotype, which creates a de novo splice site. very interestingly, c. g>t (gly val; d-negative) disrupts totally normal splicing, while c. g>c (gly ala; weak d) and c. g>a (gly asp; d-negative) only partially alter the mechanism. further visualization of amino acid changes in a d model suggests that gly asp, but not gly ala, dramatically impair rhd protein structure/folding. subsequently the global analysis of mutations in rhd exons and by msa showed that inclusion of whole exon sequence in the mature transcript is significantly reduced in / ( . %) variants, which correlates well with the quepasa-like prediction (sensibility = . , specificity = . ). additionally, while normal exon inclusion is affected by c. c>g (weak d type ), the associated leu val substitution does not seem to be deleterious to the protein. summary/conclusions: on the basis of our functional data, this work shows that splicing disruption in the presence of rhd variants is a common and general mechanism that may act independently or synergistically with alteration of protein structure through amino acid substitutions, resulting in a weak d phenotype. it also illustrates the potency of combining functional tests and in silico tools towards the phenotypic/clinical interpretation of rare variants. background/aims: monetary and non-monetary incentives may support blood services in recruiting blood donors but have also been criticized for violating ethical principles and threatening blood safety by attracting donors with a high risk for infectious diseases. although incentives for blood donors have been discussed extensively over the past decades, empirical research on this topic remains limited. the aim of this study was to describe attitudes towards incentives for blood donors in europe and show donor return rates of compensated and non-compensated blood donors in south-west germany. methods: first, we present results of a secondary analysis of the eurobarometer, a nationally representative survey in all member states of the european union. in , participants were asked to evaluate eight potential incentives for blood donations as acceptable or unacceptable. these incentives were refreshments (e.g. coffee), physical check-ups (e.g. blood pressure), free (testing) laboratory parameters, free medical treatment, complimentary items (e.g. first aid kits), monetary travel reimbursements, additional cash reimbursements, and release from work. second, we conducted a retrospective analysis of donor return patterns of . compensated and . non-compensated donors who started donating blood at mobile and fixed donation sites. compensated donors received either eur as a regular reimbursement for their expenses (at a fixed donation site), in accordance with the german transfusion law, or a singular free entrance for an amusement park (at a mobile donation site). these compensated donors were compared with noncompensated donors who started either at a fixed or mobile donation site. chisquare statistics were used to test for differences in regular donor status after , , and months between compensated and non-compensated first-time donors. results: among german participants of the eurobarometer, physical check-ups ( . %), refreshments ( . %) and free (testing) laboratory parameters ( . %) showed the highest acceptance as an incentive for blood donors. travel reimbursements and free medical treatment were rated as acceptable by . % and . %, respectively. the lowest acceptance was for release from work ( . %), complementary items ( . %) and additional cash reimbursement ( . %). interestingly, the acceptance of potential incentives varies considerably across europe. in south-west germany, donor return of first-time donors differed significantly by type of compensation. among compensated first-time donors, who received eur as a monetary reimbursement, the proportion of regular donors after months ( . %) was significantly higher than among comparable non-compensated donors ( . %). however, a non-monetary compensation (free entrance) did not increase donor return rates. conclusion: the eurobarometer survey indicates that in most european countries monetary incentives are only accepted by a small minority. refreshments, checkups, free (testing) laboratory parameters and free medical treatment were most popular as incentives for blood donors. however, results of our four non-randomized donor samples from south-west germany suggest that monetary compensation may increase the likelihood of donors returning to fixed donation sites. regular monetary reward may therefore help to recruit regular donors especially in urban settings. incidentally, non-monetary compensation by a free entrance, however, may not affect donor return. background: previous research showed that whole blood (wb) donors that are temporarily deferred on-site are at higher risk of lapsing, yet very little studies have focused on differentiating the effects that different deferral reasons (e.g., travel, hemoglobin [hb]) may have on donor lapse. in addition, donor experience (i.e., firsttime or repeat donor) has also previously been found to affect donor lapse, yet novice ( - prior donations) and reactivated donors (returning after years of not donating) may respond differently. finally, it is currently unclear how and why different deferral reasons and donor experience interact in influencing donor lapse. aims: our aims were to understand ) how deferral reasons and donor experience jointly affect donor lapse, and ) why donors may lapse after temporary deferral. methods: a mixed methods approach was used. first, we used sanquin's donor database for a quantitative analysis of return behavior of all dutch wb donors between and (n = , ). the first wb donation for each donor was identified as the target donation. lapse was defined as non-return within a followup period of two years after the target donation. target donations included % new donors, % novice donors, % experienced donors, and % reactivated donors. deferral reasons included travel, hb, medical short-term (< days duration), medical long-term (> days duration), and miscellaneous. next, we interviewed temporarily deferred donors to understand the deferral process from their perspective. semi-structured interviews were used to understand how these donors cognitively and emotionally experienced on-site temporary deferral. we analyzed the interviews (using the framework approach, cf. hillgrove et al., bmc public health, ) to identify key topics and underlying themes. results: of target donations, % were deferred, mostly for travel ( %), medical short-term ( %), and hb ( %). survival and time-to-events methods showed that the different deferral reasons and donor experience levels differentially impacted donor return or lapse. importantly, experience and deferral interacted in influencing return (rate). for instance, deferred new donors were more likely to lapse than eligible or experienced donors (ors < . , p's< . ). even though deferral also affected return of experienced donors, this effect was smaller or even non-existent for certain deferral reasons (e.g., travel-and hb-related deferrals). qualitative results showed that almost all donors experienced temporary deferral as disappointing, particularly when it was unexpected (e.g., first-time deferral). not all donors (fully) understood the aims of deferral or how to prevent on-site deferral. donor beliefs about why deferral would lead to lapse were related to recurring deferrals, (mistakenly) interpreting deferral as permanent, or feeling all the effort did not pay off. summary/conclusions: reasons for temporary deferral differently impact risks of donor lapse at different levels of donor experience. for new donors all reasons for deferral are related to higher risks of lapse, whereas some reasons for deferral seem not to affect lapse among more experienced donors. unexpected or recurring deferrals may explain why donors lapse after temporary deferral. blood banks may tackle disappointment after deferral by explicitly showing that the donor is still valued, for instance by using personalized communication or offering an alternative good deed. background: blood donors experience a temporary reduction in their hemoglobin (hb) value after whole blood donation. in the netherlands, the hb value is measured before each donation, and a too low hb value (cut-off values: . mmol/l ( g/l) for men and . mmol/l ( g/l) for women) leads to a deferral for donation, in order to prevent iron deficiency and anaemia. the minimum interval between two donations is internationally set at weeks, but over time donors exhibit iron deficiency so that blood donors are temporarily deferred from donation each year. in the us % - % of deferrals are due to low hb, especially in women (editorial, transfusion, ) . due to the recovery process after each donation and the unobserved heterogeneity of donors, advanced statistical methods are needed to model the longitudinal data of hb values of blood donors. aims: to estimate the shape and duration of the recovery process of hb until the hb value has returned to its pre-donation level, to assess whether one can distinguish between donors with fast and/or slow recovery of their hb level and to predict future hb values. methods: the study is based on data of the donor insight study, which was a prospective cohort study performed by sanquin in the netherlands from to . we employed three statistical models for the hb value: (i) a mixed-effects models, (ii) a latent-class mixed effects model, and (iii) a latent-class mixed-effects transition model. in each model, a flexible function was used to model the recovery process after donation. the latent classes identify groups of donors with fast or slow recovery times, and donors whose recovery time increases with the number of donations. the transition effect accounts for possible state dependence in the observed data. all models were estimated in a bayesian way, using data of a sample of new entrant donors ( males and females). prior information from the clinical literature (boulton, vox sanguinis ) about the recovery process three days after blood donation was incorporated into the analysis since these values were not identified in the observed data. results: the results show that the latent-class mixed-effects transition model fits the data best. we also found that the recovery process shows a concave process (initially fast followed by slower recovery). the estimated recovery time is much longer than the current minimum interval of days between donations. namely, depending on the subgroup that the donor belonged to, males showed a recovery time of to days, while the estimated recovery time for females varies between to days. these results suggest that an increase of this interval may be warranted. summary/conclusions: the analysis shows the usefulness of the sophisticated statistical models that make use of historical information to model complex processes in time, in this case the hb trajectory over time across repeated donations. in addition, our results suggest a (much) longer time lag between subsequent donations to avoid anemia. background: complications of blood donation are known to reduce donors' return for future donation. the episode study (experience success in donation) showed that water drinking shortly before donation had an effect of % reduction of selfreported vasovagal reactions (vvr) in younger novice whole blood donors (wiersum-osselton, transfusion, ) . aims: in this study we analysed the return for a subsequent donation of the donors participating in the episode study. this was a predefined secondary outcome of the episode study. methods: the episode study was conducted in young (< years) whole blood donors making their first, second, third or fourth donation in geographically selected collection centres. the study interventions were: ml water drink, ml water drink or squeezing a ball (placebo intervention) during the wait after the screening interview and before phlebotomy, and a control group without intervention. participating donors were sent an online questionnaire about their experience within a week following their donation attempt. in the netherlands donors are usually invited for blood donation in accordance with hospitals' needs; the aim is to invite eligible donors at least once a year. donors were included in the return analysis if they had received at least one invitation within days after the index donation and we analysed their return for a donation attempt within days. associations with the interventions and donors' donation status, gender and reported symptoms at their index donation were analysed by calculating return percentage of eligible donors and by binomial logistic regression. results: out of the episode participants who had received an invitation, ( . %) returned within the study period. there was no difference in donor return between the two water groups. the likelihood of return was significantly increased in both water and placebo intervention donors compared to the questionnaire group (or . , % ci . - . and . , . - . respectively). return was slightly lower in women (or . , ) and lower in first-time donors (or . , . - . ) than after a nd - th donation. a staff-recorded or self-reported vvr at the index donation reduced donor return (or . , % ci . - . and or . , . - . respectively). other symptoms following donation were also associated with a lower return percentage. summary/conclusions: in this cohort of younger new and novice blood donors, . % returned for a subsequent donation. a vvr (either staff-recorded or selfreported) reduced donor return. donors who received a study intervention, either water or placebo, were more likely to return, whether or not they had suffered a vvr. it is conceivable that the mere fact of study participation could also have increased donor return, even in de questionnaire group; this will be examined in the total population of target group donors. background: the contribution of older blood donors to the blood supply is substantial. in australia, donors aged > years contributed % of all donations made in . however, with ageing, the general health status of older donors changes relatively faster, thus progressively affecting their ability to donate. an indepth understanding of the relationship between older donors' health status, future donation patterns, and risk of iron-deficiency could be of a great value to inform the blood service to predict the number of future donations, and manage the risk of iron-deficiency. aims: to understand the relationship between self-reported health, blood donation patterns, and the management of identified iron-deficiency in older blood donors. methods: we linked the sax institute's and up study baseline data collected between and to the blood service donation records, inpatient records, and medicare records*. the data-linkage was conducted by centre for health record linkage. using these linked data, we examined the relationship between health, donation patterns, and iron-deficiency and its management. results: we followed up , active whole blood donors for , eligible person-years (average age at recruitment . years, . % female, average follow up . years per-person). after adjusting for the effect of age, sex, body-mass index, education, non-english language spoken at home, country of birth, smoking, physical activity, regular use of multivitamins, alcohol consumption at enrolment, and total number of whole blood donations in the years prior to enrolment, participants with better self-reported health at recruitment showed significantly higher rates of donation. excellent, very good, good, and fair/poor health status donors made ( % ci - ), ( - ), ( - ), and ( - ) donations per person-years, respectively. iron-deficiency was identified in . % of donors in the study (n = , % ci . - . ) . sixty percent of those with iron deficiency (n = , , % ci . - . ) visited their general practitioner (gp) within days of the identification of irondeficiency, and . % ( % ci . - . ) of those visiting gp underwent further iron status examination and monitoring. after adjusting for several potential confounders including the total number of donations made during the follow-up period, excellent self-reported health status was independently associated with lower risk of iron-deficiency (p for trend = . ). summary/conclusions: information on self-reported health status can be an effective indicator to estimate the future donation yield of an older blood donor panel, and risk of developing iron-deficiency. donors with better self-reported health had a higher number of future whole blood donations and a lower risk of iron-deficiency. donors referred to gps for management of their iron status utilised the health services as expected, however there is an opportunity to improve their contact with their gps. * medicare records was provided by australian government department of human services. anaemia is a major public health issue, affecting % of the population worldwide according to the world health organization. iron deficiency is responsible for approximately half of all cases globally, with other causes including anaemia of chronic disease, other nutritional deficiencies, haemoglobinopathies, renal impairment, malignancy and bone marrow disease. in the elderly, where anaemia is even more common, the cause is frequently multifactorial. anaemia is associated with increased mortality, decreased cognitive and physical function, depressive symptoms and fatigue, particularly in older adults. poor outcomes have also been reported in anaemic patients with underlying comorbidities such as cardiac and renal disease, and cancer. within a hospital setting, anaemia is highly prevalent. preoperative anaemia, affecting up to % of patients, is associated with poor clinical outcomes including higher in-hospital mortality, longer length of stay and higher icu admission rates. anaemia management requires a proactive and multi-faceted approach, typically involving a multi-disciplinary team in which the transfusion practitioner plays a vital role. this includes screening of high-risk patients and pre-admission clinics to identify and manage patients at high risk of peri-operative anaemia. implementation of patient blood management (pbm) guideline recommendations has been shown to be effective to prevent and optimally manage anaemia within the community and hospital settings. the transfusion practitioner has key roles in the coordination, monitoring and auditing of pbm programs. active patient involvement and engagement of all members of the multidisciplinary team, including primary care clinicians, are also key to enhance the success of such programs. tp - the role of the transfusion practitioner in anaemia assessment and management: processes, tips and resources for creating background: patient blood management (pbm) is an evidenced based integrated multi-disciplinary approach aimed to improve clinical outcomes by effectively managing and conserving the patient's own blood, thus reducing unnecessary exposure to transfusion. pbm has the patient as the central focus with the aim being to improve their outcomes and include them in the process. pbm includes three pillars: ) optimising the patient's own blood, ) minimising blood loss and ) optimising a patient's physiological tolerance of anaemia. delayed assessment/management of anaemia contributes to increased health costs and unnecessary blood transfusions, and transfusion has been recognised to be associated with increase morbidity and mortality. the term transfusion practitioner (tp) includes those known as transfusion nurses, transfusion safety officers, haemovigilance officers, or patient blood management (pbm) coordinators. a key aspect of the role is driving and influencing clinical blood management activities to help align practice to internationally recognised guidelines and standards, including pbm. aim: to demonstrate the tp role in anaemia assessment & management and discuss strategies, processes, tips and resources for creating organisational and cultural change to implement pbm. context: literature outlines the importance of a multidisciplinary team to implement pbm related changes, and tps play a fundamental role within these teams to support 'buy in'. tps are seen as enablers, pulling resources together, engaging with those involved, providing education and facilitating change. they are often the ones to conduct audits, collating data and evaluating outcomes. approaches to implement pbm should be tailored to suit individual organisations. the authors will outline different approaches, highlighting where the tp can support or lead activities. one approach to anaemia assessment is to undertake an audit, examples of available tools will be shown. with this data, the tp along with the pbm team can explore options for corrective action. these could include interventions such as developing a pathway where all or a specific group of patients are assessed and or treated either at a preoperative clinic, or with their local general practitioner; through to more complicated strategies such as establishing anaemia clinics. the skills of the tp are a valuable asset to analyse clinical specialties/patient mix who should be targeted to achieve best outcomes, they know the organisation and as such are well placed to help develop a process/concept that will suit, and they can provide education and support to promote and embed these practices. conclusion: appropriate assessment and management of anaemia requires a multidisciplinary approach. the tp plays an active and crucial role in this team. examples of processes, tips and resources to support change and embed a pbm culture across the clinical spectrum will be shared. d-s - department of hematology and central hematology laboratory, inselspital bern, bern, switzerland immune haemolytic anaemia (iha) is characterized by an increased breakdown of red blood cells (rbcs) due to allo-and/or autoantibodies directed to rbc antigens with or without complement activation. clinical and laboratory signs of haemolysis in concert with the presence of a positive direct antiglobulin test characterize iha. alloantibodies formed during pregnancy and/or after prior transfusions may cause acute or delayed haemolytic transfusion reaction after transfusion of a rbc product incompatible with the specificity of the alloantibody. autoantibodies to rbcs reduce the survival of endogenous and hamper the recovery of donor rbcs after transfusion. lymphoproliferative disease, autoimmune disease, infection or drugs often cause autoantibodies to rbc, but frequently no obvious cause can be identified. besides the antigen specificity, the isotype critically determines the biological activity of rbc antibodies in vivo. the isotype defines the affinity to fc-gamma receptors on cells of the reticuloendothelial system as well as the capacity to activate the classical pathway of complement, igm being the most effective. antibody-mediated complement activation results in the opsonisation of rbc with c bc/c d with subsequent complement receptor-mediated removal by phagocytes (extravascular haemolysis). occasionally, complement activation proceeds via the activation of c to the formation and insertion of the membrane attack complex resulting in intravascular haemolysis. there is growing evidence that the innate immune system plays an important role in the pathogenesis of iha. the process of complement-mediated haemolysis results in systemic inflammation, which contributes to morbidity and mortality of patients suffering from iha. complement activation results in the release of anaphylatoxins, which are strongly vasoactive and mediate chemotaxis, inflammation and formation of radical oxygen species. release of cell-free haemoglobin and cell-free haeme upon haemolysis induces endothelial cell activation, no-depletion, cytotoxicity, ros formation and neutrophil activation. natural plasma scavengers, such as haptoglobin and hemopexin complex with their target molecules, cell-free haemoglobin and haeme, with subsequent removal of the complexes via cd and cd -mediated phagocytosis. although being positive acute phase proteins due to consumption the plasma scavengers become exhausted during chronic haemolysis thereby failing to prevent the adverse biological effects of cell-free haemoglobin and haeme in the circulation. inducible haeme oxygenase- (ho- ) is an efficient cellular scavenger by breaking down haeme into biliverdin with subsequent formation of bilirubin, co and ferrous iron with subsequent oxidation to ferric iron and storage by the ferritin h chain. ho- has an established role in the systemic protection from systemic inflammation induced by haemolytic and non-haemolytic diseases. the lecture will emphasise the role of innate immunity with a special focus on different plasma-and cellular systems involved in the pathogenesis of systemic inflammation in patients suffering from iha. d-s - understanding erythrocyte clearance c roussel, p amireault, p ndour and p buffet research and teaching, institut national de la transfusion sanguine, paris, france the clearance of erythrocytes is essential in physiology, disease and transfusion. elimination of erythrocytes altered because of senescence or pathological processes is expected to protect the microcirculation from obstruction by adhesive or rigid erythrocytes. it also contributes to the harmful consequences of anemia and hemolysis in hereditary and acquired red blood cells diseases as well as in conditions associated with auto-or allo-immunization. immunobiology has explored in great details antibody-mediated clearance of erythrocytes but conventional approaches may not be fully operational to explain delayed hemolytic transfusion reactions. some important clearance processes are independent from the recognition of molecules or antigens on the erythrocyte surface. increased erythrocyte stiffness triggers their clearance in hereditary spherocytosis, malaria and possibly also in the context of autoimmune anemia. knowns and unknowns on the mechanisms and sites of erythrocyte clearance will be presented based on a critical review of old and recent contributions. d-s - cardiovascular and endocrine-metabolic diseases and aging, istituto superiore di sanit a, rome, italy existing literature indicates that red blood cells (rbcs), beyond gas transport, exert a complex role in human physiology, being involved in many functions essential to maintain ion, metabolic and immunological homeostasis. rbcs display an immunomodulatory activity on adaptive immune cells by promoting t cell growth and survival and inhibiting activation-induced cell death. the balance between cell death and survival controls t cell homeostasis and anomalies in this balance account for diseases linked to excessive or faulty t cell growth. rbcs are able to modulate innate immunity by binding endogenous molecules such as chemokines and mitochondria-derived dna, as well as external agents such as pathogens. rbcs can also directly modulate innate immune cell activation or tolerance by controlling the maturation of the circulating pro-inflammatory subset of dendritic cells (dcs). these cells are potent inducers of primary antigen-specific t cell responses, produce tnf-a when stimulated by lps and are the principal il- p -producing cells among leukocytes. the pro-inflammatory capacity of circulating dcs is controlled by rbcs that are able to inhibit their maturation and il- production. in diseases characterized by local th inflammatory response such as psoriasis vulgaris and rheumatoid arthritis, pro-inflammatory dcs play a role in the induction and perpetuation of inflammation. collectively, literature data indicate that rbcs exert important modulatory functions that may result in immune activation or quiescence, depending on the environmental conditions. when rbcs encounter a microenvironment characterized by an intense production of ros, the rbc defenses get overwhelmed or are unable to counteract the new pro-oxidant status and become themselves a source of ros, which cause the generation of senescent signals on rbcs. the major feature of oxidized rbcs is the clustering and/or the breakdown of band . other features are the complexation of hb with spectrin, the loss of glycophorin a, the externalization of phosphatidylserine and the reduction of the "marker of self" integrin-associated protein cd . a similar senescence phenotype has been documented in rbcs during the storage period. oxidized, senescent or stored rbcs, due to surface antigen modification and to the release of pro-inflammatory molecules, fail to control immune cell homeostasis thus contributing to the perpetuation of inflammation and to the pathogenesis of immune-mediated diseases associated to oxidative stress, such as autoimmune diseases and atherosclerosis. our research group demonstrated that rbcs from patients with carotid atherosclerosis presented a senescent phenotype similar to that acquired by rbcs from healthy subjects following to in vitro oxidation. oxidized erythrocytes fail to control t lymphocytes apoptosis and lipopolysaccharide-induced monocyte-derived dc maturation, thus representing dangerous signals for adaptive and innate immunity and contributing to the pathogenesis of atherosclerosis. in conclusion, the crosstalk between rbcs and the immune system represents a mechanism to maintain immunological homeostasis. however, in high oxidative stress conditions, that can take place during a prolonged storage period or in particular diseases, rbcs can acquire a pro-oxidant behaviour and lose their functional and homeostatic features. by interfering in immune system homeostasis, rbcs become a potential tool that can be manipulated to improve or reverse pathological situations characterized by anomalies in the control of adaptive and innate immunity. transfusion therapy remains an important treatment modality for patients with sickle cell disease (scd). transfusions are given to lower the percentage of circulating sickle rbcs, and to decrease blood viscosity and have been shown in clinical trials to reduce the risk of stroke by %. however, many indications for transfusion in scd remain controversial partly due to insufficient randomized clinical trials data and in part because of our limited understanding of the complex pathologic networks leading to diverse disease complications in scd despite the common single mutation. similarly, we have incomplete mechanistic understanding of why chronic transfusion protocols must be continued for those indications supported by clinical data. the beneficial effects of transfusion therapy in scd need to also be weighed against potential transfusion risks including alloimmunization associated with lifethreatening delayed transfusion reactions, increased iron stores associated with increased oxidative stress and exposure to infectious agents. we believe that a deeper understanding of the benefits as well as harmful effects of transfusions is crucial to optimize our current transfusion therapy protocols in scd. this knowledge may provide highly needed guidance, which is currently lacking, for expansion or limiting existing indications for chronic transfusions in scd. d-s - treatment of thalassaemia department of pediatric hematology, ege university, faculty of medicine, bornova/ izmir, turkey thalassaemia is a devastating blood disease with a significant worldwide burden. annually, , children are born with a major thalassemia. life-time rbcc transfusions and iron chelation remain standard of care treatment in thalassaemia. transfusion therapy still account for significant iron overload related morbidity and mortality despite chelation therapy which is associated with poor adherence, safety concerns and varied efficacy. higher risk for transfusion transmitted infections (ttis) exists for thalassemia patients whose transfusion exposure sustains lifelong. although, the risk of transmission for traditional viruses is exceedingly rare in the modern era, emerging infectious diseases continue to be recognized as potential threats to transfusion safety. the inadequacy of blood safety points to the necessity for an additional layer of security for the blood supply in the developing world. pathogen reduction technologies for rbcc may imply a proactive, more generalized approach against new and re-emerging pathogens in the developed world and may be an ultimate safeguard for transfusion safety in the developing countries. rbc alloimmunization may become a major challenge in thalassaemia management. prevention is the key reducing the burden of alloimmunization. while the recommendation is to transfuse thalassaemic patients with c/c,e/e,kell compatible blood, it is not universally practiced. extended molecular rbc typing may be an appropriate adjunctive test in addition to serological typing before embarking on transfusion therapy. if a complete rbc antigen profile has not yet been performed in an alloimmunized patient, genotyping is the only option for accurate detection of rbc antigens that may guide the antibody identification. allogeneic stem cell transplantation (a-sct) is the only available curative therapy in children with hla matched sibling which is available to approximately % patients. in the absence of msd, mud transplant with high compatibility criteria has still limited experience. mismatch related, cord blood and haploidentical donor scts are considered experimental. a-sct carries a substantial risk of saes and mortality, both increasing with recipient age and disease severity. dfs is % in paediatric and % in adults. gene therapy for correction of the a-globin chain imbalance overcomes the problems of donor availability and immunologic complications associated with a-sct. multicenter clinical studies on gene addition therapy by using self-inactivating lentiviral vector are currently underway. recently, gene editing by either gene disruption or gene correction emerged as a potential alternative to gene addition therapy in beta-thalassaemia. a new era of novel therapeutics is unfolding in thalassemia management. several targets have been identified that can improve alpha/beta chains imbalance, ineffective erythropoiesis, or iron dysregulation and a number of those now have agents in preclinical and clinical development. hydroxyurea may improve globin chain imbalance and be beneficial for reducing or omitting transfusion requirement in selected group of patients. ruxolitinib has shown the limited effect on pretransfusion haemoglobin and reduction in transfusion needs, but allowed steady decrease in spleen volume that may serve for avoiding splenectomy in beta thalassaemia. luspatercept may restore normal erythroid differentiation and improves anaemia and hepcidin mimetics or tmprss inhibitors may modulates ineffective erythropoiesis by iron restriction and improves anaemia and organ iron loading. background: thalassaemia major (particularly b-type) and sickle cell disease (scd) are the commonest clinically important haemoglobinopathies, representing major sources of morbidity. recommended therapy is regular transfusion of safe, good quality blood, and monitoring of related complications. thalassaemia international federation (tif) guidelines, in place since , include strategies for precautionary measures and use of scientific progress in detection, inactivation and elimination of transfusion transmissible pathogens. antigen-matching strategies to avoid alloimmunization against rbc antigens and other measures including haemovigilance are key components for safe blood, alongside voluntary, non-remunerated blood donation and laboratory quality assurance programmes. aims: we present the contribution of tif and the greek experience in ensuring safety and availability of blood for thalassaemia patients applying internationally accepted standards and recommendations. methods: tif -a non-profit, patient-driven organization with national thalassemia associations in countries -promotes national control programmes for prevention and management contributing to the achievement of final cure. the main working methods are provision of education, expert support, networking, communications and projects to support improvements in the quality of health, social and other care. in greece, technical standards for blood donor selection and testing are applied in compliance with directive / /ec as well as haemovigilance programmes and traceability procedures for recording adverse reactions and events associated with the transfusion of rbcs (directive / /ec). pre-transfusion and transfusion measures recommended by the council of europe are applied. in particular, measures for transfusion of "the right blood at the right time for the right patient", leucodepletion, rbc washing and accurate cross-matching and antigen and antibody screening for an extended matching policy are practised. fresh (up to days old) rbcs are used. molecular testing for abo and rh d is performed in cases with blood group discrepancies. haemovigilance in greece covers % of total blood supply. data on ttis in , patients with thalassaemia and scd-thalassaemia in - are analysed. results: tti prevalence in thalassaemia syndromes was: hbv . % (occult type . %), hcv %, hiv . %, htlv . %, wnv . % and hev %. most frequent adverse reactions in - were allergic (incidence : ), non-haemolytic febrile reactions : , , "other" : , , alloimmunisation : , , taco : , , tad : , , tt-hev : , . hyperhaemolysis was diagnosed in two scd patients, delayed haemolytic transfusion reaction in one thalassaemia intermedia patient. trends in - show reduced incidence of alloimmunisation against rbcs. rates of allergic and pyrexial ars remained stable. no major abo incompatibility case was reported and no fatal transfusion reaction of transfusion has been recorded. summary/conclusions: blood safety in transfusion has significantly improved in high and upper-middle income but unfortunately not in lower and low income countries. blood shortages and lack of stringent protective measures for thalassaemia patients is the reality for many developing countries. tif focuses particular attention on the provision of support and the promotion of initiatives promoting the safety and adequacy of blooda key component of the lifelong management of patients with transfusion-dependent thalassaemia. background: b thalassemia is the most common group of hereditary hemoglobinopathy diseases. affected people with major thalassemia are dependent on regular blood transfusion which leads to iron overload. hepcidin is a peptide and an important regulator of iron homeostasis. expression of this hormone is influenced by polymorphisms within the hepcidin gene, hamp. aims: this study aimed to analyze the association of three polymorphisms in promoter of hamp, rs , rs , and rs with iron overload in major b thalassemia patients who do not respond to iron chelating therapy. materials and methods: a total of samples from major b thalassemia patients were collected. genomic dna was extracted and sequenced for snps rs , rs , and rs . statistical analysis was performed on ibm*spss* statistic using independent t test and fisher test. results: our analysis revealed statistically significantly difference between the level of cardiac iron concentration and c.- a>g variant (p = . ). for rs statistical analysis was on the edge of significant relationship between minor allele and serum ferritin (p = . ). all samples were homozygous for allele t of rs . summary/conclusions: different factors affect iron overload in thalassemia. our findings and others emphasize the role of hepcidin polymorphism as a key component in iron homeostasis. ten to twenty years ago, countries in south eastern africa faced the peak of the devasting hiv/aids epidemic leading to an up to years drop in general life expectancy. with the burden of hiv/aids falling mainly on the economically active population of young and medium-aged adults, the epidemic endangered social and economic stability in nations most heavily affected. today, despite aids still being a major cause of death in south eastern africa, the epidemic has become an example of public health gains that can be achieved through programmatic, evidencebased approaches that are endorsed by globally aligned policy and funding strategies. based on his work from lesotho, where one out of four among adults is infected by hiv, niklaus labhardt will take the auditors through the history of hiv programs in south eastern africa and show how innovative, pragmatic and evidence based implementation brought the region to a stage where the goal to end the aids epidemic by might be in reach. background: in france, the deferral for men who have sex with men (msm) was reduced from permanent to months in july . since this change has not impacted the residual risk (rr) of undetected hiv among blood donations, the ministry of health is considering a greater access of blood donation to msm. two scenarios have been studied: s . deferral of msm during the months preceding the donation; s . deferral of msm who have had more than one sexual partner in the months preceding the donation, similarly to all other blood donors in france. aims: to assess the impact of these two scenarios on the hiv rr estimated over the period july -december which is the baseline rr with the current month deferral for msm. methods: baseline hiv rr was calculated with the classical incidence-window-period method, where hiv incidence was derived from a detuned assay (eia-ri) detecting recent infections (≤ days) since all hiv- antibodies positive blood donations are tested with this test. the assessment of the impact of both scenarios on the baseline hiv rr was based on (i) data obtained from surveys among msm in the general population and in blood donors (compliance survey), to estimate the number of additional msm who would give blood in each scenario, and on (ii) hiv incidence estimate among these additional donors. this incidence was estimated: for s , from msm blood donors with the current deferral policy ( months) and for s , from monogamous msm of the general population. results: from july to december , / ( %) hiv- positive blood donors tested with the eia-ri were identified as recently infected, allowing to estimate the baseline hiv rr at . in million donations [ % ci: . - . ], or in , , donations. for s , the number of additional msm donors was estimated at and the number of additional hiv positive donations at . , resulting in an hiv rr of . in million donations [ % ci: . - . ] or in , , donations. for s , the number of additional msm donors was estimated at , and the number of additional hiv positive donations at . , resulting in an hiv rr of . in million donations [ % ci: . - . ] or in , , donations. sensitivity analysis shows that if both the number of msm and the hiv incidence were multiplied by . , the risk would be in , , donations for s , and in , , for s . summary/conclusions: for both scenarios, the hiv rr remains very low. for s ( -month deferral), the risk is identical to the baseline rr and is very robust to variations in the model parameters. for s (no more than one sexual partner, months), the risk is . higher than the point estimate of the baseline rr and sensitivity analysis shows that this estimate is less robust than for s , since the risk could be times higher than the baseline rr. for both scenarios, there was a modest increase in eligible msm donating. d-s - background: recruiting safe blood donors amongst the largest hiv-positive population in the world is a major challenge for south african blood transfusion services. south african donor deferral criteria and deferral periods for perceived high risk activities have evolved over time, but current risk factors for infection have not been formally assessed. in addition, most studies have reported risk factors for prevalent hiv infection whereas risk behaviours for incident infection are more informative as donations with these infections could occur during the window periods of available screening assays. aims: to identify the demographic and behavioural risk factors associated with incident hiv infection among blood donors in south africa. methods: we conducted a case-control study with incident hiv-infected blood donors compared to infectious marker negative controls. incident hiv cases and controls seronegative for hiv, hepatitis b and c viruses and syphilis were accrued from a donor pool covering of provinces in south africa. controls were frequency matched at a : ratio to cases on race, age and geography. incident hivinfections were hiv rna positive by individual donation nucleic acid amplification testing (id-nat; procleix, grifols) but antibody (ab) negative (prism, abbott) as well as those rna+/ab+ donors with recently-acquired hiv based on limiting antigen avidity (lag) assay results with normalized optical density values of < . . eligible cases and controls completed a confidential audio computer assisted structured interview (acasi) on motivations for blood donation and behavioural factors, including behaviours in the months before donation. frequencies and measures of statistical association for risk behaviours comparing cases and controls are reported after adjusting for multiple comparisons. results: from november to january , we enrolled incident hiv cases and controls; ( . %) cases and ( %) controls were ≤ years old. there were significantly more female cases ( . %) than female controls ( . %) (p < . ). significant hiv risk factors (all p < . ) reported within the -months before donation included: having a primary sex partner who is male; reporting increasing numbers of male sexual partners for both females and males; frequency of vaginal sex; frequency of vaginal sex without condoms; use of methods to clean, dry, or tighten one's anus before sex; and having visited a traditional healer for medical care. lack of medical aid (private health insurance) and reports of injury or accident with blood loss were also associated with an incident hiv infection. summary/conclusions: our study has identified a set of novel, putative risk factors for incident hiv infection among south african blood donors while confirming a number of previously known sexual risk behaviours. not having private health insurance and being injured may be markers of socio-economic context that place individuals at higher risk rather than behaviours that directly increase hiv transmission risk. the detection of risk behaviours by acasi in donors who passed predonation questionnaires and interviews suggests that acasi has the potential to improve risk behaviour identification. background: in france from to , among male blood donors (mbds) found hiv- positive at blood donation screening, % did not disclose any risk factor for hiv infection during post-donation interviews, while % reported having sex with men (msm), and % and % reported heterosexual sex (hts) and other risk factors, respectively. aims: in order to gain new insights into the risk factors for hiv- infection in mbds, we performed an hiv- genetic network analysis, including hiv- positive mbds and patients included in the french primary hiv infection anrs co primo cohort (pc). methods: mbds, who donated blood between and , and pcs, included between and , were studied. epidemiological data were collected by the french blood service (efs) upon blood donation or post-donation interviews for mbds, and upon inclusion for cps. viral strains were sequenced and genotyped in pol gene, and a recent infection assay was performed to date infection in mbds (recent: < months). a partial transmission network was computed based on tamura-nei nucleotidic distance (threshold for hiv- s/t b = . %; for non-b s/ t = . %) and assortative mixing was evaluated for mbds epidemiological data, including risk factors for hiv infection (msm, hts, others and unknown). selfreported data were then compared to assortativity-enhanced data. results: hiv- strains from mbds and pcs were linked into clusters including at least one mbd. primo-only clusters were excluded from the analysis. compared to mbds who did not cluster, those found linked to the network were younger ( vs. year-old; p < . ) and were more likely to have a recent infection ( % vs. %; p = . ). assortative mixing indexes showed that paired individuals were more likely to live in the same area (p < . ) and to have the same risk factor for hiv infection (p < . ) compared to a random distribution. imputing msm risk factor to non-msm individuals paired with msm changed the distribution of risk factors as follows: msm: % vs. %, hts: % vs. %, other: % vs. % and unknown: vs. %. summary/conclusions: after validating the assortativity of risk factors between paired individuals, and imputing msm risk factor to individuals self-reported as non-msm (including those with no identified risk factor), up to % ( / ) of mbds could be reclassified as msm. this is a worst-case scenario, as the network analysis does not exclude the possibility of one or several persons between two paired individuals (missing link). altogether, these results could help reevaluate the hiv residual risk linked to msm mbds, especially in the frame of the evolution of blood donor deferral criteria. background: although most individuals remain asymptomatic, htlv infection can lead to adult t-cell leukaemia/lymphoma (atll) and htlv- associated myelopathy (ham). the serious nature of these diseases, evidence of transmission via non-leucodepleted blood, and concern about a high prevalence among donors originating from endemic areas led to the uk blood services introducing universal blood donation screening in . monitoring through routine surveillance commenced and htlvinfected donors were invited to participate in the htlv national register cohort study to assess disease progression. these data together with evidence from lookback to previously untested donations and cost-effectiveness analysis were reviewed by an expert working group in and . aims: to describe the epidemiology of htlv among uk blood donors and evidence of disease progression from long term follow up of asymptomatic donors. methods: uk blood donations screened, and infected donors identified are reported to a national surveillance scheme. these donors are contacted, their results explained and information about clinical history and possible sources of infection are collected. where appropriate, htlv-infected donors are consented to the register, with participants completing a baseline questionnaire about their health, flagged in registries for cancer or death, and followed up about every - years. results: in the uk - , htlv-infected donors were identified. prevalence among new donors was steady around / donations. prevalence among repeat donors peaked in ( . / donations), with most in previously untested. from to , prevalence of . per , donations (average of one positive/year) was recorded. in , prevalence among new donors increased to . / , donations ( positives), with increased numbers associated with asian ethnicity and coinciding with an increase in collections from bame groups. overall, most were women ( / , %), uk-born ( / ; %) and htlv- infections ( / ; %). mean age was years. almost all positive donations were from previously untested donors ( / ), with seroconversion within a year of previous donation confirmed for only of the previously tested donors. typically, infections were associated with endemic countries (including caribbean region, west africa, iran, india and japan), acquired through breast feeding or from their heterosexual partner originating from these countries. interestingly, three were thought to have been infected through self-flagellation. a total of htlv-positive asymptomatic blood donors have already been recruited to the htlv national register, and during over -person years follow-up, none had developed atll or ham. summary/conclusions: over years of testing, few seroconverters were identified, suggesting very little ongoing transmission among uk blood donors. the lack of disease among the cohort study was also reassuring, although it is likely too early to detect associated symptoms of a slow progressing disease. recruitment to this unique dataset continues, also outside of the blood donation setting. as a result of these surveillance data, evidence from lookback, and cost-effective analysis, in nhsbt ceased to test donations from previously tested donors unless the donation was being used to manufacture a non-leucodepleted component. finland lies in northern europe between the °and °n latitude. the length of the country is km and width km. by surface area it is the fifth largest country in eu. the population of the country is . million resulting in the lowest population density in eu ( . inhabitants/km ). the whole country is inhabited, although most of the population is packed in the south. the climate of finland is influenced mainly by its latitude, but the warm waters of the gulf stream and the north atlantic drift current also play a role. due to finland's northern location, winter is the longest season. the southern portions of the country are snow-covered about three or four months of the year, and the northern regions for about seven months. long distances, low population density and the extreme climate give logistical challenges. it is estimated that these logistical costs can be as much as - % of gdp in finland. the finnish red cross blood service (frc bs) has been the nationwide blood service provider in finland since . frc bs collects annually about whole blood units of which % are collected in fixed sites and % in mobile sessions around the country. central activities (donor recruitment, medical support, production, testing, supply chain management, digital services and administration) are located in helsinki. management of transfusion is highly dependent on the logistical arrangements from blood donation sites to the central facilities and from the central inventory to the hospitals. the logistics is outsourced to three major partners all of whom have their roots in nationwide public transportation and logistics services. posti ltd is a state owned company having its roots in the national postal and telecom office. today it is the leading postal and logistics service company having the widest network coverage in finland. blood units collected at different fixed sites and mobile sessions are transported overnight by posti ltd to the frc bs central facilities by am on the day following the blood donation. posti ltd is also used for the regular deliveries of blood products to the hospitals. the other important partner is matkahuolto ltd, which was founded in the s to maintain bus stations and to serve as a common marketing company for the bus and coach services in finland. it maintains a nation-wide package delivery system based on the scheduled bus route network. matkahuolto ltd is used to transport donor testing samples from the donation sites to the central laboratory. by this arrangement it is possible to obtain most of the donor samples to the laboratory around midnight, which significantly speeds up the completion of laboratory results. the third logistics partner is jetpak finland ltd, which operates the air freight for the national flight company finnair. blood transfusion services can be managed centrally in a large sparsely populated country in a manner that is of high quality, safe and cost effective. however, the supply chain has to be planned carefully. background: elearning is a divisive topic. it is often criticised as an inferior form of education while simultaneously being promoted as a means to provide education to large numbers of people in a consistent, cost-effective manner. bloodsafe elearning australia (bea) is a government-funded blood transfusion education program that commenced in and provides courses in clinical transfusion practice and patient blood management (pbm) including: -clinical transfusion practice ( courses) -pbm: general ( courses) -pbm: medical ( courses) -pbm: acute care and surgical ( courses) -pbm: obstetrics and maternity ( courses) -pbm: neonates and paediatrics ( courses) aims: to determine the engagement, outcomes and impact of learning of bloodsafe elearning australia courses. methods: a retrospective analysis of user registrations, course completion records, course evaluation data and red cell usage in australia to determine learner demographics, and the impact on acquisition of knowledge and application to clinical practice. results: in the period from july to january : - , people registered as learners - , , courses were completed -these learners came from countries, with , ( . %) of them from outside of australia. analysis by profession shows that: - . % are nurses and/or midwives - . % are medical - . % are laboratory, anaesthetic technicians or other. analysis of user evaluation data (n = , ) from april to january shows that these courses have a positive impact, with . % of respondents stating they gained additional knowledge, . % able to make changes to clinical practice, and . % reporting that these changes will improve patient safety and outcomes. analysis of international participants shows greater benefits with . % gaining knowledge, . % able to change their clinical practice and . % believing this will improve patient outcomes. analysis of red cell usage in australia shows that since there has been a . % reduction in red cells issued. this has been achieved through a number of pbm activities including development of guidelines, research and audits, education, waste reduction strategies, and promotional campaigns. bloodsafe elearning australia courses on pbm were released in and are one part of this pbm activity, and it is notable that these courses have the widest reach as they are undertaken by a large proportion of doctors, nurses and midwives in australia who are not directly involved with the blood sector. stakeholder feedback shows that the program provides credible, consistent education that is cost-effective, reduces duplication, is 'best-practice' elearning, is readily accessible, and allows institutions to focus on the development of practical transfusion skills. summary/conclusions: this analysis shows that elearning is a well-accepted, wellutilised form of education for healthcare workers to learn about clinical transfusion practice and patient blood management, and learners gain knowledge that can change their clinical practice and improve patient outcomes. it is also likely that these courses have contributed to better utilisation of a scarce, freely-donated resource. this approach has global reach and availability, and is a cost-effective model for improving transfusion practice in the developing world by providing education for millions of healthcare workers. d-s - prospective platelet auditing: analysis of trainee compliance with guidelines pathology, columbia university, new york, united states background: apheresis platelets are a component product with high cost and limited supply. furthermore, there is a potential for severe transfusion reactions associated with this product such as transfusion related lung injury (trali), and sepsis due to bacterial contamination. therefore, transfusion guideline compliance is closely monitored by many centers. this quality assurance analysis describes our experience with prospective platelet auditing performed by physicians in pathology residency training. aims: this study aims to evaluate the ability of physicians in training to perform prospective auditing and compare policy compliance for different levels of experience. methods: this is a quality assurance analysis of a prospective platelet audit program for a -month period (january -december ). the blood bank paged the on call physician any time an order was placed for a patient with a platelet count of > , /ll, ≥ doses of platelets with no interim repeat count, or an unknown platelet count. audit records created by physician trainees in their first post graduate year (pgy ) were compared to subsequent years (pgy > ). information collected included the total number of doses requiring approval, number of products approved, training year for the approving physician, and transfusion indication. cost analyses assumed $ for a dose of platelets. descriptive statistics and comparative analysis using a pearson's chi-square were used with a difference of p < . considered statistically significant. results: there were platelet doses requiring approval with ( %) routed to the pgy group and ( %) to the pgy > group. there were ( %) ordered doses that were in compliance with hospital transfusion policy and ( %) that were not in compliance with hospital policy. of the appropriately ordered doses, the pgy group declined release of necessitating the clinical team to insist upon release without approval, and there were zero such instances in the pgy > group. when paged by the blood bank, pgy physicians approved product release not in compliance with policy for / ( %) doses while pgy > physicians approved not indicated products for / ( %) of doses (p < . ). products not indicated by hospital policy were held from release by pgy physicians for / ( %) doses and / ( %) doses by pgy > physicians (p < . ). the ordered doses not in compliance with hospital policy had an estimated cost of $ , . of this cost, there was a calculated $ , savings of products not released due to prospective auditing. there was an additional potential savings of $ , for products not indicated but released ($ , from the pgy and $ , from the pgy > group). summary/conclusions: despite a higher number of requests being routed to the more senior pgy > group, there were a disproportionately higher number of out of compliance platelet orders being released by the pgy group in addition to withholding needed products on several occasions. potential mitigation strategies for this could include a closer level of oversight for pgy physicians, and the potential monetary savings could justify a hiring a dedicated patient blood management team or quality assurance manager to monitor compliance and provide feedback to clinicians. d-s - what can we learn from how adverse events are detected? norwegian directorate of health, oslo, norway background: the primary aim of reporting systems, such as haemovigilance systems, should be learning and improvement and to identify risk areas, not simply counting errors. to understand and learn from adverse events the description of how, where and why they occur, and how they are detected, is important. to support our understanding, we use a predetermined classification that is required for reporting to eu, supplemented by classification suggested by ihn, who and ourselves. in we started asking the blood establishments what steps they would take to prevent recurrence of the event, and we added a simple classification to tell how the adverse event had been detected. aims: this study aims to analyze how different types of adverse events reported to the haemovigilance system were detected, whether the current quality management systems used in norwegian blood establishments had effective barriers and whether new barriers should be considered. methods: adverse events reported to the norwegian haemovigilance system in and were analyzed with focus on how the adverse event had been detected. in all cases classification had been performed by the reporter of adverse events and were confirmed, or reclassified if necessary, by the haemovigilance team before analysis. for analysis based on classification we used powerbi (microsoft). results: a total of adverse events were reported from norwegian blood establishments. all had been classified according to how the adverse event had been detected. twenty ( . %) adverse events were detected because of alarms or warnings from it-systems or equipment. routine checks by blood establishment staff detected ( . %) events and formal internal or external reviews detected one event. seven ( . %) events were detected because the donor became ill shortly after donation, but the illness was not caused by the donation. sixty-four percent of events were detected in a way that did not fit our present classification and hence were classified as "other". twelve out of wrong blood in tube were detected by an alarm from the it-system or routine check, as were six of events related to blood ordering, two of seven errors in testing, six of events where incorrect blood had been transfused, and eight of events related to donor selection. in reports human error was listed as the cause of the event and of these were detected by alarms or routine checks. summary/conclusions: detection of adverse events by alarms or routine checks are highly efficient when the blood establishment has historic data to check against, as exemplified with wrong blood in tube or a patient require irradiated blood components. when no historical data exists or when the quality management systems do not require routine checks, events are usually detected by chance. further analysis is needed to see if and where the quality management systems should be improved. the wide variety of adverse events can make it difficult to select which area to prioritize in the improvement work. results: the hb measurements from the finger prick were on average . g/l ( . %) higher than from the venous blood samples. the range of the difference was - -+ g/l. these results were used in order to add novel information to determine the measuring uncertainty of hb measurement in frcbs. in . % ( / ) of the donors in this study the venous hemoglobin measurements were below the cut-off point of donor eligibility. in those measurements the difference of the finger prick and venous hemoglobin measurement was at most + g/l. % of the hemoglobin results from the finger prick were in the range ae g/l compared to the venous hemoglobin results. % of the results from the finger prick were between ae g/l (the precision of the device) compared to venous hemoglobin results. in cases the difference between finger prick and venous measurements was outside standard deviations from the mean i.e. . % from the bottom (n = ) or top (n = ) of distribution. systematic errors were seen in some nurse's results both towards too low or too high hb result in the finger prick measurement and some nurses had random errors in both directions. the batch of cuvettes, donors' age, gender or the time of sampling were not detected to have an impact on the difference between finger prick and venous hb measurements in this study. summary/conclusions: the results of the poc measurements compared to the cell counter were in agreement with published data and with manufacturers' information on the device. the practical skill test is a workable way to develop competence and operations to measure the hemoglobin from the finger prick. it offers an opportunity to give personal feedback to nurses concerning their personal performance in the use of the current hb measurement technic. it also provided data on the accuracy of the poc method in the everyday donor selection process. background: whole blood donation has frequently been related to iron deficiency. a blood donor loses per donation about % (men) to % (menstruating women) of iron stores. to replenish the iron lost by blood donation in a donation interval of days, a donor needs to absorb . mg iron per day. this amount exceeds the reported maximal amount of absorbed iron of - mg/day, eventually leading to iron deficiency, with consequences such as donor deferral and possibly iron deficiency-related symptoms (decreased physical endurance, fatigue, pica, restless legs syndrome, and cognitive functions). since hb levels do not reflect donors' true iron status, measuring ferritin is a better way to detect low iron stores in whole blood donors. studies from usa and denmark showed that on the introduction of ferritin measurement with either extension of donation intervals or iron supplementation in case of low iron stores, deferral percentages for low hb declined in both male and female donors. aims: to gain more insight in iron status of whole blood donors during their donor career, how this affects donor health and which measures may prevent low iron stores in donors. methods: in the netherlands, sanquin blood bank is currently implementing a policy with ferritin-guided donation intervals. in brief, ferritin levels are measured in all new donors and in repeat donors every th donation or in case of an hb below the deferral threshold. donation intervals are extended if ferritin levels are < lg/ l, or ≥ and ≤ lg/l (for and months respectively). we anticipate that routine ferritin measurement will ultimately result in a lower prevalence of iron deficiency, less hb deferrals and improved donor retention. this will be further evaluated in a stepped wedge cluster-randomized trial 'find'em', which may also identify subgroups of donors prone to develop (symptoms of) iron deficiency. in addition, implementing ferritin screening may lead to a decreased donor availability. for this purpose, we modeled the impact of the implementation of our ferritin deferral policy on donor availability over time, which provides insight for both the expected size of the impact of the ferritin deferral policy and the time and rate at which this impact is expected to occur. this allows the blood bank to timely plan actions to counterbalance possible donor shortage and ensure an adequate blood supply. lastly, iron supplementation can be an alternative measure instead of donation deferral. as the used and recommended dosage of iron supplementation varies widely across blood services, sanquin is planning to start a new study in whole blood donors to gain evidence on the dosage and frequency of iron supplementation and its effect on ferritin and hemoglobin levels and donor health. results: the before-mentioned studies are ongoing and results will be expected from onwards. summary/conclusions: iron deficiency is a frequent side effect of whole blood donation. to prevent iron deficiency and its consequences, like donation deferral and health issues, more evidence-based insight in iron management of whole blood donors is being generated. d-s - superdonors -genetic risk profile and risk of low hemoglobin deferral background: no reliable method exists for stratifying new blood donors into those who can maintain sufficient hemoglobin (hb) levels and those who will be deferred because of a low hb (< . mmol/l [< . g/dl] for women and < . mmol/l [< . g/dl] for men). polygenic risk scores (prss) have shown great promise in predicting complex disease risk. prss could also prove useful for identification of donors genetically predisposed to low hb levels, and, thus, to an increased risk of deferral. aims: the objective of the study was to evaluate the association between prs (modelled to predict hb level as a quantitative trait) and risk of deferral as a binary outcome. methods: the danish blood donor study (dbds) is an ongoing nationwide blood donor cohort since with more than , participants. extensive genotyping has been performed on approximately , dbds participants using the infinium global screening array (illumina â ) and extended by use of imputing based on the pan-scandinavian reference genome. based on hb and genetic data on more than , icelandic individuals (an independent discovery cohort), we constructed different weighted prss for individuals from dbds. information on the donors' whole blood donations following inclusion into dbds unto end of was obtained from a nationwide donation database, scandat. the best predictor of hb among the nine prss was chosen and used in all subsequent analyses. we performed multilevel mixed-effects linear regression analysis with hb as outcome, and prs as factorized explanatory variable with cutoffs at , , , , , , , and th percentiles, respectively. moreover, the models had a two-level clustering on donor id and donation site and an id-specific random intercept; and further adjusted for: sex(binary), age(continuous), year of donation(factorized), and time since last donation (continuous). lastly, risk of deferral was evaluated in random effects logit models with similar covariables and clustering structure. results: mean number of donations per donor after dbds inclusion was . donations. generally, we observed a statistically significant positive association between prs(hb) and current hb levels. compared with donors in the - prs percentile group, donors below the th percentile had lower (- . hb mmol/l ( % ci: - . ; - . )) and donors above the th percentile higher (+ . hb mmol/l ( % ci: . ; . ) hb levels. in the random effects logit models we observed a marked increase in deferral risk with decreasing prs percentile strata. with the - prs percentile stratum as reference, donors below the th percentile and donors above the th percentile had odds ratios of deferral of or = . ( % ci: . ; . ) and or = . ( % ci: . ; . ), respectively. summary/conclusions: we found a statistically significant positive association between prs(hb) and hb levels and a markedly increased risk of deferral with decreasing prs(hb). from a scientific point of view, it is unsurprising that a genetic score for hb from an independent cohort is associated with hb in another cohort. however, from a practical perspective, prss may be the first step in a personalized donation approach to donors and their risk of deferral. background: individually calibrated inter-donation intervals for repeat blood donors have the potential to minimize the risk of iron related adverse outcomes (e.g., hemoglobin deferral or collecting a donation from a donor with low or absent iron stores) without unduly impacting the donated blood supply. machine learning has shown promise for personalized clinical risk assessment. aims: our aim is to use machine learning to develop donor-specific, personalized inter-donation intervals that minimize the risk of adverse outcomes while maintaining or improving the adequacy of the donated blood supply. methods: using a public use dataset from the reds-ii donor iron status evaluation (rise) study (cable, transfusion, ) we defined donor profiles with physiological measures including hemoglobin, ferritin and soluble transferrin receptor along with questionnaire responses regarding diet, reproductive health indicators, and demographics. we used these profiles ( features, , donations from , repeat donors) and the time until the next donation attempt to predict iron-related outcomes of the next donation attempt. possible outcomes were no adverse outcome, hemoglobin deferral, low-iron donation (ferritin < ng/ml for women and < ng/ml for men), or absent-iron donation (ferritin < ng/ml for men and women). we trained multiple machine learning models on , of the donations and selected the model with the best performance (lowest cross-entropy loss in cross validation). we assessed the best model's performance on a hold-out test set of donations, which were not used to train or select the model. we then used our model to generate risk estimates for these test donors as a function of days since their last donation, which varied from days to days. to show individual variation, we generated graphical representations of individual donors' risk over time. results: ferritin, log ferritin, body iron, and time since last donation were most useful for predicting iron-related adverse outcomes at the next donation attempt. the estimated risk of adverse outcomes at the next donation attempt varied considerably across donors. as expected, the risk of adverse outcomes days after the last donation was lower than the risk days after the last donation for most donors (risk of hemoglobin deferral decreased for % of donors; risk for low-iron donation decreased for %; and risk for absent-iron donation decreased for %). summary/conclusions: the risk of iron-related adverse outcomes as a function of time since last donation varies considerably between donors. machine learning models trained on relevant donor profiles can effectively estimate how an individual's risk will change over time. individual risk estimates could allow blood centers to protect highrisk repeat donors while continuing to allow more frequent collections from low-risk donors. further study is needed to ensure this approach works well for donor classes that are not well-represented by the rise dataset, to assess risk prediction outside of the physiological measures collected in the rise study, and to determine the viability of assigning an optimal inter-donation interval to a first-time donor using this approach. background: iron depletion is common among repeat blood donors, who contribute a large proportion of the blood supply in many countries. exogenous iron from multivitamins with iron or iron-only supplements helps prevent donation-induced iron depletion, but whether dietary iron protects against iron depletion in repeat donors has not been rigorously evaluated. available data from the reds-ii rise study in the us (cable, transfusion, ) and from the danish blood donor study (rigas, transfusion, ) suggest minor impact of dietary iron consumption on blood donor iron status in multivariable regression models. both studies, however, analyzed food items singly, such as beef or fish, rather than in aggregate, so precision was limited. aims: to evaluate whether a composite measure of dietary heme iron consumption, weighted for frequency and iron content, was associated with incident iron depletion among repeat blood donors. methods: a re-analysis of the rise cohort was undertaken to test the hypothesis that reported levels of animal protein consumption was associated with lower risk for incident iron depletion among repeat blood donors. the six blood centers participating in rise enrolled first-time and frequent donors for - month follow-up of donation frequency and iron status. a brief checklist of food categories was administered at baseline to assess frequency of consumption of several categories of animal protein that are rich in heme iron, the biochemical form of iron most readily absorbed. an iron composite score (ics) weighted for frequency and heme iron content was derived and subjects were grouped into tertiles (thirds) of ics. iron status was assayed at enrollment and study completion and at roughly one-third of donation visits in between. modified poisson regression with generalized estimating equations was used to generate risk ratios controlling for donation frequency and other covariates. results: of enrolled donors, were iron replete at baseline and completed the food checklist. the median value of the ics for each tertile (lowest to highest) was . , . , and . mg of heme iron weekly. these values are equivalent to approximately , , and servings of beef per week, or alternately twice as many servings of chicken or pork. across follow-up visits with iron outcomes assayed, almost % of donor visits were associated with intermediate iron depletion (serum ferritin < ng/ml) and . % with complete depletion of iron stores, representing serum ferritin < ng/ml. after controlling for demographic factors and donation frequency, the lowest tertile of ics was associated with a greater than fold higher risk for complete iron depletion during all follow-up visits (rr . , % ci . , . , compared to the highest tertile). summary/conclusions: in this longitudinal evaluation of dietary iron and iron status, blood donors with low intake of heme iron had an elevated risk for developing advanced iron depletion. these results suggest that blood centers should continue to recommend iron-rich diets to repeat blood donors. background: blood donors lose approximately mg of iron with every blood donation. as a result, frequent blood donors are at risk of iron deficiency and low hemoglobin (hb) levels, which may affect their health and eligibility to donate. lifestyle behaviors such as dietary iron intake and physical activity, may influence iron stores and thereby hb levels. gaining insight into associations between lifestyle behaviors and hb levels is valuable for blood supply organizations, as lifestyle behaviors can potentially be considered to prevent hb deferrals. examining the mediating role of ferritin, a measure reflecting iron stores, in these associations will help to gain insight into whether iron stores could be the limiting or enabling factor that links lifestyle behaviors to hb recovery after donation. aims: to investigate associations between lifestyle behaviors (dietary heme and non-heme iron intake and physical activity) and hb levels, and whether ferritin mediates these associations. methods: donor insight-iii (dis-iii) is a dutch cohort study of blood and plasma donors and included , donors. participants who were pregnant, had hemochromatosis, used iron supplements/medication, got a hysterectomy or bilateral oophorectomy were excluded (n = ). hb levels were measured in edta whole blood samples using a hematology analyzer (xt- , sysmex, japan) and ferritin was measured in plasma from lithium heparin tubes (architect ci , abbott laboratories, u.s.a.). dietary heme and non-heme iron intake (grams/day) were assessed using a food frequency questionnaire adapted to measure iron intake. moderate-tovigorous physical activity (mvpa, minutes/day) was assessed using the international physical activity questionnaire (ipaq)-short form. results: in total, , ( , female) participants were included. donors with higher intakes of heme iron had significantly higher hb levels (regression coefficient (b) ( % confidence interval ( % ci)) in men and women respectively: . ( . to . ) and . ( . to . ) mmol/l), independent of age, smoking, menstruation, number of donations in previous two years, donation interval, sedentary behavior, the other lifestyle variable (i.e. (non-)heme iron intake or mvpa), and initial hb level. non-heme iron intake was negatively associated with hb levels (- . (- . to - . ) and - . (- . to - . ) mmol/l for men and women respectively). ferritin mediated associations between dietary iron intake and hb levels (indirect effect in men and women respectively: . ( . to . ) and . ( . to . ) lg/l for heme and - . (- . to . ) and - . (- . to - . ) for non-heme). more mvpa was negatively associated with hb levels in men only (- . (- . to - . )), which was not mediated by ferritin. summary/conclusions: in conclusion, higher heme and lower non-heme iron consumption are associated with higher hb levels in donors via higher ferritin levels, indicating that donors with high heme iron consumption may be more capable of maintaining iron stores to recover hb levels after blood donation. more mvpa was associated with lower hb levels, although effect sizes were small, independent of ferritin. taking a donor's lifestyle behaviors into account may be useful in preventing low hb levels in blood donors. immune thrombocytopenia (itp) is still diagnosed by exclusion of other causes for thrombocytopenia. sensitive and specific detection of platelet autoantibodies may support the clinical diagnosis and prevent misdiagnosis of itp. for example, the direct monoclonal antibody immobilization of platelet antigens (maipa) assay, performed with in vivo sensitized patient platelets, offers platelet glycoprotein specific autoantibody detection with high accuracy. a drawback is that low platelet counts demand a large blood sample to have sufficient patient platelets available for analysis. circulating platelet autoantibodies are more difficult to detect by maipa; and may demand more sensitive detection platforms, such as those using surface plasmon resonance. in general, the presence of anti-gpiib/iiia, anti-gpib/ix and anti-gpv platelet autoantibodies is investigated. all these antibody specificities have been found in patients with itp. in itp, platelet autoantibody-mediated destruction via the spleen has been proposed; but also other mechanisms leading to low platelet counts in itp may play a role. inhibition of megakaryocytopoiesis by autoantibodies or by t cells has been suggested. in mice, gpib-directed antibodies induce loss of platelet-sugar epitopes, inducing hepatocyte-medicated platelet destruction. platelet autoantibodies can cause complement activation, which may contribute to platelet autoantibodymediated destruction. interestingly, we recently found that lack of detectable platelet autoantibodies is correlated with non-responsiveness to rituximab (cd moab) treatment in itp patients. in children with newly diagnosed and often transient itp, platelet autoantibodies of igg class or not often found, but of igm class are present for short duration. in conclusion, testing for platelet autoantibody characteristics and their pathologic effect may be helpful in establishing the diagnosis of itp and in choosing the best individualized therapy for itp patients. a-s - thrombopoietin receptor agonist (tpo-ra) treatment raises platelet counts and induces immunomodulation in immune thrombocytopenia (itp) jw semple , r aslam , e speck , j rebetz and r kapur lund university, lund, sweden st. michael's hospital, toronto, canada background: itp is an autoimmune bleeding disorder in which autoantibodies and/ or autoreactive t cells target the destruction of platelets and megakaryocytes in the spleen and bone marrow. several therapeutic options e.g. corticosteroids, intravenous immunoglobulins (ivig), rituximab and splenectomy are available for patients but inadequate efficacy, side effects and/or expense can make them undesirable. for the last years, tpo-ra e.g. romiplostim and eltrombopag have made a substantial contribution to the treatment of itp patient's refractory to first-line treatments. of interest, approximately % of patients that are tapered from tpo-ra therapy show a sustained response (e.g. a stable higher platelet count than before treatment). the mechanism of how tpo-ra induce these sustained responses is unknown. aims: to analyze the efficacy and immunomodulatory properties of a murine tpo-ra (amp , amgen) in a well-established murine model of itp that demonstrates both antibody-and t cell-mediated thrombocytopenia (chow l et al., blood ) . methods: platelet glycoprotein (gp) iiia (cd ) knockout (ko) mice were immunized with cd + platelets and itp was initiated by the transfer of their splenocytes into mice with severe combined immunodeficiency (scid). the scid mice were treated with either placebo or tpo-ra weekly and platelet counts and serum anti-platelet antibodies were measured weekly. results: in an initial pilot dose escalation study, control na€ ıve scid mice treated with a single subcutaneous bolus of different concentrations of murine tpo-ra ( , and ug/kg) had significantly higher platelet counts by h post infusion. in addition, compared with untreated mice, bone marrow histology revealed significantly increased numbers of megakaryocytes. maximal platelet count increases were observed with the highest tpo-ra dose and this dose was chosen to treat scid mice suffering from itp. when scid mice were treated with weekly injections of tpo-ra, platelet counts began to increase after weeks and were fully rescued to control levels after weeks post splenocyte transfer. of interest, compared with non-treated itp mice, serum igg anti-platelet antibody production in the tpo-treated mice was significantly reduced starting from two weeks post splenocyte infusion. summary/conclusions: these results suggest that murine tpo-ra is not only an efficacious therapy for murine itp but also induces immunomodulation indicative of immunosuppression. thus, this model may be able to elucidate the mechanism of how tpo-ra's induced immunosuppression in patients with itp. background: desialylation, the loss of sialic acid content on platelets (plts) glycoproteins (gps) was recently identified to contribute in immune thrombocytopenia (itp). however, the potential impact of autoantibodies (aabs) on megakaryocyte sialylation remains unclear. aims: to investigate the effect of itp aabs on plts and megakaryocytes (mks) sialylation and the subsequent impact on plt survival. methods: aabs from well-characterised itp patients induced gp-modifications were tested using a lectin binding assay. after incubation of mks or plts with itp or control sera, glycan changes were analysed by flow cytometry (fc). to investigate the impact of desialylation on plts life-span, the nod/scid mouse model was used. results: itp sera were investigated in this study. ( %) sera induced a significant increase in rca signal on plt surface compared to control sera from healthy donors (rca-mean fold increase (rca-fi): . , range: . - . , p = . ). in addition, ( %) sera caused higher ecl binding to test plts (ecl-fi: . , range: . - . , p = . ). injection of desialylating aabs resulted in accelerated clearance of human plts from the circulation of the nod/scid mice which was significantly reduced by a specific neuraminidase inhibitor that prevents background: autoimmune hemolytic anemia (aiha) is a rare autoimmune disease characterised by hemolysis associated with the presence of immunoglobulins (igg, igm, or iga) and/or components of complement system on red blood cells (rbcs), which is usually demonstrated by a positive direct antiglobulin test (dat). depending on the presence of an underlying disorder, aiha can be subdivided into primary and secondary and, by the temperature at which autoantibodies bind optimally to rbcs, into warm antibody aiha (waiha), mixed aiha (including both warm igg and cold igm antibodies), cold agglutinin disease (cad), paroxysmal cold hemoglobinuria (pch) and dat negative aiha. a frequent finding in immunohematology is the presence autoantibodies on rbcs without clinical symptoms of hemolysis that may later develop. aims: the aim of this study was to analyse serologic findings and transfusion support in patients with aiha and also to analyse dat positive patients without clinical symptoms. methods: we included data for all adult patients with aiha and dat positive patients without clinical symptoms diagnosed and/or treated at the university hospital centre (uhc) zagreb, croatia in the period between and . the diagnosis of aiha was defined by anemia with features of hemolysis (elevated bilirubin and/ or elevated lactate dehydrogenase and/or low haptoglobin level) and a positive dat. results: the data from patients ( % women) meeting the inclusion criteria was analysed. the mean age at the time of aiha was years (range - years). the mean hg level at diagnosis was . g/l. dat results were positive mostly with igg+c d ( %) or igg ( %). most patients had warm aiha ( %). other types of aiha diagnosed were mixed aiha ( %), cad ( %), pch ( . %) and dat negative aiha ( . %). in cases alloantibodies were detected with autoantibodies in the patient's plasma. patients were treated with corticosteroids as st line therapy and some with intravenous immunoglobulins (ivig). in severe or refractory patients rituximab and/or splenectomy was applied. a total of % of patients were transfused at a mean hemoglobin level of . g/l. during this period we detected dat positive patients without clinical symptoms. summary/conclusions: most patients from our study were diagnosed with warm type of aiha, followed by mixed type aiha and cad. on the other hand, pch and dat negative aiha were very rare, which is in concordance with relevant literature. most patients were transfused despite therapy used, which is not desirable in patients with aiha and should be better controlled, especially in moderate cases of anemias, where this is rarely necessary. a significant number of patients that were dat positive without clinical symptoms may later develop aiha and should be closely monitored. background: autoimmune haemolytic anaemia (aiha) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. aiha is a rare disorder and although british society of haematology (bsh) guidelines for diagnosis and treatment were published in february , there is little evidence for clinical practice in the united kingdom. aims: to investigate the approach to diagnosis, investigation and management of patients with aiha in english nhs trusts. methods: a survey of diagnostic and management practice was designed, piloted and disseminated to clinical transfusion leads in all english acute nhs trusts from november to march . completion was by a consultant haematologist treating patients with aiha but a response that represented a departmental consensus was encouraged. results: responses represented % ( / ) of english acute trusts. median number of adults with aiha diagnosed annually was - . in the preceding years, % ( / ) recalled at least one patient who had died due to aiha. although % ( / ) undertook a bone marrow biopsy in all patients, % required additional features, mainly: neoplasia, age over or being treatment-refractory. for patients with suspected drug-induced immune haemolysis, % ( / ) would not organise confirmatory tests, either because it was considered unnecessary ( / ), or because clinicians were unsure how to access tests ( / ). when determining aiha subtype, % ( / ) indicated there were no circumstances in which they would undertake cold antibody testing (antibody titre and/or thermal amplitude), with considering this unnecessary and unsure how to access tests. in clinical scenarios of patients with aiha and dat positive to c d ae igg ae cold associated symptoms, up to % ( / ) of respondents would not test for cold antibodies. for first line treatment of primary warm aiha, mean duration of prednisolone mg/kg given before judging the patient refractory and reducing the dose was . weeks (sd . , range - weeks). second line treatment of choice was rituximab for % ( / ) of respondents and splenectomy for %. intravenous immunoglobulin and splenectomy were the most cited rescue therapies. for primary cold haemagglutinin disease (chad), first line treatment was rituximab-based for % ( / ) but single agent steroid for %. we also explored the potential for future audit and research. % ( / ) of respondents were able to identify patients with aiha who previously required transfusion. % ( / ) of respondents would consider supporting a registry of patients with aiha requiring transfusion. the key questions that respondents thought a registry should address were: morbidity and mortality, treatment response, and differences in the diagnosis and treatment of aiha subtypes. there was uncertainty over access to cold and drug-induced antibody tests and clinicians do not always conduct bshrecommended cold antibody tests for aiha with c d positive dat. initial treatment of primary warm aiha and chad broadly matched bsh guidelines although % ( / ) would continue prednisolone at mg/kg beyond the recommended days before starting a taper, with greater toxicity risk. summary/conclusions: the findings support the need for a range of research initiatives, including creation of an aiha registry. preoperative anemia is common and is associated with adverse outcomes in the peri-operative period. preoperative anemia also increases the risk of allogeneic blood transfusions, which may lead to increased perioperative mortality, increased hospital length of stay and infections. diagnosis and treatment of anemia is one of the tenets of patient blood management (pbm), along with reduction in unnecessary transfusions and diagnostic phlebotomy, as well as use of hemostatic agents to reduce bleeding among many others. effective pbm is multi-disciplinary, multi-modal, timely, individualized and patient-centered. early referral to pbm and multi-modal pbm interventions are associated with greater improvement in pre-operative hemoglobin. pbm has been shown to reduce transfusions and cost, while system-wide, multi-modal programs may also be associated with improvement in mortality. using examples from our local research and practice, i will discuss three aspects of pbm. iron and erythropoiesis stimulating agents (esa) are effective, safe and used extensively in management of pre-operative anemia. previous studies have questioned whether esa leads to increased risk of thrombosis, however, recent systematic reviews do not support these concerns. another pbm approach is to reduce bleeding during surgery by using hemostatic agents such as tranexamic acid (txa). txa reduces transfusion requirements in knee and hip arthroplasty, and is safe, widely available and relatively cheap. txa is effective in both anemic and non-anemic patients, making it an attractive universal pbm strategy. finally, recommendations and evidence-based guidelines on pbm exist, including the most recent international guidelines developed by the pbm international consensus conference. however, knowledge translation in pbm has been a problem and a number of barriers to its implementation have been identified. these include perceived or actual lack of expertise, time, and resources, as well as lack of physician and patient engagement. one way to address patient engagement is education through character driven animation and we are currently trying this approach. a-s - low vs . high hemoglobin trigger for transfusion in vascular surgery (tv): a randomized clinical feasibility trial (the tv trial) background: current guidelines advocate to limit red-cell transfusion during surgery, but the feasibility and safety of such strategy remains unclear as the majority of evidence is based on postoperative stable patients. aims: we assessed the effects of a protocol aiming to restrict red-cell transfusion during elective vascular surgery. methods: fifty-eight patients scheduled for lower limb-bypass or open surgery of abdominal aortic aneurysm were randomized to a low-trigger (hemoglobin < . g/ dl, mmol/l) vs. high-trigger (hemoglobin < . g/dl, mmol/l) for red-cell transfusion throughout hospitalization. intraoperative change in cerebral-and muscle tissue oxygenation was assessed by near-infrared spectroscopy. we used a nationwide registry to collect data on death and major cardiovascular events, which encompassed ( ) severe adverse transfusion reaction, ( ) acute myocardial infarction, ( ) stroke, ( ) new-onset renal replacement therapy, ( ) vascular reoperation, and ( ) amputation of the lower limb. results: the primary outcome, mean hemoglobin within days of surgery, was significantly lower in the low-trigger group: . g/dl vs. . g/dl in the hightrigger group (mean difference . g/dl; p = . , longitudinal analysis) as were units of red-cells transfused ( background: controlled non-hemato-oncological studies have consistently demonstrated a single-unit red blood cell (rbc) transfusion policy as well as a stringent hemoglobin (hb) rbc transfusion threshold to be safe and reduce blood product utilization. yet, it is unclear whether these conclusions also apply to the hemato-oncological patient population. aims: to quantify reduction of rbc blood product utilization by the introduction of a restrictive single-unit hb-triggered rbc transfusion policy among the inpatient hemato-oncological population. methods: under the liberal transfusion protocol, applied up till november , , standard double-unit rbc transfusion was indicated with a hb threshold ≤ . g/dl and/or anemia-related symptoms. following this date, the restrictive transfusion protocol was introduced involving a lowering of threshold to . g/dl and single-unit transfusion. for patients with an asa-score of ii-iii and iv, a hb threshold of respectively ≤ . g/dl and ≤ . g/dl applied. we evaluated rbc blood product utilization over a month period starting december , (liberal protocol) and december , (restrictive protocol) in all hemato-oncological patients admitted for chemotherapeutic treatment including hematopoietic stem cell transplantation (hsct) with an expected duration of neutropenia of ≥ days. analysis of categorical and continuous data was performed using the chi-square and mann-whitney test, respectively. results: during both observational periods, patients were admitted who in total received therapy cycles, including acute myeloid leukemia (aml) induction cycles and autologous hscts. distribution of indications of admittance, median age, duration of hospitalization and duration of neutropenia did not differ between both periods. during the restrictive period, in / ( . %) of transfusions the assigned hb trigger was adhered to. the percentage of single-unit transfusion episodes increased from / ( . %) to / ( . %) with the introduction of the restrictive protocol. overall, rbc blood product utilization per admittance did not reduce under the restrictive protocol (cumulative number of transfused rbc units . (interquartile range (iqr) . - . ) during the liberal versus . (iqr . - . ) during the restrictive period (p = . )). however, rbc blood product utilization per neutropenic day demonstrated a trend towards reduction: . (iqr . - . ) versus . (iqr . - . ) units per day during the liberal versus restrictive period, respectively (p = . ). this reduction was mainly attributed to autologous hscts during which rbc blood product utilization decreased from . (iqr . - . ) to . (iqr . - . ) units (p = . ), corresponding to a reduction from . (iqr . - . ) to . (iqr . - . ) (p = . ) units per neutropenic day. moreover, / ( . %) patients during the liberal versus / ( . %) during the restrictive period did not require rbc transfusion during admittance. consequently, stringent hb thresholds as compared to single-unit transfusions seem to more strongly impact rbc blood product utilization. summary/conclusions: a hb-triggered single-unit transfusion policy results in a strong reduction of rbc blood product utilization in the setting of autologous hsct. no utilization reduction was observed among other hemato-oncological inpatient populations receiving intensive chemotherapy. further improvement of protocol adherence rates could potentially increase the benefit of this blood saving strategy. a-s - assessment of hb content of packed red cells (prbc): is it time to label each unit with hb content? r jain , n marwaha and s sachdev transfusion medicine, aiims, new delhi transfusion medicine, pgimer, chandigarh, india background: in the current era of evidence based medicine and individualized care of patients, rbc transfusion continues to be administered on the basis of conventional wisdom and the notion of an average benefit per unit. the existing blood transfusion practice based on the "number of units transfused" ignores the fact that the total hb varies markedly among the individual rbc units. aims: the present study was aimed at estimating the hb content in packed red cell unit prepared by three different protocols from ml and ml whole blood collection in three types of blood donors: replacement blood donor (rd), first time voluntary donor (ftvd) and regular voluntary blood donor (rtvd). methods: a total of prospective blood donors were included in this study. three hundred whole blood collections were performed in each of the three groups of donors (rd, ftvd, and rtvd). within each group collections were done in double ml, triple ml and quadruple ml blood bags respectively. a pre-donation venous sample was drawn from sample collection pouch for analysis in hematology analyzer as reference method for hb concentration of donor. the hb content of packed red cell units were estimated after collection of representative sample from the blood unit. volume of prbc unit was estimated by the formula of weight of blood in prbc divided by specific gravity. the hb content in unit was estimated by the formula: hb content in unit = hb value of the prbc unit (g/dl) volume of prbc unit (dl). results: in this study the hb concentration (g/dl) was comparable among three types of blood donors except that rtvd had lower hb values when compared to rd (p = . ). hemoglobin concentration of prbc ranged from . - . g/dl; mean hb was . ae . g/dl. net hb content of prbc bag was lower in prbc prepared from rd as compared to ftvd (p = . ) and rtvd (p = . ). the hb content of prbc units prepared from ml collection ranged from . - . g and from ml collection ranged from . - . g. we observed a wide range of net hb content in the prbc units and the correlation coefficient showed the strongest association of net hb content of the prbc unit with the overall volume of prbc (r = . , p = . ).higher volume prbcs have more hb content. volume of prbc bags in the study ranged from ml to ml (including both and ml collections). summary/conclusions: the present study shows that labelling hb content of the prbc unit help in better inventory management for patients. the hb content may help in decision making for release of units for paediatric/low weight versus adults/ higher weight patients. adopting a policy of optimizing dosage of rbc transfusion could have the potential to significantly improve rbc utilization and decrease patient exposure to allogenic blood. this would help further in the clinical transfusion practices based on evidence. a-s - nv more, p desai, s rajadhyaksha, a navkudkar and n deshpande transfusion medicine, tata memorial centre, homi bhabha national institute, mumbai, india background: red blood cell (rbc) transfusion is an important medical therapy benefiting the patient in a wide spectrum of clinical setting. critically ill intensive care unit patients in particular, as well as medical and hemato-oncology patients, are among the largest group of the user of rbc. periodic review of blood components usage is essential to assess the blood utilization pattern in any hospital or health care set up. our institute is a bedded tertiary care oncology centre with approximately , to , rbc transfusions annually. these transfusions are required in various stages of patient treatment like chemotherapy, radiotherapy, surgical and palliative care and there are established guidelines by the institute to be followed by clinicians. aims: to study clinical practices of rbc transfusions based on indications and to evaluate appropriateness of rbc utilization practices at the institute. methods: this was a prospective observational study, started after approval from institutional ethics committee. total of rbc transfusion events in adult patients over a period of four months were included and analyzed as per institutional guidelines for their appropriateness. details of transfusion events in form of pre transfusion hemoglobin, indication of transfusion, type of request, number of unit requested and issued, time of issue, site of transfusion and adverse reactions, etc were obtained from department of transfusion medicine records. overall statistical analysis was descriptive using spss software. chi-square test in cross tables was applied to see the relationship between different variables and considered significant if p-value was < . . results: total rbc transfusion events for patients were analyzed. there were ( %) events in patients of medical oncology and ( %) in patients of surgical oncology. maximum transfusions were received by patients in age group of to years ( %). total % of transfusion events were appropriate as per institutional guidelines. all transfusions administered in operation theatre were found to be appropriate with p value < . . inappropriateness was more %( / ) and significant in daycare setup (p < . ). anemia was the most common indication of rbc transfusion observed in % of events ( / ). total % rbc transfusions were given as planned and % as urgent transfusions. most common adverse transfusion event observed was allergic reaction in . % of total transfusion reactions. summary/conclusions: clinical practice of rbc transfusions in our hospital was largely found to be appropriate and rational with adherence to institutional guidelines. blood utilization audits should be conducted regularly by transfusion services and results should be discussed with clinician for ensuring judicious use of the scarce resource. the concept of transfusion safety officer (tso) can be introduced for better coordination between clinicians and blood transfusion services to improve practices. a-s - paul-ehrlich-institut, langen, germany on a global scale, blood services are quite diverse in regard to aspects like organisational structure, regulatory background, donor populations, donation rates or pathogen epidemiology. the world health organization (who) recognizes blood and blood products as essential medicines and provides guidance to member states for various aspects like blood regulation, best practices in blood collection and transfusion, or screening parameters. more recently a who guideline on residual risk of transfusion associated infections has been established which may facilitate decision-making for the most appropriate screening algorithms. it emphasizes the need for regional evaluation of screening assays and regulatory control of blood-associated ivds. background: babesia, a protozoan parasite that infects red blood cells, is a leading infectious cause of mortality in u.s. transfusion recipients. babesia is usually transmitted through the bite of an infected tick but may be transfusion transmitted (tt) or transmitted from mother to child during pregnancy. babesiosis is a world-wide disease; the ticks that carry babesia have a global distribution. babesiosis has been reported throughout europe and in canada, korea, india, and japan. prospective testing of blood donations in endemic areas of the u.s. revealed . % of donors were positive for babesia dna or antibodies (moritz, nejm, ) aims: -to report results of ongoing babesia clinical trial -to explain significance of babesia as a tt infection methods: in cobas â babesia for use on the cobas â / systems, is a qualitative polymerase chain reaction nucleic acid amplification test, developed to detect in whole blood (wb) donor samples the babesia species that cause human disease: b. microti, b. duncani, b. divergens, and b. venatorum. testing began in october under a u.s. fda-approved investigational new drug application. wb was collected into a proprietary medium that lysed red blood cells and stabilized babesia rna and dna. donations were collected in states with high, low, and no babesia endemicity and screened as individual blood donor (idt) samples. reactive index donations were retested in simulated minipools of (mp ), plus idt replicates with cobas â babesia. reactive index donations were also tested with validated alternate babesia nat and for b. microti igm and igg antibodies. donors with reactive results were invited to enroll in a follow-up study to test for additional evidence of infection. results: to date, , valid donations have been screened with cobas â babesia, and ( . %) were reactive. of ( %) initially-reactive donations were confirmed to be positive for babesia with a positive alternate nat or serology result. of ( %) confirmed-positive donations was collected in a state with low babesia endemicity (pennsylvania), and ( %) was collected in a state where babesia is not considered endemic (iowa). of ( %) confirmed positive donations were collected in states with high endemicity. of ( %) confirmed babesia-positive donations were detected in late fall or winter. all ( %) confirmed babesia-positive donations were reactive in mp . serology results are available for of confirmed-positive donations: at index, of confirmed babesia-positive donations were only igg-positive, while none were only igm-positive; were positive for both igg and igm. of the confirmed-babesia positive donations were negative for both igg and igm antibodies. cobas â babesia showed an overall specificity of . % ( , / , ; % exact ci: . %> %). summary/conclusions: the cobas â babesia test successfully identified babesiapositive donations, including confirmed-positive donations with no igm or igg reactivity. donations were collected in states considered low-or non-endemic for babesia. confirmed-positive donations were collected outside of the summer babesia season, when most clinical cases occur. screening with cobas â babesia continues in several laboratories. cobas â babesia is not fda licensed or available commercially. background: babesiosis in humans is caused by the erythrocytic protozoan parasite, babesia microti which is transmitted by tick bites, but is also transfusion transmitted. although frequently asymptomatic or presenting with flu-like symptoms in a normal host, if immunocompromised infection can lead to severe complications and death. b. microti is endemic in the north eastern/upper midwest united states where partial testing of donations has been implemented. in canada, a study of~ , donors did not identify any b. microti antibody-positive samples, suggesting low risk at that time, but risk should be monitored. aims: to evaluate the prevalence of b. microti-positive donations in potentially atrisk areas in canada. methods: between july and november , , blood donor samples were selected from sites near the us border. minipools were tested for b. microti nucleic acid by transcription mediated amplification (tma) using the procleix â babesia assay on the panther â system with individual testing on reactive pools. reactive donations were also tested by b. microti-specific: american red cross (arc) igg immunofluorescence assay [ifa] and imugen ifa/pcr. a subset of , tma-negative samples, primarily from the province of manitoba and eastwards to nova scotia, were tested for b. microti antibody using the arc ifa and if positive, the imugen ifa/pcr. donor age, sex, donation status, residential location and collection site location were recorded. donors who tested reactive/positive were informed, deferred and asked about risk factors (possible tick exposure and travel within canada, the usa and elsewhere, history of symptoms) and a follow-up sample was requested for supplemental testing (tma, arc ifa). reactive donations were removed from inventory. results: the , donor samples were proportional to collections in target geographic regions. age group, sex and donation status were also similar to the donor base in the collection areas. one sample from winnipeg, manitoba was tma reactive and antibody positive on supplementary testing. the donor did not remember symptoms or spending time in wooded areas. he visited the city of fargo, north dakota, usa. the subset of , samples tested for antibody were also proportional to collections in the targeted areas. four antibody-positive samples were identified from mid-september to october , all in south western ontario near lake erie. none were tma reactive. three were interviewed and none remembered any symptoms, any likely tick exposure, or relevant travel within canada or the usa. summary/conclusions: this is the largest b. microti prevalence study in canada. the results indicate very low prevalence with only tma-confirmed-positive donation of , tested. the donor was from the only region in canada where one autochthonous human case has been reported and active tick surveillance identified b. microti positive tick populations. seropositive donations in south western ontario may suggest low prevalence in that region, but interpretation is less certain due to lack of corroborating supplementary results or case history. given the close proximity to the us border, forgotten us travel should not be ruled out. a-s - background: the protozoan parasite toxoplasma gondii is prevalent in animals and humans worldwide. wild and domestic felids are the definitive hosts, and homoeothermic animals serve as the intermediate ones. after primary infection, the parasite persists lifelong within latent tissue cysts. transmission is by ingestion of undercooked or raw meat infected with cysts, by ingestion of food or water contaminated with oocysts, or transplacentally. however, it can also be acquired by blood transfusion and organ transplantation. toxoplasmosis can be a severe disease in immunosuppressed people and neonates whose mothers have acquired primary infection during pregnancy. aims: there is no information about the specific epidemiology of t. gondii infection in blood donors in portugal. therefore, we sought to determine the seroprevalence of t. gondii and associated risk factors in the population of blood donors in portugal. methods: between september and july , blood donors who attended the portuguese blood and transplantation institute blood banks located in oporto, coimbra and lisbon, and also at regional blood collection meetings, were invited to participate in the study. a written informed consent was obtained and a questionnaire about socio-demographic and behavioural variables was answered. sera were assessed for igg antibodies to t. gondii by a modified agglutination test (mat) commercial kit (toxo-screen da â biom erieux, lyon, france). results: of the blood donors (mean age . ae . ; range - years old), . % were positive for antibodies to t. gondii. when questioned about toxoplasmosis, almost half the blood donors did not have any knowledge about the disease. the centre of portugal had the highest seroprevalence ( . %) followed by the north ( . %) and the south ( . %). blood donors living in rural areas had a significantly higher seroprevalence (p = . ) than those living in urban areas. seroprevalence increased with age, with the highest seroprevalence ( . %) found in the age group of - years old (multiple logistic regression [mlr]: or = . ; ci: . - . ; p < . ), and decreased with educational level (p < . ). engaging in soil-related activities (gardening or agriculture) was significantly related to t. gondii seropositivity (p = . ). regarding water consumption, untreated sources (even though including mineral and tap water) was confirmed as a risk factor (mlr: or = . ; ci: . - . ; p = . ). other behavioural and eating characteristics, including cats in the household, eating raw or undercooked meat, processed pork products, or not washing raw fruit and vegetables before eating, were not associated with t. gondii infection. summary/conclusions: the risk of t. gondii transmission through blood transfusion is low, and serologic testing of antibodies, with exclusion of blood donors, appears not to be feasible. immunosuppressed individuals, organ transplant patients and pregnant women, should receive t. gondii antibody-negative blood components for transfusion. this study explored the epidemiology of t. gondii in portugal thus providing useful information on the seroprevalence and potential risk factors for t. gondii transmission. information regarding toxoplasmosis and its prevention could be promoted by medical and public health authorities among blood donors, and also the general population, when addressing policies, and designing screening programs, for monitoring and controlling infection and disease in portugal. a-s - who is syphilis testing excluding? c reynolds , c pearson , k davison and s brailsford nhsbt/phe epidemiology unit, nhs blood and transplant nhsbt/phe epidemiology unit, public health england, london, united kingdom background: screening for treponemal antibodies to detect syphilis in blood donors has been in place in england since the s. there have been no reported syphilis transfusion transmissions in england since records began in part due to sensitivity of the organism to cold storage. since we have specific tests in place for other sexually transmitted infections such as hiv and hepatitis b virus (hbv), the utility of syphilis screening is often questioned. however, it may be a useful proxy for higher risk behaviours particularly following shortening of deferrals for higher risk sexual behaviours from to months in november and against a background of increasing infectious syphilis in the general population. aims: here we describe the epidemiology of recently-acquired syphilis in blood donors in england compared with hiv and acute hbv infection between and . methods: monthly donation testing results are collected from the nhs blood and transplant (nhsbt) screening centres and reference laboratory. the demographics, possible sources of infection, and compliance to donor selection in confirmed positive donors are collected by proforma at post-test discussion with the nhsbt clinical team. recent syphilis is classified as igm positive and/or recent history including a negative donation within months for regular donors. results: between and there were recent syphilis cases, hiv and acute hbv infections identified by donation screening. recent syphilis rates per , donations increased from . to . whereas hiv decreased from . to . with less than positive donations in . acute hbv rates rose slightly from . to . in . males outweighed females accounting for . %, . % and . % of cases of recent syphilis, hiv and acute hbv respectively. nearly a quarter of cases of recent syphilis and hiv were seen in donors below years old. of the male donors with recent syphilis, . % reported sex between men and women (sbmw), . % sex between men (sbm) and . % did not report a risk. this contrasted with hiv where . % of male donors reported sbm, just . % not reporting a risk. overall , and males with recent syphilis, hiv or acute hbv respectively were non-compliant to the sbm deferral in place at the time of donation. in , donors with recent syphilis aged - years (median years) were excluded from the donor pool, including non-compliant to the sbm deferral. there were fewer than hiv cases identified in , all over years old, all compliant, reporting sbmw. of the hbv acute cases in , were male, all but one in the and over age-group. summary/conclusions: over the year period demographics of recent syphilis cases appeared similar to hiv with highest rates in young males, albeit lower proportions reporting sbm. following the switch to a month deferral, hiv case detection continued at low level, while syphilis screening continued to exclude higher numbers spanning all age-groups, potentially at risk of other sexually transmitted infections, including non-compliant donors. background: globally, an estimated million blood donations are given annually. in the blood service we are obliged to monitor donor health and ensure that blood donation is safe. in recent years, large-scale blood donor cohort studies in several countries have increased our knowledge on health effects of blood donation. health concerns relate both to immediate side effects like fainting and to possible long-term health issues related to repeated blood or plasma donation. the studies have provided us with data that can now help us introduce an evidence-based individualised donor care -a parallel to personalised medicine. individualised donor care in the management of iron depletion: studies have shown that a large percentage of our frequent whole blood donors, especially young women, are iron depleted. iron depletion is a strong predictor of deferral for low haemoglobin but has also been associated with e.g. restless legs syndrome and lower birth weight in children of frequent donors. the risk of iron deficiency can be mitigated by ferritin-guided prolongation of interdonation intervals or by iron supplementation. prolongation of interdonation intervals can challenge our inventories. iron supplementation, on the other hand, may give gastrointestinal side effects and other effects have been proposed as well, e.g. the masking of malignant disease and increased iron availability with subsequent risk of infection. in a large study we found that iron supplementation is not associated with increased risk of infection. what is the optimal balance between iron supplementation and prolongation of interdonation intervals? a growing number of blood services have implemented various flavours of iron management regimens generating more results. moreover, genetic studies in e.g. the uk, us, holland, and denmark can help us to find donors at high risk of iron depletion or low haemoglobin. we can use all these data in a big data approach in the pursuit of an individualised risk assessment model. other risks for blood donors: the presentation will also cover other risks associated with donation. new studies identify predictors of fainting after blood donation and also new interventions to prevent fainting. the global demand for plasma derived medicinal products has increased severalfold the last years. plasma donors are bled up to times per year in the us. very little is known about the health effects of frequent plasma donation. we know that immunoglobulin levels decrease with frequent donation but how does this affect health? summary/conclusions: the precautionary principle mitigates risk through early intervention prior to evidence. we tolerate next to no risk of transfusion-transmitted infectious diseases. the health of the blood donors, however, has not been protected similarly. we owe to our whole blood and plasma donors to investigate health effects of blood donation and ensure their safety. while the first attempts may not be perfect, we now have the tools to construct models for individualised donor care. background: in , the isbt, aabb, ihn and eba jointly issued the standard for surveillance of complications related to blood donation which categorized donor adverse events (dae) into categories ( subcategories) defined by specific criteria. severity and imputability were briefly described but were optional. subsequent validation of these categories demonstrated consistency in categorizing reactions, but wide variation in assignment of severity. in , with international input, the aabb donor biovigilance committee developed a severity grading tool (sgt) using a recognized medical adverse event grading system in which neutral grades replace subjective terms (mild, moderate, severe). aims: a large us blood collection establishment (bce) applied the draft sgt to assess its use in real cases of dae. methods: we performed retrospective analysis of all allogeneic and apheresis needle-in donations between / / to / / . severity grading was assigned based on criteria defined by the sgt. database review of dae was performed, and each event was assigned a grade based on the type of outside medical care (omc), and on specific key search terms. search terms for omc included emergency room, emergency medical response, urgent care, healthcare professional, and hospital admission. additional specific key search terms included fracture, concussion, laceration, dental injury, surgery, and hospitalization. since duration and activities of daily living (adl) limitations were not captured in our dae database, cases in our dae claims' database were reviewed. case files of events classified as grade or higher were individually evaluated by a physician for grading accuracy. results: in , , needle-in collections, , dae were graded for severity. the majority ( , , . %) were vasovagal reactions (vvr), followed by , apheresis-related ( . %), , needle-related ( . %) and allergic ( . %) events. the majority of dae were grade accounting for . % of all dae, followed by grade ( . %), and grade ( . %). there were grade and no grade dae. among the vvr, . %, . %, . % and . % were grade , , , and respectively. grade vvrs included concussions, fractures, dental injury, and pre-faint and fainting events requiring hospitalization for work-up. two grade vvrs involved falls resulting in intracranial hemorrhage requiring immediate medical intervention. for allergic and apheresis dae, there were only and grade reactions respectively, and no grade or events. needle-related dae included . % grade , . % grade , . % grade , and no grade events. of the six grade needle-related dae, were nerve irritations lasting > months, and were dvts requiring hospitalization. summary/conclusions: the sgt provided consistent assignment of severity for the majority of dae, based on outside medical care and specific key search terms. assignment of severity based on impact on activities of daily living or on duration of injury/condition requires tracking over time making such assignments more difficult; modification of our dae tracking database and claims database to capture adl and duration should improve severity assignment for such cases. background: the international haemovigilance network (ihn) has collected aggregate data on complications of whole blood and apheresis donations from member national haemovigilance systems (hvs) since . aims: we analysed the data collected in - in order to learn from the data and consider future improvement of data collection. methods: national hvs entered annual data on donor complications in the passwordprotected "istare" (international surveillance of transfusion adverse reactions and events) online database. from the donor complication spreadsheet allowed entry of separate data for whole blood donation (wbd) and apheresis, but also provided an option for entering data for all donation types. annual numbers of whole blood and apheresis donations were also collected. the harmonised international standard definitions were implemented in . reactions were captured according to severity level (mild, moderate, severe) but without distinction between donor sex or first time vs repeat donation. extracted data were used to calculate national and aggregate donor complication rates (generally per donations). results: twenty-four hvs provided figures for donations and donor complications for one or more years (median years per country was , iqr - ). the total number of country years (cy) was , covering million donations. the overall complication rate was . / donations and the median country rate was . complications/ donations (iqr . - . ). rates were generally consistent within a hvs from year to year but showed considerable variation between hvs; this was also the case for reactions classed as severe. not all countries differentiated between mild and moderate reactions and some reported all reactions under a single severity level. vasovagal reactions were the most commonly reported complication: overall . / donations, median country rate . / donations (iqr . - . ). rare and apheresis-related types of complications such as generalized allergic reaction ( . per , , cy), and major blood vessel injury (category available since ; overall . per , , cy) were only reported occasionally. eighteen of the hvs provided separate data for complications of whole blood and apheresis donations in one or more years (total cy, . million wbd and . million aphereses, total million donations). for these hvs the median rate of vasovagal reactions was . / wbd (iqr . - . ) and . / apheresis procedures ( . - . ) . reported haematoma rates were higher for apheresis than for wbd: the median per hvs was . / wbd (iqr . - . ) vs . / aphereses ( . - . ); rates of arm pain and/or nerve injury (not separated in - ) also tended to be higher: median . / with wbd, iqr . - . , vs . / with apheresis, . - . . summary/conclusions: international reporting allows hvs to study rates of blood donation complications, to distinguish between wbd and apheresis complication rates and capture information about very rare events. variability of reporting and of severity assessment between countries impairs the feasibility of comparisons between hvs. work is needed to improve harmonisation of classification of donation complications and severity assessment for data comparison and research. background: to prevent iron related hb loss, screening with ferritin testing was implemented in stockholm county (approx. registered blood donors) during a two-year roll-out. iron supplementation is offered to blood donors but has not prevented hb deferrals resulting in control visits per year. ferritin testing is hypothesized to increase iron compliance. aims: implementation of ferritin testing for surveillance of iron levels for the entire blood donor population with specific attention to new donors, women returning after pregnancy, donors with low hb and at return visit after low hb. yearly testing of plasma and platelet donors. methods: ferritin testing, following a staff education program, was implemented for applicant donors, donors with low hb, women after pregnancy, apheresis donors, followed by screening of registered blood donors per donation site. after initial screening, donors will be tested at each th (women) or th (men) donation, and with yearly testing of young adult blood donors below years. six nurses were educated to process ferritin and blood count results. donors with aberrant ferritin were contacted by letter. results: establishment of cut-off levels and algorithms for ferritin testing and iron treatment was evidence based but met practical limitations such as number of analyses and results that could be processed per week, limitations in liss set-up, blood demand contra preferred cut-offs, iron supplementation compliance. for applicant donors, hb testing show that % of female and % of male applicants cannot be registered because of low hb ( and mg/l respectively). adding ferritin testing, a preferred cut-off level of lg/ml (male reference level), would result in additionally % female and . % male applicant donor loss. as this would threat the blood demand, cut-off was set to lg/ml for women, above the female reference lg/ml, with an acceptable % loss of female applicant donors. for registered blood donors, mg of extra iron tablets were offered at low ferritin ( - lg/ml). this was sometimes combined with prolonged intervals and often repeated before ferritin was restored above lg/ml. donors with ferritin below lg/ml (in . % applicant donors, . % registered donors) or above lg/ml ( . % applicant donors, . % registered donors) were deferred and recommended to see their physician. for hb deferral, the interval was prolonged from to months, irrespective of ferritin levels. this, together with iron supplementation, resulted in an increase from % to % approved hb at return. the team of nurses processing ferritin and blood count results ( ½ nurse fulltime weekdays) reacted to approximately donor results daily, representing % of test results. summary/conclusions: many female donor applicants have suboptimal ferritin levels although they meet required hb for donation. iron treatment was added to retain donors with low ferritin as only prolonged intervals may danger the blood supply. for implementation of ferritin testing, it is necessary to have a well-functioning and agile organization to create and apply algorithms for testing, extension of intervals and iron treatment. background: since november a new donor screening regime is introduced in the netherlands where serum ferritin levels in whole blood donors are measured periodically to further control potential iron deficiency in donors. donor deferral thresholds are set at and ng/ml, and donors are deferred for six and twelve months respectively if ferritin levels are below these values. as limited information is available on ferritin recovery in whole-blood donors, the policy is introduced in parts such that adaptations to the implementation may be considered based on intermediate results and the impact of the measure on donor well-being can be evaluated. aims: to assess the effect of donor deferral on donor ferritin levels. methods: ferritin levels are measured in new donors and at every fifth donation in repeat donors. donors with ferritin levels below the indicated thresholds are deferred and ferritin is re-evaluated at their return for donation after six or twelve months. the policy allows estimating long term trends in ferritin levels post donation in repeat donors. as ferritin levels are measured in all new donors a reference distribution of ferritin levels in healthy individuals is obtained as well. results: among repeat donors % ( % of , male donors, and % of , female donors) have ferritin levels below ng/ml and are deferred for their next donation. furthermore, the distributions of ferritin levels in repeat male and female donors are similar and each has an average ferritin level of ng/ml. in contrast, we found that only % of new female donors (n = , ) and . % of new male donors (n = , ) have a ferritin levels below ng/ml. the average ferritin level in new donors was ng/ml for males and ng/ml for females. comparing the ferritin levels in new and repeat donors, a reduction in average ferritin levels between . and . was observed in female donors and between . and . in male donors. both ratios increased with donor age. at the end of december donors with low ferritin levels returned for donation after six or twelve months deferral. repeat ferritin measurements show that on average the ferritin levels in female donors increased by ng/ml per year whereas average ferritin levels in male donors increased by ng/ml per year. summary/conclusions: in line with earlier findings in literature our results show that repeat donations substantially reduce ferritin levels in repeat donors. these range from . to . in female and from . to . in male donors, who generally have higher ferritin levels. deferral of donors with low ferritin levels seems to be effective in increasing ferritin levels in donors, however, further monitoring of follow-up in repeat donors is warranted to see whether the proposed scheme allows for sufficient donor recovery over time. there are~ different rare diseases and the genes for half have been identified. approximately . million uk citizens experience premature ill-health because of a rare disease. a conclusive diagnosis is generally not reached and on average the diagnostic odyssey lasts . years. the main aims of the , genomes project are to reduce the diagnostic delay by embedding whole genome sequencing (wgs) to accredited standards in the care path of patients with undiagnosed rare diseases. the project started in and dna samples from , nhs patients and their close relatives have been analysed by wgs. here we review the results from the nihr bioresource pilot study for the , genomes project comprising phenotype and genotype data from , individuals recruited at hospitals using approved eligibility criteria for rare disease domains. we determined the population structure including ethnicity and relatedness estimation, high level phenotypes collected using human phenotype ontology (hpo) terms and quality control and summary metrics for samples and variants. the sequence resource contains over million unique variants in the , genetically independent samples, with % of variants previously unobserved in other large scale publicly available genome datasets. we summarise the curation of gene lists and pertinent findings in , unique diagnostic-grade genes for the domains. over , reports assigning pathogenic or likely pathogenic causal variants have been issued with diagnostic yields varying between domains from . % to %, while the proportion of novel causal variants ranged between % and %. we show the power of the bayesian association test, bevimed, to recapitulate decades of clinical genetics discoveries and by identifying > novel genes and novel diseasecausing variants in the non-coding space of the genome. we show how typing data for all red cell, hpa and hla class antigens can be extracted from wgs data. we mined the data from the , genomes project and similar sequence resources to re-version the probe content of the uk biobank axiom array. we genotyped donors from england and the netherlands with this new array and observed a . % concordance when comparing , blood centredetermined antigen typing results with genotype-determined ones. for the red cell and hpa antigens that were available for , donors, the array typing provided a . -fold increase in typing results per donor ( . vs . ) and rare donors were identified. using the genotyping data we identified . times more compatible units among this cohort of donors when blood demand was modelled using referral data from , english patients with more than three red cell alloantibodies. in conclusion the , genomes project has shown the feasibility of using wgs across a universal healthcare system to deliver a diagnosis for patients with rare diseases. based on these results the nhs has commissioned the analysis of another , dna samples from patients with cancer and rare disease. with analysis of dna by wgs and arrays becoming part of routine clinical care, blood services must develop competencies to extract transfusion and transplant relevant information from clinical-grade genotyping data. next-generation sequencing (ngs) enables the sequencing of thousands of genes, the exomes, and even entire genomes by single experiments at a reasonable price. there have also been advances in cytometry: use of antibodies with different fluorescence tags enables simultaneous monitoring of the expression of dozens of antigens. however, immunological methods cannot detect every variant discovered by ngs. genome sequencing reveals not only the exome but also the regulatory elements of transcription/translation, such as promoters and enhancers. rna sequencing determines which genes and spliced transcripts are expressed. it is amazing to realize how much this novel technology has been contributing to the better understanding of various biological phenomena. since the initial cloning of the human blood group a transferase cdnas in the early s, we have been studying the abo genes, a and b glycosyltransferases, and a and b oligosaccharide antigens. various scientific disciplines including genetics, immunohematology, biochemistry, enzymology, and glycobiology have been applied to their study. we have made several important scientific contributions. we demonstrated the central dogma of abo: the a and b alleles at the abo genetic locus encode a and b transferases, which synthesize a and b antigens, respectively. we elucidated the allelic basis of the abo system. we found amino acid substitutions between a and b transferases and inactivating mutations in o alleles. we became the first who succeeded in the abo genotyping, discriminating the aa and ao genotypes, as well as the bb and bo, which was impossible by the immunological approach. we have taken a simple experimental strategy: preparation of eukaryotic expression constructs of a/b transferases and their derivatives, dna transfection to human hela cells or their sublines, and immunological detection of the a/b antigen and/or biochemical examination of the enzymatic activity. we used this to show that the codons and are crucial in determining the sugar specificities of galnac/galactose of a/b transferases. we also identified mutations in several subgroup alleles causing restricted substrate use and diminished transferase activity. we also showed that cis-ab and b(a) alleles specifying the expression of both a and b antigens by single alleles encode a-b transferase chimeras. since then, other scientists have characterized more than abo alleles. recent human genome sequencings have identified many more single nucleotide polymorphism variations. the genome sequences of many species are also available. taking advantage of those sequences and associated information, we have expanded our research to include evolutionarily related a , -gal(nac) transferases and their genes and scaled it up from the genetic to genomic level. in this talk, i would like to present the followings. : our elucidation of the molecular genetic basis of the abo blood group system (as requested by the organizer); : identification of novel abo alleles by others; : more snp data from genome sequences and potential problems for abo genotyping; : findings obtained from analysis of abo genes from other species; bacteria, vertebrates, to primates; : a , -gal(nac) transferases and their genes and the crosstalk between a transferase and forssman glycolipid synthase (fs); and : the potential causes of generation of abo polymorphism and of species variations of the gbgt gene specifying the fors polymorphism. in recent years, there has been a concerted effort to improve our understanding of the quality and effectiveness of transfused blood components. the expanding use of large datasets built from electronic health records allows the investigation of potential benefits or adverse outcomes associated with transfusion therapy. together with data collected on blood donors and components, these datasets permit an evaluation of the effect of donor and blood component factors on transfusion recipient outcomes. large linked donor-component recipient datasets provide the power to study exposures relevant to transfusion efficacy and safety, many of which may not otherwise be amenable to study for practicality or sample size reasons. analysis of these large blood banking-transfusion medicine datasets allow for characterization of the populations under study and provide an evidence base for future clinical studies. knowledge generated from linked analyses has the potential to change the way donors are selected and how components are processed, stored and allocated. however, unrecognized confounding and biased statistical methods continue to be limitations in the study of transfusion exposures and patient outcomes. results of observational studies of blood donor demographics, storage age, and transfusion practice have been conflicting. this review will summarize statistical and methodological challenges in the analysis of linked blood donor, component, and transfusion recipient outcomes. c-s - a large deletion spanning xg xg and gyg gyg constitutes a genetic basis of the xg null phenotype, underlying anti-xg a production background: the xg blood group system comprises the homologous antigens xg a and cd . the cd gene resides within pseudoautosomal region on the short arms of the sex chromosomes and thus mimics autosomal inheritance. xg, on the other hand, is x-linked and straddles the pseudoautosomal boundary; a truncated pseudogene composed of only the first exons remains on the y chromosome and therefore males carry a sole full-length copy of xg. this phenomenon manifests as asymmetric frequencies of the xg(a+) phenotype between the sexes: roughly % of women and % of men are xg(aÀ). also, whilst xg a immunization is rare, the vast majority of all anti-xg a makers reported are men. recently, we reported that the rs c variant disrupts a gata motif between xg and cd . this abolishes erythroid xg a expression and causes the common xg(a-) red cell phenotype. however, rare individuals who produce anti-xg a cannot be accounted for by this finding. we hypothesized that a structural defect in the xg coding region causes the true xg null phenotype underlying anti-xg a production. aims: we undertook to determine a genetic explanation for anti-xg a production. methods: genomic dna (gdna) was extracted from two whole blood samples and cell-free dna (cfdna) from archived plasma samples from donors producing anti-xg a ; one cfdna sample was from a female donor and the rest from males. polymerase chain reaction (pcr) experiments, sanger sequencing, and database searches were performed to identify and confirm the deletion. aliquots of gdna from four males reported to carry a similar deletion in the genomes project were also tested. results: in one gdna sample, exon-specific pcr identified a deletion involving part of xg and the downstream gene gyg . database searches indicated that the most likely deletion was the infrequent genomic structural variant esv reported in the genomes project. further analyses with a short ( bp) and a long ( bp) pcr amplicon across the suspected breakpoint determined that this deletion was approximately kb and corresponded well with esv . this finding was confirmed in the second gdna sample. given the rarity of anti-xg a producers, we decided to test for the same deletion in cfdna extracted from old archived plasma samples. of the cfdna samples, poor quality in four samples prevented amplification even from control reactions and one was contaminated with bacterial dna. in the remaining nine samples, eight could be amplified for the deletion-specific -bp short amplicon while one was negative for the deletion. sanger sequencing of the amplicons revealed a heterogeneous repetitive dna element, ltr b, hinting at a previously-reported recombination event. this deletion was not detected in the samples from the genomes project which reiterates the previously identified deficiency in data interpretation and reporting for deletions. summary/conclusions: a large deletion disrupting the xg and gyg genes accounts for the xg null phenotype underlying the majority ( of ) of anti-xg a makers. one sample remained unexplained, indicating further heterogeneity to be explored. our data help to explain why anti-xg a production is rare and has primarily been reported in men. background: s and s antigens encoded by gypb differ by one nucleotide (nt), c. c>t, p.thr met. two different genetic backgrounds are associated with silencing of s antigen and a u+ w phenotype. these include the nt change c. c>t (p.thr met) causing partial exon skipping and designated gybp* n. (gypb*ny) and c. + g>t, an intron change causing complete skipping of exon , designated gypb* n. (gypb*p ). aims: samples from three individuals, a previously transfused african american sickle cell patient (p ), a blood donor of unknown ethnicity (p ), and an african american patient (p ) (lapadat r. aabb abstract) were investigated for discrepant serologic and molecular results when determining s and s phenotype. methods: standard methods were used for rbc typing with licensed s and s reagents and rbcs from donor p were also tested with monoclonal and polyclonal anti-s and anti-s. dna was isolated from wbcs and hea precisetype performed on p and p . p was also tested by gypb*s/s as-pcr, exon pcr-rflp for c. + g>t and as-pcr for c. c>t. p was tested for gypb*s/s and c. c>t and c. + g>t changes by a real-time pcr-fluorogenic ' nuclease taqman chemistry. for all, gypb exons - were amplified and sanger sequenced and aligned to consensus using clustal x. results: rbcs of all three probands typed s-and strongly s+ while dna testing indicated c. t/c (gypb*s/s). assay for the two common gypb*s silenced alleles, c. c>t and c. + g>t, indicated all three samples had both silencing mutations previously reported to be independently associated with a sÀu+ w phenotype. hea precisetype could not interpret this novel allele combination and indicated gypb*s as pv (possible variant). samples were confirmed to be heterozygous for c. c/t, c. c/t and c. + g/t by exon specific sequencing and as-pcr, pcr-rflp and real-time pcr. by long range sequencing of gypb, all three were heterozygous c. t/g and c. a/g (p. leu/trp), c. a/t (p. thr/ser), c. a/g and c. g/t (p. glu/gly), c. c/t (s/s), c. g/t (p. val/leu), c. c/t (p. thr/met), and c. + g/t. all samples were also c. g/g (p. ser) and heterozygous for several previously recognized silent changes in exon , c. t/c, c. t/c and c. a/g. summary/conclusions: we report a novel silenced gypb*s allele that can confound gypb genotyping interpretation. the allele was found in three probands associated with a sÀs+ phenotype. in these samples, two changes previously reported to be inherited independently and both associated with silencing of s antigen are carried on the same allele. dna-based testing could not rule out that c. t or c. + t are separate and that gypb*s was also silenced. robust s+ rbc typing indicates both changes are on gypb*s. gene sequencing confirms the c. + t change is on a gypb* n. [gyp*he(ny)] background. c. c>t (rs ) and c. + g>t (rs ) have a frequency of . respectively . in the african population (exac). although we identified samples, the frequency of this novel allele is unknown. background: the lutheran blood group system currently consists of antigens. these antigens are of low immunogenicity and may cause mild-to-moderate transfusion reactions and hemolytic disease of the fetus and newborn. the activation of lu-glycoprotein/bcam on red blood cells (rbcs) and its interaction with laminin- a is thought to play a role in vaso-occlusion in sickle cell disease and other hematological disorders. the two glycoprotein isoforms lu-glycoprotein and bcam are encoded by the bcam gene which consists of exons located on chromosome q . . a number of rare lutheran phenotypes have been previously recorded in israel, including lu:- , observed among iranian jews, lu:- in one thalassemia patient and one case of lu:- . in this report, a previously transfused pregnant arab patient with b-thalassemia intermedia was investigated because she presented with an antibody to an unknown high frequency antigen (hfa), potentially related to the lutheran system. aims: to characterize a novel lutheran antigen through serological and molecular investigation of a patient with a lutheran related antibody. methods: initially, the red cell phenotype and the presence of a lutheran related antibody in the serum of the patient were detected by standard serological techniques, utilizing enzyme treated and chemically modified cells and rare cells and sera from the nbgrl collection. further serological investigations were carried out using standard iat (liss tube and bio-rad gel) technique. plasma inhibition studies were performed using soluble recombinant lu protein (srlu). eluates were prepared using acid elution method (gamma elu-kit ii). genomic dna was isolated from whole blood and all exons of the bcam gene were amplified by pcr and directly sequenced by sanger sequencing. the impact of the identified mutation on lutheran glycoprotein structure was studied by molecular dynamics calculations. results: the patient's plasma reacted with all cells tested, except for three examples of in(lu) cells and cells treated with -aminoethylisothiouronium bromide, trypsin and a-chymotrypsin. inhibition studies with srlu protein showed complete inhibition of the antibody, thereby confirming the antibody to be directed toward an epitope on the lu-glycoprotein. in addition, testing of inhibited plasma revealed the presence of underlying anti-e and anti-fy a . an eluate was prepared to isolate the patient's lu-related antibody and this eluate was found to be incompatible with examples of lu:- , lu:- , lu:- , lu:- , lu:- , lu:- , and lu:- cells, whereas in(lu) were compatible. results of serological typing of the patient's cells, for lu system hfas, could not be conclusively determined due to the patient having been recently transfused. however, results suggested (through absence of mixed field reactivity) the patient's cells to be lu: - , , , , ,- , . bcam sequence analysis confirmed the patient to be lu* , lu* and revealed a novel homozygous mutation c. a>c in exon , encoding p.lys gln in the lutheran glycoprotein. summary/conclusions: a novel homozygous mutation c. a>c (p.lys gln) in exon of bcam was identified in a patient with an antibody to a lutheran hfa. serological and genetic evidence presented here indicates discovery of a novel antigen of the lutheran blood group system, which we propose to name lura. background: lutheran glycoprotein and basal cell adhesion molecule antigen b-cam are two isoforms of a type i membrane glycoprotein residing on red cell surfaces. both isoforms are adhesion molecules with the main function of laminin binding, and both carry antigens of the lutheran blood group system (lu). the system currently comprises antigens, all encoded by mutations in the alternatively spliced single gene bcam located on chromosome . currently, isbt lists high incidence antigens in the system. aims: we report a case study of an individual with an unidentified alloantibody to high incidence antigen present in her plasma. samples from the patient and her family were investigated. we provide here serological and molecular evidence for a novel high incidence antigen of the lutheran blood group system. methods: serological investigations were performed by standard iat (liss tube and bio-rad gel) technique. plasma inhibition studies were completed with soluble recombinant lu (srlu) protein. genomic dna was isolated from whole blood of the patient and her family members; all the exons of the bcam gene were amplified by pcr and analysed by direct sanger sequencing. the impact of the identified mutation on lutheran glycoprotein structure was studied by molecular dynamics calculations. results: presence of a lu-related antibody in the patient's plasma was confirmed, reacting moderate strength by liss iat with untreated and papain treated cells. cells from the patient's mother, father and two siblings were all incompatible with her plasma, though weaker than panel cells, reflecting dosage. only in(lu) cells were compatible with patient's plasma. the antibody was successfully inhibited with srlu protein, thereby confirming the epitope recognised by the antibody resides on the lutheran glycoprotein. the patient's cells were found to be lu: - , , , , , , , , . bcam sequencing revealed a novel homozygous mutation c. g>a in exon , encoding p.val met in the lu glycoprotein. the c. g>a change appears to be an extremely rare mutation, listed in gnomad database with a frequency of . - and with no known homozygous examples. homology model of the novel lutheran glycoprotein was subjected to all-atom molecular dynamics calculations to analyse potential conformational changes. summary/conclusions: we report serological and genetic evidence for a novel antigen of the lutheran system, which we propose to name lunu. the evidence will be submitted to the isbt red cell immunogenetics and blood group terminology working party for consideration for allocation of antigen status. the absence of this high incidence antigen arises from a rare single amino acid change p.val met in the lutheran glycoprotein and the presence of anti-lunu in the patients' plasma was presumed to have been made in response to previous pregnancy. on native, papain-treated (diagast) and trypsin-treated (sigma) rbcs. genomic dna was extracted from peripheral blood cells by an automated method, amplified by sema a exon-specific primers and sequenced. results: the proband was a -year-old female patient of moroccan origin, group a, d+c+e-c+e+, k-, without transfusion history. she was hospitalized at weeks gestation for a blighted ovum requiring a manual vacuum aspiration, with a significant hemorrhage risk. a rbc antibody screening was performed by a first laboratory. the antibody reacted + by iat on all native reagent rbcs, with negative autocontrols, but was nonreactive on papain-and trypsin-treated cells. an anti-ge was initially suspected, due to the pattern of reactivity and ethnic background. new blood samples were referred to our national immunohematology reference laboratory. the antibody showed the same profile. anti-ge and anti-ch could be ruled out. the serum was nonreactive with two jmh:- and positive with two jmh:- samples. the patient was found to be jmh positive. in addition, a soluble recombinant jmh protein (jmh imusyn/inno-train) fully abolished the reactivity of the panagglutinating antibody. the antibody was an igg . overall, these results were consistent with a probable jmh variant and prompted us to perform sema a sequencing. three nucleotide changes were found, in homozygous state: a rare nonsynonymous change in exon , c. g>a (p.asp asn, rs , maf < . , sift score = ); a common synonymous change in exon , c. a>g (p.gln gln, rs , maf = . ); a rare non-synonymous change in exon , c. g>a (p.arg his, rs , maf < . , sift score = . ). the analysis of surface accessibility of asp and arg using the d structure of sema a (rcsb pdb- nvq https://www.rcsb.org/structure/ nvq) showed that only arg was predicted to be an exposed-epitope. interestingly, all other reported jmh variant phenotypes correspond to an arginine substitution. of note, we retrospectively found another individual of algerian ancestry (pregnant woman) with a pan-agglutinating igg antibody showing a similar pattern of reactivity, and with the same three changes in sema a. we unfortunately could not perform a cross-compatibility testing with the proband (no material left and unsuccessful contact). summary/conclusions: serological and molecular studies allowed us to provide evidence for a novel high-prevalence antigen in the jmh blood group system, very likely encoded by the p.arg his substitution in sema a. we propose to provisionally assign the name jmh for this antigen. interestingly, our two unrelated jmh:- individuals were from north african ancestry. background: the abo system was discovered almost years ago and the underlying structures later elucidated as carbohydrates carried by glycoproteins and glycolipids. the terminal trisaccharides galnaca (fuca )gal and gala (fuca )gal constitute the clinically important a and b epitopes, respectively. clausen et al. (pnas, ) showed that the a antigen could be extended to a repetitive glycolipid a epitope, galnaca (fuca )galb galnaca (fuca )galb glcnac-r. however, extended forms of b antigen have not been described. we encountered two related situations with unexplained serological reactivity. firstly, enzyme-conversion to group o treatment of group b (b-eco) red blood cells (rbcs) with a -specific gh family exogalactosidase (bzyme) abolishes b antigens as detected by hemagglutination and flow cytometry with all monoclonal anti-b tested. despite this, % of group o plasmas have been reported to give positive crossmatch results with b-eco rbcs. secondly, plasmas from ab and b individuals of the globoside-deficient p k phenotype contain anti-p and anti-px but react stronger with bpp-rbc than with app/opp-rbc. based on these findings, we hypothesized the presence of a bzyme-resistant, b-related glycolipid. aims: to identify the molecular basis of the enigmatic serological observations outlined above. methods: plasma and eluates from an a b individual with the p k phenotype were investigated by hemagglutination and flow cytometry, as were eluates from b p k and o plasma. rbc membrane glycolipids were extracted from two batches of pooled, expired group b-rbc units (frozen -litre reference preparation and confirmatory preparation from freshly collected units). native or enzyme-treated glycolipid fractions were analysed by liquid chromatography electrospray ionizationmass spectrometry (lc-esi/ms) and immunostaining of thin layer chromatography (tlc) plates. antigen expression in the h+bÀ human erythroleukemia (hel) cell line was analysed by flow cytometry following overexpression of selected glycosyltransferases. results: anti-p-depleted eluates made from a b p k plasma contained anti-px and antibodies of unknown specificity that reacted stronger with native or papaintreated bpp-rbcs compared to app/opp-rbcs. anti-px was removed by adsorption onto opp-rbcs but reactivity (here designated anti-extb) remained against b/bpp/b-eco rbcs. lc-esi/ms of glycolipid fractions from group b units revealed an unknown hexnac-hex-(fuc-)hex- hexnac-hex- hex heptasaccharide. upon b-nacetylhexosaminidase treatment of this candidate structure, a group b type hexasaccharide was produced, demonstrating that the terminal hexnac of the hexnac-gala (fuca )galb glcnacb galb glc heptasaccharide was b-linked. since the discovery of the anti-platelet effects of aspirin platelets have been a major therapeutic target for pharmaceutical companies and also a very profitable target. however, the effectiveness of aspirin has also been a challenge as it is an inexpensive drug and any new agent needs to show clear benefit over aspirin. furthermore the risk of bleeding from anti-platelet agents, especially cerebral bleeds, has also presented challenges. in the 's orally active gpiib/iiia antagonists were considered to be the 'super aspirin' but clinical trials showed increased mortality and ultimately this class of drugs was relegated to iv use only in high-risk patients. gpib/ix/v antagonists were also a promising drug target but no agent made it to market. the real breakthrough was the discovery of the p y antagonist clopidogrel which, in conjunction with aspirin, proved to be very effective at preventing thrombotic events and as a result it became the biggest selling drug in the world at the time. with clopidogrel now offpatent the combination of aspirin and clopidogrel is a formidable challenge to any new agent both in efficacy terms and pharmacoeonomic terms. so is there a future for new anti-platelet agents? with the growing awareness of the role of platelets in inflammation and an understanding of how the immune activation of platelets differs from the classical haemostatic activation of platelets it is now possible to develop novel anti-platelet agents that target inflammation without compromising haemostasis. it is here that we should look for the next generation of anti-platelet agent. c-s - university hospitals of geneva, geneva, switzerland platelet function defects, either congenital or acquired, are associated with increased bleeding risk, particularly in a perioperative setting. the use of platelet function assays is therefore tempting in order to tailor transfusion and limit platelet transfusion to those bleeding patients with impaired platelet function, as assessed by those assays. however, the current guidelines provide only weak recommendations supporting the routine use of these assays. indeed, there are numerous platelet function assays on the market that differ in their method of evaluation of platelet function and agreement between their results is at best moderate. the threshold values beyond which procedure-associated bleeding risk becomes worrisome is not standardized. moreover, observational studies addressing the predictive value of platelet function testing in perioperative or spontaneous bleeding are not consistent. finally, management trials with randomized patients assessing the benefit of platelet function testing are scarce. more recent data identified selected situations where platelet function testing may be useful though. i will review the different platelet function assays as well as selected clinical studies addressing the impact of platelet function testing to improve bleeding and transfusion-related outcomes. the latest recommendation will be addressed too. background: platelet refractoriness complicates the provision of platelet transfusions in management of thrombocytopenia in oncology patients. platelet refractoriness poses challenge due to alloimmunization to hla and human platelet antigens and is associated with adverse clinical outcomes. aims: a prospective study was undertaken to analyse result of platelet compatibility with post-transfusion platelet count increment and to ascertain presence of platelet antibodies as causative factor in platelet refractory oncology patients. pulmonary complication after blood transfusion is the leading cause of transfusionrelated morbidity and mortality, with an incidence reported between . - % of all transfused patients. the most important transfusion related pulmonary complications are transfusion associated circulatory overload (taco), transfusion related acute lung injury (trali) and transfusion associated dyspnea (tad). in this presentation the recent changes in the international definitions will be presented and discussed. furthermore, insights in the different underlying pathophysiologic mechanisms will be highlighted. in the past decades only for trali prevention strategies have successfully been designed and implemented. currently no evidencebased treatment strategy is available for any of these life-threatening syndromes. insight in the pathogenesis of pulmonary complications after transfusion should pave the way for future prevention and treatment studies. the issue of the impact of iron overload / toxicity on the hematopoietic stem transplantation (hct) outcome has been firstly addressed in the field of transfusion dependent thalassemia. today the concept has been extended to other diseases characterized by periods of variable duration of transfusion dependence such as myelodysplastic syndrome (mds) and myeloproliferative diseases. patients requiring regular blood transfusions certainly develop iron overload leading to tissues and organ damage. iron burden before transplant significantly impacts outcome and long-life posttransplant. it is well known that iron overload is deleterious for organs such as liver, heart and endocrine glands and it has been postulated could also increases the risk of infections and severe graft versus host disease early after hct. recent preclinical data has shown how increased production of reactive oxygen species (ros) resulting under iron overload condition, could impair the stem cells clonality capacity, proliferation and maturation. also, microenvironment cells could be affected through this mechanism. for this reason, iron overload is becoming an important issue also in the engraftment period early post-transplant. high baseline ferritin levels before hct have been shown to negatively influence clinical outcome, but nowadays, ferritin is considered a steady and not biologically active form of iron, while free iron forms as non -transferrin bound iron (ntbi) and labile plasma iron (lpi) are considered the main trigger of cell damage more representative of the dynamic tissue damage. the scientific community is moving the iron disease from a "bulky" disease, such as classically in thalassemia (based on quantitative iron parameters as ferritin, red blood cell transfusion number, mri) to a "toxic" disease (based on active and dynamic biological markers as ntbi/lpi). at this time in all the studies published on hct setting, only the correlation between direct or indirect estimates of iron overload (mainly serum ferritin) and outcome parameters has been explored, while the duration of exposure to toxic iron species has not been taken into account. the first study that explored the lpi role in relationship with outcome was published by wermke and colleagues in malignancies. they investigated the predictive value of both stored (mri-derived liver iron content) and non-transferrin-bound-iron, defined as enhanced labile plasma iron (elpi) on post-transplantation outcomes in patients with acute myeloid leukemia or mds. their prospective, observational all-ive study showed that patients who had raised elpi concentration at baseline, also had significantly increased incidence of non-relapse mortality at day ( %) compared with those who had normal elpi at baseline ( %) (p = . ). reinterpreting transplant predictive factors in the light of the current advances in understanding iron homeostasis further supports the concept that the key to successful transplantation is regular and life-long chelation therapy to consistently suppress tissue reactive iron species and prevent tissue damage in the years before hct. in transfusion medicine, the role of donor sex was long considered to be limited to the increased risk of trali observed after transfusions from female donors. this risk has been shown to be limited to female donors with a history of pregnancy and to plasma rich products (i.e. excluding red blood cell products, typically containing < ml plasma). until, in , we found that sex-mismatched red blood cell transfusions were associated with increased recipient mortality. since then, several other studies have confirmed these findings, but some studies also did not find an association. all of these studies relied on the analyses of routinely collected health care data, which was not primarily intended to be used for research. as a result, analyses are complex and often difficult to properly appraise based on published descriptions. therefore, the discussion about possible reasons for these discordant findings has largely focused on the methodological approaches of the different studies. other potential explanations include differences in donor or patient populations, production methods, or storage time of blood products. the different potential explanations are expected to be associated with different underlying biological mechanisms. therefore, further delineating which donor, patient, and product characteristics modify the observed association could provide more insight into the underlying mechanism. in , we observed that only transfusions from female donors with previous pregnancies were associated with increased mortality and only in male recipients under years. this leads us to postulate that pregnancy induced long term changes in the female immune system are transferred during red cell transfusion, with negative consequences for young male recipients. the low amount of plasma present in red cell products further lead us to assume a cellular component, like passenger leukocytes, to be involved. it has been shown that micro-chimerism of passenger leukocytes can persist for decades after transfusion, even of leuko-reduced blood products, suggesting long term immune-modulation could play a role. we hypothesized that passenger leukocytes would die during storage of blood products and the negative effect of ever-pregnant female donors, on the survival of young male red cell recipients, would therefore be attenuated by increased storage time. however, our data seem to indicate the opposite. the risk of death was increased over three-fold for young male recipients of old (> days storage) red cells from ever-pregnant donors, compared to for young male recipients of fresh (< days storage) red cells from ever-pregnant donors ( -year cumulative incidence of death . % versus . %). the negative control group (i.e. young male recipients of red cells from male donors) showed a much weaker association of mortality with storage time (i.e. . % versus . %). these findings seem to falsify our hypothesis that mortality could be caused by passenger leukocytes, establishing long term immune-modulatory effects. another potential mechanism that has been suggested could be the presence of cellfree dna in transfused blood products. this cell-free dna increases during storage. however, more research is needed both to establish if cell-free dna can also be linked to previously pregnant blood donors and by which mechanism it could negatively affect young male transfusion recipients. clinical trials (cts), the gems in clinical research for generating robust evidence in medicine and public health, are costly and complicated undertakings. in resource limited setting like sub-saharan africa (ssa) where the health systems are sub-optimal and where capacity for research is limited, the conducting of cts can be a daunting challenge. the challenges of undertaking cts in rls may be categorized based on the occurrence of the bottleneck(s) in relation to the ethics and regulatory approval process: pre-approval: protocol development: in order to develop a context-specific protocol which is subsequently subjected to an ethics and regulatory approval process, investigators need to review and ensure that the protocol is pragmatic and feasible with respect to implementation. this results into a time-consuming reiterative process of reality-checking the protocol. site selection: in light of the limited research infrastructure, investigators in rls and their developed world partners spend considerable time reviewing and selecting suitable sites for participation in the anticipated protocol for the cts. suitable sites are usually very few and with competing on-going studies. approval: institutional review board (irb) approval: the irb approval process can be quite lengthy ( - months) with considerable unpredictability in the periods between the initial and subsequent irb reviews. national regulatory approval: the requirements by national regulators are unusually innumerable with limited flexibility to accommodate specific cts. post-approval: the key post-approval challenges for cts implementation in rls are attaining appropriate participant enrolment and maintaining high retention rates. specifically, for participant enrollment, the challenge may be unforeseen competing cts targeting the same participant pool or community perspectives that may discourage participants from getting screened for the cts. retention may also be a challenge particularly where participants view enrollment as a chance to access healthcare services may therefore not have any incentive to keep in a study after the initial study visits. in conclusion, cts are complex undertakings wherever they are conducted but are doubly challenging in rls like sub-saharan africa. the bottlenecks at the preapproval, approval and post-approval stages are considerable. nevertheless, it is rewarding to perform ctus in rls given that the data generated therein is highly valued by national regulators and may hasten the registration process for medical products. background: interest in an appropriate and effective whole blood (wb) pathogen reduction technology (prt) is growing, especially in sub-saharan africa where the residual risk of transfusion-transmitted infections (ttis) remains unacceptably high and wb is still frequently used. cerus corporation, manufacturer of the intercept tm blood system, and swiss transfusion src are collaborating on a clinical development program to adapt intercept prt using amustaline (s- ) and glutathione (gsh) for red blood cells (rbcs) into an appropriate prt for wb in resource-limited settings in africa. treatment with amustaline/gsh has been shown to inactivate a broad spectrum of transfusion-transmissible pathogens in rbcs. studies with amustaline/gsh in wb have shown effectiveness against a duck hepatitis b virus (> . log reduction) and plasmodium falciparum (> . log reduction), with future studies planned. a wb prt system with amustaline/gsh also has the potential benefit of minimal electricity requirements. aims: to describe the safety and clinical objectives for a phase clinical trial using the amustaline/gsh prt system for wb in africa, and describe research and development efforts to adapt the intercept prt system for rbcs into a robust and appropriate wb system for settings with high burdens of tti and limited resources. methods: the protocol for a phase clinical trial using pathogen-reduced wb treated with amustaline/gsh in an african country is presented, as are current research and development activities related to the development of a prt system for wb. results: in the planned phase clinical trial in africa, clinically stable patients with anemia who require wb transfusion will be randomized into two study arms at a large medical center in a sub-saharan african country. enrolled patients will receive one unit of non-leucocyte-reduced wb treated with amustaline/gsh, or a unit of untreated control wb or rbcs. the primary safety endpoint will be the incidence of high-imputability transfusion reactions (swissmedic ≥grade ) within the first hours of transfusion. data will also be collected on all adverse events and transfusion reactions (all grades) and the development of treatment-emergent antibodies to pathogen-reduced wb or auto-antibodies within (ae ) days of the study transfusion. clinical efficacy will be characterized by hemoglobin increment hours after transfusion adjusted to hemoglobin dose and body weight. summary/conclusions: a prt system for wb is being developed based on the intercept prt for rbcs that is in advanced development in europe and the united states. intercept-treated rbcs have met efficacy and safety endpoints in phase clinical trials. the amustaline/gsh prt system used to treat intercept rbcs has demonstrated effective inactivation against a broad spectrum of agents that may result in ttis. a phase clinical trial using an adapted prt system for wb in africa is the first step in a clinical development program that includes additional pathogen inactivation efficacy studies and improvements to the wb prt implementation process. together, these developments and evaluations represent progress toward a realistic and appropriate prt for wb in africa and other resource-limited settings. background: in australia, demand for plasma-derived products has increased dramatically, and there is a need to increase plasma collections. first-time donor retention, including the rate at which first-time donors return, is a pressing issue. a quick return is optimal as this increases the overall plasma yield and is associated with long-term retention. however, we lack evidence of effective interventions to encourage first-time donors, particularly those donating plasma, to return and to establish a higher frequency donation routine. working from schultz's ( ) framework, this intervention study was based upon insights from interviews with first-time plasmapheresis donors. participants identified barriers such as time and lack of knowledge about plasmapheresis. facilitators included being able to help more people and to donate more frequently than allowed with whole blood. participants generally favoured donating at a frequency of every weeks. aims: the aim of this study was to test the effectiveness of three intervention conditions compared with the business-as-usual (bau) procedure on the proportion of donors returning to donate plasma and the number of plasma donations. we report on the data from months post-donation. methods: donors were randomly assigned to one of four study conditions. in conditions and , donors received an email one day after their initial donation. in the first condition, donors received the bau 'thankyou' email. donors in the second condition received an alternative email with content derived from the interview study. donors in the remaining conditions received either the bau email (condition ) or the revised email (condition ) coupled with a telephone call. the phone call was scripted to provide additional information about plasma, including how often plasma can be donated, a suggestion to donate every weeks, and a prompt to forward-book appointments. results: the final sample (n = ) comprised women ( %) and men ( %) aged - (mean = ). after two months . % of donors returned to donate plasma at least once. after controlling for gender, age, and blood group, donors in each of the intervention conditions were more likely to return to donate plasma than were donors in the bau condition. the greatest effect was found between donors randomized to condition (revised email + phone call), or = . , ci = . - . , and bau. donors assigned to the two telephone conditions (condition and ) donated plasma at a higher frequency than bau. summary/conclusions: this study tested the effectiveness of interventions designed to encourage first-time plasma donors to return to donate plasma and to establish a routine of donation. early indicators suggest that the evidence-based email and phone call elements are more effective than bau in bringing donors back to donate plasma, and the revised email combined with a phone call had the greatest positive effect on short-term plasma yield. background: healthy individuals with hereditary hemochromatosis (hh defined as hyperferritinemia and homozygous p.c y mutation), but also carriers of other hfe mutations (p.c y/p.h d or homozygous h d) with elevated serum ferritin (sf) are accepted as blood donors, if allowed by local regulations and if eligibility is fulfilled. generally, blood components are released for transfusion at normal sf levels (< ng/ml in females, < ng/ml in males). aims: prospective, two-center, randomized study comparing the efficacy and tolerability of double-erythrocyte apheresis ( rbcaph) and whole blood phlebotomy (wbph) for iron depletion in asymptomatic subjects with hh or hyperferritinemia and other hfe mutations in the setting of routine blood donation. methods: eligibility criteria included age ≥ - years, total blood volume ≥ l, bmi < kg/m , hb ≥ g/l, elevated sf levels and no end organ damage due to iron overload. rbcaph ( ml rbc) were scheduled every days and wbph ( ml) every days until sf was < ng/ml. a complete blood count and sf were measured at baseline, at every visit and at follow up weeks after completion of the study. adverse events were systematically recorded. the treatment effect was tested by poisson regression, with gender, hfe mutation, bmi and baseline sf as covariates. results: subjects ( females; mean age years) were randomized to wbph (n = ; female) or rbcaph (n = ; females). hfe mutations were p.c / p.c y in subjects, p.c y/p.h d in , and p.h d/p.h d in . at baseline, mean hb was g/l (sd . ) and median sf was ng/ml (iqr - ng/ml). procedures (wbph n = , rbcaph n = ) were completed; were interrupted (local hematoma, insufficient flow); ( wbph, rbcaph) were postponed because of low hb and for non medical reasons. there were drop-outs in the wbph arm due to depression and poor compliance, respectively. anemia (hb < g/l in males, < g/l in females) occurred after visits in wbph subjects and after visits in rbcaph subjects. fatigue was reported after phlebotomies and aphereses. only participants ( %) completed the study per protocol. blood components ( rbc concentrates and plasma units) for transfusion were obtained. overall, a median of . wbph (iqr . - . ) was needed to reach sf < ng/ml, corresponding to . times of rbcaph (median . , iqr . - . ) (p = . ). analyzing separately p.c /p.c y and p.c y/p.h d carriers, the relation wbph to rbcaph was . and . , respectively. treatment arm and hfe mutation were the covariates with significant effect on the primary endpoint (p = . and . , respectively). summary/conclusions: rbcaph is more efficient than wbph for iron depletion in healthy subjects with hh or other hfe mutations and moderate hyperferritinemia. intensive treatment schedules, generally recommended for hh, are difficult to keep because of hb drop and compliance. less intensive treatment in asymptomatic individuals with hh and their inclusion in blood donation would avoid negative effects on quality of life and benefit blood collection centers in the long term. background: serum ferritin (sf) measurements in whole blood (wb) donors demonstrated that female sex and intensity of donation are major risk factors for iron deficiency. approximately ml red blood cells (rbc) and - mg iron are lost with wb donation. double unit rbc ( rbc) collections of ml (ca. ml less than the rbc amount of two wb donations) lead to a loss of about mg iron. in switzerland, the maximal allowed donation frequency for male donors is once every months for rbc and once every months for wb donation. aims: to describe and compare the course of hemoglobin (hb) and sf in male subjects donating wb and rbc at our institution. methods: we included wb and rbc donors (n = ) who donated with the maximal allowed donation frequency over months between and , yielding , wb and , rbc donations. we excluded subjects with hyperferritinemia and known hfe mutations. hb limits were g/l for wb and g/l for rbc donation. with rbc apheresis ml rbc were collected. sf was measured on a predonation serum sample; hb was determined from finger prick samples. the donors received no iron substitution. we used generalized estimating equation models for hb and sf trajectories. results: mean age at the first blood donation was (wb) and years ( rbc), respectively. at the first donation, mean hb was g/l (sd ) in wb and g/l (sd ) in rbc donors; mean sf was (sd ) and lg/l (sd ), respectively. on average, hb and sf were higher in rbc donors ( . g/l and lg/l, respectively; p < . ). there were subjects with sf < lg/l in wb and in rbc group, and with sf < lg/l (but > lg/l) and , respectively. in rbc donors, between the first and the last donation, mean hb declined from g/l to g/l (p < . ) and mean sf from lg/l to lg/l (ns). in wb donors, mean hb dropped from g/l to g/l (p < . ) and sf from lg/l to lg/l (p < . ). similar results were found when adjusting for age and season. hb values dropped from baseline until the th donation for wb donors and until the th donation for rbc donors with an upward trend thereafter. in both groups, no hb value below the limits of blood donation and no anemia were observed. sf reached a nadir at the th donation in both wb and rbc donors ( lg/l and lg/l) and increased thereafter in rbc donors. in wb donors, sf followed a parabolic trend that peaked at the th donation, and then declined until the last donation. summary/conclusions: the maximal allowed blood donation frequency for wb and rbc male donors in switzerland is not only protective for the development of anemia, but also for deferral of blood donors because of low hb. this was observed even in subjects with low sf at baseline. background: granulocyte concentrate transfusion is a potentially lifesaving option for patients without functional neutrophils. however, recent studies have failed to demonstrate the anticipated clinical effectiveness of this procedure. granulocyte concentrates are manufactured using sedimentation agents to separate granulocytes from red blood cells and enhance granulocyte collection efficiency. high-molecularweight hydroxyethyl starch (hes) is most commonly used for this. however, authorities recently restricted the use of hes due to its unfavorable risk-benefit-profile. modified fluid gelatin (mfg) is an already used alternative sedimentation agent. as the granulocyte product contains these substances, any impact of the sedimentation agent on granulocyte function may affect the clinical effectiveness of granulocyte transfusion. aims: we tested the hypothesis that mfg is not inferior to hes in terms of the functionality and viability of granulocytes. methods: granulocytes from ten healthy donors were isolated, aliquoted and incubated in parallel for hours with either % (control), . %, % or % mfg (gelafundin %, b. braun melsungen ag) or hes (hespan %/ / . , b. braun medical inc.), respectively, and granulocyte migration, chemotaxis, reactive oxygen species (ros) production, neutrophil extracellular trap formation (netosis), antigen expression of cd b, cd l and cd b, and viability were subsequently investigated in vitro. testing was performed using live cell imaging of the cells embedded into a collagen i matrix for parallel testing of migration, ros production and netosis. in addition, flow cytometric (facs) analysis was utilized for surface marker expression, viability and respiratory burst measurement. results: granulocyte migration decreased in a dose-dependent manner in response to hes and mfg. relative to the controls, all three concentrations of hes lowered migration distances (p < . respectively), whereas only the higher concentrations ( % and %) of mfg showed lower relative migration distances (p < . respectively). track straightness was reduced with both sedimentation agents at % and % to the same extent (p < . respectively). hes resulted in lower cd b expression (p = . ) and higher cd l expression (p = . ) compared to the controls, whereas the differences for cd b did not reach statistical significance. mfg did not affect the expression of any investigated surface antigen mediating endothelial adhesion and transmigration in comparison to the controls. no significant differences in the timing of ros production or netosis, or in neutrophil viability or respiratory burst were observed. summary/conclusions: these results indicate that mfg is not inferior to hes in terms of granulocyte phenotype and function in vitro when used at equal concentrations, and that potential impairment of granulocyte function can occur with hes. background: plateletpheresis donation leads to a well-known transient decrease of donor's platelets. the question of long-term effects raised with the development of regular donations by some donors in order to satisfy a growing demand. a seminal work (lazarus, transfusion, ) stated that there is a sustained thrombopenia in frequent plateletpheresis donors, correlated with the total number of donations. aims: french regulation authorizes up to plateletpheresis donations per year, with a minimum weeks interval between them. we tried to evaluate the risk of sustained thrombopenia under these conditions. methods: we retrieved all plateletpheresis donations occurring between / / and / / from the french civilian blood donors' base and then selected a cohort of donors with at least donations during that period. in order to minimize measurement errors, platelet counts analysed were means of three consecutive donations, i.e. measures for each donor. results: the cohort includes , donors ( women and , men). mean platelet counts fluctuate between . and . platelets/ml. analysis of variance does not show any statistically significant difference (f = . ), even taking donor's sex or age in consideration. there is no difference if we consider the total duration of the donations, either. donors with the lowest first counts show a significant rise in subsequent measures and donors with the highest counts show a decrease trend, exhibiting a classical regression toward the mean. summary/conclusions: plateletpheresis french regulation does not seem to be at risk of sustained donor thrombopenia. this conclusion is in agreement with recent literature data. the primary biological role of the human leukocyte antigen (hla) system is the regulation of the immune response to foreign antigens. because of this role, hla genes and molecules have an important role in transplantation, etiology of many autoimmune, non-autoimmune and infection diseases, but also in transfusion medicine. an increasing probability of an hla non-compatible blood products, tissues or organs exists due to the extremely high polymorphism of hla genes, with more than , described alleles to date, and their different frequency distribution in various worldwide populations. the hla system, originally discovered as a result of a transfusion reaction in the s, can cause detrimental immune reactions in transfusion therapy. hla antibodies present in the patient are responsible for some of these reactions, while in other cases hla antibodies or hla reactive cells present in the transfused product are accountable for the immunoreactivity. hla antibodies form as a result of exposure to foreign hla antigens during pregnancy, transplantations and blood transfusions and can cause platelet immune refractoriness, febrile transfusion reaction, transfusion-related acute lung injury, and transfusion associated graft versus host disease. in order to avoid or reduce the development of these transfusion-related events, hla antibody negative or compatible products should be used. almost all existing methods presently used for molecular typing of hla polymorphisms are based on polymerase chain reaction, but with different resolution levels (low resolution -two digits or high resolution -four digits). in addition to providing a more precise detection of polymorphisms at hla classical loci (e.g. hla-a, -b, -c, -drb , -dqb ), molecular methods can also determine polymorphisms at hla loci which previously could not be typed by serology (e.g. hla-drb , -drb , -drb , -dqa , -dpa ). the most commonly used method for the detection of hla antibodies was until recently complement-dependent cytotoxicity (cdc) technique, but it is increasingly being replaced by a more sensitive, solid phase based method (luminex technology). in conclusion, an accurate and precise determination of both hla gene polymorphism and hla antibodies presence is essential for the safe and efficient administration of transfusion products. background: in only a minority of pregnancies complicated with anti-hpa a antibodies serious fetal/neonatal disease develops. the difficulty in predicting which mothers should be treated with ivig hampers implementation of fnait screening. we found that fc-core fucosylation and galactosylation are highly variable in anti-hpa a igg, and that these glycan features strongly affect binding to fccriiia receptor. the level of fc-core fucosylation of anti-hpa a alloantibodies was found to correlate with platelet count and outcome of the newborn, suggesting that antibodyspecific fucosylation might serve as a biomarker in fnait screening. however, at present the fc-glycosylation pattern can only be determined by complicated methods involving purification of the antigen-specific igg, and analyzing trypticly released -igg-derived-glycopeptides by tandem liquid chromatography-mass-spectrometry (ms) techniques. these methods, although powerful, are not yet suited for high throughput clinical screening. aims: our aim was to provide a simplified method to quantify the biological activity of anti-hpa- a antibodies, and possibly other alloantibodies against blood cells. methods: here we explored if cellular surface plasmon resonance (spr) imaging can replace ms, resulting in less complicated handling of patient sera and donorantigen-bearing cells. the strength of the binding of platelets to fccr on spr sensor was monitored under flow. the spr sensor was equipped with both wt fccriiia (sensitive to fc-glycosylation status) and mutant fccriiia-n a (insensitive to fcglycosylation status). in addition, the biosensor was prepared with anti-platelet cd (c ) and anti-igg to calibrate the number of injected platelet as well as to quantify igg-opsonization. the quality of the anti-hpa a glycosylation was monitored as the ratio of the binding of opsonized platelets to the wt and the mutant n a-fccriiia. platelets opsonized with recombinant glycoengineered anti-platelet antibodies with different levels of fc-fucosylation were used as standards. for validation, plasma samples with anti-hpa a antibodies, already analyzed by mass spectrometry and with known clinical outcome were tested (sonneveld, bjh, ) . results: we found that the ratio between the binding to the wt fccriiia and to the mutant n a-fccriiia correlated with the level of fucosylation of the hpa a antibodies, as measured by mass-spectrometry (r = À . ; p < . ). overall, a similar predictive value for disease severity was obtained as we previously reported for this retrospective cohort. in addition, quantitative information on antibody concentration can also be extracted using the fccriiia-n a receptor as sensor on the chip, while anti-igg gave aspecific signals, presumably because it recognized cytophilic platelet-fccriia-bound antibodies as well. summary/conclusions: in conclusion, the combined use of wt and mutant fccriiia in a label free spr assay provides both quantitative and qualitative information of platelet bound anti-hpa a antibodies, which circumvents the need for purification of specific antibodies and laborious mass spectrometric analysis. this approach might be generally applicable to determine the biological activity of cell bound antibodies not only for anti-hpa a in fnait, but also for anti-rhd alloantibodies in hdfn or anti-platelet antibodies in itp. background: immunization against the human platelet hpa- a alloantigen is the most common cause of severe fetal and neonatal alloimmune thrombocytopenia (fnait) in otherwise healthy term newborns. the screening for hpa- a antigen in pregnant women is an important tool for identification of pregnant women at risk of having a fetus/neonate with fnait. any targeted intervention depends on efficient screening methods as well as sensitive and specific methods for detection of anti-hpa- a. within the framework of the polish-norwegian project (prevfnait) we have performed hpa- a screening program in poland. aims: our aim was to assess the frequency anti-hpa- a antibody detection and the clinical outcome of newborns identified through the study. women who joined the program due to the fnait in the previous child or in the current newborn are not analyzed in this study. methods: hpa- a screening of pregnant women in - gestational weeks was performed by facs phenotyping or rq-pcr genotyping at ihtm in warsaw. hpa- a negative/hpa- b/ b women were tested for hla drb * : and for anti-hpa- a antibodies by maipa (followed up at week - , , , - and weeks after delivery). if anti-hpa- a were detected, quantitative maipa was performed. all hpa- a negative women were contacted for information concerning the newborn. if the baby had thrombocytopenia and anti-hpa- a were not detected by maipa, the look back samples were tested retrospectively by paklx test (immucor). results: hpa- a negative women were identified ( . %). anti-hpa- a was antibodies were detected by maipa in women (two delivered tweens). in addition, anti-hpa- a antibodies were later detected by paklx in further women who delivered baby with severe thrombocytopenia and/or ich. total number of immunized mothers was ( . %). they delivered babies; were boys. three women were treated by ivig: two by and injections since th and th gw respectively. the anti-hpa- a concentration in the st one was . ; . ; . iu/ml in , , gw respectively and in the nd < . iu/ml in all examined samples. the decision on treatment was based on the low plt count~ g/l in the fetus in cordocentesis. their newborns (one delivered tweens) were healthy. the rd treated woman entered the program in gw (anti-hpa- a concentration was high . iu/ml). she obtained one injection of ivig. her baby was born with mild thrombocytopenia with no ich. severe fnait occurred in / newborns: in with anti-hpa- a detected in paklx only and in with antibody concentration in maipa - st : . / . / . at / / th gw respectively; nd : . / . at / th gw respectively. ich was observed in all of them; plt count was < x in four, / in one. summary/conclusions: / the severe thrombocytopenia due to anti-hpa- a alloimmunisation in our prospective study occurred in / pregnancies / the paklx could improve anti-hpa detection in the screening program and should be considered as an additional diagnostic test, if maipa result is negative / the hpa- a alloimmunisation frequency is higher in pregnancies with male than female fetus. background: foeto-maternal platelet alloimmunization (fmpai) is mainly characterized by foetal and / or neonatal thrombocytopenia (fnait), sometimes revealed by intracranial hemorrhage (ich) or even by foetal death in utero (fdiu). the experience of the pnil milwaukee (usa) reported in that the diagnosis of alloimmunization was carried in only % of neonatal thrombocytopenia cases with a clinical symptomatology highly suggestive of an alloimmune etiology. aims: the aim of this two-year study was i) to determine the frequency of platelet incompatibilities in fnait, ich and fdiu and ii) to evaluate the frequency of detectable platelet alloantibodies (alloab) and their specificity in cases of incompatibility. methods: platelet genotyping was performed by hpa beadchip genotyping kit (bioarray solutions, immucor, warren, nj). serology investigation was carried out by different methods: complete maipa kit (apdia bvba, turnhout, belgium), pack lxtm assay (immucor gti diagnostics, waukesha, wi) and « in house » maipa. all and data were collected using the laboratory information management system. results: patient files were analyzed. no incompatibility is demonstrated in hpa- to - , - and - systems in . % (n = ). hpa- and / or and / or incompatibilities were found in cases ( . %), hpa- and / or in cases ( %). platelet alloimmunization was globally confirmed in only . % of the cases. platelet alloabs were identified regardless of clinical manifestations: anti-hpa- a ( . %), anti-hpa- b ( . %), anti-cd ( . %), anti-hpa- a and anti-hpa- b ( . % respectively) and anti-hpa- b and anti-cd ( . % respectively). alloabs were found in the context of neonatal thrombocytopenia, in ich and in fdiu, and in a follow-up of pregnancy. even if no anti-hpa- alloab could be identified, the incompatibility in this system was highly associated with fnait, ich and fdiu (n = , n = and n = on cases). summary/conclusions: this study strongly confirmed the known immunogenicity of some hpa systems and highlighted overall the severity of hpa- and hpa- incompatibilities. the definite diagnosis of fmpai is difficult to make due to the present technical difficulties in the detection of antibodies against the hpa- and hpa- systems. however, our results suggest that special attention should be paid to the management of pregnancies with these incompatibilities due to the frequency of severe foetal/neonatal adverse events. background: fetal and neonatal alloimmune thrombocytopenia (fnait) is a potentially life threatening disease caused by maternal alloantibody formation against fetal human platelet antigens (hpas), of which anti-hpa- a is accountable for the fast majority of the cases. population-based screening for fnait has been topic of debate for over decades. logistically as well as financially, the major challenge of such a screening is the typing of pregnant women to recognize the % hpa- a negative women. at present, hpa- a typing is mostly done by genotyping. for costeffective implementation of anti-hpa- a screening there is need for a high-throughput, quick and low-cost phenotyping assay. aims: the aim was to develop a high-throughput, quick and low-cost phenotyping assay in order to identify hpa- a negative pregnant women. methods: an automated sandwich elisa was developed to perform hpa- a phenotyping using a murine monoclonal anti-gpiiia as coating antibody and horseradishperoxidase-conjugated recombinant igg anti-hpa- a as detecting antibody. to ensure the applicability for high-throughput testing in a potential screening setting, ll of the uppermost plasma of - days-old stored edta anticoagulated blood tubes was used, without first swirling or spinning them. in two phases, samples of pregnant women were tested and compared to an allelic discrimination polymerase chain reaction assay as golden standard. in the first phase, samples from unselected consecutive pregnant women were tested. the second phase was part of a prospective screening study in pregnancy and confirmatory genotyping was restricted to samples with an arbitrary set od < . in the hpa- a elisa. the developed elisa was optimized to require no additional handling (swirling or spinning) of stored tubes. during phase i, consecutive samples were tested. in phase ii, the hpa- a elisa was performed in another , consecutive samples, with confirmatory q-pcr in , . the two phases combined, samples from in total , hpa- a negative and hpa- a positive pregnant women were genotyped. the assay reached a % sensitivity with a cut-off od between . and . , leading to a specificity of . %. summary/conclusions: a quick, low-cost and reliable assay for hpa- a phenotyping was developed that can be used in a population-based screening setting to select samples that has to be tested for the presence of anti-hpa a antibodies. because plasma from non-mixed or spinned tubes of three to six day-old samples can be used, this assay is applicable to settings with suboptimal conditions. background: cytomegalovirus (cmv) sero-prevalence in ireland is lower than that which is reported in many other european countries. a study of pregnant women in found that . % of irish women were cmv seropositive in comparison to % from western europe and % eastern europe and % from africa. an internal study carried out by the irish blood transfusion service (ibts) in indicated the rate of cmv seropositivity in irish blood donors was . %. therefore a significant proportion of the irish donor and recipient population are susceptible to primary cmv. this is of particular concern for patients for certain at-risk groups such as very-low birthweight cmv seronegative neonates, cmv seronegative patients undergoing transplantation and other cmv seronegative immunocompromised patients. this results in a demand for the provision of cmv sero-negative blood components. in the ibts evaluated the abbott alinity s cmv igg assay as a replacement for the cmv mastazyme eia (total ab eia). aims: to assess the performance of the abbott alinity s cmv igg screening assay in comparison to the cmv mastazyme eia (total ab eia). methods: diagnostic sensitivity was determined by testing confirmed cmv igg positive donors from an external laboratory. sensitivity was assessed using three seroconversion panels (n = ). analytical sensitivity was calculated using linear regression analysis of the who first international standard for anti-cmv igg. diagnostic specificity was determined by testing donors. further evaluation of discordant results was carried out using the architect anti-cmv igg and igm assays and vidas anti-cmv igg and igm assays. results: the diagnostic sensitivity of the alinity s anti-cmv igg assay was determined to be %. the seroconversion sensitivity reported out of samples reactive. the analytical sensitivity of the alinity s cmv igg assay was determined to be . iu/ml. the validation reported discordant results from donor samples tested with both the alinity s cmv igg assay and the current mastazyme total assay. discordant results were observed (alinity s anti-cmv igg positive/mastazyme total negative). further testing of these samples classified discordant results as positive, as negative and as indeterminate. discordant results were observed (alinity s anti-cmv igg negative/mastazyme total positive). further testing classified these samples as negative. overall the diagnostic specificity was determined to be . %. summary/conclusions: both the seroconversion and analytical sensitivities are comparable between the alinity s cmv igg assay, the cmv mastazyme total ab assay, the architect cmv igg assay and the vidas igg assay. the slight variations can be attributed to the individual assay cut-off definitions, which can vary greatly between cmv assays. it must be noted that the determination of the diagnostic specificity ( . %) does not include indeterminate discordant results. further testing will be carried out to try to characterize all discordant samples in collaboration with abbott. this evaluation did not identify any donors with isolated confirmed cmv igm antibodies in a pool of donors. based on this evaluation the abbott alinity s cmv igg assay is a suitable replacement to the mastazyme total ab assay for blood donor screening. background: africa has a unique set of challenges regarding safe blood transfusion. two of the largest contributing factors are: ) the most common disease states in sub-saharan africa (ssa) require large amounts of blood as lifesaving interventions e.g. malaria, ) the highest burden of infectious diseases transmissible through transfusion (tapko, toure, & sambo, ) is found in ssa. this has often led to the binary donor base that exists in ssa, consisting of voluntary non-remunerated blood donors (vnbd) and family or replacement donors (frd) as transfusion centres are unable to supply the demand when relying only on vnbd. voluntary non-remunerated donors are the safest blood donors as they have no incentive (other than altruistic motives) and are not under social pressure to donate, both factors that may induce individuals knowing or suspecting themselves to be infected with a blood-borne agent to donate blood. nucleic acid testing (nat) in conjunction with serological testing is the gold standard for testing, however, the vast distances and high temperatures of africa makes transport of traditional plasma samples a logistical challenge. many publications evaluating the stability, suitability, and ease of use of dried blood spots (dbs) for nat have been published. generally, results have been shown to be comparable to traditional plasma samples. dbs is being used successfully in the early infant diagnosis (eid) programs for hiv by means of pcr testing, especially in africa. aims: . to demonstrate that dbs and/or dried plasma spot (dps) testing is suitable for blood donor screening and can make nat testing more widely available in africa . to determine the diagnostic sensitivity and specificity of testing dps and dbs samples, in comparison to testing of plasma samples. methods: negative new donor samples and confirmed positive donor samples, as defined by routine blood safety screening done at western cape blood service, were screened using a dried blood spot kit. after routine testing was completed, one dbs sample and one dps sample for each blood donor were prepared and analysed with the ultrio elite assay on the panther analyser. summary/conclusions: dbs/dps can be used as a sample for screening blood donors as the invalid rate was . %, and only found on dbs samples. logistically dbs/dps is well suited for the resource-poor countries as samples are: -easy to obtain (fingerpick samples could be used.) -transport is simplified as samples will not leak or haemolyse due to high temperatures. -samples can be stored at room temperature dbs/dps demonstrated acceptable specificity. the ultrio elite performed well with regards to hiv and hcv sensitivity. sensitivity with regard to hbv was not as high but this could be due to very low and erratic viral loads. background: sanquin blood supply is responsible for the blood transfusion services in the netherlands. at the national screening laboratory sanquin (nss) annually more than . blood and plasma donations are tested, on average . samples per day. for more than years, infection serology testing was performed using the prism (abbott diagnostics), but since mid of july , serological testing for the hbsag, hiv ag/ab, anti-hcv and anti-hbc is done with abbott's alinity s system. aims: to compare the numbers of initially and repeatedly reactive results of whole blood and plasma donation samples and the rate of non-specific results leading to deferral of donations and donors for prism and alinity s assays using data from months before and months after implementation of the alinity s systems at nss. methods: initial and repeat reactive rate of the assays run by either prism (hbsag, hiv o plus, hcv) or alinity s (hbsag, hiv ag/ab combo, anti-hcv,) were calculated for january to june (prism) and august to december (alinity s). due to the lack of a true confirmatory method for anti-hbc, we only compared the rate of repeatedly reactive results for prism hbc and alinity s anti-hbc. results: the rate of repeat reactive results for prism (p) and alinity s (a) assays were as follows: ) hbsag p . % ( / . ) versus a . % ( / . ); ) hiv p . % ( / . ) versus a . % ( / . ); ) anti-hcv p . % ( / . ) versus a . % ( / . ). the rate of anti-hbc reactive samples was not significantly different between prism ( . %) and alinity s ( . %). over the study period, the rate of initially reactive samples for the three main screening assays (hbsag, hiv, hcv) was also comparable between alinity s ( . %) and prism assays ( . %), mainly attributable to a rather high number of initially reactive alinity s hiv ag/ab results. this was due to initial issues with blood collection tubes that were resolved. as a result in december, the rate of initially reactive samples decreased to . %, which was significantly lower than for the three prism assays ( . %). summary/conclusions: the introduction of the alinity s assays lead to a decrease of the average repeat reactive test results (hbsag, hiv, hcv) by . % as compared to the prism, mainly due to a lower false reactive rate of the alinity s anti-hcv assay. this will be further investigated for first time and multiple time donors. with the implementation of the alinity s at sanquin we aimed to improve not only the operational efficiency but also to further minimize unjustified disapproval of donors. these first data show that the low initial and repeat reactive rates of the alinity s assays indeed have a positive impact on unnecessary deferrals of donations and donors. background: in blood banks, testing all blood donations for markers of infectious diseases plays an important role in maintaining the safety of blood transfusions. mandatory serological testing in switzerland is performed for anti-hcv, hiv ag/ab, hbsag and syphilis. highly specific and sensitive tests with corresponding automation are essential for this purpose. aims: a comparative study was carried out to evaluate the usability of the newly launched alinity s system (abbott) and the specificity of the infectious disease parameters hbsag, anti-hcv, hiv combo and syphilis (abbott) with the currently used elisa methods on the quadriga befree system (all diasorin, formerly siemens healthcare diagnostics). methods: the study took place at the interregional blood transfusion service in berne, switzerland. the specificity of the parameters was studied on , blood donor sera from both first time and repeat donors. the samples were tested first on the quadriga be free system with enzygnost hbsag . , enzygnost anti-hcv . , and enzygnost hiv integral assays and on the pk with the newbio-pk tpha assay (newmarket biomedical). all samples were retested on the same day with hbsag, anti-hcv, hiv combo and syphilis on the alinity s. initial reactive samples were repeated in duplicate. discriminatory tests were carried out for repeatedly reactive samples using alternative screening tests and neutralisation (for hbsag) on an abbott architect i system and immunoblots (hiv-, hcv-, syphilis-inno-lia, fujirebio). for all samples, results from our routine individual donation nucleic acid testing (hcv, hiv, hbv, roche cobas system) were available. results: based on the results from testing , blood donations, the observed specificities of alinity s assays (a) and enzygnost assays ( summary/conclusions: the alinity s system was easy to use by the operators after a very short introductory training and provides good operational efficiency such as high throughput even when selective testing for samples is needed. the observed specificity of abbott alinity s versus siemens enzygnost assays is comparable in a blood donor screening setting. unfortunately, we were not able to analyse statistically the specificity data due to the insufficient number of donor samples tested in parallel. it is worth mentioning that around % of the samples included in the study derived from repeat donors who had been previously tested with the enzygnost assays but were "first time donors" for the alinity s assays. all four assays from both systems exhibit a very good specificity and are highly suitable and practicable for routine blood donor screening. background: effective screening for transfusion-transmissible infections is essential to ensure safe blood transfusions. the world health organization recommends mandatory serological testing of blood donations for human immunodeficiency virus (hiv), hepatitis b (hbv)/c (hcv), and syphilis. due to increasing demands on clinical laboratories, there is a need for reliable and accurate automated blood screening tests. the fully automated cobas e analyser can be used with elecsys â infectious disease parameters to screen donor blood samples. aims: to compare the performance of elecsys â infectious disease parameters on the cobas e analyser (roche diagnostics) with other commercially available assays for routine first-time blood donor screening. methods: we provide results from etablissement franc ßais du sang (montpellier), a blood bank which participated in a large, multicentre study of the cobas e analyser. the following infectious disease marker assays were compared: hiv, elecsys â hiv duo versus prism hiv o plus; hcv, elecsys â anti-hcv ii versus prism hcv; hbv surface antigen (hbsag), elecsys â hbsag ii versus prism hbsag; hbv core antigen antibodies (anti-hbc), elecsys â anti-hbc ii versus prism hbcore; syphilis, elecsys â syphilis versus newbio pk tpha assay. specificity was tested using residual fresh serum samples from unselected first-time blood donors, and calculated according to assay package inserts and site-specific cutoffs. samples were tested using comparator assays, then retested the same day using elecsys â assays. initially reactive samples were repeated in duplicate; confirmatory tests were conducted on repeatedly reactive samples. confirmatory tests: hiv, nucleic acid testing (nat), architect hiv ag/ab and inno-lia â hiv i/ii score assays; hcv, nat, archi-tect hcv and inno-lia â hcv score assays; hbsag, nat, architect hbsag and elecsys â /prism hbsag confirmatory assays; anti-hbc, nat, hbsag, anti-hbs, and architect anti-hbc assays; syphilis, architect syphilis tp and inno-lia â syphilis score assays. sensitivity was tested using preselected, anonymised, positive, citrate-phosphate-dextrose-plasma samples (plasmatec laboratory products) and compared with archived data for comparator assays. sensitivity was calculated according to the final nat result. results: across all infectious disease markers, specificity to detect repeatedly reactive samples using elecsys â versus comparator assays was similar ( . - . % versus . - . %; n ≥ ). in specificity analyses, there were discrepant results for hiv testing, for hcv, two for hbsag, eight for anti-hbc, and five for syphilis. sensitivity of the elecsys â hiv duo assay ( . %; % ci . - . ) was higher than the prism hiv o plus assay ( . %; % ci . - . ), but the difference was not statistically significant. sensitivities of elecsys â and comparator assays were the same for hcv ( . %; % ci . - . ), hbsag ( . %; % ci . - . ), anti-hbc ( . %; % ci . - . ), and syphilis ( . %; % ci . - . ); three hcv and six anti-hbc samples were classified negative/ indeterminate and excluded from the analyses. in sensitivity analyses, there were two discrepant results for hiv testing, three for hcv, and five for anti-hbc. summary/conclusions: elecsys â infectious disease parameters on the cobas e analyser demonstrate high specificity/sensitivity for screening first-time blood donor samples, with similar clinical performance to other commercially available assays. background: individual plasma and serum specimens from whole blood or plasmapheresis donors are tested for absence of infectious agents by serological assays prior to use for transfusion or production of blood derived therapeutics. the department of plasma analytics (pa), takeda (austria), and haema ag, grifols (germany), both labs with high throughput and a high level of automation, were seeking for alternatives to replace their current serological test systems (abbott prism next). aims: to allow a direct comparison of the two final candidate analyzers alinity s (abbott) and cobas e (roche diagnostics gmbh), a side by side evaluation was carried out by the pa and haema with support from abbott and roche (provision of instruments and reagents). the aim was to compare assay specificities as well as handling and performance of the instruments. the outcome should be used to better understand potential specificity differences and practical handling aspects (throughput, etc.) of a next generation serological analyzer. methods: the two candidate instruments were installed in the pa. from march to june , close to , aliquots from routine preselected repeat donors, provided by haema, were run on both study instruments in parallel. plasma samples were tested for hbs antigen (ag), hcv antibody (ab), hiv ag/ab, and partially for syphilis ab. serum samples were additionally tested on hbc ab. samples with repeat reactive results ("rr", two reactive results out of three tests) not confirmed by confirmatory tests were counted as false reactive. the necessary sample size was calculated based on a one-sided comparison of proportions with the aim to detect potential specificity differences (a = %) in the size of those specified by the manufacturers' instructions. two different lots were tested for the three main assays. results: out of , plasma and , serum samples, test results representing individual donations were found rr on one or both instruments. two samples were confirmed positive ( hbsag, hcv), two others were indeterminate. the sample containing low level antibodies against hcv was pcr negative and only detected by the roche system. the percentage of false reactive results for the five assays on the two systems were (alinity s/e ): hbs ag: . / . % in a total of / samples tested; hcv ab: . / . % in / , p < %; hiv ag/ab: . / . % in / , p < %; syphilis ab: . / . % in / ; hbc: / % in / . no significant difference was found between the calculated specificities in our study and the manufacturers' data. a potential influence of sample matrix and kit lots was assessed. a trend towards more false reactive results in serum vs plasma was found for nearly all assays. no clear-cut statistical difference was seen between lots. summary/conclusions: the study results are in line with the manufacturers' specificity data, showing that the alinity s hcv ab and hiv ag/ab assay show a slightly higher specificity in a population of plasma and serum samples from repeat donors prescreened by prism. a possible influence on the test specificity by the sample matrix was detected but needs further investigation. the possibility to edit complex genomes in a targeted fashion has not only revolutionized basic research but biotechnological and therapeutic applications as well. with the rapid development of genome editing tools, in particular zinc-finger nucleases (zfns), transcription activator-like effector nucleases (talens), and the crispr-cas system, a wide range of therapeutic options have beenand will bedeveloped at an unprecedented speed. therapeutic genome editing in hematopoietic cells enable new interventions in the blood and immune system, including novel approaches to treat immunological disorders, infectious diseases, and cancer. we have developed gmp-compliant protocols to manufacture gene edited cd + hematopoietic stem and precursor cells (hspcs) as well as chimeric antigen receptor (car) t cells, with the final goal to provide novel cell therapies for patients suffering from primary immunodeficiencies, chronic infection with human immunodeficiency virus type (hiv- ), and some tumor entities. despite great success in improving their specificity, engineered designer nucleases can induce genotoxic side effects by introducing mutations or chromosomal aberrations. we have established novel genome-wide assays that enable us to detect chromosomal aberrations induced not only by off-target activity but also by on-target activity, such as micro-aberrations and translocations, with unparalleled sensitivity. in toto, our developed protocols allow us to achieve genome editing in hematopoietic cells with high efficiency and to assess the genotoxic risk associated with the expression of crispr-cas nucleases and talens in clinically relevant human cells, so forming the basis for planned phase i/ii clinical studies. adoptive t cell therapy (act) has proven a potent means to treat blood-borne tumors and solid tumors. adoptive cell therapies include t cells that are genetically engineered with tumor specific t cell receptors (tcrs), or with chimeric antigen receptors (cars). in addition, tumor infiltrating cells (tils) can be isolated from tumor lesions, which are then expanded and reprogrammed in vitro prior to transfusion into the patient. the anti-tumoral efficacy of act products depends on several parameters, including the capacity of cd + t cells to produce cytokines, chemokines and granzymes, a feature that is critical for effective anti-tumoral responses. here i will discuss our efforts to develop and improve act products for future clinical use. i will present pre-clinical work on developing til therapy for non-small cell lung cancers. in addition, i will show that human cd + t cells can be divided into different subsets, and that only one of those subsets is highly cytotoxic. this finding may help improve the quality of genetically engineered t cell products, like tcr and car t cell products. background: the baltic states -estonia, latvia and lithuania have a lot in common. we are located side by side, share the baltic sea as a gate to the west, and more importantly, a common history. we were members of the ussr and suffered years of soviet occupation. we held hands in a km long human . . .chain" across the three states to express our mutual support, and later on, even joined the european union on the very same day -june st , . the three differ a bit in size, population and more in the languages spoken in each one, but that does not explain why the path towards voluntary unpaid donation varies as it does. aim: the aim is to describe the journey towards voluntary non-remunerated blood donation in the baltic states after regaining independence from the soviet union. methods: the information was collected from published and unpublished memories, annual reports and written interviews with latvian and lithuanian colleagues. results: in soviet times, all orders came from moscow and quality control was conducted from the capital city of latvia, riga. donors were mostly paid and given an extra vacation day. big factories were the best places to collect blood and people were queuing to donate. in , the soviet union fell apart and the baltic states suddenly got the freedom and responsibility to decide. in estonia the first edition of "guidelines for the preparation, use and quality assurance of blood components" was taken as guidance in . a lot of advice came from finnish colleagues. in , it was decided to move towards non-paid voluntary donations. the process took years. the first couple of years were economically difficult for the reborn state, as money had less value than food. instead of cash, donors were given rapeseed oil, sugar and pasta, for example. as the situation improved, food items were replaced by small symbolic gifts that carry sentimental value. it has been this way for more than years by now. in lithuania, the process started later, the first program for developing a framework for voluntary non-remunerated donations being carried out in - . it resulted in % of the donations being unpaid. the second program initiated in is still ongoing, aiming towards % non-remunerated donations by . by the end of , they had reached . %. in the beginning, the main obstacle was a private blood center creating unfair market conditions. in latvia, monetary compensation for blood donations still exists, but the younger generation has been encouraged to donate blood for free and some results can already be seen. summary/conclusions: a common starting point does not guarantee the same results, at least not at the exact same time. examining the circumstances leading to the different outcomes could benefit countries yet to start moving towards non-remunerated donations as well as those considering the opposite. haemoglobin (hb) was as expected significantly different between women and men (meanaesem: . ae . vs . ae . g/dl; p < . ). percentage of females with low hb < . g/dl were . %, . %, . %, . % and . %, percentage of males with hb < . g/dl were . %, . %, . %, . % and . % for the age groups - respectively. ferritin values were higher in males compared to females (median; th - th %>tile: ; - vs ; - lg/l; p < . ) and in older age groups compared to younger age groups (median; range in age groups - in females: ; - , ; - , ; - , ; - , ; - and in males: ; - , ; - , ; - , ; - , ; - respectively) . percentage of females with ferritin ≤ lg/l were . %, . %, . %, . % and . %, while percentage of males with ferritin ≤ lg/l were . %, . %, . %, . % and . % for the age groups - respectively. white blood cell counts (wbc) were slightly higher in females compared to males (meanaesem: . ae . vs . ae . ; p < . ). percentage of females with wbc > x /l were . %, . %, . %, . % and . %, while percentage of males with wbc > x /l were . %, . %, . %, . % and . % for the age groups - respectively. none had wbc < x /l. platelet counts (plt) were higher in females compared to males (meanaesem: ae . vs ae . ; p < . ).percentage of females with plt < x /l were . %, . %, . %, . % and . %, while percentage of males with plt < x /l were . %, . %, . %, . % and . % for the age groups - respectively. among the low plt counts most were caused by edta-dependent pseudothrombocytopenia. extreme deviations from normality were seldom and referred to gps for further investigations. summary/conclusions: first time donors are young with % younger than years of age and the female/male ratio was / . of the first time donors with data on ferritin available, % had low ferritin (≤ lg/l). the typical male first time donors neither had low hb nor low ferritin, even with a significantly lower ferritin in younger donors. in female first time donors the prevalence of low hb ( %< . g/dl) and low iron stores ( %≤ lg/l) is high. in all, while all first time donors are highly appreciated, campaigns could target the male population to even out the gender imbalance. blood centers must be aware of the higher prevalence of low iron stores in the youngest donors. background: the aim of assessing suitability of prospective blood donors is protection of their health and the safety of transfused patients. selection process is not always effective in obtaining all relevant information from blood donors in a timely manner. for several reasons, some risks remain undetected or they are disclosed at a future donation(s). therefore, recording and management of post-donation information (pdi) are of great importance for improvement of transfusion safety, donor counselling and education as well as overall improvement of the selection process. aims: the aim of the study was to present results of pdi management at croatian institute of transfusion medicine (citm) and the effect of education activities on their trends. methods: we have analyzed reports on pdi recorded in two-year period ( - ), according to the types of information obtained, age and sex of blood donors, total number of their donations preceding pdi, and the time of receiving the information. the effect of an information leaflet on pdi launched in november was assessed by comparing results in two study years. results: a total of pdi were recorded: in ( / donations) and in ( / donations) with the following distribution: nonsexual risk as tattoo and piercing ( . %), surgical procedures ( . %), travel history ( . %), infections/ contact ( . %), other medical reasons ( . %), endoscopy/invasive diagnostic procedures ( . %), malignancy ( . %), autoimmune diseases ( . %) and sexual risks ( . %). majority ( . %) were late pdi, revealed on the future donation(s): . % on the first next donation, . % on the second and . % after more than subsequent donations. the mean age of blood donors associated with pdi was ae years (median years), while the mean age of all donors in / was years (median years). of all pdi, . % were related to male donors ( % in total pool of citm donors). using chi-square test there were no significant difference between female and male donors in total pdi frequency and in their distribution to early and late pdi (p > . ). the median number of all donations preceding pdi was for female donors and for male donors. implementation of education leaflet for blood donors resulted in . % reduction of pdi in compared with (p > . ). the effect is more pronounced (p < . ) when comparing second and first half of (- . %). reduction is observed in all types of pdi with the exception of infections/contact (because they are mostly early pdi) and malignant diseases. the share of early pdi increased from . % in to . % in , which may suggest better awareness of blood donors on the importance to inform blood bank on changes in their health status. summary/conclusions: our study points to the importance of systematic recording and management of pdi, including education of blood donors about the need of providing all relevant facts related to their health and the safety of donated blood in a timely manner. we are planning further improvements by providing information on this topic on posters and screens on donation sites. background: currently, the transfer of data between organizations and/or computer systems is very limited, and where present is typically proprietary. in the absence of a standardized reference format individual organizations and vendors attempting to integrate disparate databases must develop unique solutions. aggregation of information from multiple sources is complex and costly, constituting a significant barrier to effective analysis of data to improve practice and inform policy. aims: to standardize the definitions and facilitate integration of key data items used in blood donation and transfusion. we report here on an initial effort to map internationally harmonized critical steps in the blood collection/donation process in order to test the approach. methods: through a collaborative process of serial conference calls and correspondence, an informal multi-national consortium of experts across the transfusion industry are attempting to create a vocabulary with sufficiently precise definitions to be usable by automated systems and that can be the foundation of a blood collection/transfusion medicine common data model (cdm), using the following steps: -define the scope of activity to be addressed and segment into key processes. -identify the set of data elements in each segment that are common to all systems. -review and consider existing standards and definitions for each data element. -develop draft definitions for each data element. -release draft to public domain for critical review and refinement with long-term goal of gaining widespread endorsement. results: a standardized approach to blood donation was mapped through identification of common pathways and core mappable data elements. denominator data associated with donor characteristics and blood collection was selected as the first segment to address. a dictionary (or vocabulary) of common terms has been created and will be presented for international comment. summary/conclusions: developing an international consensus on the core elements and their definitions across the transfusion chain is critical for data integration and automation efforts. the expected benefits of this endeavor include that it allows the establishment of algorithms to automate reporting and thus reduce hands-on staff time; reduces time and resources needed to integrate new databases; allows systems to continue to use existing concepts and definitions internally while also providing data output in a standardized format; supports the ability to consistently analyse, interpret and present information regardless of the data source; establishes data definitions against which new systems can be developed; helps to improve comparability of results by providing a common data model for researchers and policy makers; improves confidence in data integrity and reliability of the derived information as a © the authors vox sanguinis © international society of blood transfusion vox sanguinis ( ) (suppl. ), - basis for rational decision making; and reduces data gathering effort and cost thus improving opportunities for more efficient/complex data analysis. standardizing the transfusion medicine dataset is the first step in achieving the automation of data transfer and analysis needed globally to drive patient safety, research innovation, and best business practices. further steps must address the precise methods of data exchange, identification of responsible entities for maintenance and further development, and engagement of computer system developers. red blood cell (rbc) alloantibodies develop in a subset of individuals following exposure to non-self rbcs through transfusion, pregnancy, or other activities; these antibodies can lead to difficulty locating compatible rbcs, acute or delayed hemolytic transfusion reactions, or hemolytic disease of the newborn. alloimmunization is underestimated due in part to antibody evanescence, the random nature of posttransfusion antibody screens, fragmented medical care, and the lack of widespread antibody registries. factors that influence who will develop detectable alloantibodies are not well understood. transfusion burden is one risk factor for alloimmunization, though many highly transfused individuals never form alloantibodies despite exposure to many rbc units (and many non-self abo blood group antigens). individuals with sickle cell disease (scd) and myelodysplastic syndrome (mds) are more likely to form rbc alloantibodies than most other patient populations. individuals with rheumatologic and other forms of autoimmunity, though not chronically transfused, are also at higher than average risk of forming rbc alloantibodies. inflammation, in a broad sense, is one common thread among these diagnoses associated with high prevalence rates of rbc alloimmunization. reductionist murine models support some types of inflammation (including viral-like stimuli) around the time of rbc exposure as being associated with an increased likelihood of alloantibody formation. strategies other than transfusion avoidance or extended antigen matching beyond abo/ rh would be beneficial to prevent new rbc alloantibody formation, especially in patients at highest risk. background: the unique genetic makeup of the omani population makes them rich in the genetic blood disorder. % of omani populations are Àa/Àa gene carriers, % Àa/aa, and % of the population are aa/aa. around % of omani nationals carry the gene for hbs, and - % carry the gene for b-thalassaemia. recent statistics show that there are around patients with thalassaemia major and with scd in oman. the other rbc abnormality that is common in oman is g pd deficiency which is found in % of males and % of females. omanis are known to have the highest frequency of a thalassaemia and g pd reported so far in any race. although blood transfusion is one of the supporting treatments of scd, it can cause some serious complications for the patients. alloimmunization of red blood cells is one of the consequences of blood transfusion. alloimmunisation of the rbcs can cause haemolytic transfusion reactions and may trigger hyperhaemolysis, in which transfused and patient's own rbcs are destroyed. alloantibodies can cause delay in the process of transfusion, it can be costly and time consuming. high number of patients developing alloantibodies may indicate a major difference in the patient and donor population. it may also indicate lack of a controlled, generalised sickle patients management policy. in oman the decision of transfusing scd patient is left to physicians attending the patient. aims: this study is aimed to highlight the increasing number of alloimmunised sickle cell patients. in the royal hospital we get new cases of sickle patient with alloantibodies each year. the acknowledgement of these cases may help in is assessing the current practice of transfusing scd patients, or will help to define the donor and patient population difference. methods: patients were recruited in the royal hospital for this study. edta blood samples were taken for antibody screening test and in the positive cases antibody was identified, all tests done by capture technique using immucor neo machine. results: of the scd patients, % of the patients were male and % female, mean age was years, in the range of - years. % of the scd cases were positive for the alloantibodies, % were female and % were male, the age range was from - years. % of the positive were scd, % s trait and % were s/ bthal. most of the patients developed one antibody, however cases of multiples antibodies were also detected. % of the patients were with single alloantibody, % of them with two antibodies, % with three antibodies, % with four antibodies and % with five antibodies. the majority of the cases were igg against rh antigens anti-e is being the majority %, followed by anti-d %, anti-k %, anti-c %, anti-c %, anti-jk a %, anti-jk b %, anti-fy a %, anti-e %, anti-s %, antis %, anti-kp a . %, anti-fy b . % and igm being %. summary/conclusions: rbc alloimmunisation rate is high in oman majority of the patient affected are female. interestingly sickle trait patients were also transfused and % of them developed alloantibodies. the practice of transfusing rh and kell matching blood unit is implemented four years ago and still high alloimmunization percentage is achieved. background: in ghana, routine pre-transfusion investigations for patients with sickle cell disease (scd) involve only abo-d typing and immediate spin crossmatch, without screening for irregular rbc antibodies aims: determine the prevalence and specificities of and risk factors for rbc alloantibodies in multi-transfused patients with scd methods: in , a cross-sectional study in multi-transfused patients with scd, from two tertiary hospitals in ghana was performed. participants' data on demography, transfusion and medical history were recorded. antibody screening and identification tests were done at sanquin, the netherlands, with standard serology using liss as enhancer and with papain treated rbc panel cells ('enzyme only'). characterization of rhd genes was done by multiplex ligase amplification assay. logistic regression was used to determine the association of patient characteristics, i.e. sex, age at enrollment (continuous), age at first transfusion (categorized as ≤ , - , - and ≥ ), previous pregnancy, number of transfused units ( , - and - and > ), and years after last transfusion (< , - , - , > y) with presence of alloantibodies results: patients ( males and females, median age years, range . - ) were included. the median number of transfusions was (range - ). the median years after last transfusion was (range weeks- . years). in patients, anti-rbc antibodies were detected. in of them the antibodies were weakly reactive with enzyme treated cells only or pan-reactive, possibly some of them representing autoantibodies or antibodies against high frequency antigens. in seven patients enzyme-only anti-le a was demonstrated, likely naturally occurring antibodies. thus, in at least patients ( . %) alloimmunization was demonstrated or suspected; in patients the alloantibodies were 'enzyme only'. besides, the alloantibodies of known specificity ( anti-d, anti-d+c, anti-e, anti-c, anti-e, anti-k, anti-s, anti-le a , anti-go a ), three antibodies reactive only with fy(a-b-) cells and two antibodies of yet unidentified specificity were detected. in six d-patients ( had been pregnant) anti-d (together with anti-c in two patients) was found. in three out of four d+ patients with anti-d, an rhd variant gene was demonstrated ( dau-alleles and diii type or diva- ). logistic regression revealed that none of the risk factors analysed was associated with the presence of antibodies in the patients. immunobiology -red cell alloimmunity fifty-eight patients, had experienced an adverse reaction during or shortly after transfusion ( patients had dark urine). adverse reactions were associated with the number of units received (or . ( % ci, . - . ; p = . ), but not with the presence of antibodies (p > . ) summary/conclusions: in at least % of multi-transfused patients with scd alloimmunization could be demonstrated, mainly ( %) directed against rh antigens. the enzyme only reactivity, coupled with absence of antibodies in seven of patients with probable haemolytic reaction and known evanescence of especially non rh antibodies suggest possible low titre and disappearance of some clinically relevant antibodies. given the high immunization rate together with the high frequency of adverse transfusion reactions, pre-transfusion screening for rbc antibodies should be considered for patients with scd. background: rh blood group system and mainly antigen d is one of the most immunogenic, diverse and clinically important protein-based blood group. antibody anti-d may induce hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. anti-d prophylaxes become ineffective if an anti-d immunization has occurred. approximately % of the d+ population carries rhd alleles associated with reduced d antigen expression. qualitative variants, in which some epitopes are lacking and can produce anti-d antibody, are usually termed partial d. by contrast, d weak is commonly defined as a quantitative variant that have all d epitopes and should not make anti-d. del is a very weak form of d antigen and cannot be detected by routine serological tests. because some of del individuals have already developed an anti-d antibody whereas others did not this group contains both qualitative and quantitative changes. aims: investigation was prompted by finding discrepant results in typing of d antigen in a pregnant woman / rd pregnancy, st delivery, abortions in st trimester/. routine serological techniques detected d negativity and the presence of antibody allo-anti-d in clinically significant titre. the non-invasive testing of d status of the foetus from maternal peripheral blood was indicated, but this was not applicable due to presence of the rhd gene in the woman's dna sample isolated from buccal swab. our aim was to investigate the discrepancy and determine the underlying rhd genotype. methods: blood samples, dna from peripheral blood and buccal swab of the pregnant woman were investigated. routine blood grouping and antibody testing were performed by column agglutination. two anti-d sera (id-diaclon anti-d igg (cell line esd ) by biorad and anti-d duo igm+igg, clone: th + ms by immucor) were used for adsorption/elution test for identification of del phenotype. initial rhd genotyping was performed by rt-pcr (exons , , ) with the dna from buccal swab; further resolution was performed using pcr-ssp (fluogene; inno-train diagnostik gmbh); sequencing was performed by sanger analysis (inno-train diagnostik gmbh). results: genotype was identified as rhd positive by ce-certified pcr-ssp kits (fluogene). sanger sequencing of rhd from exon to revealed presence of a nucleotide deletion in position c. dela, which is specific for allele rhd* el. . this nucleotide change results in the amino acid change p.val leufs* causing the del phenotype. presence of antigen d was proved by adsorption/elution technique. titre of the anti-d was rising during the pregnancy to the level two weeks before the delivery. the newborn was delivered by s.c. without a sign of hemolytic disease. blood grouping of the newborn revealed blood group a, d negative, dat negative, testing for del was not performed. summary/conclusions: the case reported here shows that females with rhd* el. allele are able develop strong anti-d immunization, so this type of del phenotype belongs to the "partial del subgroup". presence of variant rhd gene in mother disabling antenatal fetal genotyping from maternal blood by current methods requires a more attentive approach to care for such pregnancies. supported by mh cz-dro uhkt and rvo-vfn . a-s - ea scharberg , s rothenberger , a st€ urtzel , n gillhuber , s seyboth , e richter , g rink and p bugert institute for transfusion medicine and immunohematology, drk-bsd ba-w€ u-he, baden-baden institute for transfusion medicine and immunology, heidelberg university, medical faculty mannheim, mannheim, germany background: rb a (di ) is a low prevalence antigen of the diego blood group system. it has been found in few families only. the clinical significance of anti-rb a is unknown so far. the slc a *c. c>t (p.pro leu; isbt allele name: di* . ) allele is the molecular basis of the rb a antigen. in the gnomad database this gene variant was found in only one of , sequenced genomes (allele frequency: . ). aims: to prove the frequency of the allele in our population and gain an rb a positive donor we performed a molecular screening for di in , blood donors. after our antibody screening test accidentally contained an rb a positive test cell we found out that anti-rb a is a very common antibody specificity. the frequency of the antibody in patients and blood donors was proved. methods: for the molecular screening of the blood donors we developed a pcr-ssp method. the antibody screening test in , patients and in blood donors was performed in the gel technique (biorad ahg id-cards) using a cell screening panel (drk-bsd src) including an rb a positive test cell. positive reactions with the rb a positive cell were confirmed by an additional rb a positive test cell of different source. additional antibodies were excluded or identified in the same method using an antibody identification panel (drk-bsd irc). results: the molecular screening for the di* . allele in , blood donors revealed no single positive individual. within the first weeks of usage of our antibody screening test which accidentally contained the rb a positive test cell patients with anti-rb a were found. it was . % of , patients tested in laboratories in different parts of germany. some laboratories stopped using the rb a positive lot to avoid expensive and time consuming identification and conformation tests. in of randomly tested blood donors ( . %) anti-anti-rb a was also present. summary/conclusions: despite the very low frequency of the di* . allele, anti-rb a is a very frequent unexpected antibody in patients and blood donors in germany. it is obviously naturally occurring and is even more frequent than anti-wr a and anti-vw we found in previous studies in around % of patients and donors. a-s - national blood center, ministry of health and sports, yangon, myanmar hemovigilance which detects every event not only for patient' reactions and donor's complications but also incidents and near misses definitely improve quality of blood transfusion services especially for those situations where implementation of all the standards in one time is not possible. healthcare system in myanmar is still in the stage of requiring priority for clinical professions and has limited resources for supportive roles. supportive services including transfusion service are still not a center of interest from prioritization of health care system. blood transfusion service has been practiced in myanmar since . real essence of transfusion service is hidden behind laboratory practice and transfusion is regarded as part of laboratory investigation. hospital laboratories take care of testing of blood donated by replacement donors. this kind of transfusion services under laboratory umbrella is still being practiced in myanmar except national blood center (nbc) which was established in in accordance with blood and blood product law. this law was formulated cohesively with who strategies of blood safety. in , who global data-based study sent questionnaires for assessment of safety status of transfusion service. nbc noticed that there was no data which can support corrective actions for safety. from that time onward, active retrospective review of existing data and introduction of records, prospective finding of process errors and any events from hospital blood banks were recorded and taking into actions at local level. cost of every unit of blood is supported by government. in , national blood and blood product committee was established. the steering committee is working hard to get cooperation from every service by aiming to prevent those undesirable events before establishment of national level policy, standards and guidelines for sustainable service quality. in conclusion, by using essence of hemovigilance as a tool, quality of transfusion service can be improved step by step to fulfil the gap in spite of limited resources. the system started in local, extends to regional level by getting agreement of importance from hemovigilance results and is finally approaching to national level endorsement. background: erroneous transfusion of abo-incompatible(aboi) blood almost always reflects a preventable breakdown in transfusion protocols and standard operating procedures and can have disastrous consequences, with significant morbidity and mortality. these incidents need to be investigated in a systematic manner to identify system vulnerabilities to mitigate risks and improve patient safety. since , reporters to shot have been asked to score( - ) the extent to which the cause of incidents can be attributed to key factors: staff, environmental, organisational and government/regulatory which helps recognise the key factors identified whilst investigating these incidents. aims: to understand why unintentional transfusion of aboi blood components continue to happen despite standard procedures and national guidance available. methods: retrospective analysis of unintentional transfusion of aboi blood components reported to shot between - (inclusive) was done to identify common themes and recognise areas of improvement. information provided using the shot human factors investigation tool (hfit) between - was reviewed to understand more about why the errors occurred. results: sixty-seven unintentional aboi transfusions were reported between - ; majority ( / , . %) were red cell transfusions but aboi plasma ( / ) and platelet transfusions ( / ) were also seen. most errors occurred in the clinical area ( / , %), and could have been detected at point of administration. in ( %) cases, the error could not have be detected at the point of administration with a primary laboratory error in / ( %) incidents. reviewing data from hfit for cases in - ( aboi cases), the total score for staff culpability was , compared to a total score of for all the other three organisational and system factors. this disparity is most obvious for the aboi red cell cases, all of which scored the maximum for staff culpability, i.e. / compared to / as the combined total score given to the other factors. in the preceding years ( to ), there were no hf scores available; however, the emphasis on staff-related culpability is demonstrated by cases that included an outcome of the local case review and ( . %) mentioned staff-related retraining or disciplinary procedures. the risk of haemolysis and serious harm is more likely with aboi red cells than with other components with / ( %) that resulted in death, / ( %) major morbidity and / ( %) no or minor adverse reaction. of these cases, one resulted in conviction for manslaughter and at least two staff dismissals. summary/conclusions: transfusion never events continue to occur, and it is evident that investigations into such incidents focus mainly on staff failings and do not consistently identify system wide changes that need to be incorporated to address prevalent issues. national recommendations and a safety alert to 'use a bedside checklist' immediately prior to administration were issued between - to support prevention of such errors but never events continue to persist. current approach is ineffective because it often leads to apportioning blame, rather than understanding the often-complicated and multidimensional factors contributing to the error. this must be replaced by a holistic approach which addresses local work pressures and embraces advances in automated technology like electronic prescribing and barcode scanning. of the confirmed trars, n = were possibly related to treatment, n = trars were probable, and n = were definitely related to treatment; n = trars were grade , n = were grade , and none were grade . in recipients of conventional wb, there were n = ( . %) ars, n = ( . %) fnhtrs, n = ( . %) taco, n = trali, and n = ( . %) unclassified transfusion reactions. of the confirmed trars, n = were possibly related to treatment, n = trar was probable, and n = were definitely related to treatment; n = trars were grade , n = was grade and n = was grade . there were mirasoltreated wb transfusions in pregnant women and trars ( . %), both grade and probably related. there were transfusions of mirasol-treated wb and transfusions of conventional wb in patients < years old resulting in n = ( . %) trars in recipients of mirasol-treated wb and n = ( . %) in recipients of conventional wb. summary/conclusions: timely data reporting of trars and expanding the hv infrastructure has helped to improve the hv system in ghana. of wb transfusions in routine use in ghana, there were . % trars in recipients of mirasol-treated wb and . % in recipients of conventional wb. additionally, mirasol-treated wb was safely transfused in pregnant women and pediatric patients. haematology, monash health, melbourne, australia background: transfusion-associated graft-versus-host disease (ta-gvhd) is rare and usually fatal. it can be prevented by provision of irradiated blood products to at-risk individuals, such as those receiving nucleoside analogues, alemtuzumab, bendamustine or with hodgkin lymphoma (hl). duration of risk is uncertain, so ensuring these individuals correctly receive lifelong irradiated blood components, as currently recommended by anzsbt and bsh guidelines, is challenging. in australia, platelets are routinely irradiated, but red blood cells (rbc) are not. aims: to determine whether patients receiving fludarabine, cladribine, bendamustine, alemtuzumab, or dacarbazine (for hl), appropriately received irradiated rbcs. secondary outcomes included rates of ta-gvhd after unintended exposure to non-irradiated components, factors influencing correct issue of irradiated rbcs such as transfusion management plans, and provision of adequate clinical information on blood requests. methods: we performed a retrospective audit to identify patients receiving therapies indicating risk for ta-gvhd using pharmacy dispensing records from january to october at monash health, a multi-campus university hospital in melbourne, australia. diagnosis, treatment dates, group and hold (g&h) requests, rbc transfusions, and follow-up information were sourced from laboratory and medical records. results: we identified patients who received fludarabine (n = , %), bendamustine (n = , %), cladribine (n = , %), dacarbazine for hl (n = , %) and alemtuzumab (n = , %). the median age of patients was years (range - ) and ( %) were male. median follow-up was months (range - ). post-exposure, patients ( %) received transfusions with % correctly receiving irradiated rbcs. the remaining , all from haematology/oncology, received a total of unirradiated rbcs. in patients, this was rectified on subsequent transfusions. there were no cases of ta-gvhd at median follow-up of . months (range - ) from first rbc transfusion. after medication administration, patients had g&h requests after a median of months (range - ). only % of requests had sufficient clinical information to prompt irradiation, such as hl or medication details, and only % asked for irradiated components. preventive strategies have now been employed. transfusion management plans for haematology patients were implemented in march . for audited patients, these were written from days prior to days after medication exposure. two were written following inadvertent unirradiated rbc transfusion. patients identified in this audit will have a laboratory flag generated and prospectively, pharmacy dispensing records will be sent to blood bank to identify at-risk patients. our hospital is transitioning to electronic medical records (emr). an alert will be generated in emr when ordering transfusions if there has been exposure to these medications. however, clinical awareness and documentation remain vital. additional measures include patient education, alert cards, and ongoing collaboration with medical staff to encourage transfusion planning. summary/conclusions: recognition of patients at risk for ta-gvhd remains low, even among haematology units. we are making progress on ensuring provision of lifelong irradiated blood components in patients exposed to nucleoside analogues or alemtuzumab, as well as hl patients. implementation of an emr and additional strategies in this domain is important to prevent ta-gvhd. background: blood transfusion is considered an essential element in the management of patients globally. it might be risky and transfusion related adverse reactions may occur with the less adherence of transfusion policies. standard guidelines regarding the screening of blood for infectious disease, genuine need of transfusion and abo compatibility are followed and monitored drastically. however, patient assessment during transfusion especially at patient bedside and post transfusion is also equally important. aims: we are a newly established hospital and are working towards the best possible management of patients. in this regard to minimize the transfusion errors and to highlight if any lacking being practiced during transfusion, we conducted this study to observe the compliance rate of documentation of transfusion form by the healthcare staff and also to observe the compliance of line of action taken in case of occurrence of transfusion reactions. methods: this was a observational study conducted at nibd and bmt, pechs campus from february to february . ethical approval was obtained prior to the study. transfusion form for each transfusion was filled. the form provided information on documentation of blood product receiver name, employee identity number, date and time of receiving blood product, patient name, medical record number on units, on patient's wrist band and on transfusion form. abo compatibility on the unit and on form, medical record number from wrist band, name and employee identity number of two healthcare staff started transfusion, transfusion start and completion time. time, temperature, blood pressure, pulse and initials of staff at the time of order, onset, after minutes and at the completion of transfusion were also included. transfusion reaction form was also filled by the healthcare staff. data was analyzed by using spss version . . results: a total of transfusions forms were analyzed. over all compliance rate was %. out of , ( %) forms were available in source notes and of , ( %) were partially and completely filled. higher compliance was seen in the initial months of hospital establishment than later months (p-value = . ). highest non compliance was seen in documentation of initials of duty doctors on transfusion form at the completion of transfusion( %) and highest compliance was seen in documentation of name by healthcare nursing staff at the start of transfusion( %). a total of ( . %) adverse events were reported from red blood cells and platelets. mean time of start of symptoms was hours and minutes for red blood cells and for platelets it was hour and minutes. transfusion was instantly stopped as the symptoms appeared with no delay of time and actions were taken to resolve the reactions. time of appearance of symptoms and time of start of medication were documented and error free. all blood bags were returned to the blood bank and discarded after hours as per the policy of hospital. summary/conclusions: the study was conducted to highlight the scarce practices that are being implemented by healthcare staff in context of documentation and reporting of transfusion reactions at our hospital. stringent actions should be taken for the adherence of compliance by healthcare staff to avoid morbidities and mortalities. we believe that it will also be helpful to provide baseline information in the process of preparation of a national guidelines and protocol on blood transfusion procedures. a-s - as buser, a holbro and l infanti regional blood transfusion service, swiss red cross, basel, basel, switzerland to make blood supply safer, pathogen inactivation (pi) technologies have been developed. they are based on photochemical (amotosalen/uva or riboflavin/ uv) or uv-c light treatment to reduce potential pathogens in blood components. this gain of safety might however be offset by "off target" effects of these technologies. in virtually all clinical platelet transfusion trials, it has been demonstrated that post transfusion increments with pi platelet (plt) components are lower as compared to conventional components, indicating different biological behaviour such as survival/ clearance of nontreated and treated plt. published studies have also suggested shorter survival of platelets in vivo in animal studies. additionally, data of the rates of alloimmunization and refractoriness after transfusion of pi platelets are show discrepant results. animal studies suggest a reduction of the rates of alloimmunization when transfusing (leukoreduced) pi plt as compare to conventional plt. in the clinical setting, published data, including very recent reports, showed different rates of hla class i and ii alloimmunization with the two currently available photochemical based pi technologies. while pi of plt components surely benefit patients regarding pathogen safety, the impact of potential off target effects possibly impairing efficacy of pi plt transfusions need more investigation. background: brucellosis is an endemic disease and still a major health problem in saudi arabia. ministry of health in saudi arabia listed brucellosis as a notifiable disease due to its endemicity. in the last ten years, the incidence has decreased significantly to approximately cases per , but is still higher than that in developed countries. human-to human transmission is extremely rare including breast feeding, transplacental, sexually and blood transfusion. five cases of brucellosis through blood transfusion have been reported in the literature. brucella transmission through blood transfusion is likely underreporting due to the long incubation time of - weeks (range, days to months),vagueness of clinical presentation and lack of hemovigilance systems in endemic areas. (allohsct) and ( . %) autologous (autohsct) hsct patients, with mean corrected count increments (cci) of . , . and . , respectively. mean cci decreased in a linear fashion between day ≤ and day pcs ( . , . and . at ≤ days; . , . and . at days, respectively), although the number of pc transfused on day to autohsct patients was small (n = ). background: nipah virus (niv) is a paramyxovirus (genus henipavirus) that emerged in the late s in malaysia and has since been identified as the cause of sporadic outbreaks of severe febrile disease in bangladesh and india. niv infection is frequently associated with severe respiratory or neurological disease in infected humans with transmission to humans through inhalation, contact or consumption of niv contaminated foods. nipah virus (niv) belongs to the list of pathogens identified by the who to have the potential for a global pandemic. aims: this study aimed to investigate the efficacy of the theraflex uv-platelets system to inactivate niv in platelet concentrates (pcs). the theraflex uv-platelets system (macopharma) uses uvc light without the need of any additional photoactive compound. methods: plasma reduced pcs from bcs ( % plasma in additive solution ssp+) were spiked with virus suspension ( % v/v). pcs (n = , ml) were then uvcirradiated on the macotronic uv machine (macopharma) and samples were taken after spiking (load and hold sample) and after illumination with different light doses ( . , . , . and . (standard) j/cm )). the titre of the niv (malaysia) was determined as tissue culture infective dose (tcid ) by endpoint titration in microtitre plate assays on vero cells (atcc â crl- tm ). the results of the infectivity assay demonstrated that uvc irradiation dosedependently inactivated niv. after spiking a niv titer of . (bag no. ) and . (bag no. ) log tcid /ml was received in the pcs. at a uvc dose of . j/cm and higher niv was inactivated down to the detection limit of the system ( . log tcid / ml), resulting in log reduction factors of ≥ . (bag no. ) and ≥ . (bag no. ). summary/conclusions: our results demonstrate that the theraflex uv-platelets procedure is an effective technology to inactivate niv in contaminated pcs. vs. ae . e platelets/unit, p < . ), whereas the platelet content of apheresis pc did not change ( ae . vs. , ae . , p = . ). summary/conclusions: pathogen reduction resulted in the transfusion of older pc on average, but without altering the number of pc ordered or the use of pc per patient. pathogen reduction has improved pc stock management without an increase in platelet demand, despite lower platelet content of buffy coat pc after pr implementation. donors and donation -donor adherence -are we doing the right thing? the transfusion procedure is the last step in a multi-process supply chain. the task of matching supply with demand requires donor managers to consider average consumption rates on a weekly or monthly basis, but to also have insight into variability in order distribution and possible attribute (blood groups) requirements. since hospitals and blood banks are usually not deeply interwoven and often only ex-post data is available, forecasting methods should be implemented. a thorough analysis of order pattern to set weekly target inventories and safety levels is required to close the information gap. a collection plan needs to identify possible bottlenecks which can be prevented through the planning of inter-shipping, changes in message urgency and building of reserve donor pools. constant analysis of collection and mobilization kpis allows donor managers to implement the rolling-wave planning approach and continually adapt to changing requirements, unexpected events and overall systematic variability. the variability happens on the demand side, as order quantities and their attributes, such as blood group distribution, are subject to change. however, also the supply is subject to significant variation, as donor response rates, attrition, deferrals and overall availability of donors are not constant. the data was collected with the face-to-face interview method right after the donation. first-time donors has attended to the study in regional blood centres in cities in turkey. the survey included items in accordance with the standard tpb predictors of attitude, self-efficacy, and intention. self-identity, anticipated regret, donation anxiety, paraphernalia anxiety, personal moral norm, descriptive norm, satisfaction, motivation also assessed for the first-time donors. the relation between the predictors and intention confirmed with correlation analyses. the predictors' distribution analysed by multiple linear regression. a number of goodness-of-fit indices were calculated and examined for each tested models (ibm, amos spss). the results of goodnessof-fit tests for proposed model provided a better fit to the data than these models (cmin/df = ). moreover, this result indicated that the fit between the proposed model and the data could be improved with further modifications with the inclusion of paths between motivation and attitude, self-identity and intention. moreover, inclusion the paths between donation anxiety and intention and between self-efficacy and attitude, on contrary to recent analyses suggesting opposite paths. evaluation of goodness-of-fit tests showed good result for revised model with a value of cmin/df = . , close to perfect fit. the revised model revealed that attitude was the strongest positive direct predictor of intention followed by personal moral norm, self-identity, motivation and anticipated regret (path coefficients: . , . . . , . , and . , respectively). donation anxiety was the negative direct predictor of intention (- . ). satisfaction was the strongest positive indirect predictor of intention via attitude and followed by self-efficacy ( . and . ). paraphernalia anxiety was the negative indirect predictor of intention (- . ). descriptive norm did not show any significance. our model accounted for . % of the variance in intention. summary/conclusions: these findings suggest several potential avenues for enhancing donor retention. the results obtained with this study provide important data from the standpoint of donor retention, which should be, implemented in the future strategies of turkish red crescent. background: transpose-transfusion and transplantation: protection and selection of donors, is a european consortium project, including partners from countries, reviewing donor selection and protection policies for substances of human origin (soho).one of the main issues in the current donor selection system, which transpose aims to tackle, is that for many, if not most criteria, is not evidence based. the transpose consortium therefore tries to re-assess selection criteria, revised them where needed and provide recommendations as evidence-based as possible. transpose additionally adds to the current european directorate for the quality of medicines & healthcare (edqm) guidelines by emphasizing donor safety. aims: the aim is to compare existing donor eligibility criteria throughout europe, and to compile a list of risks to consider, with evidence-or consensus-based deferral criteria to provide more uniform donor screening criteria. methods: there are three horizontal work-packages (wps); wp coordination, wp dissemination, and wp evaluation of the project, and four technical ones with specific deliverables and milestones to be regularly produced: -wp inventory of donor selection & protection practices; -wp development of risk-based guidelines for donor selection and protection; -wp development of a standard donor health questionnaire (dhq); -wp training course/workshop on the use of the guiding principles, guidelines and the dhq. the transpose project launched in september and will complete in spring . wp has completed its work in october, wp will complete its work in june , and wp and wp have recently commenced. results: with the use of the deliverables created by wp , we have created an indepth inventory of current practices in donor selection and protection, including overview of similarities and differences across european countries and across soho types. there is an agreement amongst experts that existing guidelines are often based on the precautionary principle rather than on risk assessment. consequently, in the development of wp 's guidelines for donor selection and protection, we now make an effort to also emphasize donor safety, in a more evidence-based way via the use of risk-based assessments. this will result in a standardized dhq with a common trunk and more in -depth questions per soho. summary/conclusions: the impact of the outcomes of transpose will be threefold. first, outcomes are expected to be of help in revising donor selection and protection related eu directives. second, the set of guiding principles and donor selection & protection guidelines will facilitate eu member states to take a next step in implementing donor selection and protection policies in a consistent and clear-cut way to the benefit of both donors and recipients of soho. third, a standard donor health questionnaire with carefully guided local/regional/national adjustments will become available per soho which can be used widely and will consequently enable comparisons of the prevalence of certain risks and risky behaviours throughout europe. background: transpose-transfusion and transplantation protection and selection of donors is a european consortium project, including partners from countries, that reviews donor selection and protection policies for blood, plasma, tissues, assisted reproductive technology (art) and stem cells (together soho). donor selection criteria (dsc) in europe are based on eu-directives, guidelines and countries' own additional criteria. literature shows that particular criteria are outdated or not risk-based, often leading to unnecessary donor deferral or an underestimation of risks for donors. aims: to ) provide a comprehensive inventory of current systems for selection and protection of donors and donations, ) critically review them and ) recommend an over-arching donor health questionnaire (dhq) including all necessary criteria currently used by different eu-member states (eu-ms). methods: in-depth semi-structured interviews with key stakeholders in blood collection were conducted to identify main topics for improvement in the current dsc. these formed the basis for a survey sent to professionals from collection institutions of all soho to get feedback on current systems from as many eu-ms organisations as possible. questionnaires were sent to a total of experts ( blood; plasma; tissues; stem cells; art) and ( %) completed questionnaires were received. where information was lacking, additional experts were asked to recommend upon dsc. results: for blood and plasma donation four main areas of concern in dsc were identified: risk-based selection, adaptability, flexibility and consistency. the stakeholders agreed that dsc are often outdated and lack evidence, hence leading to unnecessary deferral of donors and underestimated risks for donors. they suggested to base dsc on group risk-assessment (risk-based selection) and on conducting more research to achieve standardized risk perceptions and evidence-based deferrals, either for safety of recipient or donors. criteria could be made more detailed to fit specific groups to defer less donors (adaptability). furthermore, implementing criteria was considered easy, but abolish criteria when not regarded as a risk anymore seems almost impossible (flexibility). additionally, deferral periods are perceived too long, seen as both negative, i.e. jeopardizing donor return intention and positive, i.e. no risk for safety (consistency). changing legislation into guidance was an often-mentioned suggestion to improve dsc. specific feedback on plasma donations revealed that many whole blood topics are not applicable to plasma-only donors, e.g. parasite infections such as malaria (no deferral needed); travel history (no deferral needed), and recent bacterial and viral infections (deferral periods currently too long). a clear need for more research on plasma collection-related issues was identified. summary/conclusions: dsc are perceived redundant on a substantial number of aspects by most stakeholders. besides achieving the goal of save and sufficient soho for patients, many regulations could be improved to diminish deferrals and decrease donor risks. transpose will add to reviewing, improving and harmonising these regulations and criteria. furthermore, transpose will provide suggestions to improve directives and guidelines and a dhq, focusing on both donor health protection and safety of donations, but also removing deferral criteria that are not relevant (anymore), and offer a future research agenda to make dsc more evidence-based. background: transpose -transfusion and transplantation: protection and selection of donors, is a european commission co-funded project with participation of stakeholders from both not-for-profit and private blood collecting organizations as well as researchers and officials. the project aims to create new evidencebased donor selection criteria as well as guiding principles for risk assessment of threats to the safety of all substances of human origin (soho) except solid organs. as part of this, an inventory of current donation-related risks was performed, including an investigation of both type and number of adverse events reported. aims: we here aim to present an overview of reported adverse events in plasma and whole blood donation in europe and to compare this to the anticipated risks rated by transpose stakeholders. methods: national or local data on adverse reactions from the years - , both serious and mild, in whole blood and plasma donors was collected from the relevant stakeholders (eighteen and nineteen respectively). stakeholders were also asked to grade the most important anticipated donor risks according to severity, level of evidence and prevalence. we then compared the relevant risk categories as evaluated by the stakeholders with the categories of the provided data, as well as the heterogeneity of category numbers. results: thirteen stakeholders provided data on adverse events during whole blood donation in a given year, including in total thirty-three different categories of adverse events, ranging from only one unspecified reaction to seventeen different categories, with an average of nine categories per stakeholder. the most frequently used categories were hematoma (included by %), arterial puncture ( %) and nerve damage ( %). vasovagal reactions were also frequently included ( %); however, this was being done variably as vasovagal reactions unspecified, and acute and/or delayed vasovagal reactions. only one stakeholder reported iron deficiency. for plasma donation, seven stakeholders provided data on adverse events. a total of twenty-seven different categories were reported, ranging from one to seventeen per stakeholder, with an average of nine. the most frequently reported adverse events were hematoma ( %), citrate reactions ( %) and arm pains and nerve damage (both %, respectively). anticipated risks in blood donation were rated by nine stakeholders rating iron deficiency, vasovagal reactions and hematomas the greatest risks to donors. for plasmapheresis, six stakeholders rated vasovagal reactions, hematomas and citrate reactions as highest risk. summary/conclusions: as shown, categories used to describe adverse events in blood donation vary tremendously across europe, with some countries only being able to provide total numbers of adverse events without further specification. furthermore, there is a gap between perceived high donor risks and reported adverse reaction categories in donor vigilance for whole blood, as reports on iron deficiency are virtually absent despite being considered the most significant risk. our findings show the need for international collaboration on creating an international standardized donor vigilance system, to gather more insight into donor risks to protect the health of donors. plenary session -a glimpse of the future pl- - modern transplantation medicine has made significant progress within the last decades due to a better immunological understanding of rejection and advances in immunosuppression. however, the severe side effects of long-term, typically lifelong, immunosuppression and the shortage of donor organs remain the major restrictions in transplantation. the idea behind all research to improve transplant outcome has always been the modification of the recipient's immune system to ideally induce a specific tolerance towards the donor's graft. in fact, the immunological blindness of the recipient towards the donor's graft is achieved by a general reduction of the immune system's competence and represents a major burden for transplant patients. the idea of invisible organs is an entirely different approach to solve the problem: instead of inducing an immunological blindness of the recipient's immune system an immunological invisibility of the donor's organ is created. this is achieved by genetically engineering the transplant to eliminate the organ's immunogenicity defined by the gene products of the major histocompatibility complex (mhc) and minor histocompatibility antigens. in addition to manipulating the expression of mhc genes required for immune recognition, immune cloaking strategies are used to evade immune rejection. these approaches take advantage of creating an immunosuppressive environment and expressing immune suppressive molecules by immunomodulatory transgenes. mhc engineering and immune cloaking in an entire organ is achieved during ex vivo perfusion by lentiviral transduction of gene expression modifiers and transgenes to induce a permanent immunological invisibility of the organ. importantly, mhc engineering also prevents the presentation of minor histocompatibility antigens, which usually are not possible to match between donor and recipient, but which trigger potent immune responses and graft rejection. eliminating the targets of cellular and humoral rejection as well as creating an allograft-specific immune environment through immune cloaking camouflages the organ and equips it with a powerful set of defense weaponry. immune-engineering of transplants achieved during the inevitable ex vivo period of the allograft after explantation without the need to accept off-target effects allows keeping the recipient's immune system fully functional and capable to combat infections and cancer. in pre-clinical in vivo studies from rodents to minipigs a clear survival advantage of ex vivo engineered transplants could be demonstrated. this approach has the potential of eliminating the burden of organ rejection and immunosuppression, thereby sustainably increasing transplant survival, organ availability and quality of life. gene editing for sickle cell disease: re-expression of the fetal c-globin genes (hbg / ) could be a universal strategy to ameliorate the severe b-globin disorders sickle cell disease (scd) and b-thalassemia by induction of fetal hemoglobin (hbf, a c ). we have previously identified bcl a erythroid enhancer sequences, marked by hbf-associated common genetic variants, that are required for repression of hbf in adult-stage erythroid cells but dispensable in non-erythroid cells. recently we have optimized conditions for selection-free on-target crispr-cas editing in human hscs as a nearly complete reaction without detectable genotoxicity or deleterious impact on stem cell function. we demonstrate that cas :sgrna ribonucleoprotein (rnp) mediated cleavage at core sequences of the + bcl a erythroid enhancer results in highly penetrant disruption of gata binding motif, reduction of bcl a expression, and induction of fetal c-globin. erythroid progeny of edited engrafting scd hscs express therapeutic levels of hbf and resist sickling, while those from b-thalassemia patients show restored globin chain balance. moreover we find that hscs preferentially undergo nonhomologous as compared to microhomology mediated end-joining repair. nhej-based bcl a enhancer editing approaching complete allelic disruption in hscs appears to be a feasible therapeutic strategy to produce durable hbf induction. in this presentation, i will compare and contrast bcl a enhancer editing to other autologous curative gene therapy and gene editing approaches at various stages of clinical and pre-clinical evaluation. oxygen is vital for life. without oxygen death is assured for aerobic organisms. although everybody knows this fact a lot of medical acts forget to take care of it, leading to a lot of potential troubles. indeed, during cell respiration the glucose oxidation by oxygen gives carbon dioxide, water and energy. this energy also called atp is necessary for cellular metabolism and consequently for life. we have identified an extracellular hemoglobin coming from a marine worm, called arenicola marina, which is able to deliver oxygen to this animal living in the intertidal areas on the atlantic coast in france between the north sea and biarritz. this molecule called m was developed in the medical device named hemo life â . we have showed that this product was very efficient to protect organs before transplantation. a multi centers clinical trial performed under the supervision of pr. le meur from the chu of brest, on patients waiting kidney grafts showed a delay graft function reduced roughly by three between the two kidneys harvested on the same donor with and without hemo life â and grafted on recipients. in , a world first was realized in france by the pr. lantieri to georges pompidou hospital in paris, france. indeed, it was the first time that a patient received a second graft face. this surgery was realized with hemo life â and showed a very nice result according the pr lantieri, the anastomosis were very easy and no edema was observed. furthermore, we have developed dressing incorporating m making a product called hemhealing â . preclinical data on diabetic mice showed an increase of healing process. hemoxycarrier â , a therapeutical oxygen carrier, is also in progress of development in order to address ischemic diseases such as the sickle cells disease, myocardiac infraction and stroke. this universal oxygen carrier without blood typing, which is the ancestor of our red blood cell containing hemoglobin showed that it is able to deliver oxygen at different biological levels, cellular, tissues and organs and could address a multitude of medical applications. background: main goal of transfusion is saving life and/or improve the health status of human by "safe blood" which needs regular, voluntary, unpaid blood donors. donor recruitment is being more sensitive and challenging part of the blood supply system in actual global socio-economic conditions. achievement to enough voluntary non-remunerated blood donation (vnrbd) can be established by an efficient donor recruitment. efficiency of the donor recruitment has still close relation with blood donor recruiter although there are so many new tools. occupational specifications, rights and responsibilities of blood donor recruiter have wide range differences between countries which cannot be explained completely by the specific conditions of each country. also, a concrete document which has an international consensus was not existing on this subject. turkish blood foundation (tbf) has been organizing an international workshop since ; anatolian blood days (abd). "who is a blood donor recruiter?" was the topic of abd-vii at - march . aims: main aim of the workshop was to check and evaluate the existing systems of the participant countries. than create a model for clearly defining occupational specifications, responsibilities and rights of blood donor recruiter. methods: experts from countries participated in the workshop. those countries are albania, algeria, bosnia-herzegovina, estonia, france, germany, hungary, india, kazakhstan, lithuania, macedonia, montenegro, oman, portugal, qatar, romania, russia, saudi arabia, serbia, slovenia, sri lanka, tajikistan, turkey, uganda, uzbekistan. these countries reflect almost all religious, ethnical, social, cultural and economic situations of the world. a questionnaire which was analyzing existing systems at participant countries sent before the workshop. after country presentations different discussion groups were organized. below listed topics were announced at final declaration. results: donor recruiter: . should have university degree preferably in marketing and business administration field. . should have a certificate and/or professional experience in public relation . should have efficient skill in conversation, sociability, independence, self-confidence, reliability, resilience and conscientiousness as well as to work in a team . should get a special training which includes not only social topics such as public relation, marketing, etc. and medical topics related bb&tm before practicing alone as a donor recruiter . should be a permanent staff . should have basic salary and performance bonus might be given . is eligible to monitor and modify mobile team working period at blood drive . should participate the mobile blood drive which he/she has organized . should participate the group who will create promotional materials for national blood service . number at each blood establishment should be defined based on annual blood collection such as staffs for , whole blood collection annually in germany. summary/conclusions: in conclusion; both donor recruitment and retention are not easy tasks to undergo while public are aging, and birth rates are decreasing all around the world. dedicated blood donor recruiter whose occupational specifications, rights and responsibilities are clearly defined will be the corner stone of the success for providing enough safe blood for transfusion. ct smit sibinga and j emmanuel background: africa is a large continent with independent states and a total population of , , , (february ) . healthcare policies and strategies are developed through who's advocacy, guidance, and support from hq in geneva and the who regional offices; eastern mediterranean regional office (emro) supporting arabic speaking countries and the african regional office (afro) responsible for sub-saharan countries. population distribution is approximately . % urban. there are a large number of different local dialects and languages spoken. the main languages spoken are english, french, portuguese, spanish and arabic. countries are mainly classified by undp as being of low and medium human development index the africa society for blood transfusion (afsbt) has members in most countries, advocates for the development of sustainable and effective blood services, and has developed a stepwise level accreditation program. in emro held a consensus meeting developing a "strategic framework for blood safety and availability for - " with a set of priority interventions focusing on leadership and governance, cooperation and collaboration, provision of safe blood and blood products, appropriate clinical use of blood, and quality system management. in all member states of the african union (au) countries, in abuja, nigeria, pledged that national budget for health should be at least % of the national fiscal budget. in ministers of health of who member states endorsed that blood and blood products be included in the essential medicines list; these endorsements and who's universal health coverage (uhc), have yet to be fully implemented. aims: to analyze (gap-analysis) to what extend countries in africa implement the world health assembly resolution wha . on availability, safety and quality of blood products, which urges governments to ensure safe, accessible, affordable and available supplies of blood and components from voluntary non-remunerated blood donors, which meet clinical transfusion requirements and achieve national self-sufficiency, following who guidelines and recommendations. methods: to provide an overview of the current status of the blood supply in africa strengths and weaknesses, data from who's global status report on blood safety and availability were analyzed and used. the study has been descriptive and explorative. results: the report identified a number of areas requiring attention; principle amongst these were -inadequate funding; -lack of governance and leadership; -ineffective public education on blood donation; -absence of capacity building for clinicians on rational use of blood; -lack of haemovigilance and implementation of quality management systems; -the need for regulatory or oversight mechanisms. summary/conclusions: national authorities should address areas requiring attention if progress towards ensuring a sustainable safe and sufficient supply of blood products is to be achieved. key is the commitment and support of national governments, which should implement resolutions and recommendations agreed by ministers of health at wha and african union. background: the core function of the blood donation testing (bdt) laboratory is to screen every unit of blood collected from a donor for blood group type and infectious disease markers to ensure safety of the national blood supply prior to transfusion. the lab operates daily on two work shifts, comprising of staff on the morning (am) shift (from : to : ) and staff on the afternoon (pm) shift (from : to : ) on weekdays and staff on the am shift and staff on the pm shift on weekends. bdt lab has a staff strength of to be rostered for the work shifts. each staff is on a five-day work week and has to work pm shift and am shift per month on average. the higher number of pm shift leads to staff feedback that they do not get sufficient time in the evenings for family and social or leisure activities. a lean six sigma project was initiated to review the work rostering to improve the work-life balance of the staff. aims: the project aims to reduce the number of staff working on the pm shift without affecting the downstream processes and continues to meet the timely release of blood supply to the hospitals. methods: lean six sigma tools were used to study the bdt lab workflow process and to identify factors that contributes to the higher number of pm shifts that the staff has to take on. data on the turn-around time and the man-effort required for each screening tests performed was analyzed. a survey was also conducted to understand the preference of the staff on the acceptable number of pm shifts per month. results: the main contributing factor for more staff required to perform the pm shift is due to majority of the daily donation samples being received only in the evening. as this factor is beyond the control of the bdt lab, redeploying work from the pm shift to the am shift was eventually selected as the solution to reduce the number of staff needed for the pm shift. the screening test that was shifted was determined based on the test system that has the shortest turn-around-time and is able to allow continuous release of results. at the same time, most of the staff must be trained for that test system. a trial on the new roster involving staff on am shift and staff on pm shift was conducted. the total number of pm shift per month was reduced from to using the re-defined process. the % reduction translates to fewer number of pm shifts that the staff has to undertake and was able to meet the staff's expectation. summary/conclusions: with the adoption of the new process workflow, bdt lab was able to reduce the number of pm shifts that the staff needs to be rostered using evidence based process improvement method. most importantly, the lab has a satisfied team of staff with better work-life balance. background: preparedness of blood transfusion services for emergencies and crisis situations is an important issue concerned with patient and transfusion safety. aims: having an experience of delay in blood component supply in an emergency situation due to partial interruption of hospital information system (his), it is aimed to create a crisis kit and constitute an alternative work flow for emergency in crisis situations. methods: it is stipulated that the failure of his which is normally conducts all process for transfusion would be disabled in a disaster or crisis situation. a brain storm was made on possible challenges associated with disability of his during transfusion emergencies. according to the scenarios a kit was developed for the process management of transfusion emergencies. results: a flow chart was designed in proper with transfusion emergency definitions of who and instructions were written to explain the flow chart. all forms categorized with different colour codes are designed to fill with handwriting. the kit consists of flow chart and instructions, analysis request forms (blue coloured), blood component request forms (pink coloured), proceeding forms (green coloured), pens and blood sample tubes with edta were put into a plastic folder labelled as transfusion emergency disaster & crisis (tedc) kit. additionally, the kit is placed in a sealed clear plastic bag and delivered to all inpatient and intensive care units of pediatrics and pediatric surgery. a training programme concerned with transfusion emergency situations and usage of the tedc kit was developed for health care workers involved in blood transfusion process. pre and post-assessment tests were developed for the evaluation of effectiveness of the training programme. summary/conclusions: it's challenging to improve the response capacity of blood transfusion services during emergencies and crisis situations. abstract withdrawn. background: india is a developing country having licensed blood banks majority have manual documentation which causes inaccuracies and errors in blood bank activities. monitoring is a herculean task. computerisation is the need of the hour but this goal involves many hurdles and challenges aims: the aim of this study is to discuss the challenges faced during computerisation of blood bank activities and the remedial solutions for it methods: department of transfusion medicine, king george medical university, lucknow is one of the biggest blood bank of the country with annual collection of , blood units. two years ago, the blood bank worked on totally manual system. computerisation involved challenges associated with hardware and software installation and personnel training. hardware was installed in two phases. initially hp system but later shifted to apple imac due to frequent breakdowns. with hp server. software installation (easy software) involved erratic internet connectivity hence changed to lan. customisation involved radical changes according to our needs. at times we had to change our way of working to suit the software. biometrics linking, medical registration, cash id generation, donation, serological crossmatch, automated blood grouping, labelling, chemiluminescence & nat testing, blood component preparation and camps were all included with challenges at every level. remedial actions were taken from small to big. training of the staff was the most essential part of the implementation of computerisation who initially showed considerable resistance and at time faking ignorance due to apprehension that their mistakes will be highlighted and they will be penalised for it. it was a herculean task in creating their password protected identity and enforcing them to use it. gradually the staff realised that computerisation made their task easier as it cut down on paper work and repetition and also prevented serious mistakes from happening. hard copies at certain essential areas were still maintained to continue work in the event of major breakdown of computer results: computerisation aided us in regulating the movement of the donor which at times was repetitive due to manual entry. transfer of data ensured a safe supply and the mistakes could be retraced very easily. implementation which included installation, training and enforcement took a period of months. after overcoming all the challenges we minimised hard copies to registers and started taking printouts of the other necessary details. the turnover time for the employees due to computerisation decreased by %. waiting time for attendant decreased by %. traceability of all the units became %.supervision of the activities being carried out was % accurate. identification of the donors was easy due to biometrics which included thumb impression and iris scanning. the decision making time for donors decreased by % thus making the system more efficient. summary/conclusions: manual to computerisation involves involvement from source to supply and it is essential to anticipate the challenges and be prepared for solutions in order to make its implementation successful p- ct smit sibinga , y abdella and f konings iqm consulting, zuidhorn, netherlands who eastern mediterranean office, cairo, egypt background: who defined essential medicines (ems) as medicinal products that satisfy health-care needs of the majority of a population. they should be available at all times, in adequate amounts, in appropriate dosage forms, with assured quality and affordability. in blood and blood products (whole blood, red cells, platelets, plasma, and plasma-derived products) were added to the who model list of ems. appropriate and effective regulatory framework (legislations, regulations, etc.) and a functioning regulatory authority (ra) is crucial for management of blood products as ems. however, particularly in the less developed world, these prerequisites have barely been implemented. aims: to analyze and advise on existing legislation and regulations. existing legislative instruments of the member states of who eastern mediterranean region (emr) were collected and analysed for relevance and appropriateness for preparation and use of blood and blood products as well as use of associated substances and relevant medical devices. a literature search was done on matching combinations of regulatory system, regulatory framework, legislation, regulation, with production and use of blood and blood products, which resulted in almost exclusively references with respect to national and international legislation. benchmark: who recommendations (aide m emoires) and eu directives. methods: existing legislative instruments of the member states of who eastern mediterranean region (emr) were collected and analysed for relevance and appropriateness for preparation and use of blood and blood products as well as use of associated substances and relevant medical devices. a literature search was done on matching combinations of regulatory system, regulatory framework, legislation, regulation, with production and use of blood and blood products, which resulted in almost exclusively references with respect to national and international legislation. benchmark: who recommendations (aide m emoires) and eu directives. results: various formal legislative documents of only / countries are put in force by governments [ (egypt) till (pakistan -sindh)]. most are detailed descriptions of ra, operational establishments, and specific requirements. however, none of these legislations complies with who and eu recommended formats and contents, and will not support effective regulatory oversight to promote and enhance quality, safety and availability of these ems. summary/conclusions: government should provide effective leadership and governance in developing a national blood system (nbs, fully integrated into the national health-care system. essential functions of a nbs include an appropriate regulatory framework with legislations, regulations and other non-legislative instruments administered by a ra. these documents should spell out principles and cadres, standard setting, and organization of the blood system to ensure an adequate supply of blood and blood products and safe clinical transfusion for which a model was designed. the structure of nbs will depend on organization and level of development of the health-care system. however, all critical activities within nbs should be coordinated nationally to promote uniform standards, economies-of-scale, consistency in staff competency, quality and safety of these ems, and best transfusion practices. key is formulation of an appropriate regulatory framework administered by a ra responsible for regulating the vein-to-vein chain in the preparation and use of these ems. background: the capacity of blood transfusion service to provide adequate supplies of blood components is the issue of concern for health providers worldwide; longer term observations of trends in this respect are therefore of crucial value. aims: the study aim was analysis of some basic activities of the polish blood transfusion service in - including organizational changes, numbers of donors, donations and blood components as well as activities directed at increasing their safety. methods: retrospective analysis of data supplied by the regional blood transfusion centers (btcs). results: in the discussed period, blood and blood components were collected in regional btcs and local collection sites as well as during mobile collections. although the number of local collection sites decreased from in to in in favor of mobile collections, which increased from , to , , the former is still the number one location for donating blood. on average . % of all donations were performed in local collection sites. the total number of blood donors both at the beginning and the end of the discussed period was similar ( , in and , in ); over % of all donors were non-remunerated. however, the number of first-time donors dropped significantly (from , in to , in ). the total number of donations increased from , , in to , , in ; most frequent were whole blood collections (from , , in to , , in ) . some blood components (mostly plasma and platelet concentrates) were also collected by apheresis. most frequently prepared blood components were red blood cell concentrates -rbcs ( , - , , units per year), fresh frozen plasma -ffp ( , , - , , units) and platelet concentrates -pcs ( , - , units, with significant increasing tendency). additional processing methods such as leukocyte depletion and irradiation were more frequently applied to pcs ( - . % in respective years irradiated, . - . % leukocyte-depleted), than to rbcs ( . - . % irradiated, . - . % leukocyte-depleted). in , the pathogen reduction technologies in plasma and the pcs were implemented. up to date however the use of these technologies is limited in most btcs. in approximately . % of pcs and % ffp units issued for transfusion were subjected to pathogen reduction technologies. summary/conclusions: our study data may contribute to the assessment of some long-term tendencies observed in polish blood transfusion service and may serve practical-benchmarking. this in turn may prove beneficial to the transfusion community as a whole. background: in poland % of hospitals depend on blood for the treatment of patients; over . mln units of blood components are annually transfused. it is therefore purposeful to expand the knowledge on factors impacting on blood transfusion service (bts). the institute of hematology and transfusion medicine (ihtm) as competent authority is responsible for collection of data related to the activity of all polish blood transfusion centers (bcts). this data is exploited to a much lesser degree than the recently available statistical methods and data processing tools would allow. moreover, survey of research in the field of public health indicates a negligible share of issues related to bts. it seemed therefore necessary to "fill in the gap" with true assessment of performance of the polish bcts for improvement of bts activity. st stage of our investigation refers to collection, merging of data from different sources, their unification and preparation (big data) for further analysis to be performed using multidimensional statistical analysis and data mining methods. aims: assessment of the activity of the polish btcs over the year-period in two stages. goals at st stage: . data digitalization; scanning of paper documents. . development of a uniform template for collecting digital data from various sources. . standardization, unification and quality improvement of available data: filling in missing data, elimination of errors, duplicate records etc, that may distort the outcome of analyzes. . selection of data for analysis. methods: digitalization and big data methods for processing various types of data: a) stored in paper form ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) , b) digital stored in two file types (.doc and .xls, for the years - and - , respectively). for each data-type, a separate excel file model was created. the models were then merged into one analytical table with data processing methods. results: . pages of paper documents were scanned. . models developed for data from different sources: a. paper-data were rewritten and ascribed to its model; outcome - tables, columns, , rows. b. .doc and .xls. filesdata were ascribed to other models; outcome - tables, columns, rows. . the models were merged into analytical table to create a mb database (comparable to approx. min of music). . the data was subjected to standardization, unification and quality improvement: filling in missing data, elimination of errors and duplicate records that may distort the results of analyzes. . selection of data for analysis at nd stage. summary/conclusions: the st stage provided a set of selected data for analysis in the nd stage which will rely on multidimensional statistical analysis and data mining methods. the outcome of such analysis will contribute to optimal realization of objectives: a) gaining in-depth knowledge about the fundamental phenomena that shape polish bts, b) identification of potential changes bcts, c) development of overall guidelines for change management. aimed to touch untouched or less touched topics of bb&tm. so far workshops were organized and each year around countries were participated from almost all religious, ethnical, social, cultural and economic situations of the world. . supported realization of major changes in bb&tm in turkey; a convincing medical individuals and agencies mainly moh to give the deserved consideration to bb&tm b encouraging the recognition and establishment of national blood program c issuing a new blood act and numerous necessary bylaws, etc. d creating an appropriate standard donor questionnaire form e changing blood transfusion practice from % whole blood (at ) to %. f changing donated blood screening criteria; while anti-hcv screening became obligatory malaria, screening cancelled g preparing national guidelines h promoting haemovigilance nurse post i promoting patient blood management . around , blood bankers attended national courses, , attended national congresses, , attended nationwide symposiums. summary/conclusions: bbtst can be accepted as a sample how a scientific nongovernmental organization may give a very positive impact on developing and progressing bb&tm activities with close collaboration moh and other related organizations abstract withdrawn. background: globally there is growing investment in information technology (it) in business. this similar trend has been observed in blood establishment computerized systems (becs). the it investment can be high hence it decisions need to be properly informed. the africa society for blood transfusion (afsbt) encourages the use of its in african blood services as this optimize quality blood services, thereby improving patient's outcomes. afsbt established in an it working group (afsbt itwg) with the support of the swiss red cross (src) to spearhead the it standards among member blood services. a number of priority it thematic areas were identified. these includes it governance which focuses on creation (strategic alignment) and preservation (risk management) of business value. there is absence of published literature on how a structured it governance framework can be implemented in a resource constrained setting. a review of the national blood service zimbabwe (nbsz) it governance was done based on published it governance framework. aims: to explore how a structured it governance can be developed, implemented and monitored in a resource constrained setting. methods: a published mit-cisr framework which has six components was used to assess the strength, gaps and opportunities of the it governance. results: nbsz has been implementing an evolving structured it governance system. in terms of service strategy and organization there is a well-established it function which is reflected in the nbsz strategic plans. this ensures it annual budgetary support, which averages . % of the total budget. the it governance arrangements are such that decision rights are assigned to different it staff (executives, it specialist, and users). a range of it solutions have been embedded within the nbsz operations such as becs, financial, donor mobile application, social media, temperature monitoring, and human resources. the business performance goals are defined and are congruent across the various business units. it organization and desirable behaviors are documented in the ict policies and procedures and were needed remedial actions are available through the code of conduct. the it metrics are included within the nbsz monitoring and evaluation system which use a four colored traffic lighting reporting system. it was noted that the it accountabilities are undesirably tilted to the it specialist only hence some ict projects tend to have delayed deliverables. the it governance mechanisms are supported with tools such as service level agreements and established communication approaches. simple excel based solutions are used to track critical performance metrics such as on the interactive blood supply management status, which averages . % ( ) based on a -day projected stocking and supply levels. nbsz need to properly document the return on investments on all these ict initiatives, which is estimated ( / ) to be at . % of annual savings. summary/conclusions: blood services in resource constrained settings can implement a properly structured it governance and this will ensure maximum return on it investments. the nbsz approach will be shared and further developed in the afsbt itwg to support other blood services in improving their it governance. haematology/blood transfusion, alfred health, melbourne, australia background: in october , an integrated electronic medical record (emr) was implemented at an australian metropolitan multi-campus heath service using cerner millennium tm , aiming to achieve himss (healthcare information and management systems society) level . prior to implementation, large numbers of blood specimens were collected from patients unnecessarily and sent to pathology without a test request attached (no blood test requested -ntr). these specimens required additional processing in the laboratory. electronic specimen collection using cerner specimen collect tm allowed streamlining of specimen processing by eliminating paper requests. as part of the new workflow, individual specimen labels are printed with the specified blood test and correct tube type. this helps prevent the practice of collecting additional specimens due to uncertainty of the collection requirements. aims: • to quantify the expected reduction in ntr specimens following introduction of electronic specimen collection, and outline the benefits • to determine the impact on collection errors and wrong blood in tube (wbit) events methods: data was obtained directly from cerner millennium tm using a ccl (cerner command language) query which is run monthly by pathology it staff. this data includes all specimens registered for the month with indication of rejected specimens, wbit & ntr samples. 'rejected specimens' includes incomplete specimen and/or request certification, unlabelled specimens/requests and mismatched specimens. further information about wbit events was collated from riskman reports and staff interviews. results: data from the months prior to emr implementation was compared with months post. ntr numbers reduced from /month to /month ( % reduction), freeing up more storage space in fridges. rejected specimens due to inadequate patient request labelling reduced from a mean of /mth to /mth. wbit numbers have increased slightly: before having median (range - ), after with median (range - ). although it was hoped that wbit incidence may reduce with the new emr, of the post implementation wbits involved electronic specimen collection. departure from planned protocols involving a lack of working printers, causing staff to print patient labels away from the patient's bedside, as well as multiple patient labels printing on individual printers appear to be a main cause of the emr wbits. summary/conclusions: emr implementation has led to a reduction in ntr, and rejected specimens due to inadequate request labelling, as well as increased storage space in laboratory refrigerators. associated benefits include: • decreased financial costs of the wasted equipment • decreased staff time collecting and processing unusable specimens • decreased environmental impact of manufacture and disposal of unused specimens • decreased potential of iatrogenic anaemia work in preventing the occurrence of further wbits is ongoing, by ensuring that label printers are in working order, are in plentiful supply and easily accessible to staff; and also ensuring positive patient identification and blood collection by the patient's bedside remains a priority. jm mustaffa , k teo , s tsai , p heng , r sagun and m wong laboratory medicine khoo teck puat hospital, singapore, singapore background: khoo teck puat hospital (ktph) is a -bed general and acute care hospital, opened in , serving more than , people living in the northern sector of singapore. the blood bank of ktph department of laboratory medicine provides specimen testing and blood transfusion services for ktph as well as the neighbouring yishun community hospital (ych), one of the largest community hospitals in singapore providing intermediate care for recuperating patients including rehabilitative services. the process of ordering transfusion-related test requests in both hospitals is through printed forms. aims: in line with the hospital directive to move towards electronic patient management, the ktph blood bank intended to implement an electronic type and screen (e-t&s) system. the goal of the project is to ensure zero patient identification errors and maintain full traceability and accountability for the blood collection process in all transfusion-related testing. another aim of the system is to reduce repeated venepunctures when specimens are rejected due to missing essential patient information on the printed forms by implementing mandatory fields in the e-t&s form. methods: the e-t&s was implemented in phases. phase : an online version of the printed form was signed electronically by the ordering doctor and a witness within the electronic medical record system, sunrise clinical manager (scm) system with the doctor counter-checking by signing on the specimen label to ensure correct patient identification. phase : the ordering doctor is not required to sign on the specimen label since fingerprint biometrics are required for the electronic signin. phase : elimination of the witness step for blood collection. specimen collection and rejection data from to was analysed. specimen rejection rate was presented as percentage of rejected specimens (mislabelled, unlabelled and clerical errors) over total specimen count for each month. results: between january and march , before the implementation of the e-t&s phase , the average rejection rate for blood bank specimens was . % and . % for identification and clerical errors respectively. during phases and of implementation, rejection rate increased due to unfamiliarity to the new work processes. by february , with the implementation of the final phase of the e-t&s system the specimen rejection rate was . % and . % for identification and clerical errors respectively. rejected specimens were mostly from the few locations that had to use paper requisition due to workflow or infrastructure limitations. summary/conclusions: the e-t&s system was implemented successfully in ktph. full traceability and accountability of the blood collection process was maintained with the fully electronic system. the adoption of electronic documentation has also reduced the number of preventable repeated venepunctures that were due to incomplete order information on the printed forms. future developments in technology and full implementation of e-t&s system in all hospital locations may make zero patient identification error achievable and ensure transfusion safety in all patients in the near future. background: blood component administration represents a critical phase due to the possible occurrence of errors during the different steps from the identification of the patient to the infusion of the product. error occurrence can be reduced by the implementation of validated information systems. we tested the scweb â system at the bedside in a transfusion outpatient clinic. aims: the aim of the study is to validate a system designed to assist and to control blood administration step by step using electronic devices to ensure traceability and documentation of the process methods: the scweb â system is based on it monitored checklists which guide the personnel to follow the procedure, according to best practices; the system must initially be activated by the operator which is recognized by an auto-signing system based on bluetooth low energy which avoids the operator having to identify himself/herself beforehand. appropriate privacy protection is provided. thereafter the system takes up the task to give instructions and to verify the adherence, by asking an active confirmation of the proper fulfillment of the activities; a continuous registration and documentation is made by the system. standards and specifications for each step of the procedure have been configured on scweb â system to track in detail operator and patient identification, presence of informed consent to transfusion, blood pressure, pulse and temperature recording, vein access, verification of the blood unit. an alarm has been set after min, to ensure the control of patient's conditions. for each step, an active confirmation of the action is required and nurse and doctor direct involvement must be actively confirmed on the device by both operators. the system has been tested at the bedside on patients admitted to the outpatient clinic for red cell concentrate transfusion; compliance of the personnel and organizational impact has been recorded. results: the system required a very short training: ease of scweb â system allows its implementation without negative impact on organization of transfusion outpatient clinic and without difficulties by operators (nurses and doctors), who appreciated the help given by the it check system. the registration of the electronic check list offered a reliable tool for the traceability of the transfusion procedure, also granting a paperless and timely available documentation of the entire process through a registration in electronic format of all the operator's action in every single phase of the transfusion process. when prescribed, confirmation of the checklist was only possible in the presence and with the active confirmation of two operators (doctor and nurse). summary/conclusions: the scweb â system is useful as a barrier against the mismatch of transfusion (preventive measure), as a traceability and documentation measure and as a tool for training of personnel in blood transfusion administration; it avoids paper registration during the transfusion process, due to the timely registration of the activities performed by operators recognized by the system thanks to the bluetooth low energy auto-signing device. the scweb â system will be connected to the transfusion data management system, to monitor all the process from the arrival of the unit from the blood bank. background: he blood banks aims at reducing cost and increasing customer satisfaction by providing quality in service. the quality in service can be attained by streamlining the processes and restructuring the supply chain of the organization by implementing it tools. aims: aim is to understand the complex flow of information and processes within the supply chain of the blood bank. the requirement of such a study is a part of the integrated erp modeling for the integrated functioning of a blood bank. methods: he approach used to understand and map the sequence of processes, and the work responsibilities of each process and the operational decisions involved at each step is process mapping and data flow diagrams for front end system modeling and analysis. the processes are mapped and represented in a schematic diagram. dfd (data flow diagram) are constructed for representing the system. a context diagram is also constructed for understanding the entities interacting with the system. the emr systems aim at replacing (or supporting) the paper based medical records. the whole model of the system is divided into two parts-front end and back end. the front end design and analysis is done using epc (event-driven process chains), resource views, data flow diagram for data view. reporting was on donor selection, finance and collection of blood bag, blood collection process, component preparation, blood testing and blood distribution results: process mapping using event driven process chain generated a whole view of the processes involved. the resource view gave an organizational structure and the personnel involved. the data view using context diagram and data flow diagram gives a flow of data and amount of data involved. this framework can be used for business process reengineering for the blood banks by conducting a time study and removing non value added activities. data view helps analyze redundant data in each process. it also helps in staff training and orientation within the department. summary/conclusions: a systematic overview presented in this paper facilitates in removal of non value added processes, duplication of data, bottlenecks, reduction of cycle time and thereby improving service quality in blood banks. background: the transfusion of blood components, one of the most prevalent interventions in clinical practice is a major expenditure item in healthcare services which tend to increase in recent years. aims: it is intended to investigate the impact of transfusion associated costs to hospital costs in pediatric intensive care unit (picu) patients. methods: during a year period (january -december ) patients, females and males receiving transfusion with blood components along the stay in picu were included in the study. transfusion associated costs and total costs for healthcare services for children treated in picu was collected by using hospital information system (his). statistical analysis of data was performed by spss software (version . , spss inc., chicago, il, usa). mann-whitney u test and kruskal-wallis test was performed for comparison of independent categoric variables and numeric data; chi-square analysis was performed for comparison of two numeric variables and spearman correlation analysis was performed for associations. results: the median age of patients was . months (interquantile range-iqr ). the median length of stay was days (iqr ). in total blood components were transfused in which of red blood cell concentrates, apheresis platelet concentrates, granulocyte concentrates, fresh frozen plasmas, and cryoprecipitate and whole blood. the ratios of transfusion associated expenditures to hospital costs were categorized in intervals of percentages as < %, - %, - % and > %. most of the patients ( . %) were ranked in the lowest interval. the medians for hospital cost and transfusion associated cost were . euros (iqr = . ) and . euros (iqr = . ), respectively. a significant strong positive correlation between numbers of transfusions and hospitalization cost of picu was detected (r: . , p < . ). while it was found a significant weak positive correlation between transfusion associated cost and hospital cost (r: . , p = . ) there was also a significant weak positive correlation between the age and transfusion associated cost (p = . , r: . ). a significant difference was found between the patients with and without hematological malignancies (p < . ) for transfusion associated cost. the reason why pediatric dosages are mostly prefer is that the hospital provides healthcare for only children and splitting of the blood components was common in the hospital. but unexpectedly a significant increase on the transfusion associated costs which is related to split blood components was detected (p < . ). summary/conclusions: studies on the economics of blood transfusion have been conducted mostly in patients who require chronic or multiple transfusions. picus, specialized facilities that provide care for patients with severe life-threatening diseases are major departments often necessitate multiple transfusions. there are many variables to evaluate the impact of transfusion associated cost to hospital cost in picu patients, but the major factors are underlying conditions, admitting diagnoses and transfusion strategies. although there are unexpected data in our study demonstrated the increasing impact on transfusion associated cost originated from blood components split for pediatric usage no significant relationship was determined to explain this situation. further studies on the economics of blood transfusions have to be carried out to clarify the variables of transfusion associated costs. background: approximately . % of the transfused blood component is packed red cell (prc). over ordering of prc unit is a common practice and excessive pretransfusion testing was being wasteful of resources and have adverse consequences on cost. high crossmatch to transfusion (c/t) ratio as quality indicator of blood bank implies that crossmatches were performed unnecessarily. aims: the aims of this study were to evaluate the cost effectiveness of strategies for limiting the number of pretransfusion testing of ordering prc. methods: all prc units who ordered from dr. hasan sadikin hospital from january to december were collected in this retrospective study. number of ordering prc unit, completed pretransfusion testing of ordering prc units, and prc units that were transfused were recorded. restrictive pretransfusion testing strategies were done based on the hemoglobin level and diagnosis as transfusion indication criteria. cost effectiveness was measured by multiplying the unit cost of pretransfusion testing and number of prc unit. results: out of total , ordered prc unit, , ( . %) were subjected to pretransfusion testing and . % ( , ) of ordering prc unit which are pretransfusion testing were transfused. this means that . % ( , ) of ordering prc unit were not subjected to pretransfusion test. this showed savings of , , , rupiah. c/t ratio was . which demonstrate a good ordering pattern. however, . % ( , ) of completed pretransfusion testing of ordering prc unit were not transfused, leading to blood bank loss of , , , rupiah. summary/conclusions: strategies for limiting the number of pretransfusion testing on the good c/t ratio was still associated with saving cost effective background: blood is a precious resource for saving patient lives. the purpose of blood and blood component therapy is to provide suitable and safe blood products to achieve best clinical outcomes. nurses have an important role in ensuring safe blood transfusion. it is crucial for nurses to have sufficient knowledge about blood donation and collection, storage, component preparation, possible adverse effects of blood transfusion and necessary management and care. aims: the aim of this study was to assess the impact of an educational intervention on knowledge and awareness of nurses regarding blood transfusion services and practices. methods: the baseline study to assess the knowledge and awareness regarding blood transfusion services and practices of the nurses posted at various areas of the hospital including wards, operation theatres and critical care areas was carried out at our institute hospital which is a tertiary care teaching centre. the nurses were then sensitized and educated regarding blood transfusion services and practices during their day to day activities by referring them to the blood transfusion guidelines of the institute. subsequently, a self-developed questionnaire which was used for the baseline assessment of knowledge and awareness of the nurses was again used to reassess them. a total of questions were included in the questionnaire pertaining to: general awareness (two questions), blood donation (two questions), testing and blood component preparation related (two questions), storage of blood/blood components (two questions) and pre-transfusion checks and bed side transfusion practices (eleven questions). fifty nurses each were included for both the baseline as well as post-sensitization assessment. for different category of questions, the correct response rates were compared with those obtained in the baseline study using mann-whitney test. the entire study duration was spread over a period of three months (december, to february, . results: the overall mean percentage of 'correct' responses for questions in the baseline study was . %, whereas post sensitization it was . %. the mean percentage increase in general awareness related questions was . %, . % for storage of blood/blood components related questions, . % for pre-transfusion checks and bedside transfusion practices related questions, . % for testing and blood component preparation related questions and . % for blood donation related questions. the percentage increase in correct response was found to be statically significant for each of the five categories of questions. the overall mean percentage increase in correct response rate was also statistically significant (p < . ). summary/conclusions: this study revealed that after sensitization and educational intervention there was a significant improvement in the knowledge and awareness of nurses regarding the blood transfusion services and practices. abstract withdrawn. background: tact, introduced in the uk in to support managers, provides resource-saving, continual, 'real-time' monitoring of knowledge-based competency of staff in transfusion laboratories. tact is available online / , complementing existing practical competency schemes and external quality assessment. multiple variations on a standard pre-transfusion testing scenario are generated using constrained randomisation; logic rules for automatic assessment of sample acceptance, abo/d, antibody screen and identification (as/id), and component issue are based on bsh guidance. during , tact was offered internationally to transfusion laboratory managers to trial, and saw uptake in five countries. the core tact programme, based upon uk guidelines, is under review for programming conversion, to be customisable for the international community. aims: to assess the feasibility of tact programming conversion to meet the requirements of country-specific pre-transfusion testing guidelines, and to direct future programming in line with feedback from international users. methods: guidelines from / international users were obtained and translated where necessary. these were compared against the core assessment elements of current tact programming. international users were approached for their feedback on the current version of tact, as it compared to their local policies and practices. results: the following criteria were cross-referenced: specification of transfusion request forms, sample label acceptance criteria, reagents used for abo/d and as/id, resolution of grouping anomalies, alloantibody confirmation/exclusion, and selection criteria of blood components for transfusion-dependent patients and women of child-bearing potential. apparent differences included:-australia:--selection of red cells for patients with immune anti-d. greece:--inclusion of the name of the patient's father on the transfusion request. italy:--testing of all new patients with an anti-a,b reagent and two different monoclonal anti-d reagents. international users in the same three countries supplied feedback. this included suggestions for:-greater complexity of cases presented, provision of patient history, inclusion of follow-on tests e.g. phenotyping and cells for antibody confirmation/exclusion, broader range of reaction strength grading, and official professional cpd credits. the following differences were noted:-nomenclature used, the format and content of the request form, use of english abbreviations of patient clinical details, and the availability, provision and specification of blood components. summary/conclusions: this analysis has shown very few instances where the current tact iteration differs from the guidelines reviewed, and that it is feasible to expand the use of tact on a more international basis. the current iteration of tact has been developed to represent an abbreviated scope of pre-transfusion testing practices, which can be applied to laboratory practice outside of the uk without difficulty. further work is required to enable international users to configure tact such that the system represents all laboratory practice on an international basis. aims: these courses provide education to clinicians on patient blood management and safe transfusion in neonatal and paediatric settings in order to improve patient outcomes and increase awareness of the national patient blood management guidelines. this analysis aimed to investigate the uptake, practical use, and perceived value of the courses by learners. methods: a retrospective analysis of course completion statistics and course evaluation data. results: there have been , paediatric and neonatal courses completed from march to february with . % of learners being nurses and/or midwives. analysis of course evaluation data (n = ) showed that these courses: -provide knowledge ( . %) -improve patient safety and outcomes ( . %) -result in change to clinical practice ( . %) -are relevant to clinical practice ( . %) -are easy to use ( . %) -are readily accessible ( . %). examples that learners provided of how they can apply this learning to their clinical practice include: -"[i am now] more aware of special requirements for neonatal blood transfusion" -"[i] feel more confident especially when talking with parents" -"[i will now be] checking the patient's blood results and will speak up for unnecessary blood sampling" -"[it's good that] when there is ambiguity in clinical practice [this is] very well shown by explanation from experts in the field" -"we don't do a lot of transfusions [and this is] a reminder that transfusions are not always the first answer to the baby's clinical picture". summary/conclusions: analysis of course and user evaluation data demonstrates that these courses are being used by nurses and doctors working in the neonatal and paediatric settings and that they provide knowledge of pbm that can be applied to clinical practice, thereby contributing to improved patient care. background: blood transfusion is a high-risk clinical activity that must be mastered both theoretically and practically in order to guarantee the required result without any incident or complications. the mastery of transfusion knowledge among nurses represents a very important link in the transfusion chain. the objective of this work is to compare the theoretical and practical knowledge of transfusion among two groups of nurses divided according to their seniority. aims: this is a cross-sectional descriptive study conducted over a period of month [ st april- th april] . we selected two groups of care staff: the st group consists of students at the end of their training at the higher institute of nursing sciences. the nd is made up of nurses working in university hospitals of tunis, currently practicing blood transfusion. the evaluation's tool used was a questionnaire of simple or multiple choice questions, were related to theoretical knowledge of labile blood products and to transfusion practice. ten questions were considered "life-threatening" if their answers were false. a comparative study was made between the two groups. methods: this is a cross-sectional descriptive study conducted over a period of month [ st april- th april] . we selected two groups of care staff: the st group consists of students at the end of their training at the higher institute of nursing sciences. the nd is made up of nurses working in university hospitals of tunis, currently practicing blood transfusion. the evaluation's tool used was a questionnaire of simple or multiple choice questions, were related to theoretical knowledge of labile blood products and to transfusion practice. ten questions were considered "life-threatening" if their answers were false. a comparative study was made between the two groups. results: the participation rate in the survey was %. the nd group participants had an average seniority of years . more than half of them ( %) had seniority of less than years. only % had more than years of experience. the rate of correct answers for all items combined was . % for students versus . % for practicing nurses. the theoretical knowledge part was more mastered in the st group than that of practicing nurses ( . % vs . % of correct answers). on the other hand, the control of the transfusion act was better in nd group ( % vs . %). the overall "dangerous" response rate was % for students and . % for practicing nurses. false practical knowledge was more common in group ( . % vs. . %). summary/conclusions: the theoretical as well as the practical knowledge remains not well mastered by the care staff. our study highlighted the best theoretical mastery for young students and practical for practicing nurses. this could be explained by the freshness of knowledge in the first group and the daily practice in the second group. background: the european commission (ec) directive / /ec on blood donor selection criteria is years old. in the meantime, knowledge on risks related to blood donor selection has progressed and challenged several obligatory rules. transpose -transfusion and transplantation: protection and selection of donors, is a european commission co-funded project with participation of more than stakeholders from both not-for-profit and private organizations providing substances of human origin (soho). the project aims to provide evidence-based donor selection criteria and guiding principles for risk assessment of threats to the safety of soho. as part of this work, an inventory of current blood donor selection criteria in europe and an evaluation of the evidence behind current practice was performed by experts working on this project. aims: to identify the gap between the ec directive / /ec on whole blood donor selection criteria and a current evaluation of the clinical relevance of the criteria based on scientific literature by a panel of european experts within the transpose project. methods: in , we performed an inventory of blood donor and transfusion recipient risks in participating european countries. project members were asked to provide the existing donor selection criteria related to these risks and to carry out a risk-based evaluation for each of them. the evaluation was based on the available scientific literature and on a risk assessment template based on the abo risk-based decision-making framework, developed by transpose. all risks with divergent assessments within the panel were resolved through discussion; in all cases an expert consensus was established. subsequently we compared the results with the content of the ec directive / /ec for every risk, thereby identifying discrepancies and missing items in the directive. results: the panel identified risks considered to be significant, distributed between donors and recipients. for / ( %) of them the expert evaluation deviated from the content of the ec directive, or the ec directive provided no information about the decision making. in particular, a discrepancy was observed for / criteria concerning general health and medication, / for transfusion transmissible infections, / for high-risk behaviour and travel, and / for other diseases. summary/conclusions: our results highlight a significant gap between the whole blood donor selection criteria stated in the ec directive / /ec and the scientific evaluation performed by a panel of transpose participating experts. this gap includes both new risks not addressed in the ec directive and addressed risks that are however evaluated differently. this involves both blood donor and transfusion recipient safety, and various medical and epidemiological topics covering several aspects of the blood donation criteria. we strongly recommend a change in the european legislation, allowing a procedure to guarantee that blood donor selection criteria are updated regularly within the framework of the european institutions, to keep aligned with scientific progress, epidemiology and changes in medical practice, in order to enable an updated risk-and evidence-based framework for donor selection criteria. the risk-assessment method elaborated in the transpose project is a valuable instrument for this purpose. background: the brazilian health regulatory agency -anvisa has developed the method for assessment of potential risk in hemotherapy services (marpsh) which is based on the data collected during the inspections of blood services carried out by regulatory authorities. using marpsh any blood service can be classified in one of possible potential risk categories: high, medium-high, medium, medium-low and low risk. each category represents a different potential risk level, according to the proportion of compliance with the established regulatory requirements. marpsh has been used since , showing a trend of risk reduction on blood services evaluated all over the country. aims: this work aims to describe the utilization of marpsh as a tool for an integrated risk management model. also, it shows the main results obtained after years of data monitoring and coordination of regulatory actions and policies by anvisa, targeting quality and safety of blood products. methods: the utilization of marpsh follows a network risk management model since the inspections are carried out by decentralized organs in all states and some municipalities. the inspectors fulfill a standardized inspection guide containing the regulatory requirements, where each item is associated with a level of risk, varying from i to iii as the risk increases. at the end of the inspection, after a statistical calculation, the service is categorized. this classification gives an estimate of its quality profile, guiding the adoption of suitable measures for risk management by local authorities and services. these data are send to the states (if realized by municipalities) and to anvisa that perform consolidation in a national level. either states or anvisa use data to coordinate risk management measures in a broader spectrum. data are continuously monitored by anvisa as part of its strategical panel of indicators. anvisa follows up specially blood services in high and medium-high risk with the aim of helping or complementing local authorities' actions. additionally, anvisa periodically sends this information to the brazilian ministry of health and local governmental organs from brazilian national blood system that also support actions to improve quality in their blood services networks. results: since , when the assessment covered blood services, marpsh reached blood services in ( % of the blood services registered) what corresponded to almost % of the inspection cover in this year. over this period ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) it is possible to notice a dramatic decreasing in trend for proportion of blood services classified as high and medium-high risk, varying from % to %. summary/conclusions: marpsh generates data necessary to the categorization of blood services into five levels of potential risk. as a result, the regulatory actions are applied by local organs with the purpose of reducing or eliminating risks involved in the production and use of blood components. data have shown a significant risk reduction over years of marpsh's utilization. additionally, monitoring of this data at a national level has been permitting appropriate planning and prioritization of integrated strategies directed to risk management and strengthening of blood services in brazil. background: sub-saharan africa has the highest need of blood transfusion in the world, mainly for childbearing women and children suffering from malaria. meeting basic quality and operational requirements to provide patients with safe blood remains a challenge in these settings. aims: the aim was to identify and prioritize potential hazards for patients in blood bank practices in the democratic republic of the congo (drc). we focused on two subsets: (i) sensibilisation and selection of donors, and (ii) qualification and production of blood products, using failure mode effect analysis (fmea). methods: two risk analysis workshops were organized at the national blood transfusion centre in kinshasa, the democratic republic of the congo. in both workshops, a multidisciplinary team was invited to represent different hospitals and profiles in transfusion management in drc: quality coordinators (n = ), training coordinator (n = ), medical doctor for donor selection (n = ), hemovigilance officer (n = ), laboratory technicians performing donor sampling, blood qualification and production (n = ), biomedical scientists (n = ), microbiologist (n = ), clinical biologist (n = ), nurse (n = ). the principle of fmea was applied, which implies identification of possible hazards (failures) in a process flow, followed by scoring each hazard according to their impact, probability, detection and feasibility. in the both workshops, participants were guided by an external facilitator who guaranteed common understanding of the methodology. main focus of the risk analysis was potential harm to the transfused patient in the process of (i) sensibilisation and selection of donors, and (ii) qualification and production of blood products. all ideas were written on coloured cards and mapped on a chart according to their impact, probability, detection and feasibility score. hazards were ranked according to their final risk score by multiplying these four scores. results: in the process of sensibilisation and selection of donors, the three hazards with the highest final score for impact, probability, detection and feasibility were: (i) a paid donor is recruited after sensibilisation by family members of the patient, (ii) donor selection staff approves a non-eligible donor for blood donation because of personal/financial motivation, (iii) blood donors are not correctly informed about blood donation during sensibilisation by family members of patients. in the flow of qualification of blood products, highest scores were made for: (i) no double check for validation and sorting of qualified blood products, (ii) stock-out of reagents, (iii) no check for match between registered test result and tested blood tube. regarding production of blood products, the top three consisted of: (i) transport time between blood collection and processing is > h, (ii) storage of qualified and quarantined blood products in the same fridge (some sites only), (iii) power cuts. summary/conclusions: the risk analysis resulted in three prioritized hazards in the process of donor selection/sensibilisation and blood product qualification/production. some are very specific to the sub-saharan african setting and have been described before (power cut, family and paid donors, stock rupture,. . .). an action plan needs to be put in place to reduce their final risk score. the risk analysis needs to be continued for the remaining blood transfusion flows. background: . million of germany's population, so just under a quarter of residents, have a migration background. the majority of these has roots in regions where the population has a distribution pattern of blood group and hla-antigens that differs considerably from the predominant one in the german population. sufficient supply of these individuals with red blood cell (rbc) and platelet concentrates (tc) will continue to be a major challenge in the future, as blood donors with compatible blood group antigens are dramatically underrepresented in the local donor pools. many migrants suffer from severe hematological disorders such as b-thalassemia or sickle cell disease and will not only need compatible blood transfusions, but an allogeneic stem cell transplantation in the foreseeable future. as healthy family donors often are not available, at present suitable stem cell donors with a similar genetic background can only be found in international donor registries. aims: this project was initiated to recruit new donors with a migration background for blood donation and to increase the number of blood stem cell donors among this group. methods: serological extended blood group phenotyping was performed by automated gel card technique (fa. grifols, erytra) and included ab , rh (ccdee), kk, fy (ab), jk(ab), lu(ab), m, n, s, s. hla typing for hla-a, -b, -c, -dr, -dq, and -dp was performed by next generation sequencing. allele frequencies were analysed using genepop version . ; the rare and very rare alleles were defined according to the allele frequency database (www.allelefrequencies.net) rbc genotyping using next generation sequencing is currently being established and will include additional antigens with the most frequent distribution pattern differences between migrant and resident populations according to literature. results: so far, more than blood donors with a migration background have been recruited for a blood donation in this project. amongst this group, over blood donors from more than non-european countries enrolled as potential stem cell donors. an initial evaluation of the data revealed a very similar distribution of blood groups compared to the current blood donor population in north rhine-westphalia. of migrant donors, ten fy(a-b-) donors were identified, which corresponds to a percentage of . %. amongst hla-typed potential stem cell donors, we found ( . %) with rare and very rare alleles. summary/conclusions: blood donors with rare blood group and hla phenotypes (e.g. null types such as fy(a-b-)), are in demand for adequate medical care of people with a migration background. the technological development of blood group determination by next generation sequencing will significantly improve the supply for all blood transfusion recipients in germany. this project is funded by the european development fund - (erdf) and the european union. background: mortality due to uncontrolled haemorrhage following trauma is the most important cause of potentially preventable deaths. trauma care systems in low and middle income countries like india, are still in developing phase. also, the role of blood component therapy in improving patient outcomes has been mostly derived from combat settings. application of these protocols in an urban setup has not been well established and marked variation in practice exists. hence this study aims to identify the key components of transfusion practices to optimize the transfusion protocol in trauma settings. aims: to study the current transfusion practices in severely injured trauma patients, admitted to the red area/resuscitation bay after initial triage in ed methods: this prospective observational study was conducted over a period of year starting from june to may at the department of transfusion medicine in collaboration with emergency department at jpnatc, aiims, new delhi. the study included severely injured patients (iss ≥ ) that were admitted within h to the red area/resuscitation bay after triage. data collected included the demographics, injury, laboratory and transfusion details for these patients results: during the study period patients ( . % males) were enrolled. mean iss scores was . . mean time to hospital admission after injury was : (iqr . - : ) hours. mean time to first rbc transfusion following admission was : (iqr : - : ) hours. approximately . % ( ) patients were in shock (sbp < mm hg &/or pulse rate > /min). whereas, ( . %) patients were coagulopathic (pt ≥ . times of normal). during initial h of admission, these patients were transfused with ( . %) rbc, ( . %) ffp, ( . %) rdp and ( . %) cryoprecipitate of total blood components utilized for these patients. massive transfusion (defined as transfusion of ≥ units/ h) was given to ( . %) patients. summary/conclusions: significant quantity of blood components were required during initial resuscitation in severely injured patients. pre-hospital transfusion can significantly reduce the time to transfusion. further studies are needed to assess utility of pre hospital transfusion in severely injured patients. background: allogenic stem cell transplantation recipients are known to be the main consumers of platelet concentrates (pc). the geneva university hospital is one of the three allogeneic hematopoietic stem cell transplantation (hsct) centers in switzerland. since the blood center is also part of the hospital, data of pc consumption are easily available. as needs rose steadily since several years, with an average increase of % per year, pc supply is a serious concern for our center. aims: in this study we tried to evaluate if any pre-transplant indicator could help to forecast the number of pc needed after an allogeneic hematopoietic stem cell transplantation. methods: this observational retrospective study was conducted in geneva hospital on patients suffering from various inherited or acquired disorders of the hematopoietic system who were treated by hsct in . pc consumption was examined from january to december . the five indicators were: gender, stem cell source (bone marrow (bm) vs peripheral blood stem cell (pbsc)), donor type (hla matched ( - / ) vs haploidentical), conditioning regimen (standard vs reduced intensity), and cmv serology of the recipient. results: data for a total of patients aged from to years were analyzed; ( %) were male and ( %) female; ( %) were cmv-negative and ( %) were cmv-positive. out of a total of transplants, ( . %) were haploidentical and ( . %) hla-matched. according to the stem cell source, bm was transplanted in cases ( . %), and pbsc in cases ( . %). two patients also received a cd + stem cell boost. our analysis showed that, with a mean follow-up of days, the number of pc transfused to our patients treated by hsct ranged from to units, with an average of and a median of , illustrating a high variability. the results indicated that gender, stem cell source (bm vs pbsc), conditioning regimen (standard vs reduced intensity), and cmv serology of the recipient do not have any statistical impact on platelet consumption. however, we observed a tendency of an increased need for platelet transfusion when patients were cmv positive. our results also showed a statistically significant (p = . ) higher number of pc transfused for patients treated with a haploidentical ( ) versus hla-matched ( ) transplant. summary/conclusions: this study points out the high variability of platelet consumption after hsct, which limits the forecast of platelet production needed to support allogeneic hsct recipients. a larger cohort would be required to confirm a potentially higher platelet consumption in cmv positive patients, and to consolidate our results showing a higher pc consumption for patients treated with haploidentical transplant. abstract withdrawn. background: historically at our institution, a minimum of four red blood cell (rbc) units were crossmatched for all cardiac surgery cases regardless of surgical case-type or patient characteristics. two rbc units were packed in validated blood product coolers and brought to the operating room (or); the balance of crossmatched units remained in the blood bank. a retrospective review revealed that very few rbcs were transfused ( : % ( / ), : % ( / )). moreover, approximately products were wasted each month as a direct result of this practice. thus, we recognized an opportunity to improve inventory management in terms of personnel activities and blood component utilization. aims: the goal of this study was to reduce advance preparation of coolers in cardiac surgery cases without compromising patient care and safety. we limited our intervention to those patients who were eligible for electronic crossmatch. we maintained the aforementioned historical practice for those patients with history of and/ or those who currently demonstrated clinically significant red blood cell alloantibodies. methods: a multidisciplinary group consisting of representatives from the blood bank, cardiac surgery, cardiac nursing, cardiac anesthesia and surgery quality department was assembled in october to determine whether a modification of practice was reasonable and safe. group members evaluated site specific society of thoracic surgery (sts) cardiac surgical data between july and december to establish intraoperative red cell transfusion rates classified by type and urgency of surgery. the group's main goal was to discontinue preparation of default coolers for patients eligible for electronic crossmatch who were scheduled for all types of non-emergency cardiac surgery cases in which ≤ % of historical cases required at least one red cell transfusion. additionally, team members simulated the multiple protocols by which red blood cells could be prepared and delivered to the or and estimated the time for each scenario. results: review of sts data showed that the following cases met the criteria of ≤ %: elective primary coronary artery bypass graft (cabg), urgent primary cabg, elective mitral valve repairs, and elective aortic valve replacements. simulation showed that, in patients eligible for electronic crossmatch, preparation from receipt of order to completion of unit packing for delivery took . min using the pneumatic tube system (maximum of units per tube) and . min using delivery of a cooler using a human courier. summary/conclusions: based on the simulation results, and with consensus agreement from the multidisciplinary group, default cooler preparation for elective primary cabg, urgent primary cabg, elective mvr, and elective avr was discontinued in december . one year following implementation of the change in policy rbc units were issued to the or (a % reduction); % ( ) were transfused, compared to % in . wastage rates decreased from products a month to per month on average. summary/conclusions: the most obvious drawback of pabd is the higher cost in running the program in comparison with collection of allogeneic blood in the areas of additional patient attention and clerical input in labeling, separate storage and so on. in this audit, % of the autologous blood components were not transfused into the intended recipients and wasted; in this context, the pabd program could not be considered as a cost-effective approach in protecting blood safety. background: the national blood service zimbabwe (nbsz)'s blood supply management status (bsms) is an integral process of ensuring the availability of a safe and sufficient blood supply provision. nbsz introduced a new daily blood bank statement with improved metrics from may . the new analytics approach focuses on three interactive components of the blood bank statement; the available stock, quarantine stock (as per the desired -days stocks level), and the demand versus supply. it is imperative to have a closely monitored blood supply chain because blood has limited shelf life with uncertainties in both supply and demand. the 'blood-for-free' proclamation by the government of zimbabwe in july set more pressure on the blood demand. these metric-based analytics seek to assess if the nbsz's improved blood bank statement is a realistic model for the bsms. aims: to assess the use of the interactive metrics in monitoring the blood supply management status. methods: a prospective cross-sectional study was conducted. a total of daily blood bank statements which were submitted between may and december from each of the five branches were analyzed. the bsms which is calculated as the average of the three interactive measures of quarantine stock, available stock and demand versus supply was determined. sub-analysis of branches was done to determine individual branch performance. analysis by month was done to assess seasonal variations. findings and recommendations were shared among key stakeholders to validate the bsms methodology. results: overall the quarantine stock average was . % (sd +/- . ), the available stock was . %: (sd +/- . ) and the demand versus supply was at . % (sd +/- . ).the overall bsms was . %; (sd +/- . ) for the study period. gweru and masvingo nearly supplied all the demanded blood with . %, overall bsms of . % and . %, overall bsms of . % respectively. bulawayo supplied . % of the blood demanded with an overall bsms of . %. mutare supplied . % with a bsms of . % and harare . % and a bsms of . %. there were monthly variations but the service could supply above % of the blood demand. in the month of may the service met . % of the demand and a bsms of . %. in november and december it supplied . %, bsms of . % and . %, bsms . % respectively. august also had a below average supply of %, bsms - . %. june, october and september recorded above the average values; . %, bsms of . % and . %, with a bsms of . % respectively. summary/conclusions: the overall bsms performance was satisfactory and it was noted that branches capacitated according to demand. the new interactive analytics approach is appropriate for showing the blood bank status and assessing the performance of the branches. this new approach has optimized the decision-making process in blood supply management. the metrics are tracked using excel based model hence this approach is suitable for resource constrained settings with limited ict infrastructure . st vincent's hospital melbourne (svhm), a tertiary hospital supporting medicine, surgery and non-major trauma emergency and itu services implemented a mtp in . subsequent mtp reassessment has led to implementation of regular multi-disciplinary review of all mts to identify areas for improvement in transfusion and other aspects of support for critically bleeding patients. aims: to implement a systematic service-wide stakeholder review of mt events at svhm aiming to identify deficiencies and implement improvements in mt management. methods: a multi-disciplinary mt review team was established as a subcommittee of the hospital transfusion committee (tc) to update the organisational mtp in and subsequently continued to meet quarterly as the mt review subcommittee (mtrs) of the tc, systematically reviewing all aspects of mts at svhm. instances where or more red cell units are transfused in < h are identified from the laboratory information system and reviewed by the mtrs which includes representatives from accident and emergency, intensive care, operating suite (os) and transfusion laboratory staff; the head of the patient's treating unit is also invited to contribute. reviews include: demographics, clinical details, comorbidities, time from patient arrival to pre-transfusion specimen collection/receipt, time from blood request to release/transfusion, regularity of full blood examination (fbe)/coagulation (coag) testing, timing of blood component transfusion, total component provision/ratios, component waste, patient outcome, and communication between various clinical areas and also the laboratory. a discussion summary with actions/ recommendations is provided to the tc and some cases referred to the hospital mortality/clinical review committee. results: cases reviewed: from treating units including cardiothoracic surgery ( ) hepatobiliary/gastrointestinal/colorectal surgery ( ), vascular surgery ( ), neurosurgery ( ), orthopaedic surgery ( ), endocrine ( ) and "other" (encompassing general surgery, urology, general medicine and oncology - ). areas for monitoring/improvement identified: transfusion documentation, regularity of fbe/coag specimen submission, reducing time between patient arrival and specimen collection, reducing specimen transport time, interfacing point of care bloodgas analysers to the central pathology result management system as well as component management/waste reduction and the introduction of viscoelastometry assessment in the os. of reviewed cases involved the transfusion of emergency uncrossmatched o rhd negative red cell units. the appropriateness of the use of this precious resource is also reviewed by the mtrs. summary/conclusions: the svhm mtrs meets regularly to review mt events and formalise multidisciplinary collaboration in identifying possible improvements to support these often critically ill patients. matters highlighted include communication issues, delays in specimen delivery and blood component waste minimisation. areas for further work include minimising delay between mt events and review, and formalisation of key performance indicators for mts. background: the use of radio frequency identification (rfid) technology to manage the blood supply chain is recognized as a major enhancement to the operations of blood banks and hospital transfusion services. to facilitate optimal blood supply management, it is crucial to guarantee the integrity of rfid tags throughout the transfusion chain. since rfid tags can be affixed to blood products very early in the process, these tags undergo the same process-steps as the blood products themselves (e.g. centrifugation, label printing, shock-freezing and irradiation). aims: the goal of this study was to validate the mechanical and functional resistance of biolog-id rfid tags through different blood related processes: centrifugation, label printing, shock-freezing, intensive reading at À °c, and irradiation. biolog-id tags are passive hf ( . mhz) tags. they are compliant with is , iso - and follow the guidelines for the use of rfid technology in transfusion medicine (vox sanguinis, ). methods: biolog-id tags were evaluated using a series of rfid encoding and reading tests. before each of the processing steps, each tag was encoded with donation number, site id, product code, blood group and expiry date. the data was encoded using the isbt format. the different processing steps and conditions tested were: -centrifugation: quintuple whole blood bags, filled with ml water. centrifugation at , rpm for min. tags processed, tags per kit affixed at different positions. -shock-freezing at À °c: shock-freezer (angelantoni, sf ), units processed, reading immediately after removal from shock freezer. water, tags irradiated at gy and tags at gy results: all biolog-id tags were encoded and read with a % success rate in all series of tests. summary/conclusions: biolog-id rfid tags can be encoded and read through common processes used throughout the blood transfusion chain. their mechanical and functional integrity is not affected by centrifugation, shock-freezing, intensive reading at À °c, printing, eto sterilization and irradiation. background: the provisioning of compatible red blood cells by international cooperation is presented. the units were meant for an -year old female, with homozygous sickle cell disease (scd) and multiple complications. patients' blood group was a positive with anti-c, -e, -wr a and an antibody to a high prevalence antigen in the rh system, anti-hr b possibly combined with anti-hr b (rh ). the antibody was not reactive with rh null , -d-or hr b negative cells. the donor center put out an international request for group a or o, rh null or -d-units lacking wr a and possibly k, fy a , jk a , wr a , do a and s (the latter antigens for prophylactic matching). the patient sample had been genotyped for rhd and rhce using mlpa and sanger sequencing and the patient was found to carry rhd* /rhd* n. and rhce*cevs. / rhce*cevs. . aims: the request was sent to the american rare donor program (ardp). the ardp working with the american red cross national molecular laboratory, used the rh genotype information to identify donors carrying the same or similar rh variant alleles using the rh allele matching approach described previously (keller et al. transfusion ( s): a). methods: a recent blood sample was used to confirm anti-hr b ; no anti-hr b was detected. the patient rhd and rhce alleles were used to build punnett squares for both genes with donors carrying the same and similar alleles that would be predicted to be compatible. tier donors are those predicted to carry the same combination of rhd and rhce alleles as the patient. tier donors are those predicted to be homozygous for one of the allele combinations carried by the patient. tier donors are those predicted to carry alleles similar (but not identical) to those carried by the patient, with similar predicted phenotype. the database of donors in the ardp carrying rh variant alleles was queried against the alleles in the patient-specific punnett square. results: donors of group a or o and matched for rh alleles were identified as follows: tier , tier and tier donors. after the clinical team agreed to drop one or more of the prophylactic antigen matches, one tier unit lacking s and jk a was identified at the american red cross. while the request was being processed, the patient experienced a sickle cell crisis, red cell aplasia and recurrent aiha and her hemoglobin level dropped from to . g/dl. at that time, she was transfused the only compatible units available - of the rare -dphenotype and her hb increased to . g/dl and eventually to g/dl. the tier rh allele matched unit was shipped to amsterdam where it was frozen, and reserved for the transfusion care of this patient. summary/conclusions: this case illustrates how rh allele matched blood can be found for a highly rh alloimmunized patient, and can avoid use of the exquisitely rare -d-or rh null blood. background: blood transfusion has been a complicated and high-risky clinical procedure. any error could cause serious injuries to patients. to better assure the procedure safety. aims: we enhanced and built a blood transfusion database platform and develop inventory management strategies to better guarantee the patient transfusion safety. methods: we designed six new features of the platform ( ) assuring the patient identification with barcode techniques; ( ) designing a structured order entry; ( ) proactively reminding the physicians with patient's previous blood transfusion reaction with related precautions including the use of leukoreduction filter; ( ) automatically reminding physicians the happening of reaction and suggesting relevant test; ( ) building a complete traceability log system; and ( ) supporting data analysis. the blood transfusion safety team includes medical technologists, nurses, physicians, system analysts, and blood transporter and the whole process is electronic management. the new blood transfusion platform integrated the workflow, reduced the incidence of abnormal blood samples collected ( % after implementation, p < . ), reduced the time of call for medical technologists with blood component preparation and improved the achievement rate of emergency -min blood crossmatch ( . % after implementation, p < . ). the barcode correctly identified patients and monitored the entire transfusion process to reduce the error rate of blood component supply ( % after implementation, p < . ). summary/conclusions: after the transdisciplinary team approach with e-monitoring and a better design of clinical decision support module with barcode technology, blood transfusion database platform improve the blood supply efficiency and assure blood transfusion safety. background: in the modern world, terrorist acts are characterized by a multiplicity of combined injuries to a large number of victims. qualified medical care is urgently required for a large number of patients in one locality at the same time. it leads to increase in emergency demand for blood components, mostly red blood cells. the desire to donate blood to the victims is a natural manifestation of society's solidarity in response to tragic events. however, donor activity and patient needs do not always correlate. aims: to analyze the donor activity during the terrorist attacks. methods: a retrospective analysis of donation activity in periods of terrorist attacks in moscow ( moscow ( - . the average daily blood donations' number (dbdn) before ta compared with the number of donations in day after ta and with the dbdn during days after ta. also the number of delivered rbc units (d-rbcu) daily before ta and daily in days after were compared. results: in - , terrible ta occurred in moscow: people died and more than were injured. with the explosion in subway in / people died, were injured. the number of d-rbcus increased by % on ta-day, and by % during next days. dbdn in the st day after ta increased , times, and in the next days - , times. second explosion in subway in / resulted in died, injured. the number of d-rbcus increased by % on ta-day, and by % during days. dbdn in the st day after ta increased , times, and in the next days - , times. in (explosion on market) resulted in died, injured. d-rbcus delivery increased by % on ta-day, and by % during days. dbdn in the st day increased , times, but decreased to , times during the next week. with subway explosion in people died, were injured. the number of d-rbcus increased by % on ta-day, and by % during days. dbdn in the st day after ta increased , times, and in the next days - , times. with the explosion in airport in people died, were injured. rbcus delivery increased by % on ta-day, and by % during next days. dbdn in the st day after ta increased , times, and in the next days - , times. summary/conclusions: an increase in donor activity is observed already the next day after ta and usually lasts for days, but does not correlate with the number of victims. the rbcs' delivery from blood bank increases in all cases on the day of the ta. therefore, the guarantee for patients is the maintenance of rbcs' stock, including cryopreserved ones. it is also necessary to promptly send excess of red blood cells harvested at the peak of activity to the cryobank. background: rh system is the major blood group system besides abo system. even after proper blood grouping and cross matching there is a possibility of alloimmunisation in recipients against the rh or minor blood group antigens like kell, mnss, duffy etc. in medical colleges which cannot bear the financial burden of complete phenotyping of patient and donor, implementation of rh & kell phenotypes match blood transfusion can play a major role in preventing alloimmunisation and adverse events in multitransfusion patients aims: to evaluate the efficacy of rh & kell phenotyping as a cost effective measure instead of extended phenotyping in multitransfused patients methods: study was carried out in the department of transfusion medicine, one of the biggest blood bank of the country with annual collection of , blood units. patients of thalassemia, aplastic anemia and leukemia were taken who required multiple transfusions. complete phenotyping was done initially of all the patients before transfusion. patients were taken as control and the other were taken as cases. blood units of healthy donors were chosen ( were males and were females). in all the donor units, identification of rh & kell phenotyping was done by the antigen antibody agglutination test by the erythrocyte magnetize technology on fully automated immunohaematology analyzer qwalys. these blood units were transfused to patients who had been selected as cases. in the control group, patients were transfused blood units which were not phenotyped for rh & kell but gel crossmatching was done. follow-up was done on these patients for transfusion reactions and at the end of six months they were evaluated for any alloimmunisation. results: at the end of months, no reactions were reported in cases receiving rh & kell phenotype blood and no alloimmunisation was seen on repeat phenotyping. the control group on the other hand reported reactions in cases ( . %) and phenotype at the end of three months showed alloimmunisation with 'e' antibody. the phenotypic frequencies of rh & kell blood groups in the population were comparable with other published studies. amongst the rh antigens (e) was the most common ( . %) followed by d ( . %), c ( . %), c ( . %) and e ( . %). thus 'e' was the most common and e was the least common of all the rh types. background: the prevalence of a particular blood group has an uneven distribution in different geographic areas and is largely determined by the national composition of the population. moscow is one of the largest city of europe with population of . million. the understanding of prevalence of red blood cells antigens (rbc-ag) among the population has great importance for blood banking planning. aims: to determine frequency and distribution patterns of transfusion-significant rbc-ag among donors in the moscow region. methods: the results of immunohematological studies on ab , rhesus and kell systems were analyzed retrospectively in blood donors for years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) in moscow. data collection and processing was carried out using the regional information system for transfusiology. rbc-ag detection (ab , rh, kell) systems was performed using microplate method (automatic immunohematological analyzer "galileo neo" (immucor, inc., usa)) and "ih- " (bio-rad laboratories, usa) with diagnostic cards. results: the most frequent blood group is a (ii) . %, (i) blood group . %, b (iii) . %, ab (iv) . % (n = ). rh(d+) was established as positive in the presence of antigen d and as rh(d-) negative in its absence. donors with weak variants of antigen d (du) were determined as rh (d+) positive. the ratio of rh (d+) and rh (d-) was . % and . %, respectively. donor's phenotype detection was routinely conducted from the year, therefore the number of donors was . the most common phenotype among donors ccdee ( . %), the second in frequency ccdee ( . %), the third in frequency rhesus negative phenotype ccddee ( . %) in the studied population. the ccdee and ccdee phenotypes were . % and . %, respectively. the most rare are ccdee ( . %), ccdee ( . %), ccddee ( . %). other options: ccddee, ccdee, ccdee, ccddee, ccddee, ccdee, ccddee were detected in single cases and amounted to a total of . % (n = ). cw antigen was tested in donors and was detected in . %. cw is most commonly found in donors with ccddee phenotypes ( . %), ccdee ( . %) and ccdee ( . %), with other variants of the data phenotype, the antigen was detected in . % of the examined (n = ). antigen k was detected in . % of donors, in . % of this antigen is absent (n = ). summary/conclusions: the study of transfusion-relevant antigens distribution in population is necessary for building of effective and flexible model for blood service managing. a differentiated approach in choosing a strategy to form a long-term bank for storing blood components, taking into account the frequency of various antigen variants, contributes to improving the quality, accessibility and safety of medical care. of dislocated division of our blood establishment in orthopaedic hospital valdoltra (obv) in the number of outdated units at the hospital side dropped considerably. results: since when the issued number of red blood cell units (rbc) was the amount of issued units rose to in and then dropped more or less steadily to in . in this period the hospitals' programmes rose for % in all areas. number of donated units declined from in to in . after reorganization in the number of outdated units fell from % of stocked units to . %. after setting a dislocated unit of ctdiz on obv location the number of discarded rbc fell from to in . for transfusion specialist who is constantly in contact with the clinician in the hospital the most important day routine is when the stock availability is displayed. it happens times a day; at a.m. when the previews' day collection is released and another three times a day when the updates occur. the central base is led in ljubljana (the capital) and all centres are able to control and order the stock for the blood banking. blood wastage remained low and the traceability of the blood usage in south-western region remains high ( %). though it is not supported by an informational system the traceability form the blood bank to the patient is done on paper. this issue demands a big effort by the staff in blood bank and in hospitals. summary/conclusions: reorganization enabled better stock utilization and traceability of issued units. sometimes it is impossible to predict the peak demand of rbc especially during the summer season when the population of the area doubles and car accidents as well. transfusion specialist's effort in assuring the optimal blood stock represents the crucial daily routine. blood bank, grande international hospital, kathmandu, nepal background: voluntary non-remunerated blood donor consists of % blood donor's population in nepal. therefore demographic about the distribution of blood donors according to the age group is important to achieve % voluntary nonremunerated blood donors in nepal. aims: to explore the demographic distribution of the blood donor in different age group in the kathmandu nepal. methods: this is retrospective study conducted at nepal red cross society central blood transfusion service. data from january to january were collected from donor management software. the data includes socio demographic data. data has been process with spss version - results: during years study period, total of , blood donation happened from both mobile blood collection and in-house blood collection. out of , collection, ( . %) are from - age group; ( . %) are from - age group ( . %) are from - age group; ( . %) are from group - age group; ( . %) are from age group - and ( . %) from age group above respectively. summary/conclusions: the distribution of abo blood group varies regionally and from one population to another. in kathmandu, nepal - years age group is the most common age group encountered donating blood. the data generated in the present study and several other studies of different geographical region of india will be useful to health planners and future health challenges in the region. background: the information system on hemovigilance sihevi-ins©, coordinated by the national health institute was available in to all blood banks in the country. this software allows to centralize and record the identification data of a donor, its infectious and immunohematological tests, as well as the fractionation and final destination of each blood component obtained from a donor. aims: to describe the abo and rhd typing discrepancies in blood donors found in the blood group variables registered by each blood bank to sihevi-ins©. methods: retrospective analysis of the information registered by of the blood banks authorized nationwide between january and december . results: sihevi-ins© received information of , accepted donors, % of them with more than one donation in the same blood bank in a period of months. a total of abo or rhd discrepancies were identified in people, who made donations in blood banks (estimated risk: one discrepancy per , accepted donors). five of the blood banks implicated in these discrepancies are hospital-based (annual average collection of , ae , units, representing . % of the national collect). the remaining blood banks are distributors (average collection: , ae , units per year, representing % of the national collect). % of blood group typing discrepancies (n = ) were related to the abo group. the most common discrepancy was between a typing group and ab typing group ( %) . in % of the cases, the same blood bank initially registered in the same donor, an o blood type donation and later an a blood type (n = ) or b type (n = ). rhd typing discrepancies account for % (n = ) of the total. additionally, in three donors, a simultaneous discrepancy between abo and rhd typing was detected in the same blood bank. the results could be due to: a) failure in the warning mechanism before the release of the blood component; b) errors in typing the information of the donor registered in the system or c) failures in the identification of the donors at the time of selection. the above shows risk in the process of control of blood components release, which can impact patient safety unless abo and rhd typing blood groups are systematically verified before transfusion. summary/conclusions: despite blood banks have a verification and validation process through software to release blood components, flaws were detected. although sihevi-ins© is not a software to validate the information before the release of blood components, it was through this program that abo and rhd typing discrepancies were identified in donors who attended the same blood bank multiple times. this finding implies increasing the controls that should be used in each blood bank, to avoid lose traceability of the processes and to put at risk the life of the recipients. blood donor testing department, blood transfusion institute of nis, nis, serbia background: the ability to automate blood grouping and antibody detection procedures is a requirement for blood donor testing laboratories. mistakes in the sample identification and testing procedures could be prevented by testing on automated immuno-hematology systems. irregular antibody screening and abo/rhd grouping of blood donors are tests performed routinely in blood transfusion institute of nis. neo iris (immucor, usa) is a fully automated instrument for the abo and rh d grouping using microplate hemagglutination technique and antibody screening and identification using solid phase red cell adherence (sprca). aims: evaluation of the automated neo iris system for abo and d grouping and irregular antibody screening of blood donors in blood transfusion institute of nis. methods: during the evaluation period a total of edta-anticoagulated samples for abo and d forward and reverse grouping using microplate anti-s, anti-s, anti-jka and anti-k. in one case ih- failed to identify anti-c antibody in very low titer in sample with anti c+d antibody presence. in two samples ( . %) false-positive result were observed both on ih- system and neo iris and in two cases ( . %)only on neo iris due to nonspecific reasons. summary/conclusions: abo/rhd grouping results obtained on neo iris system, using microplate method, have a good correlation with results on ih- system as our routine column agglutination method. for antibody screening and identification neo iris showed high sensitivity for detection of clinically significant antibodies which is important step for increasing blood transfusion safety. background: continuing improvement of laboratory quality to provide accuracy test results for precise diagnosis and treatment is the mission of advanced laboratory. immunogenetic testing for histocompatibility including human leukocyte antigen (hla) typing, hla antibody detection and cytotoxicity test is critical for diagnosis and evaluation of transplantation and prognosis monitoring. in order to improve the quality of experiment competency, an external quality assurance schemes with review and education per year program was established and performed during the period from to in taiwan. aims: the proficiency testing (pt) held semiannually from to were reviewed to investigate the outcome of competency improvement of laboratories participated in the program. methods: the test items in the exercises were classified into groups, hla genotyping (including pharmacogenetics hla typing), cytotoxicity test, hla and platelet antibody. the methods of hla genotyping include ssp (sequence specific primer), sso (sequence specific oligonucleotide), sbt (sequence based typing) and either ssp+sso or ssp+sbt were used, the methods of hla antibody including elisa, flow cytometry and luminex were used and the methods of platelet antibody including sprca and elisa were used. there are four shipments of exercise materials in two years and each shipment include two positive and one negative samples for antibody detection, two each of whole blood and serum for cytotoxicity of t and b cell and three whole blood for hla genotyping. aims: this study aims to survey transfusion related laboratory tests for the quality improvement of hospital's blood bank management. methods: we analyzed survey results of kinds of routine work categories of blood banks that were registered on korean association of external quality assessment service. blood bank worker voluntarily replied this electronic survey. the categories were as follows: . characteristics of institution . the equipment of blood bank . the kinds of tube in blood bank . the present kinds of blood bank tests . abo and rh type tests . the cross-match tests . the irregular antibody tests . hemovigilance system . other blood bank tests . massive transfusion protocol . quality control issues we analyzed and compared each category data according to considering characteristics of hospitals. results: there were consensus and some differences of current blood bank tests. we presents the result of a pilot survey. especially the cross-match tests were divided by saline phase method added with irregular antibody tests or completion of rd step anti-human globulin phase according to institutional environment. automated typing machines or automated irregular antibody test devices were more increased in large-scale hospitals than small-scale hospitals. different kinds of tubes were used such as edta tube for abo and rh typing, plain tube for cross-match test. the retention segments of rbc were reserved for minimum days. most blood bank were registered and regularly listed up transfusion events to korean hemovigilance system for safety transfusion. also, a lot of institution have none or underdeveloped massive transfusion protocol. more specific survey results will be analyzed in further poster presentation. summary/conclusions: this survey will show the current status of transfusion related blood bank test. this institutional blood bank comparison will be helpful to assess the currency of individual blood bank environments. abstract withdrawn. background: we know that quality management is a continuous process, involving implementation, maintenance and improvement. aims: our purpose is to show our experience in implementing the quality management system in the whole institution and our first steps in achieving the jacie accreditation in the stem cell collection facility in order to provide our patients and donors the best possible care. methods: the institute for transfusion medicine of the republic of macedonia (itm) is the main institution in charge of blood transfusion service (bts) in the whole country, which is the national unified system. the stem cell collection facility is a part of the itm. this facility is operational since year with collections of stem cells ( in patients and collections in sibling donors) till now. we are obtaining the implementation and maintenance of qms through the establishing of the iso standardization for the whole institution (itm), as well as of implementing jacie standards in the stem cell collection facility. the two of our colleagues became the jacie inspectors and the standard operating procedures (sops) were developed, followed by regular meetings, trainings and self-evaluation of the personnel. we asked for the orientation visit from the independent jacie inspector in order to come one step closer to the jacie accreditation and to improve our overall qms. results: the institute for transfusion medicine of rm was a part of the ipa project "strengthening the blood supply system". this project aimed to ultimately bring the blood transfusion service to european union standards allowing the exchange of blood components and all other types of collaboration with other european union countries in future. the project put the basis for unification of blood transfusion standards and operating procedures in the whole country as well as set up essential education of blood transfusion personnel. although a lot of strengths were found during the orientation visit from jacie inspector, there are still a lot of areas for improvement. our strengths are motivated team and supportive institutional leadership including macedonian ministry of health. areas for improvement are: labeling of cellular therapy products and lack of laboratory for quality control. there is a national regulatory framework in place and who and world bank initiatives in macedonia which support quality in health care and accreditation. summary/conclusions: our institution has in plan to implement isbt standards for labeling of cellular therapy products and to establish a laboratory for quality control of cellular therapy, as well as to meet all the requirements to become jacie accredited facility. working by standards, following the rules and regular self-evaluations will help us to maintain the strong quality management system. every institution will benefit from a quality management system that brings you into line with international standards. ensuring the quality of our services and products is essential to keep safe and strong blood transfusion service. background: implementation of robust quality assurance program is key to high performing blood establishments. quality control and quality assurance systems together constitute the key quality systems and are parts of quality management. effective and efficient quality control policies not only provide guidance that help to increase the reliability of results but also maintains the laboratory's consistence performance overtime. aims: therefore, we established a set of qc limit using historical data which can timely identify unexpected variation in the testing systems and trigger a review of test processes in blood screening laboratories as part of quality assurance system. methods: last two consecutive years (jan, to dec, ) qc data from archi-tect i sr (abbott laboratories, chicago) was extracted using abbottlink for philippines red cross tower national blood center total of data points ( data points in & data points in ) were obtained for different qc levels for four serological blood screening assays (hiv combo, hbsag, anti-hcv, syphilis). the data was sorted for each assay/lot and qc level combination by year. qc limits were calculated using simple mean, standard deviation (sd) and coefficient of variation (cv%) and were validated and compared with manufacturer's recommendation. results: all the six positive quality control levels cv% ( . - . ) were within manufacturer's precision recommendation (within lab precision hbsag ≤ %, anti-hcv ≤ %, syphilis ≤ %, hiv ≤ %) in . five out of six positive quality control levels cv% ( . - . ) showed within manufacturer's precision recommendation (within lab precision hbsag ≤ %, anti-hcv ≤ %, syphilis ≤ %, hiv ≤ %) in except syphilis tp positive control ( . %). all four negative quality control levels showed the sd values within . - . in and . - . in respectively. summary/conclusions: excellent qc performance was observed in philippines red cross tower national blood center blood screening laboratory based on historical data and evidence-based laboratory qc limit for blood screening assays were established using historical data which takes into account total variation expected in a test system and offers a more robust and meaningful mechanism for setting control limits, for the first time. background: quality indicators (qi) in transfusion medicine (tm) are 'critically important aspects of transfusion medicine practice that are measured and utilized to gain insight for continuous quality improvement, into the degree to which the tm is capable of providing quality tm care, products or services for the aspect of practice measured following comparison of the measurement against acceptable local or international reference thresholds, benchmarks, standards, or practice guidelines'. the critical control point (ccp) selected for this study is 'administration techniques and monitoring of key elements'. this has been selected since the clinical fraternity plays a larger role in ensuring quality services in administration of blood components. there was a need to follow up compliance to standard protocol for bedside transfusion practices hence was decided to study the same with four selected quality indicators and introduce corrective measures if necessary. aims: . to assess the existing transfusion practices in the institute with specific quality indicators . to introduce corrective reforms to improve the existing practice . to assess the transfusion practices after interventions using the same quality indicators methods: to assess the existing transfusion practices in our centre, transfusions were prospectively followed up with a structured checklist. the quality indicators used were (i) verification of blood components prior to transfusion (ii)initiation of transfusion within min of release from the blood bank (iii) close observation of transfusions for the first min (iv)completion of transfusion within the right time frame for each component. as a corrective measure, a transfusion monitoring format was designed which was distributed in every ward and the nursing officers were informed to monitor and document transfusions using that. in addition, the blood bank staff was made to call up the wards and ensure that the transfusions of every component had been initiated within min of issue. transfusion practices were once again monitored by following up transfusions using the same quality indicators. results: there was significant difference in all the four variables between the two phases. . % transfusions were verified in phase i while . % were verified in phase ii (p < . ). . % transfusions were started within half an hour of issue while in the second phase, it rose to . % (p < . ). . % transfusions were observed in the first min in phase i and . % were observed in the second phase (p < . ). in phase i, . % transfusions were completed within right time while the same in phase ii was . % (p < . ). summary/conclusions: we recommend the following as quality indicators for bedside transfusion practices: background: antibody titration consists in performing antibody detection with selected red cells of different sample dilutions. the titer is reported as the reciprocal of the highest dilution that induces macroscopic agglutination. the usual applications of titration are prenatal studies and complex antibodies identification. some publications have demonstrated that more variation in antibody titer and titration score are noted upon repeat testing of the same sample when testing was performed in tubes as compared to repeat testing in gel. aims: to evaluate the efficacy of automated antibody titration versus manual method by using gel microcolumn technology. methods: edta-anticoagulated whole blood donors' and plasma frozen samples containing a known irregular (rh, kidd, duffy, mns, etc.) and regular (a & b) antibodies were selected. the titers of samples were determined in parallel by using grifols analyzers (erytra and erytra eflexis) and compared versus grifols gel manual method by using grifols gel microcolumn technology and grifols red blood cell reagents. sixty of these also processed in parallel in erytra and erytra eflexis analyzers for comparison. for the precision study, of these samples were tested in the automated systems for times ( datapoints for each analyzer) on different testing days. the hands-on (manual intervention) average time required to complete a titration was measured ( expert technicians) in different sample workload ( and samples testing). these results were compared with the same number of independent titrations performed in grifols analyzers. for the walk-away time, different sample workload ( and samples testing) were assessed in manual method ( expert technicians) and compared to timings obtained when reproduced in grifols analyzers. results provided by analyzers were reviewed and compared to manual method. results: titer obtained by erytra or erytra eflexis was equivalent to the titer obtained manually (differences ≤ titer: % ≤ . titer). the results proved that both instruments were equivalent in performing titration (differences ≤ titer; % ≤ . titer). the precision results showed no difference between titers obtained through the % of the runs performed with the grifols analyzers (differences ≤ titer: % ≤ . titer). the manual hands-on in automated system was reduced in a % compared to manual method for sample. when the number of samples was increased ( samples), the difference in hands-on in was even more reduced ( %). in addition, the walk-away was % higher in automated system compared to manual method. furthermore, when the number of samples was increased ( samples), the walk-away difference was increased even more ( %). finally, automated system software demonstrated to increase the standardization of the test as all samples, results and reagents traceability were automatically managed. summary/conclusions: grifols gel system including erytra and erytra eflexis analyzers provided a scalable and efficient solution to perform standardized titrations in the immunohematology lab. the study proved that using grifols gel system, titrations can be run in an automated reliable way (less than one-fold differences versus manual gel), thus reducing at least % the hands-on, increasing at least % the walk-away, rising the standardization and automating all testing traceability. , and fourth case of use (results) scenarios, tasks were considered "very easy" by %> % of users and "easy" and by - % of users; %> % of the users considered "sufficient" the design to ease the interaction; and %> % of users never founding any situation of not knowing how to proceed. for the fifth case of use (user roles), % of users considered tasks "very easy" or "easy"; % of users considered "sufficient" the design to ease the interaction; and % of users never found any situation of not knowing how to proceed. for the sixth case of use (maintenance plan), % of users considered tasks considered "very easy" or "easy"; % of users never found any situation of not knowing how to proceed; and % of users considered the maintenance plan similar or better than other instruments. reliability analysis ( background: quality control procedures in blood group serology for reagents, techniques, personnel working and automated equipment are essential for the accuracy of the laboratory results. the observation of high number of uninterpreted results during blood donor grouping was a motive for investigation and possible targeting the problem. aims: to identify blood group interpretation problems by analyzing the testing results obtained with the commercial quality control samples routinely used during blood grouping. methods: a microplate (mp) system for performing abo and rhd, as well as rh phenotype and kell blood group determination with two automated analyzers techno ( and ) and correspondent two mp-readers lyra ( and ) using maestro software from diamed is currently in use for blood donor typing. three types of mp are being used such as: a, b, ab, dvi-, dvi+, ctl/a , b profile for first time donors, then a, b, d ctl for repeat donors and finally, the c, c, e, e, k, ctl profile. the accuracy and safety of the blood grouping results is ensured by using the diamed q.c. system which consists of + tubes of whole blood and tubes containing serum with known specific antibodies. we analyzed and compared the interpretation of the q.c. whole blood samples' results from both of the analyzers after a new optic camera was installed on the techno /lyra system. ward to ward. methods: a prospective observational pilot study was done for around prbc unit issues which were followed in real time for understanding the tat within blood bank & from ward to ward. as per the definitions, the areas where the times are documented perfectly are understood and considered for calculations. based on the conclusions of pilot study a monitoring form has been designed and utilised to monitor the tat within bb & wtw. the data is analysed monthly and an avg tat for bb & wtw is calculated. the common causes of delay in providing the blood components were analysed and strengthened to both reduce & control the tat. results: in pilot study, total wtw tat averaged to min, with highest time taken min, where there were additional processings like leukodepletion, irradiation, saline washing of red cells and holding the transfusion. lowest wtw tat was found to be min where there was a prior information for crossmatch. after the surveillance form has been started, the average time taken for wtw tat came down to min, maximum being min (jan ), the areas where delay happened were identified as internal courier delays, technician delays, billing & other logistics delay. the concerned staff are put on regular training to maintain the tat. summary/conclusions: although ethically all the staff work for providing better care for patients, there will be few areas that delay the life supporting blood transfusion. monitoring using tat surveillance forms help in avoiding the delays and hence provide better & timely transfusion support. blood donation -blood donor recruitment p- hematological and physiological characteristics of regular blood donors with beta-thalassemia traits background: according to recent evidence, the physiological variability observed in the hematological characteristics of regular blood donors (linked -in certain caseswith genetic factors or the donor's lifestyle) may affect red blood cell (rbc) storage lesion. beta-thalassemia heterozygous (b-thal-het) blood donors represent a group of particular interest because of a) the high frequency of thalassemia mutations in specific geographical areas b) the physiology of the b-thal-het rbcs, which predisposes towards more effective management of storage-associated stress. aims: the goal of the present study was the comparative examination of the hematological and rbc physiological features of regular blood donors with or without beta-thalassemia traits before blood processing for transfusion purposes. methods: healthy blood donors of greek origin ( - years old), who met the blood donation criteria were recruited in this study. plasma/serum (uric acid, electrolytes, extracellular hemoglobin, antioxidant capacity), cellular (rbc indices) and biological parameters (corpuscular fragility, proteasomal activity etc) were measured. the results were statistically analyzed and topologically represented in biological networks for both donor groups (+/-b-thal-het). significance was accepted at p < . . results: b-thal-het represented % of the donor cohort. no differences in lifestyle (smoking, alcohol consumption, physical exercise) were observed between the two groups. nevertheless, regardless of sex and sex-dependent parameters (e.g. hct, hb concentration), b-thal-het demonstrated: a) reduced hct, mcv and mch ( % p = . , % p = . and % p = . , respectively) and b) increased rbc count ( %, p = . ) compared to the average donors. moreover, mpv platelet index was found slightly elevated (p = . ) and serum total protein concentration slightly reduced (p = . ) in the same group. a trend for higher plasma antioxidant capacity (p = . ) was evident in the group of b-thal-het, in addition to statistically significant lower levels of osmotic fragility (by %, p = . ) and hemolysis (by %, p = . ) compared to controls. finally, analysis of the three proteasome-associated enzymatic activities (n = per group) in the rbc cytosol and the membrane, revealed similar levels in the two groups (p > . ). the b-thalhet and control biological networks showed insignificant variations in respect to the amount of connections and their hub profiles. however, differences were observed regarding the number or type of connections, or even their topology in the network, in the cluster of lipids (triglycerides, ldl etc), nitric oxide, clusterin, carbonylated plasma proteins and rbc osmotic fragility (correlated with the concentration of electrolytes selectively in b-thal-het donors) between the two groups. summary/conclusions: b-thal-het who meet the criteria for blood donation are a non-negligible sub-group of the total donor population in greece. they exhibit several similarities to the general cohort, but differ in fine characteristics of rbc physiology, including resistance to hemolysis and extracellular antioxidant capacity. the differential network profile of hematological and redox parameters may be important in respect to the subsequent blood processing and storage of b-thal-het erythrocytes for transfusion purposes. background: blood service in poland is based on voluntary and non-remunerated donations. regional blood donor centre in poznan as well as other regional centres (total of ) are the only entities authorized to collect, process, store and distribute blood and its components to hospitals in the region of their activity but they are also responsible to provide sufficient amounts of blood and its components. regional blood donor centre in poznan is one of the largest blood centers in poland with the total number of donations exceeding , per year. in the recent years we have observed a growing popularity of tattoos among various age groups as well as among people registering to donate blood (first time and repeat donors) hence, it is critical to introduce suitable measures to ensure the safety of blood and its components. aims: the aim was to analyse the correlation between the increasing number of donors deferred from donating blood due to having tattoos made and the number of recorded confirmed hcv infections and the effect it may have on the safety of blood and its components. methods: the analysis was made using the data for the years - obtained from the computer system 'blood bank' which is in operation in regional blood centre in poznan, poland. we have analysed the total number of deferrals of donors due to recently acquired tattoo and the total number of recorded confirmed hepatitis c infections. we must note that the category of temporary deferrals due to tattoos is a broad one: it includes so called regular 'artistic' tattoos, permanent make-up procedures as well as medical tattoos. results: we have recorded a significant increase in number of deferrals due to tattoos from in to in (+ %). in the group of male donors this trend remained rather stable with a slight decrease: from in to in (À . %). in the group of female donors the growth was more prominent: from in to in (+ %). in terms of the recorded confirmed hcv infections a downward trend can be observed: from in to in (À . %). in the group of male donors from in to in (À %), in the group of female donors from in to in (À %). summary/conclusions: as we can conclude from the analysis the applied policy of temporary deferrals of donors with recently acquired tattoos (in the last months) proves to be a reliable method of increasing the safety of blood and its components. nevertheless, the current conduct of the qualification of the donors which requires a month deferral following the new tattoo must be complemented by various and numerous educational activities regarding the means of hcv transmission (and other bloodborne viruses such as hbv, hiv) and ways of protection from possible infections. special emphasis must be put on the group of female donors as the growth of deferrals was more prominent among them. at the same time it is vital to ensure for constant availability for all donors of well designed, concise educational materials (hard copies on the premises, articles, infographics, downloadables etc. on the website). background: a temporary deferral has a negative impact on donor retention, with many donors failing to return at the end of their deferral period. anecdotal evidence collected by the australian red cross blood service suggested that many donors do not know when they are eligible to return to donate, suggesting that a reminder message may be effective at promoting donor return once the deferral has ended. aims: the aim of this study is to evaluate the effectiveness of a reminder message on the return rates of deferred donors at the end of their deferral period. this reminder message notified donors that their deferral period was ending and encouraged them to make an appointment to donate. this study also aimed to determine the most effective time to send the message, message content, and mode of communication (sms vs email) in optimising donor retention post deferral. methods: three separate randomised controlled trials were conducted to answer these questions. data on donors' attempted return behaviour and subsequent deferrals, appointments and donations made one month after the deferral end date were collected and analysed. results: overall, . % of donors who received a reminder message attempted to return compared to . % of donors in the control group (p < . ). looking at each time point, donors who received the message week before their deferral ended were % more likely to attempt to return compared to the control group (p < . ). the week prior reminder message was particularly effective with males, with . % attempting to return to donate, compared with . % of females (p < . ). there were no significant differences in the return rates of donors who received the recipient versus non-recipient focused message, or donors who received the message via email or sms. summary/conclusions: a reminder message sent to deferred donors at the end of their deferral period is a simple, cost-effective way to promote donor retention, providing clear information regarding the date on which the donors can return to donate as well as a prompt to make an appointment background: our challenge is to provide % voluntary donation for safe blood, thus taking into account the current history of family donation, promotion of blood donation, level of awareness and voluntary donations from various institutions, the opinion of interviewees will give us a clearer idea of what we want to achieve and what needs to be improved in the future. aims: . provide % voluntary donation for safe blood. . establishing a special department within the national blood transfusion center responsible for marketing and promotion of voluntary blood donation. methods: this study was conducted as a combination of qualitative and quantitative methods. the study was a combination and identification of existing data, direct interviews with persons of different age groups, preparation and dissemination of questionnaires and analytical processing of the collected information. the study questionnaire with questions in total was divided into sections out of which questions on blood practices were answered by all interviewees. people answered questions on the blood transfusion service. questions on blood knowledge were answered by people. questions on the knowledge of the blood transfusion were answered by people, questions on blood donation were answered by people and questions on the communication channels were answered by people. results: out of interviewees, % have never donated and did not intend to donate, due to the fact that most of them were afraid of needles and infections, while the smallest part didn't donate blood because it was not allowed by the religion, % did not donate, but expressed the readiness to donate in the future, % have donated voluntarily only once, % were family donors, % regular volunteer donors, and % have donated voluntarily several times and did not want to donate anymore. from those who have donated, % have donated for one of their relatives, % have donated for thalassemic children, % have donated to benefit free check-up and % have donated because it was valuable for their health. the question as to whether they would voluntarily donate again, % have answered yes, % no and % were still not sure. this means that donation of those who have donated once did not leave a positive impression, did not increase the desire to repeat the donation once again, rather it has restrained or made it unsafe for them to repeat donation. among the causes mentioned by the interviewees were bad conditions in the donation facilities, staff behavior, inadequate treatment, they did not feel good after donation and had hematoma at the venipuncture. summary/conclusions: based on the results obtained from the study, the national blood transfusion center needs the establishment of a genuine promotion department where there is a need for a transfusion doctor who should be an active part of it. the national blood transfusion center should build up and implement a rigorous retention policy for voluntary blood donors, as the study found out that around % of donors who have donated once would like to donate again. their attraction through a donor retention policy will surely lead to self-sufficiency with safe blood. the safe blood is a public good and for this reason it is the duty of all state instances, the media and non-governmental organizations to give their support in the promotion of voluntary blood donation. background: smoking, unhealthy diet, sedentary behavior and inability to maintain adequate exercise have significant consequences for several chronic disorders, including obesity. a balanced and equilibrate nutrition may prevent the negative consequences associated to the status of obesity. in italy, overweight and obesity is increasing with adults of overweight and of obese in with a higher frequency in the south. blood centers can play a public health role in obesity surveillance and interventions. aims: since the quality of life, self-reported by the patient, related to health and adequate quali-quantitative nutrition, are becoming necessary and relevant in the field of nutrition, we conducted a demographic study to evaluate the health status of the blood donors by monitoring the nutritional habits and lifestyle. methods: a descriptive cross-sectional face-to-face questionnaire was developed. it included a item dietary assessment, reporting semi-quantitative food frequency, dietary behavior and questions on self-rated health status. normal weight was established with bmi < kg/m , overweight with a bmi ≥ and < kg/m , and obesity with bmi ≥ kg/m . obesity prevalence was standardized by sex. donors were repeat blood donors, who had made at least donations in the last years, and were eligible to donate. results: of the blood donors enrolled between july and january , were regular repeat donors, did not wish or chose not to respond at survey for several reasons (i.e. lack of time or privacy) and accepted, of which were deferred from blood donation and were excluded from the analysis. among the included participants . % (n = ) were male, age ranged from - years with a mean age of . ae . sd and . % (n = ) were female age ranged from - years with a mean age of . ae . sd. data showed that donors followed mainly a mediterranean diet and had more awareness to lifestyle, women more than men, in comparison with general population. the prevalence of overweight was found . % in men and . % in women. our survey showed that . % of the participants evaluated their health as "good", without gender difference (men, . % vs women, . %). besides, . % reported their health as "very good". summary/conclusions: overweight and obesity are common among regular blood donors and it is more frequent in men than women. our preliminary data showed that women have a better knowledge of the nutritional properties of food and consequently adopt a more balanced and proper diet. furthermore, it is clear that they are aware about the relationship between lifestyle and health putting into practice their information. unfortunately, the survey structure, of observational nature, does not make it possible to establish whether women are more alert to health to participate more in donation programs or if, on the contrary, the status of regular donor could help the improvement of knowledge and healthy lifestyle. background: donor recruitment pose an ongoing challenge to blood banks worldwide. one approach to improve the effectiveness of donor recruitment is to target influencing factors. a yearly league is conducted at the sultan qaboos university (squ) to encourage university students and faculty to donate blood. during this, the colleges are evaluated based on different measures including the number of donors recruited from each college and the efforts made by the students in increasing the awareness of blood donation in the colleges and in the society via different means including the utilization of the social media. the whole competition is organized and ran by an independent group of students. aims: this study aims at studying the impact of the yearly squ college competition on the perception of blood donation among squ students. methods: a comprehensive anonymous voluntary survey was developed and used to assess perception of students aged - attending squ and other universities (non-squ) over a two years' period. analysis was performed using ibm spss statistics . . categorized variables were presented in numbers with percentages and associations between the groups were analyzed using chi-square test. a p-value of < . was considered statistically significant. results: a total of students were surveyed ( squ, non-squ). there was no statistical difference between squ and non-squ students with regard to past history of blood donation and the number of donations made. when comparing between both cohorts, % of the squ and % of non-squ students reported the university as the main source for information (p < . ), while % of squ and % of non-squ students reported that the social media was the main source respectively (p = . ). there was no statistical difference between male and female donors on their perception of level of self-knowledge on blood donation (p = . ). about % of the youth agreed that blood donation is one of the duties toward the community. squ students reported higher rates of respond to specific requests for blood donation ( . % vs . %, p < . ). squ students reported greater influence of peers ( % vs . %, p < . ), personal knowledge ( % vs . %, p = . ) and personal experience ( . % vs %, p = . ) when compared to non-squ students. they also reported more feeling of commitment to the society ( . % vs %, p < . ). squ students reported lower influence of parents ( % vs %, p = . ), lower rates of fear from needles ( % vs %, p < . ) and lower rates of fear from blood ( % vs %, p < . ). there was no difference between male and female genders in any of the discouraging factors. summary/conclusions: these results highlighted the positive impact and important rule of the youth in the promoting blood donations among themselves through this yearly college competition; in recruiting blood donors and in the dissemination of the knowledge of blood donation. distinct promotion strategies should be adopted to increased first time and repeated blood donation among the youth. we advocate for similar initiatives in encouraging blood donation and disseminate knowledge among individuals in the community. dubai blood donation center, dubai health authority, dubai, united arab emirates background: dubai is multicultural city in united arab emirates. only about % of the population consists of uae nationals with the rest comprising expatriates from various countries all over the world. approximately % of the expatriate population (and % of the emirate's total population) are asian, chiefly indian ( %) and pakistani ( %). dubai blood donation centre is the only centre providing blood donation services in dubai. arabic is the national and official language and english is used as a second language. in order to have good quality screening, it is important that blood donors understand the educational material and questionnaire properly. aims: dubai blood donation centre receives donors (nationals, residents and gcc card holders) from various countries. the aim of this study is to analyze the multinational profile of donors and to find out the need to add any third language to meet the customer needs and expectations. methods: a cross-sectional study of blood donors was conducted in dubai blood donation centre in . the donors were asked about their country of origin, languages which they can read & understand and about the preferred mode of communication. results: a total of donors were surveyed and asked about the languages which they can read and understand and responses were obtained. the most common languages which can be read and understood by blood donors in dbdc are english (n = ; %), arabic (n = ; . %), hindi (n = ; . %) and malayalam (n = ; . %). the donors come from different countries, most common ( . %) donors are indian and ( . %) are from uae. it was found that % donors can read and understand only one language. majority ( . %) donors can read and understand either of the official languages arabic or english. however, ( . %) donors can't read and understand these two official languages, the other common languages being hindi and malayalam. the donors were asked about the preferred mode of communication, responses were obtained. the most common mode of communication were sms and telephone ( % together). summary/conclusions: based on the above findings, it can be concluded that the blood donor profile in our centre is multinational which is a unique and almost similar to the population profile of dubai. as . % donors can't read and understand arabic and english, so it has been decided that the educational material and questionnaire need to be prepared in one more language. hindi has been decided as the third language in the centre and donor questionnaire and educational materials in hindi will also be made available to the donors. further,the donors will be communicated through sms for routine messaging and disease notification while telephonic calls will be done only when the blood is urgently required. background: metabolic disorders (metds), including hypertension, dyslipidemia, hyperglycemia, and central obesity, are tightly associated with cardiovascular diseases and type diabetes mellitus. due to the sedentary lifestyle and increased consumption of high-calorie diet in modern society, metds have become serious health problems worldwide. to have a better understanding and possible improvement on blood donors' health condition, we conducted a survey of the prevalence of metds among blood donors in a blood donation station located in the hsinchu science park in taiwan. participants with metds will be provided with health education materials about metabolic risk reduction, in order to prevent the development of future complications. aims: the aims of this study were to determine the prevalence of metabolic disorders among blood donors, and to calculate how much money would be paid to identify a case of hyperglycemia, hyperlipidemia, or undiagnosed diabetes. methods: this study was approved by the institutional review board of taiwan blood services foundation (tbsf). the body weight, body height, waist circumference (wc) and blood pressure (bp) of participants were measured. blood samples were obtained to determine the values of hemoglobin a c (hba c) background: the law on blood donation supports the development of the blood service and guarantees the protection of the donor's rights and the maintenance of health during blood donation in the russian federation. national criteria for donor selection for blood donation are used in the activities of blood service establishments and are aimed at ensuring the blood products safety. the study of the characteristics of blood donors allows to predict the development of blood service and to plan the volume of blood products for transfusion and plasma fractionation. aims: the aim of this work was to study the characteristics of whole blood and apheresis donors in the blood service in the russian federation. methods: indicators of activity in the blood service establishments in the russian federation in sectoral statistical observations over the period - and the calculation of indices characterizing the whole blood and apheresis donors were analyzed. data are presented according to the administrative division of russian federation into federal districts (fd). results: the proportion of whole blood donors was . %, plasmapheresis donors - . %, blood cell apheresis, including plateletapheresis, donors - . %. for the period - , the percentage of repeated and regular whole blood and apheresis donors increased from . % to . %. the percentage of first-time donors ranged from . % to . %. the largest proportion of plasmapheresis donors was observed in the volga fd ( . %). about . % of the total plasma was collected by apheresis from donors. the percentage of plateletapheresis donors increased from . % to . %. the largest percentage of plateletapheresis donors was observed in the central fd ( . %), where a significant part of medical centers of cardiac surgery, hematology and bone marrow transplantation are located. the proportion of platelet concentrate collected by apheresis increased to . % in . actions to recruit young donors for blood donation and its components were regularly carried out in all federal districts. summary/conclusions: in the russian federation, the structure of donation is characterized by an increase in the proportion of plateletapheresis donors, stabilization of the percentage of plasmapheresis donors and an increase in the proportion of repeated and regular whole blood and apheresis donors. there are significant regional variations of donor's characteristics in the federal districts. background: shortage of blood supply despite continuous blood donation campaigns especially during local festive seasons has been a major issue in our country. thus, our faculty initiated blood donation drives in collaboration with national blood centre in order meet the demand for the blood requirements. however, the pre-donation deferral rate was relatively high among our young blood donors leading to loss of valuable blood units. understanding the causes of donor deferral provides direction on strategies for young donor recruitment and retention of future blood donation. aims: the aim of this study is to evaluate the young donor deferral pattern and to identify factors which could help in minimizing the preventable deferrals. methods: this is a retrospective study of voluntary young blood donors age between to years old recruited during mobile blood donation in faculty of medicine, universiti teknologi mara, malaysia. the study was conducted between january to december . the data were retrieved from the official reports of each mobile blood donation. results: a total of young blood donors had attended mobile blood donation during the study period. the overall pre-donation deferral rate is . %. the main causes of deferral are low haemoglobin (hb) level ( . %) followed by low blood pressure ( . %), upper respiratory tract infection ( . %) and sleep less than h ( . %). summary/conclusions: low haemoglobin and low blood pressure are the two common reasons for blood donation deferral among our young blood donors. in our study a significant proportion of deferrals are due to sleep less than h whereby this could be prevented if the donors are aware of the donor selection criteria. strategies to mitigate preventable deferrals and improve blood donor retention particularly young blood donors as source of motivation for future blood donation are urged to avoid additional stress on the blood supply. background: in the modern world, donating blood has become a humane manner for saving of patients life. but there are barriers to blood donation which are designed to ensure both donor and blood recipients' safety. anemia is one of the most common health problems in the world. based on the who estimation, nearly a quarter of the world's population are suffering from anemia, its prevalence varies among the populations and age groups. the prevalence of anemia among men is . % and in non-pregnant women is . %. aims: the aim of this study was to determine the status of hemoglobin in volunteer blood donors referring to fars province blood transfusion service and to determine the demographic status of them during the last two years. our study included blood donors for all blood donors during the last two years. methods: the study is descriptive cross-sectional and our sampling was non-random and simple sampling method. all parameters related to the donors, including age, sex and type of donation were investigated and analyzed in spss software. results: the total number of referrals for blood donation was . repeated blood donors was . % of total population and had the highest number of referrals, followed by first and lapsed donors with . % and % respectively. in terms of gender distribution, . % were female and . % were male. the highest rate of hemoglobin level less than . g/dl was found in first-time donors with . % and the lowest prevalence was observed in lapsed donors, followed by repeated donors with . %. . % of the repeated blood donors have hemoglobin higher than . . there was a significant difference between blood donation type and hemoglobin level. summary/conclusions: according to our findings, low hemoglobin levels are more common among first-time and female donors, and this requires a special training among these groups. because the high share of first time donors in blood supply and the positive impact of female donors on the blood safety, corrective action for that groups is recommended. finnish blood donor biobank j partanen, t wahlfors, m arvas, j clancy, k l€ ahteenm€ aki, e palokangas and n nikiforow background: the increasing need for large, well-characterized cohorts of healthy individuals for modern biomedical research, such as genomics or phenomics studies typically including tens or even hundreds of thousands of subjects, has posed the possibility of using blood services as an option for collecting samples and related data. the possibility to re-contact blood donors for repeated sampling or asking for additional data has further increased interest in collecting large biobanks from blood donors. there is also a need to study more thoroughly the effects of blood donation on donor health. aims: the first-phase goal is to recruit , blood donors with broad biobank consent for the finngen (https://www.finngen.fi/) project, a large publicprivate effort aiming to collect genome and health-related registry data of % ( , ) of the finnish population. ( . %), dental examination ( . %) and medication history ( . %). permanent deferral namely, risk factor involving transfusion transmitted infections and chronic disease were ( . %) and ( . %) respectively. the prime cause of permanent deferral was risk factor involving transfusion transmitted infections while the temporary deferral was bed side hypertension. gender wise, the leading cause of donor deferral in male was bed side hypertension and anaemia was the major cause in female. summary/conclusions: the findings of the survey aid to evaluate the significant causes of blood donor deferral. this study suggests that the restrictive criteria can be used for blood donor selection. this will in turn increase the blood supply of tertiary care hospital. background: donor selection is the first step towards safe blood but retaining blood donors is also very important for the blood supply. donor questionnaire and the medical interview should provide optimal doctor deferral. aims: to evaluate deferral rate in blood donors in order to identify the main reasons and to target eventual corrective activities. methods: we analysed the data concerning blood donors who were registered in the period of three years ( - ). we used data from the information system e-delphyn. background: iron deficiency (id) in blood donors is an underestimated issue in many countries and may cause symptoms to blood donors even without anemia. id prevention is mainly based on the prevention of anemia in whole blood donors, which is done by deferring donors whose haemoglobin level is under defined threshold ( g/l in women, g/l in men in france). efs (french blood establishment) studies has observed that the rate of deferral for anemia is significantly higher in women than in men, either in french west indies ( . % versus . %) or in continental france ( . % and . %). assessing the prevalence of id is of great interest since strategies to counteract it must deal with donor health and self-supply. however, data on id are missing in france. aims: to estimate the prevalence of id in french whole blood donors and to identify risk factors associated with id. methods: this non-interventional, cross-sectional, multicenter study is performed in blood donors of efs and ctsa (blood center of the french military health service). all whole blood donors who met selection criteria are potentially included. donors coming for bloodletting and donors who refuse to participate to the study are excluded. no additional sample is taken for the study, ferritin is tested after blood qualification on surplus amount. samples are selected at random within all the geographical areas and all mobile blood drives and blood centers between march and march , . results: this study ferridon has been approved by ethical research committee. nine thousand ( ) whole blood donors will be included in efs centers in continental france. to have information on donors of afro-caribbean origin and comoros origin, donations should be included in the french west indies and in reunion island. additionally, whole blood donors will be included in ctsa centers. in this study, id is defined by ferritin lower than ng/ml and iron overload is defined by ferritin higher than ng/ml. all donors with iron deficiency or overload will received a letter advising to consult their general practitioner. weights will be calibrated on age, sex and geographical area so the sample will be representative of the french whole blood donors. estimation of id prevalence will take into account the weights and logistic regression model will be used to analyze risk factors associated with id. data will be analyzed during april and may to get result at the end of may. summary/conclusions: ferridon will be the first study on id in the french blood donors. considering the french health care system and diet, it will be interesting to compare those results to results from other countries. mostly this study will allow to consider various strategies dealing both with donor safety and self-supply. background: in portugal, with an aging population of around million people, only . % are blood donors. the country has a national center of blood supply and some central hospitals with a blood donation center. despite the growing practice of the excellent concepts of patient blood management, it is imperious to attract new donors. this need has been our inspiration to use new approaches towards people, in a constant work of promotion. aims: reach the majority of our local population using radio and telecommunication as well as social networks in an attempt to raise the number of new blood donors in a central hospital of the north of portugal. methods: active communication with the population of our reference area, via the social networks facebook tm and instagram tm , through educational digital posters and messenger service to answer any kind of questions. establish contact with radio and television stations as well as with the mayor of the city, journalists, schools, town hall deputies and celebrities, through email and telephone calls. design posters, flyers and public advertising to distribute in the city. results: through the social networks it has been possible to reach a population of dozens of thousands in our city, in a daily basis. the national and world donor days were celebrated with success, in our health facility, with city mayor and journalists, and also in three television stations with national broadcast, reaching millions of people. celebrities (sport, television, music, stand-up comedy, journalists and a magician) have accepted our challenge through videos or donating blood, appealing to blood donation and sponsoring our cause. these projects and continuous availability to innovate have given our hospital a self-sufficiency of % in , instead of % in , which implied receiving less blood unities from the national center of blood supply. our most recent project involves high schools, in an attempt to educate our next generation of donors, with meetings in the town hall with deputies and district school delegates. summary/conclusions: the aging population and the low percentage of blood donors are an important issue concerning public health. nevertheless, the good will and continuous advertising and educative work towards the population, appealing to the ethical and civil responsibility since young ages have shown to improve our capacity of response as a central hospital, increasing the auto-sufficiency of blood unities and the interest of younger donors. it is of the utmost importance to understand that this is a continuous and a hard work of the professional team of our hospital, involving countless calls, emails and hours to obtain some positive response, in an endless job. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the mean interval between donations is shorter for former regular donors ( . months, p < . ) whereas donors with an interval of to months are more likely to be regular (aor ; % ci . - ). summary/conclusions: at the provincial blood transfusion centre of bukavu, the percentage of regular donors is low and there is a substantial loss of former regular donors. some factors identified to be linked to fidelity are unique to our study: female gender and a longer interdonation interval. other factors that are similar to those found elsewhere have a particular significance in our donor population which consists mainly of young people and people without income. efforts must be undertaken to ensure a supply from voluntary donations; recruitment strategies and target groups must be refined. future qualitative studies are needed to explain the various associated factors and better understand the motivations of regular and non-regular donors to improve donor retention. results: in kazakhstan, the proportion of donors is higher, especially primary. the number of blood and especially plasma donations is higher, which can be explained by the presence of several albumin and immunoglobulin production sites. increased evidence of blood transfusion rules, the development of a patient's blood management in combination with an increase in the quality of blood components cause a reduction in clinical need for red blood cells and plasma for transfusion. at the same time, the need for platelets is growing. it is difficult to assess the correctness of comparing the amount of banked whole blood. it is equally difficult to compare the number of received and distributed donor red blood cells and plasma: in russia they are measured in liters, and in kazakhstan in doses. with a certain degree of conditionality platelet extraction can be compared. in russia, they are counted in equivalent doses isolated from a dose of whole blood (at least cells per dose), and in kazakhstanin therapeutic doses (at least cells per dose). in , the estimated consumption of platelets in kazakhstan exceeded the russian indicator by . %. % of platelets in kazakhstan and . % in russia are harvested by the apheresis method. inactivation of pathogens is performed in % of platelets in kazakhstan and in . % in russia. pathogen inactivation with amotosalen allows us to abandon the examination of donors for cytomegalovirus and irradiation of platelet concentrate. the main modern trend in the use of cryoprecipitate is using it as a source of fibrinogen, a blood coagulation factor that is first depleted in coagulopathy associated with injury and massive bleeding. its production is growing in both countries, endowment in kazakhstan in exceeded the russian indicator by . %. a significant plasma percentage in both countries does not pass quarantine due to repeated non-appearance of the donor and is subject to destruction. inactivation of pathogens is performed in % of plasma in kazakhstan and in . % in russia. despite the instruction and selection of donors, laboratory screening of infection markers remains effective: russia more often identifies hiv and viral hepatitis c from potential donors, and viral hepatitis b and syphilis are detected in kazakhstan. . % of donors in kazakhstan are exempted due to the results of multiplex screening of nucleic acids of three hemotransmissive viruses. summary/conclusions: the blood services of russia and kazakhstan perform tasks to provide medical organizations with effective blood components. in conditions of decreasing demand for red blood cells and plasma, it is advisable to focus on the efficiency of resource use and improving the quality of blood components produced. blood collection including apheresis p- finnish red cross blood service, helsinki, finland background: skin disinfectant must effectively reduce microbes from the arm of the donor. as a result of poor disinfecting microbes may be transferred from skin via venepuncture to the collected blood and contaminate the blood components. aims: to ensure the efficiency of the skin disinfectant used for donor arm disinfecting by validation. the validation has two criteria which the post-disinfection samples must achieve: . no bacteria growth near the puncture spot (result cfu) in ≥ % of the samples. . total amount of bacteria on average is at most cfu/ cm . at most % of samples are allowed to have - cfu/ cm . methods: microbiological samples were taken with contact plates from voluntary persons' elbow folds before and after skin disinfection. the disinfecting was performed according to normal procedure by five nurses altogether with ethanol based disinfectant used in blood donation. on the sample plates an x was marked and this was directed to the puncture spot pointed by nurse. post-disinfection sample was taken at the moment the skin would be punctured with needle. results: the amount of bacteria varied from to above cfu/ cm in the pre-disinfection samples. disinfection reduced bacteria very well; the critical puncture spot was totally clean ( cfu/ cm ) in . % of the samples and . % of the samples had or only cfu/ cm . the average number of bacteria after disinfection was , cfu/ cm and the maximum number was cfu/ cm . most of the remaining bacteria were single colonies at the edges of the plates. summary/conclusions: both main criteria are fulfilled. the sub criteria of the second main criteria is also full filled if the not so critical colonies at the edges of the plates are not taken into account. the skin disinfectant in question is shown to be effective and can still be used in blood donation paying attention to thorough procedure performance according to the instructions and sufficient drying time of the disinfectant. background: research questions involving blood donation and recipient data often require advanced statistical methodologies. while such methodologies may appear in other medical research areas, specific tailor-made statistical tools and approaches are required for the analysis of blood-related data. these toolkits, which often require collaboration, are not always readily available to blood services, especially so in resource-limited settings. an international network of statisticians, epidemiologists and clinical researchers has been established for this purpose, which started with an invited session at the meetings of the international biometric society. aims: to exchange ideas, experience and knowledge to further improve the quality of blood sector research. methods: currently our network covers four major blood services and members from five different countries. the network has monthly conference calls about past and current research topics. we wish to extend this network further, and establish a subcommittee on statistical and epidemiological methodology with regular face-toface meetings at an international organization such as isbt. results: the monthly meetings have already demonstrated that the members share common problems and interests. for example, we are discussing techniques to analyze data with repeated measurements, e.g. eligibility haemoglobin tests, ways to assess the healthy donor effect, e.g. determining appropriate controls groups, and predictive models of blood supply and demand, e.g. stochastic processes and queuing models. the network also aims to organize training sessions in methodology either on site and/or by developing web lectures. summary/conclusions: an international network on statistical methodology for the analysis of blood donation and recipient data will improve the quality of research in the field of transfusion medicine research. expanding the network to include countries and blood services in research limited settings needs to be actively pursued. background: blood donor hemoglobin concentration (hb) is commonly measured from a skin-prick sample at the donation site, and low hb is the most common reason for temporary donor deferral. while a proportion of the deferrals do reflect true low hb, the skin-prick sample is prone to preanalytical error and variation resulting in false deferrals. aims: we assessed the applicability of a venous blood sample for second-line hb screening in blood donors failing the initial skin-prick test. methods: initial hb was measured from a skin-prick sample with the hemocue hb + (hemocue ab) point-of-care (poc) device. donors with hb < g/l for females or < g/l for males or with a decrease > g/l from latest donation were included in the study. in the study group, a venous blood sample was collected for hb measurement with the poc device at the donation site. donation eligibility was based on this hb result. venous hb was also determined with a hematology analyzer (sysmex xn, sysmex co.). the blood service's current workflow served as the control group: two more skin-prick samples were collected and the donor's final hb and donation eligibility assessed with an algorithm based on all three skin-prick hb results. results: in the study (n = ) and control (n = ) groups, the proportion of male donors ( % and %) and the mean initial skin-prick hb ( g/l and g/l) were similar. significantly less donors were deferred from donation in the study group ( %) than in the control group ( %; chi-square test p = . ). the mean difference in venous hb with the poc device versus the hematology analyzer was À g/l (range À to + g/l). two donors were incorrectly accepted based on venous sample poc result; however, in both, hb measured with the hematology analyzer was only g/l below the limit of donation eligibility ( g/l for a female and g/l for a male). interestingly, a further donors ( % of all deferred in the study group) would have been eligible for donation based on the hematology analyzer result. summary/conclusions: utilizing a venous blood sample for second-line screening of donors failing the initial skin-prick hb test significantly decreased low hb deferrals without compromising donor health. blood donors' and blood service nurses' reactions to the new workflow have been favorable. we conclude that valuable donations can be recovered and donor satisfaction increased by implementing a second-line hb screening model utilizing venous sample analysis at the donation site. background: there is a paucity of literature on haemoglobin (hb) reference values for adults above years of age. this age group has been reported to use up to % the blood supply. some studies report a decline of mean hb with age, but others have found no change with age. conflicting findings of hb levels in the healthy elderly population may be associated with challenges in accessing data from healthy older adults, small sample sizes, selection bias and recent health population data. to donate blood, each individual is assessed as 'healthy' and must meet the minimum hb criteria. however, the hb criteria across countries vary and many blood collection services have an upper age limit for donors. as many populations around world are aging, restricting the upper age limit for blood donation may potentially affect the size of the donor pool and consequently the nation's blood supply. aims: to explore the hb levels of healthy older adults, through a multi-centre retrospective observational study of blood donors aged years or older. methods: over a one-year period, hb values were collected from blood donors aged ≥ years from blood centres of four countries. the estimated proportion of blood donors aged ≥ years old for each country was . % in south korea (sk); . % in hong kong (hk), < . % indonesia (indo) and % in japan (jap). the minimum hb criteria varied between each country and ranged from . - . g/dl for women and . - . g/dl for men. hb levels were determined using point of care testing (hemocue, compolab, hemcontrol) or the xe- d sysmex dependant on the country of origin. statistical analysis of the mean, standard deviation and cumulative distribution of hb were determined by gender and age. background: medication usage is assessed to determine donor eligibility from the perspective of both recipient and donor safety. different time frames since last taken apply to different medications. assessment of medication use varies by jurisdiction, but most european centres use multiple questions. these often include a general question about recent medication use whereas the usa does not. at canadian blood services there are medication questions on the donor history questionnaire (dhq), including any medication use in the last days, vaccination and specific medications over different time frames (high teratogenicity medications). the name of each medication taken and reason for use are documented by staff at each donation attempt. assessment of the frequency with which this process occurs is the first step in improving efficiency of this aspect of donor screening. aims: to determine the percentages donors answering yes to medication questions by demographic variables. methods: all whole blood donors who completed the dhq (full length or abbreviated) in were included in the analysis. donors' answers to each of the medication questions were extracted from the national epidemiology donor database, as well as sex and age. the number and percentage of donation attempts in which a donor answered yes to each medication question were calculated. donors who answered yes to any medication question were sorted by sex and by age group, the totals and percentages calculated. results: there were , donation attempts with a completed dhq. overall, % of donors answered yes to medications in the last days, % to vaccination, and less than . % to others ( % any). slightly more were female ( vs %) of those who answered yes to any medication question, as well as by individual question. the percentage of donors answering yes to any medication question increased progressively in each age group from % of - year olds to % aged + (p < . for trend). summary/conclusions: more than one third of all donation attempts answer yes to a medication question and require further questioning and documentation. this is more common in older donors and follows a similar trend to general population medication use. comparison of ways of assessing medication use in different countries may help identify effective but more efficient approaches. in addition, the contribution to donor and recipient safety of assessing all medications should be assessed. blood center experience with trima accel and tomes software j schreier , a davison , j gambarte , y l opez , c calonge and e herranz terumo bct, lakewood, united states centro de transfusi on de la comunidad de madrid, madrid, spain background: in the madrid community, more than apheresis platelet collections were completed in , of which almost were completed in the blood transfusion center and the remainder in several hospitals in the region. trima accel was implemented to meet the productivity needs of the blood transfusion center while improving the donation experience. tomes (terumo operational medical equipment software), which enables bidirectional communication with trima accel devices, was used to connect and centrally manage all trima accel devices with automated data capture and reporting. aims: the aim of this study was to evaluate operational improvements using trima accel with tomes compared to trima accel version . methods: this was a retrospective study analyzing apheresis procedures on trima accel version during the control period from january to september compared to apheresis procedures on trima accel during the test period from september to december . this was not a paired study. operator interventions, and completed procedure rate comparisons, were analyzed using a -proportions test, whereas donor demographic data were analyzed using a -sample t-test. results: trima accel was used to collect single and double platelet products stored in platelet additive solution. operators selected either a single (target platelet yield = . or . ) or double (target platelet yield = . ) platelet donation based on desired procedure time not the maximum number of products that could have been collected per donor. no statistically significant differences were observed for donors in the test arm compared to donors in the control arm for total blood volume (control = ml, test = ml, p = . ), hematocrit (control = . %, test = . %, p = . ), or platelet pre-count (control = / ll, test = /ll, p = . ). females represented % of donors in the control arm compared to % of donors in the test arm. platelet split rate (platelet products per procedure) increased from . with trima accel version to . with trima accel ; procedure time decreased from . min to . min for single collections and from . min to . min for double collections with trima accel (these differences were not statistically significant). the percentage of procedures that completed with no operator interventions due to access alerts increased from . % to . % (p < . ) and the rate of completed apheresis procedures increased from . % to . % (p = . ) with trima accel . manual transcription of data during the procedure was discontinued with the implementation of trima accel with tomes. tomes captured procedural data and operator steps with barcode scanning and tracking of configured events. this information was transferred to tomes post procedure and printed as a final report. summary/conclusions: trima accel significantly decreased operator interventions, and automated data capture with tomes eliminated manual transcription of data. both outcomes freed operators to complete other tasks and focus on donor well-being. background: the european committee (partial agreement) on blood transfusion (cd-p-ts) of the council of europe (coe) has appointed a working group (wg) to focus on issues with plasma supply management (psm). the task of the wg is, among others, to collect and analyze data in order to fill knowledge gaps concerning donor safety in plasmapheresis. in doing so, the working group will gather evidence base data to support the upcoming revision of the th edition of coe's "guide to the preparation, use and quality assurance of blood components", the blood guide. an international survey was conducted sept-dec , distributed to blood establishments (bes) by the cd-p-ts representatives to coe's member and observer states. the questionnaire included sections covering collection practices (volume and frequency), management of red cell loss, donor panel demographics and data on donor adverse events. aims: the aim of this study was to investigate whether collection practices following the recommendations published in the blood guide for maximal collection volumes and number of donations per year were indeed associated with higher levels of donor safety and improved donor base sustainability. methods: from the total of respondents, bes collected plasma for fractionation (pff) by apheresis and the study had a dataset covering , , plasma donations in the latest fiscal year (lfy). the parameter used as marker of donor safety was the rate of immediate vasovagal reactions with loss of consciousness (vvr with loc) per , plasma collections. the parameter used as marker of donor base sustainability was the retention rate of donors, ie % donors active in the previous year returning to make a donation in the lfy. results: in the blood guide, the collection volume per apheresis is limited to % of the estimated total blood volume but maximally ml, including anticoagulant. respondents had differing practices and scale of collection program be were aligned or lower, and be had higher collection volumes. altogether reported the immediate vvr with loc rate, which mainly was lower than / collections. there was a small trend towards reduced rate with larger collection volumes than allowed by the current blood guide. saline compensation during or after collection did not affect the rate of vvr with loc. no correlation was observed between the annual donor retention rate and the rate of vvr with loc or saline compensation practices, as reported by respondents. the retention rate banded in the range of %> % (mean = %, min = %, interquartile range = %, max = %). the association between maximum allowed yearly plasma collection ( l) appears to be reasonably constant and showed no clear association with the donor retention rate. summary/conclusions: restricting the maximum collection limit according to the current blood guide was not associated with either lower vvr with loc or with higher donor retention rate. this study supports reassessment of current blood guide s limits for collection volume of maximum of ml per donation and l per year per donor. methods: serum ferritin concentrations were established from sera stored at À °c from repetitive platelet donors between and , using architect â ferritin assay chemiluminescent microparticle immunoassay (cmia). the hematimetric parameters were evaluated in a total blood sample using the celldyn â . sixteen samples were obtained from women (age: . ae . years, range: - ) and samples from men (age: . ae . years, range - ), corresponding to . % and . % of the total female and male repetitive donors of platelets by apheresis using trima accel â terumo-bct and amicus tm fresenius-kabi. the difference in the concentration of serum ferritin between the last and first donation was established, as well as the change in the predonation platelet count between the last and first event. results: in the study population, . % of women and % of men performed repetitive donations of platelets by apheresis with an interval of less than three months. the change in ferritin concentration was evaluated according to the interval between donations. in women ferritin delta was À . ae . ng/ml when the donations had an interval less than three months, vs . ae . ng/ml when the time between donations was higher (p = . ). in men the change in the ferritin levels was À . ae . ng/ml with donation times less than three months vs © the authors vox sanguinis © international society of blood transfusion vox sanguinis ( ) (suppl. ), - À . ae . ng/ml with prolonged donation times (p = . ). in women, the change in platelet count was À ae . /ul, when the donations had an interval less than three months vs À ae . /ul when the time between donations was greater (p = . ). in men, the delta of the platelet count was À ae . /ul in donation times less than three months vs À ae . with higher donation times (p = . ). no correlation was found between the concentrations of serum ferritin and the platelet count (r = . , q = . for males, and r = . , q = . for females). summary/conclusions: the data obtained suggest that repetitive donation of platelets by apheresis with intervals between donations of less than three months, significantly reduce serum ferritin concentrations in women and men, although normal levels were maintained in both groups. there was no correlation with platelet count. therefore, it is proposed to develop prospective studies to establish the minimum time interval safety for platelet apheresis donor procedures. background: the demand for platelets concentrates is increasing continuously and becomes a challenge for the blood establishments. apheresis platelet collections may be a solution for this challenge. improving apheresis collection efficiency while maintaining blood donor safety is an important goal for the service du sang of the belgian red cross. aims: our establishment evaluated the improvements of the trima accel automated blood collection system version (ta ) by comparing its routine performance with that of the previous software version . (ta ). methods: prospective, multi-site, controlled, non-randomized trial. apheresis collections were performed in three sfs sites: liege, mons and namur using the two trima software versions ta and ta sequentially. data were collected from december to april on ta and from june to july on ta . simple and double doses of platelets (respectively . , . and . , . ) were collected in platelet additive solution (ssp+, macopharma) with concurrent plasma from the same cohort of donors in accordance to donor's eligibility and preferences. in order to maintain the same final platelets content in platelets concentrates, the trima accel's tool yield scaling factor (ysf) was subsequently adjusted from . (ta ) to (ta ). platelet yield, duration of procedure, number of alarms requiring operators' interventions were recorded and evaluated. donor's hypocalcemia was avoided by giving preventively oral or intravenous calcium which was documented by the operators. results: five hundred ninety ( ) collections with ta and with ta were recorded, with % and % complete procedures respectively. mean duration of procedures was min on ta against min on ta , p < . . the mean alerts number per procedure on ta was . against . on ta , p < . whereas the maximum alerts number per procedure was and respectively. on ta , % procedures did not require operator's intervention against % on ta ,. with ta the inlet flowrate was automatically adjusted in . % procedures. the inlet flowrate was increased in response to access pressure in . % of procedures, for % of the procedures the inlet flowrate was decreased and for . % of the procedures the inlet flowrate was increase and decreased on the same procedure by the ta autoflow system. summary/conclusions: ta with its autoflow function improves apheresis donors experience while decreasing operator' interventions through a significant reduction of access draw alerts. as expected from the trima accel platform, post-donation safety remains high. a weak increase in procedure duration was observed for the same platelet yield which may be resolved with further adjustments. background: trima accel system is an apheresis platform relying on continuous flow centrifugation to collect from a donor platelets, plasma or rbcs based on donor qualification. the latest software version -trima accel (ta ) introduced the autoflow feature which allows for automated flow rate adjustments. moreover, ta leverages the mobilization capacity of the spleen increasing potential platelet productivity while maintaining high post-donation safety standards characteristic of trima accel. aims: the objective of this evaluation was to assess the impact of ta software by retrospective comparison of procedure data and potential for increased productivity with those of trima accel version (ta ) in the same cohort of platelet donors. methods: eight hundred twenty one procedures, started on ta from th january to th october were compared to procedures, started on ta from th october to st december . procedural data from the trima devices were captured using the cadence system (terumo bct, lakewood co). parameters investigated were the number of machine access pressure alerts per procedure, the potential for higher platelet yield collections and the actual collected yields within the same cohort of platelet donors. results: both donor populations (ta vs. ta respectively) were comparable and were characterized by: tbv - vs. ml; platelet count pre-procedure - ³/ll vs. ³/ll; hematocrit pre-procedure - % vs. %. gender distribution was % female with ta vs. % with ta . venous access pressure alerts were significantly improved by ta with an average of . alerts per procedure as compared to . with ta , i.e. % decrease. this decrease went down to % if only male procedures were analyzed. the maximum number of pressure alerts went down by % from alerts in one particular run in the ta cohort to alerts in one ta procedure. procedure time for single platelet products was reduced from to min and for double platelet products from to min (ta and ta respectively). donor qualification possible was % of procedures yielding single products and % of procedures yielding double products with ta . the percentage of procedures qualifying for doubles increased to % with ta . in terms of split rates, i.e. how many platelet doses could be produced per apheresis collection, potential split rates increased from . to . from ta to ta , respectively. in fact, the observed split rate rose modestly from . to . , as shorter procedures were generally selected according to donors' preferences. summary/conclusions: in comparable donor populations, implementation of ta decreased the number of access pressure alerts significantly compared to previous trima versions. the average procedure duration was also found to be slightly reduced. implementation of ta has the potential to increase productivity significantly. the observed modest actual rise in split rate suggested that factors related to donor and inventory management will determine at which extent the potential of the new software will be used. donor compared experience on trima accel to trima accel version august to october or trima accel during the test period from november to january . this was not a paired study. donors completed the survey while recovering from the apheresis procedure in the cantina. results from the paper survey were transcribed into excel for analysis. results: donors completed the survey during the control period whereas donors completed the survey during the test period. the mean number of previous donations for the control period was . (min max ) and for the test period was . (min max ). there were no first time donors during the control period and first time donors during the test period. % of donors rated their overall donation experience as good on trima accel compared to % on trima accel version . zero ( ) donor rated their experience on either trima accel device as poor. % of donors who responded to the question said they would donate on trima accel again. summary/conclusions: no significant difference was observed in donor experience between trima accel version and trima accel as both versions receive high marks. background: the trend on growth of query for donor platelet concentrates is observed in russia for past few years. as reported by edqm in , a higher number of the platelets was consumed compare to by . %. patients' with hematological malignancies treatment requires platelet concentrates transfusions during chemotherapy, immunotherapy and hematopoietic stem cells transplantation. according to the data collected in national research center for hematology (nrch) in , ( . %) of the , patients, treated within facility, received platelet transfusions as transfusion therapy. the total number transfused units is , , which is higher (by . %) comparing to . platelet concentrates production can be performed either by apheresis process or by pooling individual units recovered from the whole blood. taking into account that the nrch produces blood units for its own needs, the pooling is not suitable method for production because its implementation doesn't cover require for platelets and overproduces rbcs. that is why platelet concentrates in nrch are obtained by apheresis only. in summary, the growth on requirement for platelet concentrates and their safeness explains the need for a comparative study for effectiveness of platelet production using various apheresis systems. aims: the aim of the study is to compare effectiveness of platelet concentrate production using mcs + (haemonetics), upp and trima accel (terumo bct) version . protocols. methods: the data for protocols of platelet donations performed in were analyzed: on trima accel and on msc + . all donors were voluntary and non-paid donors with previous experience of blood donations. the choice of the platelet collection device was random; analysis of the main characteristics of donors did not reveal any significant differences between the groups. the median age of the donor was years old, height - cm, weight - kg, platelet count before donation - /l, hematocrit - %. detailed data are presented in table . student's t-test for unrelated sets was used for statistical analysis of the data. a value p of less than . was considered as significant. results: the data obtained showed significant difference (p < . ) between average number of platelets collected on trima accel ( . ae . /l) and on mcs + ( . ae . /l). while cost of consumables are comparable, trima accel demonstrated . % higher efficiency. procedure duration also was comparable and averaged within min for both devices. detailed data are presented in table . it is crucial to mention that proportion of trima accel's donations was significantly increased in nrch during and reached , % in total ( - . %). a flexible usage of trima accel's consumables for different procedures (regular platelet collection and collection in pas) allowed to change the pas/regular platelets collection ratio from . % up to . %. summary/conclusions: obtained results proved the effectiveness of the trima accel's use for platelets concentrates production. it allowed to increase the average count of platelets obtained for one procedure by . % compared to mcs + while the cost of consumables and procedure duration are comparable. the donor's comfort during procedure did not affect either. in long terms increasing of number of platelets collected is reducing the cost of platelet concentrates production. abstract withdrawn. background: apheresis collected platelet concentrate is preferable in terms of reducing the risks of adverse reactions in platelet transfusion when compared to random donor platelet concentrates. aims: the aim of our study is to present our experience in collection donation of single donor platelets with apheresis. methods: this is a retrospective study performed in the institute for transfusion medicine from till . all donors were fully informed on the donation procedure and signed an informed consent for donation. the optimal platelet count that we want to achieve was ≥ . equal to random donor platelet doses. minimum preapheresis platelet count in donors requested to start the apheresis collection was . /ll. platelet collection was performed using flow cell separators haemonetics mcs+ and trima accel. acid citrate dextrose formula a was used for anticoagulation. median precollection platelet count of donors was . /ll, with range from . /ll to . /ll. male were % of the donors and females were %. the single procedure usually took - min. the median platelet count collected was . , range - . . the median processed blood volume was ml and median used acd-a was ml. mean total volume of collected product was ml. the adverse effects included vein perforation and the numbness of the extremities as reaction of acd-a (hypocalcemia), which occur rarely and was very mild. summary/conclusions: the collected platelet count was more than the wanted optimum platelet count. the number of apheresis donors is increasing and we are working on expanding our voluntary platelet donors registry and increasing the number of typed donors in the registry. background: to determine value of hemoglobin in blood donors, there are some tools or methods used, such as: cyanmethemoglobin method that can detect of hemoglobin quantitative and methods cupric sulfate solutions (cuso ) can detect of hemoglobin qualitative. according to who (world health organization) to determine the level of hemoglobin in blood donors enough used cuso solutions with specific weight (y) . can to detect value of hemoglobin above or same with . gr/dl. but, cuso solution specific weight . can not to detect and elimination value of hemoglobin above gr/dl or polycythemia sick. because it, central blood transfusion unit (utdp) as the central of blood service in indonesia to manufacture cupric sulfate solution (cuso ) with a specific weight (y) to detect value of hemoglobin below gr/ dl and determine value of hemoglobin above gr/dl. because it, do the testing the accuracy of the solution cupric sulfate in detecting and eliminating donor with value of hemoglobin above gr/dl with the test of samples. aims: to determine accuracy and effectiveness by blood donors unit in indonesia red cross to use cuso solution with specific weight . in to detect and elimination value of hemoglobin donors above gr/dl. methods: used the method cyanmethemoglobin and cuso (y) . determination value of hemoglobin donor. test results were analyzed with spss software version using nonparametric analysis wilcoxon test. results: this research testing the accuracy and effectiveness of using a cuso solution with specific weight (y): . in detecting and eliminating hemoglobin value donors above gr/dl. from data processing using spss with the wilcoxon test p value . . summary/conclusions: it was found that the cuso solution (y): can detect hemoglobins value above gr/dl and more effective in checking the hemoglobin in blood donors. it can be seen from the data processing with spss version with the wilcoxon test p value < . . it is important to monitor the precise course by which repeated blood donation affects hb and the probability of low hb deferral. zinc protoporphyrin (zpp) is a functional indicator of body iron levels and is hypothesized to predict hb levels among blood donors. advanced statistical methods are necessary to properly analyze the longitudinal associations between zpp and hb in data with repeated donations per donor. aims: to determine whether predictions of future hb levels using current hb levels can be improved by taking zpp levels into account, and to illustrate the use of statistical models for repeated measurements of blood donors. methods: we used data from the zpp and iron in the netherlands cohort (zinc) study. we identified previous zpp levels (log-transformed) as the main predictor and adjusted for previous hb level, age, day and time of donation, donation history, bmi, blood volume and blood pressure. we used linear mixed models, which take into account missing data in the outcome and associations between repeated measurements, to investigate the longitudinal association between previous zpp and current hb levels. the longitudinal analysis with linear mixed models was contrasted with a simpler analysis based on the area under the receiver-operating-characteristic (roc) curve for the probability of low hb deferral. results: in total, whole blood donors ( , whole-blood donations) were included in the zinc study, % being female donors. previous zpp showed a statistically significant association (p < . ) with hb levels in females, but the size of the association was quite small (regression coefficient, b = À . , % confidence interval À . to À . ). the same was true for males, but the size of the association was even smaller. blood volume and age for women were significant secondary predictor variables; blood volume, age and donation interval for men. by comparison, the roc analyses showed relatively larger, but less statistically significant predictive effects of zpp on hb. summary/conclusions: zpp is a statistically significant predictor of hb levels, but the size of the effect after adjustment for previous hb and other variables is small. the results cast doubt on whether zpp is an effective predictive marker for hb level and low hb deferral, and suggest that zpp should not be included in prediction models for hb levels. by properly adjusting for associations between repeated measurements and by using all available data, longitudinal models provide less biased and more precise estimates than simpler cross-sectional analyses. background: finnish red cross blood service (frcbs) is a national blood service and is responsible for all blood collection and component production in finland. the highest age for blood donors was years until the end of year and since beginning of donors between and years have been able to donate blood. blood donation after the age is possible if the donor has donated within the last months. the upper age limit was raised up based on adverse event data from frcbs donors up to years and on published data from other blood establishments. aims: the aim of this study is to find out if the new policy from with upper age limit of years is safe. therefore donor adverse event data was analyzed in order to evaluate if the blood donors older than years have more adverse events compared to other donors. the most common donor adverse event for donors over years, haematoma, was registered times ( . %). in the other age groups haematoma was registered times ( . %) and the difference between the oldest age group compared to all other donors was statistically not significant (chi , p = . ). vvrs with loc were registered times ( . %), vvrs without loc times ( . ), and the total number of all daes was ( . %) in the age group years or older. the respective numbers in the other age group were: , . %; , . %; ( . %). the number of vvrs with and without loc, and total number of all daes in the age group years and older was smaller than in the other groups and the difference between these groups was statistical significant (chi , p < . ). summary/conclusions: donors over the age of years have less donor adverse events than other age groups. decision to raise the age limit from to seems proven to be right as the older age group has even less donor adverse events than other donors. background: deep vein thrombosis (dvt) of the donor's phlebotomy arm is a rare, but serious complication of blood donation that needs to be recognised and managed appropriately in a timely manner. post donation dvt will be classed as a 'serious adverse events of donation' (saeds)these are events that either result in donor death, hospitalisation, intervention or significant symptoms persisting for more than one-year post donation. aims: to review cases of dvt post donation reported in uk in - years and identify any common themes for improving practice methods: all data relating to saeds from the four uk blood services reported to shot in the last years ( - inclusive) were reviewed to look for reports of dvt post donation results: a total of saeds were reported in uk from approximately . million donations (whole blood and apheresis) collected during this period. three cases of upper limb dvt were reported during this time accounting for % of the saeds reported and rate of dvt of in . million donations collected. -case : a regular male whole blood donor in his early s reported worsening arm pain days following blood donation, had a painful venepuncture and a small bruise at site of donation. he was diagnosed to have an upper limb dvt extending to the subclavian and brachiocephalic vein and started on oral anticoagulants. no other contributory factor was obvious -case : a female donor in her mid- s gave her sixth whole blood donation without event. days after donation, she developed worsening arm pain in the donation arm and was diagnosed with an upper limb dvt and commenced oral anticoagulation. there was no other identifiable risk factor for the thrombosis -case : a female donor in her s developed pain, swelling, redness and itchiness in her donation arm and chest wall two days after donation. she also described prominent veins on the affected side compared to her other arm. she contacted the transfusion service one week after donation; by this time she was also breathless on minimal exertion. she was admitted to hospital and commenced on anticoagulant therapy. a diagnosis of dvt and associated pulmonary embolus was confirmed. the donor's only risk factor for thrombosis was use of the oral contraceptive pill. summary/conclusions: rare complications of blood donation, like dvt, can occur. superficial venous thrombosis may occasionally progress into the deeper veins of the donor's arm but dvt can also occur without signs and symptoms of superficial thrombosis. none of our patients had any overt evidence of superficial thrombosis. one patient in our series reported using oral contraceptive pill. no other risk factors for thrombosis was forthcoming. transfusion services should encourage donors to make early contact with the blood service if they experience arm complications so that they can be investigated and managed in a timely manner. staff dealing with such donors must recognise the possibility of this rare complication, explore other additional contributory factors and initiate prompt and appropriate management. background: voluntary blood donation is widely considered to be safe with very minimum chance of adverse reaction, which may occur during or after the end of phlebotomy procedure. aims: to find the adverse blood donor reaction among voluntary blood donors in tertiary care hospital in kathmandu methods: this is a prospective study done among voluntary blood donors at grande international hospital, kathmandu, nepal from february to march . the outlines of reported and communicated adverse donor reaction were also collected after the blood donation from voluntary blood donors in different locations including outdoor and in-house blood donation drive results: in the present study , whole blood donors were included, during the period of years, ( . %) adverse donor reactions were reported. majority ( . %) of adverse donor reactions were mild in nature such as, sweating; ( . %), light headedness; ( . %), nausea and vomiting; ( . ), allergy and bruises; ( . %), sore arm; ( . %) and hematoma; ( . %) while ( . %) were severe adverse reactions similarly, anaphylaxis; ( . %), loss of consciousness; ( . %) and convulsive syncope; ( . %). markers of the adverse donor reaction were age, sex, pulse, weight, blood pressure and donation status. age and first time status were related with significantly higher risk of adverse reaction with - years old at higher risk compared to - years old. first time donors were at higher risk compared to repeated volunteer donors. summary/conclusions: the results of the study are helpful to identify and understand the complication of adverse donor reactions though the incidence of reactions in the blood donors is lower than in other studies. donor age and donation status were strong possibilities of complications. background: blood donors with pollen-induced allergy and asthma must often refrain from donation in pollen season despite medication, because of symptom severity or similarity to airways infection. extracts of the medicinal mushroom agaricus blazei murill (abm) given orally have been found to reduce ige anti-ovalbumin levels and ameliorate the skewed th /th cytokine balance in mice sensitized to ovalbumin (takimoto, immunopharm immunotox, ; ellertsen & hetland, clin mol allergy, ). aims: the objective was now to examine whether supplementation with the abmbased extract that we used in the mouse model for allergy, could alleviate allergy and asthma in blood donors by reducing specific ige levels and basophil sensitization. methods: sixty donors at oslo blood bank with self-reported birch pollen allergy and/or asthma were recruited and randomized in a double-blinded, placebo-controlled study with oral supplementation for weeks before the birch pollen season with the abm-based extract andosan tm (immunopharma, oslo, norway). this is a water extract of the bacidomycetes mushrooms abm ( %), hericeum erinaceus and grifola frondosa. the participants filled in questionnaires for allergic conjunctivitis & rhinitis, asthma and medication. serum ige (immunocap â , immunodiagnostics, sweden) and bet v -induced basophil activation in whole blood determined by cd expression in a flow cytometer (flow cast â , b€ uhlmann lab ag, switzerland), were analyzed before and after the pollen season. (trial record: nct , clinical.trials.gov). results: there was significant reduction in allover allergy-related ailments and types of allergy medication used in the abm extract compared with placebo group during the pollen season and no side effects. also, abm treated asthmatics had fewer symptoms and used less medication than controls. in the abm group, serum levels of specific ige anti-bet v and anti-t , were significantly reduced during the pollen season as compared with levels in the placebo group. whereas the maximal allergen concentrations needed for eliciting basophil activation before the season changed significantly to lower concentrations (i.e. enhanced sensitization) after the season in the placebo group, these concentrations remained similar in the group given the mushroom extract. summary/conclusions: oral pre-seasonal supplementation with an abm-based extract for months reduced general allergy ailments, asthma symptoms and medication in blood donors with birch pollen-induced allergy and asthma during the pollen season. this was due to reduced specific ige levels and basophils rendered less sensitive to allergen activation. the study suggests that supplementation with abm mushroom extract can have prophylactic effect on aeroallergen-induced allergy and asthma in blood donors. it may therefore reduce such ailments in affected blood donors and impact blood donations in the pollen season. results: dhv started in / / . the data presented in this abstract is till / / . data is collected from total of blood donors ( . % male donors and . % female donors). repeat donors accounted for . % against . % of first time donors. of the total number of donor adverse events recorded, . % ( ) reported for male donors and . % ( ) for female donors when the donor adverse events stratified age-wise, the highest incidence reported in age group - years (male . % and female %). among age group - years, (male . % and female . %), whereas in age group - years, (male . % and female . %) data analysis of total reported and registered donor adverse events, are categorized as hyperventilation ( ), sweating ( ), dizziness (pre-syncopal ), loss of consciousness ( ), vomiting ( ), convulsions ( ), hematomas with re-bleed ( ), nerve irritation ( ) and off-site reactions ( ). many donors showed multiple forms of reactions. summary/conclusions: evaluation of donor side effects helps to improve donation process and donor compliance. most frequently recorded reaction remains dizziness (pre-syncopal). our donor vigilance data show reactions occurred more frequently in younger age, female and first time donors. repeat donation and age are predictors for low rates of adverse events. participation in dhv implies an effort to improve donor care and safety infrastructure and a desire for national and international comparisons to determine best practices and also to look into effectiveness of risk reduction strategies and follow-up trends. pre-donation hydration was implemented as an interventional tool to test the effects of hydration on pre-syncopal reactions to blood donation, specifically targeting those at highest risk such as female, first-time, high school donors. the results are awaited. background: descriptions of deferral categories and a knowledge in the percentage of deferrals in each category are of value in formulating recruitment and retention strategies. this can also help in planning more efficient recruitment strategies and thereby assist in reducing the shortage in blood supply. aims: the aim of the study is to categorize all donors who were deferred during medical checkup and find out the donor deferral rate in dubai blood donation center from january st to december st and also to find out whether there is any yearly or seasonal trend in any of the categories of deferral criteria's which can aid in forecasting and managing donor pool. methods: a retrospective study of donors deferred during last three years from january st to december st was done in dubai blood donation centre. the donors deferred during pre-donation medical check-up were categorized into categories including low hemoglobin, high and low bp, intake of antibiotic, fever and flu, taking other medications, travel history etc. the deferrals were analyzed monthly and yearly and then were compiled to find any yearly trend or seasonal trend in the donor deferral rate in any of the categories. the data were analyzed using the spss software and a p value of < . was considered significant. the assessment of donor suitability is in accordance with aabb standards and is consistently applied in every blood donation setting on each occasion of donation to all blood donors. results: during this study, , donors were registered from january st to december st and , ( . %) donors were deferred. the common reasons of deferral were low hb, high bp, travel history, intake of antibiotics and cough/flu symptoms. there was a significant decrease in deferral rate from . % ( / ) in to . % ( / )in and further to . % ( / ) in (p < . ). the specific deferral rate due to low hb also significantly (p decreased during these three years ( / in , / in , / in ), though no change was seen in the deferral due to other reasons. the reduction in the rate of deferral due to low hemoglobin may-be linked to the change in the staff performing the hemoglobin testing in dbdc (nurses instead of phlebotomist were assigned to perform hb estimation of donors). there was a seasonal variation in the deferral rate in all the three years-lowest in june ( / ) and then increasing with a peak in october ( / ) and plateauing till january. this pattern of deferral corroborated with the rate of deferral due to flu/fever and cough and antibiotics with an average of / in june and increasing to / in october (p < . ). summary/conclusions: staff competency is pertinent in accurately deferring donors. there is also a significant seasonal pattern in flu/fever and intake of antibiotics deferral rate that is reflected in the total donor deferral pattern. seasonal variation of specific category of donor deferral should be taken into account for donor recruitment and retention efforts. background: west nile virus (wnv) is a mosquito transmissible flavivirus. it has been shown (vogels thesis) that the common mosquito in the netherlands can transmit wnv in laboratory circumstances but presently does not lead to effective transmission. however, the number of outbreaks of wnv is increasing and moving from the eastern and southern european borders towards the traditionally more colder western and northern parts of europe. in order to prevent wnv transmission to blood transfusion recipients, dutch donors that travelled to regions with wnv risk are deferred for a period of days for whole blood, platelet donations and quarantine plasma in order to exclude potentially infected asymptomatic donors. aims: to assess numbers of dutch donors who are deferred for travelling to wnv risk areas within europe, and the return after onsite and offsite deferrals of donors. methods: data from to on donation attempts and deferral were retrieved from eprogesa, the blood bank information system. onsite deferral is defined as a donation that was attempted in the deferral period or in the days prior to deferral, all other deferrals are considered as offsite. a generalized estimating equation model was used to assess the association between onsite versus offsite wnv risk deferrals in - and subsequent return rates within two years (after which a donor is inactive according to domaine). results: in - , , donation attempts led to onsite deferral for wnv risk; % at whole blood donation attempts, % at new donor examinations. in total , offsite deferrals could not be traced directly to a donation, but based on the next donation more than % were probably whole blood donors. the number of deferrals peaks each year during august, the major holiday period in the netherlands, and increased from in august to in august . this increase is probably caused by the expansion of wnv risk regions. the return rate of wnv deferred whole blood donors is slightly lower than for donors who are not deferred ( % versus %); for wnv deferred new donors the return rate is % (versus % for no deferral). thus wnv deferral resulted in approximately - extra lapsing donors during these years. however wnv deferred donors, that are older (odds ratio (or) . ; % confidence interval ( % ci) . - . ), of male sex (or . ; % ci . - . ) and whole blood donors as opposed to new donors (or . ; % ci . - . ) were more likely to return to donate. there was no difference in return rate by offsite and onsite deferral. summary/conclusions: travel-related wnv deferrals are increasing with expanding risk regions, especially in the holiday season where the availability of donors is already low. although the numbers of donors who are permanently lost after wnv deferral are limited, the increasing numbers of lost donations make it important to consider alternatives to donor deferral such as wnv nat testing. background: low haemoglobin due to iron deficiency is increasingly recognized as a serious problem in many blood centers. donor education, iron supplementation, ferritin monitoring, and lengthening of inter-donation interval are currently the main mitigation measures. however, a number of factors in particular donor knowledge could impact their success. locally, iron supplementation programme was implemented since with target group of donors who have given blood within the last six months. aims: here we look at an online donor survey to gain insight on their view of the programme and knowledge. methods: donors with successful blood donation in the past six months would be given days of one tablet of iron supplementation ( mg elemental iron) since . an electronic questionnaire was sent to blood donors in to assess their view on the programme and knowledge which focused on iron store and absorption, compliance and any side effects occurred. results: donors (male to female was : . ) replied to the questionnaire. of them, received iron supplementation (male to female was : . ). most of the respondents ( %) had one or more donations in the preceding months. of the donors received iron tablets, only ( %) took all; ( %) took more than % but not all; ( %) took some but less than % and ( %) did not take any. gastrointestinal upset was reported in ( %) donors and constipation seen in ( %) among those who took at least some of the iron supplementation. most respondents answered correctly to the questions on the knowledge on iron store and absorption. when comparing those with better compliance (took more than %) to those who did not (took less than %), significantly more donors in the former knew vitamin c could enhance iron absorption (p < . ). on the other hand, no difference was seen when they were asked if ) iron can only be absorbed from meat; ) tea and coffee consumed during meal can enhance iron absorption; ) everyone can take iron supplementation on their own; and ) iron store in male is always more than female. summary/conclusions: the results suggested that there is definitely more room to enhance the blood donors' knowledge on iron store and absorption in order to improve the effectiveness of iron supplementation programme. besides, the side effects reported by the donors could be an important limiting factor that better alternatives should be explored and considered. background: vasovagal reactions (vvr) are a well-established deterrent to donor return. however, the correspondence between vvr experience and donor lapse is not perfect. in australia, for example, vvrs only reduce two-year return rates by % for whole blood donors and % for plasma donors. the elements of a vvr and the donor's interpretation of this event that protect against or encourage lapse have not yet been identified. aims: in this study we explored the views of donors on donating following a vvr, with a particular interest in their emotional reaction to the vvr, their understanding of what caused the reaction, and their intentions to return. methods: semi-structured telephone interviews were conducted with whole blood and plasma donors who had a recent vvr experience. data were analysed using the framework approach. results: donors are generally motivated to give blood to help others and to positively impact on those in their communities. they anticipate feeling good after their donation but in contrast, for many, a vvr leaves them feeling anxious, embarrassed, and disappointed. for donors, the experience of a vvr negatively influences their perceived ability to donate successfully, and many fear it will happen again. however, this effect appears minimised among donors who at least partially attributed their reaction to their own behaviour, such as poor hydration. for donors already juggling multiple demands, a vvr may tip the balance with donating becoming too much of an effort and perceived risk. however, donors appeared more confident to return if they felt supported by staff or if they could donate with family or friends. summary/conclusions: this study provides valuable insight into the vvr experience, which will aid in the improvement of donor safety and retention. the findings highlight the need to improve communication at the time of and following a vvr, to educate donors on how to reduce their vvr risk, and to intervene to help donors maintain their perceived ability to give blood in order to maximise retention following a vvr. background: frequent blood donation depletes the iron stores of blood donors. iron depletion might have negative effects on the health of the general population, but its effect on the blood donor population is not well known. aims: to investigate the iron status of finnish blood donor population and how it relates to donor health, the finnish red cross blood service set up the findonor , study in . we investigated whether there were changes in donors' selfrated health and if these possible changes could be associated with differences in iron biomarkers (ferritin and soluble transferrin receptor -stfr) or hemoglobin levels during the first study visit. methods: participants were recruited in three donation sites in the capital region of finland between may and december . participants filled out an electronic questionnaire about their health and lifestyle at the donation site during their enrollment visit. participants were asked by letter to fill out the same questionnaire electronically during the summer . we included the participants ( men and premenopausal and postmenopausal women) who completed both health questionnaires. to evaluate self-rated health we used the well-validated single question: "how would you rate your health in general?". participants were able to evaluate their health status on a five-point scale: excellent, very good, good, moderate, and poor. iron biomarkers and venous hemoglobin were measured from blood samples collected at the first study visit. we first computed the odds-ratios of reporting poorer health depending on demographic group. we then compared iron biomarker and hemoglobin levels between donors who reported improved, similar or poorer health rating. results: donors who rated their health in the first questionnaire as moderate (n = ), good (n = ), very good (n = ) or excellent (n = ) health tended to report improved ( %), similar ( %), similar ( %) or poorer ( %) health ratings respectively in the second questionnaire. pre-menopausal women reported their health poorer in the second questionnaire compared to the first questionnaire more often than post-menopausal women (pre-menopausal %, post-menopausal women %), or = . % ci . - . ). there were no differences between other groups. there were no significant differences in iron biomarkers levels (ferritin and stfr) or hemoglobin levels between donors whose health ratings were improved, similar or poorer. summary/conclusions: in this cohort, pre-menopausal women rated their health poorer at the end than at the beginning of the study more often than post-menopausal women. no association was found between changes in self-rated health and iron levels (ferritin, stfr) or hemoglobin levels. further studies about the factors relating to blood donors' self-rated health need to be carried out. background: in recent years, the blood donation business has made great achievements, but it still cannot avoid the occurrence of adverse reactions to blood donation which not only brings certain obstacles to the blood donation work, but also affects the enthusiasm of blood donors. aims: to understood the causes and other relevant factors of adverse reaction among blood donors, the information of blood donors at dai autonomous prefecture of xishuangbanna were analyzed in . methods: the data of volunteers from january to december were analyzed. the causes of adverse reactions were classified, and the incidence of adverse reactions was compared in terms of gender, frequency, age and blood type of blood donors. results: there were blood donors in , ( . %) of whom had adverse reactions and causes were induced, among which mental stress was the most common factor that accounted for . % ( cases). there was no significant difference in the incidence of adverse reactions between men and female (p > . ). from the frequency of blood donation, the incidence in the first donor was significantly higher than that in the second donor (p < . ). when it comes to age, the incidence was different and the - age group was the highest ( . %). among different blood group donations, there was no significant difference (p > . ). summary/conclusions: adverse reactions of blood donation is closely related to the psychological state and age of the blood donors. the staff of the blood center should further optimize the service, strengthen the communication and publicize the knowledge of blood donation. the ultimate goal is to increase the blood donation rate on the basis of reducing adverse reactions. background: blood loss due to repeated blood donation can lead to iron deficiency or anemia, but currently there is no management plan for the prevention of iron deficiency in korean blood donors. female and male donors are required to wait at least weeks between blood donations in korea, which is the shortest period among all northeast asian countries. female and male donors are allowed to donate whole blood up to five times per year and platelets up to times per year (if spaced more than days apart for the latter) due to the chronic blood supply shortage. these facts induce concern about the impact of blood donations on the donors' iron status. aims: this study aimed to evaluate the effect of oral iron supplementation in repeat donors based solely on donation history. methods: the high-risk group included male donors with ≥ whole blood donations or plasmapheresis or plateletpheresis donations, and female donors with ≥ whole blood donations or component donations, both within the previous year. the control group consisted of first-time or reactivated (ft-ra) donors who had no history of blood donation in the past years. the hemoglobin (hb) level, ferritin level, total iron binding capacity (tibc), transferrin saturation, and soluble transferrin receptor (stfr) of repeat donors at high risk for iron deficiency were compared to those of ft-ra donors. iron deficient erythropoiesis (ide) is defined as present if the log of the ratio of soluble transferrin receptor to ferritin was ≥ . . the repeat donors took iron supplements for weeks and the same tests were repeated after and weeks to evaluate their effects and the side effect and compliance was assessed. results: a total of male and female repeat donors were recruited, and each male and female ft-ra donors were recruited to the control group. after week iron supplementation, among male donors, the prevalence of: low hb level (< . g/dl) decreased from . % to . %; low ferritin level (< . ng/ml) decreased from . % to . %; high tibc level (> lg/dl) decreased from . % to . %; low transferrin saturation (< . %) decreased from . % to . %; and ide (stfr/ferritin ≥ . ) decreased from . % to . %. among female donors, the percentage of: low hb level (< . g/dl) decreased from . % to . %; low ferritin level (< . ng/ml) decreased from . % to . %; high tibc level (> lg/dl) decreased from . % to . %; low transferrin saturation level (< . %) decreased from . % to . %; and ide (stfr/ferritin ≥ . ) decreased from . % to . %. in total, male ( . %) and female ( . %) blood donors reported undesirable side effects related to iron supplementation. a total of male ( . %) and female ( . %) blood donors were administered iron supplementations for days. participants ( . %) answered that they were willing to take a complimentary iron supplementation. summary/conclusions: ferritin level, considered a reliable indicator of iron status, increased and ide decreased significantly after iron supplementation in female donor group, but not in male donor group, compared to the ferritin levels and ide of control donors. iron supplementation in repeat donors at a high risk of iron deficiency was shown to reduce their risk of iron deficiency or anemia irrespective of gender; however, -week oral iron supplement was not enough to restore iron storage level in the male donor group. background: c-reactive protein (crp) is an acute-phase protein and a non-specific maker of inflammation and tissue damage produced by the liver. several prospective epidemiologic studies have demonstrated that high-sensitivity c-reactive protein (hs-crp) is a predictor of future coronary events among apparently healthy men and women, hs-crp level greater than mg/l has been independently associated with a % excess risk in incident of coronary heart disease (chd) as compared with levels less than mg/l. frequent blood donation has been associated with a lower incidence of coronary artery disease (cad); however, there is a dearth of information on serum levels of crp in the nigerian donor population. aims: to investigate whether regular blood donation is associated with lower serum hs-crp level in nigerian blood donors. methods: a descriptive cross-sectional study carried out to measure serum levels of high sensitive c-reactive protein (hs-crp) and ferritin among blood donors attending the donors' clinic in lagos university teaching hospital (luth). subjects who did not meet criteria for blood donation were excluded. additional data on sociodemographic characteristics was collected using interviewer-administered questionnaire. serum ferritin was analysed using chemiluminescent microparticle immunoassay performed on the abbott architect ci (abbott laboratories, abbott park, il, usa). serum concentration of hscrp was estimated by immunoturbidimetry method using analytical kits from erba diagnostics mannheim gmbh in semi-autoanalyzer (xl , erba mannheim). data was analysed using stata version (stata corp) statistical software. results: in total of blood donors, ( . %) were males and ( . %) were females, the mean age was . ae . years. two hundred and thirty four ( . %) were first time donors and ( . %) were regular donors, serum levels of hs-crp was slightly higher in regular donors compared to first time donors ( . ae . vs . ae . mg/l, p = . ) though the difference was not significant. serum levels of ferritin was significantly higher in first time donors compared to regular donors ( . ae . vs . ae . ng/ml, p = . ). interestingly, levels of serum hs-crp were significantly higher in male than female population ( . ae . vs . ae . mg/l, p < . ) and smokers than non-smokers ( . ae . mg/l vs . ae . mg/l, p = . ). correlation analysis showed no correlation between serum hscrp and serum ferritin levels in both categories of donors while there was a weak positive correlation between hs-crp levels and white blood cells among the first time donors. summary/conclusions: this present study did not reveal any decrease in baseline levels of serum hs-crp with regular blood donation; smoking status and gender were however associated with an increase in baseline hscrp. this finding suggests that hs-crp level might not be a useful marker of future coronary events in healthy blood donors in nigeria. background: because the blood donation removes mg of iron from the donor, iron deficiency, frequently occurs in regular blood donors leading at a long term to the anemia. aims: to determine the effect of blood donations on ferritin levels in regular blood donors. methods: all prospective donors have been submitted to a physical examination and a health history assessment intended to ensure that the prospective donor is in a good general health and eligible to donate blood. the acceptance criteria are: • hemoglobin > or = . g/dl for male and > or = . g/dl for female • inter donation interval = days • donations/year for male and /year for female all eligible donors and deferred donors for all reasons except for low hemoglobin who accepted to enroll in this study and signed a consent. in addition to the medical exam, two samples have been collected one for cbc and another for ferritin. donation history, sex, age and weight have been documented. results: first time and regular donors accepted to enroll in this study. only female donors ( . %) participated to this study. . % of the participants were first time donor. % of male and % of female frequent donors are iron deficient out of male blood donors were iron deficient ( %) with serum ferritin < ng/ml. . % were repeat donors. out of female donors were iron deficient ( . %) with serum ferritin < ng/ ml, all were repeat donors. . % of repeat donors were iron deficient / of the deplete donors were first time donors summary/conclusions: frequent blood donors have higher prevalence of iron deficiency than first time donors. female donors have a slightly higher prevalence of iron deficiency than male donors. prevalence of iron deficiency in abu dhabi donor population is lower than the published data. changes need to be done on: increase inter donation interval or restrict the total number of allowable donations in a -month period for whole blood and red cells modifying donor hemoglobin requirements testing for serum ferritin iron supplementation donor education abstract withdrawn. background: haemovigilance procedures aim to guarantee not only the safety of the recipients of blood and its components but the safety of the donors as well. every adverse reaction that occurs during the donation of blood or its components can potentially be a threat to the health of the donor which can subsequently lead to the decision of the donor to resign from donating blood. aims: the aim is to analyse the type and the frequency of occurrence of adverse reactions among the donors donating blood or its components independently of the method of the donation. methods: we have analysed the number of collected donations and the number of adverse reactions in the years - in the group donors of aged - . we have specified following adverse reactions: vasovagal response without fainting, vasovagal response with fainting, vascular reactions (bruises) and other (e.g. allergic reaction to the anticoagulant, loss of blood pressure due to hypovolemia). the analysis was made using data obtained from computer system blood bank which is in operation in blood center in pozna n, poland. results: in years - the total number of adverse reactions among the donors was recorded which is . % of the total number of collected donations. % of the adverse reactions occurred in the group of donors aged - . vasovagal response without fainting was the most common adverse reaction in the total number of reactions and totalled . % of all adverse reactions. in the group of donors ages - it totalled % of all adverse reactions. the second most common type of adverse reactions was vasovagal syncope that totalled . %, in the analysed group of donors . %. vascular reactions (bruises) totalled . % of all adverse reaction, in the analysed group . %. the remaining adverse reactions totalled . %. summary/conclusions: . vasovagal reactions (with and without fainting) were proved to be most common adverse reactions in the group of donors aged - i.e. in the groups of donors just starting to donate blood. it seems reasonable to continue with further research into the reasons for the occurrence of this psychosomatic reactions. . it seems beneficial to provide constant educational activities of young donors regarding the preparation for the process of donation of blood and its components (proper nourishment, hydration as well as planning the time for scheduled donation long enough for a safe and pleasant procedure. . it seems beneficial to provide constant training for the medical staff involved in the process of donation regarding active observation of donors, proper conduct in the situation when the adverse reactions occur during the blood donation, ways to minimize the fear of donors, effective communication with the donors (explaining the process of blood donation, proper behaviour after the donation e.g. avoiding physical exercise or straining the arm). blood products -blood processing, storage and release background: the accumulation of microvesicles (mvs) in rbc concentrates during storage may be responsible for clinical symptoms such as inflammation, coagulation, and immunization. aims: our aims was to determine whether any of cd molecules responsible for important functions are present on the microvesicles, and if their expression level is dependent on the storage period of rbc units. additionally, by using cytometric analysis and phagocytosis visualization in a confocal microscope, we examined the interactions of donor monocytes with erythrocyte microvesicles, depending on their time of storage. methods: erythrocyte microvesicles were isolated from "fresh" ( nd day) and "old" ( nd day) stored rbc units. qualitative and quantitative cytometric analysis of these membrane structures was performed using the annexin v-fitc, anti-cd a-pe antibody, and calibrated beads. the microvesicles were also visualized under a confocal microscope. the expression of the molecules cd a, cd , cd , cd , cd , and of phosphatidylserine was analysed using flow cytometry. measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. results: the analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about . lm in the "fresh" and "old" blood samples. we observed a statistically significant increase in the number of microvesicles in the "old" units ( ae mvs per ll), as compared to the microvesicles in the "fresh" ( ae mvs per ll). at day , the microvesicles had elevated expression levels of cd and reduced expression levels of phosphatidylserine. significant changes were also observed in the case of cd and cd molecules. the expression of these molecules of vesicles isolated from "fresh" rbcs was lower than in the case of -day vesicles. the phagocytosis index was significantly higher ( . %) for the microvesicles isolated from -day stored rbcs than for microvesicles from the - background: platelet concentrates (pcs) are conventionally stored at room temperature with a limited shelf-life of - days. alternative storage methods, such as cold storage and cryopreservation are attractive options due to the potential for extended storage, reduced bacterial growth and improved hemostatic function. cryopreservation of human pcs has been associated with formation of more microparticles and elevated procoagulant activity compared to liquid-stored (room temperature-and cold-stored) pcs. microparticles are submicron plasma membrane particles that have been postulated as potential mediators of adverse transfusion outcomes. similarities in the size and storage-related changes up to days suggest that sheep may be a suitable model in which to investigate the effects of pc transfusion. previous research has established that room temperature stored sheep pcs contain fewer microparticles than human pcs. however, nothing is known of the effect of other storage conditions. aims: this study aimed to determine whether cold storage and cryopreservation contribute to variation in concentration and size of sheep platelet derived microparticles compared to conventionally stored sheep pcs. methods: sheep buffy coat derived pcs in % plasma/ % ssp+ were prepared with minor modifications to standard procedures for preparation of human pcs. sheep pcs were split into units (n = of each) on day and stored either at room temperature (rt; - °c with agitation) for days, cold stored for days ( - °c no agitation) or cryopreserved (À °c with the addition of - % dimethyl sulfoxide) for - days and sampled post-thaw. platelet supernatant, prepared by double centrifugation, was stored at À °c. the mean size and concentration of microparticles were measured using nanosight ns nanoparticle tracking analysis system (malvern instrument). results are mean ae standard deviation. storage associated changes overtime were determined using a one-way analysis of variance with bonferroni's post-test. paired t-tests were applied to determine the effect of cryopreservation. a p-value of < . was considered significant. results: at day , sheep pcs had a microparticle concentration of . ae . microparticles/ml with a mean size of . ae . nm. storage duration at rt sheep pcs was not associated with significant changes to microparticle concentration or size. cryopreservation of sheep pcs significantly increased the concentration ( . ae . microparticles/ ml; p = . ) and the mean size ( . ae . nm; p = . ) of microparticles post-thaw. the mean size and concentration of microparticles in the cold-stored pcs at day was comparable to room temperature pcs stored for days ( . ae . nm vs. . ae . nm; p = . and . ae . microparticles/ml vs. . ae . microparticles/ml; p = . respectively). summary/conclusions: cold storage of sheep pcs did not impact formation of microparticles over the days storage period; however, cryopreservation increased microparticle concentration and the size post-thaw. further investigation is required to determine whether these findings are influence hemostatic function. a pre-clinical sheep model of cold-stored and cryopreserved pc transfusions can facilitate mechanistic studies and complement clinical trials. background: during storage, the properties of rbc in storage solution change ("storage lesion"). for instance, ph, atp and , -dpg concentrations decrease upon prolonged storage. these changes can affect oxygen delivery by the cells. the capacity to deliver oxygen is defined as p : the oxygen tension (po ) at which % of the hemoglobin is saturated with o . an oxygen dissociation curve (odc) represents the non-linear relationship between saturated hemoglobin and po . this relationship is dependent on temperature, ph, pco and , -dpg. due to changes in these factors, the curve will shift along the x-axis. in whole blood, p is at a po of about mm hg. not much is known about p of rbcs in storage solution, and the changes during storage. aims: to determine the oxygen dissociation of rbcs stored in standard red cell additive solution sagm and in pagggm (an experimental red cell additive solution, transfusion. ; : - ). methods: rbcs were prepared in sagm (n = ) or pagggm (n = ). pagggm is designed to better maintain both atp and , -dpg during storage. rbcs were stored at - °c and sampled on day , and for (internal) ph, atp, , -dpg and p . p was determined by hemox analyzer (tcs scientific corp.). the principle of the hemox is based on the measurement of spectrophotometric properties of hemoglobin at different oxygen pressure. rbc samples were brought from oxygen-rich environment to oxygen-poor environment ( %) using n gas. p was determined from the obtained odc. results: the whole storage period, ph i of pagggm-rbcs was higher compared to sagm-rbcs. , -dpg content of sagm-rbcs decreased during storage and was below the detection limit after day . , -dpg content of the pagggm-rbcs increased the first days of storage and slowly decreased from day on. at day , pagggm-rbcs still contained . -dpg ( . lmol/g hb). p values decreased during storage from mmhg at day to mmhg at day for sagm-rbc and from mmhg to mmhg for pagggm-rbc. p values of pagggm-rbcs were higher during the entire storage period. summary/conclusions: during storage, the p decreased in all rbcs. the p was higher for the pagggm-rbcs during the whole storage period. the higher p in pagggm-rbcs seems to correlate with the higher , -dpg content in these cells. background: in belgium % of the platelets are pathogen inactivated (pi) and legislation requires a minimum platelet content of . per platelet concentrates (pc). therefore routine pools are produced with buffy-coats (bc). facing increased demand of pc and stable to slightly declining red blood cells (rbc) demand, production of whole blood (wb) derived platelets must be adapted to switch flexibly from to bc per pool. this dual pooling strategy should allow alignment between wb collection forecast, pc inventory, pc demand and pc production. aims: first develop a pooling procedure with bc and ml platelets additive solution (pas) instead of ml for bc, without changing the settings of our wb separators and platelets separators. maintain a content of ≥ . platelets with a ratio plasma/pas between to % required for pi. after validation, deploy a dual pooling strategy ( or bc/pool). methods: wb is collected with top and bottom kit (composelect; fresenius kabi) and separated (macopress; macopharma) to produce ml bc with % haematocrit (htc) and > % platelets recovery with average platelets content of . random bc are pooled with ml or bc are pooled with ml of pas-e, platelets are then extracted on tacsi pl (terumo bct) and pc are treated for pi (intercept blood system; cerus). each pc is sampled and platelet content is determined (abx pentra xl ; horiba). results: during the study bc were processed into pools ( ( . %) with bc and ( . %) with bc). before tacsi separation, bc mixture with pas-e had volumes of ae ml ( bc) and ae ml ( bc) with respectively htc of ae % and ae %. the plasma/ pas ratio was ae % in both cases. tacsi separation was performed with one same program for both types of pools. after pi, platelets content of the pools was . ae . with bc and . ae . with bc (average ae standard deviation). pools below the limit of < . were / ( . %) with bc and / ( . %) with bc. the platelets concentrations ( /ll) were ae ( bc) and ae ( bc). platelets recovery was % ae for bc and % ae for bc. summary/conclusions: bc could theoretically produce pools of bc or pools of bc. this means a maximum potential gain of + % pc. in practice during shortage periods we switched from to bc when dictated by the actual inventory levels and hospital needs. the advantage of this dual pooling strategy was a gain in production capacity to cover these shortage periods ( pc, + %). the disadvantage of pooling randomly bc is that pools contained less than . platelets per pool potentially limiting their usage to low weight or paediatric patients. a preselection of the bc based on platelet count could optimize the bc pooling procedure. background: apheresis-derived platelet concentrates (apcs) is a standard medical therapy indispensable to contrast bleeding or hemorrhage. however, bacterial infection caused by storage at room temperature (rt) still remains the major drawback. recently, we showed that cold-stored apcs are associated with better plt functionality but with accelerated clearance (haematologica , pmid: ). cold-induced apoptosis was identified as a potential mechanism of the shorter plt survival aims: to investigate the protective effect of apoptotic inhibitors during cold storage of apcs methods: apcs were collected and stored at rt and °c in the presence or in the absence of caspase- inhibitor. the phosphatidylserine exposure and the mitochondrial membrane potential (mmp) (tetramethylrhodamine ethyl ester perchlorate [tmre ] staining) were measured using flow cytometry. the protein expression was quantified by western blot results: a higher expression of the apoptotic marker phosphatidylserine was detected in cold-stored apc compared to rt (% apoptotic events meanaesem: ae % vs. ae % p = . ). to verify if the apoptotic signal, observed with phosphatidylserine, specifically involved the intrinsic pathway, the mmp was analyzed as a marker of alive cells. interestingly, after cold storage a decrease of the mmp was observed compared to rt indicating the activation of the intrinsic pathway (mean fluorescence intensity tmre meanaesem: . ae . vs. . ae . , p = . ). accordingly, a decrease of the procaspase- level after cold storage was detected by western blot analysis. however, when plts were stored in the presence of caspase- inhibitor a significant rescue of the cold-stored cells viability was observed (tmre staining: % alive cells meanaesem: ae % vs. ae %, caspase inhibitor vs. ionomycin, p = . ). this indicates that the activation of the apoptotic pathway, induced during cold storage, can be prevented using caspase inhibitor summary/conclusions: our results show that the reduction of cold-stored plt viability can be prevented by a specific caspase inhibitor. consequently, cold storage, associated with a better plt functionality, may become an efficient strategy for apc storage in combination with apoptotic inhibitors background: gamma-irradiation is used to treat red blood cell (rbc) concentrates (rccs) for patients who are immunosuppressed. this treatment is known to damage rbcs and to increase storage lesions. one of the causes of the storage lesions is the presence of oxygen. several studies have shown, based on different strategies to reduce o , a reduction of storage lesions related to metabolism, protein modifications and cell morphology. aims: the present research work investigated the effect of gamma-irradiation on rccs stored under normal condition and hypoxia/hypocapnia. methods: saturation of o (so )-and abo-matched rccs from whole blood donations, leukoreduced and prepared in paggsm (macopharma, france) were pooled and split in two identical rccs within h post-donation. one bag (treated) was submitted to oxygen and carbon dioxide adsorption (oxygen reduction bag, hemanext, usa) for h on an orbital shaker ( rpm) at °cae and then transferred to a storage bag impermeable to gas. the other one (control) was left as it is. the two bags were then stored at °c. a g-irradiation treatment ( gy, gammacell elan, theratronics) was applied at day or and the rccs (expiry dates at day or day , respectively) were stored until day . hematological parameters, glycolytic metabolites, extracellular potassium level, antioxidant power, morphology and deformability were measured. results: starting so values were of . %ae . (n = ) in control and of . %ae . (n = ) in treated bags, and reached . %ae . and . %ae . at day , respectively. as expected, an increase in glycolysis rate was observed during deoxygenation without any influence from the irradiation. potassium levels were identical in treated and control, and reached around mm at expiry with an irradiation-dependent kinetic release. antioxidant power and deformability were identical in both conditions. no difference in hemolysis was observed after irradiation on day and the values stayed equivalent through end of storage (at day , hemolysis (control) = . %ae . , hemolysis (treated) = . %ae . , p-value > . ). when irradiated at day , hemolysis was lower (p-value = . ) in treated rccs at the end of storage (day , . %ae . ) compared to control ( . %ae . ). seven days post-irradiation, two-third of the control rccs were above the limit of . % whereas all the treated rccs remain below the limit. quantification of microvesicles and morphological analysis confirmed these data. summary/conclusions: the storage under hypoxia has a beneficial effect on rbc storage thanks to a decrease in o content and to an improvement of metabolism. this benefit provided equivalent storage when rccs were irradiated at day and was an advantage when irradiated at day . importantly, the results show that combining irradiation with hypoxia/hypocapnia retained the improved hemolysis profile of o depleted rbc. in summary, the reduction of o level in rccs enables a better storage of rcc when a late irradiation is applied. background: in vitro blood circuit machines require a constant monitoring of blood flow rate which have to be maintained at a constant value. also, measuring the hematocrit of flowing blood in such machines is essential for performing real-time diagnostics. recently, acoustophoresis has emerged has a promising blood separation technology capable of replacing centrifugation for the preparation of platelet concentrate. to avoid damaging blood cells, the technique is used without infusing pumps thus increasing the need of flow monitoring. however, acoustophoresis chips performs at low flow rates, outside the range of available commercial flow meters. in addition, hematocrit measurement is of a particular interest for acoustophoresis since it is a direct indicator of the separation efficiency. aims: in this study, we present a straightforward doppler ultrasound system designed for measuring blood flow rate and hematocrit in an acoustophoresis chip [bohec et al, platelets, ] . we show that the stability of the in vitro environment can be used to obtain high level of accuracy of the doppler method using a basic and low-cost experimental set-up. this improvement allows a precise measurement of flow rates as low as . ml/min in sub-millimeter tubing. furthermore we evaluate the capability of the system to measure hematocrit of human blood samples coming from different donors. methods: the experimental set-up was constituted of an ultrasonic continuous wave doppler probe mounted on a d printed support. the accuracy of flow rate measurements between . ml/min and . ml/min was evaluated as well as the optimal measurement time. for different blood bags, the relationship linking the total energy of doppler signals and hematocrit was derived. hematocrit in a range under % was estimated from doppler signals for each blood bag. results: the system is able to acquire exploitable doppler signals for the whole flow rate and hematocrit range. flow rate estimation from the signals shows a high accuracy with a mean measurement error under % for a measurement time of s. the mean error is still under % for a measurement time of . s. hematocrit estimation from doppler signals shows a good linear correlation with reference measurements for bags , and . hematocrit estimation for bag diverges from reference for values above %. summary/conclusions: the proposed doppler ultrasound system is capable of measuring low blood flow rate in narrow medical tubing with a high accuracy. it is particularly suited for an acoustophoresis device but the versatility of the system makes it easily applicable to any in vitro blood circuit. we furthermore demonstrated that the system can be used for measuring hematocrit under % without additional developments. this finds interesting applications in blood sorting technologies but also demonstrates that doppler ultrasound is a potential simple and low cost method for measuring hematocrit of flowing blood in vitro. background: hereditary hemochromatosis (hh) is the most common genetic disorder in populations of northern european descent manifesting with high levels of storage iron (ferritin) in blood and tissues. the standard treatment is serial therapeutic phlebotomy to decrease iron overload. the collected blood is frequently discarded but some blood banks allow "healthy" hh patients to donate blood for patient use. red cell concentrates from hh donors have been reported safe for transfusion, but little or no data is available on platelet concentrates from hh donors, including the potential contribution of surplus iron to the "platelet storage lesion". aims: the aim of this study was to compare platelet quality, activation and aggregation over seven-day storage in platelet-rich plasma from patients with newly diagnosed hh and from healthy controls. methods: whole blood ( ml) was drawn into compoflow blood bags containing cpd and sag-m from healthy controls and newly diagnosed hh patients. platelet-rich plasma (prp) was prepared from whole blood and split into four compo-flex bags each containing ml prp (range - platelets/l). platelet quality tests were performed on days , , , and of storage. platelet aggregation was tested using a chrono-log aggregometer and four agonists (adp, arachidonic acid, collagen, and epinephrine). platelet expression of cd , cd b, and cd p was measured with flow cytometry while ph and metabolites were measured with a blood gas analyzer. scd l and scd p in the supernatant were quantified using enzyme-linked immunosorbent assays. results: both hh and control groups included males and females. the mean age was significantly lower in the control group, years ( - years), than in the hh group, . years ( - years) (p = . ) while ferritin levels were significantly higher in hh patients (median . , range - ) than in controls (median . ng/ml, range . - ng/ml) (p < . ). in the hh group, each had the c y/c y and c y/h d genotypes. results of prp quality control tests were comparable between the two study groups over seven days of storage (p < . ) with the exception of glucose (higher in hh patients on all time points, p < . ). platelet aggregation and the expression of activation markers (cd p and cd b) on platelets and in the supernatant (scd p and cd l) were comparable between hh and control prp units over all seven days of storage. the analysis revealed comparable and expected alterations in metabolic and platelet activation markers over seven-day storage in both groups. ph increased, glucose decreased, and lactate increased over time while cd b expression decreased and cd p increased. platelet aggregation responses decreased during storage but to a varying degree depending on the agonist, however, the decrease was comparable in cases and controls. summary/conclusions: these results suggest that high iron stores in hh do not adversely affect the quality of platelet units produced from hh patients. furthermore, the data also suggest that blood from hh patients, including platelets, can be donated for patient use. background: platelets are often shipped over long distances from collection centres to blood processing centres and subsequently to hospitals. platelet agitation facilitates oxygen transfer, thus promoting aerobic metabolism, and maintaining platelet ph. during shipment, platelets cannot be agitated continuously, which may promote anaerobic metabolism. previous studies have examined the effects of prolonged periods without agitation on apheresis platelets collected in plasma, but not platelets in platelet additive solution (pas). it is therefore important to determine whether platelet quality and function are maintained during prolonged transport or hold time in a shipper. aims: the aim of this study was to evaluate the effects of prolonged storage without agitation on the in vitro quality of apheresis platelets in pas. methods: triple dose apheresis platelets (n = ) were collected using a trima accel platform in % plasma/ % pas (ssp+). after resting for h, platelets were split equally into three components, packed into a shipper and transported immediately to the blood centre. upon arrival, one of the platelet components was removed (< h; t ), and the others remained within the shipper, without agitation. the second component was removed at h post-collection (t ), and the third was removed at . h post-collection (rounded up to h; t ). platelets were tested on day , and post-collection and in vitro quality and function were monitored. data were analysed using a two-way repeated measures anova, where a p-value of < . was considered significant. results: platelets held without agitation for h consumed significantly more glucose than those removed at h or immediately upon arrival (p < . ), even on day post-collection. this was accompanied by increased lactate production (p < . ), indicating increased anaerobic glycolysis. consequently, the ph was significantly lower in t platelets (p < . ), and on average it was . ph units lower than in platelets held in the shipper for h or less. however, the ph remained above . in all components. mean platelet volume was also reduced in t platelets (p < . ), suggesting acceleration of the platelet storage lesion. phosphatidylserine exposure, surface expression of cd p and microparticle generation were significantly higher in the t platelets throughout the storage period (all p < . ), suggesting platelet activation. release of scd p was also increased in t platelets (p = . ), whereas extended storage in a shipper did not affect release of rantes (p = . ). adp-induced activation of glycoprotein iib/iiia, measured by pac- binding, was decreased in t platelets (p < . ), indicating reduced platelet responsiveness to agonist stimulation. additionally aggregation in response to collagen (p = . ) and adp (p = . ) were significantly lower in t platelets, suggesting a decrement in platelet function after prolonged storage without agitation. summary/conclusions: significant in vitro changes were observed in platelets held without agitation for h. these results suggest that the length of time that platelets are held in a shipper should be minimised where possible. background: the shelf-life for platelet products has been restricted to days. this very limited window of time is intended to sustain the quality of platelet and to reduce the risk of bacterial growth. we have recently demonstrated that in suitable platelet bags, the platelet product quality remains high after days of storage. this was proved by examining in vitro, the quality parameters of platelets such as platelet concentration, glucose, ldh, and ph (alexopoulos k. et al., haema, , ) . our new target is to extend this research in extra days of storage. we also want to determine if there is any bacterial development in this period. aims: the goal is to investigate the capability of storage period for platelet units, from to days. methods: in this study, platelets were collected from normal blood donors in the blood bank department of general hospital of patras "agios andreas". a total of ae ml of whole blood was drawn into triple cpd/sag-m top-top bags blood container systems, lmb technologie (gmbh). the platelet concentrates were prepared by platelet rich plasma (prp) method and then they were placed in a platelet incubator with agitator (helmer pc ). samples were drawn aseptically with a needless access coupler (cair-lgl) on days , , and . platelet count was done by ceeldyn ruby (abbott all data shown are reported as mean ae standard deviation (sd). the swirling effect remained positive (+) during the seven days storage period. the bacterial screening was found negative. summary/conclusions: platelet concentration in the bag remained constant between day and day , maintaining platelet yield. the decrease in glucose and increase in lactate, along with the decreased ph, show that the platelets remain metabolically active between days and of storage. the ph remained well within the acceptable range. no bacterial contamination was reported. thus, we conclude that platelet concentrates in these specific bags may be used with an extended shelf life of days. further studies are needed with other platelet bags to confirm our hypothesis. abstract withdrawn. aims: we introduce rt-dc as a fast, robust and unbiased quality control tool for pc, rcc and hpsc. utilizing the interdependency between cell deformation and the molecular state of the cytoskeleton, we demonstrate that rt-dc is capable to assess the quality of blood products. methods: by rt-dc we assessed: i) platelets after storage at °c or room temperature (rt) over days for apheresis pcs in addition to standard in vitro platelet function assays; ii). red blood cells before and after gamma irradiation in addition to hemolysis; and iii) hpsc after cryopreservation with % or % dmso in addition to cell count, and in vitro viability. in addition we compared the regeneration time of patients' platelets and leukocytes after transplantation of hpsc products containing either % or % dmso. results: for pcs standard quality assurance tests did not show a major difference between °c and room temperature storage while rt-dc showed a highly significant difference between both start conditions (day - , p < . and day , p < . ). for red cells, we found by rt-dc no impact of gamma irradiation with gy over the entire storage period of days assessing different rcc. for hpsc, rt-dc showed that cryopreservation in liquid nitrogen resulted in a significant increase in deformation ( . for % dmso versus . for the control without dmso; p < . ). however, this did not differ to high extent whether % or % dmso were used for cryopreservation ( . and . , respectively; p < . ). hpsc viability was lower after cryopreservation using % dmso in comparison to using % dmso. overall, blood cell regeneration is comparable between % and % dmso. summary/conclusions: studying platelet and red blood cell concentrates as well as hematopoietic stem cells under different, clinically relevant, storage conditions our results demonstrate that intrinsic material properties reveal insights into cell function and allow to predict cellular state in a robust way and using small sample volumes. in order to offer more flexibility to the production process, the storage of bcs overnight ( h) has been validated in our blood center. aims: the aim of the study was to assess the platelet quality in platelet concentrates derived from overnight stored buffy coats. methods: whole blood collected at day was separated into plasma, bc and red cell concentrates either at day or at day . bcs were then stored until the pooling step at °c without agitation and pcs were prepared at day by pooling isogroup bcs. seven " h-pcs" were prepared from bcs stored for h (whole blood separation at day ) and six " min-pcs" from bcs stored for min (whole blood separation at day ). standard quality control measurements were performed during the process and the storage. in addition, the quality of the platelets into the prepared pcs was assessed throughout the period of storage by measuring the hypotonic shock response (hsr) and by measuring by flow cytometry the proportions of platelets in apoptosis (marked with annexin v), of functional platelets (marked with cd ) and of activated platelets (marked with cd the changes observed during the -h storage period appear to be limited and compatible with a further pr process using a photochemical treatment (amotosalen and uva) with intercept. summary/conclusions: leukocyte-depleted "double dose" buffy coat platelets with a high platelet content and ready for pathogen reduction can be obtained with the ipp pooling and leukodepletion set developed by kansuk. a storage period of h before applying the photochemical treatment is feasible without significantly altering the biological quality of platelets. methods: dd-bc-pc were prepared with bc and ml of pas (intersol, fresenius kabi (germany) are sterile docked to the octopus harness and combined into a ml pooling bag. the pool is centrifuged and the pc supernatant expressed through a bioflex cs leukodepletion filter into a temporary platelet storage container. the obtained dd-bc-pc were tested within h of preparation and after storage for h in the platelet storage container for volume, platelet content, residual leukocytes (wbc), plasma ratio and biological parameters, ph, po , pco , glucose, lactate, mpv, ldh, p-selectin and swirling. results: the platelet content of dd-bc-pc (n = ) was on average . ae . . in a volume of ae ml. the mean of plasma ratio was % [min: . max: . ]. all pc contain < . wbc [min: <lq; max: . ]. po decreased from ae mmhg after separation ( h) to ae mmhg after h, ph (+ °c) and pco was stable during the same time period. glucose decreased from . ae . mmol/l ( h) to . ae . mmol/l ( h) while lactate increased from . ae . mmol/l ( h) to . ae . mmol/l ( h). the vpm was stable with . ae . at h and h. ldh increased from . ae . u/l at h and . ae . u/l at h as p-selectin . ae . ng/ml at h and . ae . ng/ ml. all units have maintained swirling. the changes observed during the -h storage period appear to be limited and compatible with a further pr process using a photochemical treatment with intercept. summary/conclusions: leukocyte-depleted "double dose" buffy coat platelets with a high platelet content and ready for pathogen reduction can be obtained with the platelet pooling and leukodepletion set developed by fresenius. a storage period of h before applying the photochemical treatment is feasible without significantly altering the biological quality of platelets. background: the irish blood transfusion service (ibts) is the statutory body with the responsibility for ireland's national blood supply. in the ibts collected , whole blood units and performed , platelet apheresis procedures. blood is collected in three fixed and six mobile sites and is returned overnight in temperature controlled vans to a single processing site. in september , the ibts implemented the tacsi automated system for the preparation of buffy-coat derived platelets as an alternative to the orbisac system. aims: the aim of this study is to compare operational aspects and quality data of buffy-coat derived platelets prepared by the tacsi (t-pcs) and orbisac (o-pcs) automated systems. methods: qc data of t-pcs and o-pcs from years to were analyzed. in , the ibts prepared , orbisac platelet pools of which , were qc tested. in , orbisac and , tacsi platelet pools were prepared and tested. in , tacsi platelet pools were prepared of which were qc tested. qc data comprised pc volume, platelet content ( /unit), residual leucocyte content ( /unit) and ph at expiry. feedback from the processing laboratory operators was collected comparing operational aspects of preparation of t-pcs and o-pcs. results: from the , o-pcs tested in and , mean pc volume was ae ml. initially the t-pcs mean volume was ae ml but more recently the volumes were increased by addition of a larger volume of additive solution to ae ml. platelet content ( /unit) was significantly improved in t-pcs ( ae ) compared to o-pcs ( ae ) [p < . ] as well as residual leucocyte content ( /unit) which was lower in t-pcs ( . ae . ) compared to o-pcs ( . ae . ) [p < . ]. ph at expiry was lower in t-pcs ( . ae . ) compared to -pcs ( . ae . ) but remained within the quality requirements. from an operational perspective with two tacsi devices, five operators produce approximately t-pcs per hour compared to a lower throughput of o-pcs per hour with five orbisac devices. the tacsi system is more compatible with batch processing which aligns with our staffing arrangements in routine production and has resulted in greater productivity since its introduction. with tacsi implementation, we reverted to manual pooling of buffy coats and new staff had to undergo training to achieve optimal platelet recovery. once staff were trained during validation, we observed a % increase in platelet recovery. processing laboratory staff indicated that tacsi devices were easier and simpler to maintain in comparison to orbisac, but highlighted that some improvements to tacsi system boxes could be made to make the system more robust. summary/conclusions: pcs prepared with tacsi and orbisac automated systems both met national and european quality requirements, however we observed a significant improvement in quality markers of pcs prepared with tacsi. processing staff reported easier maintenance and processing with tacsi compared to orbisac. throughput is higher with tacsi and there is greater additional capacity to increase production in the future if required. a perez aliaga , f puente , a aranda , j domingo , l call en and a bah banco de sangre y tejidos de arag on, aragon, spain terumo bct europe n.v., zaventem, belgium background: the blood bank and tissues of arag on (bsta) is responsible for the collection, processing and distribution of blood in the region of aragon in spain. with five fixed and five mobile sites, the bsta collects and processes , whole blood units per year. to improve quality of processed units and working conditions of personnel, the bsta decided to implement the reveos automated system. validation studies were carried out in november and routine use started in july . in parallel, the bsta initiated pathogen inactivation of platelet concentrates (pc) in november to improve transfusion safety. in november , the mirasol prt system was implemented due to simplicity of this platform. aims: the aim of this abstract is to (i) describe the results of the validation studies carried out during reveos implementation (ii) present routine use data of products processed with reveos and (iii) provide information on mirasol implementation. methods: in , whole blood units were processed using reveos. quality parameters for red cell concentrates (rcc), such as: hemoglobin (hb), hematocrit (hct) and volume; plasma: volume and residual platelet content; and pcs: volume and yield, were analyzed. a control group consisting of units processed with a semiautomated platform was used as comparison. a survey was sent to the collection and processing staff for feedback on operational aspects of the use of reveos. from to , qc data of rccs (n = ), plasma (n = ) and pcs (n = ) processed with reveos were collected. number of mirasol treated pcs and hemovigilance data from to were collected. results: volume, hb and hct of rccs processed with reveos were significantly higher compared to control rccs (volume: ae ml vs ae ml; hb: . ae . g/ unit vs . ae . g/unit; hct: . ae . % vs . ae . %). control plasma units had higher volume ( ae ml) and lower residual platelet content ( ae /l) compared to reveos plasma units (volume: ae ml; residual platelet content ae /l). for pcs, volume was higher for units processed with reveos compared to control ( ae ml vs ae ), but no statistical difference was observed in platelet content (reveos: ae /unit; control: ae . /unit). we observed either an increase or stabilization in qc parameters of rccs, plasma and pcs during the years routine use of reveos. for example, percentage of rccs units hemolyzed dropped from % to % between and and platelet content in pcs increased from /unit (first six months in routine) to /unit. plasma volume increased from ml during validation to ml in . survey results from staff highlighted the user friendliness of the reveos device. number of mirasol treated pcs increased from % in to . % in with no pathogen transmission nor serious adverse events reported following transfusion of these products. summary/conclusions: with implementation of two user-friendly systems, the reveos whole blood automation system and mirasol prt system, we observed an increased standardization of our processes enabling us to provide higher quality and safer blood products to hospitals and the patients they serve. background: white blood cells (wbc) may be regarded as contaminants in blood components and potentially reduce transfusion safety. there is currently enough evidence showing that wbc reduction through pre-storage filtration reduces the incidence of three major complications of allogeneic blood transfusions: febrile nonhemolytic transfusion reactions (fnhtr), refractoriness to random-donor platelet transfusions and transmission of cmv. leukodepletion is now mandatory in canada and several european countries. aims: the aim of this study was to evaluate leukoreduction performance and the quality of the resulting red blood cell concentrate (rbc) produced with new quadruple system top & bottom imuflex crc with an integrated red blood cells filter which was used in whole blood fractionation under routine conditions of san paolo asl blood bank methods: twenty-seven whole blood donations ( ml) were collected with using quadruple blood bags top & bottom system with an integrated rbc filter (imuflex crc, terumo bct). centrifugation and processing (using t-ace ii + -terumo bct) of whole blood units followed the blood bank sop to obtain rbcs, plasma and buffy coat. rbcs were diluted with sagm ( ml) and then filtered by gravity in average - h after blood collection to obtain leukoreduced rbc. average temperature during filtration was °c. samples were collected before and after leukoreduction for calculation of hemoglobin loss through the filtration process. filtration time was recorded. final hb, hematocrit (hct), residual platelets and white blood cells were determined after filtration using standard laboratory methods (blood cell counter) results: wb donations (n = ) were performed. rbc characteristics post-filtration and separation were as follows: volume ( . ae . ml), hematocrit ( . ae . %) hemoglobin ( . ae . g/dl) and hemoglobin per unit ( . ae . g); residual platelets ( . ae . x ³/ll); residual wbcs ( À . ³/ll). the mean filtration time was ae min and no filter blocks were observed summary/conclusions: the usability and performance of the new quadruple system top & bottom imuflex â crc with integrated rbc filter was evaluated. all quality parameters complied with italian and european guidelines and requirements. in conclusion, the set was found to be a reliable and efficient collection and separation system that performs consistently background: novel devices for automated whole blood processing (reveos tm terumo bct) have been installed in regional center for blood donation and blood treatment in katowice to modernize and automate production of blood components. this system allows to separation of four whole blood units into four buffy coat depleted red blood cell concentrates (rbc), four units of plasma and four interim platelet concentrate (ipu) units in one automatic cycle. an ipu is the starting blood component for the production of pooled, leucoreduced platelet concentrates (lpc). residual leukocytes (wbc) are obtained as a byproduct and discarded. the use of this system shortens production time because it combines otherwise two separate steps: centrifugation and blood separation in press. aims: to evaluate the quality of the blood components obtained with the newly developed reveos nlr kits, which are a whole blood collection kits containing no leucocyte reduction filter for the rbcs. methods: blood [wb] was collected into nlr reveos set (terumo bct), and processed between and h from collection using the c protocol. quality control was performed on rbc, plasma and ipu. rbc were tested for hemoglobin (hb) content, hematocrit (ht), residual white blood cells (wbc), volume and hemolysis level on the last day ( nd day) of storage. the content of wbc and platelet count (plt), protein, factor viii on the day of production and after one month of storage were determined in plasma. for ipu, platelet and leucocyte counts, volume and average platelet yield index (pyi) were displayed at the device after processing was recorded. furthermore, a comparison between fully automated and semi-automated whole blood processing was done, by comparing the amount of time necessary to fractionate whole blood units using the two different processes. background: direct, single step automatic blood separation devices (reveos tm terumo bct) was installed in regional center in katowice in . the system allows for full automation of whole blood fractionation. one of produced blood components is an interim platelet unit (ipu), containing most of the platelets and limited of white blood cells (wbcs). it is then pooled through a filter to form a therapeutic unit of leucoreduced platelet concentrate (lpc). a rough estimate of the platelet yield in the ipu, the so-called platelet yield index (pyi) is calculated and displayed by the device after the end of the separation phase. it is a useful tool to optimize selection of ipu for pools to produce lpc. aims: to determine the quality of polled lpc obtained on the basis of ipu from whole blood processed up to h after collection and the usefulness of this method in daily practice. methods: ipus used for production of lpc were obtained using fully automated whole blood processing single step system -reveos tm (terumo bct). from the beginning of the application of the method, therapeutic units of lpcs were produced after overnight hold: pools of ipus and of ipus were made using platelet pooling set (terumo bct), using ml ssp+ solution (macopharma). results: therapeutic units of lpc were analyzed. the mean volume of lpc was ae ml, platelet content was . ae . x , and white blood cells (wbc) count was . ae . x , % all lpcs had wbc below . ph measurements were made on the fifth day of storage, the ph was determined in units of lpc and was . ae . . summary/conclusions: the use of interim platelet units in daily practice increases the possibility of producing high quality blood components for patients and is a great alternative to the laborintensive manual methods of lpc's production. background: the development of cold-stored platelets has been hampered by the rapid removal of blood after transfusion by apoptosis and desialylation due to platelet storage lesions caused by refrigeration. previous studies have shown that antioxidants inhibited the activation and desialylation of cold-stored platelets for days and also inhibited rapid elimination after transfusion in animal models. aims: it was tried whether in long-term ( days or longer) storage conditions, antioxidants showed similar effects inhibiting the activation and desialylation of cold-stored platelets. methods: platelet-rich plasma (about ml) were obtained from a healthy blood donors of type o blood group and divided into a ml platelet storage bag manufactured by the manufacturer, and the conditions including room temperature and refrigerated condition and various kinds of antioxidants were prepared, (ph, glucose, lactic acid) and activation index (cd p, gpiba (cd b), annexin v), degree of reactive oxygen species and sialidase activity on the th, th and th day, and phagocytosis using the hepg cell line and platelet recovery after in vivo transfusion using scid mice were compared and compared. results: cold-stored platelets showed autoaggregation on the nd day of storage, and apparent aggregation on the fifth day of visual observation but not in the antioxidanttreated group. as expected, the ph, glucose and lactate levels of the cold platelets were maintained and the platelets at room temperature changed significantly. nac (n-acetylcysteine) -treated platelets were less active on day and sialidase activity was maintained lower than that of control group on day . the degree of phagocytosis by the hepg cell line remained at the same level during the storage period, and the in vivo recovery rate in the animal model was superior to that of the antioxidanttreated cold platelets for h and h after transfusion. summary/conclusions: cold platelets could be developed as clinically effective agents for up to two weeks if appropriate preservatives were added, and antioxidants, especially n-acetylcysteine, were effective. background: the reveos c procedure produces the following three blood components for transfusion: plasma, rbcs, and a single interim platelet unit (ipu). whole blood is collected into the reveos blood bag set and held at room temperature a minimum of h before processing on the device. after processing on the reveos device, the plasma product is ready to freeze. the rbc product is ready to combine with sag-m additive solution and leukoreduce using the integrated leukoreduction filter. the ipus are rested and agitated for the recommended time. for example, when whole blood is processed within h of collection, the ipu is agitated overnight before pooling. because the ipu is made from one unit of whole blood, approximately to ipus are pooled together and leukoreduced by filtration prior to storage in a platelet storage bag. this leukoreduced platelet pool ( units) can be supplied to the demand of hospitals like a platelet apheresis product. aims: to assess the usability of the reveos automated blood processing system to collect units of whole blood and to process the units on the reveos device, producing blood components that meet product quality specifications. methods: this study will evaluate approximately units of whole blood using the reveos system and approximately units of pooled platelet products. a total of units of whole blood (wb) have been collected into the reveos blood bag set and then held at room temperature without the use of an active cooling system (or temperature control system), such as cooling plates, or in this case, ice packs. of the units, units have been processed on the reveos device using the fresh procedure (within h of collection). nine units have been processed using the overnight wbhold procedure. all units have processed successfully without alarms, resulting in three components (rbcs, plasma, platelets). we also evaluated four platelet pools created from eighteen interim platelet units (ipus). one ipu has been discarded due to a -min whole blood collection time. the leukocyte count on all component using the automatic sysmex-xn cell blood counter. results: the red blood cells meet the criteria for hematocrit, hemoglobin and residual wbc counts are less than million per unit. the plasma units meet the criteria for displayed volume accuracy compared to measured volume and also for factor viii levels. the average residual platelets in plasma levels meet the criteria. the platelet pools meet the acceptance criteria for platelet yield and volume. limited data (n = ) suggests that this criteria will be easily met, provided that wb hold conditions are suitable for platelets. summary/conclusions: final results indicate that products produced from the reveos system meet or exceed product quality expectations. staff report that the reveos system is very easy to use. abstract withdrawn. background: thorough manual mixing of overnight-held whole blood (wb) units prior to centrifugation to separate red cell (rc) from platelet-rich-plasma (prp) is currently practised at the hong kong red cross blood transfusion service (hkrcbts). the mixing step is crucial to maximise platelet yield but laborious, and may cause elbow and wrist injuries in operators. a laboratory shaker, reax (heidolph instruments, schwabach, germany), originally designed for mixing solvents, was modified to allow mixing source wb units by the supplier. to enhance the well-being of the operators in the context of occupation health and safety (oh&s) management, feasibility of the application of such blood mixer to replace the manual mixing steps in blood component preparation was explored. aims: to evaluate the in vitro quality control (qc) parameters of the blood components prepared with the use of the modified mixer. methods: fifty-eight wb collections, including units in ml quadruple bags ( q) with/without integrated leucofilter and units in ml quadruple bags ( q), were processed in accordance with hkrcbts' prevailing component processing protocols except using the motor-driven mixer in lieu of manual mixing. wb collections were divided into groups, mixed at revolutions per minute (rpm) for min (n = ) and min (n = ), then processed into rc, prp platelet and plasma components. in vitro qc assessments including haematocrit (hct), haemoglobulin (hb) and haemolysis at expiry for rc; absolute platelet count and ph for platelet concentrates (plt); volume and residual platelet count for plasma units were evaluated and compared. results: all components derived from both q and q processed with the blood mixer fulfilled the qc requirements stipulated by aabb standards and council of europe (coe) guidelines. there were no significant differences for all the qc parameters in rc and plasma components produced when mixed for or min. summary/conclusions: the overall performance of blood components processed with the motor-driven mixer met the acceptance criteria at the hkrcbts in accordance with the aabb and coe standards. higher platelet yield was observed with prolonged mixing for min. however, prolonged mixing will much delay routine component processing. further evaluation on intermediate mixing time such as , or min may help determine an optimal mixing duration to maximise platelet yield without compromising processing workflow. to conclude, using the modified blood mixer to replace laborious manual mixing in components preparation is promising to improve the oh&s for blood component preparation operators. background: leukocyte reduction is widely used to reduce the risk of non-hemolytic febrile transfusion reactions and alloimmunization by multiple blood transfusions. around and after year , universal leukocyte reduction was adopted in many developed countries. there are mainly two types of filter, one to filter whole blood and the other to filter red cell concentrate (rcc) used during pre-storage blood processing. aims: in this study, we conducted the filtration tests with sepacell tm rs for rcc in comparison with sepacell tm r-s and sepacell tm pure rc widely used in the world. background: the cardarelli hospital blood bank processes over , whole blood units yearly. in order to constantly cope with the huge demand for platelets from oncohematology and intensive care departments, a progressive review of the processing protocols for platelet concentrates (pc) from buffy-coat (bc) pools has recently been implemented to reduce the number of units assembled, from (routine in italy) to . the -bc pc units have slightly lower volume and plasma content, factors that further limit the transfusion risk. in addition, the -bc assembly allows to facilitate the production of the pc units obtained from the same blood type, in order to minimize both the adverse reactions related to the abo/rh incompatibility and the amount of bcs not used. finally, the described innovation also impacts on the workload, with reduction of assembly, centrifugation and separation time. aims: the aim of this work is to define an optimized protocol for the preparation of pc units from -bc pools, so that the platelet yield is always higher than the minimum value required by current regulatory ( . cells/unit). background: blood is a basic component for human life and hence blood transfusion is an integral intervention in the treatment of patients. the need of blood transfusion is increasing with decreasing blood supply due to limited blood donations. conversely wastage of blood products is also a topic for discussion. to ensure the availability of blood products when they are needed, wastage of blood products should be controlled and minimized. aims: the study was designed to investigate the quantity and reasons of wastage of blood products at our hospital. methods: this was an observational study based on the statistical data available from blood bank department of nibd and bmt, pechs campus, karachi, pakistan. the study period was from february to february . study was approved from institutional review board. for all the blood products wastage and reasons of wastage were evaluated. frequencies were calculated by using spss version . results: a total of blood products were available at our institute including each of platelets, pack red cells and fresh frozen plasma(ffp). the overall wastage rate was . %. pack red cells and platelets were fully consumed yet shortage of supply was observed. however, highest wastage was observed in ffp in our blood bank i.e. ( %). results of investigating the common causes of blood product wastage at the hospital revealed that it was highly associated with common causes, including the expiry of unused products ( %) followed by broken bags ( %). summary/conclusions: this study showed over all diminutive wastage rate. ffp wastage was highest among all the blood products. wastage of blood products is a genuine issue in a hospital setup, strategies and plan of action should be discussed and implemented including enhanced collaboration with other centres to outsource the blood products or even donate at times, education and training of staff for better clinical use of blood products and ensure the availability when and where they are needed most. background: in the early s, the blood service of the republic of kazakhstan functioned along the lines of the soviet period: donation of blood was approached to the place of its use (carried out at hospitals), screening of donor blood was carried out at one stage (enzyme immunoassay) on analyzers of semi-automatic type, there was no mechanism calculating the cost of blood components, the activity of blood centers was funded at the rate of liter of whole blood. donation was not popular in society, household fears of infection were common when donated. aims: research objective is to study the main stages of reforming of blood services of the republic of kazakhstan (rok). the research methods are based on the system analysis of the condition of the rok's blood services based on the indicators of production activity for the period of - . results: in order to effectively reform the blood services, the following steps were taken. the blood supply was centralized and optimized. blood collection at hospitals was terminated, more than blood collection subjects were closed. the result was a more efficient use of technical, human and financial resources, as well as ensuring the high quality of the blood components produced. today, there are blood centers in the republicone for each of the administrative territorial units of the republic. the screening of donor blood for transfusion infection markers has been transferred into a two-step principleimmunological analysis and nat testing. a fully automated analytical equipment of a closed type is used for screening, unified for all blood centers of the republic. unification of the equipment choice allowed to provide centralized service maintenance of complex equipment and its uninterrupted operation. since , the national reference laboratory has been operating in the blood service, which conducts an external assessment of the quality of laboratory research in blood centers, as well as research in controversial and complex cases. introduced pathogen inactivation of platelet concentrates in % of cases of platelets. erythrocytes are used only in the weighing solution and only after leukoreduction. the use of resource-saving blood harvesting technologies, as well as methods to further enhance the infectious and immunological safety of components, are expanding annually. the foundations for the development of applied scientific research in the field of transfusion medicine were laid, and an own school of transfusiologists was created. as a result of reforming the blood services, the mechanisms of reimbursement of blood services costs from the budget of the republic have been changed, uniform tariffs have been defined by type of blood components for all blood components suppliers in the country. summary/conclusions: the transformations made it possible to create a blood service in kazakhstan that corresponds to the best international practice and ensures the quality, efficacy and safety of transfusion therapy. further development of the blood service is aimed at deepening reforms, studying and developing cellular technologies, new properties of plasma blood components, and improving the clinical practice of using donor blood components. background: reconstituted platelet concentrate (rpc) is a component that contains platelets (thrombocytes) resuspended in plasma that is blood group compatible with the recipient. it is used when transfusion of platelet concentrate is necessary but the fresh platelet concentrates of the specific blood group are not available. group rh-(negative) platelets resuspended in the ab group plasma are of universal use as such components can be used for all recipients independently of their blood group. in the area of activity of regional blood center in poznan rpc is used most often for patients after stem cell transplant incompatible with their blood group. aims: the aim of the analysis is to evaluate the loss in the number of platelets in the reconstituted platelet concentrates in order to determine their quality and usage. methods: we used for the analysis units of pooled platelet concentrate derived from buffy coats and units of pooled platelet concentrate derived from the apheresis. for the reconstitution group ab plasma was used. we investigated the number of platelets prior to and after the reconstitution. following measures were calculated: number of platelets in pooled platelet concentrates derived from buffy coats, number of platelets in pooled platelet concentrates derived from apheresis; average values and the standard deviation as well as the loss in number of platelets due to the reconstitution. the number of platelets was calculated with impedance method using horiba pentra xl analyser. . pooled platelet concentrates derived from buffy coats the number of platelets before the reconstitution: . /unit ae . the number of platelets after the reconstitution: . /unit ae . the loss in the number of platelets: . % . platelet concentrates derived from apheresis the number of platelets before the reconstitution: . /unit ae . the number of platelets after the reconstitution: . /unit ae . the loss in the number of platelets: . % summary/conclusions: . reconstitution results in the loss of . % of platelets in comparison to the initial value of pooled platelet concentrate derived from buffy coats. . the average number of platelets after the reconstitution is . /unit, which fulfils the quality requirements. . reconstitution results in the loss of . % of platelets in comparison to the initial value of pooled platelet concentrate derived from apheresis. . the average number of platelets after the reconstitution is . /unit, which slightly deviates from the quality requirements. . the results that we obtained show that the process of reconstitution always results in the loss of number of platelets and that the quality of platelet concentrates that have not been reprocessed is always higher. because of that we recommend that the reconstitution should only be performed in special cases such as for patients after stem cell transplant incompatible with their blood group and that hospitals should be supplied with platelet concentrates derived from buffy coats and apheresis. methods: pf was extracted from human pcs by using immunoaffinity chromatography and specific anti-pf antibody on the column. hereafter, pf was purified with concentration tubes having kda molecular weight cut-off and dialyzed via dialysis tubing with . kda cut-off. pf concentration was measured by bradford method. specific enzyme-linked immunosorbent assay (elisa) and dot blot analysis were used to determine the pf specificity. molecular weight of obtained pf was determined by polyacrylamide gel electrophoresis (page). results: our data confirmed clearly that separated pf had kda molecular weight which is corresponding with a tetramer pf . in addition, elisa showed that the pf qualified true antigenicity. dot blot analysis helped us to assure the specificity of pf . summary/conclusions: we used and optimized a homemade protocol to isolate and purify the pf . nevertheless, there was no any scientific report among the databases that utilized the similar purification method. recombinant pf is more feasible and ready-to-use, but its price may discourage the researchers to study the biology of pf . unused human pcs and specific anti-pf antibody can considered as an alternative to separate the pf particularly in labs with the shortage of resources. background: whole blood is increasingly used in civilian hospitals as an alternative to blood components in trauma resuscitation with massive bleeding. however, the hemostatic effect of whole blood depends on the initial platelet content and decreases rapidly within the first weeks of storage at °c. consequently the problem arises of how to provide wb to trauma centers without losing valuable low titer group o units if the units are not used within the expiry date of use. aims: to assess the in-vitro quality of red cell concentrates (rcc) produced from days cold stored leucodepleted whole blood units filtered with a platelet-sparing whole blood filtration filter in order to save the rcc once the wb units has reached the expiry date. methods: units of whole blood were leucodepleted within h using a platelet sparing filter (imuflex wb-sp/terumo bct) and stored for days at ae °c. on the th day of storage, units are centrifuged and the rcc is supplemented with sag-m additive solution for an additional storage of days at ae °c. in vitro parameters such as hemoglobin, hematocrit, residual leucocytes, platelet content, hemolysis, glucose, lactate, atp, . dpg were tested during storage. results: the imuflex wb-sp provided satisfying leucodepletion of the wb units ( . ae . . e /u)) while maintaining % of the initial platelet content ( ae . e /u). mean volume ( ae ml), hemoglobin content ( ae g/u) and hematocrit ( ae %) was within conformance range. after days of storage, % of the initial platelet content at collection is maintained and mean hemolysis is . ae . %. rcc processing at day was uneventful, without loss of hemoglobin or noticeable hemolysis. mean volume ( ae ml), hematocrit ( ae %), hemolysis ( . ae . %) were in conformance with mandatory standards at day . after days of storage (i.e. day after collection), rcc quality parameters were maintained with however an increase in hemolysis ( . ae . %) which was above usual values routinely observed with rcc produced from day processed units. rcc out of were above the maximum limit of . % at day after collection such increased hemolysis was more pronounced at day after collection ( . ae . %) with a proportion of rcc with a non-conforming hemolysis ratio above the accepted threshold ( % of the tested units). summary/conclusions: rcc units can be readily obtained from leucodepleted wb units with spared platelet contents after days of cold storage. in vitro quality attributes of such rcc are within conforming ranges for hemoglobin, hematocrit, volume and hemolysis. hemolysis increases more rapidly than in standard rcc, thus limiting the possibility of use within days after collection. additional studies are being initiated to reduce hemolysis during storage. g hetland , , f mahmood , l nissen-meyer and i nentwich immunology and transfusion medicine, oslo university hospital immunology, university of oslo, oslo immunology and transfusion medicine, akershus university hospital, lørenskog, norway background: allergen-specific ige in donors' plasma can be transferred from blood donors to recipients via plasma-containing products and consequently sensitize recipient's effector cells bearing receptors for ige (mast cells, basophils, thrombocytes etc.). these cells can then degranulate after exposure to allergen. this can cause allergy symptoms of various degrees (johansson, allergy, ) . food allergen-specific ige antibodies, although causing mild or no symptoms in blood donors, can be of clinical importance to plasma recipients due to varying sensitization and activation grade of recipients' effector cells. aims: the aims of the study were ) to assess sensitization profiles against an array of allergens in randomly selected blood donors and ) to identify donors with high concentrations of food-specific ige antibodies that could bear risk of clinically important sensitization of plasma product recipients. methods: we tested serum samples from blood donors randomly selected on one donation day outside the pollen season. we used ll serum per sample in a multiplex ige line-blot enzymoimmunoassay for analytes (euroline atopy screen, euroimmun, germany), which comprised food allergen extracts and single allergens, inhalant allergen extracts, insect venom extracts and one carbohydrate allergen ccd. this high-throughput method enables scanning of serum samples for ige against allergens in less than h. results are expressed as east (enzymoimmunoassay) classes: class (no sensitization), - (weak sensitization), (moderate sensitization), - (strong and very strong sensitization). results: east class, allergen, number of samples. food allergens: class none detected. class , sesame seed: . class , apple ; hazelnut . class , apple ; almond ; hazelnut ; sesame seed ; soybean ; cow milk alpha-lactalbumin . class : donors weakly sensitized to one or more allergens as kiwi, apple, almond, hazelnut, sesame, soybean, wheat, mutton, beef, cow milk beta-lactoglobulin and alpha-lactalbumin. we did not find any donors sensitized to apricot, peanut, rice, rye, yeast, cow milk casein and carbohydrate allergen ccd. insect venoms: class , and none, class , wasp venom ; honey bee . lower classes ( - ) not reported in this abstract. summary/conclusions: we found considerable sensitization to inhalant allergens but such sensitization probably represents low risk of clinical allergies due to passive transfer of antibodies. sensitization to insect venoms was negligible. we disclosed only one blood donor ( %) with high ige to sesame seed with a potential to transfer clinically relevant ige antibodies via plasma products. the need for a broad screening of donors for ige sensitization remains still questionable. background: the platelet storage lesion (psl) is one of the key factors that limit the platelet shelf-life to days. the mechanisms mediating the psl are not well understood, but it is becoming increasingly evident that platelets from some donors do not store as well as others. similarly, assessment of many in vitro quality parameters has been used to predict transfusion outcomes, with varying degrees of success. donor attributes such as age, and body mass index (bmi) may contribute to these differences. aims: to characterise the variability in the quality and function of apheresis platelet donations and to examine associations between donor attributes and platelet characteristics. methods: apheresis platelets (n = ) were collected, stored at °c with agitation, and tested at day , day and post-collection. vwf and fibrinogen binding (indicators of platelet adhesion; ae ristocetin stimulation), phosphatidylserine (ps) exposure (annexin v binding) and microparticles (cd + /annexin-v + ) were measured by flow cytometry. platelet aggregation was initiated with collagen, thrombin or arachidonic acid. thromboxane a (txa ) was measured by elisa. the proportion of procoagulant platelets (% coated platelets) was assessed by fibrinogen binding following collagen/thrombin stimulation. platelet-attached glycans were measured using flow cytometry and lectins ricinus communis agglutinin- (rca- ; detecting galactose-residues) and wheat germ agglutinin (wga; detecting sialic acid and nacetyl-d-glucosamine, glcnac-residues) ae stimulation with ristocetin. donation time, donor age, blood pressure (bp) and bmi were recorded. linear regression was performed using graphpad prism software. results: for many of the parameters measured, there was high inter-donor variability. platelets from subsets of donors showed up to -fold higher vwf and fibrinogen binding, formation of coated platelets and microparticle numbers. binding of lectins was also highly variable between the donors, indicating variability in sugar cleavage. following ristocetin stimulation, binding of all lectins increased, and again there was high variability in the responses, with up to -fold higher binding in platelets from a subset of donors. of particular interest, aggregation in response to arachidonic acid was observed in only % of platelet donations tested; although platelets from non-responders aggregated in response to the collagen and thrombin. txa was also higher in the arachidonic acid responders (p = . ). there were few associations between platelet parameters and donor attributes, and these associations were typically weak. ph at day was weakly associated with donor age (p = . , r = . ) but not bmi (p = . , r < . ), with a trend towards lower ph in younger donors. summary/conclusions: these data indicate that apheresis platelet products are highly variable. whilst donor characteristics may have some influence on the quality of the donated platelet product, there is a high level of inherent inter-donor biological variability. a much larger sample size is required to confirm these findings, and determine whether they have clinical implications. background: the frequency of beta thalassemia mutations, and thus, of beta thalassemia heterozygotes (b-thal-het) is particularly high in certain geographical regions. it has been reported that a significant proportion of donors (occasionally more than %) are b-thal-het that meet the criteria for blood donation (hb > . g/ dl). red blood cells (rbcs) of b-thal-het donors are characterized, in vivo, by particular geometry and redox status. despite sporadic indications that the rbc storage lesion may be milder in b-thal-het, targeted research on this donor group is still missing. aims: the aim of this study was to investigate whether b-thal-het rbcs storage at blood banks leads to a distinctive hemolytic, physiological and redox profile, thus, making b-thal-het a unique blood donor group. methods: blood samples from healthy non-smoker donors ( b-thal-het carriers and controls) were analyzed before and after preparation and storage of leukoreduced packed rbc units in cpd/sagm at various time intervals. susceptibility in hemolysis (in the presence/absence of oxidative, mechanic and osmotic stimuli), redox status (lipid peroxidation, reactive oxygen species (ros) accumulation, antioxidant capacity), intracellular ca + and proteasomal activities were determined. for statistical analysis, significance was accepted at p < . . samples from the red cell units were collected aseptically, processed (dual centrifugation at , g for min) and stored at À °c. processed samples were thawed, and then analysed using the nanosight ns nanoparticle tracking analysis system (malvern instruments). samples from all time-points from each unit were analysed on the same day. data were analysed by one-way anova with bonferroni's multiple comparisons test. results: at d , red cell units contained an average of . ae . mvs/ ml. the mean size of these mvs was . ae . nm and the mode size was . ae . nm. the concentration of mvs increased gradually throughout storage (p = . ), reaching a maximum at d of . ae . mvs/ml. both the mean (p < . ) and mode (p < . ) size of the mvs increased during storage; however, this size increase primarily occurred in the first week of storage (d vs. d : p < . for both mean and mode). by d , mean and mode size of mvs was . ae . nm and . ae . nm summary/conclusions: nanoparticle tracking analysis demonstrated the presence of mvs smaller than nm in red cell units. both the concentration and size of mvs present in red cell units increased during the days of routine storage. the concentration of these mvs was approximately -fold higher than we had previously detected using flow cytometry (aung, pathology, ) indicating the advantages of more sensitive techniques in characterisation of mvs. background: the lack of availability of sterile saline in a format suitable for use in blood centers for manual washing has led to an urgent need for blood services to consider alternative methods. for operational flexibility it would be desirable to be able to produce a washed rbc unit that had a shelf life longer than h. aims: the aim of this study was to validate the manual method for washing rbcs using sagm solution both as wash and storage solution and to ascertain whether an extended storage period for washed rbcs may be feasible. methods: six day old leukocyte depleted red blood cells (ld-rbc) and six day old ld-rbcs were manually washed and stored in sagm, and half of the units were pre-stored irradiated ( gy). a volume of ml wash solution (sagm) was added to the ld-rbss by sterile connection. after mixing the units were centrifuged for . min at g at °c (hettich roto silenta rs) before removing the supernatant using compomat g extractor. wash procedure was repeated twice using ml sagm solution, and after removal of the last supernatant, ml of sagm solution was added. all units were immediately measured for volume, haematocrit, albumin, iga, potassium, haemolysis, haemoglobin, ph, glucose and lactate and tested again after h, days and days storage at ae °c. results: all washed ld-rbcs met european specification for haematocrit ( . - . ) and all but one for hb content (≥ g/unit). hemolysis increased during storage. the rate of hemolysis in irradiated ld-rbcs was greater over time than in nonirradiated units. all units, both irradiated and nonirradiated, met european specification for hemolysis (less than . %) days after washing. after wash, potassium levels were low and then increased during storage; increase was greater in irradiated than nonirradiated units. potassium concentration days after washing and irradiation did not exceed those levels found at the end of shelf life (day ) of standard ld-rbcs. ph decreased during storage due to the metabolic activity of red blood cells converting glucose to lactate. the ph level of the supernatant depends on the age of the unit and not on the irradiation. the glucose concentration of the supernatant after washing is high due to sagm solution. the concentration of glucose decreased and lactate increased due to the metabolic activity of red blood cells. there is currently no specification in europe or finland for iga in washed rbcs. aabb and american red cross rare donor program stipulate that level of iga should be less than . mg/dl ( . mg/l). our iga method s lower limit for detection is . mg/l and all results were below this level. total albumin were well below finnish specification (< mg/unit). background: room temperature has been the standard storage condition for platelets since the s, when it was shown that this improved in vivo survival compared to when stored at °c. however, storage at room temperature has several disadvantages, including risk for bacterial contamination and short outdating. recently, the interest in cold-stored platelets increased, especially for patients with a hemostatic need. using extensive analysis techniques, we evaluated the in vitro quality of cold stored platelets in additive solution. aims: investigation of the in vitro quality of platelets stored at - °c in pas-e. methods: three experiments were performed, in which two platelet concentrates, prepared from buffy coats and ml of pas-e (pcs) were pooled and split in equal pcs. pcs were stored for days at - °c, one of each pair with agitation on a flatbed shaker and the other without agitation. various parameters were analyzed to study the in vitro quality during storage and compared to routine room temperature storage. results: during cold storage, the swirling phenomenon disappeared within one day. due to the lower temperature, metabolism of the platelets was lower as compared to room temperature storage. the metabolic conditions were acceptable with ph d -d : . - . with platelet count /u and glucose still at mm at least until days of storage. platelet activation maintained acceptable levels with cd p expression < %, while ps exposure increased rapidly; > % after days of storage. aggregation tests showed functional platelets until days of storage. agitation during storage had no effect on any of the tested parameters. summary/conclusions: during storage of platelets at - °c, the hematological parameters and ph met routine requirements, while swirling phenomenon disappeared already at the first day. the functionality of the platelets did not decrease during cold storage, indicating that the swirling phenomenon is not a good surrogate marker under these conditions. the strong increase of ps exposure might be involved in the observed short survival of cold-stored platelets. platelet concentrates stored at - °c are potentially suitable as a hemostatic agent for patients with a bleeding in need of platelets, but more studies are needed. aims: the goal of the study is the reinforcement of platelet reserves for case of emergency events and increasing their availability for treatment, preferentially in patients with massive bleeding. methods: we performed a comparative study with cpp and fp in vitro. buffy coatderived pooled leukoreduced platelets rhd negative were frozen in - % dmso and stored at À °c for months. cpp were thawed at °c, then reconstituted in platelet additive solution ssp+ and compared to fp. we measured these parameters: platelet content, platelet concentration, platelet loss during preparation process, coagulation properties, volume, ph, dmso concentration, titres of anti-a and anti-b antibodies. results: the average platelet loss after the process of freezing and reconstitution was %. the amount of platelets and platelet concentration in unit was lower in cpp compared to fp, but high enough (amount /unit, concentration . /unit). both types of plts (either pcc or fp) maintained an acceptable ph during storage. swirl was on value in fp and on value in cpp. the average plasma content in fp was % compare to . % in cpp after reconstitution. measured titres of igm anti-a and anti-b antibodies were very low ( - : ). cpp had faster clot initiation (rotem clotting time (ct) in cpp . s, fp . s). cpp contributes to a sufficient clot (rotem maximum clot firmness (mcf) in cpp . mm, fp . mm). summary/conclusions: our results shows, that cpp have higher procoagulation activity and simultaneously lower clot firmness. thawing and reconstitution of platelets are easy and fast processes if platelet additive solution is used. this method helps to increase the availability of platelets in emergency medicine. low plasma content in cpp enables their use as washed platelet product in specific groups of patients. methods: after donation, the whole blood was stored in room temperature overnight before separating next morning by reveos â system. seven abo compatible ipus, each with a target volume of ml, were selected and then they were connected to the pooling set provided by terumo bct. prior to the pooling of ipus, ml of additive solution (t-pas+ provided by terumo bct) was added and distributed evenly between the ipu bags. the pooling set was then kept h on bench in room temperature followed by h on agitator at ae °c. after filtration, the pool might be manually adjusted if its volume exceeded the maximum of ml to meet the requirements by the intercept tm blood system. the final products were two pathogen-reduced platelet units with a shelf life of days. results: during validation of the method, pathogen-reduced platelet units were controlled, in addition to the platelet count, for ph, glucose, po , pco and lactate on day , and of storage. the platelet count was . ae per unit on day . the ph value was . on day , . on day , and . on day . the glucose concentration decreased from . to . and . mmol/l on day , and , respectively. the mean po level was . , . and . kpa while the mean pco was . , . and . kpa and the lactate concentration was . , . and . mmol/l on day , and , respectively. since routine implementation of the method in april , regular quality controls showed an average of platelet count of . ae (n = ) with a volume of ae ml per unit. summary/conclusions: the validation of the method and the following two years of experience in routine shows that the pooling of ipus processed in reveos â system meet the requirements needed for intercept tm ds processing set for pathogen reduction of platelets. the results from the quality controls of the final platelet units were in accordance with the local and eu guidelines. methods: data was analyzed from published and unpublished clinical studies that performed both primary and secondary testing of platelets using the bta system. the studies included apheresis and whole blood derived buffy coat platelets and tested - ml sample volume per culture bottle. the studies classified results based on aabb bulletin - definitions with some modifications. the following assumptions were made including: • data was summarized as total number of positive tests, observed by the total number of tests performed on each day post collection; • it was assumed that one test was performed per platelet unit; • all units eligible for secondary testing were negative by the primary test the data needed to demonstrate a benefit for the use of the bta d systems for detecting contamination that was not revealed by previous bacterial testing as well as clinical specificity. results: a total of , platelet units from the studies where secondary testing of platelets was performed were analyzed. platelets were tested on day , , or ≥ , and represented . %, %, and % of the units tested, respectively. true positives were detected in platelet units representing . % of the total platelets tested. the majority were reported from platelets tested on day ≥ with a total of . data showed the bta d system used for secondary testing detects the most prevalent contaminates reported, staphylococcus spp., in ≤ h with the majority detected in ≤ h after incubation, allowing for interdiction of the units prior to transfusion. instrument specificity was reported in of the studies for platelets tested at days and ≥ days with a total false positive rate of . % (range of - . %). instrument sensitivity when used for secondary testing could not be determined since subculture of negative bottles is not performed during routine use. during previous validation testing of lrap and lrwbpc, , culture bottles were confirmed true negatives by subculture. summary/conclusions: data from the studies that tested platelets at to days post collection provided evidence that the bta d with bpa & bpn detects contaminants missed in previously tested platelet units. the data supports that the bact/alert d system is an effective safety measure for secondary testing of platelet products to extend platelet dating beyond day and up to day when testing is performed using the test parameters described in the bpa and bpn bottle ifus and according to the fda draft guidance. background: magnetic nanoparticles have recently shown great potential in nonradioactive labeling of platelets. platelet labeling efficiency is enhanced when particles are conjugated with proteins like human serum albumin (hsa). however, the optimal hsa density coated on particles and the uptake mechanism of single particles in platelets remain unclear. aims: we characterize the interaction between single particles and platelets and determine the optimal hsa amount required to coat particles. methods: ferucarbotran iron oxide nanoparticles were coated with hsa in different amounts ( . - mg/ml) and we confirmed successful hsa coating by addition of a crosslinking hsa antibody (dynamic light scattering). we labeled platelets from pooled platelet concentrates with mm ferucarbotran coated nanoparticles and analyzed labeled platelets for iron content (atomic absorption spectroscopy) and particle localization (transmission electron microscopy). single-molecule force spectroscopy was used to determine binding forces of nanoparticles to platelet compartments. we applied hsa-particles via linkers of different length (i.e. short~ nm, medium nm and long~ nm) on the cantilever tip and let them interact with a platelet provided on a collagen surface. after interaction we determined the rupture force required for platelet retrieval. results: the iron content per platelet reached a maximum at . - . mg/ml hsa coated particles with . ae . and . ae . pg/platelet, respectively. however, the . mg/ml hsa coating resulted in~ -fold higher binding affinity to platelets than particles coated with . mg/ml hsa. depending on peg length between tip and particle, particles interacted differently with platelets as shown by one, two or three force distributions of , , and pn, which correspond up to three different binding pathways, respectively. the results indicate that a particle can interact with three targets including platelet membrane, open canalicular system, and platelet granules. summary/conclusions: our results reveal mechanism of platelet-particle interaction on a single particle level and provide an optimal hsa concentration coated on particles to gain maximal platelet labeling efficiency. labelling of platelets by magnetic nanoparticles may substitute radioactive labeling. results: the activation/lesions on total platelets and small and medium-sized platelets platelet population was detected on storage day , by the increased expression of cd . the percentage of cd -positive cells among the population of large platelets did not change during storage. on the day , increased expression of cd b and cd p was detected, but only on large platelets. small and medium-sized platelets had increased cd p expression only on day . the expression of cd a on total platelets increased significantly on day , and stayed unchanged until day . the same pattern of cd a expression was detected for small and medium-sized platelets, whereas on large platelets the expression continued to increase until the end of storage. a decreased percentage of cd -positive cells was detected among the total platelets and populations of medium-sized and large platelets. the storage induced externalization of phosphatidylserine on total platelets or on any of the platelet populations was not detected. the levels of tgf-b and p-selectin in the pc-bc supernatants were unchanged during the -day storage period. increased annexin and pf concentrations were detected on day . the concentration of b-tg increased on day of storage, and continued to rise until the end of storage. summary/conclusions: the evaluation of expression of activation markers on different platelet populations could be used as an additional analysis in quality control of platelet concentrates, and in the assessment of novel approaches to platelet concentrate manipulation i.e. for testing new additive solutions, cryoconservation protocols, and cryoprotectants. background: the primary goal of autologous blood transfusion is to reduce the risks related to allogeneic blood transfusion, including transfusion-associated graft-versus-host disease (ta-gvhd). although downward trends in rates of autologous blood transfusion have been reported in europe and the americas, it still plays a role in eliminating risks related to allogeneic blood transfusion in japan, especially ta-gvhd. since february , the transfusion service in our hospital has managed autologous blood conservation techniques and helped to prevent mistransfusion by employing a bar code-based electronic identification system. aims: the objective of this study was to determine the use of types of autologous blood components in a single institution over an approximately -year period. methods: between february and december , we retrospectively analyzed autologous blood transfusion, including perioperative autologous cell salvage (pacs), pre-operative autologous blood donation (pad), and acute normovolemic hemodilution (anh). we investigated the use of autologous blood components and the rate of complying with electronic pre-transfusion check at the bedside in the operating rooms. we also determined the adverse reactions to autologous blood transfusion, which were categorized according to the definitions proposed by the international society of blood transfusion (isbt) working party on haemovigilance. results: a total of , patients ( % of whom received operations) received blood transfusion, of which , ( %) received autologous blood transfusion alone, , ( %) both autologous and allogeneic blood transfusions, and , ( %) allogeneic blood transfusion alone. the rate of autologous blood transfusion among patients who received blood transfusion was %. pacs units were transfused to , patients ( %), pad units to , patients ( %), and anh units to patients ( %), and multiple blood conservation techniques were used for one patient. the overall compliance rate with electronic pre-transfusion check at the bedside in autologous blood components was . %. adverse reactions were observed only with pad transfusion and not pacs nor anh. the number and rate of adverse reactions to pad transfusion were and . %, respectively, of which the most common was febrile non-hemolytic transfusion reaction at ( %), followed by allergic reactions at ( %), and hypotensive transfusion reaction at ( %). the severity of adverse reactions to pad transfusion was grade (non-severe) in all cases, and no serious adverse reactions were observed. among pad units, the rate of adverse reactions to whole blood pad units was . %, that to frozen pad units was . %, and that to autologous ffp units was . %. summary/conclusions: our observations indicate that the rate of autologous blood transfusion among patients who received blood transfusion was %, when all types of autologous blood conservation techniques were included. to accurately determine the rate of autologous blood transfusion in a hospital, it may be better for the transfusion service to manage all types of autologous blood conservation techniques. they are now clinically available as a blood product. the residual plasma level of this product, which is prepared using the automated cell processor acp (haemonetics corp.), is approximately %. recently, a retrospective multicenter study was reported that this product was effective and safety for prevention of recurrent or severe transfusion reactions. plt products are generally stored with continuous agitation to maintain their quality. the interrupted agitation of plt suspended in additive solution (plasma carryover: - %) for up to h was previously found to have only a slight impact on in vitro plt properties. however, in some small hospitals with no agitator, if the initiation of transfusion is delayed by an emergent change in a patient's condition, the interruption of agitation may be prolonged. aims: the aim of this study was to evaluate the effects the interruption of agitation for h (shelf life of wpc in japan) on the in vitro qualities of plt. methods: leukoreduced apheresis platelet concentrates in % plasma were washed on day one after blood collection using the automated closed-system cell processor acp (n = ). wpc, which were rested h after preparing, were divided equally into control and test units using polyolefin containers on day one. control units were agitated from days one to seven. test units were stored without agitation from days one to three, and agitation was subsequently performed until day seven. both units were stored at - °c. in vitro plt quality was examined on days one, three, four and seven. results: the plt concentration of prepared wpc was . ae . ( /l) and the volumes of the control and test units after the division were ae and ae (ml). the ph values of the test units on day three were lower than those of the control units; however, the ph of both units were maintained at higher than . during the seven-day storage period. swirling was well maintained and no clumping was visible in both units during storage period. no significant differences were observed in plts concentrates, mpv, hsr, aggregation response. the pco , po , bicarbonate, glucose and lactate mean values of test units were slightly lower or higher than those of the control units on days three or four. the levels of cd p expression were significantly higher in the test units than in the control units on days three ( . % ae . vs . ae . , p < . ); however, this difference decreased in a time-dependent manner after agitation resumed. the levels of cd b expression of test units were relatively lower than those of the control units until day seven, but no significant difference between the two units. background: monitoring residual white blood cells (rwbcs) is a requirement for quality monitoring (qm) the production of leucocyte depleted blood components. although flow cytometry is widely used for monitoring rwbc, there are no widely accepted methods to accurately and consistently measure rrbcs in blood components. sysmex have developed a novel algorithm, termed the blood bank (bb) mode for their xn-series of haematology analysers which is specifically designed to quantitate the levels of rwbcs and rrbcs in blood components. aims: we have previously assessed the linearity, accuracy and reproducibility of the bb mode on spiked samples in an r+d lab. we sought to further assess the performance of the bb software in a routine, high throughput blood component manufacturing department. methods: units of plasma, platelets (pcs) and red cell concentrates (rccs) were produced according to standard uk specifications within nhs blood and transplant (nhsbt). qm of residual cells was tested using the bb mode whilst rwbc was additionally analysed by flow cytometry using bd leucocount kit. results: during a -month field trial over , data points were collected representing all types of manufactured component. for some pcs, bb mode results from some sample tubes that did not contain edta gave very high rwbc values, indicating a potential large number of ld failures. the results were significantly different from those obtained from pcs using edta samples (p < . ) which did not show the same high values. for rccs or plasma, the range of results from plain and edta tubes were not significantly different (p ≥ . ). the analyses of ld platelet and plasma concentrates by either bb mode or flow cytometry both show more than > % of ld components have less than rwbc/unit. for ld failures (n = ) there was a good correlation (r = . ) between flow cytometry and bb mode measurements. spiking studies suggested that the limits of detection and quantitation of rrbc were around and rrbc/ll respectively. residual red cell counts from manufactured components showed a wide variation in their numbers between units. as expected platelet production methods also showed a significant difference (p < . ) in rrbc contamination, with lower levels in apheresis platelets (median = rbc/ll, n = ) compared to those produced from buffy coats (median = rbc/ll, n = ) in our hands, although the time taken to analyse samples is similar for flow cytometry and bb mode, considerable time can be saved on manual handling and the processing of samples for flow cytometry (approximately - h for - samples). summary/conclusions: we have been able to embed the sysmex bb mode into a routine production environment and confirm that its performance in spiked samples is mirrored in routine use. for platelets, sample collection in edta is essential. the bb mode offers an opportunity to reduce operator time compared to flow cytometry whilst gaining additional information on rrbc. abstract withdrawn. results: in our experiment, the typical size of a spectrin matrix section (l) was to nm (without oxidation). the heights (h) of dips were to nm. due to oxidation, the junctional complexes between spectrin and membrane proteins can rupture. only % to % of the spectrin surface has the same structure as in the control group. the values of l and h vary significantly depending on the intensity and time of exposure. we observed significant changes in the spectrin matrix after exposure to uv radiation in a model experiment. the local topological defects in the membrane arise from the action of oxidizing agents on the red blood cells. the mechanism of their appearance is connected mainly with the distortion of the spectrin matrix. as a result of oxidation processes, the spectrin molecules can be damaged. there is a transformation of tetramers to dimers. additionally, it can be easily seen with the afm, that spectrin network structure was essentially destroyed. most parts of the spectrin matrix have damaged structures with mesh breaks and dips after uv irradiation. also the results of network distortions in response to temperature changes were obtained. there are presented preliminary results of spectrin matrix change during long-term storage of prbc. summary/conclusions: atomic force microscopy in direct biophysics experiment allows to observe and to quantitatively measure the disturbances in the spectrin matrix nanostructure in response to oxidation processes in rbcs. these studies are important for the fundamental research of interactions of rbcs on the molecular level during redox processes and the consequences to their structure and function on the cellular level. this is important for the advancement of transfusion medicine, intensive care medicine, and molecular and radiation biology. methods: blood samples were taken from donors during a prophylactic examination and collected with edta-filled microvettes (sarstedt ag and co., germany). all experiments were carried out in accordance with the institute guidelines and regulations. the polylysine-coated glass was used to perform the sedimentation method for formation of native rbc smears. it is important that any fixatives weren't used. the stiffness of rbc membranes was studied in native rbcs (control) and native cells after the application of modifiers: glutaraldehyde, hemin, zn + (heavy metal ions). local stiffness was studied by atomic force spectroscopy (afs) (ntegra prima, (nt-mdt, russian federation). results: experimental kinetic curves i(z) were measured. nonlinear fitting method was used to determine the young's modulus. the experimental dependences of membrane bending were approximated by the hertz model to a depth up to nm. the young's modulus e = ae kpa for control rbc. it was shown that some natural oxidants (hemin), membrane fixatives (glutaraldehyde) modifiers, heavy metal ions (zn + ) significantly increased the absolute value of the young's modulus of rbc membranes up - times. the biophysical parameter hertz depth (h hz ) was determined for each curve. under the influence of modifiers the hertz depth h hz was changed from nm to nm. there are presented preliminary results during long-term storage of blood. summary/conclusions: the blood rheology is determined by rbc deformability, associated with membrane stiffness. the young's modulus can be used as a quantitative criterion to estimate the membrane state of a native cell. the results of the work can be used in clinical practice, in assessment of the quality of donor blood during storage before transfusion, in biophysical studies of rbc state. abstract withdrawn. immunohemotherapy, centro hospitalar universit ario são joão, epe, porto, portugal background: transfusion of blood and blood components is an essential resource in modern medicine. a proper use of human blood, being an irreplaceable resource, is necessary in order to achieve minimal wastage. blood wastage may occur for a number of reasons, like expiry date, haemolysis, seroreactivity or low volume. monitoring wastage of blood product during collection, testing and processing of blood is used as a quality indicator. aims: to determine the annual rate of discarded blood components due to expiry date in a portuguese university hospital blood bank (bb) from january to december , in order to implement appropriate measures to minimize the number of discarded blood to a reasonable rate. methods: we retrospectively analysed the rates of blood components discarded after meeting their expiry date of a portuguese university hospital blood bank from january st to december st . results: a total of , whole blood units and , apheresis platelets were collected during the study period. of the , blood components (packed red cells, whole blood pooled platelets and apheresis platelets) prepared during the study period, a total of , ( . %) blood components were discarded, of those . % due to expiry date. the rate of discarded packed red cells, according to this component production, decreased considerably over the years, in was . %, in was . % and . % in . similar tendency was shown in the pooled platelets for consecutive years with . % ( ) and . % ( ), but with an increase in ( . %). the rate of apheresis platelets had a more variable behaviour from to with rates of . %, . %, and . % respectively. summary/conclusions: blood transfusion is an essential part of patient care. for this reason, the implementation of a quality system and continuous evaluation of all activities of the bb can help to achieve maximum quantity and quality of safe blood. we identified that date expiry was the main reason of discarded blood components, although there was a significant decrease in the rate of discarded packed red cells over these three years. properly implemented blood transfusion policies, donor screening and training staff as well as implementation of automation also helps to improve the process, reducing the discarding rates of blood and blood component. background: storage performance of platelets (plt) is associated with age of the donor. the risk for plt with poor storage performance, characterized by high lactate production and rapid acidification of a plt concentrate (pc), shows a positive correlation with age. we wished to explore whether high lactate production was associated with donor health issues. aims: to investigate high lactate production by stored platelets in relationship to donor health. methods: single-donor pc were collected by apheresis or prepared from buffy coat and donors were evaluated who could be marked as 'rapid acidifiers'. in total, apheresis pcs and pcs from whole blood were included in four studies. information about donor health was obtained either from the blood bank information system or using questionnaires. in some donors, the lipid profile was measured from plasma, and the diabetes marker hba c from red cells. triglyceride levels > . mmol/l and hba c levels > mmol/mol were defined as high. results: twenty two percent ( / ) of the donors were marked as 'rapid acidifiers' and % ( / ) of these donors had health issues. 'rapid acidifiers' were of age , - (median, range) years. three groups of donors can be distinguished: a) donors affected by metabolic syndrome, prediabetes and type diabetes, indicated by high cholesterol and/or triglycerides, high hba c and/or the use of medication to treat diabetes. b) donors affected by vascular diseases who reported or used medication to treat high blood pressure. c) "other" donors who used other medication to treat various other conditions. the remaining 'rapid acidifiers' ( %) did not have high triglyceride or hba c levels and did not report health issues. summary/conclusions: pcs with rapid acidification by high lactate production are mainly collected from older donors with health issues. we postulate that high lactate production by stored plts is associated with health issues, and we will combine detailed donor information (health and behavior) with in vitro quality for a significant number of donations. background: the development of applied biotechnologies requires a search and creation of new methods of cells' functional completeness analysis. the instrumental assessment of platelets quality for the selection of the most effective donors, quality assessment of platelet concentrates for short and long-term storage and for the selection of platelets for cryopreservation is in demand in blood service. assessment of platelets morphofunctional status is possible using morphological studies of various platelet granules fractions (makarov, med. alfavit, ). among the biologically complete platelets there is a special population of cells, the so-called granule-rich platelets (grp). these cells contain the largest number of cytoplasmic granules (more than visually distinct granules). it is established that grp have increased viability and functional activity. earlier we found a correlation between the grp level in blood plasma and shift of the redox potential value in blood plasma after cryodestruction of platelets (tsivadze et al., doklady physical chemistry, ). it was suggested that the shift of the redox potential may be partly due to the release of the low molecular weight antioxidants contained in functionally complete platelets outside the cell. in turn, the concentration of low molecular weight antioxidants can be estimated using the cyclic voltammetry. aims: the aim of the study was to estimate of cyclic voltammetry method possibilities for quality assessment of platelets. methods: the functionality of platelets was examined in platelet concentrate (pc) obtained by apheresis ( . ae . /ll). voltammetric analysis in pc before and after the platelets cryodestruction was carried out on platinum electrode in the potential range from À mv to + mv using a potentiostat ipc pro l and saturated ag/agcl electrode as reference. for the morphofunctional analysis platelets were vitally stained with fluorochrome stains trypaflavin and acridine orange. microscopic examination of platelets was carried out using confocal microscope nikon eclipse i. the following parameters were evaluated: concentration and percentage of platelets with granules and concentration and percentage of grp. results: voltammetric studies in pc show that there are two oxidation peaks of low molecular weight antioxidants on voltammogram at potentials + mv and + mv. analysis of pc before and after the cells cryodestruction showed that changes in the height of oxidation peaks occur, indicating an increase of antioxidant content in blood plasma. at the same time a correlation between the changes in the height of oxidation peaks and the grp content in the sample was found. in samples with reduced initial grp content (less than %) after the cells cryodestruction significant changes in the height of oxidation peaks were not observed, regardless of the total number of cells in the pc. summary/conclusions: in conclusion, voltammetric analysis allows to indirectly estimate the population of functionally active platelets that in combination with other methods of analysis can serve to assess the quality of platelet products. background: determination of hemoglobin derivatives in blood is one of the most important studies in clinical laboratory diagnostics, especially during the storage of donor blood and its transfusion. concentration of hemoglobin derivatives can be changed during redox process. aims: to show the possibility of using non-linear fitting method to calculate concentrations of hemoglobin derivatives during reduction-oxidation processes. methods: for this we performed model biophysical experiment, in vitro. blood samples were collected into edta microvettes from healthy donors (sarstedt ag and co., germany) during prophylactic examinations. all the donors gave their consent to participate in the study. a suspension of erythrocytes was prepared in pbs buffer with ph . . we used ultraviolet (uv) irradiation of blood or nano as oxidizing agent. the drug cytoflavin (stpf "polisan", russian federation) was used as an antioxidant. in our study we used digital spectrophotometer (unico , usa) to measure the absorption and scattering of light ( . nm step). the method of nonlinear fitting was used to find the concentrations of hemoglobin derivatives. the empirical spectrum d l (k) exp was approximated by the theoretical curve d l (k l ) theor , which fits the experimental curve in the best way. under approximation the light absorption by different hemoglobin derivatives was considered in model. simultaneously effects of rayleigh light scattering on structures with size d<< k (coefficient s) and light scattering on particles with size d≥ k (coefficient k) were taken into account: d l (k l ) theor = e hbo ,l c hbo l+ e hb,l c hb l+ e methb,l c methb l+ e hbno,l c hbno l+ e methbno -,l c methbno -l+ e methbno,l c methbno l+k+s/k l ( ), where e h,l is the molar absorption coefficient for each hb h derivative at given wavelengths k l , c h is the concentration of the derivatives hb h , l is the thickness of the solution layer, d l (k l ) is the optical density of the substance, k and s are the parameters of the model. results: we determined the concentrations of hemoglobin derivatives without any additional chemicals in blood. there were measured experimental spectra for different agents action on blood. it was shown that concentration of methb increased after uv irradiation and nano action (up to %). there were calculated c h for each hb h derivative. it was established that theoretical curves coincide with experimental data with good accuracy (r = . ). incubation of rbcs with cytoflavin leads to reduction of methb to hbo . summary/conclusions: the determination of hemoglobin derivative concentrations by the method of nonlinear fitting (without adding special chemical agents to blood) can be used for measurement of carboxyhemoglobin in blood during toxic state of organism. also it is important for assessment of rbcs quality before blood transfusion. background: the use of in line leukoreduction filters have been highly expanded in iranian blood transfusion centers within the last decade in order to provide sufficient leukoreduced blood fractions from healthy safe frequent blood donations to be supplied to the leukocyte sensitive patients. leukoflex lcr , the dominant brand of such filters procured by iranian blood transfusion organization, is the most updated generation of the filters used around the world. aims: in this study, it is tried to recover the trapped leukocytes from this novel filter by different buffering systems and having optimized the elution mode, the cell differential of the viable recovered white blood cells were determined by flow cytometry. methods: having passed the routine virological tests, eight leukoflex lcr leukoreduction filters freshly used in tehran blood transfusion center were daily collected and each were back flushed by a self-designed mechanical system (a peristaltic pump, a triple junction with regulator part and an air pump) using various conditions and additives for pbs buffer at different phs in order to find the highest recovery yield for leukocytes. the optimized elute was characterized by flow cytometry for subcellular profile to be determined. results: it was illustrated that a system consisting of pbs (without cacl and mgcl ) in ph . containing mm edta and %(w/w) dextran without additive amounts of triton x was the most optimized buffering system for lcr filter back flushing. total cell content was also determined as . * granulocytes, . * lymphocytes and . * monocytes using auto hemoanalysis and flow cytometric methods. summary/conclusions: in addition to partly compensating of the overhead expenses inflicted by application of leukoreduction filters on healthcare system, the results will assist blood organization system to be more classified in rational profile design, future cell therapy strategies and exceptional blood management. also, the recovered cells could be of significance in stem cell science, cellular interaction studies as well as novel molecular developments in drug discovery. vox sanguinis ( ) results: three lines of strategy are in place to pursue self-sufficiency of the largest number of pdmps. first strategy line: maximizing the yield of driving proteins, represented by immunoglobulins (ig) and albumin; this was assured by csl behring with a yield of . g ig ( % intravenous -privigenand % subcutaneous -hizentra) and g albumin (alburex) per kg plasma fractionated, corresponding to . g ig and . . g albumin; based on present demand, this represents % and % self-sufficiency, respectively, for naip regions. second strategy line: ensuring other products from plasma fractionation; the fractionator granted . g fibrinogen (riastap) and . . iu vwf/fviii (haemate p) per year, which corresponds to the present demand of naip regions for both products, but it is under the full potential of plasma, thus providing a high margin of safety in case of increased demand (now the case of fibrinogen). third strategy line: exchanging cryoprecipitate, fibrinogen and vwf with italian regions whose plasma is fractionated by other companies to obtain prothrombin complex concentrates (pcc -kedcom) and antithrombin (atked) as to satisfy naip regions demand; this strategy allowed a supply of . . iu at and . . iu pcc, capable of ensuring self-sufficiency for naip regions until . summary/conclusions: in italy, differentiation of plasma contract manufacturing among companies with different portfolios allowed naip regions to obtain a significant contribution to self-sufficiency from vnrd plasma for a variety of pdmps by different and complementary strategies consisting in maximizing the yield and the portfolio of proteins from the fractionator and exchanging products among regions for other pdmps at high demand but not included in the portfolio of a single fractionator. plasma check system: a valuable tool for plasma freezing validation and monitoring. background: the validation of plasma freezing processes may result problematic in the monitoring/control of critical process parameters (cpp). in in italy , litres of plasma were produced and frozen. aims: in order to assist plasma freezing validation and cpp monitoring, of the italian bes performing plasma freezing utilize the plasma check system (pcs), a system able to record, store and certify the temperature (t) detected at the core of "surrogate" bags during the entire freezing session, consistently with gmp requirements. pcs is patented and commercialized by expertmed srl, verona, italy (http://www.expertmed.it). methods: pcs consists of parts: a) "surrogate" bags (check-bags) of and ml corresponding to the average standard volumes of the real products, containing a fluid validated to simulate the thermal behaviour of plasma; b) a mobile probe (cryo-med) positionable at the core of the check-bags; c) a dedicated software (memo-track). plasma freezing session data are tracked via barcode/rfid and can be consulted by the pcs that associates blast freezer code, operator code, cryo-med and check-bag. data on plasma freezing are stored in a shared folder and transferred to the be information system. the pcs can also be used to check and monitor the out-of-storage variations of core t of frozen plasma unit, i.e. during labelling and packaging procedures, thus allowing to establish optimal timeframes and operations and suitably validate these procedures. in the period - , at the pievesestina be of emilia romagna region , plasma units were frozen so as to allow complete freezing within to a temperature below À °c, in , freezing sessions, using the pcs both for process validation, change control and for the systematic monitoring of core t at each freezing session. furthermore, at the bologna be tests on the out-of-storage conditions of plasma units were carried out to revalidate the procedures of labelling and packaging. results: out of , freezing sessions carried out at the pievesestina be, ( . %) were detected to fail to reach À °c at the core of the check-bags within . of the latter, in most cases ( %) a technical error in the activation of the cryo-med was identified. in addition, the pcs was systematically utilized for periodical revalidation of the freezing procedures. the tests performed at the bologna be to validate the out-of-storage procedures of frozen plasma labelling and packaging allowed to modify the operating procedures in place so as to establish optimal timeframes and operations. this prompted corrective actions regarding: i) number of units to be taken out of storage sites at each labelling session (< units), ii) labelling time (< ), iii) optimal storage t (À °c vs. À °c), iv) optimal time between two openings of storage sites (> ). summary/conclusions: the pcs is a valuable system for plasma freezing validation and monitoring, as well as to perform monitoring and control of the whole pathway of frozen plasma in the be. it is a technologically advanced, easy-to-use and costeffective tool that can efficiently replace other traditional methods commonly used for the above-mentioned purposes. assessment of blood group matching quality using six sigma metrics background: six-sigma metrics provides a general methodology to evaluate a process performance on a sigma scale. implementation of six-sigma for quality assurance can benefit the health care sectors. one of the most important health care sectors is blood transfusion service. for that reason, maintaining a high quality in blood transfusion service is required. pathogen in activated plasma is one of the main products that are provided by the blood transfusion service. the process of producing pathogen inactivated plasma involves blood group matching step. the quality of this blood group matching is extremely significant for the delivery of plasma that satisfies the recipient need. aims: the aim of this study is to assess the quality of blood group matching of pooled plasma units using six sigma metrics, and to clarify the potential implementation of six sigma metrics as a quality management tool. methods: this retrospective study was conducted in the component preparation lab of kuwait central blood bank. the twelve months (january -december ) data of pooled fresh frozen plasma units were recruited and examined. the data was separated to data without double check ( months) and data with double check ( months). data statistics and analysis were conducted by the use of six sigma metrics. results: in a sample size of from the first six months a mismatch was found which equals dpmo and . sigma metric. and in a sample size of from the second six months a mismatch was found which equals dpmo and . sigma metric. out of the whole pooled units were found to be mismatched. some of which were found to be discarded as abo discrepancy, broken, or expired. other was still available in the system, while the rest of the mismatched units were issued. summary/conclusions: using the six sigma principle the study presents a successful assessment of blood group matching quality. as a . sigma metrics obtained from the first months, were shifted to a sigma metrics of . in the second months, after the addition of a double-checking step to the blood group matching of pooled plasma process. the implementation of these metrics in our laboratory quality management has been shown to be very beneficial. in which six-sigma metrics were able to clarify the reduction in blood group matching errors. although six-sigma benefits in major quality improvements and helps to reach an error free laboratory services, yet it presents a new challenge to laboratory practitioners. currently, the hemophilia a patients treated with factor viii concentrated as the first line of therapy but it is more expensive and the supply is not sufficient so for now they have not used factor viii concentrate as prophylaxis therapy. for some cases, hemophilia patients in indonesia depend on subsidy from the world federation of hemophilia. the first handicapped concentrated case is just for therapy not for prophylaxis. big blood centers in indonesia produce routinely fresh frozen plasma (ffp) and cryoprecipitate-anti hemophilic factor (ahf) as replacement therapy for hemophilia a, but its content and safety of factor viii from ml ffp need to be improved. nowadays, there is an available kit for producing minipool cryoprecipitate (mc) that has better safety and quality but it is available as liquid products, stored in very strict and specific temperature (À °c). prophylaxis therapy for hemophilia patients needs a stable product, easy to use and convenient treatment for patients. aims: to analyze the content and safety of f viii with minipool cryoprecipitate (mc) and lyophilized mc for home therapy. methods: we produced mc; mc as the control, mc were lyophilized with excipient and mc without excipient. we analyzed the number of factor viii, the safety, and stability. we count the erythrocyte, leukocyte and platelet residual in mc using flow cytometry. we also measure the ph, osmolality, solubility to learn its stability after storage at days at room temperature ( - °c) and blood bank refrigerator temperature ( - °c) at central blood transfusion services (cbts). results: we found the content f viii with excipient is higher ( . iu/ml) than without excipient ( . iu/ml) and the storage at blood bank refrigerator ( - °c) is better than at room temperature ( - °c) . in both group, there were no residual cells and bacterial found in mc. no significant difference in the ph, osmolality and solubility in both groups. summary/conclusions: the lyophilized mc with excipient stored at blood bank temperature ( - °c) is better than room temperature. this experiment will be continued to know its stability in extended storage time. background: peptic ulcer disease (pud) is a multifactorial and complex disease, and it affects a wide range of people in the world. however, a perfect therapy for pud has not yet been available at present. therefore, we provided a novel therapeutic approach for pud patients and observed its effect in this study. aims: we provided a novel therapeutic approach for pud patients and observed its effect in this study. methods: in this randomized controlled trial, pud patients residing in chongqing were enrolled from to . they were randomly assigned to two groups: (a) a control group used only rabeprazole, and (b) a platelet-rich plasma (prp) group that received a combined therapy of autologous platelet-rich plasma (aprp) and rabeprazole. the aggregation rate of aprp was measured via aggregation remote analyzer module. the therapeutic effect was assessed via the ulcer size and the symptom score. all data were recorded and analyzed statistically using spss. results: a total of patients were included ( patients as control group) and ( patients as prp group) in the analysis. we found that the aggregation rate of aprp is not affected in ph . after treatment with pepsin. our results showed that there were no significant differences between the prp group and control group before the treatment, and there was also no significant difference in healing time between the two groups in different variables. however, regression analysis revealed that the healing time was . d less in the prp group than in the control group, and the patients with higher symptom scores in the initial examination need more time to heal in treatment. summary/conclusions: this study showed an encouraging preliminary result that aprp has a positive result in the peptic ulcer patients, and it seems to be a better choice for refractory pud patients. despite the further follow-up studies are needed to determine the duration of efficacy of aprp, the approach will be helpful for improving the pud treatment in clinical. background: the croatian institute of transfusion medicine (citm) collects, produces and distributes blood components in an area of . million habitants. annually, it collects about , whole blood and , apheresis donations. platelet concentrates (pcs) are more inclined to bacterial contamination due to storage conditions that favor bacterial replication. the citm decided to evaluate the mirasol pathogen reduction technology (prt) system as it offers the possibility to work with a non-toxic, non-mutagenic compound that upon uv illumination induce nucleic acid damage, reducing the risk of septic transfusion. aims: the study objective was to evaluate quality of pcs treated with the mirasol prt system for platelets and stored in tpas+ for days at °c on a platelet shaker. methods: pcs were produced according to the citm's s.o.p., either through pooling of bc with tpas+, "wbd", or through apheresis collection using two devices: the fresenius amicus, "ad" and haemonetics mcs+ system, "mcd". pcs were stored in % plasma and % pas and mcd were subsequently evaluated also in % of plasma and % pas. identical pcs were produced with a pool-split protocol to be prt-treated or serve as untreated control. pcs were treated with the mirasol system according to manufacturer's instructions. qc parameters, such as yield, ph and swirl were measured at days , and . bacteria sterility test was performed at day for a sample of all treated platelets. protein content of pcs produced routinely at the citm was determined to assess accuracy of plasma carry-over calculations for all processed pcs. results: mirasol-treated wbd (n = ) and ad pcs (n = ) stored in % plasma showed at day an average ph ≥ . ; swirl ≥ . and yield = . . their untreated counterparts showed average values for ph ≥ : , swirl ≥ . and yield . - . . mcd stored in % plasma (n = ) that underwent prt showed at day average values for ph = . , swirl = . and yield = . . control mcd showed average values for ph = . , swirl = . and yield = . . mcd stored in % plasma (n = ) that underwent prt showed average values for ph = . ., swirl = and yield = . . their untreated counterparts had average ph = . , swirl = . and yield = . . total protein content in pcs derived from wbd (n = ), ad (n = ) and mcd (n = ) was g/l, g/l and g/l, respectively. while the coefficient of variation of wbd and ad ranged from % to %, plasma products respectively, the one of mcd reached %. all prt-pcs were negative for bacterial growth at day . summary/conclusions: mirasol treated wbd and ad produced according to citm current s.o.p. were quite similar to untreated controls at expiry, on day and passed the requirements of the eu guidelines ( th edition). quality of mcd units met eu criteria at day ; swirl decreased significantly at day which might be explained by the variability in plasma content of mcs+ -derived platelets, challenging the accurate calculation of illumination index for the mirasol treatment. all mirasol treated pcs showed minimal platelet loss at the end of storage. as the implementation of pr had to be cost-neutral it could only be implemented for~ % of the annual produced buffy coat platelet concentrates (bcp) (~ . bcp/year) and required a change in the bcp production method. the primary aim of the implementation was to offer increased blood safety to our most vulnerable patients. the secondary aim was to ensure that we built-up enough routine experience with pr to enable us to quickly ramp-up the production of pr-bcp to % if there were an outbreak of an emergent pathogen in the madrid region. aims: to verify if we could produce~ % pr-bcp without increasing the overall production cost (opc) for bcp. also evaluate the impact of pr on overall scrap rates of bcp, outdate rates and usage of other safety measures. methods: we compared opc for bcp between the pre-pr period ( ) . this cost was offset by substituting a semi-automated production method for bcp, which was used in to produce . % of bcp-units. a manual double dose buffy coat production method (dd-bcp) in combination with pr enabled us to reduce the bcp-disposables cost by . %. despite the moves from a semi-automated to a manual production method the overall scrap rates during production decreased in by . %. the extension of max. storage time from to days for % of the bcp-units that were pr resulted in decreasing our overall outdating rates by % (versus ). this reduction in outdating rates reduced our opc in by . %. in we gamma-irradiated . % fewer bcp-units, but this had only a minimal impact on the opc. summary/conclusions: results of this study confirmed that we reached our initial objectives of producing~ % pr-bcp without increasing the overall production cost (opc) of bcp. it enabled us to offer increased blood safety to the most vulnerable patients. we built-up enough routine experience with pr so we could quickly rampup the production of pr-bcp to % if there were an outbreak of an emergent pathogen in the madrid region. background: irradiation of red cell units is undertaken to prevent transfusion-associated-graft-versus-host-disease (ta-gvhd) in immuno-compromised patients. while irradiators using radioactive c-ray sources are primarily found in blood establishments, they require regular recalibration and supplementary safety measures. xirradiation has been shown to have similar biological effectiveness to c-irradiation and does not require a radioactive source. there is international interest in moving away from gamma sources to reduce vulnerability to terrorism. although damaging, impacts of irradiation on red cells are well recognised. only a limited number of studies have compared red cell component quality following cand x-irradiation for both standard volume red cell concentrates (rcc) and neonatal red cell splits (rcs). aims: to compare the in vitro quality of rcc and rcs when subjected to cor xirradiation on day of storage then stored for a further days. rcs were also irradiated on day of storage as that is most common practice in nhs blood and transplant (nhsbt). methods: four rcc were pooled and split into arms on day of storage, with units in each arm. all units received an irradiation dose of . - . gy. two arms remained as standard volume rcc and were either cor x-irradiated on day of storage. the other two arms were both split into rcs on day of storage before being irradiated on day (early arm) or day (late arm) of storage. for each replicate in these arms, splits were c-irradiated and splits x-irradiated. all arms were tested a day prior to irradiation and , and days post-irradiation for red cell quality parameters: haemolysis, intracellular atp and , dpg, supernatant potassium, glucose and lactate, ph and red cell microvesicle release. the rcc arms were sampled over storage; while for the rcs arms, split was tested on each testing day post-irradiation. a -way anova was used to detect statistical differences over storage between cand x-irradiation for the same components. results: all components produced were within nhsbt specification for volume, haemoglobin and haematocrit. there were no significant differences in red cell in vitro quality parameters studied over storage between cand x-irradiated units, for standard volume arms or neonatal arms and whether rcs were irradiated early or late in storage. moreover, all arms were within haemolysis specification for the end of storage (> % of units with < . % haemolysis) and % of units had atp levels above the recommended minimum for acceptable post-transfusion survival ( . lmol/ghb). both haemolysis and potassium levels at the end of storage for the standard c-irradiated rcc were comparable to our laboratory's historic data for the same component. summary/conclusions: in summary, the storage quality of rcc and rcs post-xirradiation did not differ from c-irradiation in this study, providing reassurance that either method could be used in routine manufacturing. a pajares herraiz , c coello de portugal , m morales , f solano , c perez parrillas , a rodriguez hidalgo , t diaz rueda and m flores direccion, regional transfusion center toledo-guadalajara transfusion service, toledo hospital complex, toledo transfusion service, general university hospital of guadalajara, guadalajara transfusion service, hospital nuestra señora del prado de talavera de la reina, talavera regional transfusion center, regional transfusion center toledo-guadalajara, toledo, spain background: the regional transfusion center of toledo-guadalajara (rtc) manages the collection, processing and distribution of blood components for the hemotherapy area of castilla la mancha (spain) that serves general hospitals (hospital complex of toledo (hct), university general hospital of guadalajara (ughg) and hospital nuestra señora del prado de talavera (nspt)) and the needs of , inhabitants. by also managing the hct transfusion service, it facilitates the handling of stocks. since , rtc has initiated pathogen inactivation (pi) for a part of its platelet components(pc) with the intercept blood system (cerus) using a photochemical treatment with amotosalen and ultraviolet-a. this system allows the inactivation of a broad panel of pathogens and leukocytes, extending the shelf-life of the cp from to days. this affects the expiry and discards of this blood component, allows a better management of the inventory and has an influence on production costs. aims: the objective was to evaluate the influence of pi in the production of cp at rtc and the expiry in the hemotherapy area during the last years divided into four periods ( results: pc were predominantly obtained from whole blood collections with % of bc platelets/ % of apheresis platelets. % of the available bc were used in production for period a and % for periods b, c and d. after wastes of approximately . %, the distribution of pc was stable for the periods studied. pc were distributed for period a, pc for b, pc for c and pc for d. the % of pi platelets with -day shelf life available in the hospitals was limited to % during period a. it was then increased to . %, . % and % for periods b, c and d respectively. the percentage of wastes was stable at . - . % but the discards due to expiry went down from . % (period a) to stabilize at . % in periods b and c and . % in period d. in the general hospitals the expiry went down from % to . %(hct), . % to . % (ughg) and . % to . %(nspt) respectively. summary/conclusions: greater control of pc stocks through historical analysis and consumption projection, together with it tools and the use of pi pc with -day shelf life allowed reducing discards for expiry from . % to . % in the last period analyzed at rtc and the major hospitals of the hemotherapy area. this has a great value in cost-reduction and improves inventory management and the efficiency of the processes. background: blood centers are faced with many challenges including availability of concentrate platelets as well as ensuring highest quality of the product. overcoming the shortage of platelet apheresis by using pooled platelet derived from whole blood units separated using automated standardized system, which can assist blood banks to meet the increase demand in platelets. the pathogen inactivation (pi) technology can improve the quality of the product by mitigating the risk of transfusion-transmitted diseases (ttd) and residual white cells, resulting in minimizing non -hemolytic transfusion reactions. however, the pathogen inactivation treatment must not impact the platelet quality and functionality significantly, as well as the patient safety. aims: evaluate the quality of pooled platelets derived from whole blood (five interim platelet units), separated using reveos automated blood processing system (terumo bct), pooled in % donor plasma and pathogen inactivated by amotosalen/uva technology. methods: five interim platelet units (ipus) produced with reveos device (terumo bct) from single whole blood donations, were pooled with a platelet pooling set (terumo bct) and leucodepleted with a lrf-xl filter (haemonetics). thirty pools have been included in this study, the units were treated using a large volume cerus intercept processing set for platelets according to the manufacturer's instructions and stored until day . the swirling was determined by visual inspection. the volume and yield content were assessed preinactivation and after treatment by pathogen inactivation with a cell counter (dxh- , beckman coulter), rbc contamination was also measured preinactivation with a cell counter (beckman coulter), bacterial contamination was assessed by automated blood culture with a bact/ alert system (biomerieux). the ph of the platelet units was assessed with a phmeter (jenway), and residual amotosalen levels were assessed by an hplc assay. results: the impact of amotosalen/uva pathogen-inactivated pool platelet products quality were assessed. the pre and post-inactivation of the units showed a swirling score of - . the average volume per unit of the pre-inactivation was ml ( - ml) and post inactivation was ml ( - ml), with average volume loss during inactivation was ml ( - ml), corresponding to % ( - %). the average platelet yield per unit pre-inactivation was . ( . - . ) and post inactivation . ( . - . ) with an average platelet loss of % ( - %) . the average rbc contamination per unit ( . - . rbc/ml). the culture tests were negative, the average ph at day was . ( . - . ), average ph at day / was . ( . - . ). the average residual amotosalen concentration post treatment was . lm ( . - . lm). summary/conclusions: the quality of pathogen-inactivated pool platelets tested, met the criteria set by aabb guidelines. the volume and platelet loss were in acceptable range, in alignment with previously published data. a residual amotosalen concentration below lm is considered safe and acceptable by french and german authorities. the evaluated data support the reasonable assurance of quality and effectiveness of the device when used in accordance with indication for use. background: the implementation of a pathogen inactivation process (pi) allows the redesign of processes to obtaining safe blood components by reducing the need for additional testing for pathogens detection, minimizing the residual risks (such as the infectious window period for those pathogens that are detected as usual), eliminates the need for selective tests (eg cytomegalovirus serology test) and complements gamma irradiation given its ability to inactivate white blood cells. in addition, the routine implementation of pi reduces the incidence of bacterial infection in recipients of blood components and allows blood services to proactively protect the blood supply against future emerging infections. aims: to verify the functional integrity and viability of platelet concentrates after being inactivated of any pathogenic agent, to be used as safe and functional components for transfusions. methods: a total of independent platelet concentrates were studied. platelets are donated through a process called plateletpheresis according to the established norms, after the process, platelet concentrates were submitted to pi on the intercept blood system tm platform with uv-a illuminator; an immediate sampling of each donation of platelet concentrates was carried out taking a sample of ml pre-inactivation and another sample post-inactivation ( h after pi). the platelet viability of each sample was evaluated by demonstrating the cd p expression marker by flow cytometry. once processed, platelet concentrates were released as safe components for donation. compiled the experimental data of the platelet count with platelet activation marker with respect to the total platelet, a comparative, nonparametric test of wilcoxon was carried out between two measurements (pre vs post) and the platelet viability after pi was determined. results: a total of independent platelet concentrates were studied, where the average percentage of pre-inactivated platelets with expression of the cd p marker, was %, while the percentage of functional platelets post inactivation was %, this result only shows that the functionality of the platelets is not being altered after the inactivation process. the wilcoxon test confirms that there is no significant difference between platelet activity pre-and post-inactivation, with a % confidence level. summary/conclusions: the process of photochemical treatment with amotosalen hydrochloride and long-wavelength ultraviolet light (uva) applied to platelet concentrates provides functional products without alterations in platelet function to be transfused. background: treatment of platelet concentrates (pcs) with pathogen reduction technologies is widely implemented in blood establishments to reduce the risk of bacterial contamination and to face the presence of new emerging agents in blood components. aims: the reduction of antioxidant power (aop) could be a quality control test to prove the complete viro-inactivation treatment. this evaluation has the goal to study the feasibility of the method from "abonnenc et al., transfusion, " in another blood service, assessing the aop of platelet units treated by intercept technology. methods: the aop is expressed in edel value, one edel being equivalent to lmol/l ascorbic acid. repeatability, intermediate precision and accuracy were determined. linearity was evaluated using the linear regression and the calculation of pearson's coefficient (r²). limit of quantification (loq) was determined by measuring aop using nacl samples to define the background. roc curves were used to determine a threshold to discriminate pcs before and after treatment. a distinction was realized between men and women and between apheresis (a) pc and buffy coats (bc) pcs. a one-year evaluation was assessed on pcs before and after treatment on the routine production. results: the coefficient of variation for the repeatability was less than %. for the intermediate precision, the coefficient of variation was less than %, but for the pcs after treatment, this result rose up to %. the r² value for the linearity was . %. the detection limit corresponded to a result of edel and the loq (equal to xsd) is edel. concerning roc curves, the men apcs threshold was . edel compared to women apcs with . edel. the threshold for bcpc was edel. all of these results had % of specificity. below this threshold, intercept treatment was considered to be executed. about the one-year experience on routine pcs production, apcs ( women and men) and bcpcs were tested. all of the bcpcs and women apcs were under the threshold after treatment. concerning men apcs, . % of the pcs after treatment were not under the threshold. summary/conclusions: the device validation was satisfied. for the one-year evaluation and concerning men group apcs, the threshold found by abonnenc et al. was edel. our study showed a threshold with % specificity and % sensitivity at . edel which is much lower. specificity was favored compared to sensitivity but the analysis should be revised to adapt the threshold to get higher sensitivity. this can lead to reduce the non-conformity and allows measuring the aop only after treatment. for women, our threshold was found at . edel compared to . edel for abonnenc et al. concerning sex in apcs, results were statistically lower in women group than men group before and after treatment. and for bcpcs, the two populations (before and after treatment) were very distinguishable and our threshold ( edel) was lower than abonnenc threshold which was at . edel. in conclusion, edel threshold enables the segregation and depends on the preparation process adapted in each blood service. aims: this study has the goal of measuring antioxidant power (aop) level in plasma units treated by mb technology. the aim is to use such a test as a quality control assay for documenting the execution of pathogen inactivation treatments during the preparation of plasma units. methods: aop measurements were performed using a potentiostat electrochemical analyzer. a -ll volume of sample is deposited over the electrodes on a single-use microship. the aop is expressed in edel value, one edel being equivalent to lmol/l ascorbic acid and reflects the redox status of the plasma units. different protocols were established to understand the role of mb, the illumination and the filtration on the aop variation measure: ) complete treatment, ) plasmas with mb without illumination, ) plasmas without mb with illumination and ) plasmas without mb without illumination. ten dosages on men donor samples, except for protocol where n = , were realized during the viro-inactivation process, t corresponds to a dosage of plasmas before treatment, t the plasma after the mb dry tablet passage, t is the time after illumination and t corresponds to the final product (after filtration). results: in each protocol with mb, an increase was observed after addition of mb before illumination. after illumination, the edel values decreased for about less than %, which was expected because of the degradation of mb in its photoproducts during the illumination. in the series and , the illumination seemed to have an effect by itself, with or without mb because the aop increased. the final filtration has the goal to eliminate the residual mb and its photoproducts. after this step, the aop values fell down. the series was a confirmation of the efficacy of the filter to remove the mb as shown by the decreased aop in t ( ae edel at t and ae edel at t ). however, in the absence of mb (series and ), the results at t and t were not statistically different. summary/conclusions: the filtration decreases the aop rate, except when there was no mb. the results of non-complete viro-inactivation treatment allow concluding that the measure of aop rate may not indicate that the treatment was completed or not since significant differences before and after treatments were found in the non-complete treatment series. vox sanguinis ( ) background: the intercept blood system (ibs), a photochemical treatment with amotosalen and uva, is used to inactivate pathogens and leukocytes in plasma. the intercept tm plasma processing set (cerus bv, netherlands) was modified to incorporate plastic containers in non-pvc materials sourced from alternate suppliers and connecting parts and accessories in non-dehp pvc formulations, making the system dehp-free. the final storage container was modified with a higher contact surface with plasma to limit the thawing time. proportion of units with a fibrinogen concentration ≥ . g/l was % (> % required). mean recovery fviii fibrinogen after ibs treatment and frozen storage were % and %, respectively. residual platelets were < /l, leucocytes < /l and red blood cells < /l. all units had a protein content > g/l. residual amotosalen was below lm in all post-cad samples. the concentration of tat complexes was slightly reduced after treatment and frozen storage. concentrations of c a and c a were significantly reduced with the cad treatment. the plasma thawing time in a water bath at °c was consistently short ( - min). summary/conclusions: pathogen inactivated plasma units (ffp-a-ibs and ffp-wb-ibs) prepared with dehp free intercept processing sets retained in vitro characteristics which meet the quality standards for therapeutic plasma. the process did not activate coagulation or complement. reducing ffp thawing time from routine - to - min is an important benefit for emergency use. background: plasma coagulation factor concentrations usually differ for individual donors, therefore pooling of whole-blood derived plasma units moderates high or low coagulation factor concentrations and ensures transfusion of more standardized blood components. moreover, pooling contributes to dilution of reactive antibodies and may reduce the risk of non-hemolytic transfusion reactions and trali. additionally pathogen inactivation reduces the risk of transfusion-transmitted infections, and non-hemolytic transfusion reactions as well as gvhd through inactivation of residual lymphocytes. aims: assessment of the impact of plasma pooling and pathogen inactivation on the standardization of blood components and plasma quality. methods: the study included experiments. for each experiment male-donor, abo-compatible whole-blood derived plasma units (≥ ml) were collected from different donors and pooled using the donopack optipool plasma pooling set (cerus europe b.v.). each of the -unit pools were split into equal minipools which were subsequently treated with the in intercept blood system (cerus europe b.v.). then, each minipool was split into (≥ ml) therapeutic units. samples were collected before and after pooling as well as after inactivation to assess the coagulation factor content (fviii, fix, fibrinogen, vwf antigen using elisa) and coagulation time (aptt, pt). the study-analysis included samples from five pools from single plasma units respectively ( background: biotin (bio) is an alternative to radioactive red blood cell (rbc) tracers which allows one to concurrently track in vivo multiple cell populations labeled at different bio densities. in american clinical trials, multi-labeled biorbc have been transfused in man to assess their survival (mock et al, transfusion, ) . in these studies, the different biorbc populations were monitored by ex vivo flow cytometry analysis using streptavidin. so far, the biotinylation reagents biosulfonhs was not complying with good manufacturing practices (gmp). moreover biorbc, with bio ≥ lg/ml, have induced immunization of the recipient, in rare cases (schmidt et al, transfusion, ) . this represents an obstacle regarding the regulatory european authorities. aims: the aim of this study is to describe a procedure of biotinylation of rbc intended for clinical trials while refining the levels of bio ≤ lg/ml. methods: sterile status is met throughout the process. rbc are taken from standard rbc concentrates and treated with biosulfonhs of gmp-grade ( to lg/ml) recently commercialized. washing buffer is of injectable-grade. biotinylation efficacy is controlled by flow cytometry with streptavidin conjugated to different fluorochromes: phycoerythrin (pe) or brilliant violet (bv ). results: labeling with biosulfonhs of gmp-grade or non gmp-grade is comparable and populations of rbc could be easily distinguished between themselves and from unlabeled blood cells. biosulfonhs (lg/ml): (mfi . ), (gmp mfi ; non gmp mfi . ), (gmp mfi ; non gmp mfi ), (gmp mfi ; non gmp mfi ). streptavidin-bv brighter than streptavidin-pe is a promising tool because it amplifies by . the signal of fluorescence and allows a good differentiation of the populations of rbc treated with only , , and lg/ml biosulfonhs. summary/conclusions: this preliminary study explores the feasibility of multilabeled biorbc production for clinical trials. the benefits of this approach are to overcome the need for non-radioactive tracers, to follow simultaneously various populations of rbc and consequently to limit the number of volunteers, and to reduce the risk of immunization using bio ≤ lg/ml. background: rejuvenation is aiming to revert ageing-related disease development. heterochronic parabiosis studies revealed eotaxin in young and old murine blood as a regulator of brain aging and neurogenesis. umbilical cord blood (ucb)-borne factors including tissue inhibitor of metalloproteinases (timp ) and neonatal immune cells also contributed to rejuvenation in animal models. human platelet lysate (hpl) is commonly used by us and others for highly efficient cell propagation in vitro (burnouf et al., biomaterials, ) . published data indicate only limited differences between adult and ucb-derived hpl, partly questioning enigmatic rejuvenation effects. aims: to verify candidate regenerative factors in neonatal blood products we compared protein contents of neonatal and adult plasma and platelets, respectively. methods: heparinized ucb samples (n = ) were centrifuged within h to collect neonatal platelet rich plasma. aliquots from apheresis platelet concentrates (n = ) were used as adult counterpart. platelet concentration was adjusted to - / l. plasma supernatants and platelets were obtained by centrifugation and platelet pellets were re-suspended in saline. after two freeze/thaw cycles at À °c/ °c for platelet lysis (npl; apl) the platelet fragments were removed by centrifugation. the protein content was analyzed with a proteome profiler tm array. nine samples of each group were pooled to avoid individual donor variations. a threshold of , au spot density was defined as cut-off. data were analyzed by graphpad prism using two-way anova. results: semi-quantitative evaluation of analytes per array revealed significant differences. in plasma samples and platelets and analytes were detected above cut-off, respectively. in neonatal plasma we found more highly prevalent proteins (> , au spot density) compared to adult plasma ( / vs. / ). thirteen proteins were significantly elevated in neonatal plasma including growth/differentiation factor (gdf ), platelet derived growth factor aa (pdgf-aa) and serpin e (p < . ). more highly prevalent proteins were detected in npl ( / ) compared to apl ( / ), and proteins were significantly elevated including vascular cell adhesion molecule- (vcam- ), platelet factor (pf /cxcl ), epidermal growth factor and lipocalin- (p < . ). in adult samples only proteins were significantly higher in plasma and three proteins in apl compared to the neonatal groups (p < . to p < . ). summary/conclusions: we detected significant differences in regenerative growth factor and cytokine contents of neonatal and adult plasma and platelet samples, respectively. additional experiments are underway to further characterize their impact in distinct functional readouts. background: the production and storage conditions of platelet (pl) products intended for transfusion are constantly evolving and need sometimes in vivo evaluations in clinical trials to ascertain whether the platelets have retained their ability to survive in the circulation. this requires that the transfused platelets can be distinguished from the recipient's circulating platelets. labeling of platelets with biotin (bio) affords to track in vivo and concurrently, multiple cell populations covered with various biotin densities as already described for red blood cells (mock, transfusion, ) . surprisingly, there is only one study describing the transfusion of human biopl (stohlawetz, transfusion, ) . so far, the biotinylation reagent bio-sulfonhs was not complying with good manufacturing practices (gmp), which represents an obstacle regarding the regulatory authorities. aims: the aims of this study are ) to describe a procedure to label injectable human platelets with densities of biotin, ) to evaluate the impact of biotinylation on platelet functions, ) to track human biopl in the circulation of the mouse. methods: platelets are taken from standard platelets concentrates and treated with . and lg/ml biosulfonhs of gmp-grade, recently commercialized. main platelet functions are assessed in vitro. human biopl survival is evaluated in immunodeficient nsg-mice treated with liposome-clodronate to eliminate macrophages and to prevent rejection. circulating human biopl are detected ex vivo by flow cytometry with streptavidin phycoerythrin. results: using trap ( lm), p-selectin externalization reveals a normal capacity of secretion for all biopl. gpiba and gpiibiiia expression is not affected by the biotinylation process. biopl have the ability to aggregate: using arachidonic acid ( mm), amplitude of aggregation is . ae . % (bio ); . ae . % (bio . lg/ ml); . ae . % (bio lg/ml). using collagen ( . lg/ml), amplitude of aggregation is . ae . % (bio ); . ae . % (bio . lg/ml) . ae . % (bio lg/ml). the biopl populations could be easily distinguished between themselves and from unlabeled blood cells in the mouse circulation during more than h. after h, the mean fluorescence intensities are . ae . for unlabeled circulating mouse platelets, . ae . and . ae . for circulating human biopl covered respectively with . and lg/ml biotin. summary/conclusions: this labeling approach should be helpful to evaluate new platelet products in vivo and represents an alternative to radioactive tracers. it allows to follow simultaneously different platelet populations and consequently limits the number of volunteers in clinical trials. background: severe ocular surface diseases, dry eye syndrome, persistent and recurrent corneal epithelial defects and diabetic or neurotrophic keratopathy are mainly successfully cured by standard treatment protocols. however, not rarely does refractory to these usual treatments appear, especially with serious forms of disease. in military medical academy, autologous serum eye drops -auto seds and autologous platelet lysate -apl eye drops have been being applied in the treatment of ophthalmological patients in these categories, who were previously resistant to standard therapy. aims: to show the achieved results of therapeutic use of autologous blood products (auto seds and apl) in the treatment of ophthalmological patients who previously had not responded to conventional therapysingle center experience. methods: auto seds are prepared by taking autologous blood into tubes (bd vacutainer, cat, ml) and apl in tubes with anticoagulants (greiner bio-one, acd-a, ml). control on tti of every patient and sterility of every series has been conducted. before and after the treatment, subjective ocular discomfort (ocular surface disease index -osdi), objective parameters of the tear film (schirmer's test, rose bengal, tear breakup time -tbut) and measuring of epithelialization zone were analyzed. apl, obtained from platelet-rich plasma which had been frozen, unfrozen and diluted with nacl solution, up to %. auto seds were administered in the form of % eye drops. results: auto seds have been applied to ophthalmological patients ( men and women), previously resistant to standard therapy. in total treatments were performed (each lasted days). for successful curing, one or two treatments per patient, in average, were applied. apl has been used multiple times to one patient with sj€ ogren syndrome and severe multiple tropical corneal changes. all ophthalmological patients had subjective improvements (the average pre and post treatment osdi scores were . and . respectively). also, objective progress was present in % of all patients (p < . ). summary/conclusions: the use of auto seds and apl in the treatment of ophthalmological patients, previously resistant to standard therapy, is in constant increase, because of its simplicity and low expenses. apl has turned out to be better than auto seds for patients with severe trophic changes, because apl contains larger amounts of the nerve growth factor, tgf-b, vegf and platelet derived growth factor. however, a larger number of clinical cases is needed for future conclusions. background: whole blood (wb) has recently regained favor in treatment of massively bleeding patients in military and civilian settings. platelets (plts) are a vital component in clot formation. as a component of wb, it is critical that they maintain functionality throughout storage. red blood cells (rbcs) stored in hypoxic/ hypocapnic conditions preserve high level of , -dpg while reducing storage lesions stemming from oxidative stress. , on the other hand, effects of steady hypoxia (pco ~ - mmhg) on plts contained in leukoreduced wb is poorly characterized. aims: examine the effects of hypoxic conditions on plt function and microvesicle (mv) formation in wb stored hypoxically (h) and conventionally (c) for -week storage at - °c. methods: units of wb were collected at mayo clinic rochester blood donor center from normal healthy volunteers into ml cp d. wb was leukoreduced using plt-sparing filter (terumo wb-s) then split into control (c) and hypoxic (h). h-wb was processed by the oxygen-reduction bag (hemanext, lexington ma) and unit was stored in o -free bag. ml of wb were collected from each unit at day , weeks , , . plt counts, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation, nonactivated and agonists activated plt surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac- binding), and microvesicles (mv) were measured by coulter counter and digital flow cytometer. paired student t-tests were used to analyzed differences in degradation rates; significance: p < . . results: h-plt counts declined to~ % by the o -reduction process, while similar decline was observed after week in c, and thereafter remained steady. plt activation (ps) increased over time (h >> c after processing; c increasing more rapidly during storage). p-selectin increased over time (h < c), while pac- showed large increase after week, then remained steady (h << c). plt activation by trap or adp declined modestly over weeks (~ %) while h-plt showed additional~ % reduction for all time points. collagen activation for c-plt increased after week ( %) and gradually increased to % after weeks (~ % reduction with h compared to c). plt-derived mv (cd and cd /annexin v) increased~ -fold over storage time; day mv levers were significantly higher for h, but subsequent increase rates were similar or lower. total number of plt-derived mv (cd a) in wb supernatant increased -fold after weeks for c, while h suppressed increase to -fold. (majority of the trends described above showed significant differences between h and c.) summary/conclusions: plts were activated over -week period when stored at - °c in leukoreduced wb, accompanied by a modest loss of agonist-induced activation. oxygen reduction treatment initially activated h-plts, while subsequent increase in activation rates were suppressed compared to c-plts. wb plts retained activatability, and hypoxic condition showed only modest further reduction on the activatability. hypoxic wb may provide higher quality wb for trauma patients if the levels of initial plt activation can improved during oxygen reduction procedure. methods: after informed consent, eligible patients were randomized to either first receive autologous followed by allogeneic seds or first receive allogeneic followed by autologous seds. each sed treatment phase was one month, separated by one month of patient's standard treatment (wash out period) between sed treatment phases. the patients each donated ml whole blood from which the autologous seds were prepared. allogeneic seds were prepared from blood from never-transfused male donors with blood group ab. all serum was diluted : by adding saline, and aliquoted in an eye drop dispensing system (meise, schalksm€ uhle, germany). at each visit, the osdi was determined using a validated questionnaire, with higher scores reflecting poorer outcomes. the results were analyzed intention-to-treat, and a random effects linear mixed model for cross-over design was used. results: in total, patients were enrolled, of whom were excluded because they failed the autologous blood donation. background: the following blood components for non-transfusional use (bcntu) are produced in our transfusional center (tc): ) allogeneic platelet gel (pg), derived from buffy-coats (bc) and human cord blood platelet gel (cbpg); ) autologous serum eye drops (sed). the creation of both types of platelet gel started in but only in we confirmed the process for daily production: these blood components are used to treat pediatric patients with epidermolysis bullosa. the sed, produced from , is dedicated to treat patients with dry eye syndrome. aims: production and storage bcntu. methods: the whole process production of bcntu is traced on the transfusional informatic system (emonet-insielmercato), under the same conditions of another blood transfusional components. the process takes place in closed circuit using the laminar flow hood. ) pg production starts from the bc resuspended in plasma that are not used for daily platelet concentrates, instead the cbpg is produced using cord blood units that are not used for hematopoietic transplant. both have a platelet concentration between - /ll and negative blood cultures, required by the italian law; the units are frozen at À °c and last -year. pg and cbpg must be activated with calcium gluconate or batroxobin to be used. ) the ophthalmologist's patients, with dry eye syndrome, donate ml of autologous blood; the serum is separated and after the dilution with a balanced saline solution ( %) are divided in boxes containing single-dose vials each: they are stored at À °c and they last one year. negative blood culture was evaluated. results : background: candida albicans is the most common pathogen detected in fungal infections. aims: in this study, we aimed to evaluate the in vitro antifungal activity of volunteerderived platelet rich plasma (prp) against c. albicans atcc strain and the possible effects of certain chemokines, kinocidins that might play a role in this activity. methods: prp from nine volunteers were derived by using magellan prp â kit. % calcium gluconate was used to obtain autologous thrombin. c. albicans isolates with a final yeast concentration of cfu/ml and cfu/ml were inoculated on sabouraud dextrose agar at the st , nd , th , th and th hours of incubation to reveal the antifungal activity of autologous thrombin-activated prp. the colonies were counted after - h of incubation at °c. chemokines and kinocidins (platelet factor- , interleukin- and thymosin-b ) were also measured simultaneously by elisa method. results: compared with the pbs-control group, the prp- group showed that the antifungal activity was still going on at the th hour. the difference in colony production between the two groups at th hour was statistically significant (p < . ). it was observed that the antifungal activity continued at the th hour, decreased at the th hour in the group prp- group. although the same amount of prp was used and the same amount of chemokine and kinocidins were released in both groups, the concentration of c. albicans was considered to be important in the detection of more effective prp- group. although there was an increase in il- levels by hours in the two prp groups by elisa method, no antifungal effect was detected against c. albicans. it was observed that decrease in tmsb values results from the antifungal activity on the advancing hours in the prp groups. whereas pf- did not act an antifungal activity on prp- and prp- . summary/conclusions: even in our study group where the highest platelet counts were obtained at the lowest concentration, c. albicans reproduction could not completely eliminated as mentioned in the literature. repeated doses of prp applications, such as drugs used in patients, may have longer duration of action and even complete repression of reproductive outcomes. background: generally, blood is available in developed countries for transfusion. sometimes, transfused or previously pregnant patients form alloantibodies to red cell antigens and rarely, to antigens of high prevalence. this case focuses on a twoyear-old girl, of pakistani descent, diagnosed with neuroblastoma stage iv with anti-in b and -e. although the publications indicate that % of the pakistani, indian or iranian populations are in(b-), it was discovered that this blood type is exceedingly rare. an international search was required to ensure blood product availability for chemotherapy and autologous hematopoietic progenitor cell transplants aims: illustrate the response of the public to a powerful story of a child needing rare blood for treatment and international collaboration for provision of very rare units. methods case report: a two-year-old patient's sample was referred for antibody identification. the patient had received four transfusions ( ml of red cells) in the preceding -day period. hgb level fluctuations were consistent with decreased transfused red cell survival. following the last transfusion of ml, the hemoglobin decreased from . to . . anti-in b , and a ficin-only reactive anti-e was identified in the serum and anti-in b in the eluate. the monocyte monolayer assay predicted the anti-in b to be clinically significant ( % reactivity). transfusion of antigen neg units once obtained, resulted in a stable transfusion response. although it was expected that in(b-) blood would be more easily sourced, only two donors in the usa were in (b-) e-. as - units of blood were requested for the post-transplant period, a national and international search was initiated, as was a robust media appeal to donors resulting in many donors for an intense domestic screening effort in the usa. the search of the who international rare donor panel by the international blood group reference laboratory revealed three known in(b-) e-donors; two british and one australian. they were contacted, recruited, collected and shipped to the usa with the work of the american rare donor program (ardp) staff and the isbt working party on rare donors (isbt wprd) members in each of the countries. results: the intense media coverage of oneblood (the florida blood center collaborating on treatment with the hospital) included online news outlets (youtube, facebook) resulted in over , responses from national and international potential donors to be tested for in b . isbt wprd members were sent the web information of potential donors identified in their countries by the ardp. over , samples from blood centers and associated laboratories tested with anti-in b by oneblood. two new in(b-) donors were discovered ( . %); but both typed e+, thus were not a match for the child. summary/conclusions: this intense media coverage and the overwhelming donor response was unprecedented in our experience. the coordination and cooperation among the numerous blood centers reflect the deep dedication of the blood banking community to the well-being of special patients in need. this case illustrates the response potential that a powerful story and a medical appeal for exquisitely rare blood utilizing social media and other online news outlets can generate. background: blood platelet units are generally stored in blood banks for - days, afterwards they are discarded. prepared infusible platelet membrane (ipm) from fresh or outdated human platelets correct the prolonged bleeding times in thrombocytopenic animals such as rabbits. infusible platelet membrane (ipm) as a platelet substitute may be the most feasible approach to reach the target market. our previous experiments have shown that ipm has a hemostatic efficacy to shorten bleeding time without any adverse effects in rabbits. aims: abnormal toxicity is the european pharmacopoeia standard for assessment of biological products which the test material is administered to the mice. in this study, abnormal toxicity of ipm was evaluated in experimental animal model such as mice to assure the safety of ipm without any evidence of serious toxicity. methods: in this experimental study, infusible platelet membrane (ipm) was prepared from outdated platelet concentrates. platelet concentrates were pooled, disrupted by freeze-thaw procedure, pasteurized for h to inactivate the possible viral or bacterial contaminants with a sodium caprylate stabilizer, formulated by sucrose and human serum albumin and finally lyophilized. at first, the test for sterility is carried out under aseptic conditions for ipm vials and then we injected . ml of ipm ( mg/kg) intravenously between to seconds into each health mice, weighing - grams. these tests were performed according to eu pharmacopeia monographs. results: in the sterility test no evidence of microbial growth in our product is found. the abnormal toxicity test will be passed if none of animals die during h after injection. if more than one animal dies, the preparation fails the test. if one of the animals just dies, the test is repeated. in our experiment all five mice were alive after h of ipm injection. summary/conclusions: in this research the results showed that ipm as a platelet substitute is free of abnormal toxicity with adequate safety and it may be used in human clinical trial studies as a feasible approach to develop a platelet substitute in the future. however, further studies are required to confirm the different aspects of its safety as well. the success of such investigations may affect patients' care in transfusion medicine in the future. a substantial number of infants, especially premature infants, are unable to receive adequate amounts of their mothers' milk for a variety of reasons. the world health organization recommends that infants, especially preterm and ill infants are fed with quality-controlled donor milk if they cannot be fed with their own mother s milk. due to the possible transmission of the human immunodeficiency virus many human milk banks closed in the s, therefore the availability of donor milk has decreased. aims: we analyzed the processing of donor milk and the required laboratory tests to establish a human milk bank within our blood donation service in cooperation with the department of neonatology at the frankfurt university hospital. methods: based on the recommendations for promoting human milk banks in germany, austria, and switzerland (efcni) we evaluated the manufacturing steps and the quality controls require to establish a human milk bank. background: for patients suffering from severe ocular surface disorders treatment with blood derived serum eye drops (sed) is a highly effective therapy. autologous sed, prepared from the patient's own blood, is used preferably. for this approach we have more than years of experience. if auto-sed cannot be manufactured due to medical reasons allogeneic sed present an alternative. since years, the allogeneic approach is well established in our center. aims: retrospectively evaluation of our experience with allo-sed. methods: in germany manufacturing of allo-sed is only possible as an "individual healing attempt". for each patient experienced regular ab -identical male donors without blood borne disease, who never received blood products and not taking any kind of medication are selected. additionally, donors must pass a questionnaire excluding any form of dry eye syndrome. allo-sed are manufactured directed for each individual patient according to the process for auto-sed in a closed system. patient files of our serum eye drops donors were screened for patients receiving an allogeneic treatment. data concerning indication for allo-sed, contraindication for phlebotomy, problems with donor selection and manufacturing, as well as serological and microbiological testing results were obtained. clinical results were evaluated © the authors vox sanguinis © international society of blood transfusion vox sanguinis ( ) (suppl. ), - by ocular surface disease index (osdi) and patient's questionnaire, asking for subjective benefit, symptom reduction, possible side effects, consumption and comparison with artificial eye drops or, if applicable, with auto-sed. furthermore patients are undergoing regular ophthalmologic examination within a special consultation for dry eye syndrome at our hospital. results: patients were identified receiving allogeneic sed, patients had been treated autologous previously. in total, allogeneic sed have been produced times since june . indications were ocular gvhd (n = . %), neurotrophic keratopathy (n = . %), mucous membrane pemphigoid (n = . %), sj€ ogren syndrome (n = . %) and secondary keratoconjunctivitis sicca by virtue of chemotherapy, meige syndrome, rosacea, morbus bruton (n = . %). contraindications for autologous donation were underlying disease (n = . %), poor venous access (n = . %), low haemoglobin (n = . %), low body weight (n = . %), very young age (n = . %), circulatory disturbances (n = . %) and lack of response to auto-sed (n = . %). some patients presented more than one contraindication. manufacturing problems were: lipemic donor plasma (n = . %), high donor haemoglobin (n = . %) and unspecific positive serological findings (anti-hbs n = . %). microbiological testing was sterile every time. as side effects one case of allergic reaction, suspected as serum protein allergy, appeared. clinical outcome can be considered equivalent to ased. subjectively, all patients benefited from the therapy and reported an alleviation of their symptoms. for some indications (highly active gvhd) allo-sed might even be the better option. summary/conclusions: considering our previous experience, allo-sed seem to be a safe and equally effective alternative to auto-sed for patients unable to donate blood. in case of urgent indication, timely supply can sometimes be difficult. to overcome this disadvantage licensing allo-sed as a new blood product with the possibility of production and storage in advance would be a desirable goal. in addition supply would become even safer by preparing allo-sed according to a quarantine principle like ffp. abstract withdrawn. background: vernal keratoconjunctivitis is a chronic, recurrent bilateral inflammation of the outer ocular layer. mostly affected are children and young people and the condition is more common in boys. the disease presents with eye pruritus (itching eye), photophobia (sensitivity to bright light), excessive tearing and foreign eye syndrome. severe cases manifest with diffusion of overgrown papillae usually of the upper eyelid, bursting of the connective tissue barriers and appearance of giant papillae that press on the cornea. corneal ulceration is a severe complication of vernal keratoconjunctivitis that may induce scarring, corneal neovascularization and occasionally perforation. treatment of keratoconjunctivitis mainly relies on steroids, mast cell stabilizers, antihistamines, immunosuppressive drugs (cyclosporine), artificial tears, contact lensdressing, cryotherapy and surgical papillae removal. we present the case of a year-old girl with corneal ulceration who was applied artificial tears after traditional methods of treatment proved unsuccessful. aims: the aim was to share our experience on artificial tears therapy applied in ophthalmic disorders. methods: autologous blood ( ml) was collected into disposable, sterile transfer bags used for routine blood component preparation (no anticoagulant) and incubated for h at °c. the clot was then removed by centrifugation and the serum containing erythrocytes was press extracted. centrifugation was applied again to obtain serum free of cellular components. the serum was then divided into . ml segments (capsules)and the artificial tears applied to the left eye daily. results: ulcer healing was reported after weeks of therapy with artificial tears. the dosage was reduced to daily. no recurrence of corneal ulceration was observed after subsequent weeks. summary/conclusions: artificial tears are a safe and effective therapy for ophthalmic disorders in children. background: arv non-disclosure among hiv-positive donors who tested hiv antibody (ab) positive but rna negative (ab+/rna-), so-called false elite controllers, was previously described by our group in south africa, with > % of ab+/rnadonations since testing arv positive. the extent of undisclosed arv use at time of donation represents a significant risk to blood safety in a country with a growing treated hiv population. aims: to establish the prevalence of arv non-disclosure among four subgroups of hiv-positive donors in south africa along with demographic correlates of non-disclosure. methods: south african blood donors are screened by a self-administered questionnaire, which includes questions on current hiv status and arv use, followed by a semi-structured personal interview. specimens for hiv, hepatitis b and c testing are collected at time of donation. based on id-nat (procleix, grifols) and antibody (prism, abbott; western blot) testing, hiv-positive blood donations were classified as acute (ab-/rna+), recent (ab+/rna+, limiting antigen avidity [lag] odn ≤ . ), longstanding (ab+/rna+, lag odn > . ) and potential elite controller (ab+/rna-) cases. stored plasma from these donations were tested for four arv drugs using qualitative liquid chromatography-tandem mass spectrometry (detection limit . lg/ml). chi-square tests were used to assess associations of hiv case type, gender, ethnicity, age, donor type, and donor clinic (fixed, mobile) type with arv non-disclosure. results: during , donors tested hiv-positive of whom had samples available that were tested for arvs. the overall prevalence of undisclosed arv use was . % (n = ) with efavirenz most frequently detected ( ), followed by lopinavir ( ) and nevirapine ( ) . potential elite controller cases had the highest proportion of detectable arv ( / ; %) (p < . ) followed by longstanding ( / ; . %) and recent ( / ; . %) infections. none of acute hiv cases tested positive for arvs. there were no associations between arv use and gender or ethnicity. however, older ( to years) hiv-positive donors ( / ; . %) were significantly more likely to test positive for arv than younger ( to years) donors ( / ; . %) (p < . ). arv use was more frequent among first time ( / ; . %) than in lapsed ( / ; . %) or repeat ( / ; . %) donors (p < . ). donors at mobile clinics had significantly higher arv non-disclosure than donors at fixed sites ( . % vs . %; p = . ). summary/conclusions: the . % prevalence of undisclosed infection and arv use among hiv-positive south african blood donors is alarming. higher rates of nondisclosure among first-time donors was expected, but non-disclosure among repeat and lapsed donors suggests failure in donor education and assessment. the . % prevalence among concordant ab+/rna+ cases may suggest sub-optimal viral suppression. lack of detection of arvs in acute cases should be qualified because the samples were not tested for tenofovir, the most common drug used in pre-exposure prophylaxis. donor motivation for non-disclosure of known hiv infection and arv use needs further investigation, since early arv initiation or infection while on prep could lead to low ab and rna levels, failure to detect hiv-infected donations and transfusion-transmission of hiv. blood bank, rotary blood bank, new delhi, india background: voluntary blood donation ensures safe blood transfusion. careful blood donor selection is of importance to provide safe blood to patients, although new methodologies have also been adopted by blood centers for blood safety and to minimize risk of transmitting infections through blood transfusion. the quality and the availability of blood components depend on the willingness to donate and reliability of the information given by the donors about their own health, including risk behaviour. blood donor history questionnaire is designed to evaluate donor's history in accordance with the guidelines laid down by the fda. donors, once deferred by the blood bank, will be less motivated to return for donation if he is not counseled effectively. it is important to reduce the number of deferrals by good donor comprehension and the centre should have a mechanism to recall temporarily deferred donors aims: the aim of the study is to analyse donor history and test results of those who donated blood with past history of jaundice. based on their history which suggested the type of viral infection they had, these donors were accepted or deferred. data was collected from voluntary blood donors who were screened for blood donation in the year . methods: in this study, donor history was analysed with reference to history of jaundice. jaundice in donors after the age of yrs, history of surgery, blood transfusion, body tattoos and acupuncture treatment within past one year of donation, history of multiple sex partners and related history and intravenous drug abuse history was taken into consideration. donors who revealed past history of jaundice were asked in detail about their illness and recovery. blood was donated by donors from whom the history of jaundice was elicited and it was understood that the type of virus which caused jaundice was not hepatitis b or c. those who could not give the correct history or were not sure of the cause of hepatitis, those individuals were deferred. aims: to assess the performance of this follow-up program in terms of donor participations, successful confirmed positivity rates, and potential reentry rates. methods: eligible donors were tested for hbsag, hcvab, hivab/ag, and tpab with two eias for each marker. samples reactive with at least one assay were tested further with electro-chemiluminescence assay (eca) and reactive samples were considered repeated reactive (rr). tpab reactive donations were re-tested with particle agglutination assay (tppa). samples eca or tppa non-reactive were considered non-repeatable reactive (nrr background: the blood donation service in suhl processes more than . samples annually from whole blood and apheresis donations, testing on average around samples per day. for the last years, serology screening was performed on the architect instruments (abbott) (arc), but will be changed to the alinity s system (aly) by middle of . although the design of the aly assays is based on those of the arc assays, we undertook a thorough evaluation of the four mandatory screening assays detecting hbsag, hiv ag/ab, anti-hcv and anti-hbc. aims: to validate the mandatory screening assays on the new aly system in our lab in terms of sensitivity and specificity, also including samples with known falsereactive results. determine the rate of false reactive results for hbsag, anti-hcv and anti-hiv that may lead to deferrals of donations and donors. methods: for sensitivity, we used known positive samples confirmed by immunoblot or nat. known unspecific positive samples for arc not confirmed by immunoblot or nat were testes for aly also. close to . unselected samples (edta plasma) from routine blood and apheresis donors were tested in parallel on both systems, arc and aly to determine the rate of initial and repeat reactive results. results: all known confirmed positive samples were identical detected by aly. samples with known unspecific reactive results were retested by aly with the following results: / anti-hcv, / hiv ag/ab and / hbsag were found reactive by aly to. one donation from an acute hiv infection in the early seroconversion period was detected by both methods in routine testing. there are no reactive results for aly not already known for arc. the specificity for the screening assays on aly versus arc assays were as follows: ) hbsag aly . % ( % ( / % ( ) vs arc . % ( % ( / ; ) hiv aly and arc . % ( / ); ) anti-hcv aly . % ( % ( / % ( ) vs arc . % ( % ( / . the number of anti-hbc reactive samples did not differ between aly and arc. summary/conclusions: while the switch to the new system is mainly driven by operational efficiency, obviously, the high specificity of the alinity s assay will reduce unnecessary deferrals of donations and donors. abstract withdrawn. background: blood donor selection is the cornerstone for blood transfusion safety, designed to safeguard the health of both donors and recipients. donor safety is targeted by reducing the risk of complications associated with blood donation and transfusion safety by reducing the risk of transfusion-transmitted infections (tti) and other preventable transfusion reactions. there is always a compromise on blood donor safety as well as blood safety during outdoor mega blood donation drives due to various reasons, mainly due to more number of donations within a stipulated time. aims: to compare the blood donor selection patterns between in house blood donations and donations at mega blood donation drives and its influence on donor safety and blood safety in a tertiary care hospital in india. methods: a retro prospective study was done to audit and compare blood donor safety and blood safety over a period of years from january to december . blood donor safety was analyzed by two indicators: donor health questionnaire (dhq) monitoring and blood donor reaction rates and blood safety through tti positivity rates. ( ) during mega blood donation drive. summary/conclusions: a good donor selection is a lengthy process which involves pre-donation information and advice: this is usually provided in a leaflet, especially about transfusion-transmitted infections (and the associated risk factors) and the potential risks of donation, filling of dhqs by the donor himself, donor interview: conducted by a qualified medical specialist trained in donor selection process and health assessment at the end of the interview to declare if the donor is eligible to give blood or deferred temporarily or permanently. it was observed that seroprevalence rates, number of donor reactions and incompletely filled dhqs were more among blood donations at mega blood donation drives when compared to blood donations during in house collections. this is mainly due huge number of blood donations with in a stipulated time where there is limited time spent on proper donor selection. stringent implementation of who strategy: "safe donor safe blood" is the only way for blood donor and transfusion safety. background: safety of blood transfusion is a great concern especially in crisis countries and during humanitarian emergencies. transfusion transmitted infections (ttis) are one of the major health problem in yemen that are associated with blood transfusion complications. aims: the aim of this study is to determine the prevalence of ttis among blood donors at national blood transfusion and researcher center (nbtrc this contributed to an additional reactivity of . %, thereby total reactivity being . %. % ( / ) of these were hcv reactive & % ( / ) for hbv. the nat yield was in and the viral loads of nat reactives ranged from - x iu/ml for hcv & all the hbv yields had an extremely viral load of < iu/ml. / nat reactive showed sero-conversion after - months with follow-up eclia screening, and of these were hcv reactive and hbv reactives. summary/conclusions: incidence rate indicate that the current risk of transfusion transmitted viral infections attributable to blood donation is relatively high in our country. parallel use of both serology and nat screening of donated blood in countries that have high seroprevalence can improve the blood safety. at our centre, by using best in class serology and nat technologies, we were would add an extra layer of safety to blood supply by interdicting samples from donor with recent infections. abstract withdrawn. abstract withdrawn. ( / , ) . the both hiv-rna and hcv-rna detected donors by nat were identified in the window period. summary/conclusions: in this study, we found that nat could detect infected cases with hbv-dna, hiv-rna and hcv-rna which were forgotten by serological methods therefore, nat is a sensitive screening method to detect low viral load and shorten the window period of the virus infection to ensure the safety of blood transfusions. service du sang, croix rouge de belgique, namur, belgium background: due to enhancement of kits specificity and machines throughput, roche elecsys â technology is a potential partner for blood donations screening laboratories. aims: the aim of the study was to assess the performance of the elecsys serology assays on a cobas e equipment for clinical specificity, analytical sensitivity and reproducibility. background: deceased donors are the primary source of organs and tissues for transplantation but the risk of infectious complications in the recipient is high and is the main cause of morbidity and mortality after transplantation. to minimize the risk of infections by organ or tissue transplantation, donors should be tested for anti-hiv- / , hbsag, anti-hbc, anti-hcv, and syphilis. further laboratory tests may be required depending on the history of the donor and on the tissue properties. certain grafts can be donated after circulatory death of the donor; however, the absence of the heartbeat may change dramatically the blood composition by e.g., haemolysis and proteolysis. this may have an impact on test performance and lead to false results. therefore, an assay validation is needed for testing of cadaveric samples. aims: a validation study was performed to demonstrate the suitability of elecsys hbsag ii, anti-hbc ii, anti-hcv ii, hiv combi pt, hiv duo, syphilis, htlv-i/ii, and chagas for the use in cadaveric samples from non-heart beating donors. methods: as the basis for validation, we followed the recommendations of the paul-ehrlich-institut (pei) "proposal for the validation of anti-hiv- / or hiv ag/ ab combination assays, anti-hcv assays, hbsag and anti-hbc assays for use with cadaveric samples". comparison of spiked samples from living donors and cadaveric donors was used to demonstrate accuracy. to determine precision, two cadaveric specimens were tested in several replicates. acceptance criteria were implemented according to the pei recommendations. results: results were found to be within specifications requested by the pei recommendations for all tested assays summary/conclusions: the evaluated results support the extension of the use of these assays with cadaveric specimens. background: in developed countries, blood donors are routinely screened for a range of blood borne viruses (hiv, hbv, hcv and htlv) using highly sensitive screening tests. this has dramatically improved the safety of blood supply. however, transmission by transfusion of unknown or unsuspected viruses remains a continuing threat. this is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. in developed countries, blood donors are routinely screened for a range of blood borne viruses (hiv, hbv, hcv and htlv) using highly sensitive screening tests. this has dramatically improved the safety of blood supply. however, transmission by transfusion of unknown or unsuspected viruses remains a continuing threat. this is particularly relevant considering that a significant proportion of transfused patients are immunocompromised and more frequently subjected to fatal outcomes. aims: in this context, metagenomic analyses of viral content in blood donations collected in geographical zones recognized as "hotspot" for viral emergence represents a suitable approach without any a priori for the identification of a potential emerging viral risk that may compromise blood safety. methods: in the framework of a viral discovery program founded by the french national agency for medicine security (ansm in french), more than plasma samples collected in sub-saharan africa countries ( ) ( ) and the amazon region of brazil ( ) have already been analysed by metagenomics. results: although no viral sequence could be described as novel (i.e. new species or even a new genus), we unexpectedly identified a feline bocavirus in two donors from mauritania. a large diversity of known viruses that are not part of the regularly monitored agents were also observed, among which anelloviruses, hpgv- (formerly known as gbv-c), papillomaviruses, herpes viruses, parvovirus b , chikungunya virus, enterovirus, and various small circular viruses (circo-, cycloand gemycircularviruses). while no significative differences was observed in the higher classification of detected virus (above families/genera) between africa and brazil, we observed variations at the sequence level allowing better resolution of the genetic diversity for several viruses (for example characterization of hpgv- genotypes). summary/conclusions: overall, the absence of novel viruses in blood samples collected across countries of two distant continents is reassuring regarding threats emergence. however, continuous monitoring of prospective blood banks should be continued. summary/conclusions: after the high peak observed in during the first period, this study shows that the decrease in the seroprevalence of viral markers is continuous over the next five years. the second period is marked by an irregular evolution of seroprevalence but with lower levels than the first period. the recruitment of new donors allows a quantitative increase in donations. however, improving the quality of blood products essential condition of transfusion safety is achieved through retention of recruited blood donors. background: in blood screening laboratories, samples may be transferred between automated serological and molecular instruments, and the potential for sample contamination is a serious risk to the integrity of nucleic acid testing (nat) results. the sensitive limit of detection (lod) for hiv and hcv nat assays combined with the high viral titers encountered in specimens from patients with acute infections presents a challenge for maintaining the sample integrity of negative specimens. at additional cost per test, this risk can be reduced with single-use filter pipette tips. aims: we evaluate the efficacy of applying induction heated washes to a non-disposable pipettor on serology instruments-alinity s, alinity i, and architect i sr (abbott diagnostics)-to preserve the integrity of samples transferred to a downstream molecular instrument, the m realtime (abbott molecular diagnostics), which amplifies viral nucleic acid targets exponentially. methods: in this application of induction heating, the metallic pipettor warms under its own resistance to coil-induced electrical currents. by sweeping the pipettor through an induction coil, temperatures on the pipettor are elevated throughout its length. single donor high viral titer hiv genotypes a ( . log iu/ml), b ( . log iu/ml), c ( . log iu/ml), crf ( . log iu/ml), crf ( . log iu/ml), and urf ( . log iu/ml), as well as single donor high viral titer hcv genotypes a ( . log iu/ml), b ( . log iu/ml), a ( . log iu/ml), ( . log iu/ml), q ( . log iu/ ml), and t ( . log iu/ml) were used as potential sources of contamination; these genotypes account for the majority of hiv and hcv infections worldwide. on serology instruments, one high viral titer hiv or hcv specimen and three consecutive susceptible negative samples (hiv/hcv rna negative human plasma, abbott molecular diagnostics) were tested on an hiv ag/ab combo or anti-hcv immunoassay (abbott diagnostics), and this schema was repeated four times per positive specimen. induction heated washes occurred between all samples processed on the serology instruments. the first susceptible negative from each testing block, with approximately ml of residual sample volume, was then tested using the . ml abbott realtime hiv assay (lod copies/ml) or . ml abbott realtime hcv assay (lod iu/ml) and an hcv ag immunoassay (lod . fmol/l; abbott diagnostics). study acceptance criteria required that any susceptible negative sample had no detectable level of hiv or hcv rna. results: all first susceptible negative samples (n = per platform per virus schema) run on alinity s, alinity i, and architect i sr using induction heated washes after a high viral titer hiv specimen or hcv specimen were hiv ag/ab combo nonreactive (< . s/co) and reported no detectable level of the hiv rna target, or were anti-hcv nonreactive (< . s/co) and reported no detectable level of the hcv rna or core antigen targets. summary/conclusions: while precautions should continue to be taken for samples run on molecular instruments, the integrity of samples originally tested on the alinity s, alinity i, and architect i sr was preserved for downstream molecular testing through the use of induction heated washes. aims: increasing the safety of blood and blood products -motivating the blood donors to be regular donors methods: national reporting system showed the high prevalence of ttis among first blood donors in compares with the regular donors. in per . . donations, % % of confirmed positive hiv, % of hcv, and % of hbv cases has been reported among first blood donors. in the end of a national program named "pre-donation screening tests "has been developed and has been implemented in high prevalence provinces in whole country. based on this program, all first blood donors who accept in donation sites, if after donor selection process are eligible to donate blood, they refer to give just a blood sample for screening ttis tests. after months, the invitation letters and smss send to the donors who have negative results for all screening ttis tests, and they can be eligible to donate blood after another donor selection process. in , about . % of all donations have been rejected because of at least one of hiv, hcv, or hbv confirmed positive results, while this reject rate in was . %, which shows a significant decreasing the ttis prevalence among blood donation from to . the prevalence of hiv, hcv, and hbv among donations has been decreased significantly in compared with the . prevalence of hiv among donations reduce from . % in to . % in , for hcv and hbv the same results have been experienced, respectively from . % and . % in reduce to . % and . % in . it seems this applied study could effectively scale up the safety of national blood supplies. in addition this intervention could support iranian blood transfusion service to increase the proportion of regular blood donors from . % in to . % in . it means that with increasing the regular blood donor population sizes, the safety of iranian blood and blood products will be more and more scaled up. summary/conclusions: evidence based reports show there is a high rate of prevalence of transfusion transmitted infections (ttis) among first blood donors. so an effective intervention which can reduce the risk of unsafe first blood donation can effectively increase the safety of blood and blood products. pre donation screening tests program in iran can support the national program to decrease the rate of ttis among blood donations from . % in to . % in . abstract withdrawn. abstract withdrawn. background: despite the universal application of viral inactivation and elimination technologies during the preparation of plasma-derived products, the exclusion of infectious donations before any other procedure remains the first essential step as well as the major determinant for the safety of untreated labile blood products. current selection and screening techniques have reduced the risk of viral transmission to very low levels, but there is still a very low but quantifiable risk of transmission through donations beyond routine detection, particularly during the " seroconversion window". "of an infection in a blood donor that is to say during the period when the recently infected donor has not yet developed a serological response. the level of residual risk, which must be as low as possible, is mainly conditioned by the rates of the infections concerned (hiv and hepatitis b virus (hbv) and c (hcv)) to blood donors. summary/conclusions: the evolution of serologic markers is generally satisfactory with continued regression, which has improved particularly for hiv. on the other hand, hepatitis b is still a concern because of its still high rate among new donors. it is desirable to initiate a regular donor vaccination program to protect against hepatitis b. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hcvab, havab igm and havab igg essays using abbott alinity s when compared to architect i sr. determine repeatability of these tests in alinity s essays. methods: during months a study was conducted where several samples (minimum samples per test) were randomly sorted and tested using alinity s and architect i sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of samples were tested ( for hcvab, for havab igm and for havab igg) using alinity s and architect i sr, and it was ensured that there were no statistically significant differences between results (p > . ). using samples and a total of essays we found the %cv hcvab ranged from to . %. samples were tested for havab igm in a total of essays and the %cv ranged from to . %. havab igg was tested in samples during essays and the %cv ranged from to . %. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hcvab, havab igm and havab igg. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hbsag, hbsab, hbcab, hbeag and hbeab essays using abbott alinity s when compared to architect i sr. determine repeatability of these tests in alinity s essays. methods: during months a study was conducted where several samples (minimum samples per test) were randomly sorted and tested using alinity s and architect i sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of samples were tested ( for hbsag, for hbsab, for hbcab, for hbeag and for hbeab) using alinity s and architect i sr, and it was ensured that there were no statistically significant differences between results (p > . ). using samples and a total of essays we found the %cv hbsag ranged from - . %. samples were tested for hbsab in a total of essays and the % cv ranged from - . %. hbcab was tested in samples during essays and the %cv ranged from - . %. using samples and a total of essays we found the %cv hbeag ranged from . - . %. samples were tested for hbeab in a total of essays and the %cv ranged from - . %. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hbsag, hbsab, hbcab, hbeag and hbeab. background: blood centres require high throughput assays with a high level of reproducibility to assure consistent results and minimize unnecessary retesting of samples and deferral of donors. in addition, continued economic pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. aims: evaluate the reproducibility of hivag/ab, syphilis and htlv i/ii essays using abbott alinity s when compared to architect i sr. determine repeatability of these tests in alinity s essays. methods: during months a study was conducted where several samples (minimum samples per test) were randomly sorted and tested using alinity s and architect i sr, results were compared using ibm spss statistics â . in order to evaluate repeatability, at least samples with different reactive degrees (high, intermediate and low) per test were repeated, using alinity s, an average of times per sample and it was determined the percentage of coefficient of variation (%cv). results: a total of samples were tested ( for hivag/ab, for syphilis and for htlv i/ii) using alinity s and architect i sr, and it was ensured that there were no statistically significant differences between results (p > . ). using samples and a total of essays we found the %cv hivag/ab ranged from to . %. samples were tested for syphilis in a total of essays and the %cv ranged from to . %. htlv i/ii was tested in samples during essays and the %cv ranged from . to . %. summary/conclusions: the new automated equipment alinity s system demonstrated no statistically difference when compared with architect i sr and repeatability was ensured. this demonstrates the precision of results generated by this fully automated blood screening analyzer, which helps assure consistent results for the testing and retesting of blood specimens for hivag/ab, syphilis and htlv i/ii. , human immunodeficiency (hiv) and hepatitis c (hcv) viruses' infection in blood donors were . %, . % and . % respectively. consecutive positive results for hbv were . % ( / ), for hcv were . % ( / ) and nil for hiv. there was no sample carry over in this. out of consecutive reactive donors were donated for same patients and were related with infected patient which were statistically significant (p < . ). summary/conclusions: among all tti reactive donors . % ( / ) were consecutive reactive. the reason for the same may be process related like sample carry over or reagent carry over or donor related. donor related reasons may be, one of the close relative is reactive for virus and that is transmitted to other family members. in our study reactive donors either had close contacts with persons with history of infective disease or were their first degree family relatives. these findings were found statistically significant (p < . ). this study recommends that in analysis of consecutive positive results in elisa along with looking for procedure/sample error, there is also a need to take retrospective history of donors for close contact with infected patient. background: screening for transfusion-transmitted infections (ttis) is critical in ensuring safety of blood products. transmission of infections through transfusion remains a major source of viral hepatitis especially hbv and hcv. the effectiveness of rapid immunochromatographic test (ict) devices for screening of blood is a concern and needs validation through advanced methods like chemiluminescence immunoassay (clia) and polymerase chain reaction (pcr). aims: the current study was conducted to evaluate the performance and screening effectiveness of commercially available rapid screening kits in comparison with clia and pcr. methods: this single centre, cross sectional study was conducted at the department of blood transfusion services, shaheed zulfiqar ali bhutto medical university, islamabad, from january -june . a total of ten commercially available ict devices and one clia kit (liaisonr xl) were tested for their sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and accuracy using positive and negative samples each for hbv and hcv respectively, in comparison with the values determined by pcr. the ict kits included hightop, rightsign, wondfo, accu-chek, fastep, abon, immumed, insta-answer, biocheck and ctk. results: the sensitivities and specificities of ict kits for hbsag were % and % (hightop), % and % (rightsign), % and % (wondfo), % and % (accu-chek), % and % (fastep), % and % (abon), % and % (immumed), % and % (insta-answer), % and % (biocheck) and % and % (ctk) respectively. similarly the sensitivities and specificities of different ict kits for hcv were % and % (hightop), % and % (rightsign), % and % (wondfo), % and % (accu check), % and % (fastep), % and % (abon), % and % (immu-med), % and % (insta answer), % and % (biochek) and % and % (ctk) respectively. the sensitivity and specificity of diasorin liaison murex assay for both hbv and hcv were found to be %, when compared with pcr. the ppv, npv and accuracy were determined accordingly. summary/conclusions: rapid testing ict devices for both hbv and hcv available in pakistan were found to have a variable degree of sensitivity and specificity, when compared with pcr. comparatively expensive but quality methods are more reliable as compared to rapid devices. the data generated will help policy makers to prepare future plan of action and introduce the concept of quality control in blood centres. the analysis has shown that the population of blood donors also included people infected with syphilis. in reference to the number of the tested samples this number is quite significant. the analysis proved the increase in the number of syphilis infections among the blood donors which is consistent with the general trend in the population. summary/conclusions: we have proved that testing blood donors for the treponema pallidum infection increases the safety of recipients of blood and its components and that obligatory testing of donors is fully justified. , and , the use of third or fourth generation serological assays is mandatory for screening of blood donor units for hbv and hcv infection before transfusion. routinely, blood banks in india screen the units by the elisa testing. nat is not very common due to cost constraints. aims: the aim of this study is to determine the frequency and load of hbv dna and hcv rna in hbs and hcv reactive blood donors respectively, and hence it was intended to contribute to determining whether routine hbs and hcv screening of blood donors, using elisa method alone, provides any concrete benefits with regard to hbv and hcv risk reduction or whether the implementation of nat will be of great benefit to low-resource countries like india, which has high prevalence of hbv and hcv. abstract withdrawn. , donors were routinely tested for hbv dna by using cobastaqscreen mpx- and mpx- (roche) or procleix ultrio and ultrio plus id (grifols) assays. obi was confirmed by repeat dna testing and by performing additional serological and molecular investigations on index and follow-up samples. anti-hbs concentrations were determined and anti-hbc antibodies were tested with three distinct commercial clia assays (anti-hbc elecsys roche, architect anti-hbc ii abbott, and hiscl anti-hbc assay sysmex). hbv pre-s/s, precore/core and bcp regions were pcramplified after viral particle concentration and viral amplicons were sequenced. results: hbsag-/dna+ donors ( : , ) including confirmed obi were identified ( : , prevalence). among obi donors, ( . %) tested anti-hbc+/anti-hbs-, ( . %) were anti-hbc+/anti-hbs+, ( . %) were anti-hbc-/anti-hbs+, and anti-hbc-/anti-hbs-primary obi ( . %). anti-hbc-/anti-hbs+ obi donors were significantly younger (mean: years [range: - years]) than those with anti-hbc+/anti-hbs+ (mean: years [range: - years]) and anti-hbc+/anti-hbs-(median: years [range: - years]) profiles (p < . ). hbv vaccination was documented for ( %) of these donors and was reported in one donor but without definitive evidence. extremely low hbv dna loads (range: < - iu/ml) were transiently detected in seven donors during follow up. genotypes identified were genotype b (n = / ) and genotype c (n = / ). preliminary analysis of core protein (n = ) and bcp (n = ) sequences showed no particular genetic feature that could be associated with altered antigenicity or core gene expression. follow-up was available for / anti-hbc-/anti-hbs+ donors ( - samples/donor; range: - months). anti-hbc remained undetectable with all clia assays in these donors except one. low transiently detectable levels of hbv dna were observed overtime with anti-hbs levels fluctuating between and , iu/l. no significant difference in hla-a, -b (except hla-b* more frequently detected in anti-hbc negative obi), and -drb *. summary/conclusions: overall, the . % prevalence of anti-hbs-only in hbv dna positive obi carriers ( : , of total donors) in dalian blood donors confirmed previous reports from south east asia. this phenomenon was not related to core antigenic variations but was significantly associated with younger age of carriers. a particular route and natural history of the infection may be considered. the hypothesis of acute-phase vaccine breakthrough was ruled out in / donors by the over months stability of this serological profile. breakthrough in immunized donors may still be suspected. further studies are needed to evaluate the potential infectivity of anti-hbs-only/hbv dna+ obi carriers, and to characterize the potential viral and immunological mechanisms responsible for this unusual hbv infection profile. confirmatory laboratory, hungarian national blood transfusion service (hnbts), budapest, hungary background: vaccination against hepatitis b virus (hbv) is an effective tool to avoid the infection. in hungary, population born after is considered to be immunized, because inoculation has been mandatory for children as campaign vaccination since . hbv vaccine is strongly recommended for healthcare workers, moreover trips to endemic countries and awareness of individuals could also be reasons of vaccination in immunologically na€ ıve age-groups. since the hbv vaccine contains surface antigen, a recent inoculation can cause reactivity of hbsag screening assays and positivity of confirmatory tests for several days resulting in deferral of donors from blood donation. the former regulation of hnbts, which was valid until december , , allowed the re-entry of donors whose immunization records and negative hbv confirmation of the second blood samples proved that the previous vaccination had resulted in the hbsag confirmed positivity. aims: the aim of this study was to strengthen that vaccination against hbv before blood donation had resulted in the reactivity of hbsag screening and confirmatory assays between and . background: in brazil, the introduction of nucleic acid tests (nat) for hbv-dna detection in the routine screening at public blood banks is relatively recent. at fundac ßão pr o-sangue/hemocentro de são paulo (fps-sp), about , blood donors are submitted to serological screening tests (hbv, hcv, hiv- / , chagas disease, syphilis and htlv- / ) and nat for hiv, hcv and hbv per year. approximately % of the blood donations are discarded due to some reactive result; of these, the hbv discard rate was . % in . aims: our study aims to determine the potential infectious cases among samples that had one or more hbv-reactive screening results (anti-hbc, hbsag and mp-nat-hbv) and verify the different categories of hbv infection (acute, chronic, occult hepatitis b infection (obi) and immunological window). furthermore, to characterize the distribution of hbv genotypes, drug resistance and escape mutations and analyze the risk factors. methods: we carried out a cross-sectional study of roughly , donations from may to december . hbv antibodies and antigen screening were performed using cmia kits architect â -abbott/hbsag and architect â -abbott/anti-hbc. nat screening was performed in minipools (mp) of six samples using kit nat hiv/hcv/ hbv -bio-manguinhos (sensitivity % lod iu/ml for hbv). reagent samples (n = ) that presented one or more hbv-reactive screening results (anti-hbc, hbsag and/or mp-nat-hbv), were submitted to individual nucleic acid extraction and "in house" quantitative real-time pcr-hbv (id-pcr-hbv) targeting the hbsag region (sensitivity - ui/ml). the hbv genotypes and mutation analyses were determined by direct sequencing of the hbv pol-gene/surface-gene and use of the online analysis tool geno pheno [hbv] . . socio-demographic and epidemiological data were also analyzed. financial support: fapesp / - . results: among the hbv reactive samples, were reactive for anti-hbc only ( . %), for hbsag only ( . %) and were reactive for both markers and hbv dna ( . %). routine testing and id-pcr-hbv identified ( . %) samples of active infections that had all hbv reactive/positive tests results. no hbv dnayield samples or hbsagyield or obi were observed. viral loads for active infections samples ranged from . e+ to . e+ cop/ml (median, . e+ cop/ml). hbv sub-genotypes a , a , c , d and f were found in %, %, %, %, and % of the donors, respectively. no reverse transcriptase inhibitor-resistant variants were detected. escape mutations in small hbsag protein shb region were detected in % ( / ), with the following main substitutions c ( x), r, n and g. the mean age of donors with active hbv infection was years, mostly donors were males ( %), mixed ( %) or white ( %) and had concluded high school ( %). summary/conclusions: discard rate due to isolated anti-hbc is high but no obi was found in the blood donor population studied. in addition, no case of immunological window for hbv or hbsagyield was detected. there was a predominance of subgenotype a and mutations associated with escape were found in % of hbv-dnapositive samples. continuous research and surveillance about hbv prevalence among blood donors are needed to maintain and evenly increase blood safety in brazil. background: screening for anti-hbcore antibodies in blood donors is considered to contribute significantly to blood safety, since it reveals donors with occult or probable occult hepatitis b, with variable results in molecular screening, due to very low viral load. however, universal anti-hbcore testing in blood donors, might exclude a considerable number of blood donors in countries with high hbv prevalence and even in countries with low to moderate prevalence, like greece. aims: the aim was to investigate the percentage of blood donors with natural hbv infection (confirmed positive anti-hbcore) or hbv immunization due to vaccination (anti-hbs+ only, due to vaccination) and predict the impact of generalized anti-hbcore testing. methods: during the period - november , all blood donors were asked to give their consensus for additional screening for hepatitis b anti-hbcore and anti-hbs antibodies, besides the obligatory serological and molecular screening, the samples of few donors who disagreed, were not examined. all samples with repeated positive anti-hbcore results, were further examined for anti-hbcore igm and anti-hbe antibodies. furthermore, a new donor sample was requested, to confirm reactivity. the serology results were recorded in an excel spreadsheet. additional data, including age, sex, nationality, number of previous blood donations, abo blood group, family history of hbv infection, hbv vaccination, were also recorded and statistically evaluated. donors were informed of the positive results. results: a total of edta samples were tested using the architect anti-hbcore, anti-hbs, nti-hbcore-m and anti-hbe assays (chemiluminescent microparticle immunoassay (cmia). repeated anti-hbcore(+) occurred in ( . %) samples, among which ( %) were also anti-hbe(+), while anti-hbs was found > m iu/ml in ( . %), between and miu/ml in ( . %), and < miu/ml in ( %). among anti-hbcore positive donors, / were foreigners ( . %) and were greeks, while foreigners consisted , % ( / ) of donors examined. so, anti-hbcore was found positive in , % of foreigners ( / , all from countries with high prevalence for hepatitis b infection) and in , % of greeks ( / ). in total, ( . %) samples had anti-hbs > miu/ml (considered seroprotective for the donor). summary/conclusions: almost half of our blood donors ( . %) were immunized, by vaccination and ( . %) by natural infection. the incidence of natural infection was significantly higher in foreigners ( . % versus , %). if not all anti-hbcore+ donors, % with anti-hbs < iu/ml, might be potentially infectious, especially for immunocompromised patients. if we choose to screen all blood donors for anti-hbcore and reject those with positive results, regardless of the anti-hbs levels, we would probably lose a significant number of donors and jeopardize blood sufficiency. alternatively, we could reject only those with anti-hbs < or < , or choose to selectively screen pre-donation blood donors from countries with high prevalence of hbv infection. following this pilot study, the prevalence of immunization against hbv in large numbers of blood donors from various parts of greece, must be investigated, in order to decide whether to introduce such screening. aims: the aim of this study was to perform phylogenetic analysis of the donor samples with hcv found in the neighbouring villages to determine the nature of transmission. methods: altogether, approximately million blood donor samples were screened with anti-hcv immunoassay (architect anti-hcv, abbott gmbh, wiesbaden, germany) and reactive results were confirmed with anti-hcv line-immunoassays (inno-lia hcv score, fujirebio europe, gent, belgium). based on lia positivity, in samples an association of hcv infection was supposed, because the residence places of donors were in three neighbouring villages situated less than km to each other. pcr was positive in samples. from these samples, hcv sequencing and phylogenetic analysis were performed. fourteen hcv infected samples of general population and of ivd users were also included into the study. results: phylogenetic analysis detected genetic relationship among the hcv virus sequences in donor samples. the most abundant was the a subtype, and it formed two different groups on the phylogenetic tree. according to their genetic distance, a more distant mutual ancestor could be supposed. two samples with b subtype originated from the same village, and their difference was only nucleotides. three hcv from the ivd user control group showed close genetic relationship with the viruses detected in the donor samples. summary/conclusions: based on our phylogenetic analysis, hcv transmission in blood donors could be the consequence of the ivd use and the origin might be related to or primary human sources. during and , a significant increase in the hcv seroprevalence among the ivd users was observed, which was approximately threefold in the rural areas of hungary. our recent findings highlight the importance of the proper donor selection, which can identify the typical signs of the ivd use. moreover, enhancing awareness of blood donors with education is a further significant issue in order to reduce the risk of transfusion transmitted infections. abstract withdrawn. background: in china, the residual risk of transfusion-transmitted hcv has been declining since screening of blood donors for anti-hcv and/or hcv nat from . however, many high sensitivity reagent, using to test blood donors' samples, lead to false-positive results and donors loss. aims: this study intended to establish a donor reentry procedure for hcv screening reactive donors in china. methods: from march to december , there were blood donor samples which were screened reactive or belonged to "grey zone" by elisa and/or reactive by nucleic acid test(nat) at the local blood centers were collected from chinese blood centers. all these samples were sent to institute of blood transfusion (ibt) national reference laboratory where anti-hcv and hcv individual nucleic acid test (id-nat) were conducted. if the results were reactive for anti-hcv, then the samples were tested with a recombinant immunoblot assay (riba). results: based on this study, of donors in the study who could be classified into two categories for hcv status: ( . %) true positive and ( . %) false positive. a total of of donors lost to follow-up, their hcv status cannot be determined with certainty. based on these data, a reentry procedure for hcv screening reactive donors was proposed. summary/conclusions: based on our proposed donor reentry procedure for hcv screening reactive donors, a majority of screening false-positive donors ( . %) can re-entry safely. abstract withdrawn. background: providing safe blood for transfusion in sub-saharan africa (ssa) is a particular challenge due to a combination of factors; limited resources and infrastructure, suboptimal diagnostics and a high prevalence of the major transfusion-transmissible infections (ttis). average seroprevalence data estimates from the ugandan and kenyan blood transfusion services (bts) for hepatitis c (hcv) currently stand at . % and . % respectively. between january and december , in mbale (eastern uganda) the hcv prevalence amongst blood donors was an alarming . %. with no provision or funds for confirmatory testing, the bts are unable to confirm or refute a diagnosis of active hcv. this results in large quantities of blood wastage, unnecessary anxiety in potential donors and high donor deferral rates limiting the donor pool. aims: we aim to determine the true prevalence of active hcv infection amongst seropositive donors in bts in uganda and kenya. in addition, we aim to compare the performance of locally used serodiagnostics and best available alternative tests and to examine the feasibility of cost-effective additional or alternative tests to help provide accurate results on the infectious status of blood. methods: hcv seropositive blood samples from bts study sites (kampala, mbale, mombasa) will be re-tested using the local serology screening test (abbott architect anti-hcv), an alternative who pre-qualified rapid antibody test (sd bioline) and a confirmatory test (hcv core antigen test). where there is discrepancy in the results or need for clarification, samples will be tested on the cepheid xpert platform by reverse-transcriptase pcr to obtain a quantitative rna result. s/co (specimen to cut-off) values for false positive samples (by screening serology) will be analysed and presented. pre-analytical factors (centrifugation speed, haemolysis check, time delay between collection and testing) will be controlled for and documented. results: pilot data from re-testing quarantined hcv seropositive donor blood (mbale bts) in uganda demonstrated that / seropositive blood ( %) was rna pcr negative. in december , / ( %) of seropositive samples (by screening anti-hcv serology) in kampala bts had s/co values between . - . ( . is the cut-off indicating a positive sample). data from the re-testing of seropositive samples as true representation of active hcv will be demonstrated and s/co values for the study period concomitantly with a retrospective analysis of january to december . preanalytical factors, cost analysis comparisons of the diagnostic platforms coupled with costs of the donor deferral process in false positive cases will be presented. summary/conclusions: for the bts in ssa there are significant resource and financial implications, as repeat testing and donor deferral counselling is required. evaluating and introducing new and appropriate diagnostics and algorithms in the screening of hcv is crucial in improving the supply of safe blood transfusion services in east africa. background: in november , the blood services of england, scotland and wales reduced donor deferral to three-months for commercial sex workers and individuals with higher risk sexual partners, including sex between men. the change was recommended after a detailed review by an external expert committee (sabto) which recommended that a shortened deferral of months would allow detection of recently acquired infection and maintain residual risk (rr) at a tolerable level. recommendations were accepted by government but with a government commitment to explore a more individualised approach. aims: to assess the impact of a -month deferral on blood safety in terms of epidemiology of infections in blood donors and compliance with donor selection criteria, and to explore evidence required to develop a more individualised approach to donor selection policy methods: routine uk blood donation surveillance data for - ( : preliminary) were reviewed. annual prevalence and incidence of hbv and hiv infection were estimated, with a poisson regression models to test for trends. incidence was calculated from donors seroconverting within -months, and/or microbiological and clinical evidence of recent infection. for donors positive in , compliance with the -month deferral was determined. uk hemovigilance data were scrutinised for evidence of transfusion transmitted infections (tti) associated with newly eligible donors. results: from to among new donors, annual hiv prevalence decreased significantly by an average of . % each year (p = . ) to . / , donations in ; no significant trend was observed for hbv. annual hiv incidence among repeat donors also decreased significantly by an average of . % each year (p = . ) to . / , -person years (pyrs) in (based on seroconverters). there was no significant trend in hbv incidence over the study period, however between and incidence increased from . / , pyrs to . / , /pyrs (based on and seroconverters respectively). with the information available to date, none of the seroconverting donors were non-compliant, and there was no reported confirmed ttis associated with the policy change. summary/conclusions: hiv prevalence and incidence has continued to decline. hbv incidence in repeat donors increased in although initial analysis suggests this is not associated with the policy change. monitoring continues, and residual risks will be re-estimated as data post-change accumulate. these data are reassuring, and therefore it is appropriate to scope the evidence for, and feasibility of, a more individualised approach to selection policy. a multidisciplinary steering group has been convened including representation from patient and stakeholder groups. gaps in knowledge are being defined, and a package of work is in development under the project of fair (for the assessment of individualised risk), using the abo rdf for guidance. background: permanent deferral of men who have sex with men (msm), established in the s, primarily to minimise the risk of hiv transfusion-transmitted infections is increasingly challenged. accordingly, blood services in many countries have changed to time-based deferral. in canada, a -year deferral was implemented in , reduced to -months in ; a -month deferral is now being considered. aims: to estimate the risk of undetected hiv among screened blood donations under a -month deferral since last sex between men. methods: the applied model combines features of previously published english and canadian models to estimate hiv risk under a -month deferral. three scenarios varying hiv incidence, prevalence and non-compliance under a -month deferral were modelled. assumed constants were the hiv nucleic acid window period, testing procedure error rate and assay sensitivity. model inputs were incidence under the current -month deferral, calculated as hiv positive donors with a previous negative within months divided by number of person years, numbers of hiv positive donations, hiv positive msm, hiv msm incident cases and newly eligible msm donors (from donor surveillance and compliance surveys). the risk with a -month deferral was estimated for three scenarios, one determined "most likely", where msm donor non-compliance, hiv incidence and hiv positive donations do not change and msm newly eligible to donate are estimated from compliance surveys. this scenario is based on data from two sequential policy changes in canada. an "optimistic" scenario where non-compliance halves and a "pessimistic" scenario where msm hiv incidence, hiv positive donations, non-compliance and new msm donors double were also used. the median hiv residual risk was used as the final estimate. the uncertainty in this estimate was assessed with the . th and . th percentiles over the simulation ( % ci). results: incidence, per , donations, was estimated to be . , . and . for the "most likely" "optimistic" and "pessimistic" scenarios, respectively. for the month deferral "most likely" scenario, hiv residual risk was predicted to be in . million donations ( % ci: in , million to in . million). for the "optimistic" scenario, hiv residual risk was estimated to be in . million donations ( % ci: in , million to in . million). finally, for the "pessimistic" scenario, hiv residual risk was estimated to be in . million donations ( % ci: in , million to in . million). with these residual risk estimates, based on the number of donations in canada, one hiv infectious donation would be in inventory every years for the "most likely" scenario, every years for the "optimistic" scenario and every years for the "pessimistic" scenario. summary/conclusions: the risks of hiv entering the blood supply in canada for a -month msm deferral are predicted to be very low for all modelled scenarios, including a "pessimistic" doubling of hiv incidence post change. background: safety of blood and blood products is a major concern in pakistan. the prevalence of transfusion transmitted infections among multi-transfused thalassaemia patients is high (above %). the hiv epidemic in pakistan is following the asian epidemic model where after establishment among the high risk groups, its transmission to general public is rapid. fear, stigma and ignorance have contributed heavily to hiv transmission in pakistan. the hiv detection among blood donors is on the rise and reports occur in media repeatedly. aims: to investigate the possible transmission of hiv through blood transfusion in punjab, pakistan and to highlight the steps being taken to reduce further transmission of infections methods: in september , a report of hiv transmission through blood transfusion was reported in the media where a mother and her newborn acquired hiv after blood transfusion from a hiv positive donor (confirmed later). the case was referred to and investigated by the punjab blood transfusion authority (pbta). the pbta team took blood samples of both recipients (mother and her newborn) and the blood donor who was a family relative. the samples were tested by highly sensitive chemiluminescence immunoassay (clia). the clia results confirmed the presence of hiv in both recipients and the blood donor. due to maternal hiv antibodies transfer through the placenta, the infection status of the newborn was not re-confirmed as he died within two weeks. the donor informed that he had donated times in the past few years. the pbta was able to trace only one earlier donation three months ago. the recipient (a female) was found, tested by clia and was found to be hiv positive. all these cases occurred in unlicensed private blood banks that were screening for hiv on rapid manual devices. the blood banks were sealed by the authority and infected cases were registered by the provincial aids control programme and are being treated. summary/conclusions: the main reasons for hiv spread through blood transfusion is the use of sub-standard rapid screening devices which are not evaluated and validated at a national level. in addition, the existing system relies on the family/replacement donors. the national safe blood transfusion programme, is implementing blood safety system reforms as recommended by who. under the reform agenda, the blood transfusion authorities have been made functional and grant licenses to only those blood banks with proper systems to ensure quality and safety of blood products. the programme is developing a national system for the evaluation, selection and validation of all assays used for screening of blood in close coordination with the drug regulatory authority of pakistan. to promote the culture of voluntary blood donations, the programme has taken concrete steps initiating with the formulation of a national blood donor policy, interaction with celebrities, celebration of world blood donor day and more recently the launch of blood donation feature through 'facebook'. the promotion of voluntary blood donation concept along with regulation of blood sector will reduce the risk of hiv transmission through blood transfusions in pakistan. mianyang blood center, mianyang urumqi blood center, urumqi, china rti international, rockville national heart, lung, and blood institute, bethesda stanford university, stanford, united states background: the incidence of hiv infections has increased substantially over the past decade in china, especially among young people, who represent nearly half of the chinese blood donor population. this upward trend in hiv infections underscores the importance of monitoring hiv prevalence and incidence in chinese blood donors. aims: to estimate hiv prevalence and incidence rate (ir) among chinese blood donors using blood donation data from five geographically-disperse blood centers in - participating in the recipient epidemiology and donor evaluation study-iii (reds-iii) china program. methods: western blot confirmatory testing was done on samples of blood donations reactive for hiv- / on one or both rounds of routine elisa tests or positive by nucleic acid amplification testing (nat). multiple imputation was used for samples with missing confirmatory test results. hiv prevalence was calculated among first-time donors. to estimate hiv ir in first-time donors, single-well lag-avidity eia testing was conducted with first-time hiv recent (incident) infections defined as being infected within approximately days based on avidity of hiv antibodies. a novel model was derived to estimate hiv ir among infrequent repeat donors who had provided only one donation in the - estimation interval. to derive an overall hiv ir for repeat donors, this estimate was combined with the classical-model ir estimated for repeat donors who had given at least donations in the estimation interval. multivariable logistic regression model was used to examine factors associated with hiv infection. results: a total of , , whole blood and apheresis platelet donations with postdonation screening results were collected at the five blood centers between and , including , donations from first-time donors and , donations from repeat donors. hiv prevalence among first-time donors was . per , donors ( % ci, . - . ). hiv ir was estimated to be . per , person-years ( % ci, . - . ) among first-time donors and . per , person-years ( % ci, . - . ) among repeat donors. hiv prevalence and ir varied across regions with an increasing trend observed at some blood centers. among first-time donors, being male, older than years, minority ethnicity, less than college education, and certain occupations (commercial services, factory workers, retired, unemployed, or self-employed) were associated with positive hiv confirmatory testing results. summary/conclusions: although hiv prevalence and incidence remain low among chinese blood donors, it is important to monitor hiv epidemiology in blood donors on a continuous basis, especially among populations and regions of higher risk. further donor screening and education strategies need to be developed and evaluated to reduce these risks. the ir methods used in this study for first time donors as well as repeat donors who donate very infrequently is readily applicable to other countries who have similar donation patterns. background: in thailand, the national blood centre is responsible for blood donation service which includes follow-up and blood donor counseling in order to indicate the infection status, especially hiv-positive blood donors. currently, although the epidemic of hiv infection in thailand is in decline, the hiv-positive cases still have been found in blood donors screening. thus, monitoring of hiv infection status in blood donors and post-blood donor counseling are important for providing the hiv-positive infected donors lead to access the hiv treatment immediately. aims: to study the hiv follow-up cases on serological testing over years for assessment of the hiv infection in thai blood donors. the retrospective analysis of hiv follow-up cases on serological testing (cmia, ics and western blot) was conducted during to at thai national blood centre. results: a total of , , voluntary blood donations over years, the repeated reactive results on hiv serological screening were , ( . %) cases and only half of these hiv reactive donors returned to follow-up testing for ascertaining their hiv status. for hiv follow-up process, the hiv reactive screening donors must be followed for months and tested by using the different three principles of hiv serological testing. a total of , hiv reactive results were separated to three patterns including hiv positive results, inconclusive results and negative results which the number of each group was , ( . %) cases, ( . %) cases and , ( . %) cases respectively. out of , hiv positive results, we found that , ( . %) cases were positive with all hiv serological testing for the first-time follow-up and ( . %) cases were tested and become to positive results after follow-up more than one time. in cases of inconclusive results, ( . %) cases were reactive only or testing(s) which these donors did not return to confirm again leading to temporarily deferred donors in blood donor system. in addition, ( . %) cases of inconclusive results could not conclude the hiv result although they were repeated several times. for the last pattern, , negative results cases showed ( . %) cases were negative results after follow-up over months while ( . %) cases were inconclusive results before changing to negative results which almost cases of this group were reentry as blood donor after deferral period is over. summary/conclusions: the number of repeated reactive results on hiv screening was constant over years of which returned to follow-up only half of hiv reactive donors leading to accumulation of temporarily deferred donors in blood donation system. hiv follow-up positive cases were informed and counseled immediately then referring to anonymous clinic for treatment. the problem and challenges of hiv follow-up were inconclusive results that were unclear and some of these did not return to retest lead to loss of re-entry donor who might be changed to negative result afterward. hence, the effective counseling and follow-up system need to be taken urgently to encourage the temporarily deferral donors returned to retest for reducing stigma of deferred donors in hiv follow-up cases. . we only analyzed the information that had non-reactive results for infectious markers reported by blood banks to sihevi-ins©, because they represent a risk for blood recipients. results: when loading the information of sivigila in sihevi-ins©, donors were found ( % men); of these people donations were obtained ( % whole blood). donors ( %) had a reactive result for hiv being subsequently reported in sivigila. in addition, five of them were reactive simultaneously for hbv in blood banks and took on average ae days to be reported in sivigila. donors ( . %) had an hiv reactive result notified by sivigila and subsequently they were reactive in blood banks. this behavior may suggest an attempt to spread the disease. donors ( % men) despite being initially reported in the sivigila database, presented a non-reactive result in a blood bank for hiv; one of them was reactive for syphilis and hbv and only one for hbv. this pattern may suggest false positive or negative results in one of the two databases analyzed. fourteen donors had negative test in blood banks for hiv and in a range of up to months they were reactive by sivigila ( % of them donate whole blood). this conduct may suggest that accepted donations were in a window period and therefore warrant further investigation. considering that two blood components could be obtained on average from each donor, a potential risk is estimated for recipients. summary/conclusions: the donors reported first in the blood banks through sihevi-ins© and later in sivigila allow to estimate an adequate orientation to the health services. the information from general epidemiological surveillance programs could improve the selection of donors and transfusion safety. background: it is assumed that bacterial contamination of blood products most often takes place during the donation process. the number of bacteria at this time point is estimated to be around - cfu per bag. little is known about the growth behavior of different bacteria species in whole blood (wb) units during storage and the distribution of bacteria to the different blood products. aims: aim of the current study was to determine the growth of different bacteria species in contaminated wb units and to study the distribution of the bacteria to the different blood components. methods: whole blood (n = - per species) was inoculated with approximately cfu of different bacteria species (escherichia coli, klebsiella pneumoniae, pseudomonas fluorescens, staphylococcus aureus, staphylococcus epidermidis, streptococcus dysgalactiae, streptococcus pyogenes) and stored for to h at room temperature before centrifugation and separation into red blood cells (rbc), buffy coat (bc) and plasma. bcs from spiked wb were each pooled with random bcs to prepare plasmareduced platelet concentrates (pc). samples were taken from wb after storage and from the blood products (rbc, bc, plasma and pc) right after preparation, and the bacterial titer was determined. sterility of pcs was tested by bact/alert after seven days of storage. results: bacterial growth in wb varied remarkably between donations and bacteria species. the highest titers in wb were detected for the streptococcus species, whereas staphylococcus aureus, staphylococcus epidermidis, escherichia coli and pseudomonas fluorescens did not multiply. bacteria preferably accumulated in the bcs during separation, reaching titers of up to . cfu/ml in bcs and up to . cfu/ml in the corresponding pcs right after preparation. in total, out of pcs tested positive for bacteria at the end of storage. the results were dependent on the species used: e.g., / pcs tested positive after spiking with streptococcus pyogenes, while only / pcs tested positive after spiking with escherichia coli. bacterial contamination of rbc and plasma units was much less frequent and associated with higher bacterial titers in the parental wb units. summary/conclusions: the growth and distribution of bacteria during processing of wb into the different blood products is species-dependent and remarkably varies between donations. results: both patients were male ( yo and yo) with a history of acute myelogenous leukemia status-post haploidentical stem cell transplant. the patients were thrombocytopenic and underwent simultaneous transfusion of irradiated, non-pr, day platelets stored in platelet additive solution, from a single apheresis collection. the blood supplier's primary pre-release bacterial cultures were negative, and the on-site point of release secondary safety measure pan genera detection (pgd) testing was negative for both gram positive (gp) and gram negative (gn) organisms. both apheresis units also passed visual inspection prior to release from the blood bank. during transfusion, both patients displayed signs of septic transfusion reaction including rigors, fever, hypoxemia, tachypnea, tachycardia, and hypotension. transfusion reaction evaluations were initiated, and both patients were admitted to the medical intensive care unit and started on broad-spectrum antibiotics. gram stain of one platelet unit demonstrated gram negative rods (gnr) and gram positive cocci (gpc) in clusters, and the second platelet unit demonstrated gnr only. repeat secondary safety measure pgd testing of both units was negative for both gp and gn organisms. direct bacterial cultures of both platelet units grew both gnr and gpc identified as a. baumanii and s. saprophyticus after h of incubation. colonies on the initial bacterial plates were too numerous to count (tntc), and subsequent re-plating of the platelet units showed: unit : a. baumanii tntc and s. saprophyticus with . cfu/ml unit : a. baumanii . cfu/ml and s. saprophyticus . cfu/ml blood cultures collected from both patients became positive within h with gnr on gram stain, and both blood cultures ultimately grew both a. baumanii and s. saprophyticus. the primary pre-release cultures at the blood supplier remained negative. after days on antibiotics and pressors, both patients stabilized and were discharged home. the blood donor was interviewed, and he was well. no cultures were collected. summary/conclusions: this case documents failure of both primary pre-release bacterial testing and secondary on-site point of release pgd testing to identify two pathogenic organisms. a. baumanii and s. saprophyticus are unusual causes of septic transfusion reactions, and it is possible that these organisms have different limits of detection in the pgd assay compared to other organisms. rapid attention to clinical signs during transfusion and prompt initiation of antibiotics is critical for the management of septic transfusion reactions. we are currently evaluating ways to further reduce septic transfusion reactions, including increasing the utilization of pathogen reduced platelets. background: transfusion-associated infections due to the transmission of bacteria still represents a major risk in developed countries nowadays. despite the refrigerated storage of red blood cells (rbc), fatal reactions of patients receiving contaminated rbc units are repeatedly reported. in order to further increase the safety of blood transfusions, new strategies and measures have to be developed. in this context, transfusion-relevant bacteria reference strains can serve as a valuable tool for the validation, comparison and interpretation of these new developments. aims: conducting a collaborative study to establish the first repository for red blood cell transfusion-relevant bacteria reference strains. methods: six bacterial strains (serratia liquefaciens, serratia marcescens, pseudomonas fluorescens, listeria monocytogenes, yersinia enterocolitica a- and yersinia enterocolitica a- ) were distributed from the paul-ehrlich-institut to laboratories in countries for enumeration, identification and growth measurement in a -day interval for a total of days after low-count spiking of rbc bags ( - colony-forming units (cfu)/rbc bag). results: except for s. marcescens, all other strains showed good-to-excellent growth in rbc. s. liquefaciens, p. fluorescens, y. enterocolitica a- and y. enterocolitica a- achieved > cfu/ml at day and cfu/ml at day . growth of l. monocytogenes was lower reaching a maximum concentration of > cfu/ml at day . in out of laboratories, s. marcescens showed no growth at all. summary/conclusions: five of the six tested strains showed robust growth in rbc independent of donor variability and are promising candidates to be adopted as official rbc transfusion-relevant reference strains by the world health organization. background: the samplok sampling kit (ssk), itl biomedical, is used by nhs blood and transplant (nhsbt) for sampling of platelet components (pc) for bacterial screening using the bact/alert d system. inoculation of bact/alert bottles is performed immediately after sampling. validation of delayed inoculation, with retention of the sample within the ssk devices, would allow a contingency for transport to other screening sites in the event of an incident that prevented testing at the sampling site. ssk maintain a closed system for sampling of pc and are compatible with standard blood collection bags. a graduated chamber ( or ml) ensures that only the required sample volume is collected and an integrated needle allows inoculation into bact/alert bottles. aims: the national bacteriology laboratory, nhsbt, evaluated the impact of prolonged storage of pc samples in ssk devices with regard to bacterial viability and detection. methods: four reference species were assessed: staphylococcus aureus (atcc ), streptococcus agalactiae (atcc ), escherichia coli (atcc ), clostridium perfringens (atcc ). a pool and split method was used with apheresis pc suspended in plasma. units were screened using bact/alert d prior to spiking to prove the absence of contamination. pc were spiked with a single species (range - . cfu/ml) and tested on bact/alert with a ml inoculation into anaerobic and aerobic bottles (positive control). enumeration was performed to confirm the bacterial concentration. each unit was sampled using two ml ssk, which were held for a period of h at - °c. the process was repeated with a -h period. once elapsed, ml of each ssk was inoculated into an aerobic and anaerobic bact/alert bottle, one ssk per atmosphere per species and the remaining sample was enumerated. all bottles were incubated on the bact/ alert system for a maximum of days ( ae . °c) and subcultured if positive. results: positive controls had detectable growth by bact/alert, excluding aerobic bottles with c. perfringens. this was expected as it is an anaerobic organism. after the storage periods, all bottles had detectable growth by bact/alert. s. aureus showed an increase of . -log after h ( . to . cfu/ml) and . -log after h ( . cfu/ml to . cfu/ml). s. agalactiae increased by . -log after h ( . cfu/ml to . cfu/ml) and . -log after h ( . cfu/ml to . cfu/ml). c perfringens increased by . -log after h ( . cfu/ml to . cfu/ml) and . -log after h ( . cfu/ml and . cfu/ml). for e. coli, the concentration after h was reduced by . -log ( . cfu/ml and . cfu/ml), however this was possibly a result of inherent counting errors. at h, an increase in growth by . -log ( . to . cfu/ml) was obtained. summary/conclusions: storage of pc samples in ssk devices for up to h does not have a negative effect on bacterial viability and detection using the bact/alert d system. background: the intercept tm blood system for platelets efficiently inactivates pathogens and leukocytes in platelet concentrates (pc). the system utilizes amotosalen and uva light and is available for the treatment of apheresis and whole blood (wb) derived platelets (mostly buffy coat pools) in europe in plasma or platelet additive solution (pas), and the treatment of apheresis platelets in the us (trima tm in % plasma or amicus tm for % intersol pas/ % plasma). acinetobacter baumanii and staphylococcus saprophyticus strains were isolated from a saline flush taken h after successful and complete transfusion of an apheresis intercept-treated pc in % pas/ % plasma, from a patient with a suspected septic reaction that occurred h post transfusion. bacterial isolates, and a sample of a gram stain-negative and culture-negative sister split pc were submitted for evaluation. we report here the in vitro inactivation of the fast growing, gram negative bacterium a. baumanii and slower growing gram positive s. saprophyticus. the sister unit was assessed for amotosalen break down products as an indication of successful inter-cept treatment. aims: the objective of the study was to evaluate bacterial inactivation of a. baumanii and s. saprophyticus in apheresis platelets, assessed immediately after treatment and with re-culture at the end of a day shelf-life. methods: a double apheresis pc collected in % pas/ % plasma was split into three equal components, yielding approximately ml of platelets/pc. a. baumanii and s. saprophyticus were grown in lb broth and each pc unit was inoculated with either bacterial strain or the combination of both strains, each at~ log colony forming units/ml (cfu/ml). after inoculation, the contaminated units were treated in small volume (sv) intercept kits. samples were taken: pre and post-inactivation treatment, and at , and days of storage. the samples were analyzed by plating on lb plates ( ll- ml). residual amotosalen levels were assessed by hplc. results: initial bacterial titers were . - . cfu/ml. following the inactivation treatment, no viable bacteria were detected by plate culture. no bacteria were detected after , and days of storage, corresponding to > . log inactivation of both bacterial strains. performance of the intercept treatment process was confirmed in the sister pc unit as evidenced by levels of amotosalen and its byproducts characteristic of intercept treatment, as well as by review of the process documentation. summary/conclusions: amotosalen/uva effectively inactivated a. baumanii and s. saprophyticus individually and together below the limit of detection after days storage. no bacteria in the sister pc by gram stain and culture, and the presence of amotosalen byproducts suggested that the pc collection involved in the septic reaction was sterile at the time of intercept treatment and was successfully illuminated. the possibility of only one-of-two split pc being contaminated due to biofilm formation is minimized in the intercept system which decants platelets into virgin storage bags at the end of treatment. contamination of the source pc container likely occurred after intercept treatment, possibly at the time of spiking for transfusion. background: studies in sub-saharan africa have documented bacterial contamination of blood products for transfusion varying between , %> , %, up to times higher than in the north. published data from central africa are lacking. aims: the aim of this study was to determine the proportion of blood products contaminated with bacteria in three hospitals in the democratic republic of the congo (drc). to assure aseptic sampling, we used a closed system of sampling bags. in addition to the presence of contamination, we assessed semi-quantitative colony counts. methods: from july to december , a total of blood products were sampled, of which in hôpital provincial g en eral de r ef erence, kinshasa (hpgrk), in hôpital p ediatrique kalembe lembe, kinshasa (hpkll) and in hôpital saint-luc, kisantu (hslk). after compatibilization of blood products, ml of blood was transferred from the primary blood bag to an attached sampling bag. sampling bags were sealed off, stored in the fridge and transported once daily to the bacteriology laboratory. using the adapter connected to the sampling bag, ml of blood was inoculated in a blood culture (bactalertpf, biom erieux) and incubated at °c for days. cultures were checked daily for signs of growth. in addition, ml of blood was equally distributed on the cled and macconkey agar-coated sides of a dipslide (meus s.r.l.). dipslides were incubated h for semi-quantitative culture, expressed as colony-formingunits (cfu) per ml. in case of blood culture growth, bacteria were identified and a second blood culture was inoculated to exclude contamination during processing. bacteria grown on semi-quantitative culture were also identified. results: a total of . % ( / ) of whole blood and red cell concentrates were contaminated with bacteria. in hpgrk, . % ( / ) of blood products were contaminated, in hpkll . % ( / ) and in hslk . % ( / ) . the proportion of contaminated blood products was significantly higher in hpgrk compared to hslk (p = . ). there was no significant difference between the other sites (p = . and p = . ). majority of isolated bacterial species were coagulase-negative staphylococcus spp./micrococcus spp. ( . %) and bacillus spp. ( . %). the remaining % of bacterial isolates were identified as non-fermentative gram-negative rods, klebsiella pneumoniae, staphylococcus aureus, mould, listeria spp., corynebacterium spp./coryneform bacteria. the concentration of all isolated bacteria was lower than ³ cfu/ml, except for one coagulase-negative staphylococcus spp. found in hpgrk at ³ cfu/ml. summary/conclusions: to our knowledge, we were the first to reach a sample size of more than blood products for bacterial culture and the first to use a closed system of sampling bags in sub-saharan africa, which guarantees aseptic sampling of blood cultures. this might explain the low bacterial contamination rate ( . %) of blood products in three hospitals in drc compared to previous studies in other sub-saharan african countries. moreover, bacterial concentrations in the contaminated blood products were low (< ³ cfu/ml). the different proportions of contamination between study sites suggest that different environments and practices play a role in the risk for bacterial contamination. background: although the screening of the treponema pallidum (tp) is mandatory in blood donations, its necessity is controversial, because there have been no transmissions by blood products documented in the developed countries in the last few decades. aims: based on laboratory markers, active and early tp infected donors (aeid) were determined. the demographics of aeid and the frequency measures of cases were compared with that of early infected syphilis cases (syc) notified from the to -year-old general population registered at the nphc. methods: altogether, , to -year-old donors were screened with anti-tp immunoassay (architect syphilis tp, abbott, wiesbaden, germany) between and . reactive results were confirmed with immunoblots (viramed biotech ag, planegg, germany), which discriminated both specific anti-tp (igg, igm) and non-specific vdrl antibodies in five dilutions. meeting the criteria of anti-tp igg positivity with a vdrl titer of ≥ : and anti-tp igm positivity, donors were considered as aeid. they were stratified by age, gender and residence regions. the proportion of aeid (paeid) and syc (psyc) were calculated in first time (ft), and repeat tested (rt) donors and in the to -year-old general population, respectively, in each year studied. the period prevalence (pp) of aeid and syc was estimated in the populations at risk , across - . statistics: weighted proportions and one-way anova with tukey post-hoc test and z score test were applied. statistical significance was defined as p < . . results: anti-tp reactivity was confirmed in blood donors. aeid was proved in cases with ft and rt donors. in that period, syc were notified. both in aeid and syc, the age group of - years with approximately % and % of individuals was the dominant. the proportion of men was % and % (p = . ) in the aeid and syc, respectively. paeid estimated in ft donors was significantly higher ( . %; . %; . %, p < . ) than that of rt donors ( . %; . %; . %) and the proportion of syc ( . %; . %; . %) in the general population. pp of aeid showed a roughly homogenous distribution in the regions ( . %- . %), however, pp of syc had a significant ( . %; p < . ) central hungary dominance in relation to the other regions ( . %- . %). comparing the pp of aeid to syc, central hungary indicated a significant difference ( . % vs. . %, p < . ), however, other regions showed no substantial differences. summary/conclusions: donors with anti-tp confirmed positivity are referred to the healthcare system. based on the laboratory markers tested, aeid could be separated and their demographical characteristics are pretty similar to that of syc notified from the general hungarian population. the proportion of early and active infection in ft donors is significantly higher than that of rt donors and the proportion of syc in the general population. given the considerable number of tp infection in background: quality assurance and safety of hematopoietic stem cells (hsc) with emphasis on prevention of bacterial and fungal contamination are the prerequisites for any transplantation procedure. bacterial contamination is of particular significance as it occurs relatively more frequently and bacteria are gradually gaining more antimicrobial resistance. aims: the aim was to determine the incidence rate of bacterial and fungal contamination during processing of transplantation material at the institute of hematology and transfusion medicine (ihtm) taking into account the hsc sourceperipheral blood (pbsc), bone marrow (bm) or cord blood (cb). methods: analysis involved both autologous and allogenic components collected at ihtm and other hospitals and dedicated for ihtm patients. in all, the analysis comprised donations, including bm ( . %), pbsc ( . %) and cb ( . %) donations processed in our laboratory in the years - . bm was collected in operating theatre-conditions, pbsc with cell separators -cs- (baxter), cobe spectra (gambro) and trima accel (terumo bct) and cb was acquired from umbilical vein at obstetrics and gynaecology wards. aerobic and anaerobic bacteria contamination was determined at various incubation temperatures (room temperature and °c) using a variety of culture media. pbsc and bm were tested using samples with trypcase-soy broth (tsb-t) and with schaedler + vit k (biomerieux). cb was tested using bactec peds plus/f and bactec lytic/ /anaerobic/f (becton-dickinson). results: in the - period contaminated products were found: pbsc ( . % of all tested pbsc units) and cb ( . % of all tested cb units). no infected bm products were determined. the overall percentage of contaminated products was estimated at , %. in , three ( ) products were found contaminated with staphylococcus epidermidis; all came from one patient with central venous catheter and were collected on consecutive days. other products were contaminated mostly with staphylococcus epidermidis ( . %). detailed results to be presented on the poster. summary/conclusions: according to ihtm policy no contaminated product is admitted to clinical use. the outcome of our study identifies processing experience of the staff as the main indicator of product quality. important is also proper assessment of donor health and condition of the injection site as products are usually collected from central venous catheter. the closed system is an additional safeguard against contamination during processing. the sample collecting procedure should help to avoid false positive results. background: syphilis is considered a global public health problem. the world health organization (who) estimates that there are annually around million new cases of syphilis in the world, more than % occurring in developing countries. despite significant decrease in syphilis transfusion transmission. the recent increase in worldwide incidence associated with the risk of transmission through platelet concentrates (cp), which are stored at room temperature, have called attention to the potential residual risk of syphilis transmission by transfusion. between and we observed in our institution a significant increase of % in positivity of syphilis among blood donors from . % in to . % in and . % in (p < . ). aims: to determine the prevalence of active syphilis in blood donors and characterize the serological profile of syphilis positive donors. methods: each positive sample in a treponemic chemiluminescence assay (cmia, abbott architect) performed during blood donor screening in was submitted to a treponemic elisa anti-treponema pallidum igm (euroimmun) and a non-treponemic test (antigen-omega diagnostics). samples with positive results for one or two of these tests (indicating recent syphilis) were submitted to a real-time pcr for syphilis. the inno-lia syphilis-fujirebio immunoblot test was also performed for samples that presented a positive result for elisa-igm alone. financial support: fapesp / - . results: among , samples screened in , ( . %) presented a positive result for cmia -syphilis. of these, ( . %) were included in the study. a total of samples ( %) showed vdrl+/igm+; ( %) vdrl+/igmand ( . %) vdrl -/elisa igm+. the inno-lia syphilis test was performed as a confirmatory test in ( . %) samples that presented positive results for elisa igm and vdrl negative with ( . %) positive results, ( . %) undetermined and ( . %) negative. none of the samples showed the presence of treponema dna by real-time pcr. the prevalence was . %, the incidence was . % in the year , and the incidence in relation to the total positivity was . %. both, prevalence and incidence were higher in men, white, not married, aging - years and high school educational level. we observed a % a-hbc seroprevalence in the elisa igm-syphilis positive samples and a prevalence of . % htlv coinfection. summary/conclusions: we observed a significant increase in prevalence of syphilis in ( . %) with an incidence of . % of the total of cases initially positive in the cmia test. according to our data, we could identify a risk of syphilis transfusion transmission in blood banks that exclusively use the vdrl for donor screening, once we found ( . %) cases with negative vdrl and elisa igm and inno-lia positive. continuous monitoring of the profile of donors infected with syphilis at this time of reemergence of the disease is useful and important not just for blood banks, as it reflects the epidemiological situation of disease in community, and can contribute to the definition of health policies. background: transfusion related sepsis is a serious concern limiting platelet storage time to days at room temperature. while most units are screened for bacterial contamination when collected, bacterial monitoring methods can take up to days to detect contamination. thus, cold storage of platelets represents an attractive alternative for improving platelet safety. in this study, we assessed bacterial growth in platelets stored either at room-temperature (rt; °c) or refrigerated (cs; °c). aims: the aims of this study were to ) assess the effect of storage temperature on platelet function and bacterial growth in "contaminated" platelet units, and ) identify factors contributing to bacterial growth during rt storage. methods: apheresis platelets in plasma (plt) were obtained from healthy donors using the terumo trima accel automated blood collection system (terumo bct). fresh plasma (fp) was collected similarly. aliquots of plt or fp were transferred to ph safe minibags (blood cell storage, inc) and "contaminated" with acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, staphylococcus aureus, staphylococcus epidermidis, or pbs (uninfected control). minibags stored at rt were agitated using an orbital shaker set to rpm while cs aliquots were stored under static conditions. bacterial growth was monitored daily through dilution plating. lactate levels were assessed by istat (abbott) cg + test cartridges. plasma glucose levels were assessed using blood glucose testing strips (germaine laboratories). platelet activation and aggregation were assessed on days , , , and by flow cytometry and multiplate platelet aggregometry, respectively. results: bacterial growth progressed rapidly over the first - days post-collection in all plt aliquots stored at rt except those challenged with s. epidermidis. significant growth of s. epidermidis was not detected until day . bacterial numbers remained unchanged in refrigerated aliquots through day . rt storage resulted in significantly (p < . ) decreased platelet aggregation over time which was exacerbated by bacterial challenge. plt function was largely preserved with refrigeration regardless of challenge. bacterial growth was significantly reduced, or at least delayed, in fp. fp challenged with gram-negative pathogens exhibited a significant (p < . ) delay in bacterial growth at day . while growth of e. coli and p. aeruginosa recovered by day , growth of a. baumannii was significantly (p < . ) inhibited throughout. fp challenged with gram-positive pathogens exhibited significant (p < . ) reduction in bacterial growth relative to plt aliquots. bacterial growth correlated with plt lactate production. lactate levels in plts challenged with e. coli showed diminished significantly after day , indicative of lactate utilization. with exception of fp challenged with s. aureus, bacterial growth was restored in fp supplemented with lactic acid in all challenge groups. summary/conclusions: refrigeration preserved platelet function while both inhibiting bacterial growth and lactate production. conversely, the opposite was observed with rt storage. these data demonstrate that bacterial growth can be controlled through refrigeration without loss of function and rt storage may potentiate growth of certain bacterial strains through accelerated plt metabolism. background: bacterial contamination of peripheral blood progenitor cell (pbpc) for transfusion has been the cause of serious sepsis and life-threatening infections. however, a standard procedure or choice of test sample(s) has not been established to screen pbpc products for microbial contamination, because these products are not large enough to facilitate inoculation of the recommended volume for the automated blood culture systems. samples taken from by-product plasma and low volume pbpc product were cultured in routine sterility test. aims: to evaluate the residual risk of microbial contamination in pbpc products for transplantation, we cultured sufficient post-thaw inoculation volumes from pbpc products which were discarded for various reasons in our blood center. methods: in routine sterility test, a -ml sample of by-product plasma collected with pbpc product was inoculated into bact/alert bpa and bpn culture bottle ( ml each) within h after collection. the bottles were then placed in the bact/ alert system and incubated for at least days or when a positive reaction was indicated by the automated liquid-media culture system. moreover, a -ml postthaw sample would be cultured before transplantation performed. in the residual risk investigation, discarded pbpc products were thawed, and then a -ml and a -ml aliquot were taken and cultured with the same method. all positive bottles were subcultured for bacterial isolation and identification. results: in september and march , after maintaining in liquid nitrogen for to years, pbpc products collected from patients, which was preserved in a volume between and ml, were discarded. all of these products had been cultured negative in routine sterility tests with plasma samples. these final products were thawed and cultured with both the -ml and the -ml aliquot. one of these pbpc products had the positive culture result with the -ml retested samples. nevertheless, the same pbpc product had the negative result with the -ml post-thaw pbpc sample and the -ml by-product plasma sample. propionibacterium acnes was isolated from the bpn positive bottle. summary/conclusions: the residual risk of microbial contamination in pbpc postthaw products still exist after routine sterility test with the plasma sample and the -ml volume of pbpc sample. the bacterium isolated from pbpc product was normal skin flora bacterium. an optimal screening method of pbpc products merits further study to increase the safety of the blood supply. background: hospital hygiene tools that serve as a proxy for assessment of microbial contamination are increasingly used. they include adenosine triphosphate (atp) bioluminescence and air particle counting. however, their use for microbial monitoring of blood banks remains underexplored. they could be of particular interest in a sub-saharan african setting (temperatures, dust) to circumvent bacterial culture and provide direct results usable for monitoring over time. aims: the aim of this study was (i) to quantify environmental bacteria in the air and on surfaces that are regularly in contact with blood products, and (ii) to evaluate atp bioluminescence techniques and particle counts as a predictor for bacterial contamination, in three blood banks in the democratic republic of the congo (drc). methods: samples were taken in three blood banks in the democratic republic of the congo: hôpital p ediatrique de kalembelembe (hpkll) ( surfaces, air), hôpital provincial g en eral de r ef erence (hpgrk) ( surfaces, air) and the national blood transfusion centre (cnts) ( surfaces, air). surfaces that are regularly in contact with blood products were selected (sealer, fridge, donor chair,. . ..). regular surfaces were sampled using rodac contact plates ( . cm²) containing cled and macconkey agar, irregular surfaces using swabs (nrsii, medicalwire). atp was measured on the same surface (pd , kikkoman), expressed as relative light units (rlu) per cm². air was sampled by active sampling ( liter; spinair, iul) on cled and macconkey medium. in parallel, particles > . lm and > lm were counted using a particle counter ( , liter; metone a). culture media were incubated for h at °c before counting colony forming units (cfu). results: for regular surfaces, the median (range) viable bacterial count was ( - ) cfu/rodac, ( - ) cfu/rodac, ( - ) cfu/rodac for hpkll, cnts and hpgrk, respectively. at hpkll, highest viable counts were found in the sink (plain growth) and cool boxes ( and cfu/rodac). in cnts the blood processing bench, the donor chair arm support and washing basin showed the highest counts (plain growth). whereas in hpgrk, most bacteria were found in a fridge (plain growth), blood bag trolley (plain growth) and manual separator ( cfu/ rodac). gram-negative bacilli were isolated from water basins and sink in cnts and hpkll, but also surfaces close to donor chairs at hpgrk. the median (range) of atp per cm² was . ( - . ) rlu at hpkll, ) rlu at cnts and . ( . - . ) at hpgrk. atp results and total viable count were not correlated (n = , p = . ). median (range) bacterial count in the air was ( - ) cfu/ l for all sites together. there was no correlation found between total bacterial count and particles > . lm or > lm (r = . and r = . respectively; p < . ; n = ) summary/conclusions: total viable bacterial count of surfaces varies over blood bank sites. according to our results, atp and particle counts did not correlate with bacterial counts on surfaces and in the air, respectively. bacterial isolates from blood bank environments in drc need to be identified and seasonal variations need to be evaluated. background: the risk of transfusion-transmitted hepatitis e virus (tt-hev) infections in line with the question of the necessity of hev-nat screening of blood products is currently subject to an ongoing debate on the importance of timely introduction of hev screening of blood donors and the impact of blood safety. different countries have chosen different regulatory approaches. just recently, the german federal authorities have introduced mandatory testing of all therapeutic blood products beginning from january st . however, we already decided to voluntarily test all our blood products since january . aims: in this study, we present our results of a % screening of therapeutic blood products for hev rna including four years of active surveillance of hepatitis e infection among blood donors in germany. methods: from january to december , a total of , allogenic blood donations from , individual german blood donors were screened in a minipool format of samples of for the presence of hev rna (realstar hev rt-pcr kit, altona diagnostic technologies (adt), hamburg, germany). nucleic acids were extracted from . ml plasma using the chemagen msm-i extractor (viral k, perkin elmer chemagen gmbh). the % lod of the assay was determined to . iu/ml ( iu/ml per single donation). the presence of hev-specific igm and igg antibodies was determined using the anti-hev igm/igg elisa (euroimmun, luebeck). hev rna concentrations were quantified using the first who international standard for hepatitis e virus rna for nat-based assays. all hev rna positive donors were deferred from donation for months. follow-up samples were tested for the presence of hev rna and hev-specific antibodies. genotyping was performed by sequencing of the hypervariable region (hvr) and orf . results: in total, hev rna positive donors were identified. of these, hev rna-positive donors, were nat-only positive donations ( . %, negative for anti-hev igm and anti-hev igg), three donors had a positive igm titer ( . %), donors showed reactive igm and igg titers ( . %), donors already had isolated igg titers ( . %). median values of viral loads were approximately twice as high in index donations that were antibody negative. merely donors showed elevated alt levels ( . %), mostly within a double increase within the reference range ( . %), only . % of donors had even further elevated alt levels. significantly higher alt values were found in donors with a viral load > , iu/ml compared to the group with viral loads between and iu/ml. available follow-up samples confirmed igg seroconversion for all donors, however we also observed long-term igm positivity in some donors. genotyping revealed genotype in all cases. the month-dependent incidence ranges from : to : , blood donations with a peak in june and july. summary/conclusions: the high number of identified hev rna positive donors emphasizes the need for hev-nat screening to increase the safety of blood products. this study further confirmed that hev infection is common in german blood donors. background: zika virus (zikv) is a mosquito-borne virus that has caused outbreaks in central and south america in february , and has threatened the safety of blood transfusion globally. there is a high risk of zikv transmission by whole blood and blood components transfusion. it was reported that, zikv rna in infected patients plasma can only be detected within to weeks. however, in whole blood, zikv rna might present positive up to day after the symptoms appear in some patients, even if the clinical symptoms disappeared with zikv rna negative in plasma. this phenomenon suggested that the presence of zika is associated with red blood cells (rbcs). moreover, another report showed that viral load in whole blood of type a west nile virus (wnv) patients was higher than type o, implying that the binding of virus to rbcs may be related to blood group glycoprotein. both of zikv and wnv are member of the flavivirus genus. the study is intended to explore whether zikv have the same adherence mechanism to rbcs as wnv. aims: to investigate the distribution of zikv in blood components and adherence of zikv to different blood types of rbcs in whole blood. methods: five units for each blood type of a, b, o and ab whole blood were randomly selected. each unit of ml whole blood was divided into two half-unit. zikv was added to one half-unit in a certain proportion, and incubated at °c for days. each component of whole blood was collected for viral load detection. in the other half-unit,rbcs were suspended in the same type pools of plasma with equal volume after the plasma removed from the whole blood after centrifugation. zikv was added with the same certain proportion, and then incubated at °c for days. the whole blood samples and the upper plasma by centrifugation were collected detected for zikv rna. meanwhile, rbcs were washed and resuspended with normal saline followed by viral load detection. results: zika rna of these samples which extracted from whole blood, rbcs, and plasma were determined in a quantitative reverse transcription pcr, and viral rna of each component was all up to copies/ml. although, zikv rna loads did not show significant difference in distribution between rbcs and their corresponding plasma components, zikv rna quantification were significantly higher than those in plasma (p < . ) in type o rbcs and lower than those in plasma (p < . ) in type ab rbcs. summary/conclusions: in our study, we detected high viral rna loads in rbcs. it was demonstrated that zikv adheres to erythrocyte in whole blood, and the blood type may have influence on the adherence. background: hong kong is not endemic for dengue virus (denv) with most of the documented cases being imported. the presence of sufficient number of mosquito vectors, aedes albopictus, in the territory has led to two self-limiting indigenous outbreaks affecting residents from to . during august to september , another outbreak of confirmed cases of autochthonous dengue fever were reported to the department of health, linked to two epidemiological clusters, one in lion rock park near wong tai sin (wts) district ( cases) and the other in cheung chau, an outlying island ( cases). aims: we assessed the risk of dengue transmission from blood donors during the outbreak using a simplified version of the probabilistic model developed by biggerstaff and petersen (b-p) and the european up-front risk assessment tool (eufrat) model (oei, transfusion, ) . methods: patient demographic and general population data were obtained from the centre for health protection and the department of census and statistics of the hong kong government for the number of -to -year-old patients in the outbreak and residents of the same age range in hong kong and wts district as at mid- respectively ( - years old being the eligible age range for first time donation). to apply the b-p model, we estimated denv incidence among donors in hong kong territory and in wts with confirmed denv infection during august to september after correction for clinical:subclinical infections ratio, the mean length of asymptomatic viraemia and the probability of collecting blood from asymptomatic donors as described previously (seed, transfusion, ). to estimate the risk using eufrat model, outbreak and blood donation variables were entered into eufrat's web-based interface (https://eufrattool.ecdc.europa.eu/), which provided automatic calculation of risk-related output parameters. results: while using the b-p model, the estimated risk of collecting a denv viraemic donation was one in , ( , - , , ) territory-wide for the -day study period but increased to one in , ( , - , ) in wts. similarly while applying the eufrat model, the risk of encountering a viraemic donor was in , ( , - , ) territory-wide and in , ( , - , ) in wts during the same period. the eufrat also predicted a territory-wide issue of . unit of denv-contaminated labile blood component during the outbreak period. summary/conclusions: like many mosquito-borne infections such as denv, the risk is characteristically localised and varies geographically and seasonally during outbreaks. the average predicted risk of collecting a denv-viraemic donation territory-wide is low at in , during the outbreak based on the b-p model, which was generally considered as tolerable. however, the risk increased by folds when blood donations were collected from wts residents, who had higher chances of paying visits to lion rock park in close proximity. it was then justifiable to institute risk mitigation policies such as geographically-based deferral and/or fresh component restriction, enhanced post-donation reporting, etc. to protect against blood safety. background: hev is a developing threat to blood safety following the reporting of several cases of transfusion transmission hev (tt-hev). transfusion-related hev infection has been reported in several countries but its true frequency is probably underestimated because it is often asymptomatic and testing of blood donors is infrequent. pakistan is classified as a highly endemic region; with sporadic cases of hev occurring throughout the year, mainly affecting the adult population. to the best of our knowledge, no studies have been reported from pakistan on the epidemiology of hev in blood donors. aims: to assess the epidemiology of the hev specific antibodies and serum alt levels in blood donors of capital twin cities of pakistan. methods: this cross sectional study was conducted from july to december at three blood banks in the capital twin cities (rawalpindi and islamabad) of pakistan. the blood donors were equally distributed across the three blood banks. only donors who tested negative for hiv, hbv and hcv were included in the study. serum alt levels were analyzed by using automated clinical chemistry analyzer (selctra pro m) using merck kits. all samples were tested for hev-specific antibodies (igm and igg) by using enzyme linked immunosorbent assay (elisa) kits (adaltis, italy). statistical analyses were performed using spss software version . (ibm). results: in our study population there were ( . %) males and ( . %) females. the mean age of recruited blood donors was . (sd ae . ), with a range of - . younger donors were more common with a frequency of - year olds of ( . %). we found an overall hev igg prevalence of . % and an hev igm prevalence of . %. there were ( . %) blood donors who were positive for both igg and igm antibodies. our results revealed that the hev specific antibodies (igg, igm) prevalence increased with age. the mean value of serum alt was . (sd ae . ) with a range of - iu/l. the serum alt levels were elevated (> iu/l) in ( . %) blood donors. there was significant correlation (p=< . ) between serum alt level and hev specific antibodies for igg and igm. summary/conclusions: this study shows that a significant proportion of blood donors at our blood centers have been infected with hev and may be able to cause tt-hev. as we have not yet measured hev rna, we have used igm antibodies as a proxy for donors who have active infection. hev is generally asymptomatic, so it is debatable whether mandatory hev screening in blood donors should be required. results of this pilot study show that there is a need to conduct a larger study at national level with highly sensitive assays before making screening for hev mandatory in pakistan. background: hepatitis e virus (hev) is a zoonotic virus. who estimates that there are million hev infections, million acute hev cases and thousands hevrelated deaths worldwide each year. in recent years, the prevalence of hev in european and american countries has increased significantly. the survey results show that the positive rate of hev igg in blood donors is respectively . % in new zealand, . % in britain, . % in denmark, % in the united states and . % in the netherlands. hev has become a global public health concern. in addition to the food route of infection, several cases have been reported that hev can be transmitted via blood products. aims: to investigate the prevalence of hepatitis e virus (hev) infection among voluntary blood donors and potential impact on blood safety in guangzhou china. methods: blood samples from blood donors were collected from april to april at the guangzhou blood center and were tested for anti-hev igg antibody (hev igg), anti-hev igm antibody (hev igm) and hev antigen (hev ag)by enzyme linked immunosorbent assay (elisa). hev rna detection was performed on hev antigen positive samples by rt-pcr. the association of age, gender, ethnicity, occupation and alt with hev igg and igm were analyzed by chi-square test. multivariate logistic regression analysis was applied to identify the independent risk factors of hev infection. results: the positive rates of hev igg, igm and hev ag were . % ( / ), . % ( / ) and . % ( / ), respectively. no positive hev rna was detected. age and ethnicity were independent risk factors for hev igg and hev igm. the rate of hev antibody increased significantly with age (igg or = . , p < . ; igm or = . , p < . ). donors who were zhuang minority ( . %, . %) showed higher anti-hev than those who were han ethnicity ( . %, . %), and the difference was statistically significant (igg or = . , p = . ; igm or = . , p < . ). in addition, we found that occupation was an independent risk factor for hev infection, where students showed the lowest anti-hev rate. summary/conclusions: the results indicate that the positive rate of hev antibody among blood donors in guangzhou is high, and the infection status differs in different populations. our study provides basic data for the estimation of risk of transfusion-transmitted hev. background: human cytomegalovirus (hcmv) belongs to the viral family of herpesviridae. it is an enveloped double-stranded dna virus, widely distributed in the human population ( - % seropositive subjects worldwide) and cause of severe disease in immunocompromised patients and upon infection of the foetus. in normally healthy subjects, hcmv persists lifelong without clinical manifestation undergoing alternating phases of active viral replication and latency. since hcmv can be readily detected in blood, as free virus as well as associated to neutrophils and monocytes, hcmv transmission is a complication of blood transfusion. even though leukoreduction of blood products has been shown to significantly reduce the risk of hcmv transmission, higher inactivation standards may be required for high-risk, immunocompromised groups of patients. aims: in this study, murine macrophages infected with murine cytomegalovirus (mcmv) were used as a model to study the inactivation cell-associated cmv in human plasma using the theraflex mb-plasma system (macopharma). methods: mcmv expressing the green fluorescent protein was used to infect murine macrophages. infected macrophages were harvested h after infection, washed and used for spiking of plasma. plasma units (n = , ml) were spiked with infected cell suspension ( % v/v) and treated with the theraflex mb-plasma system according to the manufacturer's protocol using the macotronic-b illumination device (macopharma). samples were taken after spiking (load and hold sample), after illumination with different light doses ( after addition of mb, , , and [standard] j/cm ) and after blueflex filtration. mcmv titers were determined by endpoint titration and large volume plating on murine fibroblasts. infectious virus, which expressed gfp in infected cells, was detected using a fluorescence microscope. results: the results of infectivity assay showed that the treatment of human plasma by the theraflex mb-plasma system inactivated cell-associated mcmv in a dosedependent manner. after spiking with mcmv infected macrophages a mcmv titer of . (bag no. ) and . (bag no. ) log tcid /ml was achieved in the plasma units. in hold samples, a mcmv titer of . (bag no. and bag no. ) log tcid /ml was determined. the illumination step of the theraflex mb-plasma treatment procedure efficiently inactivated mcmv. already three-fourths of the standard light dose decreased infectivity of cell associated and remaining cell-free mcmv to infectivity levels below the limit of detection (≥ . log). further investigations would be needed to evaluate the log reduction capacity of the blueflex filtration step for cell-associated mcmv. summary/conclusions: the results with the murine model virus suggest that the theraflex mb-plasma system is an effective technology to inactivate cell-associated cmv in human plasma units. background: the use of pathogen inactivation (pi) technologies for platelet concentrates and plasma is slowly but steadily increasing. methods for treatment of red blood cells (rbcs), the most commonly used blood component, are still under development. aims: a novel approach for pi in rbc units employing uvc light was developed. methods: pi treatment was applied to full-scale rbc units after leukodepletion. the pi capacity of the uvc-based method was evaluated by bacteria and virus infectivity assays. a panel of in vitro assays to measure quality, metabolism, functional, morphologic, and blood group serology variables was applied to a pool-and-split approach in which pathogen-reduced rbcs were investigated in comparison to untreated rbcs. results: uvc treatment caused dose-dependent inactivation of bacteria and enveloped and non-enveloped viruses in rbc units. at a full dose, the mean log reduction factors ranged from . (bacillus cereus) to . (serratia liquefaciens) for the tested bacteria, and from . (emcv) to ≥ . (vsv) for the tested viruses. uvc treatment did not alter rbc blood group antigen expression. quality of uvc-treated rbcs was maintained during storage, e.g. hemolysis in uvc-treated and untreated rbcs were well below . % until day of storage. summary/conclusions: the data obtained until now show that uvc irradiation is a potential new method for pi in rbcs and justify further development of this process. background: histo-blood abh antigens are the mayor allogeneic antigens in human and they are widely distributed in almost all tissues. the expression of a- , -fucosyltransferase (fuct ), encoded by fut gene, determines the secretor status of an individual. about % of caucasian population have a functional copy of fut (secretor gene) expressing abh blood group soluble antigens in organic fluids such as saliva and seminal plasma. this individuals are known as "secretors". soluble abh blood group antigens have been associated with several metabolic and infectious diseases as well as reproductive failures. the incidence of infertility related of both male and female factors continues to rise despite many advances in reproductive technologies. it is well known that abo antigens are expressed on sperm membrane and in seminal fluid of secretors as well as abo antibodies are present in cervical mucus. in previous studies we observed significant loss in progressive motility of spermatozoa of non-secretors compared to secretor ones caused by specific cervical mucus antibodies in abo-incompatible couples. in addition, sperm cells are haploid cells, so that a heterozygous individual has two sperm subpopulations, each expressing the corresponding allele. the specific antibody of cervical mucus will attack only its complementary sperm. aims: to evaluate the prevalence of secretor character in men belonging to fertile and infertile couples in order to investigate a possible association with reproductive success. methods: samples of semen, from infertile men and from fertile controls were studied. comprehensive infertility evaluation was performed in all patients according to who criteria. secretor phenotype was evaluated in seminal plasma by inhibition of hemagglutination assay using saline erythrocyte suspensions, monoclonal antibodies anti-a, anti-b and lectin from ulex europaeus (anti-h). to distinguish between abo genes, genomic dna was extracted by an enzymatic digestion method. pcr was designed with two sets of oligonucleotides that allow to amplificate two different regions of the transferases without use of restriction enzymes. by comparison of bands of the pcr products, the individual genotype was determine. cervical mucus antibodies of their female partners were titrated with the corresponding red blood cells. results: results were analysed in both groups. in infertile couples with abo incompatibility, the frequency of non-secretor phenotype of male partners ( . %) were significantly higher than those from fertile couples ( . %) (p < . ) the results obtained by pcr in sperm cells correlated % with red cells phenotypes. summary/conclusions: incidence of infertility continues to increase. several factors have a negative impact on men's reproductive health. immunological implications are now being studied and considered as a cause of failure in sperm-egg interaction, even among normal gametes. secretor phenotype in male partners could help reproductive success by blocking cervical abo antibodies. furthermore, if the male is heterozygous, cervical mucus antibodies will only affect the corresponding sperm. we propose to evaluate abh antigen expression on sperm membrane and seminal plasma as well as abo antibodies in cervical mucus to contribute to the diagnosis and treatment of human infertility. background: the h blood group contains one antigen, the h antigen, which is present on virtually all red blood cells (rbc) and is the acceptor substrate of both a and b gene-specified glycosyltransferases. in blood group o the h antigen remains unmodified and therefore its rbcs show the highest and the rbcs of blood type ab the least amounts of h antigen. individuals with the so called bombay phenotype carry homozygous h null alleles (h | h) and do not produce any h antigen. the para-bombay phenotype retains some h antigen on rbcs either induced by a weakly active (h+ w | h+ w ) or completely silenced fut gene (h | h), mandatory linked with an active fut gene. aims: in this study, we aimed to develop an adapted flow cytometric method to quantify the relative amount of h substance present on rbcs in order to distinguish different abo phenotypes in routine diagnostics as well as to capture rare h-deficient phenotypes. methods: analyses were performed on a flow cytometer (facs canto ii, bd biosciences, ch) and measured with identical instrument settings. list mode data were evaluated and visualised using bd facsdiva software. rbcs were incubated with increasing concentrations of monoclonal anti-h antibodies (bric -pe and a : mixture of bric -pe/bric , ibgrl, uk). after rinsing the cells with pbs, micro-aggregates were mechanically dissolved. rbcs from blood donors with different abo phenotypes (o ( ), a ( ), a ( ), b ( ), a b ( ), a b ( )) and patients with genetically confirmed bombay and para-bombay phenotype were assessed. results: saturation of h antigen binding sites on type o rbcs was achieved only upon use of a : antibody mixture (bric -pe/bric ) covering approx. half of the h-binding sites by unconjugated bric . in contrast, non-o type rbcs reached saturation of h-binding sites using pure bric -pe. rbcs coated with bric -pe at saturation revealed a distinct pattern of mfi (mean fluorescence intensity) depending on the abo phenotype. in addition, mfis of rbcs upon staining with bric -pe did discriminate bombay-and para-bombay type rbcs, respectively. summary/conclusions: adapted flow cytometry is able to distinguish variant expressions of rbcs h antigen. thus, our flow cytometric method may complement traditional serological and genetic analyses in routine abo phenotype cases or, more intriguing, when the bombay or para-bombay phenotype is suspected. it will be of interest to further prove this method by investigating additional rare h-deficient phenotype cases. s chen , , x xu , , x hong , , k ma , , j he , , j chen , and f zhu , blood centre of zhejiang province zhejiang provincial key laboratory of blood safety research, hangzhou, china background: weakened a and b antigen expression results in abo typing discrepancies. h gene controls the development of h substance from which a and b antigens develop. depressed a and b antigen expression and strengthened h antigen expression are always simultaneously observed in abo subgroups. there are other possibilities for weak antigen expression of abo system such as leukemic change and pregnancy. it is undiscovered whether abnormal expressions of a, b and h antigen stand for abo subgroups in hemopathic patients. aims: the aim of this study is to explore the role of enhanced reactions with anti-h in direction to abo subgroups of hemopathic patients. methods: samples from blood donors and hemopathic patients with nonconcordant abo typing by serological tests were collected after consent information. the agglutination strength of these rbcs with anti-h reagent was recorded. enhanced reactions were determined by comparison with the results from normal abo groups. the genomic dnas of samples were extracted and genotyped for abo system. this work was sponsored by the medical science research foundation of zhejiang province ( rc ). results: samples in blood donors showed increased expression of h antigen, of which were identified as abo subgroups. there were enhanced reactions in hemopathic patients. however, were finally confirmed as normal abo genotypes. no statistical significance ( . % vs . %, p > . ) in the frequency of strengthened h antigen expressions was observed between donors and hemopathic patients. the total number of subgroups is and respectively in blood donors and hemopathic patients. extremely significant statistical differences ( . % vs . %, p < . ) existed in the frequency of subgroups with enhanced h antigen, which meant the possibility of subgroups in hemopathic patients samples was less. summary/conclusions: the expression of h antigen is comparably enhanced in subgroups and hemopathies. but most of hemopathic patients with strengthened h antigen expression present normal abo genotypes. as a result, the enhanced reaction with anti-h is necessary but not sufficient for serological identification of abo subgroups in hemopathic patients. background: although the use of automated blood bank analyzer with the advantages of speed and efficiency has recently increased, the abo discrepancies in automated blood bank analyzer have caused the reporting delays of the results and increase of the task. aims: we analyzed the causes of abo discrepancies in automated blood bank analyzer and suggested a solution strategy based on the causes. methods: from november to january , cases ( . %) of abo discrepancies among , abo blood type tests performed using the -min reaction mode of ih- in chonbuk national university hospital blood bank were identified. we compared the test results of -min mode with results of immediate mode using different red cell reagents, and analyzed the causes of discrepancies by performing additional tests such as microscopy, auto-control, antibody screening and identification, anti-a and abo genotyping. results: in the immediate reaction using different red cell reagents, cases ( . %) of discrepancies disappeared and cases ( . %) remained discrepancies. all abo discrepancies observed in the -min reaction were due to serum side causes, and one case ( . %) was due to both of serum and red cells side cause. nonspecific response ( cases, . %), cold antibody ( cases, . %), rouleaux formation ( cases, . %), cis-ab ( cases, . %), and abo subtype ( case, . %) were analyzed as causes of discrepancies. one discrepancy due to cis-ab was disappeared in the immediate reaction using different red cell reagents, abo subtype was changed to totally different blood group, a. on the other hand, in cases of the discrepancy corrected by the immediate reaction using different red cell reagents, the intensity of the positive results still observed in immediate reaction was not different from the -min reaction. summary/conclusions: ih- , an automated blood bank analyzer, was considered useful for automation of abo blood typing, and some observable abo discrepancies are expected to be mostly addressed by reexamining with immediate reaction mode using different red cell reagents. abstract withdrawn. background: abo blood group antigens mainly expressed on red blood cells, but along with that they also present on many organs and tissues like epithelia, platelets, vascular endothelia and neurons etc. the importance of abo antigens extends beyond transfusion medicine by association with various systemic diseases like cardiovascular diseases, gastric diseases, cancers, infectious diseases etc has been proven previously. previous researchers also tried to find out the involvement of abo antigens in neurological diseases like alzheimer's disease, parkinson diseases etc. but association with neurological tumours is less explored. aims: this study aimed to analyse the association of abo blood group antigens with neurological tumours. methods: a retrospective study in a tertiary care institute in india analysed the years data from jan to dec . the carcinoma patient's admitted in neurosurgical department during study period were included in our study. their diagnosis and abo blood groups were collected from hospital information system. data were analysed into microsoft excel and spss (version ). results: during study period a total of patients with neurological tumours were admitted in our hospital. the blood group frequency of these patients were . %, . %, . %, . % for a, b, o and ab respectively. the common neurological tumours found in our study were glioma ( . %) followed by pituitary adenoma ( . %), meningioma ( . %), schwannoma ( . %), cavernoma ( . %), neuroma ( . %) and space occupying lesions ( . %). the prevalence of abo antigens was almost similar in all neurological tumours except in neuroma. neuroma was found in . % o group patients as compared to other blood groups which was found statistically significant (p < . ). summary/conclusions: in this study we tried to analyse the association of neurological tumours with abo blood groups antigens. we found there is no association of neurological tumours with abo blood groups because the prevalence on abo group in general population is almost similar in patient with neurological tumours except neuroma. neuroma group of tumours like neurofibroma, neuroblastoma, nerve tumours etc. were more common in o group of patients while in our population frequency of b blood group antigen ( . %) is more common as compared to o blood group( . %). background: rhd and rhce represent homologous genes in head-to-head position on chromosome (chr , p . ). they encode for the proteins rhd resp. rhce which compose together with rhesus associated glycoprotein (rhag), band and ankyrin the ankyrin complex (ac) linking the red blood cell (rbc) membrane to aspectrin of rbc cytoskeleton (s.e. lux, blood, ). cooperatively, the proteins of ac are important for maturation and physiologic properties of rbcs. many proteins of the rbc membrane express blood group antigens on their extracellular surface and are therefore of concern in transfusion medicine. cepellini et al. described weakened hemagglutination reactions of rhd+ rbcs in the presence of an rhc+ antigen (cepellini et al, pnas, ) . we attempted to further elucidate the expression of rhd/rhag proteins in various rhce/rhce pheno-/genotypes using a sophisticated flow cytometry approach. aims: in this study, we investigated a flow cytometric method for measurement of the antigen-density of various rhce-phenotypes. methods: analysis was performed on a flow cytometer (facscanto ii, becton dickinson (bd)) using bd facsdiva software and identical instrument settings for all samples. optimized number of rbcs was incubated with saturating concentration of pe-conjugated anti-rhd antibodies brad- /brad- /fog- (ibgrl, bristol, uk). debris was excluded by rbc gating in fsc/ssc plot. quantibrite-pe beats (bd) were applied according to manufacturer's instruction to quantify the relative expression of rhd epitopes. in addition a representative number of samples from common phenotypes were assessed for expression of rhag using bric- pe (ibgrl). results: a total of samples from healthy blood donors with serologically defined rhcde phenotypes were included into this study (rr( ), r r( ), r r ( ), r r( ), r r( ), r r ( ), r r ( )). variant expression of rhd by different rhce phenotypes using brad- -pe was shown. rhd was weakly expressed in presence of rhc antigen (cepellini effect). effect of rhd gene dose on rhd protein expression is mitigated by rhc/c genotypes. when only samples with molecularly confirmed phenotypes were assessed, the rhdce genotype predicts consistently the strength of rhd protein expression. outlier samples ( ) were retrospectively genotyped and revealed rhdce genotypes as expected from the strength of rhd expression falsifying serological rhcde phenotypes. in contrast, rhe/e polymorphic site does not correlate with rhd expression. in addition, rhag protein is equally present across all rhcde phenotypes. similar results were obtained by using alternative anti-d antibodies such as brad- -pe and fog- -pe, although different antibody's avidity precludes quantitative comparison of antigen expression on rbcs. summary/conclusions: sophisticated facs methods reveal different expression of rhd on rbcs according to rhce/rhce phenotype/genotype. rhc/c polymorphic sites (c. g>c, c. a>g, c. a>g of exon , exon resp. and intron ) are in linkage with rhd expression, confirming the observation by cepellini et al. in contrast, rhe/e (c. c>g, exon ) is not in linkage with rhd expression. based on epigenomic signature it is conceivable that altered transcription factor binding sites (tbs) of rhd mirrored by homologous rhc/c may cause variant rhd expression. rhe/e snp mirroring the homologous sequence of rhd in exon is not recognised as tbs. in addition, although ac comprises all three rh proteins (rhd, rhce, rhag), their transcriptional regulations seem to be distinct. red cell reference laboratory, australian red cross blood service, perth, australia background: the rh antigen was first described when an antisera thought to contain a potent anti-c did not react with all c+ cells. these non-reacting c+ cells were classified as c+, rh:- , and the antibody specificity anti-rh . most polyclonal anti-c contain anti-c and anti-rh . previous studies have shown of monoclonal anti-c reagents are actually anti-rh . these reagents will not detect the c antigen where the red cells are rh:- . aims: the australian red cross blood service investigated a phenotype discrepancy in a blood donor. the donor's historic phenotype c+ (r r) was inconsistent with the current donation phenotype c-(r r ). we aimed to investigate the cause of the discrepancy so the donor could be assigned the correct phenotype, identify the root cause of the discrepancy and implement any corrective actions. methods: the donor's red cells were phenotyped with all available anti-c reagents as per the manufacturers product insert across both manual and automated testing platforms. following variable results and weaker reactions with some reagents, dna was extracted from the edta sample and was genotyped using immucor bioarray tm hea precise and rhce beadchip tm . targeted dna sequencing of rhd and rhce was also performed using the trusight tm one sequencing panel. a review of the historical phenotype results, including the testing platform and reagents used at the time was also performed. results: on the current sample the donor's red cells gave a + reaction by tube with bio-rad seraclone â ( ) [clone ms ] and immulab epiclone tm [clone anti-c reagents. the sample tested negative on the beckman coulter pk using beckman coulter anti-c [clone ] blood grouping reagent and tested positive ( ) reaction on the immucor neo using immuclone â ( ) anti-c [clone . immucor bioarray tm hea precise beadchip tm predicted a c+ phenotype and no variants were detected with the bioarray tm rhce beadchip tm . the trusight tm one sequencing panel genotyped the donor as rhd* /* n. and rhce* . /* with a predicted phenotype of c+, c+ w , d+, e-, e+, rh: (locr+), rh:- . a review of the donor's historical records indicated the donor tested as c+ on the pk , which at the time was being used with an in-house bromelain preparation (sigma-aldrich) and diagast olymp pheno anti-c reagent [clone ms ]. summary/conclusions: results indicated the phenotype discrepancy was caused by the c+ rh:- variant associated with the rhce* . allele. reagents containing clones ms- and ms correctly phenotyped the donor as c+, with the manual tube reagents showing a weaker reaction which may alert the operator to a possible variant which is important in the patient setting. the beckman coulter pk and associated anti-c [clone ] failed to detect the c antigen. this reagent appears not to detect the c antigen where it is associated with the rh:- phenotype, which is in contrast to the previous report by faas et al, transfusion, where it was demonstrated that clone reacted with c+ rh:- bromelain treated red cells. abstract withdrawn. background: although serological rhd typing has always been challenging due to variation of techniques and variable sensitivity of anti-d reagents, most individuals are unequivocally typed as either rhd positive or rhd negative. however, variants of d (weak d and partial d phenotypes) may present typing difficulties. individuals with partial d (missing epitopes of the d antigen) must be typed as rhd negative as blood receivers, but as rhd positive, as blood donors. aims: the aim of our study was to evaluate the algorithm used since at ahepa university blood center, to resolve rhd typing problems among first time donors. methods: since automatic analyzers may type variants of rhd as rhd+, our practice is to routinely perform two different typing methods in first time donors: an automated microplate method on the neo analyzer (immucor) and the slide test, using a potent reagent (anti-d blend-immunodiagnostika). in case of negative, weak, slow or mixed-field reaction, further testing with an automated microplate weak d method [immucor-modified indirect antiglobulin (anti-igg) test] follows. the next step of the protocol consists of testing with the commercial id-partial rhd typing kit (bio-rad) comprising a panel of monoclonal anti-d reagents, in an indirect coombs gel test assay. the patterns obtained with this kit can distinguish between d weak and partial d and can also differentiate between categories ii, iv, v, vi, vii dfr, dbt and dhar. the last step of our algorithm consists of molecular testing (immucor bioarray rhce and rhd beadchip assays) at the hellenic national blood transfusion center, in case of remaining uncertainty. results: we applied the above algorithm in samples: a) by using the partial d kit, samples were typed: four samples were characterized as "partial d" ( dfr, diii) and as "weak d". four of the weak d samples (all from women of reproductive age) were confirmed by molecular typing ("weak d type " three samples, "weak d type . or . " one sample). b) the nine ( ) remaining samples that showed atypical serological pattern, were sent for molecular testing, which characterized samples as "weak d type ", one sample as "weak d type " and another as "weak d type ". results are pending for samples. summary/conclusions: in our experience some partial rhds may be mistyped as rhd+ if the technologist does not inspect the pattern of the reactions and only takes into account the assignment by the automatic analyzer as d+ or d-. by use of our algorithm, serological characterization was sufficient to distinguish between weak d and partial d in , % of cases. molecular typing was necessary in the rest. the integration of molecular techniques improves the quality and accuracy of d typing of blood donors. if applied to patients, it also allows administration of d positive blood without compromising safety to those carrying prevalent weak d types that have not been reported to produce anti-d. furthermore, it permits withholding rhig in case of pregnant women carrying such weak d types. background: rhd antigen is one of the most clinically significant blood group antigens. except d positive and d negative phenotypes, there are over rhd variants, which represent as serologic weak d phenotypes (swd). patients with certain swd can make anti-d alloantibodies. by serology testing it is not possible to clearly distinguish among different swd. in croatian institute of transfusion medicine (citm) patients and pregnant females with swd are mostly reported as rhd negative and generally did not refer for confirmation, because molecular testing was not part of the algorithm. that remains the risk of shortages of rhd negative blood and overuse of anti-d immunoprophylaxis for pregnant females. according to uk guidelines patients with swd who are likely to require chronic transfusion support and females ≤ years are treated as d negative and refer for confirmation of d type. people who are rhd genotyped as weak d type , or are not susceptible for rhd alloimmunisation. one study showed that in croatian population the most frequent variants are weak d type , and . aims: the aim of this study is to estimate the prevalence of swd in patients and pregnant females and to find out serologic and molecular characteristics of swd referred for confirmation. methods: from / / to / / we analysed . samples of patients and pregnant females. rhd typing was performed by anti-d igm monoclonal reagents in direct agglutination micromethod on tango (bs , bs ) (biorad, dreieich, germany), swing maestro [lmh / (ldm ) + - and th- + rum- + ldm ] and ih- [lmh / (ldm ) + - ] (id-card, biorad, cressier, switzerland). cut-off value for tango was determined as ++ and for gel microtyping as +++. the samples with results below the cut-off were reported and treated as rhd negative, all except those which gave discrepant results at current testing or with historical data. these were sent to rhd genotyping for confirmation. dna extraction was done by qiaamp blood mini kit (qiagen, hilden, germany) and rhd genotyping by pcr-ssp kits ready geneweak d and ready genecde (inno-train, kronberg im taunus, germany). results: from . samples ( , %) were swd. / ( %) were referred to rhd genotyping. / ( %) samples were weak d type , or , while / ( %) were weak d type and partial d variants vii and va. serologic reactions with monoclonal igm anti-d reagents showed different pattern for weak d types , and . clearly negative serologic reactions were given in / samples with bs and bs , in / samples with lmh / (ldm ) + - and in / samples with th- + rum- + ldm . summary/conclusions: the prevalence of swd in this study is rather low ( , %). after rhd genotyping % of referred samples were finally reported as d positive. serologic determination of d variants is inconsistent and only rhd genotyping can resolve rhd status in swd. to define the permanent rhd status of swd female of childbearing potential and patients who are likely to be chronically transfused we will introduce rhd genotyping in the new algorithm. background: among all blood group systems, the antigens of the abo system are by far the most clinically significant. comes second in importance is the antigens of the rh system, which comprise d, c, e, c, and e antigens. another clinically relevant antigen is the k of the kell blood group system, which is known to be involved in both htr and hdfn. the distribution of the major blood group antigens, such as rh, and kell, is well-studied among populations of developing countries. in contrary, a relatively few studies have addressed their frequencies in saudi arabian population this is also the case in jazan province, where only two published studies have analysed the prevalence of abo and d antigens, while the frequency of other clinically important antigens, such as rh and kell antigens, is yet to be explored. aims: to determine the frequency of the following clinically relevant blood group antigens; rh(d, c, e, c, e) and k among saudi blood donors in king fahd central hospital in jazan province. methods: a retrospective, cross-sectional study was carried out in the blood bank of king fahd central hospital in jazan province. the red cell phenotyping records for blood donation of randomly selected saudi donors, who donated blood between january and june , were reviewed to identify the prevalence for the following antigens: d, c, e, c, e and k. the hospital blood bank routinely performs rh/k phenotyping for all blood donation using either bio-rad or ortho diagnostic column agglutination technology (cat) platforms. phenotype frequencies were expressed as percentages. results: this study included a total of saudi voluntary as well as family replacement blood donors. the d antigen was found to be positive in . %, while k antigen was positive in . %. among other studied rh antigens, e was the most common ( . %) followed by c ( . %), c ( . %) and e( . %). dce/dce ( . %) and dce/dce ( . %) were the most common phenotypes amongst d-positive and dnegative donors, respectively. surprisingly, dce/dce phenotype was significantly prevalent ( . %) with almost times higher frequency compared that reported in caucasians ( . %). the rare phenotype dce/dce was found in donors ( . %), while dce/dce and dce/dce phenotypes were found in only one donor each. summary/conclusions: this study is the first to determine the frequency of rh and k antigens in saudi blood donors in jazan province. determination of the frequency of these clinically significant antigens in our geographical area will facilitate the selection of antigen-matched red cell units for transfusion in recipients with multiple alloantibodies. it will also help in the management of blood donation processes and planning the estimated need of blood stock of different blood group phenotypes to meet the patient's needs. abstract withdrawn. background: the gerbich (ge) blood group system includes several high-frequency antigens located on glycophorin c and d. with only few reports published on the clinical significance of antibodies directed against these antigens, it is unclear whether blood transfusions have to be antigen negative in the presence of an anti-ge antibody. the monocyte monolayer assay (mma) is an in-vitro method used to estimate the clinical significance of alloantibodies. aims: to illustrate the role of the monocyte monolayer assay (mma) in the transfusion management of a patient with an anti-ge alloantibody. methods: the clinical and transfusion history was retrospectively retrieved from the patient's medical records. serological investigations were performed by indirect antihuman globulin test. papain and trypsin treated cells were also used. the clinical significance of the antibody was assessed by mma. genomic dna was isolated from whole blood and the samples were further characterized by pcr. results: a -year-old male patient with lung cancer without previous transfusions was admitted ( / ) for surgery. his hemoglobin was . g/l. an anti-ge antibody was detected and it was decided to transfuse ge-positive packed red blood cells (prbcs). however, no blood transfusion was needed. in july , the patient was admitted for colon cancer surgery with a hemoglobin of , g/dl. the anti-ge alloantibody was still detectable and a ssp-pcr revealed the genotype ge* .- . an mma performed on the pre-transfusion sample revealed a monocyte index (mi) of . % and the antibody was considered not to be clinically relevant. the mi was interpreted as following: - % not significant; - % inconclusive; > % clinical significant. however, due to the clinical background of the patient it was decided to transfuse ge-negative prbcs, which were obtained from etablissement francais du sang (efs), paris, france. two days after surgery, the patient received units of ge:- ,- prbcs without any transfusion reaction. one and a half year later ( / ), peritoneal carcinomatosis, as a complication of colon cancer, was diagnosed. the patient's hemoglobin was g/l and he had a passage disorder, symptoms of deterioration and an adynamia. based on the mma results from july indicating no clinical significance of the antibody, it was decided to transfuse ge-positive prbcs. in the following days the patient received a total of units of gepositive prbcs no immediate or delayed transfusion reaction were observed following these transfusions. two further mma's, performed on samples drawn on december th and th ( days after transfusion of a total of three and two days after two further prbcs respectively), showed a mi of . % und % respectively and the anti-ge antibody was considered still not to be clinically significant. summary/conclusions: we report the case of a patient with an anti-ge antibody transfused with ge-positive prbc. as ge-negative prbc are not available in switzerland and not easy to obtain internationally the mma can help in the decision on how to transfuse. in this case, the clinical course confirmed the mma-based prediction. transfusions of ge-positive prbcs were tolerated without signs or symptoms of immediate or delayed transfusion reactions. background: abo grouping discrepancies occur when the results of forward grouping are not corroborative to those of the reverse grouping. these may be due to weak subgroups of a and b, missing or weak abo antibodies or red cell alloantibodies. determination of correct abo blood group of a donor is essential for preventing abo incompatible transfusions and to avoid hemolytic transfusion reactions in the recipient. aims: to determine the frequency of abo discrepancies and their resolution to correctly identify the blood group of the donors. we also determined the frequency of 'weak d' positivity in rhd negative donors. methods: this was a retrospective study on donor samples collected from st april, to th september, (two and a half years). for discrepant samples, the abo and rhd grouping was repeated using tube technique using commercial antisera {anti-a, anti-b, anti-ab and anti-d (igm), anti-d blend (igm+igg), anti-h and anti-a lectins}. adsorption-elution testing was done for detecting weak subgroups of a and b. antibody screen ( -cell) and identification ( -cell) was done by gel technique (bio-rad, switzerland). 'weak d' testing in rhd negative donors was also performed by gel technique. antibody titration was done using tube technique. the donor details including name, age and the registration/unit number of the donation were also checked for all the discrepancies to avoid repetition while data analysis. results: we detected ( . %) abo discrepancies out of the total donor samples tested during the study period. out of these, ( . %) were rhd positive. the most common cause of abo discrepancies was weak anti-b antibody ( / ; . %), followed by weak anti-a antibody and weak subgroups of a ( / each; . % each) and weak subgroups of b ( / ; . %). the remaining . % ( / ) discrepancies were due to agglutination with o cells in reverse grouping. the overall frequency of weak subgroups of a and b collectively was . % ( background: detection of unexpected red blood cell (rbc) antibodies before transfusion is critical for prevention of hemolytic transfusion reaction. ideally, unexpected rbc antibody detection is carried out within days after receiving a patient's sample. however, in some cases, retests could be performed after more than days for evaluation of any transfusion reaction, quality control or research. therefore, it is necessary to determine the stability of antibodies after refrigeration or freezing for a certain period of time. aims: we carried out antibody identification test with fresh, refrigerated and frozen samples using automated analyzer ih- and manual tube methods to evaluate the stability of antibodies after storage and compare the results between the two methods methods: antibody identification tests were performed using ih- (bio-rad, cressier fr, switzerland) and manual tube methods. fifty samples showing positive results in antibody screening test by both methods were divided into three and tested immediately, week after storage at °c and month after storage at À °c. the specificities and reactivities of antibodies at each storage state were recorded and compared between the two methods. results: specificities of antibodies identified were concordant between ih- and manual tube methods irrespective of the storage state. the results were as follows: anti-e/e+c, ; anti-le a , ; anti-di a , ; anti-c+e, ; anti-m, ; anti-d, ; anti-c, ; anti-k, ; anti-jk a , : anti-xg a , ; unidentified antibody, ; autoantibody, cases. with regard to the changes in reactivity owing to storage, ( %) samples (anti-e+c, ; anti-m, ; anti-di a , ; anti-d, ; anti-c+e, ; anti-le a , ; anti-c, : autoantibody, ; unidentified antibody, ) showed identical reactivities after week and month storage by both ih- and tube methods. however, ( %) samples, comprising unidentified antibodies, anti-le a , anti-c+e, anti-e, anti-e+c, and autoantibody, showed decreased reactivities after storage in both methods. three samples, comprising anti-di a , anti-e+c and anti-k antibodies, showed increased reactivities after storage. one sample with anti-jk a showed increased reactivity only after month storage, while one sample with anti-xg a showed decreased reactivity only after month storage. higher reactivities were observed in all samples detected using the ih- analyzer than manual tube methods (p < . , wilcoxon rank sum test). summary/conclusions: the specificities of unexpected antibodies detected by ih- and tube methods were the same in all storage states; however, reactivities were higher in ih- than in the tube method. twenty-six ( %) of samples showed identical reactivities after week refrigeration and month freezing. nineteen ( %) samples showed decreased reactivities after storage; however, ( / , %) of them were nonspecific antibodies, unable to identify using commercial id panels. therefore, it is suggested that retests for evaluation of transfusion reaction, quality control or research could be reliably performed after more than days, if stored appropriately in refrigerated or frozen states. abstract withdrawn. t gleich-nagel , d huber-marcantonio , n rufer , g canellini and c niederhauser unit of transfusion medicine, interregional blood transfusion src, lausanne laboratory diagnostics, interregional blood transfusion src, bern, switzerland background: a positive direct antiglobulin test (dat) is mainly found in patients with warm/cold autoantibodies or alloantibodies directed against transfused erythrocytes. the identification of antibodies fixed on red cells is important for the clinician, allowing the further evaluation of a patient's clinical situation including their current medication. in immunohematology the elution of a positive dat remains a tedious and expensive procedure. the blood transfusion service src (bts src) has derived a flow chart that indicates in which situation an elution of dat positive samples should be performed. in order to follow the bts src guidelines, it is mandatory to obtain additional data related to the patient's condition, such as haemolytic parameters and recent transfusion history. currently, our laboratory is not always able to apply the recommended flowchart, since information is often unavailable. aims: here, we performed a comparative study between the algorithm provided by bts src and our in-house strategy, which is based on the qualitative changes of a positive dat, without the need for additional patient and biological information. methods: details of dat positivity and the patient's transfusion history was taken from the software eprogesa (mak-system) and analysed in excel. we analysed a total of ' dats and evaluated them for their positivity, whether an elution was performed or whether antibodies were detectable in the eluate. furthermore, we performed an additional analysis on those samples, that were derived from recently transfused patients (< days). results: a positive dat was found for igg and c d in out of ' ( . %) samples, a level similar to previous reports of positive dats for hospitalized patients. among these positive samples, ( %) were eluted because of a qualitative change in their positivity according to our in-house algorithm. identification of warm autoantibodies or alloantibodies occurred in only . % ( / ) of the cases. from the patients transfused within the last days and having a positive dat, ( %) were eluted according to our in-house algorithm. the same samples would have been analysed if the swiss transfusion guidelines had been applied. however, this comparative study reveals a significant discrepancy in regards to overall sample numbers that should have been eluted according to the two algorithms ( versus samples). this is mainly due to the fact that the swiss transfusion based algorithm does not recommend an elution of positive dats from patients who did not receive a transfusion within the last -days, except if there is a significant clinical suspicion (e.g. haemolysis). summary/conclusions: this comparative study indicates that our elution-based algorithm was performed on all clinically relevant samples as recommended by the bts src guidelines. qualitative changes in the dat positivity represent our main parameter for selecting those samples to eluate. besides ensuring that no clinically relevant samples were missed, this strategy also led to a large number of unnecessary elution analyses. in conclusion, a significant reduction in the laboratory workload and economical savings arises if the relevant clinical information and patients history is known prior to laboratory analysis. background: novel anti-cd monoclonal antibodies, such as daratumumab (dara) and isatuximab, used in treatment of multiple myeloma, interfere with routine blood bank serologic tests. as part of the strategies to manage these patients, it is recommended to perform extended phenotyping to provide matched units (aabb association bulletin # - ). many investigations have focused on the interference with iat for the screening and identification of underlying alloantibodies and how to overcome them, but less has been published on the potential interference with extended phenotyping techniques. aims: the purpose of this study is to compare different technologies to type the most important antigens in myeloma patients before and during the treatment with therapeutic anti-cd antibodies. vox sanguinis ( ) (suppl. ), - methods: edta-anticoagulated whole blood samples coming from patients in different stages of treatment with daratumumab and with isatuximab have been typed in parallel with dg gel microcolumn (grifols) and mdmulticard technology (grifols). the results are also compared with genotyping results obtained with id core xt (grifols). direct coombs, autocontrol and antibody screening has also been performed as complementary tests. results: the study provides that four patients had positive dat and/or ac before therapeutic cd antibodies treatment. in these cases, of negative antigens (fy / jk and/or s) turn to positive in gel technology but mdmulticard showed % agreement with genotype id core xt results. focusing in the data obtained during the treatment, negative antigens were type as positive in gel technology ( % of the tests). mdmulticard agreed with genotype in % of the analyzed antigens. as complementary data, of patient-treated samples had dat or ac positive and showed panagglutination. summary/conclusions: the results demonstrated that mdmulticard is an effective method to type cd -directed cytolytic antibodies treated samples in addition to dat and or autocontrol positive samples. background: antibody titration is a semi-quantitative method to estimate the strength and concentration of antibodies present in plasma or serum sample. titration methodology should be validated together with clinical data to evaluate the relevance of the titer value in each application. the titer of an antibody depends on different parameters: the antibody concentration in the sample, the density of the corresponding antigen expressed on the red blood cells used, the affinity constant of the antibody-antigen and other parameters regarding the technique used (e.g. gel cards or tube test). gel cards technology reduces the intra and inter-laboratory variation in titration studies comparing with the tube technique. aims: to evaluate the suitability of dg gel coombs, dg gel anti-igg and dg gel neutral (grifols) for titrations using two sample volumes ll and ll. methods: twenty frozen plasma samples containing unexpected antibodies from different specificities (anti-jk a , -fy a , -k, -d, -e and -c) were titrated in dg gel coombs and dg gel anti-igg cards and donor fresh plasmas with natural occurring antibodies (anti-a and -b) were titrated in dg gel coombs and dg gel neutral (saline technique). the titer of the antibodies was determined by testing two-fold dilutions of the plasma with selected red blood cells depending on the antibody tested. plasma samples were diluted in dg gel sol. selected red blood cells serascan diana, serigrup diana or donor blood were added into the card ( ll at . %). further, sample dilutions were dispensed into the card ( ll or ll). subsequently, cards were incubated min, °c (coombs technique) and min, - °c (saline technique), centrifuged in dg spin and the results read. agglutination intensity was graded visually according to the instructions for use of dg gel cards. the reciprocal of the highest plasma dilution that gives macroscopic agglutination was interpreted as the titer. results: titers obtained with dg gel coombs and anti-igg (n = titrations, titer ranged - ) were compared for each sample with unexpected antibodies. no differences were found between gel cards types (differences were ≤ . titer in the % of the cases). differences between dg gel coombs and neutral (saline technique) (n = titrations, titer ranged - ) were observed when anti-a and -b antibodies were titrated using the same sample. the titer was similar or higher in coombs in comparison to the saline technique. coombs titers may be a mix of igm antibodies reacting at °c and igg antibodies. differences were > titer in % of the comparisons and ≤ titer in the rest of the cases ( %). comparing sample volumes of ll and ll in all cards (n = titrations), higher titers were observed using ll, as expected. differences were titer in the % of the comparisons, < titer in % and > titer in the % of the cases. background: autoimmune haemolytic anemias (aiha) are characterized by production of antibodies directed against red blood cells and destruction by the mononuclear phagocytic system or complement system. aiha observed in paediatrics is usually self-limiting and often precipitated by viral infections. in some, the condition is secondary to autoimmune diseases, drugs, infections or underlying primary immune deficiencies. appropriate immuno hematological evaluation to characterise the underlying autoantibody can help identify the type of aiha to aid in diagnosis & treatment of these cases. aims: retrospective analysis of immune-hematological evaluation, treatment and outcome of aiha in paediatrics. methods: patients aged - years, diagnosed with aiha, between april -december ( months) were included in this analysis. aiha was defined as positive direct coombs' test (dct) with anemia associated with corroborative evidence of haemolysis in the form of raised indirect hyperbilirubinemia, raised ldh, raised reticulocyte counts or red cell agglutination on peripheral smear. further monospecific dct and evaluation for the specificity of autoantibody was done for all patients using biorad gel cards and panel cells. steroids were given as first line in all; second line agents included cyclosporine and rituximab. red cell transfusion was given in those with severe anemia with cardiac decompensation. results: patients were diagnosed during the study period with autoimmune haemolytic anemia. haemoglobin at presentation ranged from . to grams/dl. the initial presentation was severe anemia in children and mild-moderate anemia with thrombocytopenia (evan's syndrome) in . the trigger for haemolysis was infection in children. rheumatological evaluation was performed for children out of whom were diagnosed as evolving lupus. primary immune deficiency evaluation was advised for and one child was diagnosed as suffering from combined immunodeficiency. dat was positive in out of aiha patients as one of the infant had dat negative iga mediated aiha secondary to viral infection. two out of dat positive cases had igg & c d positivity on monoclonal dat testing whereas rest had only igg coating the red cells. dat titration was more than : in patients; where only of these patients had both igg and igg coating and rest had only igg . alloantibody screen was negative in all. specificity of autoantibody was found only in one case, which was against rh blood group antigen (anti e). all patients received prednisolone as the primary treatment. three children attained remission following a - weeks of steroids. in those who were steroid dependent, cyclosporine was used as the second line agent in and rituximab was used in . out of these children children are in sustained remission and off any medication, whereas the rest are on low dose steroids with cyclosporine. summary/conclusions: aiha is not an uncommon problem in children and can vary in its clinical severity. the proper diagnosis and management involves efficient immuno-hematological evaluation, as detailed characterization of the autoantibody coating the red cell is very important in guiding the clinician for management and prognosis. abstract withdrawn. background: drug-induced immune hemolytic anemia (diiha) is rare and has only been described once with dexchlorpheniramine (polaramine tm), an antihistaminic agent widely used in the treatment of a variety of allergic reactions. we report a case of diiha complicated with acute renal failure associated with antibodies to dexchlorpheniramine. a -year-old woman with no history of transfusion, was treated semimonthly with a combination of chemotherapy and targeted therapy for metastatic colorectal adenocarcinoma. her chemotherapy regimen consisted of oxaliplatin and -fu with leucovorin rescue (folfox). panitumumab (monoclonal antibody anti-egfr) was used as targeted therapy. premedication with dexchlorpheniramine iv was systematically given at the beginning of each cycle of treatment to reduce the risk of perfusion reactions mainly associated with panitumumab. the patient developed chills and febrile agranulocytosis during the first and second infusion respectively. the third infusion was not performed due to pyrexia, chills, general discomfort experienced by the patient at the beginning of chemotherapy. probabilistic antibiotherapy was administered and the patient recovered rapidly. during the next infusion (day ), following premedication with dexchlorpheniramine, a more "impressive" reaction including all the above mentioned symptoms occured along with back pain and dark colored urine. the infusion was halted and no chemotherapy was delivered. bacterial infection at the implantable port was first thought to be the cause of this adverse event but was not confirmed. additional laboratory findings revealed biological signs of inflammation associated with iha and acute renal failure. the patient was treated with hemodialysis (day ), two units of rbcs (day ) and was discharged one week later in stable condition. dexchlorpheniramine was then suspected and samples collected on day were sent for a diiha laboratory workup. aims: the aim of this study was to support a clinical diagnosis of diiha. methods: laboratory workup included direct and indirect antiglobulin tests (dat and iat). drug antibodies investigation was performed by incubating patient's serum and eluate from patient's rbcs in the presence of drug against normal donor rbcs that had not been previously treated with the drug (i.e., by the so-called "immune complex" method). control tests were performed in parallel. drug was diluted in pbs and tested at and mg/ml. results: dat was positive (anti-igg + , anti-c d + ) and no unexpected rbcs antibodies were detected by iat in patient's serum and eluate without the in vitro addition of the drug. an antibody directed against untreated (titer ) and enzymetreated (titer ) normal donor rbcs was demonstrated only in patient's serum in the presence of the drug tested at mg/ml by the gel method. the pool of normal sera did not react in the presence of the drug. summary/conclusions: the multi-drug treated patient described in this study was demonstrated to have dexchlorpheniramine dependent antibody detected by the "immune complex" method. the key to the diagnosis was the observation of positive dat with negative eluate tests which prompted a reexamination of the medications administered in temporal relationship with the hemolytic event. although rare, this case report should alert physicians to the need to investigate the possibility of dexchlorpheniramine induced hemolytic anemia in any patient who develop unexpected anemia after hematologic or oncologic procedures p- singapore, singapore, singapore background: daratumumab is a monoclonal antibody against cd used in the treatment of multiple myeloma and has been known to bind to cd on rbc's and interfere with indirect antiglobulin based serologic tests such as red cell antibody screens and crossmatch compatibility testing. in order to negate the interference of daratumumab, our reference laboratory follows the daratumumab protocol recommended by the aabb which uses dithiothreitol (dtt) treated reagent red cells in red cell antibody screening and identification test in patients known to have received daratumumab. aims: the objective of this study is to determine the impact of daratumumab in the turnaround time (tat) for red cell antibody screening and identification. methods: a retrospective review of the tat for red cell antibody screening and identification samples of patients known to be treated with daratumumab from october to december was performed. turnaround time is defined as the time the sample is received up the time the results were reported. the tat for routine red cell antibody screen and identifications were also reviewed during the same period and was compared with the tat of samples from patients treated with daratumumab. results: a total of patients on daratumumab had samples sent to our reference laboratory for red cell antibody screen and identification during the study period. information on daratumumab treatment was not provided to the reference lab prior to the start of testing in of the patients while the use daratumumab was mentioned in the serology request form of the other patients. the median tat for red cell antibody screen and identification is min (range: - ) if information on daratumumab was provided prior to start of testing and min (range: - ) if information was not provided prior to testing. the median tat for routine testing is min (range: - ). using wilcoxon rank-sum test, turn-around time for antibody screening and identification for daratumumab treated patients was observed as statistically not significant when compared to routine samples (p value . ). however, tat for serologic tests requests with appropriate medical history compared to the testing requests without relevant information was also observed to be significantly difference (p value . ). summary/conclusions: there is no significant impact in the tat of red cell antibody screen and identification in patients known to receive daratumumab as compared to routine testing. however, there is a significant difference in the tat if information on daratumumab treatment is not provided prior to testing. this highlights the importance of providing the relevant medication information in the request form in order to prevent delays in testing and provision of blood to patients on daratumumab, which can result in improved organizational efficiency and have positive impact on cost and resource savings. background: daratumumab, an anti-cd monoclonal antibody, has been shown to be highly efficacious in the treatment of multiple myeloma (mm). cd is a glycoprotein highly expressed on plasma cells and, to a less extend, on the surface of red blood cells (rbc). when bound to cd on rbc, daratumumab interferes with the pretransfusion tests, with positive antibody screening and crossmatch. anti-cd interference is an important challenge as many mm patients will require blood transfusions during their treatment. dithiothreitol is a reducing reagent with multiple applications in blood bank testing. treatment of rbc with dithiothreitol irreversibly removes cell surface cd tertiary structure, avoiding the binding and testing interference by the anti-cd daratumumab. aims: to demonstrate the efficacy, safety and celerity of the protocol between the blood bank (bb) and haemato-oncology of our institutions, using just the crossmatch. methods: a retrospective research was used for the evaluation of the results obtained from the implemented protocol. this comprehends a previous contact by haemato-oncology that leads to a study of the patient before the beginning of daratumumab treatment, and consists in: abo/rhd grouping; rh and kell phenotyping, and other clinically significant antigens; antibody screening; and direct antiglobulin test. genotyping may be required for some patients who received previous blood transfusions. before the beginning of the therapy, a blood sample of the patient is sent to the bb to perform laboratory tests and frozen after. this frozen sample is used for crossmatching in patients that already started therapy, did not have a blood transfusion in between, and have a positive antibody screening and/or crossmatch. in further transfusions, in case of positive tests, the dithiothreitol-treated donor rbc is applied. the donor rbc antigens are always selected accordingly to patients negative clinically significant antigens, when transfusional support is needed. the laboratory tests are executed in gel column agglutination technique. results: since , patients were studied, from which were transfused with blood units, according to the protocol. there were no immunizations or adverse reactions to transfusion registered within the transfused patients, neither delay on the availability of blood units. patient blood sample collected and frozen prior to the beginning of the treatment, has shown to be a good strategy by reducing significantly the waiting time for the blood unit in the first transfusion. summary/conclusions: this protocol, which defines the communication among the involved professionals, has shown to be a secure and effective way of reducing interferences caused by daratumumab. it ensures the previous study of the patients and their transfusion with rbc respecting the patients negative clinically significant antigens. if not adopted, the mitigation measures described in this protocol, delays in the availability of the rbc requested and alloimmunizations, may and will possibly occur. a good communication between the bb and the haemato-oncology is crucial for a good time management when a transfusion is requested for these patients. three methods were used to resolve this dara interference. reagent rbc's were treated with dtt, which know to denature cd and then tested with patient plasma. allo-adsorption study was performed using a certain ratio of red cells to plasma. in addition, a selection of phenotyped cord cells were used as an antibody screening panel. results: dtt treatment of reagent red cells was successful at eliminating dara interference and allowing for the presence of underlying antibodies to be identified. in this case, underlying antibodies were not detected by using reagent dtt treated red cells or phenotyped cord cells. adsorption technique was ineffective at elimination the reactivity. summary/conclusions: dara is the first commercial fda-approved therapeutic monoclonal antibody used in treating multiple myeloma patients. • since cd is weakly expressed on normal red blood cells, dara attachment to red blood cells can interfere with pre-transfusion iat testing. • dtt treatment of reagent red blood cells and cord cells can abolish the interference of dara to test for the presence of underlying alloantibodies. • to prevent delays in issuing red blood cell units to patients, hospitals should send patient samples to be tested before receiving dara treatment to ensure that clinically significant alloantibodies are not being masked. background: antibody screening (as)is considered superior to antihuman globulin (ahg) cross match during pretransfusion compatibility testing. in spite of knowing the utility and superiority of as, it has not been adopted uniformly in india. therefore, scarce data is available from this subcontinent in terms of optimisation of red cell antibody detection during pretransfusion testing in form of "type and screen" aims: the main objective was to study the benefits of performing simultaneous antibody screening along with the blood grouping during the first hospital visit to the hospital. other objectives were to study the prevalence of clinically significant antibody among the indian population and to follow up the patients who were transfused antibody screen negative but cross match incompatible blood. we also studied some other relevant quality indicators related to efficiency of blood transfusion services methods: this prospective study was carried out at a tertiary healthcare centre in india between july and dec ( months). the study protocol was submitted to institutional review board and permission was granted. blood grouping and as were done during patients' first hospital visit, which we called "type and screen". when the patient got admitted to the hospital and required blood transfusion, a blood request form was generated by the user and sent to blood bank. depending upon the results of antibody detection, further course of action was chosen. if patient was found to have no antibody, immediate spin test (ist) cross match compatible blood was issued and transfused. in such cases the procedure of ahg crossmatch testing was continued even after issue of blood. cases where ahg cross match test was found negative no further follow-up of the patient was done whereas when ahg cross match was found positive, patients were followed after the transfusion results: a total of patients were "type and screened". majority were from hemato-oncology, bmt, liver transplant, paediatric cardiac surgery, and medical icu units. clinically significant allo-antibody was detected in patients and autoantibody was detected in patients. alloantibody was detected mainly against rh and kell blood group systems. in diagnosed aiha cases, majority were in the form of warm aiha ( %) and % of aiha cases were having hidden single or multiple alloantibody. significantly higher proportion of patients in as positive group required blood transfusion than as negative group ( % vs %, p < . ). in both the groups, in planned cases, most of the time blood was issued immediately within the defined turnaround time except in where either transfusion was delayed or surgery was postponed. it happened only in trauma or surgical bleed cases. expiry of blood was decreased significantly due to no usage of blood ( . % vs. %, p < . ). during the period of study we obtained cases where the ist cross match was compatible but the ahg cross match was incompatible. during follow up none of the cases demonstrated any sign of hemolysis summary/conclusions: in developing countries like us, optimization of as during pretransfusion testing increases operational efficiency and significantly decreases the expiry of blood. results: during the period when absc was performed on pk , , donation samples were tested and , ( . %) were found absc positive. antibodies to red cells were identified in donations out of , ( . %) absc positive samples and in the rest, no irregular antibodies were detected. the prevalence rate for atypical antibody was . %. the top most frequent antibody specificities were: nonspecific cold antibodies ( . %), anti-e ( . %), anti-mi a ( . %), anti-m ( . %) and anti-le a ( . %). a total of , donations were screened for atypical antibodies by ih- and , ( . %) were screened positive. among these, anti-red cell antibodies were identified in , samples ( . %), which was significantly higher than those identified in pk screened positive samples (p < . ). the prevalence rate for atypical antibody as screened positive by ih- and with confirmed red cell specificities was . %, which was also significantly higher (p < . ). the top most frequent antibody specificities were: anti-mi a ( . %), anti-m ( . %), anti-le a ( . %), anti-e ( . %) and non-specific cold antibodies ( . %). anti-fy b was detected in cases, which would be missed detection by enzyme treated reagent cells on pk system. summary/conclusions: the performance of the ih- system using a -cell screening panel including one cell with mi(a+) expression and column agglutination technology with iat phase was superior in comparison with that of pk in the context of higher sensitivity in detecting more true positive results and higher specificity in detecting more true negative and less false positive results. this has translated into the advantages of reduction in workload of reference laboratory in performing less antibody identifications in those false positive samples as well as enhanced transfusion safety by removing more irregular red cell antibody positive plasma-containing components from the issuable inventory, which may potentially lead to haemolytic transfusion reactions. the prevalence of irregular red cell antibodies of . % in healthy blood donors in hong kong reflects more the true statistical figure. background: chronic red blood cell (rbc) transfusion is the upfront therapy for thalassaemia patients, however this therapy is featured by several adverse events including rbc alloimmunization. phenotype matched products transfusion policy can prevent alloantibody formation, but it makes routine transfusion more difficult for both the donor center and the transfusion service. a recent systematic review (franchini et al, blood transfus ) reported a rbcalloimmunization prevalence of . %, with a higher incidence against rh and kell systems in thalassemia intermedia patients. aims: the aim of our retrospective study is to evaluate the rbc alloimmunization prevalence in thalassemia patients transfused in a single center over a years period with limited phenotype matched rbc (rh and kell system antigens) units. methods: from to thalassaemia patients, with a minimum follow up of year and transfused with more than > rbc units, were included in our study. patients were studied for: blood group and rh / k phenotype determination, direct antiglobulin test (dat), irregular antibodies research (abirr). cross-match and detection of alloantibodies were performed using the indirect antiglobulin test by the column agglutination method. six-monthly dat and antibody screening were performed using the indirect antiglobulin test and enzymatic papain-treated rbc test. results: overall patients ( females, males) were included in our retrospective analysis: patients were affected by thalassaemia major and by thalassaemia intermedia. rbc alloimmunization prevalence was . % ( patients): patients were found to be positive for rbc alloantibodies, four with alloantibodies and autoantibodies. eleven alloantibodies were detected ( anti-h, anti-cw, anti-e, kpa, anti-jka, anti-jkb, anti-m and anti-lua). in out of alloimmunized patients we found an anti-e antibody reactive in enzymatic papain-treated rbc test only, in the third alloimmunized patient anti-kpa and anti-lua antibodies were detected, while in the remaining patients, in which auto and alloantibodies were detected, a severe autoimmune hemolytic anaemia (aea) requiring therapy was diagnosed. in these cases the appearance of alloantibodies is concomitant with the presence of autoantibodies. among the patients positive for alloantibodies, were affected by major thalassemia and one by intermedia thalassaemia summary/conclusions: in our experience a limited phenotype matched rbc transfusion policy showed a rbc alloimmunization prevalence similar to literature data: . % vs . %; we didn't find higher alloimmunization prevalence in thalassemia intermedia patients may be due to the low patients number. we believe that introduction, in our department, of an extended-phenotype matched transfusion, including antigens of the main group systems (fy, jk, mns) and the main rare antigens (cw, kp, lu), could reduce the risk of red blood cell alloimmunization in thalassemia patients. abstract withdrawn. background: undoubtedly, preventing alloimmunization has an advantage over overcoming its consequences. however, the high cost of technical and organizational aspects of preventive measures requires their scientific substantiation confirmed by clinical and laboratory data. selection of donors of the rhesus (d, c) and kell (k) antigens for the red blood cell transfusions to hematological patients has been regulated in the russian federation since . recipients with the phenotype c+c-transfuse red blood cell only with the same antigenic combination. for transfusions red blood cell obtained from k-negative donors are used. the compatibility of the donor and recipient with the antigens c, e, e, c w (rhesus system) and k (kell system) is additionally taken into account from april . that is, transfuse red blood cell that do not contain antigens in the phenotype that are not in the recipient's phenotype. aims: to evaluate the efficiency of red blood cell donor selection using antigens of rhesus (c, c, e, e, c w ) and kell (k, k) systems for the prevention of the recipient alloimmunization. methods: immunohaematological studies using equipment and reagents of biorad (usa) were performed in patients of the hematology clinic. non-hodgkin lymphoma was diagnosed in patients, acute leukemia in , multiple myeloma in , chronic lymphatic leukemia in , chronic myeloid leukemia in , aplastic anemia in , hemophilia in , myelodysplastic syndrome in , and other hematological diseases in . the frequency of detection of antibodies to antigen c ( . % vs . %) and to antigen e ( . % vs . %) decreased four times. the frequency of detection of antibodies to the c w antigen has not changed significantly ( . % vs . %, respectively). selection of antigens c (rhesus) and k (kell) has been carried out in the clinic since , therefore the immunization index for these antigens remained unchanged and amounted to . % vs . % for anti-c antibodies; . % vs . %for anti-k antibodies. alloantibodies to the antigens e (rhesus) and k (kell) were not detected for the entire observation period. summary/conclusions: research verified the effectiveness of alloimmunization prevention of recipients by selecting red blood cell for antigens c, c, e of the rhesus system and k (kell). the study concluded that selection of red blood cells for the antigens c w , e (rhesus) and k (kell) does not affect the level of alloimmunization of patients and is not clinically justified. background: in the russian federation, there is an order according to which patients requiring multiple transfusions, who are at high risk of immunological complications are to typed for red blood cell antigens: abo, d, c, c, e, e, cw, k, k. selection of erythrocyte-containing blood components is carried out taking into account the donorrecipient compatibility according to all the listed antigens. aims: analysis of results of immunological evaluation of patients of hematological clinic. methods: the study included first time patients of hematology clinic in - . typing of antigens of abo, rhesus, kell systems, screening and identification of antibodies were carried out using equipment and reagents from bio rad (usa). results: interpretation of results of immunohematological screening was complicated in ( . %) patients. the total number of complex cases was . the double population of red blood cell was most often determined in antigens of the rhesus system ( . % of the total number of patients) as a result of previous transfusion therapy. of those, chimera for the antigen e was detected in cases ( . % of patients with the chimera for rhesus and kell antigens), cin ( . %), sin ( . %), e - ( . %), cw - ( . %), k - ( . %). in such cases, donor red blood cells were chosen not carrying chimeric antigen for transfusions, in the presence of chimeras in both paired antigensred blood cell transfusion with the cc phenotype and / ee. chimera for abo antigens was detected in . % of the examined individuals. the discrepancy between the direct and reverse blood grouping of the abo system in patients ( . %) was due to a decrease in the production of antibodies - cases and the appearance of extra agglutinins - case. autoantibodies were detected in . % of all patients, including . % of patients, when they caused panagglutination phenomenon. upon detection of autoantibodies that complicate the individual selection of donors, transfused red blood cells that are compatible with antigens of abo, rhesus, kell, duffy, kidd, mns systems. alloantibodies were detected in . % of patients, including specific anti-din ( . %), anti-dcin ( . %), anti-kin ( . %); antibodies of unidentified specificityin ( . %), polyspecificin ( . %). summary/conclusions: the complexity of interpreting immuno-hematological tests in hematological patients is due to intensive transfusion therapy, changes in red blood cell antigens and appearance of nonspecific antibodies due to underlying disease. red blood cell for transfusion in these patients should be selected taking into account the expanded red blood cell antigen profile. abstract withdrawn. background: blood transfusion is an essential part of therapy for many patients. although life-saving for many patients, blood transfusion is not without risk. the main goal of blood transfusion services is that transfused blood should be compatible with the patient. the clinical and serologic evaluation, which allows for the transfusion of the most compatible (or "least incompatible") blood, requires a joint effort between the clinician and the transfusion medicine physician. aims: root cause analysis of incompatible cross matches in patients. methods: in this prospective study, total of , , crossmatches were performed over period of last four & half years, out of which units were found incompatible by column agglutination method-cat in polyspecific (anti-igg+ c d) gel media. a root cause analysis protocol was formulated to resolve incompatibility to ensure safe transfusion. results: on the evaluation of , , crossmatches, only units were found to be incompatible ( . %). the major cause for incompatibility found in patients was aiha-( . %). other causes of incompatibility were infections ( . %), multiple transfusions ( . %), trauma ( . %), evan's syndrome ( . %), rh negative mother ( . %), sca ( . %) & incompatibility due to dat positive packed red cells ( . %).the most common antibody found were anti-'c', anti-'s' & anti-'m'. summary/conclusions: the rca protocol involves a thorough evaluation of the patient's clinical condition and underlying pathology to identify the cause. a logical stepwise approach will enable provision of safe transfusion to the patient. background: antibodies to high-frequency antigens (hfas) are a transfusion hazard, as compatible blood is often very difficult to obtain. other clinically significant alloantibodies represent an additional transfusion risk. in patients treated with allogeneic bone marrow transplantation (bmt) recipient red cell alloantibodies may cause acute or delayed haemolysis of donor red blood cells (rbc) and contribute to morbidity and mortality. aims: the aim is to present the case of a patient with myelodysplastic syndrome (mds), multiple "common" alloantibodies and an additional alloantibody to a highfrequency antigen, treated with allogeneic bmt. methods: a forty-one-year-old caucasian patient with mds (raeb- ) was admitted to our hospital in january for unrelated allogeneic bmt. she previously received myeloablative conditioning therapy according to the flu / bu / atg protocol ( days of mg iv. fludarabine, days of busulfan mg iv, days of mg iv. antithymocyte globulin). the indirect antiglobulin test (iat), done in august and december of , was negative. according to anamnestic data, the patient had two pregnancies. she received red cell transfusions during childbirth and platelets in december . results: the patient's blood group was o rhd positive, iat positive. the donor blood group was a rhd positive, iat negative. phenotype of the recipient's rbcs, as well as the donor rbcs, was also determined. anti-e and -c w were found in the patient's plasma, but an additional alloantibody was suspected. the autocontrol was negative. column agglutination technology (cat) and tube technology were used to identify rbc antibodies. plasma was tested with pheno-matched rbcs, papain-and . m dithiothreitol-treated rbcs, as well as cord and autologous rbcs. adsorption and elution tests were done, excluding other "usual" clinically significant alloantibodies, and the patient received three incompatible (xm in iat, cat) yt(a+), e-, c w -, k-red cell units. the sample was urgently sent for an antibody investigation at the international blood group reference laboratory (bristol, uk). in the reference laboratory, anti-e, -c w and an alloantibody to a high-frequency antigen were confirmed, whose specificity was determined to be anti-yt a . anti-jk b was also suspected and later confirmed. before the patient was discharged from the hospital, she received eight more red cell units (yt(a+), e-, c w -, jk(b-)), during which she was serologically closely monitored. summary/conclusions: the results of the antibody investigation in this case study indicate the presence of multiple alloantibodies in a patient who has previously received immunosuppressive myeloablative conditioning therapy. in addition to the "common" alloantibodies (anti-e, -c w , -jk b ), an alloantibody to a high-frequency antigen (anti-yt a ) was detected in the patient. this patient was transfused with incompatible red cell units (yt(a+)) in an emergency, with no ill effects. although anti-yt a is rarely a clinically significant antibody, according to literature, it can cause immediate haemolytic transfusion reaction. additional risk were "common" clinically significant alloantibodies, especially anti-jk b , which was in this case extremely difficult to detect and had further complicated the selection of blood. background: the identification of an antibody against a high-incidence antigen always introduces a challenge due to the difficulty in finding compatible units of red blood cells (rbcs). in patients needing surgery it is important to minimize their transfusional needs by implementing patient blood management programs (pbm). tests that predict the clinical significance of antibodies, such as monocyte monolayer assay (mma) are also useful in guiding clinical decisions. kell blood group system contains highly immunogenic antigens. antibodies against these antigens are immunoglobulin g, and can cause severe hemolytic transfusion reactions and fetal anemia. results: case report we report the case of a -year-old female, with non-hodgkin lymphoma, chronic anemia and scoliosis with severe neurological compromise, proposed for lumbar spinal stabilization surgery. she had a total hip replacement surgery in , with unknown transfusion history. her obstetric history was g p a . the patient had no history of thromboembolic or hemorrhagic events. during pre-transfusional tests, she was typed as a rr and had a positive antibody screening test. the identification studies were suggestive of an antibody against a highincidence antigen, so the surgery was delayed until clarification of these results. she was also referred to a pbm appointment where her hemoglobin was improved from . g/dl to . g/dl by administration of ferric carboxymaltose iv and darbepoetin sc. the patient was phenotyped as kp(a+b-) with anti-kpb, an antibody against a highincidence antigen (> % prevalence worldwide). it is a rare antibody with variable reactivity, causing from none to moderate/delayed transfusion reactions. to access the clinical significance of this antibody, a mma was performed, resulting in a reactivity of . %, suggesting no clinical relevance, however it could be altered after transfusion of kpb+ blood. in order to find compatible rbc's, several family members were phenotyped, however they were all positive to the kpb antigen. in portugal there were no rr kp(b-) blood donors, as it is extremely rare, so we searched in the international rare donor panel (irdp) and two donors were found in spain. two units of compatible rbc's were requested prior to the surgery, which was performed successfully four months later without transfusional support. summary/conclusions: anti-kpb is a rare antibody that in some cases can cause hemolysis of the transfused kp(b+) red blood cells. the combination of kp(b-) and o rr, an extremely rare phenotype, presented a challenge in finding compatible rbcs. this case illustrates not only the complex transfusional and logistic problems that an antibody against a high-incidence antigen can pose, but also the importance of an efficient pbm programme to mitigate the transfusional needs in these patients. background: blood transfusion is an integral part of the supportive care for patients with sickle cell disease (scd). allo-immunization is a recognized complication to red blood cells transfusion (rbc) in those patients. this may result in difficulties in providing compatible blood, and may be associated with the risk of acute of delayed hemolytic transfusion reactions. aims: to describe transfusion management in a patient with scd who has multiple alloantibodies with difficulty in obtaining compatible blood, in order to highlight the importance and clinical consequences of this complication and suggest a possible management approach methods: an -years-old female patient with scd presented to our hospital with hemoglobin level of g/dl secondary to acute splenic sequestration. she had a history of multiple previous admissions and many previous rbc transfusions. blood grouping and pre-transfusion compatibility testing were performed in addition to phenotyping of the patient's red cells. screening was done using column agglutination technique by automated machine (ortho; usa) and antibody identification was performed manually using commercial cells identification panel. phenotyping for the patient was done using haemagglutination technique with mono-specific anti sera (bio-rad; switzerland). results: the patient was of group o rhd (positive). antibody screening was positive and antibody identification revealed probable anti-e and anti-fya with possible development of anti-k allo-antibodies, in addition to recent development of autoantibodies; giving pan-positive reactivity with the identification panels. phenotyping of the patient's rbcs was found to be r r and k-negative. other masked allo-antibodies of undefined specificities were suspected and no compatible blood was found. the clinical condition warranted a blood transfusion, so least incompatible phenotypically matched rbc unit was released. the patient developed acute hemolytic transfusion reaction with drop of the hb level to . g/dl. despite screening hundreds of rbc units, no compatible units were identified, and no transfusion was given. the patient was managed conservatively using hydration, analgesics, hydroxyurea, erythropoietin, intravenous immune globulin (ivig), steroids, and rituximab. hb level increased to g/dl in weeks, and the patient was discharged from the hospital. the sample of the patient was sent to a reference lab (institut fur klinische chemie und laboratoriumsmedizin-regensburg -germany) for further investigations, clarifications and advice for compatible transfusion in case of need. the report of the reference lab revealed the development of additional anti-m and anti-s with confirmation of the presence of anti-fya, anti-k and warm auto-antibodies. phenotyping of rbcs was confirmed by molecular diagnostic testing done in the reference lab; as r r, k-neg. summary/conclusions: finding compatible blood may be extremely difficult in patients with scd who develop multiple alloantibodies. it is therefore essential to perform an initial extended red cell phenotyping for the patients at diagnosis and to have on shelf ready phenotyped blood units for issuing to the patients, to minimize allo-immunization. transfusion may occasionally be avoided in allo-immunized patients, utilizing alternative options of treatment and reducing the risk of serious complications such as hemolytic transfusion reactions. background: red blood cell (rbc) antigens that are present on less than % of most populations are known as low incidence antigens and those present on more than % are known has high incidence antigens. the mns blood group system consists of antigens carried on glycophorin a (gpa), glycophorin b (gpb) or on hybrids of these glycophorins. there are low incidence and high incidence antigens in the mns blood group system. an individual that is homozygous for gp.mur will be negative for the high incidence jenu (mns ) antigen. anti-jenu was first described in a thai patient with thalassemia where only compatible units were found following screening of units. the jenu negative phenotype is a rare phenotype with an estimated frequency of . %. a male patient with a history of previous transfusion presented with an anti-e and a weak auto antibody with no apparent specificity. a donor unit selected for cross match (group o rhd positive, c+e-c-e+, k-) was incompatible with a reaction grade of + by column agglutination technology. the patient's sample and donor unit were referred to the red cell reference laboratory for investigation for a possible antibody to a low incidence antigen. aims: we aim to characterize the phenotype of the incompatible donor unit. methods: standard serological procedures were used to identify the antibody specificities in the patient's sample. blood group phenotyping of the patient and donor was performed by standard serological procedures. genotyping and zygosity testing was performed using polymerase chain reaction (pcr) high-resolution melting (hrm) assay. gp.mur is a gp(b-a-b) hybrid glycophorin resulting from a gene conversion event between gypa and gypb . this disruption to gpb impacts s expression. the donor was negative with anti-s moab (albaclone), positive with anti-s polyclonal (immulab) and negative with anti-s monoclonal antibody (immulab). this s and s phenotype was consistent with the previously reported examples of gp.mur homozygote jenu negative individuals. molecular testing was consistent with serology supporting gp.mur homozygosity and jenu negative phenotype. summary/conclusions: this donor has been added to our rare donor panel and their red cell donations are cryopreserved for future use in our rare donor frozen inventory. there is limited anti-jenu antiserum available to confirm the jenu negative phenotype. we currently rely on the serological profile of red cells presenting with the gp.mur phenotype, s negative and the discrepant s phenotyping to identify jenu negative donors. this case has highlighted the importance of following up unexplained serological incompatibilities. the development of a monoclonal antibody directed against jenu antigen would provide an opportunity to screen for suitable donors for this rare phenotype. background: molecules expressed on tumor cells are a target of interest for drug development by the use of monoclonal antibodies or blocking proteins. however these drugs have the potential to interfere in pretransfusion testing when the target molecule such as cd is also expressed on red blood cells (rbcs). recently, many drugs targeting cd have been developed but appropriate mitigation strategies and approach to selecting rbcs for safe transfusion is still an obstacle. aims: we describe a case of delayed hemolytic transfusion reaction (dhtr) by anti-jk a in a patient treated previously with cd targeted high affinity sirpa fusion protein alx . methods: a -year-old woman diagnosed with nasal cavity squamous cell carcinoma was enrolled in an alx clinical trial. her blood type was group ab, rhd positive, and the antibody screening test was negative for the past months. she had no previous transfusion history during the past two years. after two infusions of alx , two units of apheresis platelets were requested for transfusion. the blood bank noticed that the antibody screening was positive and further investigation was proceeded. results: antibody screening showed trace positivity in both panel cells (i & ii) at room temperature (rt) and °c albumin phase, and + at anti-human globulin (ahg) phase by tube method. the auto control was negative at rt and °c albumin phase, but + at ahg phase. antibody screening ( cells) and identification ( cells) all showed + at ahg phase using gel cards. direct antiglobulin test was + for anti-igg and + for anti-c d using gel cards. two units of rbcs were requested for transfusion after hemoglobin decrease to . g/dl. rbc genotyping was unavailable at the moment. as her previous antibody screening was negative (anti-jk a not detectable), e-, c-, fy b -rbcs were given as a second best option, considering the phenotype distribution of major blood groups in the korean population. the hemoglobin level was well sustained between . - . g/dl but it decreased again to . g/dl twenty days after rbc transfusion. further laboratory investigation was consistent with a dhtr. the patient was no longer being given alx , and antibody screening and dat decreased to - + reactivity. we presumed that antigen typing results would be reliable after chloroquine dissociation and cell washing using antisera that did not require ahg for testing. serologic phenotyping showed that the patient's cells were c+, e+, c+, e+, jk a -, jk b +, fy a +, fy b -, s-, s+, m+, n + . antibody identification using papainized panel cells revealed anti-jk a antibody. we concluded that the dhtr was due to anti-jk a , and jk a -, fy b -, s-rbcs were issued for further transfusion requests. the patient's hemoglobin level recovered to . g/ dl. the patient's genotype was later identified to be the same as serologic typing. summary/conclusions: communication with the physician and blood bank to perform adequate pretransfusion testing before administration of drugs targeted to cd is important to achieve safe transfusion for patients. serologic phenotyping using antisera which do not require ahg for testing can be used as a second option when genotyping is unavailable in a timely manner. background: transfusion is still a key treatment for sickle cell disease (scd) patients. as a result, these patients are much more exposed to transfusions' risks, the most feared one being a delayed hemolytic transfusion reaction (dhtr). we investigated a female scd pediatric patient with no known antibody, who was referred to us for a suspicion of two dhtrs. three transfusion episodes were reported (a total of four units collected from four donors). for the last transfusion, a premedication with rituximab was done. the patient was planned to undergo a bone marrow transplant with her brother as her donor. aims: to describe the molecular and serological workups needed to investigate a dhtr in a scd patient. methods: antibody identification and crossmatches were performed by iat gel testing with red blood cells/panels, which were used raw, papain-treated and trypsintreated. rbcs' phenotypes were determined by conventional techniques. semi-quantitative phenotypes were conducted by serial dilutions with a monoclonal anti-jk a (ms /seraclone â ). dna was extracted using the magna pure compact instrument (roche). sequencing of jk exons - was carried out by in-house techniques. results: the antibody identification showed a very weak anti-jk a , which was only reactive on papain-treated rbcs. autologous control was also only positive in this technique. dat and the eluate were negative. as the patient had recently been transfused (less than four months earlier), on this first sample we were neither able to perform autologous adsorptions, nor verify her jk a /jk b phenotypes. in order to rule out the imputability of an anti-lfa in the dhtr outcome, crossmatches with her donors' rbcs were undertaken. three out of the four donors were tested. apart from the anti-jk a reactivity, none of them was reactive. because the patient had previously been phenotyped as jk(a+b+), her jk gene was sequenced. her genotype was determined as jk* ( a, a, a, g)/jk* . to confirm this jk a variant allele, a family study was conducted. all her siblings were found to harbor the same genotype. her mother's and father's genotypes were jk* ( a, a, a, g)/ jk* and jk* /jk* , respectively. subsequently, autologous adsorptions were performed, which proved the anti-jk a to be an autoantibody. considering the weakness of this antibody, internal controls were used, in order to evaluate a possible dilution effect of this technique. finally, serial dilutions with the anti-jk a reagent showed a weakened jk a expression encoded by the jk* ( a, a, a, g) variant allele. this finding is consistent with the fact that the crossmatches between the proband's serum and her brother's rbcs were weaker than those performed with (jka+b+) rbcs. summary/conclusions: about a third of dhtrs are reported to happen in patients with no previous history of immunization. performing sensitive serological techniques in order to identify antibodies is necessary to select the most appropriate units. molecular work and extra serological testing can be useful to determine whether an antibody is an allo or autoantibody. even though in this case the anti-jk a was the only antibody identified, because it was proven to be an autoantibody, it is difficult to conclude if it was the cause of the dhtr. nevertheless, jk(a-b+) blood was issued, and no adverse events have been reported. luckily, the patient's bone marrow donor harbors the same variant allele. background: according to the aabb, a pre-transfusion sample must be obtained within days of transfusion if a patient has been transfused or pregnant in the preceding months. despite this safeguard, high risk patients (i.e. those recently transfused with a history of pregnancy or transfusion) may develop antibody during this day window. to avoid issuing incompatible red blood cells (rbcs) to these patients, a new antibody screen (abs) sample should be drawn and tested shortly before anticipated transfusion. aims: we report a case of a y/o man who presented to the ed (hospital day , hd ) with a post-fall intracranial hemorrhage and multiple fractures. anti-e and anti-jka were identified after admission on a new specimen prior to current specimen expiration (< days). methods: specimen # (s ) was sent on hd for type & abs (t&s) and crossmatch (xm) of rbcs. abs and immediate spin xm were negative; there was no patient history. by hd , he had negative t&s specimens (hd : s ; hd : s & ; hd : s ) and had been transfused rbcs (hd : ; hd : ) via electronic xm (exm). at hr on hd , rbcs were requested and could have been issued via exm since s was not expiring until midnight. however, given recent transfusions, bb staff first called the patient's nurse to review history. patient was uncommunicative, but had scars suggesting past trauma or surgery. s was requested and received at hr. results: s showed anti-e and anti-jk a in plasma and eluate. his hemoglobin/hematocrit (h/h) decreased from . ( . - . g/dl)/ . ( . - . %) on hd to . / . on hd . during this period, he underwent several surgeries without unexpected bleeding, documented jaundice or dark urine. two e-jk(a-) rbcs were transfused on hd , which he tolerated well with an increase of hemoglobin from . g/dl to . g/ dl. he did well post transfusion with stable h/h between . / . . to . / . . he was discharged on hd . repeat abs on s was negative. of the rbcs transfused before s , one was e+ and four jk(a+). the family reported that he was injured years prior and had been admitted to hospitals, but was unaware of transfusion. hospital # (h ) reported admissions years ago ( rbcs transfused) and years ago; all abs were negative. h admission was years ago with positive abs and inconclusive workup. h admission years ago showed negative abs. summary/conclusions: the patient developed a significant antibody response in less than days from the specimen collection, likely a secondary immune response to sensitization from a transfusion years earlier. a new specimen was requested prior to transfusion even though the existing sample (which was abs negative) had not expired. this approach identified new antibodies, preventing transfusion of incompatible rbcs, and a potentially serious hemolytic transfusion reaction. this case suggests that for high-risk patients, abs more frequently than every days may be beneficial. it is important to increase clinicians' and laboratorians' awareness of this issue. background: red cells with partial d antigen have historically been classified as such, based on the fact that the red cells type as d positive, but individuals make anti-d antibody when exposed to conventional d antigen. a definitive confirmation of the variant of d antigen is obtained after the rh d genotyping. aims: to present a case study of the patient's alloimmunisation with the present d partial antigen type dnb, most likely on previously received transfusions. methods: the patient's pretransfusion testing included the determination of the abo blood group and rhd type (id card diaclon abo/d dv+, dv-, reverse grouping, monoclonal antibodies, gel method), antiglobulin crossmatch, additional phenotyping (gel and tube methods), antibody screening, identification of the specificity of irregular anti-erythrocyte antibodies by commercially available red cell panels (id dia-panel bio rad gel method, panocell immucor, tube method) through an indirect antiglobulin test (iat) and enzymes. after routine rhd typing we continued further characterisation of the rhd antigen by serologic assay (bio-rad id-partial rhd typing),and finally by rhd antigen molecular genotyping (fluogene method on fluo vista machine). results: our patient is a year old woman with a diagnosis of tu mammae who was preparing for total mastectomy surgery. she had a history of blood transfusions twenty years ago, and she also had two births. the blood group typing was: o, ccdee, k-, fy (a-b +), jk (a+b +), ss, mn, le (a+b -). the agglutination reactions that we tested with anti d serums were strong ( +). the compatibility test with rhd positive donated blood units was positive. the presence of anti-d and anti-fya antibodies in the serum of the patient was determined. we prepared one compatible blood unit, rhd negative and fya negative, for a surgery. interpretation of the id-partial rh d typing set indicated that this is a diii category of d partial antigen. a sample of blood of our patient was sent to the blood transfusion institute of serbia, where molecular typing of d antigen was performed and the presence of partial form of antigen d, dnb type, was found. summary/conclusions: rhd positive patients or donors with anti-d antibody presents in their serum should be tested for d genotyping. the recommendation for further transfusions of our patient with dnb d partial and her alloimmunisation is to prepare d negative, fya negative erythrocytic blood components, and as a possible blood donor it would be labeled as rh d positive. background: the jr blood group system consists of jr a (jr ), a high frequency antigen expressed by the abcg gene. the individuals with jr(a-) phenotype are mainly found in the japanese population and may develop anti-jr a when stimulated by blood transfusion or pregnancy. anti-jr a is a dangerous antibody for pregnancy, but also could cause mild or moderate neonatal jaundice. aims: to conduct the antibody specificity identification of the high frequency antibody in a pregnant woman with history of pregnancy but no transfusion. methods: abo, rhd and some special blood group antigens were identified by tube method in saline. antibody screening and blood group specific antibody identification were performed by indirect antiglobulin test (iat) in gel column. the reagent cells treated with trypsin, chymotrypsin and papain, were used to test the antiserum to obtain the characteristic of antibody reaction. the antibody titer in the patient's serum was detected. dna sample was extracted and exons and adjacent intronic sequence of the abcg gene were sequenced. the sample of one family member was collected for testing. results: the blood groups of the patient were b, rhd(+), lu b (+) and kp b (+). the negative reaction of the serum reacted with all reagent cells were tested in saline, but positive ( +) in iat test, while the self-control was negative. the antiserums reacted strongly ( + in iat test in gel card) with the papain-treated cells, but kept the same reaction strength ( +) with trypsin-and chymotrypsin-treated cells, which indicated the possible existence of anti-jr a . the titer of igg antibody in serum was . in cross matching test, the red blood cell of the patient's brother with the same abo and rhd blood group with the patient was successfully matched with the serum of the patient. the sequencing analysis of the abcg gene in the patient and her brother revealed one homozygous nonsense mutation in exon (c. c>t, p.gln x). after the delivery of the pregnant women, no pathological jaundice was seen in the newborn. summary/conclusions: in the condition of the anti-jr a reagent was unavailable for the identification of jr a antigen in the patient, having an indication with anti-jr a by serological test, the alternative genotyping method was used. the most common silencing jr allele reported in asian population, especially in japanese population, was identified to indicate jr(a-) phenotype. immunohemotherapy, centro hospitalar vila nova de gaia/espinho, vila nova de gaia, portugal background: if the investigation of irregular/unexpected antibodies reveals a pattern in which all or most screen and panel cells are positive, with reactions in the same phase and with the same strength, along with a negative autocontrol, an irregular antibody to a high-prevalence antigen may be suspected. high-prevalence antigens are those that are present in almost all individuals ( % or more). fortunately, because these antigens do occur so frequently, it is not common to find a patient with an antibody to one of them. however, when it happens, it may become a troubling situation. aims: clinical case report of panagglutination in assessment of irregular antibodies. methods: collection of clinical data in scl ınico â and sibas â applications. results: woman, years old, o rhd+, previously transfused with red blood cells concentrates in , was proposed to a correction surgery of a periprosthetic hip fracture. pretransfusion serologic tests were requested and irregular antibodies were detected ( + in all the screening cells). in order to identify the specificity of the antibody, a panel of cells was tested; the result was considered inconclusive, due to positive reactions ( +) with all test cells in liss/coombs and atypical positivity with dragging in all cells in enzymatic environment. autocontrol and direct antiglobulin test were negative. it was decided to send two blood samples to the reference laboratory for a more complete immunohematological study. compatibilization of red blood cells to this patient was also requested. during the waiting period, haematopoiesis was optimized. although the patient did not present anaemia at admission, the analytical study revealed iron deficiency; therefore, supplementation with intravenous iron was performed. the reference laboratory also obtained a panreactive panel ( + with all cells) in liss/coombs and weak positivity in papain. after allo-adsorption, the search for irregular antibodies was negative. an anti-yt a , apparently without clinical significance (negative igg and igg ) was then identified. transfusion was not needed either during or after the surgery, with a good recovery of the haemoglobin value in the postoperative period. summary/conclusions: yt a , which belongs to cartwright system, is a high-prevalence antigen in all populations. anti-yt a , an igg antibody stimulated by pregnancy or transfusion, is not as uncommon as we may think, which suggests that it is reasonably immunogenic. these antibodies are not generally considered clinically significant, but there are reported cases of acute and delayed haemolytic transfusion reactions in which anti-yt a has been implicated. therefore, although the described pattern of panagglutination in assessment of irregular antibodies may suggest the presence of an alloantibody directed against a highfrequency antigen, it is very important to confirm that hypothesis, recurring to a reference laboratory if necessary, to identify the antibody and to determine its clinical relevance. even if the identified antibody is associated with rare haemolytic transfusion reactions, it is crucial to optimize haematopoiesis when it is not an emergent procedure, in order to minimize transfusion and its associated risks. both for emergent and elective procedures, the creation of a national database of patients with already identified irregular antibodies would facilitate the administration of red cells concentrates without the implicated antigen. aims: to investigate the frequency and explore the genomic characterization of jk (a-b-) phenotype in blood donors in harbin, china. methods: all samples were screened for jk(a-b-) phenotype using a direct urea lysis test. and the results were confirmed with by iat using anti-jka and anti-jkb with a standard tube test. additionally, polymerase chain reaction amplification and sequence analysis of the jk gene were performed. results: from blood samples, four donors with jk (a-b-) were selected, at a frequency of . %. among these four samples available for sequencing jk gene, a total of two genotypes were discovered: heterozygote of ivs - g>a combining with heterozygote of g>a (gly glu) and heterozygote of g>a (gly glu) combining with heterozygote of c>t(thr met). summary/conclusions: the frequency of jk(a-b-) phenotype in blood donors in harbin area was lower than the reported data from the populations in other areas of china and in finland, but higher than that in japan. ivs - g>a, g>a and c>t were common mutations in the before reports, while g>a was reported first time. in addition, it is an effective measure which establish the jk(a-b-) phenotype donors in this region, to solve the blood transfusion problem in patients with anti-jk . background: blood types, indicating the type of blood group antigen expressed in the red blood cells, is determined by the type of allele at the blood group gene locus. therefore, when allele frequency of each blood group gene is determined, it is possible to predict the frequency of a specific blood type donor with a homozygous allele. it is also possible to estimate the proportion of donors within a particular blood type through combination of specific alleles. and because the ratio of blood group allele differs between ethnicity and race, this can be used as basic data for population genetics and anthropology. therefore, we present a study that examined the allele frequencies of blood group systems in the korean population through blood group genotyping. aims: the purpose of this study is to determine the frequencies of blood group alleles in the korean population, to predict the proportion of homozygous donors, and to obtain the basic data of population genetics. methods: , blood donors from age to were recruited at korean red cross blood centers located nationwide. acquired samples were examined by blood group genotyping methods using the rbc genotyping system id core xt (progenika biopharma). for each donor, genotypes of blood group systems, excluding abo and rhd, were identified. calculation of the frequencies of blood group alleles in the korean population was done. results: we conducted molecular genotyping of the rhce, kell, kidd, duffy, mnss, diego, dombrock, colton, cartwright, and lutheran blood group systems. the allele frequencies of these blood group systems in the korean population were estimated as follows. -rhce*ce . %, rhce*ce . %, rhce*ce . %, rhce*ce . % -kel*k_kpb_jsb allele % -jk*a allele . %, jk*b allele . %, jk*b_null allele . % -fy*a allele . %, fy*b allele . % -gypa*m allele . %, gypa*n allele . % -gypb*s allele . %, gypb*s allele . %, gypb*mur allele . % -di*a allele . %, di*b allele . % -do*a allele . %, do*b allele . % -co*a allele % -yt*a allele % -lu*b allele % summary/conclusions: the significance of this study is accumulation of data on the allele frequencies of blood group genes through highly accurate genotyping method in the east asia region. this enables the prediction of the proportion of donors with a combination of specific blood group alleles in the korean population, which accounts for a decent percentage of the population in this region. background: in donors from arabian countries only little is known about blood groups other than abo and rhesus. during the last years increased migration to central europe has put a focus on the question how to guarantee blood supply for patients from these countries, particularly because hemoglobinopathies with the need of regular blood support are more frequent in patients from that region. aims: blood group allele frequencies should be determined in individuals from syria, other arabian countries, and iran by molecular typing. methods: as most blood groups are defined by single nucleotide polymorphisms (snps) we introduced a maldi-tof ms assay to detect alleles encoding blood groups including kk, fy (a/b), fy null , c w , jk(a/b), jo(a+/a-), lu(a/b), lu ( / ), ss, do (a/b), co(a/b), in(a/b), js(a/b), kp(a/b), rhce*c. c>g, and rhce*c. c>g. additional blood groups and polymorphisms like yt(a/b), s-s-u-, vel null , co null and rhce*c. g>t were tested by pcr-ssp. a total of probands including individuals from syria, from iran, from the arabian peninsula and from northern african countries were included. results: % of the donors were homozygous for the fy null (fy*- t>c, fy* n. ) mutation, . % carried the heterozygous mutation. . % of the syrian probands were heterozygous for the do* c>t mutation (both, do*jo and do*jo ; jo(a+/ a-)) that is virtually unknown in caucasian donors. . % of the syrian donors heterozygously carried the kel* . allele coding for js(a) (phenotype js(a+/ b+)) that is very rare in caucasians. however, no homozygous kel* . carriers were identified. . % of the syrian and . % of all donors were negative for yt*a, which is definitely more frequent than in europeans. one donor from northern africa homozygously carried the gypb*c. c>g, intron + g mutation, inducing the s-u+ w phenotype. . % of all and . % of northern african donors were heterozygous for the rhce*c. c>g substitution, . % of the syrian donors carried rhce*c. c>g (heterozygously) and . % of all donors were heterozygous for rhce* g>t. heterozygosity for vel deficiency (vel*- ) was detected in individuals ( %; of them from syria) whereas only one syrian donor carried the homozygous mutation. none of the donors carried the aqp *c. delg (co* n. ) mutation that induces the co null phenotype. summary/conclusions: the study provides a first overview on a number of different blood group alleles in blood donors from arabian countries. some blood group alleles that largely are lacking in europeans but had been described in african individuals are present in arabian populations at a somewhat lower frequency. in single cases it could be challenging to provide immunized arabian patients with compatible blood. methods: three unrelated individuals ( blood donors and one pregnant woman) of polish origin who were typed as ab group with a very weak a antigen and normal b antigen expression were subjected to extended abo typing. in one case family studies were performed (blood samples from donor's mother, father and sister). sequencing analysis of this donor dna was performed three times (from two blood samples and buccal swab). serologic investigations were performed with standard methods: /gel cards diaclon id abo/d (anti-a: clone a , anti-b: clone g / , anti-a,b: clone es , es + birma + es ; bio-rad) and diaclon id abd-confirmation for donors (anti-a: clone m / = la- ; bio-rad); /tube techniques with: anti-a (birma ; a- h , a s.pa m , c. d ), anti-b (lb- , b- f , c. a ). genotyping was determined by rbc fluogene abo basic kit (inno-train, germany) and by sequencing of + . -kb site of abo gene to cover sequences ranging from the end of intron to utr of the abo gene. additionally sequence of exon of the abo gene was analyzed. results: abo typing showed normal b and a very weak a antigens on rbcs of all three individuals ( blood donors and one pregnant woman). the a antigen was detected by tube technique only using anti-a clones: birma ( + to +), a- h ( + to +) and c. d (weak+ to +); negative reaction of a antigen typing by gel cards was observed. the sera of all individuals contained anti-a antibodies. commercial pcr-ssp kit revealed three heterozygous a/b genotypes (absence of delc typical for abo*a alleles). in all these individuals abo sequencing of . -kb fragment confirmed the heterozygous genotype with polymorphisms characteristic for abo*b. allele ( a>g; c>g; c>t; g>a; c>a; g>c; g>a) and detected a novel abo*a allele sequence with duplication-based insertion of bp after position (abo*a c.dup _ ; gcaggacgtgtccatgcgccg). as a consequence, the online protein translation predicts an in-frame duplication of seven amino acids after codon (p.dup_ _ qdvsmrr), with synonymous change of the repeated codon (cgc>cgg) and (cgg>cgc) but both coding arginine (r). inheritance of abo*a c.dup _ allele was confirmed by family studies of one donor: his father and sister had a/b genotype associated with normal a and b antigens expression; his mother had normal a antigen expression. she carried abo*a . allele and the same abo*a c.dup _ allele as a son. summary/conclusions: a novel a weak allele at the abo gene detected in three unrelated polish individuals is an in-frame insertion of seven amino acids to the wild-type glycosyltransferase a. the stability of the encoded protein may be affected causing the weak a phenotype. the inheritance of this mutation was confirmed in the family studies. background: since the cloning in of cdna corresponding to mrna transcribed at the blood group abo locus, polymorphisms and phenotype-genotype correlations have been reported by many investigators. although many subgroups have been explained at the genetic level, unresolved samples are still encountered in clinical practice. we report here the result of an abo investigation of a sample from a swedish blood donor that showed a very weak agglutination of rbcs with anti-a in routine forward typing. aims: to elucidate the genetic basis of the apparent weak a subgroup. methods: routine abo genotyping by pcr-asp and pcr-rflp including pcrbased analysis of the upstream cbf/nf-y-binding enhancer region was carried out. further genetic analysis was performed by dna sequencing of abo exons - (including base pairs of the adjacent introns) and the proximal promoter. flow cytometric testing of rbcs was performed with monoclonal anti-a, anti-b and anti-h. results: the weak agglutination of erythrocytes with anti-a was accompanied by the expected lack of anti-a and anti-a in plasma. abo genotyping gave the genotype abo*a . /o . usually consistent with normal expression of a antigen. enhancer analysis resulted in an amplification product corresponding to the expected single cbf/nf-y binding motif. flow cytometric testing of the sample showed a antigen expression with an almost chimeric pattern where the majority of the cells (approximately %) expressed the a antigen at a very low level, marginally distinguishable from the group o control. the remaining approximately % of the cells displayed an a antigen level ranging from normal to very weak. genomic abo sequencing showed an abo*a . -like allele except for a novel mutation located in intron , c. + g. the o allele had an additional snp, c. g>a, consistent with the abo*o . allele variant summary/conclusions: a previously unreported variant, c. + a>g, likely effecting the -donor splice site of intron was found in an a weak sample. this type of mutations is expected to decrease mrna stability and/or cause skipping of the preceding exon in the mrna. however, small amounts of full-length enzyme might still be made, being able to give rise to the weak a antigen expression seen in this individual. interestingly, this mutation is very similar to the genetic variant underlying the weak a subgroup a finn . in this case, however, the c. + a>g mutation is located in the -end of intron and is predicted to cause partial skipping of exon . strikingly, the a finn phenotype also results in a pseudochimeric pattern by flow cytometry but with only approximately % positively staining erythrocytes. due to the well documented lack of a-allele-derived mrna in peripheral blood, further transcript studies could not be undertaken. further studies are needed to investigate the exact mechanisms underlying the pseudochimeric pattern observed by flow cytometry in these two interesting genotypes/phenotypes abstract withdrawn. background: abo is the clinically most relevant blood group system in transfusion and transplantation medicine. abo genotyping is potentially useful in clarifying serologic blood grouping discrepancies. this scenario includes inherited subgroups alleles, temporary acquired variant abo phenotypes in disease or pregnancy, and chimerism due to exchange of progenitor cells early in fetal life or after blood progenitor cell transplantation. aims: to investigate the molecular basis for abo discrepancies detected in clinical samples, including donors and patients, sent to our reference laboratory during the past years. methods: if routine abo grouping showed weak agglutination or forward vs reverse typing discrepancy, further abo typing studies were performed manually. adsorption-elution tests were also performed in some cases with polyclonal anti-a and anti-b to confirm whether a or b antigens were weakly expressed on the rbcs membrane. a pcr approach using sequence specific primers for a , b, o and o alleles was used for initial genotype determination. the full abo coding region was analysed as previously described in selected samples for which abo discrepancy was still unexplained. allele specific fragments spanning exon , intron and exon were amplified using a forward primer targeting the g nucleotide (to exclude o alleles amplification) in combination with either b, a or a generic reverse primer. analysis was carried out by sanger sequencing. results: a total of samples with suspected inherited abo subgroup alleles were selected for further molecular studies by sequence analysis. a subgroup alleles: in out of samples with suspected a subgroup alleles, the c. insg insertion was detected corresponding to the abo*ael. allele. the abo*aw . - variant, a hybrid a -o v allele, was found in cases. in case we found the c. g>c change, previously reported associated with weak a antigen expression. finally, a novel c. c>g change was detected in an a allele. b(a) or cis-ab suspected alleles: the abo*b(a) variant carrying the c. a>g change was found in of samples with bo genotype but a weak antigen expression. in the remaining cases, a consensus b allele was detected, thus pointing to a potential chimerism as the cause of the results observed in abo grouping. finally, we have identified an abo*b . allele carrying the nucleotidic change c. a>g in the context of an abo phenotype vs genotype discrepancy. summary/conclusions: the sanger sequencing approach applied in this study have proved to be informative and helpful to determine the molecular basis of abo grouping discrepancies with suspected inherited subgroups. we found mutations, within exon of the abo gene, in out of samples, including novel alleles. chimerism was suspected in cases of a antigen expression in samples with b o genetic background carrying an apparent normal b allele. we are evaluating at the moment a deep sequencing approach by next generation sequencing to determine the presence of a small amount of a minor allele in the presence of a large surplus of the other two alleles. background: recently, the multiple pregnancy rate has been increasing due to advances in artificial fertilization including in vitro fertilization-embryo transfer. most dizygotic twins have dichorionic placenta, but % of them share the placenta. monochorionic dizygotic twins can have blood chimerism, leading to double rbc populations in routine abo serologic typing. recently, more sensitive and objective column agglutination tests with automated systems are being widely used. therefore, blood chimerisms in dizygotic twins can be detected more easily by routine abo blood typing. aims: we report congenital blood chimerism in monochorionic dizygotic twins of triplets, found incidentally during abo serological testing and confirmed by abo genotyping and str marker analysis. methods: a -year-old male (one of triplets) was admitted to the hospital for medical checkup. he did not have any history of transfusion or bone marrow transplantation. routine abo blood grouping test was performed using automated blood bank system ih- ; however, it showed abo discrepancy. the red blood cells showed double cell populations in a gel column with anti-a and anti-b. we carried out abo genotyping both from the blood and from a buccal swab. for the further evaluation, we performed abo serologic testing, abo genotyping, and str marker analysis in his family members. results: among the triplets, blood chimerism was demonstrated in the patient and his brother. they both showed a b phenotypes in the serologic test and tri-allelic abo genotypes in the blood, a /b /o . however, in buccal swabs, the patient showed a /o and his brother showed b /o . other members of the family (father, mother, and dizygotic sister) had regular abo blood types in the serologic test. we performed str analysis in the triplets and parents. eleven loci (d s , d s , d s , csf po, th , d s , d s , d s , d s , d s , and fga) revealed more than one additional allele in the blood sample, apart from those in the buccal swabs. str marker analysis showed that his brother too had blood chimerism. summary/conclusions: we found blood chimerism in monochorionic dizygotic twins of triplets during routine abo blood typing, and this was confirmed by str analysis. as the application of assisted reproductive technology increases, the incidence of blood chimerism will also increase. blood chimerism can often create confusion during abo serologic typing and microchimerism can be overlooked in routine methods. therefore, it is helpful to use an automated blood bank system to improve sensitivity and blood chimerism should be considered if abo blood grouping reveals double populations. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron enhancer. here we describe five new alleles with singlenucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial host. results: case # is a patient with an unclear abo phenotype: forward type b, reverse type ab. sequencing of genomic dna and cloned abo exon detected variant c. - t>g in heterozygosity on an otherwise common a allele, and in trans an abo*b. allele. case # is a caucasian donor with an abo discrepancy: forward type aweak/o, reverse type a. sequencing also detected variant c. - t>g in heterozygosity on an a background, and in trans an abo*o. . allele. given that this variant is located near the intron splice acceptor site, abo* - g transcripts are postulated to undergo altered splicing, leading to an aweak phenotype. case # is a prenatal sickle-cell disease patient with an abo discrepancy: forward type aweak, reverse type a. dna sequencing detected variants c. c>t (pro leu) and c. t>a (tyr asn), both in heterozygosity on an otherwise common a allele, with an abo*o. . allele in trans. thus, the data establish an association of allele abo* t, a with an aweak-like phenotype. case # is a donor with an abo typing discrepancy: forward type o, reverse type a. sequencing detected variant c. c>g (pro arg) in heterozygosity on an a background, and in trans an abo*o. allele. an interpretation of the data is that variant c. c>g weakens the activity of the a transferase, with allele abo*a ( g) encoding the aweak-like phenotype detected by serology. case # is a year-old patient with an abo discrepancy: forward type o, reverse type ab. sequencing of genomic dna and cloned abo exon detected variant c. g>c (gly ala) in heterozygosity on an a background, and in trans an abo*o. . allele. the serology and molecular results suggest that allele abo*a ( c) encodes a cisab weak phenotype. case # is a caucasian donor with an abo typing discrepancy: forward type o with a weak agglutination with anti-ab, reverse type o. dna sequencing detected variants c. g>a (glu lys) and c. g>a (asp asn), both in heterozygosity, in trans, and on a backgrounds. variant c. g>a by itself constitutes allele abo*a . . the phenotype encoded by abo* a is uncertain. summary/conclusions: molecular characterization of abo alleles can help in their future identification and discrepancy resolution. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron enhancer. here we describe five new alleles with singlenucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial plasmid vector. results: case # is a year-old pregnant female with an abo typing discrepancy: forward type o, reverse type a. pcr-rflp predicted abo*a /abo*o . sequencing detected variant c. insg (val gly>fs ter) in heterozygosity on an otherwise common a allele, and in trans an abo*o. . allele. it is unclear how the early truncation of the a transferase encoded by allele abo* insg still allows for some residual enzyme activity, as suggested by the reverse a type. case # is a recently-transfused year-old black patient with an unresolved abo type. sequencing detected variant c. a>g (silent) in homozygosity and variant c. c>t (ala val) in heterozygosity, both on an o background, with an abo*b. allele in trans. although variants c. a>g and c. c>t are likely of no consequence to the abo phenotype of this patient, they are reported here as components of a new abo*o ( g, t) allele. case # is a year-old prenatal female with a rhd typing discrepancy. failure to yield an abo genotype on blood-chip (progenika), a genotyping microarray that interrogates polymorphic positions in rhd and abo, prompted dna sequencing. sequencing of genomic dna and cloned abo exon detected variant c. c>t (arg cys) in heterozygosity on an abo*b allele background, and in trans an abo*o. . allele. the phenotype encoded by allele abo*b( t) is predicted to be b, as evidenced by forward typing on immucor neo and reverse manual typing. case # is a prenatal black patient with an abo typing discrepancy: forward type o in gel, a + mixed field (mf) in tube. reverse type on a cells + in gel, / + in tube. sequencing of genomic dna and cloned pcr products covering exons - detected variant c. g>c (asp his) in heterozygosity, and in trans an abo*o. . allele. case # is the newborn baby of case # , with a forward type a + mf in gel, a + mf in tube. sequencing of the baby's dna detected variant c. g>c (asp his) in heterozygosity, and in trans an abo*b. allele. from these results it is inferred that the phenotype encoded by allele abo* c is a -like. case # is an year-old hispanic donor with an abo typing discrepancy: forward type a, reverse type o. sequencing of genomic dna and abo exons - and - detected variant c. c>t (gln ter) in heterozygosity, and in trans an abo*o. . allele. the truncation of the a transferase at such a relatively late position is consistent with the retention of some enzyme activity, explaining the forward a type encoded by allele abo* t. summary/conclusions: molecular characterization of abo alleles can help in their future identification and discrepancy resolution. background: expression of abo transferase genes can be affected by genetic variants located within the coding sequence, at splice junctions, in the proximal promoter and in the intron enhancer. variants reported to date in the intron enhancer include large deletions, small deletions and single-nucleotide substitutions. here we describe four new alleles with single-nucleotide substitutions found in samples with discrepant or unusual abo serology. aims: to resolve serological discrepancies or unusual serological findings in the abo blood group system by molecular methods, in particular by sanger sequencing. methods: forward and reverse abo phenotyping was performed by the gel or tube methods. adsorption-elution by the heat elution method and testing for h and a substances in saliva were performed by following the procedures in the aabb technical manual. genomic dna extracted from whole blood was pcr amplified to cover the entire abo coding sequence, splice junctions, proximal promoter and intron enhancer. amplification products were sanger sequenced directly or after cloning in a bacterial plasmid vector. background: inactive alleles of the fut could be decreased or aborted the activity of the fucosyltransferase, which results in to form the bombay or para-bombay phenotype with weak or no h antigen expression on erythrocytes. now many para-bombay individuals have been found in the chinese population. according to names for h blood group alleles v . of red cell immunogenetics and blood group terminology working group of the isbt, fut alleles were identified for bombay or para-bombay phenotype around the world. aims: the study was explored the distribution of fut alleles for the chinese individuals with para-bombay phenotype. methods: the samples were come from the blood donors or the patients. the a, b, h antigens were determined using conventional serological method according to the manufacture's instruction. the sequences of the full coding region for fut was amplified, then amplicon was purified with enzymes digestion and used as template for sequencing bidirectionally. all nucleotide sequences obtained were analyzed and compared with standard fut sequence. results: nineteen chinese individuals with para-bombay phenotype were identified. ten of them were the donors and nine individuals were come from the hospital. the rbcs had a very weak agglutination reaction with anti-h in the most of the individuals. fut homozygous mutations were found in the individuals and fut heterozygous changes were existed in individuals after bidirectionally sequencing. . %, . %, . %, . %, . %, . %, . %, . % respectively in the individuals with para-bombay phenotype. according to our previously reports, the fucosyltransferase activity of fut * n. (c. _ delag), fut * w. (c. c>t) and fut * w. (c. c>t) were abolished in vitro assay, while fut mrna levels of them had no effect compared with wild type. summary/conclusions: the fut mutations in the para-bombay individuals were various. the most common fut allele in the chinese individuals with para-bombay phenotype was fut * n. (c. _ delag). background: the regulatory mechanism of the abo gene is complicated and has been investigated extensively.variation in a antigen expression was recognized very early in the twentieth century and the a blood group was divided into a and a . later the a blood group was subdivided further based on characteristic reactivity with human polyclonal antisera, i.e., strength of reactivity and presence of mixed field agglutination; presence of anti-a , and whether a or h blood group substance was present in the saliva of secretor subjects. mutations critical for abo blood group phenotypes have predominantly been found in exons and of the abo gene, both of which encode the catalytic domain of abo glycosyltransferase. in our case report we show how mutation ranging from single nucleotide in the intron enhancer element can alter the efficacy of enzyme and alter antigen expression. aims: this study aims to investigate the molecular basis of discrepant results of abo forward/reverse typing in blood donor. methods: the abo typing was performed using tube technique and column agglutination tests (bio-rad, grifols). standard tests were completed with adsorption-elution study using o plasma as a source of anti-a, and with saliva testing for presence of a and h substances. we performed quality control for these methods. abo group genotyping was performed using pcr with sequence-specific primer by commercial kit (abo-variant; bag healthcare, lich, germany). pcr-amplified exons and intron enhancer were subject to bi-directional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results: standard serological forward tests identified blood group o, however, only anti-b iso-agglutinins were present. anti-a in adsorption-elution study was successfully adsorbed and eluted from the investigated cells. a and h substances were detected in saliva. abo genotyping using pcr-ssp indicated genotype o v/a . dna sequence analysis showed result abo*a ( + a), abo*o. . . the specimen was revealed as an a subgroup, probably a m with an unusual genetic variant in the intron region of the abo gene, the enhancer of the gene expression. summary/conclusions: we report the first case of abo*a ( + a), the mutation located in the enhancer region of gene expression in allele a, that causes discrepant results not only in abo forward/reverse typing but also in molecular blood grouping tests. based on our serological findings, this subgroup is considered as a m . background: a chimera is a single organism composed of cells with distinct phenotypes and/or genotypes. several different types of chimeras are described: artificial, twin and dispermic. the artificial chimerism can be seen following hematopoietic stem cell transplantation, or more transiently following blood transfusion. the second type may also be inherited most commonly through blood exchange in utero between twins. dispermic chimerism is induced by the fertilization of two maternal eggs with two spermatozoa and their fusion into one body. this one is also called tetra-gametic chimerism. in transfusion medicine, chimeras are often detected when mixed field reactivity is observed in abo/d typing or, less commonly, when phenotyping for other blood group antigens. aims: this investigation was prompted by finding a double population of erythrocytes in a surgery patient with no transfusion history. our aim was to investigate the chimera and determine the underlying abo genotype of this patient. methods: routine blood grouping was performed by column agglutination. separation of the double cell populations was performed by differential agglutination with igm anti-d (immuclone, anti-d fast igm, clone: d - , immucor). initial abo genotyping was performed by pcr-ssp (fluogene; inno-train diagnostik gmbh); further resolution was performed using in-house pcr-asp and pcr-rflp methods. next generation sequencing (monotype abo; omixon using illumina sequencing platform) and sanger sequencing analysis were also performed. identification of reference alleles was investigated by fragment analysis of short tandem repeats (str) polymorphisms. results: double population was found in column agglutination in tests with anti-a and anti-ab, and subsequently when typing for d and c antigens, with approximately % of o d+c+ cells. the patient's genotype was identified as abo*o. /*a by ce-certified pcr-ssp kit (fluogene). routine pcr-asp and pcr-rflp could not resolve the patient's genotype possible abo*a /*o genotype was detected by pcr-rflp, but the pcr-asp analysis gave an apparent abo*a homozygote result. sanger sequencing of abo exons and also gave anomalous reactions: no abo*a allele was detected. homozygosity for c. delg was observed as well as heterozygosity for c c/a. this result therefore suggests the patient's genotype is abo*o. /*o. . . next generation sequencing (omixon) revealed the same result. however, when pcr amplification of the cbf/nf-y enhancer vntr -region was performed, possible heterozygosity was observed, i.e. a weak band representing a single copy, and one representing copies of the enhancer region were present. presence of a single copy of the -bp cbf/nf-y enhancer vntr region is unique background: del is a very weak form of d antigen with low density expression of d antigen on the surface of red blood cell, which is generally typed as d-blood group as couldn't form agglutination in routine rhd blood group testing and could only be detected by the non-routine adsorption-elution test. in the east asian and southeast asian population, - % of the individuals with serologically apparent d-phenotype are not these with truly d-phenotype, but del phenotype, which is very rare in caucasian and black ethic groups. and the rhd* el. (rhd* a) is most prevalent (> %) in del people in these regions, so the del carried this allele was commonly known as "asia type" del. in previous studies, no alloanti-d was observed in a large cohort of chinese "asia type" del pregnant women with d+ fetus to indicate no occurrence of alloanti-d immunization against d+ red cell in "asia type" del individuals. aims: to conduct genotyping analysis in the chinese patients having serologically apparent d-phenotype simultaneous with alloanti-d to confirm the existence of the "asia type" individuals to produce alloanti-d or not. methods: from to , the blood sample of the patients or pregnant women identified with alloanti-d in our reference lab were collected. d antigen was confirmed again using the blend anti-d reagent (clone th- /ms- , igm/igg) by tube method in saline and indirect antiglobulin test (iat) in gel card. the zygosity of rhd gene was detected by hybrid rhesus box pcr with psti digestion. for the samples with d or dd genotypes obtained by rhd zygosity analysis, multiplex ligation-dependent probe amplification (mlpa) genotyping was conducted for rhd genotyping analysis. results: a total of serologically apparent d-chinese patients (female, n = ; male, n = ) with alloanti-d were identified. different titers of alloanti-d from : to : (≤ : , n = ; > : , n = ) were detected including few cases with mixed antibodies (anti-d mixed with anti-c, n = ; anti-d mixed anti-e, n = ). serological rhd typing confirmed the serologically apparent d-phenotype. rhd* n. / n. (homozygous rhd gene deletion) genotype was identified in majority of them ( / , . %) by rhd zygosity analysis, while rhd* n. / n. genotype (n = ) and rhd* n. / n. genotype (n = ) carried the rhd non-functional hybrid alleles were detected by mlpa. summary/conclusions: compared with the distribution of average % frequency of "asia type" del in serologically apparent dpopulation in guangzhou of china, no one case of "asia type" del was identified in the cohort of serologically apparent d-patients with alloanti-d in this study. this also provides evidence to confirm no occurrence of alloanti-d immunization in "asia type" del individuals. aims: a serologically rhd-negative donor was found to be rhd-positive in the routine rhd screen. to solve the discrepancy between serology and molecular screen, the sample was sequenced on dna and rna level. methods: phenotyping on id/iat-cards (bio-rad) was done using commercial anti-d antibodies. the adsorption-elution analysis was performed using an in-house pool of polyclonal anti-d antibodies. furthermore an antibody d-screen was performed (diagast). for rhd genotyping rh-type and partial d-type assays (bag health care) were carried out. the sample was further characterized by exon sequencing including flanking intronic regions. rna was extracted from whole blood, reverse transcribed and the cdna sequenced. for amplification and sequencing, both published (gassner, transfusion, ; legler, trans. med., ; richard, transfusion, ) and in-house primers were used. results: repeated phenotyping of the sample with commercial as well as, in-house anti-d antibodies confirmed the rhd negativity. in addition, the adsorption-elution analysis showed a negative result. however, genotyping, using commercially available kits, yielded a rhd positive result and no variants were detected. to investigate this discrepancy, all rhd exons were sequenced. the sequencing data revealed the mutation c. + delt in the splice donor site of exon . to confirm the effect of the splice site mutation on transcription, rna from a fresh whole blood sample was analysed. as a positive control, gypb was amplified and sequenced from the same cdna. wild-type gypb (mns ) was found. with rhd specific primers, no product could be amplified. summary/conclusions: we present a serologically rhd negative case, that was identified as rhd positive by standard commercial genotyping kits. sequencing revealed the new splice site mutation c. + delt. rna sequencing yielded no detectable product. the donor was classified as rhd negative. this case of a discrepant result between serology and genetics shows the importance of a profound and highly sophisticated genetic investigation of conflicting laboratory results. j stettler, s lejon crottet, h hustinx, c von arx, f still, j graber, c niederhauser and c henny interregional blood transfusion src berne ltd., berne, switzerland background: one of the most immunogenic and clinically significant blood group antigens in transfusion medicine is the rhd antigen. variant rhd phenotypes with weakened or absent antigen expression pose a challenge for rhd status assignment in blood donors. to ensure patient safety, it is necessary to fully characterize these variants at the molecular level. aims: samples from two donors were investigated in our laboratory due to discrepancy in rhd typing. methods: rh blood group phenotyping was done by standard serological column agglutination testing (id-system, biorad). further rhd characterization was performed by an anti-d antibody panel containing monoclonal antibodies (d-screen, diagast) and an adsorption-elution test using an in-house pool of polyclonal anti-d antibodies. molecular investigation was initially performed by ssp-pcr detecting common rhd variants (rbc-ready gene cde inno-train; rh-type bag health care). rhd sequencing was done on either dna or rna using published and inhouse primers for amplification and sequencing. results: by tube testing, the rbcs of donor were predicted to be rh:- ,- ,- , , . however, all ten exons of the rhd gene could be detected by routine genotyping. sequencing of rhd revealed a homozygous mutation c>g at position which is the second last nucleotide of exon and thus might have an influence on exon splicing. by cdna analysis a transcript with a correctly spliced exon was identified. the mutation c. c>g leads to the amino acid substitution t r located in the twelfth transmembrane domain of rhd using the model of flegel (transfus apher sci., ) as reference. adsorption-elution testing using a pool of polyclonal anti-d showed a weak positive reaction, re-classifying the donor as rhd positive. this novel allele, rhd* g, could thus be categorized as a del allele. serological results displayed an almost normal rhd antigen expression for donor . further serological determination of the rhd antigen with different antisera, however, showed a reaction pattern typically observed with a weak d variant. with commercial available kits no rhd variant could be detected. rhd sequencing revealed a novel homozygous mutation c. g>c in exon . this mutation causes a p.a p exchange in the sixth membrane-spanning domain of rhd. based on serological data, the donor is rhd positive and in case of transfusion the patient would be treated as rhd negative. summary/conclusions: here we report two novel rhd missense mutations c. c>g and c. g>c harbouring an amino acid substitution within a transmembrane segment. the c. c>g variation displayed an unusual low rhd antigen reactivity and would have been mistyped as rhd negative without extensive genotypic testing. molecular analysis of variant c. c>g suggests that the t r exchange causes a del phenotype rather than a miss splicing event. this was also confirmed by adsorption-elution testing. interestingly, variant c. g>c could only be detected due to comprehensive serological and genetically investigation. background: the rh blood group system is highly polymorphic and one of the most clinically relevant systems in transfusion. actually d antigen is of critical importance due to its involvement in hemolytic transfusion reaction and hemolytic disease of the fetus and newborn. rhd gene variants are common in africans and mostly related to partial d phenotype. aims: rhd gene sequence was investigated in two african brazilian samples. we further attempted to take advantage of combining the molecular data and the available in silico tools for the functional interpretation of the variations, in order to get insights into the clinical phenotype that may be predicted a priori from genotyping. methods: sample #id is a d-negative donor self-declared as african descent. sample #id is a patient with sickle cell disease (scd) typed as d-positive with anti-d in his serum. serologic d typing was determined by manual gel test and by microplate in an automated instrument. sample #id was also submitted to adsorption/elution test. after genomic dna extraction, all ten rhd exons and flanking intronic regions from sample #id were pcr-amplified with rhd-specific primers and analyzed by sanger sequencing. sample #id was investigated by next-generation sequencing on the miniseq platform (illumina) by using a previously published, custom (selected blood group genes) ampliseq panel. a reported three-dimensional ( d) structural model of the rhd-rhd-rhag heterotrimer was used to visualize the position of variations and predict their putative functional/clinical effect. results: in sample #id , a single nucleotide missense change, i.e. c. c>g in exon , was identified. this transversion is thought to replace a threonine by an arginine residue at amino acid position (p.thr arg) of the rhd protein. analysis in the d model clearly suggests a dramatic impact of the p.thr arg substitution occurring in a functionally-critical, conserved motif in terms of interhelix interaction, which is supposed to be highly deleterious to the stability of the protein, and potentially impairs totally its expression at the red blood cell plasma membrane. this predicted functional effect is definitely in accordance with the d-negative phenotype reported in sample #id . in sample #id , the single c. a>g transition was found in exon leading to a threonine-to-alanine substitution at amino acid position (p.thr ala). amino acid is located in rhd protein extracellular loop , and is thus thought to alter d antigen structure, resulting in a partial d phenotype. this hypothesis is in accordance with anti-d found in the serum of sample #id . summary/conclusions: for the past years, due to the advent of next-generation sequencing and the subsequent identification of numerous rare variants, bioinformatics prediction and modelling tools have evolved and currently help physicians in diagnostics, clinical management and genetic counselling. we took advantage of some of those in silico methods to predict retrospectively the effect of two novel variant rhd alleles, including one d-negative and one partial d alleles. although phenotype and clinical symptoms remain definitely the standard determinants to assess the effect of genetic variations, use of those approaches may soon become valuable for guiding subsequent investigations in immunohaematology. abstract withdrawn. alleles of the weak d type and diva cluster. in africans, the most frequent were typically associated with alleles of the weak d type (including dol and rhdpsi), diva and dau clusters with f v occurring in > % of alleles; in addition the key mutations of weak d type and dii and two inactivating mutations (c. _ inst and c. delg) not reported in rhb were among the first polymorphisms. in east asians, rhd( g>a) at . % was most frequent, followed by dfv, weak d type , dbo- , key mutations of diva and weak d type cluster as well as rhce-like substitutions and the mutations of weak d type , type , type , rhd(a v), dvl- , weak d and rhd(n s). weak d type and rhd(t r) were frequent in south asia but not elsewhere. summary/conclusions: data from tgp and gnomad add relevantly to the knowledge on rhd alleles; tgd discloses linked intron polymorphisms, gnomad frequency data not biased by the likelihood of serologic detection. current typing strategies usually start with serology later complemented by molecular typing. in the future, molecular methods will gain importance and frequent alleles currently not distinguished from "standard rhd" may need a rational transfusion strategy. in this respect, the high frequency of weak d type and type in europeans was surprising, might warrant confirmation by alternative methods and should trigger discussion on rational transfusion strategies for these alleles. consistent with an r haplotype and probable dc-. two siblings that were abo compatible including the dc-sibling were incompatible at iat phase. reactivity could be completely adsorbed from the serum using r r , r r , and rr rbcs indicating the antibody is probably a single specificity. the donor returned in and to continue autologous donations. the aim of this case study was to examine the genetic framework of the rhd and rhce genes and to characterize the rh epitope recognized by the antibody. aims: the donor returned in and to continue autologous donations. the aim of this case study was to examine the genetic framework of the rhd and rhce genes and to characterize the rh epitope recognized by the antibody. methods: serologic testing was performed by manual tube testing using ahg in the indirect antiglobulin phase. rbc phenotyping was performed by standard tube hemagglutination testing from edta anticoagulated blood. rhd and rhce exons were sequenced using genomic dna and standard sanger dideoxy method with the bigdye terminator v . cycle sequencing kit. sequence data was aligned to rhd_ng_ . . rhd zygosity was performed using pcr-rflp with mspi. background: according to recent findings in molecular immuno-hematology, rhd genotyping is strongly indicated in rhc+ and rhe+ donors classified in routine as d-negative. among these, one could find a non-negligible share of entirely new genetic alterations or even del alleles, which are often not identifiable with routine serological methods due to the low number of antigenic sites. aims: the present study reports the genotyping data of rhd on rhc+ and rhe+ caucasian donors classified serologically as d-negative, all enrolled by a single transfusion center in italy methods: rhd serological typing was carried out in microplate direct agglutination tests (iris, immucor) by using different anti-d igm clones (clone , dvi+: ldm +esd m; clone , dvi-: rum- , th ) and different anti-d igg clones (clone : ms ; clone : d e ). all donors with d-negative results (n = , divided into subjects with rhc+, with rhe+ and with both rhc+ and rhe+) were addressed to genotype analysis with rhd beadchip molecular test (immucor), pcr-ssp (bagene, inno-train) and/or rbc-fluogene (inno-train). the discrepant results between serology (d-neg) and molecular biology (wild-type or full-length rhd gene) were further investigated by bi-directional sequencing of the rhd coding regions. results: one-hundred donors have been analyzed retrospectively, as part of a pilot study. following the data obtained in this first phase, the analysis methods described above have been implemented in routine, allowing to include further donors, studied prospectively. in . % of donors (n = ), the molecular analyses showed the complete deletion of the rhd locus, while in cases ( . %) a genetic status was found with "non-deleted" rhd. over all, bi-directional sequencing on these donors revealed the presence of negative and weak-d variants. the list of rhd alleles we have identified at the molecular level is as follows: rhd* n. ( cases), rhd* n. ( ), rhd* n. ( ), rhd* n. ( ) , rhd* n. ( ), rhd* el. ( ), rhd* el. ( ), rhd* el. ( ), rhd* w. ( ) . moreover we found a donor with a lack of signal encompassing exons - of the rhd sequence (bioarray rhd beadchip), while additional cases are currently under investigation. summary/conclusions: our study confirms that a non-negligible number of caucasian subjects, classified serologically as d-negative, present rhd gene alterations that differ from the common total deletion. in line with the literature data, we also found a frequency of about in cases ( subjects out of ), in which a donor re-classification as d-positive (weak d type) was necessary. hence, a wider use of molecular typing methods is desirable in order to achieve the correct genetic characterization and the appropriate phenotypic classification of "apparently" d-negative donors. background: without evidence of abnormal serological d antigen expression there will be no quest for weak d, partial d or d variant on the red blood cells. according to our blood donor registry we found that out of serologically typed donors, . % were d+, . % d-and . % weak d. aims: to compare different weak serological reactions of the d antigen to the rhd genotyping. methods: molecular rhd typing using isolated dna and rbc-ready gene cde and rbc-ready gene d weak kits was performed in blood donors, who were serologically typed as weak d using monoclonal blended igm/igg and dvi-and dvi+ anti-d reagents by slide and microplate (mp) technique respectfully, as well as by the antiglobulin test (iat) in gel and with the set of monoclonal partial d typing reagents (biorad). in addition, rhccee phenotyping and genotyping was also performed. results: all of the donors with serologically weak reactions were confirmed to be weak d variants by genotyping except one donor whose iat was false positive due to rbc autoantibodies. the frequency of d variant genotypes was as follows: % weak d type , % weak d type , one donor was typed as weak d type and another one as weak d type . these weak d types were associated with different degrees of serologically determined weakness ranging from negative to weak positive reactions concerning slide and mp. all of them gave positive reaction ranging from + to + with iat, except for the weak d type with the score of < + which gave negative reaction by slide and mp and inconclusive result with the set of monoclonal anti-d reagents for partial d typing. the percentage of donors, who, at serological typing were only found to be d positive in the iat was %. one of the weak d type donors was negative with dvi-and positive with dvi+ reagent in the mp. the additional rh phenotype (genotype) was ccee in all of the donors except in the one who was genotyped as weak d type , as well as in the d negative donor, being ccee. summary/conclusions: further rhd genotyping is required to estimate the actual frequency of d variants in our blood donors. in practice, current serological methods are sufficient to detect almost all variant d phenotypes. there is a consensus that routine molecular d antigen screening in d negative donors in order to detect del variant when ddccee phenotyped red blood cell transfusion is practiced in all d negative patients does not seem to be cost-effective. background: rh null or rh mod -the so-called rh-deficiency phenotypes-are characterized by a null or severely reduced rh antigen expression (including d, c/c and e/ e), respectively. molecular genetic studies showed that these phenotypes are transmitted in an autosomal recessive manner. rh null phenotype originates from two different molecular events giving rise to the amorph type and the regulator type. the former is caused by homozygosity for silent genes at rhd and rhce loci, caused by inactivating mutations in rhce and deletion of rhd. on the other hand, the regulator rh null type as well as the rh mod phenotype are attributed to mutations in rhag gene when in homozygous state or when in heterozygosity with another rhag allele containing an inactivating mutation. a functional rhag is essential both for the correct rh complex assembly and rh antigen expression in the erythrocyte membrane. aims: the aim of this study was to investigate the molecular genetic basis of an argentinean proband with no detectable d, c, c, e and e antigens by standard serological techniques. methods: blood samples were collected from the proband, her parents and sister. the proband was a year-old young woman with parameters of hemolytic anemia: low hemoglobin level ( g/dl), reticulocytosis ( %), hyperbilirubinemia, increased ldh and marked spherocytosis. the d, c, c, e and e status was determined by standard serologic hemagglutination techniques using specific monoclonal antibodies. genomic dna was isolated using a modified salting-out method. dna samples were initially screened for the presence of intron and the untranslated region of the rhd gene using pcr strategies. rhc/c, and rhe/e alleles were studied by allele-specific pcrs to determine the rhce genotype. rhd zygosity was analyzed by pcr-rflp. rhd exon polymorphisms were studied by rhd exon scanning procedure based on pcr-ssp. rhag gene was investigated by exon-specific pcr amplification and sanger sequencing. results: no d, c, c, e and e antigens were detected in the proband's erythrocytes. the father and sister rh phenotype was: d+, c+, c+, e+, e+ whereas the mother rh phenotype was: d+, c+, c-, e-, e+. rh genotyping confirmed the rh phenotypes for all family members except for the proposita who genotyped rhd+, rhc+ and rhe+. all samples showed an homozygous status for the rhd gene and all rhd exons were detected by exon scanning. sequencing analysis revealed an homozygous c. c>t mutation in rhag exon in the proband whereas the rest of the family showed an heterozygous state in the same nucleotide position. the c. c>t mutation is responsible for the p.ser phe amino acid substitution predicted to be in the th rhag glycoprotein transmembrane segment. summary/conclusions: this study described the molecular background responsible for an rh-deficiency phenotype in an argentinean proband. we identified the novel missense mutation c. c>t in the rhag gene which results in the ser to phe single amino acid substitution that shows to be critical for rh antigen complex assembly within the erythrocyte membrane. further studies are being performed in order to determine whether the proband is rh null or rh mod . background: rh blood group system is the most immunogenetic blood group system and blood donor typing should account for all expressing antigens in order to prevent anti-d alloimmunization. aims: the objective of this prospective study was to investigate rhd alleles among blood donors who typed d-by serologic methods and positive for c and/or e. for this reason we developed an easy-to-perform dna-based screening method for the detection of rhd gene and positive samples were further characterized by two commercial pcr-ssp kits. methods: of individual blood donors within a month period, ( . %) typed as d-with standardized immunohematologic methods including the indirect antiglobulin test (iat). residual edta-anticoagulated blood samples were used to isolate genomic dna using the qiaamp dna blood kit (qiagen, germany) from out of ( . %) c/e+ and serologically d-donors. all dna samples were tested individually for the presence of rhd-specific dna sequences in the rhd promoter, intron , exon and exon by a multiplex pcr-ssp method. the reaction was conducted in a final volume of ll with primers that were applied as described by f. wagner et al. (bmc genetics, ) except antisense primer for exon and the two primers amplifying an hgh gene fragment as internal control, designed by our laboratory. pcr products were visualized by electrophoresis on a % agarose gel with ethidium bromide staining. in case of a positive reaction the sample was analyzed by pcr-ssp d weak and pcr-ssp cde (inno-train, germany). results: out of d-individuals analyzed, were ddccee, ddccee, ddccee and one had a ddccee phenotype. molecular analysis showed that ( . %) were negative for all four rhd dna regions. among the other samples, all of ddccee phenotype, three were found to be positive for rhd promoter, intron , exon and exon , three for rhd promoter and exon , and two for exon alone. further genotyping revealed five hybrid rhd-ce-d alleles [ rhd-ce( - )-d and rhd-ce( - )-d], one allele represented the del(m i) genotype, while the remaining two samples did not show an allele that could be determined with the pcr-ssp kits. summary/conclusions: serotyping is the standard method to assign transfusion strategies but it is not always capable to correctly define all samples that show weak reactions in d. a rhd genotyping strategy is needed to confirm d-blood donors and thus to avoid anti-d immunizations. for these reasons we suggest the implementation of an easy and possible cost-effective method. background: more than weak d types have been described to date. transfusion recipients with weak d type , , or are not at risk for forming allo anti-d when exposed to conventional rh d-positive rbcs. molecular analysis of weak d offers a more reliable basis than serotyping to determine the prevalence of weak d types and optimal d transfusion strategies. background: the d antigen, which consists of a mosaic of epitopes, is determined in all the blood donors and patients. most people are either rhd-positive or rhdnegative, but there is a certain number of people who have a variation of the d antigen, which are called weak d, partial d and del phenotypes. aims: the objective is to use molecular methods to determine whether blood donors in republika srpska (with whom a serological weak d antigen has been detected) really have the weak d antigen. in addition, determine whether blood donors, who have been determined as persons who are rhd-negative, with the phenotypes c and/ or e, who have the rhd gene and d antigen on the erythrocyte membrane, so weak that it could not be determined by serological techniques. methods: blood samples were used from regular blood donors, who have been determined as persons with a weaker d antigen, as rhd-negative or as c and/or e positive (based on the agglutination strength) using serological techniques, the test tube method, the microplate method and the gel method. gp.mur was also modelled and shown to closely resemble the tertiary structure of glycophorin a. the predicted structure is anti-parallel b sheets arranged in a "b barrel" also referred to as an ob-like-fold. the regions in which blood group antigens were identified in the predicted stable dimeric structure. summary/conclusions: ob-like-fold structures typically to bind oligonucleotides or oligosaccharides and are associated with cold shock proteins. further modelling is in progress to predict the structure of gpa/gpb heterodimers as a basis for understanding the presentation of blood group antigens. of interest, this finding is consistent with a previous report showing that this gpa binds to carbohydrates. this model serves as a foundation for future work regarding the properties of gpa, which includes identifying locations of specific interactions between gpa and other rbc surface proteins such as gpb and band , as well as identifying structural features of antigenic regions on gpa. . even though no significant differences were found among the groups studied, haplotypes containing the mcc b and sl polymorphisms were identified in d samples but were not found in tb and l groups. summary/conclusions: this preliminary data obtained suggests that cr polymorphisms and haplotypes, especially those containing mcc b and sl snps, could be involved in the disease pathogenesis of tuberculosis and leprosy. the entrance of mycobacteria into macrophages is mediated by complement receptors that facilitate their uptake by host cells so the combined haplotypes could be enhancing parasite phagocytosis and inflammation. further studies are being carried out to establish whether cr polymorphisms are risk or protective factors and whether other genetic variations in this receptor are also involved. abstract withdrawn. background: the dombrock blood group system consists of two antithetical antigens, do a and do b , and three high-prevalence antigens, gregory (gy a ), holley (hy), and joseph (jo a ). the rare do null or gy(a-) phenotype lacks all dombrock antigens, and the do null alleles vary with both do* and do* backgrounds. here we report the molecular basis of a novel do null allele in a gy(a-) brazilian patient with anti-gy a . aims: case presentation: an alloantibody to a high-prevalence antigen was detected in the serum of a year old woman from the northeast brazil with a history of pregnancies but no history of previous transfusion. she required transfusion because of a schedule for total thyroidectomy surgery due to a large compressive nodular goiter. the antibody did not react with the autologous rbcs but reacted by the indirect antiglobulin test in liss with all panel rbcs and other rbc samples tested except with the gy(a-) phenotype. the corresponding antigen was resistant to treatment with papain but sensitive to dtt and trypsin. these results suggested that the antibody recognized an antigen in the dombrock blood group system. the purpose of this study was to identify the antibody specificity and to determine the molecular basis of the phenotype detected. methods: the red cells phenotype and the presence of the dombrock related antibody in the serum were detected by standard hemagglutination techniques. rbcs and antibodies were from our in-house collection of rare samples. genomic dna was prepared from peripheral blood of the patient. dombrock genotyping was performed by id-core xt platform (grifols, spain). the exons of the do gene were amplified by pcr and directly sequenced. experimental immunohematology and diagnostic immunohematology diagnostic immunohematology experimental immunohematology, sanquin, amsterdam, netherlands background: typing of blood group antigens is essential to prevent transfusion reactions or haemolytic disease of the foetus and newborn. to date, the isbt recognises blood group antigens. most antigens ( ) belong to one of the blood group systems. since the genetic basis of these systems is known, genotyping of these antigens is possible. the molecular background of antigens is unknown and can only be determined serologically. one of these antigens is sd a (sid), first reported in .~ % of the population carry sd a on erythrocytes, but this frequency might be higher since identification is difficult due to variability in expression. in % of individuals sd a is present in urine. cells with a high expression of sd a (cad/sda++) are used for detection of antibodies. recently, a -cells antibody detection panel of bio-rad contained a sda++ cell and many individuals with anti-sd a were detected. the b galnt gene has been implicated in the synthesis of sd a . we collected individuals with and without anti-sd a to elucidate the genetic background of the antigen. aims: elucidation of the genetic basis responsible for loss of the sd a antigen on red blood cells. methods: routine diagnostics to identify antibodies in patients was performed using a bio-rad -cells panel, containing donor with high expression of sd a . additionally, pregnant women were screened for anti-sd a . dna of eight samples with anti-sd a and eight samples without anti-sd a was isolated for further analysis. sanger sequencing was performed on b galtnt exon - . results: sequencing of b galtnt revealed two homozygous mutations which are present in all eight individuals with anti-sd a , but not present in controls. the remaining two controls are heterozygous for these mutations. the first mutation within exon , c. t>c (enst . , rs ) changes a cysteine to arginine at position of the protein. the second mutation in exon c. a>g (rs ) does not change an amino acid. both snps have a maf of . and therefore we expect that . % of the population is homozygous for the minor allele. genotyping of a large population of pregnant women and the serological detection of anti-sd a in women with a homozygous mutation is in progress. summary/conclusions: the high frequency antigen sd a has not been linked to a blood group system because the molecular basis for loss of the antigen has not been elucidated. the b galtnt gene has been associated with sd a synthesis and therefore we analysed this gene for mutations in individuals with antibodies against sd a . a single homozygous mutation within exon causing an amino acid change was found in all individuals with anti-sd a , and no individuals without antibodies were homozygous for this snp. from population studies we expected~ % sd a -negatives, but either this frequency is an overestimation because of difficulties to detect low expressed antigens or mutations in other genes are interfering with sd a synthesis. a larger study of individuals with homozygous mutations in b galnt and linkage to sd a -negativity and presence of antibodies will be performed before sd a can be assigned to a new blood group system. abstract withdrawn. abstract withdrawn. background: erythrocyte duffy blood group antigen can scavenge chemokines in whole blood. duffy blood group gene consists of two major alleles: fy*a and fy*b. however, little is known regarding the association of duffy blood group polymorphisms with the red blood cell (rbc) chemokine scavenging. aims: the aim of this study was to determine the association of duffy blood group polymorphism with the rbc chemokine scavenging. methods: the duffy blood group were genotyped by ˊ-nuclease assay in healthy chinese han individuals, while erythrocyte chemokine scavenging function and duffy antigen expression from the same samples were measured using erythrocyte chemokine binding assays and quantitative flow cytometry respectively. results: rbc chemokine scavenging of cxcl was significantly lower in the individuals with the fy*a/fy*a genotype compared to those with fy*a/fy*b genotype (p = . ). similar result was also observed in rbc chemokine scavenging of ccl (p = . ). the expression of duffy antigen on rbc surface in the individuals with the fy*a/fy*a genotype was significantly higher compared to those with fy*a/ fy*b genotype (p = . ). summary/conclusions: duffy blood group polymorphism is associated with the differential rbc chemokine scavenging. it is probable that a change in duffy antigen structure caused by duffy blood group polymorphism is responsible for the differential rbc chemokine scavenging. background: individuals with p-phenotype can develop a naturally occurring anti-pp pk and has clinical significance, causing hemolytic transfusion reactions or hemolytic disease of the fetus and newborn. finding and procuring blood units of pphenotype is a challenge because of its rarity throughout the world. therefore, acute normovolemic hemodilution (anh) can be an on hand tool in the perioperative successful management of patient with rare blood group. however, this approach has not been commonly used aims: n/a. methods: n/a. results: a -year-old korean woman was referred to samsung medical center for surgical management for gallbladder malignancy. her blood type was group a, d-positive. the patient had no known history of transfusion. however, antibody screening and identification test using the column agglutination method (bio-rad, cressier, switzerland) showed panagglutination with negative reactions to autologous red blood cells, indicating the presence of alloantibodies to high frequency antigens. the specimen obtained from the patient was sent to the central laboratory of the swiss red cross (bern, switzerland) and confirmed as anti-pp pk. at first, the transfusion team of our hospital recommended the surgical team to postpone the surgery. however, anh was planned because postponing surgery was not preferred and the patient's preoperative hemoglobin was . g/dl. ml of blood was withdrawn through a radial arterial catheter in two ml blood bags containing citrate-phosphate-dextrose-a solution after anesthetic induction. equal volume of % hydroxyethyl starch solution was infused during the procedure. the patient underwent radical cholecystectomy and liver wedge resection with lymph node dissection, and two units of autologous blood were returned to the patient during surgery. she was then discharged h later with a hemoglobin level of . g/dl. later, the family study was performed with the standard serologic method using the proband's plasma containing anti-pp pk and sequencing of the a galt gene, which were conducted according to the protocols by koda et al.(transfusion. ) . the proband and her brother were homozygous for c. dupc, indicating a rare p phenotype. summary/conclusions: we experienced that autologous blood transfusions via anh is an alternative to allogenic rbc blood transfusion in patients who have no blood available because of high alloimmunization antibodies against rare blood groups. " and the third sample as "gypb*s_gyp*[ a], gypb*s_null(ivs + t)" with a predicted phenotype: s-s+ mi a + and s+s-mi a +, respectively. the gypa specific primers used for discrepancy resolution detected the nucleotide substitution, gyp.c. c>a, in gypa-b-a hybrid associated to gp.hut allele, thus confirming the id core xt result. the expression of mi a for one of these samples was confirmed using non-commercial anti-sera. hence, these three samples were not gp.mur but gp.hut phenotype. both alleles codify for the expression of mi a antigen since it is expressed on several hybrids between the usual forms of glycophorin a and b. two of these three gp.hut samples are african-american donors. gp.hut was reported in white people with a frequency about . % and in thais with . %. these three gp.hut cases found by id core xt in this study point to a higher frequency of this glycophorin variant and also to the presence in african american population. summary/conclusions: id core xt was able to detect two glycophorin phenotypes, gp.mur and gp.hut, which codify for the expression of mi a antigen. standard molecular methods should be implemented in pre-transfusion testing and obstetrical care routine to detect the most clinically relevant glycophorin variants in mns system. background: serf(+) is a high prevalence antigen in the cromer blood group system, which is encoded by a crom* allele. the lack of the serf antigen, serf(À) on red cells is caused by a single nucleotide polymorphism, c. c>t in exon of the decay-accelerating factor, daf gene. alloanti-serf has been found in thai pregnant woman with serf(À) and a serf(À) individual was found among thai blood donors. anti-serf is not a marketed product; hence, a molecular technique has to be implemented to genotype for the crom* allele among blood donors. aims: this study aimed to identify the crom* allele among thai blood donors leading to predicted serf(+) and serf(À) phenotypes. methods: dna samples obtained from , central thai blood donors were genotyped for serf allele detection using in-house pcr with sequence-specific primer (pcr-ssp) and confirmed by dna sequencing. results: the allele frequencies of crom* (+) and crom* (À) among , central thais were . ( , / , ) and . ( / , ), respectively. the homozygous of crom* (À/À) alleles was not found in this study. additionally, the pcr-ssp technique was validated by dna sequencing using randomly chosen samples together with heterozygous crom* (+/À) samples and the results were in agreement. summary/conclusions: our results confirm a high frequency of the crom* (+) allele in the thai population and their frequencies were similar to those formerly reported among thai blood donors. this study would be beneficial to predict the serf antigen from genotyping results due to unavailability of commercial antiserum. background: there is increasing interest in the use of molecular methods for predicting abo grouping. though nextgen and sanger sequencing have both been used to predict abo type, predicting abo type from buccal swab-derived dna and from deceased donors benefits from a quick and reliable method. besides a pcr-rflp that has been used by many labs for more than years, there is a commercially-available research use only (ruo) kit, and both interrogate nucleotides associated with o , o , a and b with a representing the ancestral allele. aims: the aim of this report is to compare two low-resolution polymerase chain reaction (pcr)-based methods, for investigation of samples submitted to a reference molecular immunohematology laboratory for abo typing discrepancies. fifty-six peripheral blood samples were tested, from patients and from blood donors. methods: genomic dna was isolated from peripheral blood mononuclear cells. background: del is the weakest known d positive phenotype in the rh blood group system and detectable only by adsorption and elution tests. the rhd g>a change is an important marker for del phenotype in east asians. a rapid and efficient pcr method for rhd gene g>a genotyping is useful in east asian countries. aims: the aim of this study was to develop a method for rhd g>a genotyping by using single-tube pcr with melting temperature(t m )-shift primers. methods: two allele-specific primer for rhd g>a and a common primer were designed and synthesized. two gc-rich tails of different lengths were attached to ends of the allele-specific pcr primers. single-tube pcr with t m -shift primers was carried out with the three primers. after pcr, melting curve analysis was performed. rhd g>a could be genotyped by differences of the t m s of the pcr products. all of genotyping results were compared with those obtained from conventional pcr-ssp. for the discordant results, rhd exon sequencing was performed to determine rhd g>a genotype. results: a total of samples were genotyped for rhd g>a by pcr with t mshift primers. samples were typed as a+/g-, samples were typed as a-/g+, samples were typed as a+/g+ and samples were typed as a-/g-. two samples typed as a+/g+ by pcr-ssp but a+/g-by pcr with t m -shift primers were confirmed as a+/g-by rhd exon sequencing. summary/conclusions: the single-tube pcr with t m -shift primers for rhd g>a genotyping is simple, rapid, accurate, and it is superior to conventional pcr-ssp. abstract withdrawn. background: the rh blood group system has numerous variant alleles, which may affect rh antigen expression, including rhd-rhce (d-ce) hybrid genes. these variant alleles are frequently found in people of african descent, and typically result in either d-negative (d-) phenotype, or partial d antigen expression, including silencing of high-frequency antigens and/or expression of low-frequency antigens. patients carrying those alleles are particularly at risk of alloimmunization, suggesting that their identification is important in diagnostics. quantitative multiplex polymerase chain reaction (pcr) of short fluorescent fragments (qmpsf) has proven successful for genotyping those dna samples carrying d-ce hybrid genes by assessing both qualitatively and quantitatively rhd and rhce gene exons. aims: the aim of this project was to genotype both rh genes in a cohort of brazilian patients with sickle cell disease (scd), which are known to be of african descent, by using the qmpsf approach and report hybrid gene variability in this population. methods: one-hundred fifteen dna samples were selected for the study and analyzed prospectively by the rhd-qmpsf and rhce-qmpsf approaches to investigate the copy number of all exons in both rh genes. genotypes were further confirmed or investigated by sanger sequencing and conventional pcr-rflp assays. results: in the dna samples, ( . %) exhibited a "wild-type" profile by qmpsf analysis. hybrid genes involving exon , which is functionally not relevant as reported before, was found in samples, including and samples carrying respectively rhd-ce( )-d and rhce-d( )-ce (two homozygous each). except two samples that require additional studies ( . %), rhd zygosity was resolved successfully: (n = rhd gene copies; . %), ( ; . %) and ( ; . %). clinically relevant, i.e. partial d, genotypes were identified in four hemizygous samples ( / , . %) carrying rhd*dau , rhd*dv. , a rhd*diiia-like allele, and a novel rhd*d-ce( :g h-y s-n i)-d allele, as confirmed by sequencing. other hybrid alleles, such as rhd* n. and rhd*diiic, were also found in trans with a normal rhd* allele. in rhce, c/c genotype could be resolved. the rhce*ce (rhce*ce ( c)-d( )-ce) allele, which is commonly cis-associated with rhd*Ψ, was observed in four samples. however the clinically relevant polymorphisms in variant rhce alleles, such as those involved in cemo, cear, ceag, and ceti, were mostly identified by other standard methods. summary/conclusions: although most of the brazilian patients with scd investigated in this study did not carry rhd-rhce hybrid genes, qmpsf analysis has been shown to be an efficient tool in the whole genotyping process to investigate rh gene variation. as previously reported, it has been conclusive for characterization of rhd zygosity and identification of rare, as well as novel, variant alleles. additionally, our results show a large diversity of hybrid genes among the brazilian patients with scd. therefore, we suggest that qmpsf may be used as a complementary screening approach for assessing rh genotype in selected patients and donors. = ) vs. non-bleeding (n = ) patients. platelet, pmp and cp phenotype and function were evaluated by flow cytometry: activation and granule release were examined by antibodies against granulphysin (cd ), p-selectin (cd p), activated gpiib/iiia (pac- ) and phosphatidylserine (ps) (lactadherin) unstimulated and adp, trap or collagen stimulated. coated platelets were identified as a highly granulated independent cell population appearing following collagen stimulation, gated on side scatter and gpiba (cd b). normal healthy reference levels were available. results: the platelet count in bleeding ( /l) and non-bleeding ( /l) patients was comparable (p = , ). bleeding patients had a higher bat score compared to non-bleeding patients ( vs. , p < , ). the proportion of cps was normal in all patients. however, in non-bleeding patients the proportion of ps+cps and per cell ps expression (mfi) ( , % and , mfi) were higher, compared to bleeding patients ( , % and , mfi, both p < , ), and the proportion of ps+cps correlated negatively with bat score (r = , , p < , ). cd + cp was higher in non-bleeding ( , % and , mfi) compared to both bleeding patients ( , % and , mfi) and significantly higher than the reference level ( , % and , mfi, both p < , ). finally, the proportion of ps+pmps was normal in bleeding patients, but their pmps expressed higher than reference ps per cell, both unstimulated and for all agonist ( , mfi unstimulated vs , mfi reference, p < , ). summary/conclusions: patients with it exhibited different bleeding tendency despite comparable thrombocytopenia. in non-bleeding patients the proportion and per cell level of ps+ were higher, indicating that generation of cps with high ps expression is a critical factor determining bleeding phenotype. the finding of high pmp ps per cell level in bleeding patients could represent an inadequate compensation for lack of cp function, indicating that procoagulant pmps may be less important than cps for thrombocytopenic bleeding. quantification and characterization of cps may be a useful tool for future assessment of bleeding risk as well as a therapeutic target in it and other conditions with bleeding diathesis and/or thrombocytopenia. more studies investigating this field are warranted. background: alloantibodies against human platelet antigens (hpas) and human leukocyte antigen (hla) are implicated in several immune-mediated platelet disorders. detection of these antibodies is crucial in the diagnosis and management of these disorders. aims: to establish a method detecting hpa- , hpa- , hpa- , hpa- and hla antibodies using luminex bead technology. methods: monoclonal antibodies specific for platelet glycoproteins and hla class i molecules were separately coupled to the luminex microbeads. positive anti-hpa- a, anti-hpa- b, anti-hpa- a, anti-hpa- a samples were used to validate the specificities of the luminex assay. the anti-hpa- a, anti-hpa- a standard samples were used to evaluate the sensitivities of the luminex assay by serial dilutions (from neat to / ). results: samples collected from patients or isbt platelet workshop were tested by the luminex assay. the results showed that luminex assay could detect antibodies against hpa- a, hpa- b, hpa- a, or hpa- a successfully from the known samples. the sensitivities of the luminex assay detecting anti-hpa- a, and anti-hpa- a were : and : , respectively, using the standard samples. no cross-reactivity was observed in the samples containing multi-platelet antibodies, or mixture antibodies against hpa and hla. the results of samples with platelet disorders were agreement with those of monoclonal antibody immobilization of platelet antigens (maipa) assay. summary/conclusions: luminex beads coupled with monoclonal antibodies could be successfully used to detect hpa and hla antibodies with high sensitivity. background: platelet transfusion is important in clinical treatment. the expression of abo antigen on platelet surface is differential, so it is usually need to ensure the consistency of the abo antigen in clinical transfusion. but in many cases, it is difficult to find the platelets that the abo blood type matched between the recipient and donor, and abo-incompatible platelet infusion is required in these cases. to data, the expression of abo antigens on platelets in normal blood group individuals is rarely reported in chinese population. aims: to understand the differential expression of abo antigen on platelet surface in population of zhejiang province, china. methods: total of individuals with normal abo groups ( group a, group b and group ab individuals, and group o as negative control of abo antigens on platelets) were analyzed. the expression of abo antigens on platelets was determined by flow cytometry using monoclonal antibodies: fluorescein isothiocyanate (fitc)-conjugated mouse antihuman blood group a and pe-conjugated murine igg anti-b antibody ( pe bgrl ). flow cytometric parameters were statistically analyzed by the mann-whitney test or the kruskal-wallis test to observe the difference in two or more groups using graphpad software v . . the correlation and regression analysis between a and b antigen in the platelets and rbcs were also performed by the software. population studies were reported as the mean and standard deviation (sd), and p values less than . were considered statistically significant. results: according to mfi values of abo antigens expression on platelets, the samples were divided into three groups: low expression (le), high expression(he) and moderate expression (me) according to the background mfi observed in group o samples. it was found that about . % of the individuals had a weak expression of abo antigen on the platelet surface in zhejiang province. there was a significant difference in the intensity of antigen expression between these three different groups of the same blood group. for each blood group, there was a positive correlation between the intensity of abo antigen expressed on the platelet membrane and red blood cells of the individuals. results: cases were found with antibody positive. among them, cases ( %) were only anti-hla-i positive, cases ( %) were only anti-hpa positive, cases ( %) were both anti-hla-i and anti-hpa positive. cases were found without anti-hla-i or anti-hpa. among the cases with anti-hpa positive, the distributions of anti-gpiib/iiia, anti-gpia/iia, anti-gpib/ix, anti-gpiv were . %, . %, %, . %, respectively., hla antibody positive rate in the female patients was higher than that in the male and hpa antibody positive rate in the female was lower than that in male, but there was no significance difference between them (p > . ). summary/conclusions: in ptr patients, the platelet antibody was mainly hla-i antibody combined with hpa antibody. background: human neutrophil antigens (hna) are polymorphic structures located on surface membrane of human neutrophils. alloantibodies against hna are implicated in a number of clinical conditions, including immune-mediated neutropenia and transfusion reactions. genotyping for human neutrophil antigen (hna) systems is an important in the diagnosis of disorders involving alloimmunization to hna. aims: the aim of this study was to investigate the hna allele frequencies among blood donors and hematological patients undergoing blood transfusions and to estimate possible hna incompatibilities and risk of hna alloimmunization. methods: a total of blood donors and hematological patients from the north-west region of the russian federation were recruited. dna samples were obtained and typed for hna- , - , - and - systems using polymerase chain reactions with sequence-specific primers (pcr-ssp). specific primers for hna were designed and the polymerase chain reaction amplification conditions were optimized. the v test was used to test for the hardy-weinberg equilibrium for the hna systems. the probabilities of the incompatibility and the potential risk for alloimmunization against different hna systems after random transfusions were estimated based on the hna allele and genotype frequencies. results: in blood donors, the frequencies for the fcgr b* (hna- a), fcgr b* (hna- bd), and fcgr b* (hna- bc) alleles were . , . and . ; for the slc a * (hna- a) and slc a * (hna- b) alleles, . and . ; for the itgam* (hna- a) and itgam* (hna- b) alleles, . and . ; for the itgal* (hna- a) and itgal* (hna- b) alleles, . and . , respectively. in hematological patients, the gene frequencies for hna- a/ bd/bc, - a/ b, - a/ b, and - a/ b were . / . / . , . / . , . / . , and . / . , respectively. no statistic significant difference between genotypes in these groups was observed. since the allele frequencies of hna - , - - for hematological patients and donors did not have statistically significant differences, possible hna incompatibilities and risk of hna alloimmunization were estimated based on the obtained data on the allele and genotype frequencies of hna in a group that combines donors and hematological patients (n = ). the predicted risk of hna- , - , - , - incompatibilities in this cohort were . %, . %, %, and . %, respectively. the possible risk of hna- a, - bd, and - bc alloimmunization were . , . , and . , respectively; of hna- a and - b alloimmunization, . and . ; of hna- a and - b alloimmunization, . and . ; of hna- a and - b alloimmunization, . and . , respectively. summary/conclusions: the information about hna gene frequencies can be used not only in blood services for detection and identification of hna alloantibodies in donors and assessment of alloimmunization risk but also for anthropological studies. background: non-invasive fetal rhd genotyping is performed using circulating cell-free fetal dna from maternal plasma sample and real-time polymerase chain reaction. this antenatal routine dna test is used to target rh-ig administration to prevent hemolytic disease of the newborn. aims: the aim of this study is to characterize maternal rhd variants responsible for indeterminate results during fetal rhd genotyping due to early amplification of at least one of the exons ( , or ) of the rhd gene. methods: samples were tested from / / to / / using free dna fetal kit â rhd. samples ( , %) yielded a premature signal for one or more exons of the rhd gene. after extraction of maternal cellular dna, the maternal rhd was characterized using rhd beadchip assay (immucor/bioarray). rhdiiia-ce( - )-d summary/conclusions: greater diversity is observed in the caucasian population rather than in the afro-caribbean. % of the identified variants are rhd negative alleles including alleles leading to partial rh antigen expression. unexpected alleles are found such as weak d type , , or . these data underline the benefits of maternal rhd genotyping when abnormal early signals are detected during noninvasive fetal rhd genotyping. background: a considerable number of rhd alleles responsible for weak d phenotypes have been identified. serologic determination of these phenotypes is often doubtful and makes genetic analysis of rhd gene highly desirable in transfusion recipients and pregnant women. dna-based methods are useful for enhancing immunohematology typing in doubtful d phenotypes at pregnant women. aims: determination of the rhd gene in a cohort of pregnant women with doubtful d phenotypes. methods: determination of the rhd phenotyping was performed with microagglutination technique biorad and ortho diagnostic simultaneously. rhd genotyping was performed on cases with d typing serological discrepancies with ready-to-use inno-train rbc-ready gene cde and rbc-ready gene d weak test kits based on polymerase chain reaction with sequence-specific priming (pcr-ssp) to unclear serologic findings. results: molecular analyses showed of ( %) pregnant women were rhd*weak d type and not at risk for anti-d. rhd*weak d type were typed in cases ( %) and case was rhd*weak partial . and potentially at risk for being alloimmunized producing anti-d allo-antibodies. summary/conclusions: appropriate classification of rhd phenotypes is recommended for correct indication of rhig in pregnant women. however, the serologic differences between rhd-negative and rhd-positive pregnant women is a real problem for unnecessary application of rhig prophylaxis in pregnant women with d variants. conclusion: antenatal rhig prophylaxis is useful in rhd negative pregnant women. with genotyping we found that % of serological doubtful rhd negative women was d variants that not produce anti d antibodies. in that cases those rhig prophylaxis was unnecessary and harmful as a product of human origin. on other hand there is a save up of a stock of rhig which is any way in deficit. is it time to think about cost benefit of rhig prophylaxis and genotyping in pregnant women. background: in may , uk neqas (btlp) created an external quality assessment (eqa) sample designed to mimic a feto-maternal haemorrhage (fmh) bleed of ml. all material used passed pre-acceptance serological testing; samples were dispatched to participants in countries. post-dispatch testing by flow cytometry (fc) using an anti-d marker showed a bleed volume of . ml so an investigation was initiated. aims: to determine the cause of the unexpectedly low bleed volume and what lessons could be learnt. methods: production methodology and results of pre-acceptance testing were reviewed. fc testing was repeated, plots examined, and the fmh scientific advisory group consulted for advice. further fc testing was performed at wbs using alternative markers, and the material used was investigated at ibgrl. participant results were examined to determine if the sample should be withdrawn from scoring. a questionnaire on how results were managed was sent to the participants using fc with an anti-d marker. results: a material production methodology review showed no obvious cause of the erroneous in-house result. review of pre-acceptance testing images showed no issues, further d-typing of the cord showed + reactions vs. two reagents by tube, cf. + with two different reagents by column agglutination technology. repeat fc testing using the anti-d marker gave similar results; however, closer examination of the plots showed a left shift in the positive peak, indicating reduced fluorochrome binding, possibly due to reduced d antigen density on the cord cells. further fc testing at wbs demonstrated a marked reduction in fluorescence intensity with an anti-d marker. further investigation using an anti-hbf marker showed a bleed volume of . ml, indicating the correct proportion of cord material had been used during sample production. additional serology at ibgrl on the cord material showed reactions which were weaker than the control with / anti-d reagents. overall, the investigation supported the hypothesis that the cord material was d variant. a review of results submitted by participants mirrored the fc investigation and the sample was withdrawn from scoring, as the fc median result is used to calculate scores and the d variant cord was clearly affecting testing with an anti-d marker. the questionnaire showed that all respondents examine fc plots and the gating used, but not all act on them before reporting results, and not all have a back-up plan for anti-d ig dosing in a similar situation. later sequencing of the d gene revealed the cord donor to be dvii which can have a lower than normal d antigen density. summary/conclusions: the use of a d variant cord in an eqa sample was not planned, but allowed uk neqas to highlight some important learning points: -thorough examination of fc plots is essential to avoid underestimation of fmh; a controlled procedure should be in place if modification of gates is required -access to cord/neonatal blood to allow serological investigation may be useful in a similar clinical situation -it is important to have a back-up plan for issuing anti-d ig in the event of an uninterpretable fmh result background: allo-antibodies against fetal blood group and platelet antigens produced by antigen-negative pregnant women can cause hemolytic disease of fetus and newborn (hdfn) and fetal and neonatal alloimmune thrombocytopenia (fnait). prediction of the fetus antigen status in immunized women is important for making decisions concerning further management of pregnancy. nipt is widely used for determination of fetal blood groups but determination of proper specificity in the real-time amplification of a single nucleotide polymorphism (snp), such as k or hpa- a, requires modified protocols. droplet digital pcr (ddpcr) permits detection of low-grade fetal chimerism in maternal plasma dna with higher specificity using allelic discrimination pcr protocols. aims: to establish ddpcr protocols for non-invasive prenatal diagnostics (nipd) of clinically important blood group antigens. methods: dna was isolated from plasma samples of pregnant women and donors with known genotypes (easymag, biomerieux). allelic discrimination protocols for determination of k/k (n = ), s/s (n = ), hpa- a (n = ), hpa- (n = ), hpa- (n = ), hpa- (n = ) genotypes were performed using ddpcr method with droplet digital tm (biorad). the results of allelic discrimination performed using ddpcr were concordant with the already known phenotype/genotype of donors and pregnant women. ddpcr enabled the detection of - , reads for total dna from plasma in tested samples. all fetal results were in agreement with antigen positive genotype of the neonates and the fetal chimerism was from , % to , % (one case was for advanced pregnancy - week of gestation). in / tested samples false positive results were detected at the level of or unspecific reads. summary/conclusions: the implementation of allelic discrimination protocols for ddpcr allowed detection of fetal-maternal incompatibility in k/k, s/s and hpa- a, - a/b, - a/b, - a/b antigens encoded by snp. background: in france, for pregnancies complicated by anti-d (rh ) and anti-c (rh ) allo-immunization, the tests currently used to quantitate maternal antibodies are tube method titration and continuous flow analysis determination of the antibodies concentration. recently, an automated assay was developed using the column agglutination technology on the ih- system (bio-rad â). aims: we wanted to evaluate the score, calculated from the agglutination profile of the antibodies on the ih- system, as a quantitative data to appreciate the level of maternal antibodies. methods: titers from samples containing anti-d and containing anti-c have been established using the semi-automated tube method performed since decades in our lab and the fully automated gel method on the ih- system. scores were calculated manually in both cases. antibodies concentrations were also determined for all samples by continuous flow analysis on our auto-analyzer device (evolution iii ams alliance). we looked for a possible correlation between anti-d and anti-c scores and the corresponding concentrations using the spearman correlation test. results: anti-d tube and gel scores were significantly correlated with the anti-d concentration values (p < . , r = . and p < . , r = . respectively). anti-c scores were also significantly correlated with anti-c concentration values (p < . ) but gel scores have a better correlation coefficient than tube scores (r = . versus . ). it was easier to extrapolate gel score thresholds than tube score thresholds from the autoanalyzer values, with the aim of triggering fetal monitoring by ultrasounds and measurements of the peak systolic velocity in the middle cerebral artery only for risk pregnancies. the determined gel score thresholds were and , corresponding respectively to ui/ml ( uchp/ml) of anti-d and . ui/ml ( uchp/ml) of anti-c. conclusions: calculating the score from the hemagglutination profile displayed by the ih- system provides added values compared to the sole reading of the titer. for anti-c immunization, gel scores are more discriminant than tube ones and better correlated to the concentration values established by continuous flow analysis. the proposed score thresholds to trigger fetal antenatal monitoring need, however, to be confirmed on more samples and to be clinically documented. background: hdnf is due to maternal igg alloantibodies directed against fetal antigens that cross the placenta during pregnancy, causing hemolysis in the fetus, anemia that can lead to edema, ascites, hydrops and, in some cases, death. the diagnosis and management of hdnf is based on maternal screening, and middle cerebral artery (mca) doppler monitoring. in severe hdnf intrauterine blood transfusions (iuts) and or exchange transfusion (et) after birth are necessary to correct anemia, to prevent and treat fetal hydrops. aims: we report eight years of experience in our immunohematology reference laboratory (irl) to highlight the importance of red cell antibody detection as a fundamental parameter to identify pregnancies with high fetal risk and to drive a correct treatment. methods: we report laboratory data from pregnant women with a positive indirect antiglobulin test (iat) referred to our irl from january to december . we performed antibody screening and identification by indirect antiglobulin test (iat) in microcolumn method with biovue system (ortho-clinical diagnostics, raritan, usa), and the title of antibodies in iat by tube method without additive. follow-up tests were also performed in the presence of significant red cell antibodies in order to check antibody title and begin clinical monitoring. threshold values were ≥ : for anti kell antibodies and ≥ : for other specificities. results: out of women, ( . %) displayed clinically significant antibodies, ( . %) clinically insignificant antibodies and ( %) natural antibodies of different specificities. among women with clinically significant antibodies the most frequent was anti-d ( . %) also in combination with other rh antibodies ( . %), while anti-k accounted for %, anti-e for % and antibodies against high-incidence antigens for . %. anti-m and anti-le a antibodies were also found ( . % and % respectively) but they were not clinically significant. among women with clinically relevant antibodies, showed a critic antibody title and they underwent gynecological and obstetric monitoring. fetuses resulted affected by hdfn, displaying anti-d in cases and anti-kell in . fetuses with severe hdfn (anti-d in and anti-kell in ) required iuts, were treated with et, received red blood cells units at birth. summary/conclusions: the mother screening program led to important improvements in the outcomes of hdfn. the identification of women with clinically significant antibodies allowed an appropriate monitoring program and therapy. background: the hemolytic disease of the fetus and newborn (hdfn) is a severe disease, resulting from maternal erythrocyte alloantibodies directed against fetal erythrocytes. alloimmunization in pregnant women has been found to range from , % to , % worldwide. there are over erythrocyte surface antigens, of which more than have been reported to be associated with hdfn. although anti-rhesus d was once the major etiology of hdfn, the universal introduction of antenatal and postpartum rh immunoglobulin has resulted in a marked decrease in the prevalence of alloimmunization to the rhd antigen in pregnancy. consequently, alloantibodies other than anti-d emerged as an important cause of severe hdnf, in particular anti-k and anti-c. however, there are other antigens that have also been found to be associated with hdfn. aims: retrospective identification of erythrocyte antibodies in pregnant women in hospital de braga in and . methods: this study was planned to assess the prevalence of erythrocyte antibodies responsible for alloimmunization, excluding abo-immunizations, in pregnant women attending the antenatal clinics of hospital braga during years, from january to december . in this study, we retrospectively evaluated the erythrocyte antibody screening results of pregnant women. women with positive erythrocyte antibody screening also underwent identification with gel card system following the manufacturer's instructions (diamed â ). the outcomes of infants, whose mother's indirect antiglobulin tests were found to be positive, were examined. direct antiglobulin tests, jaundice and phototherapy history, transfusion and mortality of the newborns were recorded. results: during the study period, pregnant women were attended in hospital de braga. the laboratory registered positive erythrocyte antibody screening tests. the prevalence of positive erythrocyte antibody screening was , %. anti-d was the most common antibody found ( , %). anti-d prophylaxis given during pregnancy was responsible for of cases and maternal antibody titer levels did not exceed among these cases. the prevalence of non-rhd immunization was %. anti-e ( , %) was the most frequent alloantibody other than anti-d followed by anti-m ( , %) and anti-c ( , %). multiple maternal antibodies were found in pregnant women. four women had types of alloantibodies: anti-c and anti-e; anti-c and anti-d; anti-k and anti-cw; anti-e and a non-identified antibody. one pregnant had types of alloantibodies: anti-d, anti-c and anti-e. of all cases of newborns whose mothers had a positive antibody screen tests, icterus occurred in % of them and phototherapy was given in %. summary/conclusions: the prevalence of positive erythrocyte antibody screening in hospital de braga was , %. the erythrocyte antibody screening showed that anti-d was the most common antibody found ( , %) in most of the cases because of anti-d prophylaxis. the prevalence of non-rhd immunization was %. the other most frequent alloantibodies were anti-e ( , %), anti-m ( , %) and anti-c ( , %). an increasing prevalence of non-anti-d alloimmunization was found and there are currently no preventive strategies. in contrast to rhd alloimmunization, the main risk factor for non-anti-d alloimmunization is a previous transfusion therapy. thus, it is important to minimize the exposure of women to incompatible erythrocyte antigens through unnecessary transfusions when possible. background: the mns blood group system is one of the most complex blood group systems. although alloanti-m is a common antibody observed in pregnant women and could also be found in the serum of individuals who have not been exposed to m positive erythrocytes, it is rarely clinically significant and has been regarded as an unimportant antibody to cause hemolytic disease of the fetus and newborn (hdfn), especially in caucasian and black ethnic groups, for a long time. however, an increasing number of cases of severe hdfn resulting in fetal hydrops and recurrent abortion caused by alloanti-m have been reported mainly in the asian population, especially in the japanese and chinese populations. aims: to summarize the characters of serological testing in preterm twins newborns suffered with severe hdfn. methods: the blood sample of two newborns with severe hdfn and the mother, who had the history with three hydrops fetus, were collected. abo, rhd, rhce, and mn blood group typing of the twins newborn and their mother were performed in saline with tube or gel card. direct agglutination test (dat), elution test, antibody specificity identification and antibody titer detection were conducted by iat method in gel card. results: o, rhd(+), and ccee blood groups were identified both in the mother and the twins newborn. background: in france, since may , the legislation does not promote anymore the use of the reference tube method for titration of anti-red blood cells antibodies. this opened the way to the use of newly developed automated anti-red blood cells antibodies quantitation by column agglutination technology. aims: we wanted to assess the performance of titration and scoring by the id-gel test on the ih- system (bio-radâ) and to compare it with the performance established for the reference tube method, used in our lab since decades. another objective of the study was to determine titer thresholds for the gel method, to trigger fetal monitoring by ultrasounds and measurements of the peak systolic velocity in the middle cerebral artery. methods: an home-made internal quality control (iqc) prepared and calibrated using the international anti-d standard ( / ) was used to determine the intraassay and interassay imprecisions, regarding the score and the titer results. patients samples for testing were chosen during the -months assay period, regarding the specificity of the antibodies and the tube titer in order to cover a wide range of have lower values. the highest differences (more than to dilutions higher) were seen for antibodies directed against rh system antigens. among the other specificities, anti-k (kel ) and anti-m (mns ) antibodies show the most samples with equal or lower titers compared to the tube method. conclusions: automated anti-red blood cell antibodies titration by column agglutination technology on ih- system shows better intra and interassay cvs compared to the tube method. it is explained by the fully automated process that includes the reading step. titer results are almost always higher with the gel technology. thus, it seems possible to safely extrapolate the titer thresholds defined for anti-red blood cells antibodies by the tube method to the gel method. however, based on future clinical studies and fetal/neonatal outcomes, it would probably be necessary to increase these thresholds, at least for anti-rh antibodies, in order to avoid heavy, expensive, stressful and useless monitoring of some pregnancies. results: the first case was a -day-old female infant, yellowish skin developed the next day after birth. her capillary bilirubin level was mg/dl, the evidence favored neonatal hyperbilirubinemia and the clinical manifestation revealed hemolysis symptoms. her laboratory findings showed elevated reticulocytes ( . %), ldh ( iu/l) and g pd ( . u/ghb); dat (+/-), iat (-), anemia (hb . g/dl, hct %), and blood smear showed anisocytosis, spherocytes, and polychromatic rbc. her mother blood typed o, d positive, while her blood type was b, d positive and anti-b was found from her elution rbcs ( + ). due to rarely severe anemia with abo incompatibility, maternal plasma was analysed for abo igg antibodies and showed high antibody a and b titre with : and : . the female infant received one unit washed-prbcs for anemia and intensive phototherapy for hyperbilirubinemia. her clinical condition improved significantly, hb rose to . , bilirubin level was within normal range, she was discharged. another -days-old male infant was our second case. on the third day after birth, yellowish skin discoloration developed and bilirubin level was mg/dl. two days later, his transcutaneous bilirubin (tcb) measurement data was high and laboratory findings also showed raised reticulocytes ( . %), dat (+/À), iat (À), hb . background: anti-indian b is a rare alloantibody against the high frequency antigen in b . individuals with the in: ,- phenotype (in(a+b-)) are observed with a frequency of < . % in the indian population and have not been described in caucasians. the majority of anti-in b antibodies have been reported in individuals without previous transfusions, indicating the possibility of a naturally occurring antibody. anti-in b is considered clinically significant and haemolytic reactions after in b -incompatible transfusions have been reported. haemolytic disease of the foetus and newborn (hdfn) due to anti-in b has not been described. however, a positive direct antihuman globulin test (dat) may be observed. aims: to describe the challenges of managing a pregnancy and childbirth of a woman with an anti-in b . methods: serological investigations were performed by iat (tube and column agglutination). papain and trypsin treated cells were also utilised. soluble recombinant in blood group proteins (in-rbgp) (inno-train, germany) were used in neutralization tests. the clinical significance of the anti-in b antibody was determined by monocyte monolayer assay (mma). genomic dna was isolated from whole blood and the samples were further characterized by pcr amplification and sanger sequencing of exon of cd . results: in a -year-old pregnant (para ) woman of indian origin without previous transfusions, an alloantibody of the specificity anti-in b with a titer of : was detected by iat (negative with papain-treated cells) at gestational week (gw) and . the mma, performed in duplicate on samples taken at these dates, showed a mi of . %/ . % and . %/ . % respectively. the mi was interpreted as follows: - % not relevant; - % inconclusive; > % clinical significant. the patient's parents were typed heterozygous, in: , whereas her husband was homozygous, in:- , . due to the husbands phenotype, the fetus was predicted to be in b positive. doppler flow measurement of the peak systolic velocity in the middle cerebral artery of the foetus was normal. delivery took place at gw without increased bleeding. the neonate presented no clinical manifestation of hdfn. neither the mother nor the baby required blood transfusions. summary/conclusions: we report the case of a pregnant woman of indian origin with an anti-in b alloantibody. the first mma, performed in gw , was inconclusive whereas the second mma, performed in gw , indicated that the antibody was clinically significant. if the mi-increase is only due to the pregnancy or has also a clinical significance, cannot be stated. in b negative blood components were not available and the patient's relatives were all in b positive. therefore, measures to avoid transfusions, including optimised peripartal management of haemostasis, was of utmost importance. with only few cases published, the risk of hdfn could not be excluded with certainty. an intrauterine investigation by doppler was performed to exclude relevant anaemia of the fetus. no transfusion was needed at delivery as there were no haemorrhagic complications. the neonate presented no clinical signs of hdfn. background: hemolytic disease of the fetus and newborn (hdfn) is a disease which if untreatedcan cause perinatal mortality and morbidity with a substantial risk for long-term sequela. in albania we lack of studies in this field. aims: the aim of this study is to determine the predictive value and the reliability of the "critical titre" during the evaluation of red cells alloantibodies ability to cause the hemolytic disease of fetus and newborn. methods: we conducted a descriptive, cross-sectional study. the data were collected in the university hospital for obstetrics and gynecology in albania. in the study were included immunized pregnant woman for anti-d antibodies and their newborns which were affected from the hemolytic disease of fetus and newborn. the data belong to the period and . results: the "critical titre" in our study was , meaning that this was the minimal value of the titre antibodies that could cause hemolytic disease of fetus and newborn. our study concluded that only newborns were born without the hemolytic disease of fetus and newborn and the titre values were less than . moderate hemolytic disease of fetus and newborn were caused between the titre values - . the summary/conclusions: the titre values of the mothers are a predictive option of the high risk of giving birth to a child with the hemolytic disease of fetus and newborn. it is recommended that in this cases the mother should be followed with doppler ultrasonography to measure the blood flow of the middle cerebral artery. also the doctors should recommend in pregnant women with positive coombs test not only the identification of the anti-d antibodies but also the identification of the other antibodies such as anti-e, anti-c, anti-k. background: rhd-negative pregnant women with allo-anti-d are at risk of having a fetus affected by haemolytic disease of the fetus and newborn (hdfn) where the fetus is rhd-positive. the rhd allele is highly polymorphic and many rhd variants give rise to an array of partial d phenotypes. the clinical significance for many partial d phenotypes is not well-established. rhd genotyping by non-invasive prenatal testing (nipt) to assess the fetal rhd status determines whether the fetus is at risk for hdfn. nipt tests also include strategies for detecting maternal rhd variants to provide for accurate reporting. however, the presence of a paternal rhd variant, while having the potential to confound nipt interpretation, is often not recognised. we report a "trio" family study triggered by a request for nipt for an rhd-negative pregnant mother, weeks gestation, who presented with allo-anti-d and anti-jk a antibody. subsequent paternal and fetal rhd genotyping was conducted and revealed a novel variant rhd allele. aims: we aim to characterise the paternal rhd allele and review clinical case features. methods: rh phenotyping was performed by standard serological procedures. nipt tested for fetal rhd exons , and . rhd genotyping on whole blood/cord blood dna was performed on the immucor bioarray rhd beadchip kit which predicts a rhd phenotypic variant of best fit. dna sequencing was performed using the illumina trusight one sequencing panel. copy number variation (cnv) analysis was used to assess the rhd exon structure and zygosity. results: the paternal red cells typed as group o rhd+c-c+e-e+, (ror). nipt genotyping detected fetal rhd signals for all exons, predicting rhd-positive. no maternal rhd sequences were detected consistent with homozygosity for the rhd deleted haplotype. for both paternal and cord genomic dna (gdna), beadchip genotyping predicted a rhd variant "diiia/cehar". furthermore, signal drop out was observed at nucleotide positions (c. , c. , c. ) located in rhd exon suggesting exon was either deleted or rhce-replaced. paternal and cord gdna sequencing detected out of snps (c. g>t, c. c>t, c. a>c, c. c>g) associated with diiia phenotype plus additional snps (c. g>a, c. g>c) on the rhd gene. both were rhd hemizygote by cnv analysis. no rhce variants were detected. clinical case features: the maternal anti-d quantitation increased from . iu/ml ( weeks gestation) to iu/ml ( weeks gestation). the fetus required intrauterine transfusions during the pregnancy to manage the hdfn. summary/conclusions: both father and fetus carry an rhd allele that does not align with alleles encoding diii phenotypes. this putative novel rhd variant allele comprises snps associated with diiia and with a possible exon deletion/rhcereplaced. a similar allele was reported in literature, although based on sequence analysis only, with no phenotype data. the variant allele here encodes rhd-positive phenotype and we predict that there may be a loss of d-epitopes. notwithstanding, the clinical presentation shows that maternal anti-d against this rhd phenotype (presumed partial) is associated with a severe hdfn and that such rhd blood group phenotypes are of clinical significance for alloimmunised pregnancies. abstract withdrawn. background: cd is a glycosylphosphatidylinositol (gpi)-anchored protein with apparent molecular mass of kda. in addition to being expressed on human plts, cd is expressed on activated t-cells, endothelial cells, cd + hematopoietic stem cells as well as on progenitor cells. in the chinese population, the calculated allele frequencies of hpa- a and - b are . and . , respectively. based on these data, the risk of alloimmunization against hpa- alloantibodies due to incompatible plt transfusion or pregnancy is expected to occur in relatively high frequency. however, until today there is no report of hpa- alloimmunization in the chinese population. in this study, we analyzed sera from hydrop fetus cases by maipa technique and icfa. aims: to detect the anti-hpa b alloantibodies by maipa and icfa. methods: a -year-old mother, gravida /para . the mother in the first pregnancy was diagnosed hydrop fetus at pregnancy weeks by ultrasound. in the second pregnancy, fetal hydrops was observed by ultrasound at pregnancy weeks. the mother's irregular antibody test was negative. the maternal platelet specific antibodies and hla antibodies were negative. blood routine and morphological examination of fetal umbilical cord blood showed that plt count dropped to . /l, wbc count dropped to . /l, including neutrophil %, lymphocyte %, mononuclear %, eosinophil %, basophil %, red blood cells were normal, hb was g/l. screening for hla and plt-specific antibodies was performed using a elisa-based plt antibody kit (pakplus, gti diagnostics) as recommended by the manufacturer. plt antibodies were detected by icfa and maipa.hpa genotyping was detected by cpr-ssp. results: the fetus's genotype was hpa- a/a, - a/a, - a/a, - a/a. - a/a, a/a, a/a, a/b, naka (+) and the maternal was hpa- a/a, - a/b, - a/a, - a/a. - a/a, a/a, a/ a, a/a, naka (+). the paternal genotype was hpa- a/a, a/b, a/a, a/a, a/a, a/a, a/a, a/b, naka (+), which was the only incompatible antigen compared with the maternal hpa. samples were tested using the fresh plt panels consisting of hpa- aa and - bb homozygous donors. the reactivity of the negative control and the mother's sera with the plts from hpa- a/a (donors ), hpa- a/b (donors ) and hpa- b/b (donors ) donors by maipa. the mother's serum showed no reactivity against a/a plts, weak positive reactivity against a/b plts (od values . ), but strong reactivity against b/b plts (od values . ).this finding could be confirmed by one of the reference plt laboratories (japanese red cross kanto-koshinetsu block blood center, japan) using freshly isolated plts from hpa- genotyped donors (anti-hpa- b average value . ). summary/conclusions: in this study, we found anti-hpa- b in a case of fnait (patient hpa- aa, blood group o) using the maipa technique. we were able to detect the presence of hpa- b alloantibody in one case of nait. background: fetal and neonatal alloimmune thrombocytopenia (fnait) occurs in : live births in caucasians. serological and molecular human platelet antigens (hpa) genotyping tests are performed to investigate and conclude to fnait diagnosis. however, in few cases and particularly in case of suspicion of private platelet antigen, some specialized analyzes must be performed in the laboratory (lab). these analyzes can range from sanger or ngs sequencing to platelet serology with transfected cells. aims: the aim of our study was to explore where the frontier between research and care takes place in the field of platelet immunology through the prism of the fnait investigations carried out by the platelet immunology laboratories. methods: a two-part electronic survey have been sent to foreign platelet immunology experts (pie) from platelet immunobiology working party (piwp) members and espgi board members (n = ). the first part focused on the lab practices and regulatory environment regarding to accreditation, contact with patient, informed consent and patient results. the second part stressed on the investigations performed to discover new platelet antigen and more precisely on the perceived status of these analyzes ( background: haemolytic disease of the fetus and newborn (hdfn) can occur when maternal red cell antibodies, directed against red cell antigens present on the fetal red blood cells, cross the placenta and enter the fetal circulation. in a "traditionally" conceived pregnancy, when hdfn occurs, it is as a result of maternal antibodies directed against fetal red cell antigens in the heterozygous state, whereby the antigen is inherited from the father only. with the advent of donor oocyte (do) in-vitro fertilisation (ivf), the addition of a third person into the reproductive equation allows for the possibility of a more severe form of hdfn when fetal red cell antigens are present in the homozygous state (one copy from father and one copy from donor) and maternal antibodies are directed against these. antigens expressed in the homozygous state will have more antigens sites per red blood cell and therefore are at an increased risk of red cell destruction from the maternal derived cognate antibodies. aims: to raise awareness of increased severity risk of hdfn in donor oocyte conceived pregnancies. methods: we describe two unusual cases of hdfn in our institution of two women whose pregnancy was induced using a donor oocyte and their offspring requiring transfusion support in the postnatal period to treat hdfn. results: the first is a case previously reported (doyle, quigley, fitzgerald et. al. transfusion medicine, ) of protracted hdfn due to anti-c, managed with phototherapy initially, then intervention with red cell top-up transfusion at weeks post-delivery. the second is an unusual case of severe abo hdfn requiring exchange transfusion therapy (pre-publication). summary/conclusions: given the increased number of pregnancies conceived using do we recommend that antenatal guidelines are reviewed to create awareness regarding the potential increased risk of hdfn in do pregnancies complicated by allo-immunisation. critically, antenatal testing guidelines should highlight that the predicted outcomes associated with quantitation/titres can only be used when do has not been used to obtain the pregnancy. it is also essential that clinicians inform the blood transfusion laboratory when do has been used. abstract withdrawn. %) are deceased due to organ rejection, and / patients ( %) are deceased due to disease not related to rejection. summary/conclusions: the use of therapeutic plasma exchange for the treatment of antibody mediated rejection in solid organ transplant is safe and effective when used along with other treatment modalities. further studies will help determine whether it can be reproduced in larger cohorts and whether it is more effective in certain organs. background: extracorporeal photopheresis (ecp) is an important cellular therapy for the treatment of several (auto-)immune diseases such as graft-versus-host disease. the international standard for the ex vivo treatment of the leukapheresis product is the application of -methoxypsoralen ( -mop) and irradiation with uv-a light. however, the addition of -mop to the illumination bag is associated with a potential risk of contamination. aims: the basic principle of the ecp is the induction of apoptosis in the leukocytes. our aim was to find an alternative for the conventional apoptosis induction without the need of external substance application. the objective of the study was the investigation of the apoptosis levels and kinetics in leukocytes after treatment with -mop+uv-a compared to uv-c treatment without additional -mop. methods: we used an in vitro h cell culture approach with human mononuclear cells from healthy blood donors. untreated control cells were compared with , lg/ ml -mop plus j/cm uv-a treated cells and j/cm (effective dose) uv-c treated cells. apoptosis in several leukocyte sub-populations was detected daily with annexin v and -aad flow cytometry standings. results: the apoptosis analysis of cd cd t-helper cells, cd cd cytotoxic tcells, cd b-cells, cd monocytes, cd neg cd nk-cells and cd cd nkt cells revealed no statistical differences in almost all of these cell types after treatment with -mop/uv-a or uv-c light. the apoptosis kinetic as well as the final apoptosis after h were similar in both treatment groups. summary/conclusions: the addition of -mop to the photopheresis irradiation bag is a risk for potential infections. the main effect of the -mop/uv-a treatment is most probably the induction of apoptosis in the leukocytes. here, we provide information that this induction of apoptosis can also be achieved with uv-c irradiation without the need of -mop addition. the apoptosis patterns in most leukocyte subpopulations are very similar after treatment with uv-c compared with -mop/uv-a treatment. future in vivo studies are needed to prove the therapeutic effect of uv-c treated cells in the ecp setting. abstract withdrawn. background: therapeutic plasma exchange (tpe) is performed to remove the implicating substances from the plasma causing the disease. a periodic appraisal of tpe data is important to get insight into the procedural related effects and toxicities and overall outcome in order to have a guided future approach. aims: the purpose of this study is to observe the overall profile and outcome of the patients receiving the tpe in the medicine intensive care unit (micu) of a tertiary care hospital in south india. methods: a record based audit was conducted for the all the patients who were admitted to our tertiary care hospital of south india with bedded micu and received tpe therapy between june, and december . all the tpe procedures were performed using haemonetics multicomponents system (mcs) + ln apheresis system based on intermittent flow centrifugation. we audited our tpe for: number of treatments, clinical indications, treatments prescribed and administered, any procedural or patient complications, and adherence to current best practice recommendations. results: sixty nine patients had undergone tpe procedures. among them, thirty were female patients ( %). the median age ( - ) years. guillain-barre syndrome (gbs) was the most common indication ( %) followed by cases of thrombotic thrombocytopenic purpura, diffuse alveolar hemorrhage, myasthenia gravis, autoimmune encephalitis and hypertriglyceridemia respectively. the tpe regimens received by patients in this icu were not always prescribed in accordance with current best practice recommendations. there were ( %) episodes of patient related complications during the tpe treatments. in ( %) procedures, technical error in the machine was encountered. summary/conclusions: the findings of this audit have identified differences between the current prescription recommendations for tpe and those applied. the infrequency of the therapy and the different indications may present a challenge for medicine intensive care clinicians to provide best care in all cases. background: microangiopathic hemolytic anaemia (maha) encompasses a spectrum of disorders characterised by widely disseminated thrombosis in small blood vessels resulting in formation of schistocytes and concomitant thrombocytopenia. plasma exchange (pe) needs to be considered as empirical and urgent life saving therapy in these disorders irrespective of waiting for specific testing like adamts levels in thrombotic thrombocytopenic purpura (ttp) or complement levels or factor h antibodies in atypical hemolytic uremic syndrome (ahus). aims: to assess the efficacy and safety of plasma exchange in patients diagnosed as having microangiopathic hemolytic anaemias. methods: a retrospective analysis of all pe procedures performed in patients diagnosed as having maha was done over a period of years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . procedures were done on apheretic device (cobe spectra, terumo bct, lakewood co. usa). patients' pre and post procedural hematological and renal parameters were analyzed by applying paired t test. adverse event if any was recorded. results: pe was performed in patients with diagnosis of maha ( -ahus, -ttp, each of post stem cell transplantation drug induced thrombotic microangiopathy (tma), post thyroidectomy tma and post-partum tma). the mean age of patient was . ae . years with m:f as . : . number of procedures per patient varied from to . post pe recovery was observed within - days with statistically significant increase in mean platelet count from . ae . to . ae . /l (p = . ) and significant decline in mean lactate dehydrogenase level from . ae . to . ae . lkat/l (p = . ). there was also significant decline in mean percentage of schistocytes in peripheral smear from . ae . % to . ae . % (p = . ). the mean serum urea changed from . ae . to . ae . mmol/l and creatinine from . ae . to . ae . lmol/l (p = . and . respectively) with significant increase in urine output from . ae . to . ae . ml/kg/h (p = . ). adverse events were observed in patients ( %), allergic reaction to replacement fluid (n = ) being the commonest followed by hypotension (n = ), rigors and chills (n = ). overall survival rate at months was %. summary/conclusions: pe had proven its safety and usefulness as life-saving first line treatment modality in maha. prompt and aggressive treatment helps in achieving early and complete remission in these patients. background: neuromyelitis optica (nmo) also known as devic's disease or devic's syndrome is a rare demyelinating disease of the central nervous system that most often results in selective involvement of the optic nerves (optic neuritis) and spinal cord (myelitis)and has female preponderance. neuromyelitis optica (nmo) attacks are poorly controlled by steroids and evolve in stepwise neurological impairments. assuming the strong humoral response underlying nmo attacks, therapeutic plasma exchange is an appropriate technique in severe nmo attacks. aims: to study the effect of tpe in neuromyelitis optica. methods: a year old female in the medicine department, civil hospital, ahmedabad admitted with chief complains of weakness and numbness in the arms and legs, blurred vision, reduced sensation, difficulty in controlling bladder and bowels, uncontrollable vomiting and hiccups since - days in the medicine department, civil hospital, ahmedabad. attacks were treated with short courses of high doses of intravenous corticosteroid -methylprednisolone intravenous. but there was no clinical improvement. results: clinician advised for the trial of tpe in this patient. the procedure was performed by automated device with continuous flow centrifuge machine fresenius kabi-com.tec using double lumen femoral catheter. after obtaining informed consent from the relative of the patient, cycles of tpe were performed on daily basis. after cycles, both subjective and objective clinical response to tpe was estimated by three different sources (the patient, a transfusion medicine physician, and the treating neurologist). [ ] for motor performance, patient was assessed on a disability scale ( = healthy; = minor symptoms; = able to walk meters without support; = able to walk meters with support; = confined to bed or wheelchair; = requiring assisted ventilation; = dead).patient's motor performance was increased to scale (upper limb) and (lower limb) from scale , deep tendon reflexes were normal. visual function began to improve week after the treatment. visual acuity was / after weeks. summary/conclusions: assuming the strong humoral response underlying nmo attacks, therapeutic plasma exchange is an appropriate technique in severe nmo attacks. this suggests that tpe is beneficial in nmo patients during acute attack if there is no response to corticosteroid treatment. background: babesiosis is a tick borne infectious disease caused by the protozoa babesia. while most infections with babesia are asymptomatic, some patients present with a symptomatic infections and rarely this can be a severe life threatening illness. treatment is primarily with antibiotics but red cell exchange (rce) has been used in the more severe cases which are characterized by high grade parasitemia, evidence of severe hemolysis and or multi-organ failure involving the kidney, lung or liver. a threshold parasite level of % has arbitrarily been applied as an indication for rce, however, this threshold is not evidence based. aims: to report on patients with babesiosis and high grade parasitemia who were treated with antibiotics only without rce methods: data were collected from july to july . a case was defined as a patient diagnosed with babesiosis for whom rce was requested on the basis of a parasitemia of > % but on clinical evaluation it was considered that rce could be withheld and the patient monitored awaiting response to antibiotics. results: three cases of severe babesiosis in which the use of rce was requested on the basis of a parasite level of greater than %, but was not performed. the rce was deferred on account of the good clinical state of the patient and the absence of renal failure. levels of parasite at diagnosis were . %, % and %. all patients were followed daily until discharge. two of these patients had been splenectomized and each received a single unit of red blood cells during the hospitalization. the third patient had a long history of refractory lymphoma and was pancytopenic requiring multiple transfusions during the years before the diagnosis of babesiosis. she had transfusion transmitted babesiosis from a red blood cell transfused days prior to the diagnosis. all three patients responded well to antibiotics and were discharged between - days with undetectable parasites. summary/conclusions: this small case series suggests that requests for rce solely on the basis of an arbitrary level of parasitemia should be questioned and the clinical state and evidence of organ failure considered in the decision to perform rce. abstract withdrawn. chronic transfusion program (ctp) remains the gold standard therapy for stroke prevention and for patients with a severe disease who have inadequate response to hydroxyurea treatment. aims: to evaluate the safety, efficacy and cost between scd patients on ctp that underwent both aet and partial manual exchange transfusion (pmet) procedures. methods: retrospective observational cohort study of patients with scd on ctp that have switched between pmet and aet. this study was carried out from / / to / / in a hospital in portugal. data on patient history, haematological values, duration of the procedure, intervals between them, adverse events as well as the cost of material and working hours were collected and compared between both procedures. results: a total of patients met the inclusion criteria described. however, patient was excluded from our study because of the lack of attendance to the ctp. during the study, we recorded exchange procedures ( pmet and aet), both on peripheral venous access. from all those procedures the major concern was the poor venous access, which was the reason why patients had returned to pmet. no major complication or alloimmunization was observed. the indications for ctp were cerebral vasculopathy (n = ), stroke (n = ) and recurrent vaso-occlusive crisis with multiorgan failure (n = ). for both procedures, target values were to obtain a pre-exchange hbs level ≤ % for stroke and cerebral vasculopathy and ≤ - % for other indications. the median hbs level before pmet was , % ( , - , ) and , % ( , ) before aet. we documented a higher hbs level prior to the next procedure in , % of patients (n = ). despite that all patients remained stable without any major scd related event. both procedures were well tolerated and iron overload was well controlled (median ferritin level pmet: , vs. aet: , ng/ml). the duration of the exchange procedure was longer and the intervals between procedures were shorter with pmet (median pmet: vs. aet: min and pmet: vs. aet: weeks, respectively). annual rbc requirements per procedure were superior (median vs. units) and the overall costs related with aet were , times higher - . , € and . , € aet and pmet, respectively (estimated cost per session aet: , € and pmet: , €). summary/conclusions: our study shows, that the hbs level before both procedures, performed during the same interval, was similar. we verified that pmet has a comparable efficacy with aet in terms of preventing the development or progression of chronic complications and that the cost per procedure is significantly higher with aet. however, in a clinical situation where it is important to rapidly reduce the hbs level, and/or where the control of the target hbs is stricter so that the patients are clinically controlled without an increase in hospital visits, aet is preferred. we conclude that aet is more effective in the rapid reduction of hbs and ferritin levels, as well as being less time consuming. despite this, for the reasons described above, it is more cost-effective to maintain both aet and pmet procedures. background: erythrocytapheresis/red blood cell (rbc) exchange, involves removing of a large number of rbcs from the patient and returning the patient's plasma and platelets along with compatible allogenic donor rbcs. typical indication for rbc exchange is sickle cell disease and its related complications. however, one of the miscellaneous indications of rbc exchange is for the patients of methemoglobinemia who are refractory to treatment by methylene blue. acquired methemoglobinemia is more common than any genetic causes. acquired methemoglobinemia is caused by toxins that oxidize heme iron, notably nitrate and nitrite-containing compounds. for patients failing to respond to standard treatment with methylene blue or in whom its use is contraindicated; hyperbaric oxygen or rbc exchange is indicated aims: case reports on use of rbc exchange in methemoglobinemia are few and indications are based on anecdotal reports. methods: exchange was performed on the cell separator machine, com tec by fresenius kabi. results: we report a case of acquired methemoglobinemia where patient was admitted with peripheral capillary oxygen saturation (spo ) of % on air. the patient did not show improvement in spo level with effective emergency treatment of methylene blue. since, the patient was refractory to treatment with methylene blue, the decision was made by clinician to proceed with rbc exchange. the patient improved significantly after two cycles of one rbc volume automated rbc exchange, and was discharged with spo of % on air. summary/conclusions: automated rbc exchange can be used in patients of acquired methemoglobinemia successfully when methylene blue is ineffective, and may be superior to manual one. background: therapeutic plasma exchange (tpe) is known to disturb the ph and electrolyte status. patients with compromised liver functions may be at a higher risk of electrolyte imbalance due to metabolic abnormalities. aims: the aim of this study was to analyze the variation in ph, ionized calcium, sodium, potassium, and bicarbonate in liver disease patients undergoing tpe. methods: patients with liver disease undergoing tpe during the period from july to august were included in the study. data on patient demographics, details of the tpe procedure, blood gas analysis report and adverse effects of tpe (if any) were collected and analyzed. results: one hundred and seven procedures were done during the study period; of these ( %) were done on the mcs plus (haemonetics corporation) and rest ( %) were done on the spectra optia (terumo bct). the percentage change in ionized calcium, sodium, and potassium due to the procedure was found to be statistically significant (p = . ). the systolic (p = . ) and diastolic ( . ) blood pressure also changed significantly with the procedure. the predictors for the change in ionized calcium were found to be pre-procedure ionized calcium (p < . ), the age of the patient (p < . ) and the pre-procedure ph (p = . ). procedurerelated complications occurred during procedures of which complications ( . %) were categorized as features of hypocalcemia. no association was found between hypocalcemic manifestations and pre-procedure calcium, change in calcium, age or gender of the patient. summary/conclusions: the tpe procedure in liver disease patients causes remarkable changes in ionized calcium, sodium, potassium and bicarbonate ions. the decrease in ionized calcium during the procedure is predicted by pre-procedure ionized calcium levels, ph and age of the patient. monitoring of these parameters and appropriate corrective measures are imperative to patient safety. background: therapeutic plasma exchange (tpe) in pediatric age group is technically demanding because of low blood volume, difficult venous access and poor cooperation of the patient during the procedure. we here present our experience of tpe in pediatric patients from our centre. aims: to assess the challenges during tpe in pediatric patients and formulate appropriate strategies. methods: we did retrospective analysis of all tpe procedures performed in pediatric patients over a period of years ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . tpe procedures were done on two different apheretic devices (cs plus, fenwal usa and cobe spectra, terumo bct lakewood, colorado) daily or on alternate days depending on clinical condition of the patient. for all procedures, kit was primed with compatible packed red cells. adverse events during the procedure were noted and analyzed. results: a total of tpe (range - /patient with mean of . procedures) were performed for pediatric patients with different indications like atypical hus (category i as per american society for apheresis (asfa) in total patients, neuromyelitis optica (category ii) in patients, rapid proliferative glomerulonephritis (category i), c glomerulopathy in patients each and one patient of infective hemophagocytosis. the average age of patient population was . yrs ( . - years) . the male:female ratio was : with an average weight of . kgs. adverse events were observed during ( . %) procedures. most commonly observed adverse events were allergic reaction to replacement fluid ( . %) followed by hypotension ( . %), line occlusion ( . %), vasovagal, endotracheal tube blockage and symptomatic hypocalcemia was observed in one procedure each ( . %).there was no corelation observed between physical parameters of patient with adverse events. all adverse events were managed as per departmental standard operating procedures (sops) and procedures were completed successfully except in one where the procedure was abandoned. no mortality was observed during the procedures. background: the hemoglobin (hb) content of packed red blood cell (prbc) units is heterogenous. the patient's blood volume is also variable which can be calculated based on the weight, height and body surface area (bsa) of the patient. the efficacy of a transfusion episode can be assessed if the hb content of prbc is known and the patient's post-transfusion hb increment is determined. aims: this prospective study was performed to compare the efficacy of the transfusion of prbcs based on hb content versus the standard transfusion practice in thalassemia major patients. we also determined the correlation between hb increment and the hb content of prbc units transfused. methods: a total of registered thalassemia major patients of our institute were included in the study after excluding the patients who had allo-or auto-antibodies. the study was approved by the institute ethics committee. the enrolled patients were randomly divided into two groups: group i (n = )they received abo/rhd identical prbcs suspended in additive solution (saline, adenine, glucose, mannitol: sagm-prbcs) after determining its hb content (units with hb content ≥ g); and group ii (n = )they received randomly selected abo/rhd identical sagm-prbcs. the hb estimation of the randomly selected units in group ii was blinded. following tests were done on pre-transfusion sample: hb estimation using the hematology analyzer (orion , ocean medical technologies, india), blood grouping using tube technique, anti-human globulin (ahg) crossmatch and direct antiglobulin test (dat) using gel technique (biorad, switzerland), antibody screening (abs) using a fully automated immunohematology analyzer (neo, immucor, usa). on the posttransfusion sample collected h after transfusion, hb estimation and dat were performed. results: there was no significant difference among the patient characteristics of the two groups. the mean hb content of the sagm-prbc units was significantly higher (p = . ) in group i (mean ae standard deviation: . ae . g; range: . - . g) than group ii ( . ae . g; range: . - . g). the mean hb increment in group i patients ( . ae . g/dl) was significantly higher (p = . ) than the group ii patients ( . ae . g/dl). in both the groups i and ii, there was a significant negative correlation between hb increment and weight (p = . in groups i and ii), age (p = . for group i; p = . for group ii), body surface area (bsa) (p = . for group i; p = . for group ii) and blood volume (p = . for group i; p = . in group ii). in both the groups i and ii, there was a significant positive correlation between hb increment and hb dose adjusted for bsa and the hb dose adjusted for blood volume (p = . in both groups i and ii for both the parameters). summary/conclusions: the efficacy of transfusion is more when patients are transfused with sagm-prbcs having hb content of g or more as compared to those who are transfused with randomly selected units. for optimal hb increment in thalassemia major patients, the transfusion strategy should be based on the hb content of the sagm-prbcs. background: in male transfusion recipients under years of age, receiving red blood cells (rbcs) from an ever-pregnant blood donor has been associated with increased mortality, compared to receiving a product from a male donor. although it has been suggested that older units of rbcs could be associated with increased mortality, there are significant methodological challenges in these studies. other studies indicated the freshest units of rbcs could be associated with increased mortality among transfusion recipients. we hypothesize both the association between ever-pregnant donors, and fresh units, with mortality could be caused by passenger leukocytes in the transfused rbc units, which decay during storage. aims: to quantify modification of the effect of ever-pregnant donors on mortality in young male rbc transfusion recipients, by storage time. methods: data on transfusion recipients receiving their first-ever rbc transfusion in one of six major dutch hospitals between / / and / / was collected. for the current study, male transfusion recipients under years receiving only transfusions from one donor sex exposure category were selected and followup was censored at three years after transfusion. differences in storage time between groups were estimated by linear regression, adjusted for total number of transfusions, patient age, blood group, transfusion year and month. in a single-unit, single-transfusion cohort, cumulative mortality was estimated separately for patients receiving transfusions from ever-pregnant or male donors and for 'fresh' (< days storage) or 'old' (> up to days storage) rbcs. results: for recipients of only blood from male donors, the storage time of the freshest unit was . day shorter when comparing the patients who died, to , patients who survived (ci: À . to . ). for recipients of only blood from ever-pregnant donors, the storage time of the freshest unit was . day longer when comparing the patients who died, to patients who survived (ci: À . to . ). in the single-transfusion cohort, , patients received a fresh rbc transfusion from a male donor, of whom died; patients received a fresh transfusion from an ever-pregnant female donor, of whom died. patients received an old transfusion from a male donor, of whom died; patients received an old transfusion from an ever-pregnant female, of whom died. the -years cumulative incidence of death among young male recipients was . % (confidence interval (ci): . % to . %) after a fresh transfusion from a male donor and . % (ci: . % to . %) after a fresh transfusion from an ever-pregnant female donor. the -years cumulative incidence of death was . % (ci: . % to . %) after an old transfusion from a male donor and . % (ci: . % to . %) after an old transfusion from an everpregnant female donor. summary/conclusions: prolonged storage of rbcs from ever-pregnant donors was not associated with decreased mortality at years. contrary to our expectations, our results indicate older units may potentiate the effect of ever-pregnant donors. however, due to limited sample size the observed differences were not statistically significant. background: according to the literature review, there was limited impact of premedication (antipyretics, antihistamines and steroids) before transfusion on the prevention of adverse transfusion reactions (atrs). however, the necessity of premedication remains controversial. the premedication before transfusion is still a common clinical practice in pacific-asian countries, along with the premedication rate ranging from to %. in our previous investigation, we found that premedication rate was . % in the outpatients in , which was much higher than the reported rate in asia. aims: to investigate the incidence of atrs and decrease premedication rate without increasing the rate of atrs via education and evidence-based clinical practice. methods: the incidence of atrs from april to december, was retrospectively surveyed. evidence-based clinical practice was initiated since january, . clinical data of the outpatients receiving transfusion therapy were requested and analyzed from january to september, . the incidences of atrs and premedication rates in and were compared using chi-square test. a p value less than . was statistically significant. besides, feedback of the incidence of atrs and premedication rate was given quarterly to the clinicians during the investigation. results: from april, to september, , a total of , blood units were transfused in the outpatients with , transfusion events. of these, cases of atrs, including febrile nonhemolytic transfusion reactions (fnhtr) and minor allergic reactions were reported. the overall premedication rate in the outpatients was . % in , and was significantly decreased to . % in (p < . ). it was reported that the incidences of atrs in and were . % and . % per unit, respectively. there was no remarkable difference between the incidence of atrs in and (p = . ). summary/conclusions: via education and evidence-based clinical practice, we successfully reduced premedication rate without increasing the rate of atrs in the outpatients. furthermore, introduction of computerized provider order entry (cpoe) and clinical decision support system (cdss) could be considered and be expected to prevent unnecessary premedication before transfusion, increasing the compliance with optimized transfusion strategies in the future. methods: a retrospective analysis was done over a period of one year to evaluate clinical efficacy of granulocyte transfusions in hemato-oncology patients with febrile neutropenia. mobilization of granulocyte donors was done as per standard protocol, which included subcutaneous injection of granulocyte colony stimulating factor (g-csf) - lg/kg and tablet dexamethasone mg, - h prior to granulocyte harvest by apheresis. all granulocyte products were gamma irradiated before transfusion. patient parameters like white blood count (wbc), absolute neutrophil count (anc), hemoglobin and platelet count were recorded pre-and post-granulocyte transfusion. infection related mortality (irm) within days of granulocyte transfusion was also recorded. results: minimum adequate granulocyte yield of per unit was fulfilled in % of granulocyte harvests. clinical indications for granulocyte transfusions were fever, an absolute neutrophil count (anc) < /ll, evidence of bacterial and/or fungal infections (i.e. clinical signs of infection, positive cultures and radiological evidence) and unresponsiveness to appropriate antimicrobial therapy for at least h. effects of clinical, microbiological and granulocyte transfusion related variables on infection-related mortality were investigated. the post transfusion anc (within h) increased significantly (median value: /ll) as compared to baseline levels (median value: /ll) (p < . ). infection related mortality was observed in only % ( out of ) of patients. patients became afebrile within - days and culture negative within - days after granulocyte transfusion. for analysis purpose granulocyte transfusion episodes were grouped according to doses of granulocyte transfusions, based on european guidelines (standard dose: . - . cells/kg and high dose: > . cells/kg background: hsa's blood services group (bsg) is singapore's national blood service. in , we conducted our pilot national pbm audit to promote pbm practices. it was agreed that the audit would be performed annually with incorporation of a new indicator to continue promotion of pbm and sharing of good practices. aims: to provide an update on the second national pbm audit for . results are compared to the pilot audit and summarized below. methods: we collected data on performance indicators from acute public care hospitals for weeks each in march and august (the pilot audit covered weeks in ). the performance indicators were: ). percentage compliance to documentation of red blood cell transfusion indications ). percentage of patients screened for pre-operative anaemia, to days before surgery ). peri-operative transfusion rates ( days before to days after surgery) for commonly performed surgeries: coronary artery bypass graft surgery (cabg), total knee replacement (tkr), total hip replacement (thr), nephrectomy, colectomy and hysterectomy. the first two indicators assess pbm efforts and were measured in the pilot audit. indicator ) was added to the second audit to assess impact of pbm practices on transfusion in surgical patients. it was an appropriate time to incorporate this indicator as the hospitals would have been familiar with pbm since its introduction in . results and recommendations were shared with the senior management and hospital transfusion committees of the participating hospitals. results: for indicator ), hospitals had a compliance of - %, the remaining had a compliance of - %. all hospitals incorporated electronic blood ordering but the usage was not compulsory in some. hospitals which mandated electronic ordering performed better as doctors could only order blood products after entering the transfusion indication. we saw compliance increase from % in to % in a hospital that had newly mandated electronic ordering. for indicator ), results ranged from % to %. hospitals made notable improvements when compared to , achieving % and % respectively. they had implemented pre-operative workflows screening all elective surgical cases for anaemia at least weeks before surgery. one hospital also started an outpatient intravenous iron service which reduced pre-operative anaemia rates. for indicator ), mean number of transfused units for each surgery ranged from . to . units per patient, lowest being thr and highest being cabg. this suggests that some transfusions were potentially avoidable with more robust pbm practices. the rate of perioperative transfusions was highest for cabg at % and lowest for tkr at %. summary/conclusions: the annual national pbm audit increases pbm awareness, allowing hospitals to share and learn good practices and implement measurable improvements. based on this audit, a recommendation to mandate electronic ordering of blood products to improve adherence to red cell transfusion indications and implementing pre-operative workflows with consideration for intravenous iron support was made. this audit was more representative than the pilot, with a longer duration of data collection and incorporation of indicator ) showing impact of pbm practices. background: autoimmune haemolytic anaemia (aiha) is a decompensated acquired haemolysis caused by the host's immune system acting against its own red cell antigens. aiha is a rare disorder and although british society of haematology (bsh) guidelines for diagnosis and treatment were published in february , there is little evidence for clinical practice in the united kingdom. aims: to investigate the approach to the diagnosis, investigation and management of patients with autoimmune haemolytic anaemia (aiha) in english nhs trusts. methods: we designed and distributed a survey to the clinical transfusion leads at all english nhs trusts between november and march . the survey requested information on detailed, simulated clinical scenarios. the first simulated scenario described a young patient with active aiha months after an allogeneic stem cell transplant, who has received multiple transfusions in the last weeks and is hypotensive, tachycardic, with a falling haemoglobin (hb), currently g/l. the second scenario describes a young man with a new diagnosis of warm aiha who has an initial hb of g/l and returns to clinic at a -week interval with symptoms of fatigue. he is actively haemolysing and commenced on mg/kg prednisolone. results: there was a % ( / ) response rate by trusts. faced with a - h delay for allo-adsorption studies, % ( / ) of respondents would instead transfuse acutely with abo, rh and k matched red cells negative for any previously detected alloantibodies, % ( / ) would transfuse with o rh d negative red cells and % ( / ) would wait for completion of allo-adsorption studies before transfusing. in this first scenario, a quarter of respondents appeared to delay a potentially lifesaving blood transfusion. british society of haematology guidelines recommend that when anaemia is life-threatening in the time required for full compatibility testing, abo, rh and k matched red cells should be transfused. in the serious hazards of transfusion (shot) report, the most serious and fatal of cases of preventable delayed transfusion was a patient with aiha who died untransfused with an hb of g/l, while awaiting alloadsorption studies. a key shot message was that if clinical harm to patients from withholding blood outweighs safety concerns over a possible delayed haemolytic transfusion reaction, emergency blood is essential and should be offered. the second scenario also identified considerable variation in transfusion practice. it can take several weeks for patients with aiha to respond to prednisolone so a transfusion threshold < g/l after an hb fall of at least g/l in the previous weeks is perhaps overly conservative. summary/conclusions: the overall findings support a need for studies to explore barriers to uptake of guidelines, and to identify areas for further audit and research to guide safe and appropriate transfusion practice in aiha. background: balance between supply and demand of o d negative red cells remains a challenge for almost every blood service. with this re-audit, we wanted to collect objective and comprehensive information regarding usage of o d negative red cells supplied by nhs blood and transplant (nhsbt) to private and nhs hospitals in england. aims: the aim was to understand hospital practices, actual needs and possible avoidable usage of o d negative red cells. where possible, comparisons were made with two previous audits ( ) ( ) ( ) . methods: participating hospitals were asked to determine the fate of all group o d negative red cells they received between th and th may excluding substitutions and complete an organisational survey regarding activities, policies and stockholding practices with respect to o d negative blood. participating hospitals were asked to provide (if available) the prevalence (as a percentage) of o d negative patients in their population. this information, in conjunctions with hospital activities, will be used to estimate appropriate o d negative stockholding levels. background: o rhd-negative (neg) red blood cells (rbcs) are a precious resource, are often in short supply and transfusion of these units in emergency settings carries the potential risk of transfusion-related adverse outcomes such as haemolytic reaction due to minor blood group incompatibility. as such, their use should be closely monitored within health services. most recent australian guidelines ( ) for their use in emergency settings include pre-menopausal females of unknown blood group (mandatory indication) or while the blood group is being established; use should be limited to or less units where possible before a switch to group-specific rbcs (acceptable indication). aims: audit of use of emergency uncrossmatched o rhd-neg rbcs against national guidelines in our institution (an australian tertiary metropolitan public hospital providing acute medical and surgical, emergency and critical care services). methods: use of emergency uncrossmatched o rhd-neg rbcs units over a six-year period was retrospectively reviewed. we collected information about rbcs transfused and discarded, adverse outcomes, patient characteristics, clinical indications and whether use met national guidelines or could have been avoided. results: episodes of emergency uncrossmatched o rhd-neg rbcs were identified, encompassing transfusion of rbc units to patients and the discard of rbcs (due to incorrect transport). of the episodes, episodes ( %) involved an eventual switch to group-specific rbcs (range of emergency units, - units). the main requester was the emergency department ( %). the most common clinical indication for transfusion was acute gastrointestinal bleeding ( %). of the episodes, episodes ( %) did not meet the guidelines for emergency use because > units of emergency uncrossmatched o rhd-neg rbcs were issued. episodes ( %) were flagged as potentially inappropriate as the patients were clinically stable according to documentation in the medical records. episodes ( %) were identified as potentially preventable due to delay in pre-transfusion sample collection (defined as > h elapsed between patient arrival and group and screen sample collection) in the setting of acute bleeding ( %), receipt of an unsuitable pretransfusion sample requiring sample recollection ( %), delay in pre-transfusion sample processing ( %), no valid pre-transfusion sample being available at the time of the bleeding episode despite having a planned elective procedure or being an inpatient with recent clinical bleeding ( %). only one patient was investigated for potential transfusion-related adverse outcome ( %) which was thought likely due to concurrent sepsis. summary/conclusions: over six years, episodes utilising emergency uncrossmatched o rhd-neg rbcs were identified with rbcs issued and rbcs discarded. a significant proportion of episodes ( %) were potentially avoidable if there had been a valid pre-transfusion sample available in the transfusion laboratory at the time of the episode. efforts to minimise use of this precious resource are ongoing, and include feedback to clinical units regarding importance of valid pretransfusion samples prior to applicable invasive procedures and in bleeding patients, ongoing education to medical and nursing staff, and continuing audit of use of this blood component in the hospital haemovigilance programme. abstract withdrawn. abstract withdrawn. background: platelet transfusions are often given prophylactically to thrombocytopenic hematology patients. to which extent platelet function improves after transfusion, and how this improvement correlates with an increase in platelet count, is not well studied. flow cytometry has been used to evaluate platelet function after transfusion in a few studies and can be performed even at low platelet counts. rotational thromboelastometry (rotem) represents a more physiological measure of platelet function in whole blood that has not been extensively used in transfusion settings. we used these methods to investigate if platelet transfusion improves platelet function in hematology patients and if improvement correlates with increased platelet counts. aims: the aim was to evaluate the relationship between response to platelet transfusion, measured as corrected count increments (cci), and platelet function in thrombocytopenic patients with hematological disorders. methods: blood samples (sodium-citrate anticoagulated) were collected from unselected hematology patients receiving prophylactic platelet transfusions, after informed consent had been obtained. samples were taken at three time-points: within h before transfusion, h after and - h after transfusion (via a central venous catheter or a subcutaneous venous port). for each time-point, platelet response to adenosine diphosphate (adp) and thrombin receptor-activating peptide (trap- ) was assessed by flow cytometry by measuring p-selectin and pac- expression on single platelets. rotem analysis was also performed on all samples, using intem and extem reagents. results: an interim analysis was performed after inclusion of patients. the mean platelet count before transfusion was /l (range - /l). h cci was /l and - h cci was /l, but response was highly variable. pselectin expression after stimulation with adp and trap was significantly higher at h after and - h after transfusion compared to before transfusion (p < . ). pac- expression after stimulation with adp was significantly higher at - h after transfusion (p < . ), but not at h after transfusion. in rotem, clot amplitude at and min (a and a ) as well as maximum clot firmness (mcf) improved after transfusion (p < . ). a significant correlation between absolute platelet count and p-selectin expression after trap and adp stimulation was found (r s = . and . respectively, p < . ). absolute platelet count was also significantly correlated with mcf (r s = . , p < . ), where % of patients with a platelet count of more than /l reached mcf values within the reference interval. summary/conclusions: platelet function generally improves after transfusion and was in our patient population correlated to the absolute platelet count, but was also seen at the single platelet level in flow cytometry. a post transfusion platelet count of more than /l might be sufficient to significantly improve coagulation in heavily thrombocytopenic patients, but larger studies are needed to confirm this conclusion. abstract withdrawn. abstract withdrawn. background: sickle cell disease (scd) is a genetic disorder that is frequently referred to as a hypercoagulable state. hydroxyurea (hu) is known to decrease the frequency of vaso-occlusive complications and need for blood transfusions in severely affected individuals. although cross-sectional studies show that treatment with hu is associated with decreased coagulation activation, there are no prospective studies evaluating the effect of hu on coagulation activation. aims: to assess the effect of hu on markers of fibrinolysis (d-dimer) and endothelial activation (soluble vascular cell adhesion molecule- [soluble vcam- ]) in patients with scd in their non-crisis, "steady state." methods: patients, at least years of age, with documented hbss or hbsb-thalassemia, eligible for treatment with hu were studied in this prospective, observational study. laboratory investigations were obtained at baseline, prior to commencement of therapy with hu, with repeat evaluations at three and six months of therapy. non-parametric test was applied to observe the association between hu therapy and the biomarkers of interest. results: twenty-five patients with scd (hbss: , hbsb thalassemia: ) were enrolled (females: [ %]), with a median age of years (iqr: ). following months of hu, median values for wbc count ( . /l vs. . /l, p = . ) and d-dimer ( . ng/ml vs. . ng/ml, p = . ) were significantly lower than baseline values, while the mean corpuscular volume ( . fl vs. . fl, p = . ) was significantly higher than the baseline value. no significant differences from baseline were observed in the median values for hemoglobin ( . g/ dl vs. . g/dl, p = . ), platelet count ( /l vs. . /l, p = . ), lactate dehydrogenase ( u/l vs. . u/l, p = . ) or soluble vcam- ( . ng/ ml vs. . ng/ml, p = . ) following months of hu therapy. summary/conclusions: this exploratory study confirms that treatment with hu is associated with decreased coagulation activation in patients with scd, although no effect on endothelial activation was observed. by decreasing coagulation activation, hu may decrease the risk of thrombotic complications in scd. abstract withdrawn. abstract withdrawn. transfusion medicine, apollo gleneagles hospitals, kolkata, india background: reduction of immune responsiveness through blood transfusion has been documented by previous authors. breast cancer is considered as one of the commonest cancer globally and the second main cause of death in females transfusion of allogeneic blood in breast cancer surgery is variable and differences of transfusion incidence have been observed in the literature. where the maximum surgical blood ordering schedule (msbos) dictates cross matching and reservation of blood before surgery, factors deciding their utilization are varied and numerous. our hospital protocol guides that every patient planned for elective breast cancer surgery should routinely have a blood sample sent for reservation of one unit of compatible packed red blood cell (prbc) in the blood bank. aims: in this prospective study we aimed to audit the blood utilization in patients undergoing elective breast surgery and thereby optimize the blood ordering schedule, economic burden and loss of clinical resources. methods: the study included confirmed breast cancer patients planned for elective breast surgeries from january to december . patient and disease details like age, stage, tnm status, estrogen receptor (er) and progesterone receptor (pr) status, human epidermal growth factor receptor (her - ) expression, triple negative breast cancer (tnbc) status, reproductive and treatment status were documented. patients were divided into younger group [≤ years] and older group (> years). before surgery blood samples for compatibility testing were sent to blood bank for blood reservation. details of test, blood issue and blood transfusion were documented in the blood bank. approximate loss of time in minutes and wastage of resources in terms of money (inr) in the blood bank were noted. all results were calculated as mean ae sd and a 'p' value of < . was considered statistically significant. results: of the total patients most underwent wide local excision of the breast and modified radical mastectomy. a total of patients received units of blood and blood components in all categories of surgeries. only were younger women (≤ years) with mean age of years. non-transfused patients were significantly more than transfused ones (p < . ). frequency of blood transfusion was more in young patients ( . %). seven ( . %) of the total stage iv patients received blood transfusions. frequency of blood transfusion was more in patients undergoing surgery after chemotherapy ( . %). a significant loss of time and loss of revenue was observed. summary/conclusions: we conclude that routine compatibility test is not justified for all patients undergoing breast surgery. a more targeted approach is needed to reduce blood demand and associated cost to patient and blood transfusion services. background: blood transfusion guidelines are not only essential for the optimal use of blood products, but also help reduce transfusion-related adverse reactions and improve patient outcomes. the korean national transfusion guidelines were developed in and fully revised in by the korean centers for disease control and prevention and the korean society of blood transfusion. in our hospital, which is a -bed university hospital, a transfusion-indication data-entry program based on the national transfusion guidelines was developed in . it was applied to the electronic medical record system and all transfusion orders, except emergencies, have been performed through this program since then. aims: we planned to record and analyze the reasons for transfusion in order to monitor blood product usage and provide feedback to clinicians. furthermore, we intended to contribute to patient safety through the appropriate use of blood products. methods: we classified transfusion-indications by the blood product requested and created a pop-up window listing these indications, which would appear at each regular transfusion order. indications for transfusion with each blood product were as follows: red blood cells (rbcs)acute blood loss, chronic disease (sub-classified as hb ≤ g/dl, cardiovascular disease, cerebrovascular disease, peripheral vascular disease, respiratory disease, age ≥ years, age ≤ months, chemotherapy), surgery/ procedure, transplantation and 'other'; platelets (plts)present bleeding, bleeding prevention (sub-classified as hematologic disease, solid tumor, peripheral blood stem cell transplantation, disseminated intravascular coagulopathy, infant), surgery/procedure, massive transfusion and 'other'; fresh frozen plasma (ffp)bleeding in coagulopathy, bleeding prevention in coagulopathy, massive transfusion, plasma exchange and 'other'. transfusion indications entered into the data-entry program from sep to feb were analyzed. results: the number of transfusion-indications analyzed was for rbcs, for plts and for ffps. the most common indications for transfusion were chronic disease for rbcs ( / , . %), bleeding prevention for plts ( / , . %) and 'other' for ffp ( / , . %). 'hb ≤ g/dl' was the most frequent sub-indication of chronic disease ( / , . %), and hematologic disease was the most frequent sub-indication of bleeding prevention ( / , . %). many clinicians entered transfusion indication as 'other': rbcs ( / , . %), plts ( / , . %) and ffp ( / , . %). however, the free-text supplied by the clinician when 'other' was selected, often corresponded to an indication already categorized in the transfusion-indication data-entry program; . % of rbcs and % of plts. of the indications entered as 'other' in ffp, . % were surgery/procedure-related. summary/conclusions: in our hospital, the release of blood products has been dependent on the data-entry of transfusion indications (except in emergencies) since sep . transfusions of rbcs and plts were most common for chronic disease and bleeding prevention, respectively, but many cases entered as 'other' could have been categorized as existing indications in our data-entry program. therefore, we conclude that additional training is needed for clinicians regarding the determination of transfusion-indications and correct use of the transfusion-indication dataentry program, in order to use blood products more appropriately. methods: this was a prospective cohort designed study. subjects were children aged - years with indication of platelet transfusions in sardjito hospital yogyakarta indonesia. the patient samples were collected before and h post-transfusion, the expression of cd p on platelet was determined by flow cytometry method. results: there were subjects who were divided into two groups. fifty-one subjects received non-leukodepleted pcs and the other fifty-one transfused by pre-storage leukodepleted pcs. the mean of pre-transfusion platelet cd p for nonleukodepleted and leukodepleted groups were . % and . %, and the mean increase of post-transfusion platelet cd p for non-leukodepleted was . % and the mean decrease of leukodepleted groups was . %. it was shown the increase of post-transfusion platelet cd p for non-leukodepleted group, and it was significantly (p < . ) higher than in the leukodepleted groups. summary/conclusions: there was an increase of post-transfusion platelet cd p expression in patients received non-leukodepleted, but a decrease in leukodepleted pc transfusions. background: preoperative anaemia is a common finding in patients undergoing surgery and often neglected in our country. aims: the objective of this study was to evaluate hb(values and the identification of cardiac patients who entered operation with anaemia. and also to study the correlation between hb values and the number of rbc (red blood cell) transfused unit methods: this is a retrospective, descriptive and analytical study. the data for this study was collected from the files in the statistic's service at qsut (university hospital center "mother teresa"). the object of our study were the files of patients hospitalized in the period january -may in the cardiac surgery ward, which were subjected to cardiac surgery. from the files were collected data on age, gender, primary diagnosis, accompanying diseases. we also collected hb, rbc, htc (hematocrit), mcv (mean corpuscular volume), mch (mean corpuscular hemoglobin), mchc (mean corpuscular hemoglobin concentration). from the transfusion service at qsut and from the files were pulled out the transfused patients and the number of transfused units. results: based on the who definition for anemia (females < g/dl and males < g/dl), from the patients included in the study, ( %) were anaemic. from males in the study, ( %) of them were anaemic based on hb lab values, whereas from women in the study anaemic were found to be ( %) of them. from the anaemic patients in the study, ( . %) of them with mild anaemia, ( . %) with moderate anaemia and ( . %) with severe anaemia. in the total of anaemic female . % are under , while . % are over/or years old. in the total of anaemic males, % are under , while % are over/or years old. it is noticed that most of them are with normochromic normocytic . %, normocytic hypochromic anaemia . %, hyperchromic microcytic anaemia . %, macrocytic normochromic anaemia and macrocytic hypochromic anaemia respectively . % and microcytic normochromic anaemia . %. the average value of preoperative hb decreased from . g/dl before surgery to . g/dl after surgery, so there is a decrease of approximately . g/dl of hb value. in our patients, % ( ) were transfused and the remaining % ( ) were not transfused. from transfused patients ( %) patients were anaemic. the correlation between the values of hb, rbc, htc and the number of transfusions shows that with the decrease of these values the number of transfused units increases. summary/conclusions: the diagnose of anaemia is underestimated before surgical intervention in our country and investigation of hb low values do not take the proper importance to find probable cause and correct it before surgical intervention. the lower the hb values, the greater the chance to be transfused and the number of rbc transfused units. failure to correct hb values before surgery results in unnecessary transfusions for the patients or which could have been avoided, eliminating also the risk of transfusion complications. background: alloimmunization after red blood cell transfusion is affected by various factors. it is known that the incidence of alloimmunization increases in certain diseases. extended red blood cell matching can be used to prevent the development of alloimmunization in diseases which the rate of alloimmunization is increased. in asia, extended red blood cell matching is not actively implemented. aims: we tried to investigate whether there is a difference in the disease categories between unexpected red blood cell antibody positive and negative groups. methods: from january, to december, , the diseases of the patients who had undergone unexpected red blood cell antibody identification test at dong-a university hospital was examined through medical records. from january to december , the diagnosis was made on patients who had two or more unexpected antibody screening tests. we analyzed the frequency difference of disease category between two groups. results: a total of patients were performed with unexpected antibody identification tests. of patients who underwent more than screening tests, ( . %) were positive. were consistently unexpected antibody negative. the patients with solid tumors (n = , . %) and those with hematologic diseases (n = , . %) had a higher incidence in unexpected antibody positive group. the patients with myeloid malignancy had a significantly higher frequency than lymphoid malignancy (p = . ). the frequency of patients with liver cirrhosis was significantly higher in the unexpected antibody positive group ( / , . %) than in the negative group ( / , . %) (p = . ). the incidence of non-hodgkin lymphoma was significantly higher in the unexpected antibody negative group ( / , . %) than in the positive group ( / , . %) (p = . ). summary/conclusions: there was a difference in the distribution of diseases between unexpected antibody positive group and negative group. the patients with liver cirrhosis were more frequent in unexpected antibody positive group, suggesting that extended red blood cell matching would be considered. background: in hematological patients with multiple platelet transfusions (pc) often develop immune response to human leukocyte associated antigens (hla-i) and human platelet-specific associated antigens (hpa). besides, platelet associated immunoglobulins (paig) and complement components (pac) are found on platelet. this leads to increased platelet destruction and development of refractoriness to transfusions of donors' platelets. transfusion therapy using an individual selection of platelets and plasmapheresis, contribute, in the majority of cases, to the realization of efficient transfusion by pc. but, in difficult cases, there is a need to use intravenous immunoglobulin, which may promote the efficient transfusion of pc. aims: evaluate the algorithms of using the complex therapy of refractoriness to transfusions of donors' platelets with additional application of intravenous immunoglobulin (ivig). methods: in there were three female patients in the clinics of the centre for observation, age between and years (me = ) with the ineffectiveness of complex therapy for overcoming refractoriness to transfusions of donors' platelets due to selection and plasmapheresis. the diagnoses were as follows: aplastic anaemia (aa)- , acute myeloid leukemia (aml)- . individual selection of platelets was carried out by the adhesion method on the solid phase (immucor "galileo neo"). paig and pac / were evaluated by the method of flow cytometry (bd facscanto ii) by the method of double staining with cd a. the density of fixed paig, pac was © the authors vox sanguinis © international society of blood transfusion vox sanguinis ( ) (suppl. ), - evaluated by the median fluorescence intensity (mfi). the two patients with aa received ivig-igg therapy in the standard dose , g/kg per day, for days. one patient with aml received ivig-iggam therapy in the standard dose , ml/kg/day for . results: under pressure of the complex therapy with the use of ivig in the standard dose there was are decrease in mfi over time in the case of two patients: aa- mfi-paigg reduced from to ; while the patient with aml: paiga reduced from to , and paigm from to , pac from to , pac from to . the patient with aa- over time, regardless of the treatment, there was an increase of mfi, but the effect of pc transfusions was achieved under pressure of complex therapy. under pressure of complex therapy all the patients also reduced the frequency of reaction of alloantibodies when resorting to an individual selection and increasing the frequency of compatible couples "donor-recipient". summary/conclusions: delivery of complex therapy and the additional application of ivig enables an adequate transfusion therapy of pc, neutralize hemorrhagic syndrome and continue the treatment of the main disease. detection and monitoring of paig/pac during the development of refractoriness to transfusions of donors' platelets are additional markers for prescription of ivig therapy. anaesthesia, tan tong seng hospital, singapore, singapore background: blood transfusion is quite prevalent in paediatric cardiac surgical procedures. we hypothesized that the routine use of rotational thromboelastography (rotem) to guide transfusion decisions would reduce the overall proportion of patients receiving transfusions in paediatric cardiac surgery aims: the aim of the study was to find if the use of blood and blood products in pediatric cardiac surgical cases in a single centre is affected due to rotem. methods: sixty paediatric cardiac surgical patients undergoing cpb were included in this study. thirty patients (study group) were prospectively included and compared with thirty procedure and age-matched control patients (control group). in the study group, rotem, performed during cpb guided intraoperative transfusions. perioperative transfusions of blood and blood products, postoperative blood loss and hemoglobin levels were compared between the two groups. results: the patients in the control group received fewer transfusions of packed cells ( % vs %) and fresh frozen plasma ( % vs % p<o.oo ). more patients in the study group received platelets ( % vs %) and cryoprecipitate ( % vs %). the postoperative blood loss was lower in study group compared to the control group. the postoperative hemoglobin did not differ between the two groups. summary/conclusions: the results suggest that the routine use of rotem in paediatric cardiac surgery to guide transfusions is associated with reduced proportions of patients receiving transfusions and is evidence based practice. abstract withdrawn. abstract withdrawn. abstract withdrawn. background: mycoplasma pneumonia (mp) infection has often been implicated in respiratory tract infections. although this agent has also been associated with other non-respiratory diseases, hemolytic anemia is the most common hematologic complication of mp infection. episodes of sudden onset of severe anemia are rarely seen in beta thalassemia. we report a -years-old female with beta thalassemia major who was diagnosed to have severe anemia secondary to autoimmune hemolytic anemia induced from mp infection aims: to report a case of patient with beta thalassemia major who was diagnosed to have severe anemia secondary to autoimmune hemolytic anemia induced from mp infection and how it was managed with minimal transfusion. methods: a -year-old female case of beta thalassemia major who developed autoimmune hemolytic anemia following mycoplasma infection. the patient is on monthly blood transfusion since the age of years at dubai thalassemia center. she was admitted to the hospital with days history of dizziness, severe low back and joint pain and her travel history indicated a four-week visit to pakistan. initial investigations showed a hemoglobin level of . g/dl the direct and indirect coombs test were positive. patient was transfused with two units leuko-depleted compatible prbc (o, rh +, negative for e and kell antigens.) and discharged next day. three weeks later, the patient was still with lower limbs pain and the hemoglobin was . g/dl with a positive dat with both igg+ and c + , patient was not transfused as it was difficult to find matching blood; therefore she was given one week follow up. four days later, patient reported to the center with fever, generalized body pain and fatigue. she had significant drop in hemoglobin . g/dl, mcv . , platelets count , crp . , retic count . , blood culture no growth, total bilirubin . , malaria parasite smear was negative, urine culture and microscopy were normal. mycoplasma pneumonia antibody showed positive with igg . and igm . , she was started on azithromycin for days and received units prbc least incompatible leuko-depleted (o, rh +, negative for e and kell antigens.). after which her hemoglobin increased to . g/dl. she did not show any improvement in her condition over the following weeks and repeat mycoplasma pneumonia antibody showed an increased titer, which denoted resistance to azithromycin; hence, we started her with second line of treatment levofloxacin for days. screening for collagen disease were all negative apart from high esr of mm/hr. patient remained afebrile and transfused with least incompatible available blood, post transfusion hb was . g/dl. three weeks later, during her routine follow up, she was doing well with esr dropping to mm/hr and hemoglobin . g/dl, which indicated gradual settling of hemolysis, although the last dat remained positive. subsequent visits showed hb drop of . g/dl/week which is acceptable for transfusion dependant thalassemia. results: with conservative management patient condition improved. summary/conclusions: hemolytic anemia associated with mycoplasma pneumonia is usually self-limited and resolves spontaneously after mycoplasma infection elimination. packed red cell transfusion in transfusion dependent patient may be troublesome and can aggravate the condition; therefore it should be utilized and limited to significant symptomatic anaemia background: early plasma transfusion in women with severe postpartum hemorrhage is often instituted to prevent and treat coagulopathy, but whether maternal outcomes improve with this treatment is unclear. aims: to quantify the association of plasma transfusion within the first min after diagnosing persistent postpartum hemorrhage and adverse maternal outcome. methods: we performed a retrospective cohort study of women with persistent postpartum hemorrhage, defined as postpartum hemorrhage refractory to first-line measures to control bleeding. women were enrolled from january , to january , in dutch hospitals. in a time-dependent propensity score-matched analysis, women with transfusion of fresh frozen plasma within min following the diagnosis of persistent postpartum hemorrhage were matched to women without plasma transfusion or plasma transfusion at a later time point during hemorrhage. plasma transfusion was not guided by coagulation tests. the main outcome measure was adverse maternal outcome, defined as a composite of mortality, hysterectomy and arterial embolization to control bleeding. we performed sensitivity analyses with initiation of plasma transfusion within and within min following diagnosis persistent postpartum hemorrhage, compared with other plasma transfusion strategies. results results: the cohort consisted of women with persistent postpartum hemorrhage, with uterine atony as cause of hemorrhage in women ( . %). in this cohort, seven maternal deaths ( . %), hysterectomies ( . %) and arterial embolizations ( . %) were observed. plasma transfusion within min in women was matched on time-dependent propensity scores with women with no or later plasma transfusion. rate of adverse maternal outcome was similar between women with and without early plasma transfusion, . % versus . %, adjusted odds ratio . ( % confidence interval . - . ). results of sensitivity analyses were comparable to the primary results. summary/conclusions: among women with persistent postpartum hemorrhage, initiation of plasma transfusion within min following this diagnosis was not associated with improved maternal outcomes, compared with no or later plasma transfusion. whether other transfusion strategies or identification of women with high risk of coagulopathy will improve maternal outcomes remains to be studied. background: preoperative evaluation of increased risk for perioperative bleeding is essential for cardiac surgery patients. platelet aggregation, as a laboratory test for assessment of platelet function, is very efficient for optimal antiplatelet treatment and also for assessment of the increased risk of postoperative bleeding and perioperative application of allogenic red blood cell transfusions. aims: the aim of this study was to establish whether preoperative platelet function testing for the presence of residual effect of acetylsalicylic acid (asa) on platelet aggregation can be correlated with an increased risk for intraoperative bleeding during cardiac interventions. methods: a retrospective study included patients who underwent surgical procedures (cabg ii and cabg iii) at the department of cardiovascular surgery, clinical center in ni s in period november -july . patients were previously taken asa at a dose of mg per day, but therapy was discontinued days before surgery. platelet aggregation was measured on the day of surgery using impedance aggregometer multiplate (multiplate platelet function analyzer, roche), using trap and aspi tests. patients were divided into two groups: control group (cg), which included patients with preserved function of platelets (aspi - au*min), and examined group (eg), which included patients with inhibited platelet aggregation in aspi test (< au* min background: hemophilia b is an x-chromosome-linked inherited bleeding disorder resulting from reduced levels or an absence of factor ix clinically represented by recurrent prolonged bleeding. mutations can be inherited or acquired de novo. de novo mutations arise in about one-third of patients with haemophilia. the disease affects primarily males, but approximately % of carrier females have factor ix clotting activity lower than % and are at risk for bleeding. aims: case report of a hemophilia b carrier. methods: the clinical history of the patient was thoroughly studied by analyzing medical records and analytical results using the software available in the health institution and paper records. results: year old woman, with no history of abnormal haemorrhage, healthy, up to when, after a dystocic delivery, presents with subcutaneous and ocular haemorrhage and dispersed bruises. a basic coagulation study is performed and a level of % fix is found. genetic analysis reveals a mutation in fix gene and she is diagnosed as a carrier of hemophilia b. except for a son, no other direct family members carry the mutation. in , after total hysterectomy with bilateral adnexectomy due to an invasive carcinoma in the uterine cervix, the patient suffers a haemorrhagic shock with multiple transfusion support and around six months later undergoes an ureteronephrectomy also with the need for post-surgery transfusion support. the patient is referred to the immunohemotherapy department where she has regular appointments as an outpatient since . in , she requires surgery for the removal of a thyroglossal duct cyst, which is performed under prophylactic treatment with iu recombinant fix (rfix) daily, for four days, and up to h after discharge maintaining basal levels of fix of % and with no major complications reported. in , the patient is admitted to the er in haemodynamic shock and a history of black stools and syncope. at admittance, she receives a bolus of iu of rfix and requires severe transfusion support due to active bleeding. she is diagnosed with colonic angiodysplasia and undergoes surgery. daily doses of iu rfix are administered from day (d ) and up to d maintaining fix levels around - %. the patient is operated at d under the cover of iu of rfix, twice per day, and up to d after surgery reaching fix levels of around - %. at d the dose was diminished to iu of rfix daily and at d she is discharged with no rfix replacement therapy necessary in ambulatory. since then, no complications or new occurrences have been reported. the patient is under prophylactic administration of rfix prior to minor invasive procedures (nodule biopsy, dental extractions and colonoscopies). summary/conclusions: hemophilia b carriers usually do not require any major health care for their daily routine or invasive procedures. however, in the case reported, basal fix levels conditioned any minor intervention to be performed. thus, this carrier must be considered as a moderate hemophilia b patient and mandatory prophylaxis with rfix must be performed prior to any invasive procedure. background: blood transfusion is a life saving procedure but transfusion associated complications are equally important that need to be addressed. with limited resources of blood donations and screening, excessive transfusions must be avoided to prevent risk and economic burden on patients. on the other hand discussion on appropriate use of blood product is a matter of debate in medical literature. it is recommended to restrict the liberal use of transfusion. aims: in this regard we aimed to observe the frequency of genuine and non genuine transfusion at our hospital and also to compare the outcomes of liberal and restrictive transfusions. methods: it was a cross sectional study carried out at nibd and bmt, pechs campus, karachi, pakistan, from february to january . the study was approved from institutional review board. pack red cells (prc) transfusion in case of < g/dl hemoglobin (hb) with cardiac history and < g/dl hb with symptomatic anemia was considered genuine. platelets transfusion in case of sepsis and count < /l, sepsis with bleeding and < /l, patients on active chemotherapy without bleeding but platelet count < /l and in patients presented with bleeding was considered genuine. patients who were presented with bleeding, deranged coagulation profile and with sepsis + bleeding considered genuine for fresh frozen plasma (ffp) transfusion. spss version . was used for descriptive and inferential statistics. results: a total of transfusions were evaluated for genuine and non genuine transfusion. male predominance was high in our data ( %). out of , ( %), ( %) and ( %) prcs, platelets and ffp were transfused respectively. out of platelets, ( %) were platelet apheresis. / ( %) prcs and / ( %) platelets were non genuinely transfused as per the criteria described in methods. however, ffp and platelet apheresis were genuinely transfused. in non genuine group of platelet transfusion, all patients received more than unit platelet and the increment of platelet count post transfusion was found statistically non significant (p = . ). thirty six ( %) of transfusions were done with more than units of prcs in non genuine group. the mean hb level of patients received unit and more than units prcs was statistically same (p = . ) and it was found out that there was same increment in hb in both the groups post transfusion. (p = . ). summary/conclusions: we concluded that although blood transfusion is an integral procedure for saving life yet correct use of blood products is necessary in the era where blood donations are scarce and blood transfusion has its potential associated hazards. the approach towards the appropriate use of blood products will not only minimize the risk of transmission of infectious diseases but also will reduce the economic burden. longitudinal studies on large sample size are needed to validate this study. uncertain. an ovine model enables comparisons of; (a) invasive and non-invasive methods to measure oxygen delivery and utilisation at a tissue level, and of (b) the efficacy of various resuscitation fluids and transfusion protocols in treating haemorrhagic shock. aims: to develop an ovine model of haemorrhagic shock. methods: sheep (n = ) were anaesthetised, ventilated and instrumented. instrumentation included blood flow, perfusion, and microdialysis probes placed in the brain, kidney, liver and skeletal muscle. haemodynamic, tissue oxygenation, tissue flow and respiratory data were recorded continuously by data capture software. non-invasive assessments, including brain and muscle oxygen saturation by nirs, and visualisation of sub-lingual microvascular perfusion, as well as arterial blood gas measurements and plasma/serum/urine collections were made at specific timepoints. after a baseline period ( h) sheep were bled~ % of their estimated blood volume (estimated at ml/kg) to a mean arterial blood pressure (map) of - mmhg. sheep were then resuscitated with either plasmalyte â (up to ml/kg/ hr; n = ) or transfused with sheep red cells (n = ) to achieve a map > mmhg. sheep were euthanised h after resuscitation. data are presented as mean ae standard deviation. results: sheep were haemorrhaged an average of . ae . ml blood which combined with iatrogenic blood loss (~ ml) corresponded to an average . ae . % blood loss. two out of the four sheep met clinical criteria for haemorrhagic shock (map = - mmhg, lactate > mm, svo < %). across all four sheep the nadir map averaged . ae . mmhg, lactate peaked at . ae mmol/ l, and nadir svo was . ae . %. all sheep survived to the end of the experimental protocol. summary/conclusions: these data demonstrate the successful induction of haemorrhagic shock in an ovine model. further experiments are planned to improve the protocol and to achieve % incidence of haemorrhagic shock, and then to compare invasive and non-invasive measures of oxygen delivery and utilisation as well as the efficacy of different resuscitation fluids and red cell transfusion. adverse events, including trali p- bilirubin were recorded within the -day period. the clinical parameters were compared against the reaction strength of the antibody reactions. the automated strength was measured by solid phase. the manual testing consisted of a -min incubation using liss and adding monospecific igg. the dat was performed manually by adding poly-specific igg and then testing with monospecific igg and c d. the rh group and non-rh group had and cases performed manually, and results were + or weaker further indicating the manual strength did not correlate with the clinical hemolysis. likewise, in / ( %) the dat was negative, and did not show any correlation with clinical hemolysis. however, when ldh and bilirubin were measured, the two parameters increased as the automated strength of the antibodies increased. summary/conclusions: most of the dshtr investigation was not associated with overt accelerated red cell destruction. a strong correlation was observed only between the automated immunohematology testing results and other laboratory markers of hemolysis. in our experience, the direct antiglobulin test and manual strength showed no correlation. background: numerous transfused patients present severe, sometimes critical clinical conditions. the occurrence of adverse transfusion reactions (atr) may induce deterioration in the clinical condition with a worsened clinical course and a lifethreatening or fatal outcome as is the case with nervous system impairment. in france, in , out of , notified atrs, ( . %) and ( . %) were life-threatening and death respectively. aims: our aim was to evaluate the notified atrs with neurological signs that occurred in transfused patients over a period of six years and six months in hospitals in the auvergne rhône alpes area. the study included patients with reported atrs in hospitals in this area from january st to june th . each atr was registered in the national haemovigilance database system. two signs observed at the time of the atr were analyzed: unconsciousness and convulsions. stroke was excluded. the type of atr, its severity, the blood product involved and its imputability were studied. results: during the period under study, , atr were reported, of which included unconsciousness and/or convulsions ( . %). of these patients, were females ( . %) and males ( . %). unconsciousness alone was frequently observed ( reports, . %). convulsions were notified in reports ( . %) and were associated with unconsciousness in of them. the diagnosis of seizure, with no other clinical signs, was established in cases ( . %). unconsciousness and/or convulsions were present in allergic reactions ( . %), cases of transfusion-associated circulating overload ( . %), cases of suspected transfusion-transmitted bacterial infections and hypertensive reactions. in allergic atrs, unconsciousness was notified in cases and unconsciousness associated with convulsions in one. twelve atrs were severe ( . %), were life-threatening ( . %) and in cases, they resulted in the death of the recipient ( . %). of the allergic atrs, were severe and life-threatening. red blood cell concentrate was involved in atrs ( . %) and platelet concentrate in ( . %), including cases with apheresis platelet concentrate and cases with pooled platelet concentrate. fresh frozen plasma was involved in atrs ( . %). nevertheless, the imputability of the blood product was excluded or unlikely in atrs ( . %). in the suspected transfusion-transmitted bacterial infections, the imputability of the transfusion was ultimately excluded after a negative result was obtained in the bacterial culture of the blood product. the imputability of the blood product was probable or possible in and atrs respectively, but was certain in only atrs. summary/conclusions: unconsciousness and/or convulsions were rarely observed in atrs notified in transfused patients. nevertheless, the presence of these signs highlights the seriousness of the atr ( ars, . %). lastly, the imputability of the blood product was often excluded or unlikely. in the multivariate cox model for the effect of lpi on overall survival, adjusted for age and ipss-r category, elevated lpi levels were associated with inferior overall survival (hr . , % ci . - . , p = . ). this effect was most pronounced in the td-rs subgroup (hr . , % ci . - . , p < . ). similarly, elevated lpi levels were associated with inferior pfs (hr . , % ci . - . , p < . ) for the whole study population and the td-rs subgroup (hr . , % ci . - . , p < . ). in total patients received iron chelation during the sample collection period ( patients deferasirox, patients desferrioxamine). lpi levels were normal in out of the samples collected during deferasirox treatment and in out of samples collected during desferrioxamine treatment. summary/conclusions: transfusion dependency is associated with the presence of toxic iron species and inferior overall and progression-free survival in lower-risk mds patients. in td-rs patients the effects were most pronounced indicating ineffective erythropoiesis leading to additional iron toxicity. background: post-transfusion immunomodulation has been reported to contribute to poor patient outcomes. clinically relevant transfusion models are needed to improve our understanding of underlying mechanisms. sheep transfusion models are of increasing importance in blood transfusion research as they provide several advantages over small animals, including their size, anatomy, physiology and similar blood volume compared to human. a current limitation of sheep transfusion models is the lack of characterisation of the sheep immune system. understanding the sheep immunology is necessary to advance sheep transfusion models, identify mechanisms that contribute to post-transfusion immunomodulation and facilitate the translation of findings into clinical settings. aims: to characterise the sheep leukocyte inflammatory responses to in vitro lipopolysaccharide (lps) challenge in edta and heparinized whole blood. methods: edta and heparinized sheep whole blood (n = of each) was cultured with rpmi media ( °c, % co ) alone or with the addition of lps ( - lg/ml; derived from escherichia coli : b ). the inflammatory response was assessed after h (h), h, h, h, h and h. supernatant was harvested at each time point and stored at À °c. inflammatory cytokine/chemokine production was determined using sheep specific in-house elisa (il- b, il- , il- and il- ). twoway analysis of variance with bonferroni's post-test was used to measure the effect of incubation time and concentration compared to no lps matched samples. results: when edta was used as an anticoagulant, addition of lps resulted in production of sheep il- b and il- but not il- or il- . il- b production was significantly increased following stimulation of lg/ml lps for h (p = . ) and declined following h incubation. release of il- was significant h post-lps stimulation with lg/ml (p = . ) and reached a maximum at h. the use of heparinized blood resulted in a different immune profile as all inflammatory markers tested were detected following stimulation with much lower concentrations of lps ( lg/ml), although the incubation times differed. il- b was significantly increased following h incubation (p = . ), with increasing levels observed up to h post-lps stimulation. il- production was evident from h and reached significance at h post-lps stimulation (p = . ). il- was significantly increased following stimulation of lg/ml lps for hr (p = . ) with lower concentrations of lps resulting in il- production at h (p = . ). release of il- was significant after h of lg/ml lps stimulation (p = . ), with lower concentrations of lps resulting in il- production at h (p = . ). in heparinized whole blood an lps concentration-dependent effect was evident for all cytokines. summary/conclusions: using a time-and concentration-approach our findings indicate that sheep are more tolerant and have a delayed response to lps stimulation compared to previous research using similar human in vitro whole blood culture models. in addition, data suggest that sheep have greater immune responses using heparin as anticoagulant for the collection of blood samples. improving our understanding of sheep immunology and development of relevant sheep transfusion models will provide a bridge between sheep models of transfusion and clinical settings. . rhdig inappropriately administered (unnecessary exposure) (n = , %) administered to: -rhd positive woman (n = ) -rhd negative mother with rhd negative neonate (n = ) -woman with immune anti d (n = ) -administered in error (instead of other ig) (n = ) rhdig delayed/omitted/wrong dose (risk of sensitisation to the d antigen) (n = , %) -omitted (n = ) -delayed (n = ) -inadequate dose (n = ) administration without correct patient identification (n = , %) storage & handling (n = , %) failure to check the maternal and neonatal blood groups prior to administration was identified as a source of error. misinterpretation of blood results also led to women receiving product inappropriately. e.g. reading a negative antibody screen as the mother being rhd negative. patient identification was raised as an issue. rhdig is often stored in satellite blood fridges for easy access. collection from these areas did not always require confirmation against patient identifiers and there was no register of women who received product or link to the batch number to ensure traceability. two incidents involved the administration of rhdig when the prescription for other immunoglobulin products was not clear, leading to a child and a baby receiving rhdig instead of the intended immunoglobulin. summary/conclusions: these incidents indicate problems with the processes of appropriate identification of women who need rhdig, the use and interpretation of pathology tests and requirements for prescription and administration. these resulted in omitted and inappropriate doses of rhdig. blood matters has made a number of recommendations regarding rhdig administration: -all health professionals involved in rhdig administration should be appropriately trained in the use of rhdig -confirmation of the maternal rhd status is essential prior to prescription or administration -positive patient identification must be used prior to administration of rhdig -health services should consider regular auditing to identify areas for improvement relating to rhdig blood matters continues to work with maternity care providers to improve practice. centro comunitario de sangre y tejidos de asturias, oviedo agencia gallega de sangre, organos y tejidos, galicia banco de sangre y tejidos de cantabria, cantabria banco de sangre de la rioja, la rioja banco de sangre y tejidos de navarra, navarra banco de sangre y tejidos de arag on, aragon fundaci on de hemoterapia y hemodonaci on de castilla y le on, castilla y leon fundaci on banco de sangre y tejidos de las islas baleares, islas baleares, spain terumo bct europe nv, zaventem, belgium background: hemovigilance, a long-term monitoring process made mandatory by national and supranational regulations, begins with a systematic whole blood or blood component collection and ends with an examining period after transfusion of blood components into the patients. in spain, organized in autonomous regions, the hemovigilance system is structured in three levels: ( ) the local level comprised of transfusion centers and hospital based transfusion services that monitor and collect all transfusion related adverse events (ae) and level them up to ( ) the regional hemovigilance coordinator, who communicates all the region's data to the ( ) spanish ministry of health which issues an annual report and corresponds with european institutions. to ensure safer blood supply, pathogen reduction technology (prt) was approved and implementation started in spain in . the mirasol prt system for platelets and plasma was introduced in and is currently being used in of the spanish regions. aims: to monitor the safety of the system, a passive hemovigilance study on mirasol treated products was initiated in the region of asturias and collaboration was extended to other regions (baleares, galicia, la rioja, cantabria, navarra, castilla y leon and aragon). methods: collected data included allergic and febrile reactions, trali and all other adverse event observed. severity of the event and level on imputability of the transfusion were also assessed using the who grading scale. hemovigilance data of mirasol treated products (platelets or m-pc and plasma or m-p) are included from to as blood centers started to apply the technology in routine. results: increase adoption of the mirasol system is observed between , when , mirasol treated blood products were issued to hospitals and with , mirasol products issued. due to low number of transfusions of mirasol-treated blood components in and , notification rates began to be analyzed in , showing ae rates of . %, similar to reports at the national level. stable transfusion reaction rates were observed with m-pc (around . ). rate of ae after transfusion of m-p is fluctuant between . and . . this fluctuation could be due to the inconsistent numbers of m-p transfused from one year to the other. most of transfusion reactions (around %) were of grade i severity and grade ii level of imputability. allergic reactions accounted for most of the adverse events, with g&i > reactions in and of respectively . and . no bacterial nor viral transfusion transmission was recorded on mirasol products during the study period ( ) ( ) ( ) ( ) ( ) ( ) . at the national level, nine cases of bacterial transfusion transmission (with g&i > ) were reported. these transmissions were probably due to transfusion of non-pathogen reduced products. summary/conclusions: the observed notification rate of ae is similar to the national rate but allergic reactions with g&i > is inferior with mirasol treated products. also, we found no reports of transfusion transmission infections nor cases of transfusion associated graft-vs-host disease, demonstrating safety of mirasol treated products. were attributed to human error ( %) with the lowest frequency in equipment failure ( %), compared to % and %, respectively, in the following three years. root cause analysis demonstrated failures in the quality management system including failures in administration, inadequate staffing for blood collection as well as in distribution and processing, and failures arising from institutional constraints and system failures in hospital management. high numbers of "other" aes ( %) in distribution and whole blood collection call for further investigation to indicate measures necessary for prevention and correction. errors related to incorrect blood component transfused (ibct) in - were in , , blood units ( / , ) issued for transfusion. these resulted in serious reactions ( %) ( fatal, life-threatening) . another ( %) were related with ibct that did not cause a reaction. near misses (component not transfused) were ( %) summary/conclusions: our data demonstrate increasing compliance with reporting requirements. questions about the initial factors for deviations in certain activities specifying failures in equipment and materials due to system as well as human errors, highlight the need for further specifications beyond "other" and "human error". background: the weakest link in the transfusion chain currently is the handling of blood components after their issue and the bedside blood administration practices. aims: to evaluate compliance with standard procedures for bedside blood transfusion practices by analysis of the "transfusion feedback forms" in a tertiary care multi-specialty hospital setting. methods: during the study period of months, the transfusion feedback forms received from various clinical areas of the hospital were studied with special reference to the transfusion times. the data was categorized based on the patient's location as well as the time of transfusion, whether done in routine or emergency hours. results: , blood components were issued during the study period, while transfusion feedback forms for , components ( . %) were received in the transfusion medicine department. delay in starting the transfusion (more than min after issue) was observed in transfusion events ( . %). the component transfusion time exceeded the permissible limits for component ( . %).the overall total permissible time for completion of components exceeded permissible limit in ( . %) of transfusion events. the pediatric ward ( . %), icu and ot complex ( . %) were found to be the most non-compliant delay in transfusion, transfusion time and total transfusion time. amongst the delayed transfusions after issue, ( . %) were during the routine hours i.e. between am to pm and ( . %) were in the non routine hours i.e. between pm to am. summary/conclusions: the audit of bedside blood transfusion practices has given us a good insight into various areas of noncompliances as well as the predominant locations in the hospital where the practices need to strengthened further. focused training program on safe blood administration practices for all staff involved in handling and transfusion of blood components is now planned to combat this issue. background: the international surveillance of transfusion adverse reactions (ars) and events (aes) (istare) of the international haemovigilance network (ihn) collects aggregate data from member national haemovigilance systems (hvs) in order to estimate the morbidity and mortality of blood transfusion in a holistic approach. the ultimate goal is to contribute to improving the safety of transfusion by close monitoring throughout the chain "from vein to vein". aims: we analyse recent istare data on suspected transfusion transmitted infections (sttis) for - in comparison to previous years of surveillance, [ ] [ ] [ ] [ ] [ ] [ ] [ ] methods: annual aggregate data from ihn member hvs on transfusion associated bacterial, viral and parasitic infections collected online in istare are analyzed by incidence in blood components (bcs) issued for transfusion, by severity and imputability as well as by blood component. ars with definite, probable or possible imputability were included in the analysis. trend analysis is performed to allow comparisons and to collect information on established and newly emerging infectious threats of blood transfusion. results: for - sets of annual aggregated data from countries covering , , bcs issued were analyzed. all ars totalled , and infectious ars amounted to ( . %). the overall incidence of the infectious ars was . / , units of bcs issued. bacterial infections were the most frequent ( , %), next viral ( , . %) and then parasitic ( , . %). serious were % and there were fatalities ( . %, incidence . / , ). nine deaths were attributed to sepsis and the other two were associated with non-malarial parasitic pathogens. one geotrichum clavatum fungal infection associated with apheresis platelets was reported as a free text comment. this very rarely recognized fungal pathogen caused a very severe infection in a patient but the route of transmission is inconclusive. the viral sttis included hbv ( %), hcv ( %) and hiv ( . %). the recorded as "other" ( . %) including cases of hev, one case of parvovirus b , one cmv and one ebv. no case of tt-malaria was reported. other stt-pi were (two fatal). the prevailing bcs were in general rbcs followed by platelets. comparison with corresponding data for - shows a consistent overall incidence in total sttis ( . vs . / , ). however, considerable differences were seen in separate categories, such as bacterial infections (significantly increased rate in - , p < . ) and an almost doubled rate of parasitic infections (p < . ). compared to the earlier period, there were many fewer hbv infections ( vs ) and many more hev. a similar reduction in the rate of hcv and hiv was observed in - in comparison with previous years. this may be explained by the fact that nat testing for hcv/hiv/hbv has been implemented in many countries in the last decade. summary/conclusions: the infectious risk of transfusion overall remains very low. the rate of bacterial cases has increased and among other viral sttis the frequency of hev is increasing. the mortality of transfusion due to sttis is lower than in the previous period of surveillance. abstract withdrawn. background: one of the main aspects of haemovigilance system in hospitals is following of adverse events and reactions related to blood transfusions. aims: it was intended to analyse the adverse reactions related to transfusion of blood components in pediatric patients. methods: over a four year period (january -december ), the haemovigilance records of all patients receiving blood transfusions procedures were reviewed and transfusion reactions were analysed. statistical analysis of data was performed by spss software (version . , spss inc., chicago, il, usa). majority of blood components were provided by regional blood center organized by national red crescent society. but granulocytes collected by apheresis after donor mobilization and reconstituted whole blood for exchange transfusions were prepared in the transfusion center of the hospital. results: the median age of patients who developed transfusion reactions was months (interquantile range-iqr ). the median for the numbers of individual transfusions in children in a year was (iqr ). the median for the numbers of blood components individually transfused to patients was (iqr ). patients, anaphylactic transfusion reactions in patients and transfusion-related lung injury (trali) in a patient. the overall incidence of transfusion reactions was estimated at a rate of . per units. summary/conclusions: it was reported that adverse effects related to blood transfusion, especially allergic reactions and fnhtrs are common in pediatric patients than adults. in a multinational study concerned about the transfusion reactions related with red cell concentrates, allergic transfusion reactions and fnhtrs were reported at a rate of and in units and in units, respectively. while the incidence of transfusion reactions in children was found . % in a study from the u.s.a., the overall incidence of transfusion reactions in our study which was estimated at a rate of . per units represents a lower rate. hospital gran canaria dr. negr ın, gran canaria hospital general universitario, ciudad real, spain hospital nostra senior de meritxell, andorra, andorra banco sangre y tejidos, santander banc de sang i teixits, barcelona, spain fundaci on hematol ogica colombia, bogot a, colombia centro regional de transfusi on de almer ıa, almeria complejo hospitalario de navarra, pamplona fundaci on banc de sang i teixits illes balears, palma de mallorca hospital de cabueñes, gij on, spain background: root analysis cause is defined as the cause of an error that, if it is treated, eliminates the repetition of the error aims: describe types of human and latent errors detected by a work group in the root analysis cause of transfusion incidents, analyze the concordance between the individual responses of the members and propose recommendations in order to improve transfusional safety. methods: in fifteen participants (nurses and hematologists dedicated to transfusion and component preparation) studied some incidents of administration of nonirradiated components and tried to approach the root causes by applying the classification of errors in mers-tm transfusion medicine. they transferred the answers to a questionnaire (simple or chain error, initial process affected, human and latent errors and measures derived from the analysis to correct the errors). the communication was made by mail and by the spanish transfusion society web forum, which contained the consultation documents. data and percentages are exposed for each type of error and the answers of the participants are tabulated. results: cases corresponded to patients. two patients of years of age diagnosed of acute myeloblastic leukemia (case and ) and chronic lymphatic leukemia (case ). in one case, the hematologist of the transfusion service canceled an irradiation prescription; in another, a patient with fever was transfused in the emergency room without the irradiation requirement and it was later discovered that he had received a transplant of hemopoietic progenitors month earlier; in the last case, neither the requesting doctor nor the laboratory technician nor the following doctor (prescriber) detected the alerts located in their respective computer applications. in all cases, the story was judged as sufficient for analysis. the majority of reviewers ( %) diagnosed a chain of errors. there was agreement of % with respect to the initial process affected. the initial error was communication ( %), monitoring ( %) and compliance ( %), in cases , , and . - human errors were detected per case (average: . , . and . errors respectively) and - latent errors per case (average: . , . and . , respectively). the latent errors most punctuated were: failures in the quality of the protocols ( %), in the transfer of important knowledge ( %), in the available technology ( %) and in the information to the patient ( %). all the participants contributed feasible measures of improvement according to root causes: ) improve the quality and drafting of work procedures and their compliance, including procedures of effective communication between professionals, ) train staff in knowledge important for safety, ) communicate with computer application providers to improve the effectiveness and visibility of the alerts and ) involve the patient with essential information to ensure transfusion safety. the measures were processed later as recommendations. summary/conclusions: the root analysis shows agreement between participants and allows the elaboration of useful recommendations to increase patient safety. this strategy can contribute to the comprehensive prevention of errors. background: in transfusion-associated circulatory overload (taco), pulmonary oedema develops primarily due to volume excess. data from the uk haemovigilance scheme, serious hazards of transfusion (shot) suggest that either the incidence of taco, or the recognition and reporting of taco, has increased over time. from to , reports of taco increased from to ; deaths from to , major morbidity from to . known risk factors include pre-existing cardiac and/or renal dysfunction, low body weight, extremes of age (eg, < years, > years), concomitant fluid administration, positive fluid balance, peripheral oedema and hypoalbuminemia. in a small subset of cases reported to shot, taco developed following transfusion for severe anaemia in the absence of other risk factors. this may be an under-recognised independent risk-factor. aims: to raise awareness of severe anaemia as an under-recognised risk factor for taco and is potentially life-threatening transfusion. methods: cases of taco submitted to shot over the last years were reviewed to identify cases where transfusion for severe anaemia was a key identifiable patient risk factor. results: the following are illustrative cases: -case : a patient in their s weighing kg was prescribed six units of red cells for iron deficiency anaemia after being admitted with hb g/l. the patient had no risk factors for taco except for profound anaemia. during transfusion of the fifth unit the patient became dyspnoeic, hypoxic and hypertensive. the patient recovered after diuretic therapy and had a post-transfusion hb level of g/l. -case : a patient in their s presented with a -week history of weakness and dizziness and had felt unwell for months. the hb was g/l, ferritin lg/l and ecg showed cardiac ischaemia. two units of red cells were transfused. after the second unit oxygen saturations fell despite supplemental oxygen, post-transfusion hb of g/l. a third unit was transfused over min and the hypoxia worsened with dyspnoea and crackles on chest auscultation. the chest x-ray showed an enlarged cardiac silhouette and pulmonary congestion. the patient improved with diuretics. -case : a patient in their s with severe megaloblastic anaemia, hb g/l and peripheral oedema developed taco after transfusion of units and recovered with diuretic therapy. summary/conclusions: chronic and acute anaemia are associated with compensatory cardiac changes irrespective of the aetiology of anaemia. this is further compounded by the underlying cause of anaemia particularly haematinic deficiencies (iron/b deficiency) that independently affect myocardial function. hyperdynamic circulation related to anaemia increases the load on the heart, causing myocardial ischaemia and hypoxia and if the anaemia is not corrected, eventually leads to heart failure. clinicians need to make an accurate diagnosis and avoid excessive transfusion in patients with severe anaemia with or without other additional risk factors. patients with chronic iron/folate/b deficiency without haemodynamic instability should be given the appropriate haematinic replacement. haematinic deficiency responds rapidly to appropriate vitamin/mineral. blood transfusions are to be given only when clinically indicated and even then, only the minimum volume needed for symptomatic relief transfused with consideration for diuretic therapy. methods: legal forms for reporting transfusion reactions were used in the retrospective analysis, which were adjusted by the department of quality assurance and quality control in the electronic form and distributed to clinics using blood components. clinicians were trained to report transfusion reactions through the hospital's transfusion board and through the "service for improvement of the quality and safety of health services" at the clinical center of sarajevo. analysis of the reported reactions in the institute include immunohematological and microbiological examination based on which the guidelines for further treatment with blood components are being made. users of registered services are obliged to report since . results: total of , different blood components were applied in the period between - . department for quality assurance and control has received serious adverse reactions, serious adverse event, reports of transfusion reactions, of which ( %) were inadequately filled, in the same period. from the above, ( . %) were transfusion reactions to erythrocyte blood components which were applied, ( . %) to platelet components and ( . %) were transfusion reactions after the application of fresh frozen plasma. the analysis has shown that the most frequent were febrile non-haemolysis reactions ( or . %), followed by allergic reactions ( or . %). two transfusion reactions ( . %) were characterized as circulation overload. summary/conclusions: the frequency of serious adverse reactions and events was . % ( of , ) and . % were reported transfusion reactions ( of , ). with the establishment of the hospital transfusion board and with the increase of collaboration with the clinical center, significant progress has been made. it is necessary to increase awareness among clinicians in regards to the safe transfusion practice. reporting transfusion reactions should be a mandatory procedure, a path to the proper selection of blood components, monitoring adverse reactions, and for us, transfusiologist, guide to the safest, most efficient blood components. j garc ıa-gala, e martinez-revuelta, a caro-g omez, c castañ on-fern andez and i fern andez-rodriguez hospital universitario central de asturias, oviedo, spain background: elderly patients are the main group of transfusion recipients in our country. given their comorbidities are a risk group for the development complications related to transfusion. aims: analyze the incidence of adverse effects related to transfusion in the elderly population and to assess what factors may influence its appearance methods: transfusions were reviewed in patients > years old. the variables analyzed were: sex, age, diagnosis/reason for transfusion, pre-transfusion hemoglobin (hb), number of transfused units, infusion rate and transfusion side effects, as well as the measures used to prevent or treat the transfusions. effects of circulatory overload results: a total of patients were analyzed ( women, men), with a median age of years ( - ). in total, ch were transfused. patients received ch, patients ch, patients received ch. patients were transfused at two different times. the median hb prior to transfusion was . g/dl. in the patients who received ch was . g/dl, those who received ch: g/dl and those who received ch: . g/dl. the infusion time could be estimated in % of the patients. in those who received ch was . min; . min in those who received ch and . min in those who received ch. patients ( % of the total) suffered some type of adverse effect related to the transfusion. in patients there was an increase in posterior ta, in an increase in hr, in an episode of hypotension and in another one episode of acute respiratory failure. % of those who had an adverse effect were older than years. patients with aht after transfusion, % received ch and the remainder ch. among their background, % had a history of ischemic heart disease. % also had a positive balance. the average previous bp was / mmhg and the subsequent one was / mmhg. % of patients did not receive diuretic treatment. in the case of the patient with acute respiratory failure was in oligoanuria, with positive balances. ch was transfused in total. she was treated with oxygen therapy and with intensification of the diuretic treatment, recovering later. summary/conclusions: -patients > years have a higher risk of suffering some type of adverse effect related to transfusion because they have pre-existing risk factors such as ischemic heart disease or heart failure. -we see that the risk of suffering some type of adverse effect in the elderly population is greater when we transfuse ch than ch. -we have appreciated that in those patients receiving ch, the infusion rate is higher. -the study highlights the lack of methods to prevent the development of circulatory overload. background: iron deficiency anemia is the commonest cause of anemia worldwide. weakness, fatigue, reduced physical activity and difficulty in concentration are the symptoms which are associated with its deficiency. the forefront treatment available is oral iron replacement therapy which is convenient, cost effective and has substantial outcome. another option is intravenous (i/v) iron when oral is not tolerable. despite of potential transfusion associated hazards and limited availability of blood due to shortage of voluntary blood donations, it is insisted by the patients prior to iron therapy. aims: the aim of conducting this study was to observe the impact of administration of oral iron, i/v iron and transfusion on hemoglobin levels in patients presented with iron deficiency anemia. methods: this was an observational study carried out at nibd and bmt, pechs campus, karachi, pakistan from february to december . the study was approved by the institutional review board. diagnosed ida patients presented at our hospital were recruited for analysis who were given oral iron, i/v iron and transfusion for the correction of anemia. informed consent was taken from the participants. descriptive and inferential statistics was applied by using spss version . . results: a total of ida patients were analyzed in which ( %) were females and ( %) were males. the most common symptom in females and males was fatigue followed by body aches in females ( %) and pallor in males ( %). menorrhagia was present in ( %) of females of reproductive age. surgical history was present in ( %) of females while there was no surgical history in males. mean hemoglobin, mch and mcv of females at baseline was . ae . , . ae . , and ae . while in males it was . ae . , ae . and . ae . respectively. sixty two ( %) females were advised oral and i/v iron and ( %) received transfusion. however, in males ( %) received transfusion and ( %) were advised oral and i/v iron therapy. it was observed that the increment of hemoglobin after oral/iv iron at average of months follow up in males and females was same as that the transfusion (p > . ). summary/conclusions: in our society where blood donations are scarce especially voluntary blood donations that are considered to be the safest type of blood donation. we would like to draw attention towards the alternatives to correct anemia such as oral and i/v iron replacement therapy as our results revealed that there was no difference in the increment of hemoglobin between the two groups. we need to educate our society especially the older age adults and young women who are more vulnerable of getting ida to opt oral and i/v iron therapy. it will be cost effective, convenient and also has less risk than transfusion. cellular therapies -stem cell and tissue banking, including cord blood background: the differentiation of megakaryocytes plays an important role in the production of platelets. however, the underlying mechanisms regulating megakaryocytes differentiation have rarely been studied. aims: to identify candidate genes involved in megakaryocytes differentiation and investigate the potential regulatory mechanisms of megakaryocytes differentiation from human cord blood hematopoietic stem cells in vitro. methods: cb-derived cd + cells were isolated using density gradient centrifugation and magnetic activated cell sorting (macs). cultures were stimulated with only recombinant human tpo ( ng/ml). after , and days, the mk fraction was selected by immunomagnetic sorting from the non-mk fraction using an anti-cd a monoclonal antibody. rna-seq-derived gene expression data was performed on uncultured samples (day ), cultured but unselected samples (day ), and cultured, selected samples (day , and ) by using the next-generation sequencing (ngs) platform, and rq-pcr technology was used to verify the expression of transcription factors. results: the comparison of the transcriptome profiles among the five stages showed that a massive gene expression change occurred in megakaryocytes differentiation. a total of genes were detected, of which showed up-regulation and down-regulation. among these genes, differentially expressed genes (degs) (fold change ≥ ; false discovery rate < . ) were selected were further validated with rq-pcr, including gabre, cdhr , wasf , pkhd l , thbs , pf v , lrrc and lgals . the rq-pcr result indicated that the mrna expression level increased with the prolongation of culture time. however, pf v mrna expression level was highest at day , lgals was highest at day . summary/conclusions: conclusion: our study identified a series of genes that may participate in the regulation of megakaryocytes differentiation. these results should serve as an important resource revealing the molecular basis of megakaryocytes differentiation and thrombocytopoiesis. preoperative anemia and blood transfusion requirement during hip and knee surgery rambam health care campus, haifa, israel background: blood transfusion (bt) is independently associated with increased morbidity, mortality and hospitalization length across different patient populations. due to bt-related risks, the concept of "patient blood management" (pbm) has been introduced to clinical practice. the three pbm pillars are: optimizing red cell mass, minimizing blood loss and optimizing physiological reserve. bt indications during orthopedic surgery include excessive bleeding or hemodynamic instability and not the hemoglobin (hb) level. in most clinical scenarios, a restrictive transfusion threshold (hb level: - g/dl) appears to be non-inferior to the liberal transfusion strategy in terms of blood use, morbidity and mortality. similar results are observed in highrisk patients after hip surgery. we hypothesize that preoperative anemia may lead to blood product overuse and its complications. aims: evaluating potential correlation between preoperative anemia and bt requirement during hip or knee surgery. methods: we reviewed medical files of patients who underwent hip or knee replacement surgery at rambam between - . patients with hb level measurement within days pre-surgery were included. receiving > blood unit was considered a surgery complication and such patients were excluded. patient demographic and clinical data, including comorbidities, surgery type, length of hospital stay, were collected. we created a synthetic data cohort using mdclone healthcare data sandbox, an environment enabling fast data extraction and producing synthetic data for analysis that does not require irb approval. results: during the evaluated period, patients underwent hip or knee surgery; were excluded from the analysis due to receiving > blood unit. hb measurement within days pre-surgery was available for patients. hip or knee surgery was performed in ( %) and ( %) patients, respectively. women comprised % (n = ) of patients who underwent hip surgery. in the hip-surgery group, . % of patients required bt, with this need being slightly higher among women ( . % vs. . %; p-value=ns). only ( %) patients were transfused during knee surgery and this cohort was not further analyzed. patients receiving bt had a significantly lower mean hb level than those who didn't require it ( . g/dl versus . g/dl for women and . g/dl vs. . g/dl for men; p-value < . ). hospitalization was longer in transfused patients compared to non-transfused ones (mean . vs. . days, p-value = . ) and in patients with a low hb level (female < , male < . ) than in those with a high hb level, irrespective of receiving bt (p-values < . ). patients with at least one of the following diagnoses: diabetes, renal failure, ischemic heart disease, were significantly more likely to have a lower preoperative hb level (p-value < . ). no other factors (e.g., patient's weight, rdw value or blood pressure) were predictive of transfusion need. the probability of a need for blood unit was . in the hb g/dl group and . in hb g/ dl group ( %>reduction). summary/conclusions: anemia presence before elective hip surgery is a risk factor for bt requirement and longer hospitalization. diagnosis and management of anemia using timely pre-surgery consultations may minimize intraoperative bt, particularly in women and patients with comorbidities. real-patient data and prospective trials are warranted. abstract withdrawn. abstract withdrawn. background: cd , known as platelet glycoprotein iv, belongs to type b scavenger receptor and is related to the pathogenesis of many diseases. type i cd deficiency was cd not expressed on platelets and monocytes. individuals with type i deficiency can produce homologous antibodies and cause related immune diseases. in recent years, it has been reported that cd deficient individuals cause fetal immune thrombocytopenia with fetal edema syndrome in asia. cd is not expressed in mature rbc, but exists in hematopoietic stem (progenitor) cells. anemia is an important cause of edema. in view of the phenomena of clinical and animal experiments, cd + hematopoietic stem (progenitor) cells were cultured in vitro to observe the effect of anti-cd monoclonal antibody on cd + hematopoietic stem (progenitor) cells. aims: to investigate the effect of anti-cd monoclonal antibody on proliferation and differentiation of human cd + hematopoietic stem (progenitor) cells in vitro. methods: choose healthy full-term maternal women without various obstetric complications, take cord blood ml. after density gradient centrifugation of ficoll cell separation solution, cd + hematopoietic stem (progenitor) cells were sorted by flow cytometry and cultured for - generations. mtt was used to examine the effect of anti-cd monoclonal antibody on the growth of hematopoietic stem (progenitor) cells. flow cytometry analysis was used to detect the apoptosis and cell cycle of cd + hematopoietic stem (progenitor) cells. the effect of anti-cd monoclonal antibody on the formation of cfu-e/bfu-e in hematopoietic stem (progenitor) cells was analysis by cfu-e/bfu-e account after - days culture. results: after umbilical cord blood was isolated by ficoll to obtain mononuclear cells, the hematopoietic stem (progenitor) cells of cd + were sorted by flow cytometry, and about . % of cd + hematopoietic stem (progenitor) cells were isolated. different concentrations of anti-cd monoclonal antibody and hematopoietic stem (progenitor) cells were cultured in vitro. the od value of value ( . ae . ) of anti-cd monoclonal antibody group ( mg/ml) was decreased than normal group ( . ae . ) (p < . ), and the od value ( . ae . ) was significantly decreased at the cd monoclonal antibody concentration of mg/ml (p < . ). there was no significant difference between the hematopoietic stem (progenitor) cells culture group and the igg control group (p > . ). in the annexin v flow detection, the apoptotic rate of anti-cd monoclonal antibody group ( mg/ml) was statistically increased than the normal group (p < . ). anti-cd monoclonal antibody significantly induced hematopoietic stem (progenitor) cells to undergo s phase cell reduction, g phase cells increased, and g /s phase cell arrest occurred. the number of cfu-e/bfu-e clones in the normal group was ae , the number of cfu-e/bfu-e clones in the control group was ae , and the number of cfu-e/bfu-e clones in the anti-cd monoclonal antibody culture group was ae . the number of colonies formed by hematopoietic stem (progenitor) cells in the anti-cd monoclonal antibody culture group was significantly lower than that of the other groups (p < . ). summary/conclusions: anti-cd monoclonal antibody can reduce the proliferation of human cd + hematopoietic stem (progenitor) cells and reduce the ability of erythroid differentiation. background: recently the new modern collection techniques were introduced in the apheresis procedures. cobe spectra system was replaced with spectra optia, and it was necessary to verify the efficiency of spectra optia in pbpc collections. aims: the aim of the study was to evaluate and optimize the new cmnc protocol spectra optia v. (terumo) for pbpc collections in patients with haemato-oncological malignant diseases. methods: the results of autologous pbpc collections were evaluated in: (a) well mobilized patients with precollection cd + cells concentration in blood higher than /ll, (b) from only the first collections, which were performed either by the use cmnc spectra optia v. or cobe spectra v. , v. , terumo (c) collections were performed in the standard and large volume leukapheresis regimen, lvl. engraftment data in transplanted patients were assessed. results: standard collections were performed in patients. the yield of cd + cells was high, and no significant differences were found between the numbers of cd + cells prepared from spectra optia , ( , - ) and cobe spectra , ( , ) /kg b. w. (a = , ; pval , ). the dependence of cd + cell yield on the precollection concentration of cd + cells in blood can be considered as linear with high correlation coefficients in cmnc spectra optia r = , , and cobe spectra r = , . lvl collections were performed in of patients, and there were no significant differences between the numbers of cd + cells prepared by cmnc spectra optia , ( - , ) and cobe spectra , ( , - ) /kg b.w. (a = , ; pval , ). the relations between the precollection cd + cells concentration in blood and the numbers of cd + cells from collections can also be considered as linear with the correlation coefficients in cmnc spectra optia r = , , and cobe spectra r = , , respectively. in lvl, the median platelet loss was significantly lower in cmnc spectra optia ( %) than in cobe spectra ( %). a group of patients was transplanted by means of pbpc prepared in the standard regimen. median time in the neutrophil reconstitution was in cmnc spectra optia as well as cobe spectra days, while in platelets from cmnc days, and from cobe spectra days, respectively. the number of patients obtained pbpc from lvl. the median time in neutrophils and platelets reconstitution was in cmnc spectra optia as well as cobe spectra the same, and corresponded with and days, respectively. summary/conclusions: cmnc protocol spectra optia is a modern, efficient and the safe system, which can be used for both standard and lvl procedures. in well mobilized patients the sufficient dose of cd + cells for transplantation could be prepared from one standard or one lvl procedure. no serious adverse reaction have been observed. background: peripheral hematopoietic stem cells are collected from patients/donors after mobilization with g-scf. the aim of the collection is a fixed number of cd + cells/kg. this number depends on the pre-apheresis cd + number, the blood processed and the collection efficiency of the procedure. the aim should be to collect all the requested cells in day, whenever possible. this is in order to reduce the dose of g-csf given to donor/patient and the resources used in the collection centre. the only parameter that can be adjusted is the volume of blood processed, if this is increased, the likelihood of collecting the requested amount of cells is increased, but only if the pre-apheresis cd number is high enough. therefore, you need to know, when it is feasible to increase the volume and thereby increasing the time of the procedure with the intention to collect all the requested cells in day. it can also show if it is possible to reduce the volume of blood processed, thereby reducing the time of the procedure. aims: to develop an easy tool to calculate the volume of blood processed in order to collect the requested cells in day. methods: the mean collection efficiency (ce) was calculated. ce is calculated as cd + cells collected/(pre-apheresis cd + number*processed volume)* %. based on the mean ce, an excel sheet was generated to calculate the volume of blood that should be processed in order to collect all the requested cells. the excel sheet is designed so the user enters the pre-apheresis cd + number, patient weight and the requested number of cd + cells. the ce is fixed according to the mean ce calculated. the result is the volume of blood processed in order to collect the requested yield. based on that result, the apheresis machine will provide time for the procedure, thereby it is possible to evaluate if the collection can be finished in day or not, e.g. by increasing the volume of blood processed. spectra optia â was used for all collections, cmnc for allogeneic donors and mnc for autologous patients. results: mean ce = % (n = ). a ce of % was chosen as the cut-off for the cd calculation tool. using the cd calculation tool: allogeneic donors (n = ): mean ce = %, mean blood volume processed = . tbv, mean time: min, donors were finished in day collection ( %) autologous patients (n = ): mean ce = %, mean blood volume processed = . tbv, mean time = min, patients were finished in day ( %). the calculation failed in only case ( . %). in this case the volume of blood processed was reduced according to the calculation, but because of unexpected low ce, the requested number of cells was not achieved. summary/conclusions: the cd calculation tool based on an excel sheet has shown to be simple and easy to use in order to personalize the stem cell collection. immunotherapy products: blood product, pharmaceutical, or a new category all together? from till . all donors were hla typed and matched; they were fully informed on the donation procedure and signed an informed consent for donation. minimum dose required to ensure successful and sustained engraftment was /kg cd + cells and /kg mono-nucleated cells (mnc). pbsc harvesting was performed with continuous flow cell separator baxter c , cobe spectra and spectra optia using conventional-volume apheresis processing the - . total blood volumes per apheresis. a femoral catheter was used for harvesting and acid citrate dextrose formula a is used for anticoagulation. recombinant human granulocyte colony-stimulating factor (g-csf) is used to mobilize pbpc for collection. harvesting of pbsc is usually performed after to days of g-csf subcutaneous administration at a dose of lg/kg body weight. results: all the donors were siblings of the patients treated at the university hematology hospital. there were apheresis procedures performed in healthy sibling donors. there were males and females, aged - . one to two apheresis procedures were required to collect adequate graft. the single procedure usually took - h and the volume of collected stem cells was - ml. the needed number of mnc and cd + cells was successfully collected by . apheresis. there were abo incompatible donors. procedures for mobilization and collection of pbpc from healthy donors are generally well tolerated. the only adverse effects of the apheresis procedure were bone pain as reaction of g-csf and numbness of the extremities as reaction of acd-a (hypocalcemia), which occur rarely and were very mild. the collected pbsc were used in allogeneic stem cell transplantation in patients with: acute myeloid leukemia - patients ( . %), acute lymphoblastic leukemia - patients ( . %), chronic myeloid leukemia - patients ( . %), myeloproliferative disorders - patients ( . %), myelofibrosis - patients ( . %), severe aplastic anemia - patient ( . %), non-hodgkin lymphoma - patient ( . %), multiple myeloma - patient ( . %), chronic lymphoblastic leukemia - patients ( . ), hodgkin disease - patient ( . %). summary/conclusions: the apheresis collection of pbsc in healthy donors is an effective and safe procedure. we are developing our national stem cell donors registry as a part of bone marrow donors worldwide. in that way we hope we will help widen the world network of stem cell donors and enlarge the possibility for each patient to find the right match. background: leukocyte-removing filters for blood are being used widely as a universal leukocyte reduction policy is being adopted progressively throughout the world. filtration is one of the most effective methods for preventing various adverse transfusion effects caused by leukocytes included in blood components, such as febrile reaction, alloimmunization, and transmission of leukotropic viruses. aims: the goal was to evaluate whether the new domestic blood filter finecell, developed by kolon industries, gumi, korea, is appropriately suited to the international standards. and to reveal its efficacy and safety in the settlement of leukocyte reduction system in korea. methods: thirty-two units of packed red blood cells obtained from ml whole blood collected from healthy donors were used. this was done by analyzing the filtration time, residual leukocyte count, rbc recovery, and hemolysis rate during a storage period of days after leukoreduction. results: the standards commonly used for the evaluation of leukocyte-removing filters are set by the food and drug administration of the usa and the council of europe. the results of our study satisfied these international standards. summary/conclusions: the newly developed domestic leukoreduction filter was, thus, found to be efficient and will contribute to the improvement of the quality of isolated blood components used in korea. faculty of science, humanities and education, technical university of liberec, liberec, czech republic background: as polymeric fibrous scaffold fabrication techniques strive to create structures that more closely replicate tentative extracellular matrix form and function, the need for increased scaffold bioactivity becomes more pronounced. the fibrous structure made from biocompatible and nontoxic polymers ensures mechanical stability, however cell proliferation requires further stimulation. platelet-rich plasma, which has been shown to contain over bioactive molecules, has the potential to deliver a combination of growth factors (gfs) and cytokines capable of stimulating cellular activity. aims: the presented work deals with the preparation of nanofibrous materials with platelet growth factors incorporated into the internal fiber structure. polyvinyl alcohol (pva) was used for the preparation a material providing a progressive release of native gfs without need of subsequent crosslinking. methods: materials were prepared from pva (mw , , % of hydrolysis) using electrospinning technology (nanospider tm ws u). platelet lysate (pl) was prepared from thrombocyte rich solution (obtained from regional hospital in liberec, the concentration of - x plt/ml, freeze-thaw method with subsequent centrifugation). nanofibers were electrospun from % pva solution using water: ethanol ( : ) solvent system. materials with proteins were electrospun from solution containing % of pva and % of pl. morphological analysis was performed by scanning electron microscopy. protein release was monitored using spectrophotometry (bradford method) and chromatography. results: the prepared fibrous materials consisted of random oriented end-less fiber with smooth surface with minimal defects in structure. the morphology of materials was not altered by the addition of proteins. the average fiber diameter was: ae nm for pristine pva fibers and ae nm for pva with incorporated proteins (pva/pl). pva/pl layers contain - mg of protein per gram of pva. % of the proteins are released during the first day (burst release) followed by a gradual release of up to weeks. summary/conclusions: nanofibrous pva-based nanofiber materials were prepared with native growth factors. the process used for the preparation of solutions and subsequent spinning does not affect the activity of the incorporated proteins, which are being gradually released. therefore, we believe that the developed material has great potential for use in tissue engineering e.g. to promote healing of chronic wounds. acknowledgements: supported by the czech health research council, project no nv - - . background: human a-defensins are small cationic peptides with antimicrobial and anticancer activity. up till now, six a-defensins have been described in humans. they include the human neutrophil peptides (hnp) , and which present in large amounts in neutrophil azurophilic granules and differed from each other only in the first amino acid. a fourth defensin, termed hnp- , comprises less than % of the total defensins in neutrophils and has a distinct sequence from hnp - . the other two, human defensin and , are synthesized mostly by intestinal paneth cells. neutrophil defensins (hnp - ) are . kda peptides that are characterized by three disulfide bridges. the pattern of disulfide bonds in the mature forms is crucial for the functional properties. due to this structural feature, synthesis of defensins using the chemical and recombinant approach presents quite a challenge. moreover, purification from the natural source can be very difficult because the large number of neutrophils is needed to obtain a sufficient amount of protein. in blood banks, leukocyte reduction filters are used to remove leukocytes from blood components in order to prevent adverse transfusion reactions. leukofilters contain high numbers of normal human cells and discard after use. aims: the aim of this study was to purify a-defensins from neutrophils trapped in leukocyte reduction filters. methods: blood bags from healthy blood donors were collected after written consent. all donors were screened for infectious diseases (hbv, hcv and hiv) and negative samples were included in the study. blood bags were filtered at °c by leukoflex lst- filters. the cells were extracted from the filters by back-flushing with cold phosphate buffered saline (pbs), ph . , without mgcl and cacl , containing mm edta and . % sucrose. the pbs was homogenized with the filter content and then collected in a sterile tube. neutrophils were separated from mononuclear cells by ficoll. isolated neutrophils resuspended in pbs x at a concentration of cells/ml. for degranulation, cells were stimulated with nm of formylmethionyl-leucyl-phenylalanine (fmlp) for min followed by stimulation with lm of cytochalasin b for min. supernatant was collected by centrifugation at g for min. supernatant was incubated with mouse monoclonal antibody to hnp - and purification of hnp - was performed by lmacs protein g microbeads system. the presence of protein was confirmed by western blot. results: the presence of the . kda band was confirmed by western blot, which corresponded to the size of the a-defensins. summary/conclusions: the development of defensins as therapeutic products requires access to a steady supply of neutrophils. our results indicated that lst- filters are economical source for purifying a-defensins. anatomical sciences, abadan school of medical sciences, abadan, iran background: epigenetic reprogramming of terminally differentiated cell can modify somatic cells to a pluripotential state. there are several approaches that induce pluripotency in somatic cells. exposure the cells with the embryonic stem cell extract is an easy way, and some investigations were done on fibroblast cell line. however, its efficiency was low aims: the purpose of this study was to increase the number of reprogrammed granulosa cell as a full differentiated cell into pluripotential state methods: the human granulosa cells were cultured in the medium containing -aza-deoxycytidine and trichostatin a. then, the cells were exposed to mouse escs extract and co-cultured with mouse embryonic fibroblast in the presence of leukemia inhibitory factor (lif). alkaline phosphatase test and also immunohistochemistry staining for oct , sox and nanog were performed after and h and week results: the results indicated that after h the granulosa cells were revealed a round and expanded morphology. the cells in all groups except in negative control, were showed alkaline phosphatase activity. the cells that were cultured in medium containing -aza-deoxycytidine and trichostatin a and exposed to the extract had the most numbers of alkaline phosphatase positive cells. immunocytochemistry showed the granulosa cells that were cultured in medium containing -aza-deoxycytidine and trichostatin a with extract expressed oct with weak intensity after h. no expression of oct , sox and nanog were observed in other groups at the same time. after h, oct , sox and nanog were over expressed in the cells that were treated with -aza-deoxycytidine, trichostatin a and extract. furthermore, there was high expression of oct in the granulosa cells that were cultured in medium containing dmso and exposed to the extract. after one week, the expression of oct and sox in the granulosa cells that were cultured in medium containing dmso and exposed to the extract was continued while its expression ceased in the other groups. the expression of nanog were ceased in all groups after one week summary/conclusions: present study revealed that the inhibitors of the methyl transferase ( -aza-deoxycytidine) and histone deacetylase (trichostatin a) could delete the epigenetic markers and improved cells reprogramming by administration of the extract abstract withdrawn. abstract withdrawn. abstract withdrawn. background: mesenchymal stem cells (mscs) are adherent spindle shape cells expressing different surface markers. they show special criteria including, paracrine effects, differentiation to several tissue cells, migration, immunomodulatory and regenerative potentialities. mscs are isolated from different sources and employed as therapeutic tools to treat several diseases and injuries. however, some of mscs properties including secretion of growth factors and migration ability are controversial especially during remission or in presence of tumor. interestingly, msc-derived compartment could be used as practical tools in term of diagnosis, follow up, management and monitoring of disease instead of intact mscs. exosomes are kind of extracellular vesicles (evs) characterized via their size and releasing mechanism. usually they defined as less than nm in diameter vesicles. they secreted from different cells and are also found in urine, blood, breast milk, cerebrospinal fluid and other body fluids. exosomes contain genetic material including dna, mrnas, micrornas (mirnas) and other biomolecules. mirnas are single stranded non-coding rnas transcribed from dna. immature mirnas are subjected to two known cleavages to modify to mature mirna that involve to either mrna degradation and gene expression process or cell-cell interaction and communication via secretion as the part of exosomes. aims: this study was aimed to discuss some aspects of exosomal micrornas derived from mscs in progression, diagnosis and treatment of some diseases. methods: different scientific data bases including pubmed, google scholar and scopus were used to find and review related articles. results: evs play important role either in intercellular communication related to pathological and physiological situation or intracellular communication, angiogenesis, immune system modulation and metastasis progression. mirnas could regulate expression of multiple mrnas then they play important role in different biological processes and contribute cell-cell interaction as well as influence in the progression of different disease. exosomal mirna-derived mscs are involved in cancer procession, tumor growth, angiogenesis and metastasis. they are used as diagnosis and therapeutic tools to treat different diseases such as renal failure, liver fibrosis, myocardial infarction. summary/conclusions: due to controversial aspect of using of intact mscs especially during remission or in presence of tumor, msc-derived exosome could be used as practical tools in term of diagnosis, follow up, management and monitoring of disease instead of intact mscs. aims: the aim of study try to use sybr green i based real-time pcr to identify homozygous, heterozygous, gene deletion or wild type for rhd exon , , and a. methods: for this study, we used real-time pcr with high resolution melting curve mode, and matrix mix containing sybr-green i were used for sequence specific primers of g>a and rhd exon , , . samples with rhd gene deletion homozygous/heterozygous, g>a heterozygous with rhd gene deletion and normal rhd, normal rhd homozygous/heterozygous and rhd -rhce( - )-rhd homozygous/heterozygous were enrolled in our study. all samples were screened using rhd exon genotyping, sanger sequencing and rhesus box analysis. concentration and mass of dna samples were in alleles of normal rhd/rhd gene deletion. the tm ratio of rhd exon ( °c) to internal control ( °c) were . in alleles of normal rhd/rhd -rhce( - )-rhd , . - . in alleles of rhd gene deletion/normal rhd, . - . in alleles of normal rhd and < . alleles of rhd gene deletion. the tm ratio of rhd exon ( °c) to internal control ( °c) were . in alleles of normal rhd/rhd -rhce( - )-rhd , . - . in alleles of rhd gene deletion/normal rhd, . - . in alleles of normal rhd and < . alleles of rhd gene deletion. the tm ratio of rhd exon ( °c) to internal control ( °c) were . in alleles of normal rhd/rhd -rhce( - )-rhd , . in alleles of rhd gene deletion/rhd -rhce( - )-rhd . - . in alleles of rhd gene deletion/normal rhd, . - . in alleles of normal rhd and < . alleles of rhd gene deletion. summary/conclusions: using the tm ratio of sequence specific primers to internal control is an effective way to detect the rhd gene deletion or rhd weak d types , and not detected") were tested with a method based on next generation sequencing (ngs) using the illumina miseq platform to detect a possible rhd variant not interrogated by id rhd xt. results: in total dna samples were tested in pools. fifteen ( ) pools ( samples) gave rhd deletion genotype and seventy two ( ) pools ( samples) resulted to the presence of rhd gene. the positive pools were also analyzed individually. the genotype results obtained were: rhd exon no amplification ( ), rhd exon and the genotype results obtained with id rhd xt (in pools and individually) were concordant with the results provided by the centers. hence, % accuracy was obtained using id rhd xt with dna pooled samples. the results of rh ngs for the samples with inconclusive results by id rhd xt showed rhd variants previously described: sample rhd* - inst (del), sample rhd*ivs + g (del), samples rhd*weak d type (partial d), sample rhd*weak d type (weak d), sample rhd*weak d type (weak d) and not described: sample rhd*del - (unknown) summary/conclusions: id rhd xt is a high accurate tool for genotyping the most common rhd alleles associated to weak d and d negative phenotype in up to pooled dna samples. use of rhd genotyping improve rhd typing in blood donations variant rhd alleles generate qualitative/quantitative alteration in serological expression of d antigen such as weak and partial rhd phenotypes which are clinically important in transfusion setting. population studies have shown varied distribution of the variant d alleles in caucasians, africans, east asians and indians. many countries have developed their own population-specific strategy for identifying d variants. our previous study in indian population showed absence of weak d type , , and which are commonly found in caucasians d variant individuals. instead, a novel population-specific pattern i.e.~ -kilobase duplication event, including exon , was predominantly identified in . % d variant samples. functional analyses showed that this genetic variation results in the expression of several transcripts, including a wild-type product. commercial genotyping assays available, mainly detect common d variants found in caucasians and africans, thus limiting its usefulness in india. hence, based on our findings we have designed a multiplex pcr assay specific for indian population that can be easily implemented at the laboratory level for genotyping variant rhd. aims: to characterize rhd variants using "indian-specific, rhd genotyping assay". methods: seventy samples referred to our laboratory for molecular characterization of rhd variants were included in this study. all rhd variant samples were serologically typed for results: out of the rhd variants included in this study, samples ( %) showed presence of indian specific allele i.e. exon duplication. seventeen rhd variants samples showed presence of both exon and . qmpsf analysis of these samples excluded involvement of rhd-rhce-rhd hybrids. sixty of the seventy d variant individual had r r genotype this assay thus can be used routinely in indian laboratories to identify and characterize rhd variants. - non-invasive fetal kel genotypes from allo-immunized anti-kel women were done ( positive confirmed fetuses, undetermined, positive non-confirmed, negative non-confirmed and negative confirmed). - non-invasive fetal rhc genotypes from allo-immunized anti-rh women were done non-invasive fetal rhe genotypes from allo-immunized anti-rh women were done ( positive foetuses, undetermined for , % of the allo-immunized women, the pregnancy was compatible and no specific antenatal monitoring was necessary summary/conclusions: non-invasive fetal red blood cell genotype is a powerful tool to diagnose a feto-maternal red blood cells incompatibility and allows to legitimize a costly and heavy specific antenatal monitoring s purchla-szepioła , m krzemienowska , m pelc-kłopotowska , m jurkowska , m debska , m uhrynowska and e brojer the test developed by ihtm has been offered to clinicians and pregnant women since but it is not covered by the health care system. rhd nipt is not informative for mothers with rhd variant. in such cases further analysis of the molecular background is offered to exclude from immunoprophylaxis the women with weak rhd type , and . aims: summary of a -year period of routine rhd nipt available at ihtm. methods: cffdna isolated using easymag, biomerieux from plasma of pregnant women determined with standard serology as rhd-negative (in - week of gestation) was examined for the presence of exons and of rhd and ccr by realtime pcr using lc ii (roche). maternal dna from whole blood was tested for identification of rhd variant using rbc fluogene rbc-dweak/variant (inno-train, germany) or the home-made protocol. results: in cases the rhd gene was not detected in cffdna and the administration of rhig was not recommended. in seven cases ct-values for rhd and ccr indicated a maternal d variant (d ct ccr -rhd > ); the genetic follow-up of six of them identified: rhd* w. in cases, rhd* w. and rhd* . in cases the rhd nipd results indicated that a fetus is rhd positive and rhig administration was recommended it was recommended in the remaining % of mothers. in . % cases with maternal d variant rhd nipt was not possible. however, in / such cases the test is unnecessary because follow up analysis revealed maternal rhd variant of the weak d type and in switzerland extended antigen-matching for duffy, kidd and mnsbesides rhesus and kell -is recommended for sickle cell disease (scd) patients. the ethnic diversity of red blood cell (rbc) antigen polymorphism engender that these patients are often transfused with rbcs from donors of african origin. this strategy, however, increases the likelihood of being exposed to certain low-prevalence antigens, such as rh (d w ), as these are almost exclusive to african populations. rh is encoded by several types of rhd*dv as well as by dau- . anti-rh is associated with delayed hemolytic transfusion reactions (htr) aims: here we report a specific low-prevalence antibody newly formed by the same patient, meanwhile gravida , para , causing positive crossmatches with the rbcs of two of the four selected donors. subsequently, advanced serologic and genetic workup and close international collaboration enabled optimal patient care. methods: standard serological methods were used for antibody specification (biorad, cressier, ch and in-house). crossmatches were carried out by indirect antiglobulin test at °c. molecular typing of donors' and parental blood group antigens was performed by further serological analysis (institute national de transfusion sanguine, paris) revealed an anti-rh in addition to anti-fy , anti-e and anti-jk a . genotyping of the two donors causing positive crossmatches presented heterozygosity for rhd* . which encodes rh . the newborn's phenotype was a r r k-, fy(a-b+) and most likely rh -and jk (a+b+), considering both maternal and paternal (a r r, k-k+, fy(a-b+), jk(a+b-), rh -) predicted phenotypes. the neonatal serum contained maternal anti-a , anti-rh and anti-e. the direct antiglobulin test was positive but elution only showed nonspecific reactions with papain-treated cells. latter might have been caused by anti-fy during her present pregnancy we were able to demonstrate that two positive crossmatches of two former compatible donors were caused by a new alloantibody against a low-prevalence antigen, namely anti-rh , derived from several rh + rbc transfusions during the previous pregnancy. despite this challenging blood supply we were able to support the patient with a total of ten antigen compatible and crossmatch negative rbc units from french and swiss donors until delivery with increasing age, the relative number of women in the study population raise from % in the patients younger than years to % in the patients older than years. our study showed that cardiovascular diseases were the commonest indications for warfarin use in older patients with %. only , % achieved target therapeutic range while the risk of thromboembolism and the subsequent need for proper anticoagulant therapy increases sharply with age, the bleeding risk rises as well. older patients are more sensitive to any given dose of warfarin and need a significantly lower total weekly dose. a well-informed patient provides one of the best defenses against bleeding complications. recent data demonstrate doacs advantages over warfarin, especially for older population: more predictable dosing, fewer drug interactions and reduced risk of intracranial bleeding although vast majority of fh cases are caused by mutations in ldl-r gene %- % patients do not harbor genetic cause in the known loci. patients with homozygous/severe heterozygous fh are unresponsive (ldlc above mg/dl with diet and drug therapy) and require additional extracorporeal therapy to reduce ldlc concentrations to prevent the development/progression of cad. ldl apheresis techniques remove apolipoprotein b-containing lipoproteins from blood and include heparin-induced extracorporeal ldl precipitation(help), immunoadsorption, dextran-sulfate adsorption methods: a y iraqi male visited cardiac-opd. ct coronary angiogram showed cad-dvd. he had multiple tendinous xanthomas and xanthelasmas. family history was significant for death of elder brother from coronary event at y, a sister with similar profile age y and one sister apparently normal. he was taking medical treatment for dyslipoproteinemia (ecosprin mg od, ticagrelor mg bd, rosuvastatin mg od). despite dietary and medical treatment his dyslipoproteinemia was refractory. therefore cascade-filtration was planned with evaflux a plasma fractionator. one procedure of cascade plasmapheresis was done on com.tec apheresis system (fresenius kabi, germany) separating patient's plasma as the first step and passing it through a pore sized based filter column a (evaflux, kawasumi, japan) as the second step. a total of . x plasma volume( ml) was processed. the patient was given continuous calcium infusion. the flow rate of ml/min was maintained immunoglobulins) were not assessed. summary/conclusions: the procedure successfully met the requirement of reduction of cholesterol by %. the patient became responsive to the medical treatment. follow up of the patient up to a year has been uneventful with no additional procedure requirement actions included development of major haemorrhage protocols with improved communication and required instances of delayed transfusion to be reported to the uk national haemovigilance scheme (serious hazards of transfusion, shot) methods: delayed transfusion data have been collected from . hospitals identify incidents and report them via an online database. mh may also result in avoidable or overtransfusion. reports are analysed and collated data published in the shot annual reports in july each year. results: the total number of reports of delayed transfusion has increased with time: , , in the last years. delayed transfusion in relation to mh was reported for cases - contributing to death in patients %) miscommunication was noted between clinical different teams, between emergency departments, porters and the transfusion laboratory, failure of bleeps, failure to communicate the urgency, failure to confirm the patient location. failures to follow mhp correctly occurred in / ( . %): incorrect activation including failed alerts to porters, wrong contact telephone details and wrong components in the mhp packs most transfusions included red blood cells (median, units); % of women were transfused with fresh frozen plasma (median, units) and % with platelets. mean pre-and post-transfusion hemoglobin levels were . g/dl and . g/dl, respectively, representing an increment of . g/dl per rbc unit transfused ( . g/dl in soweto and . g/dl in durban). indications for transfusion included obstetric hemorrhage ( %), chronic anemia ( %), surgery or anesthesia ( %), other ( %) and not specified ( %). transfusion for chronic anemia (vs. hemorrhage) was associated with gestation ≥ weeks (odds ratio = . , % confidence interval . - . ). surgical blood loss was a common indication in trimester ( %) that declined to % then % in trimesters and . summary/conclusions: hemorrhagic complications accompanying spontaneous abortions and ectopic pregnancies in the first and second trimesters were the most common reasons for antenatal transfusion surveillance and analysis of blood transfusion reactions represents inseparable part of hemovigilance. aims: summarization of data on reported cases of transfusion reactions. methods: analysis of serious undesirable reactions to blood products administration in the czech republic (cr) during period - . results: there were evaluated , of blood products administrations in , patients in the cr during defined three years period. announced , ( , %) transfusion reactions including severe transfusion reactions ( adjudged with grade ). the most frequent types of severe transfusion reactions: anaphylaxis , trali x, taco x, hcv x, hbc x, bacterial infection x, delayed hemolysis x. transfusion reactions incidence according to administered bp: red blood cells products: , administrations, transfusion reactions (fnhtr x, allergy x, circulatory overload x, anaphylaxis x, trali x, hbv x, hcv x) platelets: , thrombocyte administrations, including transfusion reactions (allergy x, fnhtr x, anaphylaxis x, circulatory overload x, delayed immune hemolysis x, acute circulatory overload x. granulocytes: administrations, transfusion reactions plasma: , administrations, reactions reported (allergy , fnhr , circulatory overload , anaphylaxis x, trali x, hbv x, ards x. summary/conclusions: conclusions: comprehensive analysis and data processing help to appropriate prospective setting of blood products (bp) production and hemotherapy. concrete outputs from processed data triggered undermentioned changes in many departments in the cr: . plasma for clinical uses from male blood donors, . prestorage of leucocyte reduced blood products, . production of platelets in additive solutions, . implementation of pcr testing method for blood donors screening. background: skae's basic activities include epidemiological surveillance of all adverse events (aes) associated with deviations in the collecting, testing, processing, storage and distribution of blood and blood components that may affect quality and safety near misses" and "uneventful transfusion errors" are collected to identify preventable causes. incorrect blood component transfused (ibct) events are reported following ihn instructions. results: a total of they were mainly associated with deviation in processing ( . %) and attributed to equipment failure and materials ( %) whole blood collection, materials and distribution, as a result of product defect, equipment failure, human error and other. trend analysis showed a significantly increasing (p < . ) annual rate of total aes by % ( % confidence interval - ) ) % fibrinogen-depleted phpl or ( ) % fibrinogen-depleted phpl plus heparin. internalization of fluoresceinamine-labeled heparin in stcs was investigated by flow cytometry and immunocytochemistry. all stromal cells were subjected to whole genome expression analysis (affymetrix human gene . st array) and data were analyzed using r/bioconductor and panther analysis tools. confirmative qrt-pcr was performed and protein levels of selected pathways were analyzed by a bead-based western blot system (digiwest â ). immunophenotyping, in vitro differentiation, longterm proliferation and colony forming units (cfu) assays were done for all cell types. results: in vitro exposure of heparin induced differential internalization and lysosomal accumulation in stromal cells, as well as regulation of distinct gene sets, both in a tissue-source dependent manner. affected signaling cascades were mainly involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis. the influence of heparin on protein expression and phosphorylation of four pathways (wnt, pdgf, notch and tgfbeta) was further analyzed, revealing most alterations in bm-stcs. independent of origin and medium composition, flow cytometry analysis revealed the characteristic fibroblastoid phenotype profile (cd +/ +/ + and cd -/ -/ -/ -/hla-dr-). in addition a comparable osteogenic and adipogenic differentiation capacity was found summary/conclusions: internalization of heparin in lysosomes by stromal cells, differential gene and protein expression and phosphorylation changes were observed in a tissue-source dependent manner. however, stromal cell characteristics as immunophenotype pattern, long-term proliferation, clonogenicity and in vitro differentiation were unaffected, putatively by post-translational protein modifications. in this respect, application of porcine heparin is compatible with efficient manufacturing of stromal cell based medicinal products abo incompatibility may have no effect on the clinical outcome after allogeneic hematopoietic stem cell transplantation. however, it carries additional risks of hemolytic reactions, delayed red blood cell (rbc) engraftment and incidence of graft-versus patients were categorized according to abo compatible and mismatched groups; these were further sub-categorized into major, minor and bidirectional. direct coombs test (dct) was performed when hemolysis was suspected in the post-transplantation period along with serum lactate dehydrogenase (ldh) %) were male and ( . %) female. mean age of abo matched and mismatched groups were ( . ae . ) years. most common indications for transplant included beta thalassemia major ( . %), aplastic anemia in ( . ) and pure red cell aplasia ( . %). source of stem cell was bone marrow in and peripheral blood patients abo matched while abo mismatched group comprised of ( . %) patients with further subdivision into major (n = ), minor (n = ) and bidirectional in the post transplantation period, packed red blood cell and platelets were transfused in matched group (n = ) and (n = ) comparably(n = ) and (n = ) in mismatched group. primary and secondary graft failure in matched group was . % and . % patients while in mismatched group graft failure was observed in ( . %) patients respectively. positive dct in abo matched group in ( . %) patient, whereas ( . %) patients with major and minor abo mismatch group with raised ldh levels and deranged lfts were found. episodes of acute and chronic gvhd in abo compatible and incompatible groups were insignificant. overall survival in abo summary/conclusions: these results indicate that abo incompatibility does not seem to influence outcome of the patients undergoing allogeneic hematopoietic stem cell transplantation. careful monitoring of patients can help detect problems early and treat them efficiently, thus, reducing the number of life threatening events a picascia , c sabia , f cavalca , g nicoletti and c napoli in our routine work with one lambda sab class ii reagents, we observed non-specific reactivity with some beads bearing dr and dr in patients without sensitizing events. this pattern was not confirmed by testing same sera with screening-and pra-beads suggesting non-specific reactions. aims: here, we sought to determine if fetal bovine serum (fbs) treatment would be effective in reducing/eliminating non-specific reactivity. methods: we tested sera pre-treated with fbs from non-sensitized patients that showed the dr /dr pattern. in particular, ll of fbs was added to ll of patient serum; incubated for min at °c; centrifuged and subsequently tested in the sab assay. as controls, we treated sera from patients with documented dsa including dr /dr and patients without hla antibodies. results: dr /dr non-specific reactivity was eliminated or significantly reduced after fbs treatment. we found that patients with dr and dr dsa had no change in mfi values and additional reactivity was not observed in negative fbs treated sera transfusion medicine, national blood transfusion centre transfusion medicine, national blood transfusion center transfusion medicine, national blood transfusion centre, tirana, albania background: abo blood group, has been associated with many diseases, although the explanation for abo's blood group association and some illnesses is still unclear. aims: to find the distribution of cases by blood groups in patients with malignant pathology compared to donors in order to assess the presence of the abo blood group as an epidemiological indicator to identify populations exposed to different malignant pathologies methods: we conducted a case-control study. abo blood group and diagnosis of all patients have been studied. the control sample was collected from , healthy donors from which group a ( , %), group o ( , %), b ( , %) and group ab ( , %) resulted. the study was conducted in patients who have been transfused and submitted a request to determine the blood group at the blood bank at qsut during the period - results: among the patients, when all malignant pathologies were taken together, the highest frequency was seen in blood group a ( . %), followed by ( . %), b ( . %) and ab ( . %). group a frequency was higher and o was lower compared to controls. a high incidence of blood group a is seen in: pancreatic cancer a ( %), in gastric cancer a ( %), colorectal cancer a ( , %), breast cancer a ( %), cervical cancer a ( %) and ovarian cancer a ( %) versus a ( . %) in the control group. a high incidence of blood b is seen in multiple myeloma b ( %) and cervical cancer b ( %) versus b ( . %) in the control group. blood group ab has a high incidence in malignant lymphoma ab ( %) versus ( . %) in the control group summary/conclusions: it appears that individuals with blood groups a, b and ab are more at risk of developing malignant pathologies and individuals with blood group o are more protected. background: the high homology and opposite orientation of rh genes promote many rearrangements between them and generate a large number of rhd and rhce variants which can be inherited together. several studies have shown that those rh variants in patients with scd represent an additional risk for alloimmunization and delayed hemolytic transfusion reactions (dhtrs), but little clinical or biological evidence related to alloimmunization and dhtr are presented for all the rh variant alleles. it is well established that transfusion recipients with the most common weak d types , and , are not at risk for forming alloanti-d when exposed to conventional rhd-positive rbcs. aims: we report here a case of a -year-old patient typed as weak d type , receiving d+ rbc units who presented anti-d in his plasma detected three weeks after the last transfusion. methods: rhd beadchip (immucor, nj, usa), was performed to identify the rhd variant allele associated with the weak expression of d. rhce genotyping was performed by laboratory developed tests. sequencing of rhd, rhce and rhag were performed to determine if there were other mutations that could explain the production of alloanti-d. serologic testing was by standard hemagglutination methods. the clinical significance of the antibody was evaluated by monocyte monolayer assay (mma). results: serological analysis showed a negative dat and the presence of anti-d in plasma ( + by gel). anti-lw was ruled out. rhd genotyping revealed the patient was rhd*weak d type . rhce genotyping predicted the d+c+c+e-e+ phenotype. sequencing of rhd, rhce and rhag found no additional changes and confirmed the presence of rhd*weak d type . mma showed the anti-d was clinically significant (> %). summary/conclusions: we report the production of alloanti-d in a scd patient with rhd*weak d type allele. weak d type patients are not considered to be at risk for allo anti-d but our results show that there are exceptions and that these anti-d can be associated with clinically significant rbc destruction. background: the mns blood group system is located on glycophorin a (gpa), glycophorin b (gpb) and hybrid glycophorins on the surface of the red blood cell (rbc). these glycoproteins are involved in complex structures interacting with other rbc surface proteins including the band /diego protein. the glycophorins are heavily glycosylated and contains multiple clinically significant blood group antigens. it has proved difficult to model the gpa extracellular structure due to its heavy glycosylation, and lack of homology with existing modelled proteins. aims: to develop an in silico model of gpa as a basis for improved predictions of structure function relationships methods: prediction of secondary structure and disorder: . . predictprotein (https://predictprotein.org); . . spider (http://sparks-lab.org/server/spider /); . . dsc (discrimination of protein secondary structure class): using an in-house implementation; . . jpred (http://www.compbio.dundee.ac.uk/jpred /); . . raptorx (http://raptorx.uchicago.edu). prediction of secondary structure: . . robetta (http://robetta.bakerlab.org/submit. jsp); . . falcon (http://protein.ict.ac.cn/treethreader/); . . itasser (https://zha nglab.ccmb.med.umich.edu/i-tasser/) threading methods to evaluate the quality of protein structures: . . verify d (http://servicesn.mbi.ucla.edu/verify d/); . . prosa (https://prosa.services.came.sb g.ac.at/prosa.php) protein-protein docking: . . gramm-x protein-protein docking web server (http://vakser.compbio.ku.edu/resources/gramm/grammx/); . . gramm (http://va kser.compbio.ku.edu/main/resources_gramm . .php) results: using in silico modelling we derived a stable tertiary glycosylated structure for gpa both as an individual protein and a homodimer. the hybrid glycophorin background: non-invasive prenatal testing of fetal antigen using cell-free fetal (cff) dna from maternal plasma of immunized women is widely implemented into clinical routine but the sensitivity and specificity of the method, especially for genotyping antigens encoded by single nucleotide polymorphisms such as k antigen, is limited by low proportion of cffdna in maternal plasma dna. according to literature reports detection of circulating tumour (ct)dna can be improved by selection of short ctdna fragments using automated electrophoresis methods. aims: the aim was to assess the feasibility for enrichment of cffdna fraction in maternal plasma dna by size selection using the pippin prep gel selection system. methods: plasma dna isolated using easymag (biomerieux) from rhd negative and k-negative pregnant women (n = ) carrying fetuses with known genotype was loaded into % agarose gel casette ( % df marker q , sage bioscience) and size selection of fraction was performed on a blue pippin tm (sage bioscience) with the elution from min to h min of electrophoresis. results for real-time pcr detection of fetal rhd, kel* and ccr (as a marker of total plasma dna) in dna fraction after gel selection were compared to results obtained from non-processed plasma dna. results: the total dna level (measured by ccr ) was significantly lower in dna samples tested after gel selection (from . to . geq/pcr) versus the level obtained from non-processed plasma dna (from to geq/pcr). the results for fetal fraction (measured by rhd) from dna samples of rhd-negative pregnant women carrying rhd positive fetus tested after gel selection were from , to . geq/pcr versus . - . geq/pcr for non-processed plasma dna. results for kel* detection in plasma dna from k-negative pregnant women carrying k-negative fetus were kel* -negative in dna samples tested after gel selection comparing to nonprocessed dna samples were false kel* positive amplification was observed. however, kel* detection in plasma dna from two k-negative pregnant women carrying k-positive fetus gave false kel* -negative results in dna samples tested after gel selection comparing to non-processed dna samples were kel* positive genotype was obtained. the total dna level in samples from k-negative women was from . to . geq ccr /pcr after gel selection versus from to geq ccr / pcr in non-processed dna samples. summary/conclusions: using the pippin prep gel selection system increases the proportion of cffdna fraction in total plasma dna by retaining long maternal dna fragments in the gel cassettes, but the protocol of gel separation dilutes the separated material decreasing the concentration of fetal dna and leading to false negative results of nipt. anti-rh quantification assay using ih- (bio-rad â ): promising results for monitoring rh:- pregnant women j beaud, h delaby, c toly-ndour, a mailloux and s huguet-jacquot centre national de r ef erence en h emobiologie p erinatale (cnrhp), hôpital saint-antoine, paris, francebackground: the generalization of immunoprophylaxis by anti-rh immunoglobulins since complicates the interpretation of the anti-red blood cell antibodies screening during pregnancy. to distinguish an alloantibody from a passive one, many laboratories in france use anti-rh microtitration. it is a column agglutination technology using red blood cells rh: , - , - , , (r r) . it permits to quantify low levels of anti-rh in comparison to a range of an anti-rh standard. performed since at the cnrhp and automated on evo clinical base tecan in (dilutions and distribution), anti-rh microtitration is well adapted to rh prophylaxis. aims: the aim of this study was to evaluate this technique on the ih- system from bio-rad â . methods: on ih- , the reactivity of the bio-rad â reagents was compared with the cnrhp reagents (red blood cells r r, anti-rh standard). the performances of the method were evaluated using three internal quality control (icq) ( cnrhp home-made at and ng/ml and bio-rad â at ng/ml) on papainized r r (plc) and native r r (nlc). a comparison of results from patient sera ranging from . to ng/ml was done between ih- and evo clinical base tecan. results: the results of the qci are similar between the different reagents used. there is no significant difference between the types of red blood cells except for the limit of detection: . ng/ml in plc - ng/ml in nlc. for the qci, the intra and interassay imprecision based on the dilution degree show coefficients of variation between and %, similar to those found with the evo clinical base. the correlation with the cnrhp technique performed on samples in plc and samples in nlc was satisfactory (deming plc: r = . y = . x + . -nlc: r = . y = . x- . ). summary/conclusions: the anti-rh microtitration on the ih- offers similar performances to the method conducted at the cnrhp. the ih- allows automated reading of gel cards. however, it does not have a calculation or interpretation algorithm and does not directly give the concentration of anti-rh . this final part remains manual and requires trained staff. background: haemolytic disease of the foetus and newborn (hdfn) due to maternal-foetal incompatibility has been perfectly framed for decades from the etiologic, pathogenetic and therapeutic point of view. the anti-d alloantibody is most frequently responsible for the most serious form of hdfn due to rhd incompatibility (rhdi hdfn). although immunoprophylaxis (ip) has reduced the number of cases of rhdi hdfn, this disease continues to occur and red blood cell alloimmunization still remains the most common cause of foetal anaemia. hdfn due to abo incompatibility (ab i hdfn) is currently the most common neonatal haemolytic disease in the western world. however, only in about . - % of cases haemolytic disease demands transfusion support. aims: analysis hdfn from to . methods: the hdfn's transfusional support is: intrauterine transfusion (iut) in the antenatal period; exchange transfusion (et) for severe hyperbilirubinaemia and neonatal transfusion of small volumes red cells for the newborn's late anaemia in the postnatal period. our policy for iut, for et and for the neonatal transfusion requires the use of a concentrated blood cells (ec) preferably group rh negative (cde/cde) or negative for any red cell antigens if the mother has antibodies, fresh (< days), preferably cmv safe. for iut, the ec must be compatible with mother's plasma, must have hematocrit + %, and irradiated. the unit for et must be compatible with the newborn's plasma, whit hematocrit % - % and irradiated. the ec used in post-natal transfusions is usually divided into rates of ml, hematocrit ae %. results: in last years, we calculated neonates with hdfn ( males and females): with rhdi hdfn, with ab i hdfn and with hdfn due to incompatibility for other red blood cell antigens. we have produced iut: for our hospitalized patients and for patients located in other hospitals. of these patients, who received iut, were immunized: showed anti-d antibody and antibodies different from anti-a and anti-b. , of the infants with rhdi hdfn, were transfused in utero. neonates on ( . %) have performed et: with ab i hdfn and with rhdi hdfn; the latter had also been transfused in utero. neonates on were transfused after birth: with rhdi hdfn, with ab i hdfn and with hdfn due to incompatibility for other antigens. summary/conclusions: our case studies reflect the literature data. neonates with rhdi hdfn are the most numerous ( . % of the total) and are those who have requested the highest blood supply both in the antenatal period ( . %) that postnatal ( . % performs et, . % performs postnatal transfusions). neonates with aboi hdfn are . %: nobody has received iut, only one has been subjected to et, and % has transfused after birth. patients with hdfn due to other antigens are %, have undergone iut . % and were transfused after birth . %. background: according to british guidelines on neonatal transfusion, since we shared with neonatologists a transfusion protocol for preterm babies. patients are anemic premature newborns with a gestational age ≤ weeks and/or a birthweight lower than g, until months of age. aims: reduce the incidence of iatrogenic anemia. methods: pre-transfusion tests were based on ab direct test, rh phenotype, direct and indirect antiglobulin test (dat, iat). a second blood sample was required for ab /rh confirmation. blood transfusions were performed with negative kell negative ( cde/cde/kk) cmv negative irradiated erythrocyte concentrates (ec) of less than days. ec were subdivided in ml aliquots with a hematocrit of ae %. according to the definition of "small volume transfusions", our protocol established that further four transfusions had to be delivered free of testing. after the fifth ec transfusion the supplementary release of ec was provided of type and screen (t&s) test with h of validity. serological investigation and full compatibility testing were applied in the presence of a iat and/or dat positivity and in the case of mother alloimmunization. results: from october to the end of , premature newborns received ec transfusions within their first months of life. in % of cases (n = ), transfusion requirement was comprised within the 'small volume transfusions'. another % of cases (n = ), requiring further ec administration, was requested of a blood sample for t&s determination and % (n = ) for a cross-match test. in . % of newborns (n = ), being transfused within the " small volume transfusions", blood requirement of ec was fulfilled by the initial blood test ( blood samples). . % of newborns (n = ) received more than transfusions ( - ; median = ) accounting for ec released and for this group blood samples were required. summary/conclusions: with the exception of babies requiring crossmatch test, blood tests were performed to sustain infants transfused with units. the alternative option of omitting crossmatch test (otherwise suggested by italian directives), allowed a reduction of % of blood drawn without any adverse effect or incident reported. due to the relevance of anemia in premature babies, we suggest the application of this transfusion policy in all newborns in the first months of life. background: glucose- -phosphate dehydrogenase deficiency (g pdd) is a sexlinked enzymopathy which is usually asymptomatic unless individuals are exposed to oxidative stress agents. the g pd genotype is the most common g pd genotype in sub saharan africa (ssa). some studies have linked blood from g pdd donors to poor outcome of a transfusion. however, there are no genetic screening programmes for blood donors in the region hence the contribution of g pdd to the donor pool in the ssa setting had not been described.aims: this study aimed to describe the prevalence of g pdd genotype among donors in two regions in uganda. it also described the effect of g pdd and the coinheritance of haemoglobin s and a-thalassaemia on the haematological quality of blood. methods: , blood samples from donor packs were utilized in a transfusion trial conducted in uganda, were genotyped for g pd , haemoglobin s and a-thalassaemia. haemoglobin and haematocrit measurements for the donor units (packs) at the time of transfusion were used to describe the effect of g pd and co-inheritance with a-thalassaemia (n = , ) and haemoglobin s (n = , ) on the haematological quality of blood packs. a subset of donor blood packs was utilized to determine the sensitivity and specificity of the carestarttm rapid diagnostic kit (rdt) for g pdd. results: based on g pd genotyping, . % (n = ) of the blood samples used in the trial were deficient for g pd enzyme while . % (n = ) were heterozygous. significant lower hemoglobin values were observed in red cell concentrates (p = . ) and whole blood (p = . ) donations of heterozygous g pd genotype. co-inheritance of g pdd and a-thalassaemia resulted in significantly lower haemoglobin levels. the carestarttm rdt test was . % sensitive and . % specific for detecting donor blood packs with g pdd. summary/conclusions: the prevalence of g pdd among ugandan blood donors was similar to that in the general population. the heterozygous genotype resulted in lower haemoglobin concentration of the blood units. the use of carestarttm rdt for screening of stored blood units was not as efficient in this study hence further testing for the determination of g pdd needs to be done on fresh samples from donors. transfusion medicine, jaypee hospital, noida, india background: during last two decade there has been a continuous remarkable improvement in desensitization therapy in high risk hla sensitized kidney recipients. in india there has been a tremendous increase in the number of kidney transplantations in patients having anti-hla antibodies (hla sensitized) with excellent success rate. aims: in present study, we are describing successful role of desensitization in hla sensitized patients having preformed donor-specific hla antibody (dsa). methods: all patients were preconditioned with combined modality of a standard dose of rituximab, therapeutic plasma exchange (tpe) and low dose ivig. tpe was started using com. tec (fresenius kabi, germany) after days of administration of rituximab. complement dependent cytotoxicity cross match (cdc-xm), luminex cross match with donor lysates (lm-xm, immucor inc., ga, usa) and flow cytometry cross match (fc-xm, bd facs canto ii).) was done in all cases. if any of the three tests was positive, single antigen bead assay (sab) was performed. desensitization therapy was given in all cases where dsa was detected. pretransplant tpe procedures were done until dsa (mfi < ) and cdc-xm became negative. cdcxm labeled positive at ≥ %. t-cell fcmx was considered positive above mfi and b-cell fcmx was considered positive above mfi. lmxm was considered positive above mfi. sab was performed using lifecodes single antigen (lsa) class i and class ii kits (immucor, usa). if the specificity of anti-hla antibodies was against donor hla antigen(s) it was called as donor specific antibody (dsa). results: present study demonstrated the diagnostic and clinical superiority of adding fc-xm and lm-xm in pretransplant compatibility testing algorithm over cdc-xm. cdc-xm alone was not able to detect anti-hla antibody in patients ( . %). among the three pretransplant compatibility tests, fcxm demonstrated highest sensitivity. among the cases initially screened showed dsa positivity in sab. desensitization was done in those cases only. in our study, sab was positive for class ii alone in ( %) while in remaining ( %) cases it was positive for both class and class ii. the number of pre transplant tpe procedures required was . ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . the mean number of post-transplant tpe sessions required was . (range, - ). during pretransplant and post transplant tpe procedures, five ( . %) patients presented with allergic or hypotensive reactions which were managed conservatively. most of the patients were discharged after seven days of hospital stay whereas patients who required post-transplant tpe were discharged after a relatively longer hospital stay (mean- . , median- days). after three months, protocol biopsy was done in those cases only where post transplant tpe was required. protocol biopsy showed normal findings. in present study, the mean duration of follow up was approx months with the longest duration of follow up of months. summary/conclusions: in a country like india where there is a huge gap in the demand and supply of kidney and a large no. of patients waiting for a suitable organ, transplant across hla barrier could a good doable option. thorough pretransplant compatibility and tpe are essential tools to make these transplants program successful background: most transfusion-dependent chronically anemic patients are managed by simple red cell transfusions. however, some patients are not able to tolerate the additional volume associated with simple transfusions and are at a high risk of developing transfusion associated circulatory overload, if transfused with multiple red cell units. plasma-to red cell exchange (prx) is a modified procedure wherein an apheresis machine is used to remove patient's plasma, while simultaneously replacing with donor rbcs. this procedure allows for a rapid euvolemic transfusion of rbcs to patients that are severely anemic and intolerant to excess fluid volume. others as well as our group have previously described this procedure. we now summarize our institutions nearly seven years of experience performing this procedure on a routine basis. aims: to document patient experience with prx. methods: we performed a retrospective chart review of all patients that underwent prx at our institution in the last seven years. our protocol for prx has evolved during this period. currently, we perform the procedures using spectra optia (terumo bct, lakewood, co) machine using the plasma-exchange program and tubing set. if the patient's pre-procedure hematocrit (hct) is < %, we custom prime the tubing set with % albumin. the number of red cell units transfused to the patient depends on their pre-procedure hematocrit and is individualized to the patients. results: we have treated four patients with prx procedure. patient # is a -year-old transfusion-dependent male with beta-thalassemia major. the patient had experienced multiple congestive heart failure exacerbations secondary to simple transfusions and we consequently performed prx procedure, every weeks, starting in . the patient has completed procedures with - units of washed red cells transfused to achieve a target hct goal of to %. he tolerated all procedures without any volume overload issues. he continues on this transfusion regimen. patient # was a -year-old female who had symptomatic anemia secondary to sickle cell disease (hb ss complicated by end-stage renal disease (esrd). she had progressively become intolerant to simple transfusions, including an episode of severe dyspnea, which required intubation. she underwent prx procedures with - units of washed red cells. patient tolerated the procedures without any significant complications. however, during a different surgical procedure, she experienced cardiac arrest and subsequently passed away. patient # is a -year-old transfusion-dependent male with severe anemia secondary to sickle cell anemia (hb ss). he was intolerant to excess fluid because of esrd and congestive heart failure. he has undergone prx procedures with - red cell units transfused to achieve a hct goal of %. he tolerated all procedures without any volume overload issues. he continues on this transfusion regimen. patient # is a -year-old male with a sickle cell disorder (hb ss) complicated by esrd, heart failure and chronic hypoxemic respiratory failure. the patient has undergone two prx procedures with - red cell units. other than an episode of non-bloody emesis that was symptomatically treated, he tolerated both procedures. he continues to be managed on this regimen. however, the patient remains noncompliant with treatment. summary/conclusions: prx is a safe and efficient method to transfuse multiple red cell units to volume-intolerant anemic patients. background: transplanted organ failure due to antibody mediated rejection in abo-compatible organs is a serious complication with a bad prognosis. the goal treatment in these cases encompasses the following strategies: adjustment of the immunosuppressive medications, ivig infusion, antibody removal by therapeutic plasma exchange, and/or the use of target-specific monoclonal antibody medications to lymphocytes, plasma cells, and/or complement. the american society for apheresis has assigned a category i to the use of therapeutic plasma exchange for the treatment of abo-compatible antibody mediated rejection in kidney, but a category iii to all other abo compatible organs: liver, lung, and heart. at our institution, a standardized approach for the use of therapeutic plasma exchange as a supportive intervention for abo-compatible immune mediated rejection, regardless of the organ type, has been in place since . aims: a retrospective review was performed to evaluate our patient outcomes using therapeutic plasma exchange for the treatment of antibody mediated allograft rejection in abo-compatible solid organ transplantation. methods: patients used for the retrospective review were selected from an existing therapeutic apheresis list. the therapeutic plasma exchange protocol consists of: adjustment of the immunosuppressive medications, ivig infusion, antibody removal by therapeutic plasma exchange, and/or the use of target-specific monoclonal antibody medications to lymphocytes, plasma cells, and/or complement. it is performed as follows: one plasma volume exchange is performed on days , , , , , along with one or more of the above strategies followed by an ivig infusion. cases with allograft rejection in which plasmapheresis was not used were excluded. and t devos aims: this study aimed to explore the possible causes of the decreased transfusion rate for all adult cardiac surgery patients. methods: data were collected from adult cardiac surgery patients during the mentioned time frame and were extracted from electronic patient files and a database of the department of cardiac surgery. a set of variables was defined as possible confounders by a panel of experts. after discussion, global variables (age, gender, duration of surgery, use of cpb (cardio-pulmonary bypass), american society of anesthesiologists (asa) risk score, type of surgery, urgency, attending cardiac surgeon and attending anesthesiologist) and cpb-related variables (administration of cardioplegia yes/no (cpg), duration of cpb, circulatory arrest, hypothermia, duration of aortic cross-clamp, baseline hemoglobin and cpb-priming volume) were retained. negative binomial models for counts were used for data analysis. all analyses were performed with spss. results: patients were extracted from databases and further analyzed. the mean age of this group was , years (sd +/- , years) and . % of them were male. the mean duration of surgery was min (sd +/- , min). the decrease of perioperative rbc transfusion rate over four years was statistically significant (p < . ). in , the mean use was , units per operation (sd +/- , ), which changed to , units (sd +/- , ) in . three variables (urgency, attending cardiac surgeon, attending anesthesiologist) changed significantly over years and were used in a multivariable model as confounders together with rbc transfusions and year. even after adjustment for these factors, the decrease in rbc transfusion rate was still statistically significant (p < . ). in the specific group of patients undergoing cardiac surgery with cpb (n = ), the use of rbc was also significantly reduced (p < . ). in , the mean use was , units per operation (sd +/- , ) and this changed to , units (sd +/- , ) in . after correction for the cpb variables that notably changed over the years (cpg, priming volume and hypothermia) and the three previously defined confounders (urgency, attending cardiac surgeon and attending anesthesiologist) the reduction of rbc transfusions over years still remained statistically significant (p < . ). summary/conclusions: our study shows evidence for a decreased rbc transfusion rate in adult patients undergoing cardiac surgery between and . this tendency was also seen in the subgroup of patients undergoing surgery with cpb. possible explanations of the decrease are implementation of various established parts of patient blood management. however, a unique reason could not be identified in this study. background: growing worldwide demand for immunoglobulin products such as intravenous immunoglobulin (ivig) and subcutaneous immunoglobulin (scig) is driving plasma collection. patients with primary immunodeficiency (pid) or secondary immunodeficiency due to haematological malignancy or its treatment (sid) rely on these products to maintain therapeutic serum igg levels to minimise recurrent infection. efficacy of immunoglobulin replacement therapy (irt) in pid is well established but information on sid is limited. the different aetiologies of hypogammaglobulinaemia between pid and sid raised the question of whether sid patients on irt experience similar clinical and quality of life (qol) benefits as reported in pid patients. aims: to assess whether sid patients experience similar clinical and qol benefits while on irt as pid patients. methods: following ethics approval, data on dosage, serum igg trough levels and infection (bacterial, viral and fungal requiring treatment such as antibiotics) was collected from adult pid and adult sid patients from medical records and pathology reports, for their last months of ivig and their first months of scig. the starting and maintenance dose was . g/kg/month for ivig, transitioning immediately to . g/kg/week for scig without a washout period. a study specific questionnaire was developed to gather data on patient perceived side effects, treatment satisfaction and impact of irt on social/family life, work/study and their overall qol. paired t-test was used for parametric data and the wilcoxon signed-rank test for non-parametric data. results: sid patients were significantly older with a mean age of . years versus . years in pid patients (p = . ). a mean of three training session was required to reach competency in scig administration in both cohorts. there was a trend of reduced side effects on scig for pid and sid patients compared to ivig, with a significant reduction of headaches in the pid cohort (p = . ). the majority of patients experienced infusion site reactions, which were predominantly perceived as manageable. % of infections were respiratory tract infections. pid patients had slightly higher mean serum igg trough levels with scig ( . g/l) compared to ivig ( . g/l), and fewer infections on scig than ivig (mean annual infection rate of . vs . respectively). sid patients had higher mean serum igg trough levels on scig ( . g/l) than ivig ( . g/l) (p = . ) but experienced more infections while on scig versus ivig (mean annual infection rate of . vs . respectively). the number of hospitalisation due to infection decreased in both cohorts with scig. pid patients perceived that switching from ivig to scig improved their health and qol. in contrast sid patients perceived no improvements in health and qol. summary/conclusions: data from this pilot study suggests that the clinical and qol impact of irt in sid patients is different to that of pid patients. to support evidence based irt management and effective use of this limited and expensive blood product in sid, larger studies which account for different stages of malignancy and associated treatment regimes are required. background: there is an increasing platelet transfusion for treatment and prophylaxis of bleeding in patients with hematologic disorders and malignancies. because of limited resources, leukoreduced platelet concentrates is not yet implemented in most indonesian hospitals. in vitro platelet activation may cause morphology, functional, and ultrastructure changes. those changes will reduce the platelet viability, in vivo functions, and clinical efficacy. high platelet cd p expression is the cause of faster platelet destruction in the reticuloendothelial systems. post-transfusion in vivo hemostatic efficacy can be determined by the measurement of corrected count increment (cci), recovery, and platelet cd p expression. aims: to analyze the increase of platelet cd p expression in patients of non-leukodepleted compared to pre-storage leukodepleted pc transfusion.background: haemorrhage is a leading cause of preventable death not only in the military trauma care, but also for civilian population suffering accidents or bleeding injuries in regions with low population density where health services should reach people in remote areas. resuscitation using blood products and limited infusion of normal saline improves survival for critically bleeding patients. nowadays there are hems programs (helicopter emergency medical system) carrying blood products around the world. the hems in castilla-la mancha, with physician and nurse, is the first out-of-hospital emergency service in spain that provides packed red blood cells (prbc) transfusion where the accidents happen without delaying the transport to the proper hospital for definitive treatment. this program has been developed between the blood center of ciudad real and the hems team ('gigante ', emergency service castilla-la mancha). the goal of the designed protocol was to preserve the properties of the product to be transfused in out-of-hospital environment by hems teams. aims: to describe the process for out-of-hospital prbc transfusion in hems of ciudad-real. the protocol for out-of-hospital blood transfusion was developed according to criteria of medical indications and security, monitoring, and tracking of the product. methods: data for the observational retrospective study were collected from june to august . the medical helicopter (ec t ) was provided with two prbc o rh(d) negative. the shock index was selected for the indication for transfusion according to the literature revised and as a simple rate to obtain out-of-hospital data. to achieve the feasibility and preservation of the prbc a prospective monitoring of volume was established, haematocrit, haemoglobin, leucocytes, coulter, hemolysis and microbiological culture. blood center established two groups: the case group for the prbc kept in the hems base and helicopter and the control group for the units remaining in the blood center with standardized blood conservation. for both groups, control and comparison of immediately obtained hematologic analyses, and days after collection, were performed. the statistical analysis used spss . version (significance p < , ). results: prbc samples were evaluated, , % ( ) from case group and , % ( ) from control group. analyses were tested day and day after collection. haemolysis was not observed. all cultures were negative. although significant differences were found between the parameter in the value of before-after in the value of the hematocrit, leukocytes and coulter, there are no differences between the prbc that flew and those conserved in the transfusion-service. all results comply with current legislation and blood transfusion standards. there have been administered prbc transfusion to patients during out-of-hospital advanced medical assistance. no post-transfusion reactions have been registered. prbc units have a -day rotation to allow the use of the units in the hospital after achieving their optimal status. summary/conclusions: the out-of-hospital transfusion protocol designed to transport blood (prbc) in the helicopter for hems has demonstrated to keep the standard conditions and properties of the product to be considered useful in the treatment for critically bleeding patients in the out-of-hospital. background: early and adequate treatment of major bleeding is important for survival and a good outcome. blood and plasma are given increasingly early including before hospital admission in trauma based on successes reported from combat experience. in the national patient safety agency issued a rapid response report requiring national health service hospitals in england to take actions to improve provision of blood in an emergency including provision of major haemorrhage protocols (mhp) and drills. the national reporting and learning scheme had identified reports of deaths and instances of harm due to delay over a -year period. aims: the aim of the study was to monitor the acid-base status of the patient by means of abg and to administer the blood component therapy based on teg results. methods: this study was a prospective observational study of adult patients over a period of months. serial monitoring of the abg in the intra-operative period was done. teg guided resuscitation was performed in all cases. results: the abg analysis of all patients showed decrease in the ph, increase in pco , decrease in serum bicarbonate level and elevation in negative base excess. these components of metabolic acidosis can be attributed to massive transfusion. increased lactate, an independent parameter, which reflects poor tissue perfusion or shock and predicts need for massive transfusion was observed in all patients. all the cases showed a decrease in ionized calcium levels which could be a result of citrate related toxicity. increased glucose was observed in all patients which may be due to increase in the catecholamine release as a response to haemorrhagic shock. electrolyte correction was given depending on results of the abg analysis wherever appropriate. two out of cases showed an increase in r time indicating deficient coagulation factors, which was corrected with fresh frozen plasma (ffp). three cases showed elevation in k time indicating deficient fibrinogen levels, which was corrected by ffp. fresh frozen plasma was also given in cases, which showed decrease in the alpha angle, indicating deficient fibrinogen, and cryoprecipitate was given in cases. platelets were transfused in patients showing a decrease in the maximum amplitude (ma), which indicates deficient platelets. summary/conclusions: teg as poc testing is an important tool in appropriate blood component therapy in massive transfusions. serial monitoring of abg helps in monitoring acid-base status of the patient and therefore is a guide in the correcting electrolyte level in patients undergoing massive transfusion. background: massive blood loss is encountered in various situations like trauma, major surgeries, gastrointestinal bleeds and obstetric haemorrhages. haemorrhage is an important cause of mortality and morbidity in massively bleeding patients. early recognition of haemorrhage and intervention is essential for survival. massive transfusion (mt) of blood is required to replenish blood losses and is a lifesaving treatment in these patients. a variety of haemostasis and pathophysiological changes occur during massive haemorrhage and massive transfusion. all of these changes contribute to the vicious cycle of progressive coagulopathy due to the 'lethal triad' of refractory coagulopathy, progressive hypothermia and persistent metabolic acidosis. aims: the aims of the study included understanding management of cases of massive blood transfusion in surgical patients, impact of mt of blood components on patient outcome, evaluating post-operative complications of massive transfusion and the development of institutional massive transfusion protocol (mtp).methods: this prospective observational study commenced after institutional ethics committee (iec) approval. it comprised of adult surgical oncology patients who received massive transfusions and was conducted for a period of months. every case of a massive transfusion was studied under the following headings ( ) patient's details ( ) patients base-line laboratory tests ( ) resuscitation with transfusion ( ) intra-operative laboratory tests ( ) thromboelastography (teg) ( ) post-operative complications ( ) duration of stay in the hospital ( ) day mortality rate. results: complete blood count, serum electrolytes, arterial blood gases, coagulation profile and teg were used to monitor transfusion therapy in the intraoperative period. intraoperative laboratory parameters of patients showed dilutional coagulopathy, metabolic acidosis, hypocalcaemia, hypomagnesaemia, hyperkalaemia and hypokalaemia, increased lactates and glucose. electrolyte correction was done based on the derangement. the derangements were on a decreasing trend in the postoperative period and returned to baseline level by nd or rd post-operative day with no requirement of correction in the post-operative period. the post-operative outcomes were evaluated in terms of the surgical site infection (ssi) as per the centers for disease control (cdc) criteria, surgical complications as per modified clavien-dindo classification and respiratory complications. a total of ( . %) patients had ssi, ( %) had surgical complications and ( %) patients had respiratory complications. the length of the stay in the hospital was longer for patients who had postoperative complications. despite complications, owing to excellent peri-operative care, ( %) patients were discharged alive. summary/conclusions: surgeries associated with massive transfusion are at an increased risk of ssi as well as morbidity and mortality. complications associated with rapid transfusions of blood, acute haemorrhage and associated risk of the surgery lead to a prolonged icu stay and increased length of stay in the hospital. a well-developed massive transfusion protocol optimizing the ratio and dose of the blood component therapy results in excellent patient outcome with minimal postoperative morbidity and mortality. background: despite the introduction of new oral anticoagulants (dabigatran, rivaroxaban, apixaban), vitamin k antagonists (vka), such as warfarin and acenocoumarol are still the most widely used oral anticoagulants for the treatment of non-valvular atrial fibrillation (nvaf). the use of vka must be regularly and often laboratory controlled in order to ensure the adequacy of therapy and to avoid subdosing or drug overdose. the most commonly used test for the control of oral anticoagulant therapy is the prothrombin time (pt), expressed in inr system, which provides an internationally standardized monitoring of the treatment. time in therapeutic range (ttr) represents a measure of the quality of the anticoagulant effect of vka and estimates a percentage of time a patient's inr is within the desired therapeutic. aims: the aim of this study was to evaluate of the effectiveness of vka therapy in patients with nvaf and to identify factors affecting the anticoagulation efficacy. methods: a retrospective study was conducted on a population of outpatients with nvaf, treated with vka and followed in blood transfusion institute of ni s from january to december . laboratory control of inr was done from capillary blood of patients on thrombotrack solo (axis shield, norway) and thrombostat (behnk elektronik, germany). targeted ae . %, but . % of patients had a ttr less than %. patients were at high risk of thrombosis in . % of time (inr < . ) and high risk of bleeding in . % of time (inr > . ). the most significant independent factors affecting the quality of vka therapy are gender, arterial hypertension, diabetes mellitus and the use of amiodarone and antiplatelet drugs (aspirin, clopidogrel). summary/conclusions: the ttr is undoubtedly useful indicator of the effectiveness of vka treatment. the most important predictors of poorer efficacy of vka therapy are arterial hypertension, diabetes mellitus, patients' gender and the use of amiodarone and antiplatelet (aspirin, clopidogrel) drugs. to improve the quality of vka therapy, education of patient and better collaboration with them, as well as a successful teamwork with clinicians are also imperative. background: an estimated . million deaths per year result from haemorrhagic blood loss. at a cellular level, haemorrhagic shock develops when oxygen delivery is insufficient to meet oxygen requirements to maintain aerobic metabolism. successful resuscitation prevents further oxygen debt and repays the prior oxygen debt. this includes the administration of fluids and blood components (e.g. plasma, red cells and platelets). measurement of oxygen delivery and utilisation at a tissue level requires invasive monitoring not possible clinically, meaning that surrogate markers such as lactate and venous oxygen saturation (svo ) are used instead. new technologies such as incident dark field imaging and near-infrared spectroscopy may offer a non-invasive alternative; however their utility in haemorrhagic shock remains background: transfusion-induced red cell alloimmunization is still a major challenge in transfusion practice. besides logistic problems for the transfusion laboratory, it may compromise available blood supply, and when undetected and unanticipated, it may risk haemolytic transfusion reactions. knowledge about risk factors can help to optimize preventive matching strategies. severe renal failure and subsequent renal replacement therapy influence the immune system and could therefore modulate the risk of alloantibody formation against foreign red cell antigens subsequent to transfusion. aims: this study aims to quantify the association between renal failure, according to its degree and its treatment with renal replacement modalities, and transfusioninduced red cell alloantibody formation. methods: we performed a multicenter case-control study within a source population of patients receiving their first and subsequent red cell transfusion between and in the netherlands (risk factors for alloimmunization after red cell transfusion, r-fact study). using a conditional multivariate logistic regression, we compared first-time transfusion-induced red cell alloantibody formers (n = ) with two similarly exposed non-alloimmunized control recipients (n = ) during a five-week alloimmunization risk period. degree of renal function was categorized as: 'no renal failure' i.e. glomerular filtration rate (gfr) > ml/min/ . m , 'moderate renal failure' i.e. gfr ≥ - ml/min/ . m during a continuous period of minimally seven days, 'severe renal failure' i.e. gfr < ml/min/ . m and/or use of any type of renal replacement therapy during at least one day of the alloimmunization risk period. odds ratios were interpreted and presented as relative risks (rr). adjusted rrs were conditioned on the matching variables and identified confounders. results: no renal failure was observed among ( . %) cases versus ( . %) controls; moderate renal failure among ( . %) cases versus ( . %) controls; and severe renal failure among ( . %) cases versus ( . %) controls. among the latter, cases and controls underwent renal replacement therapy. moderate renal failure and severe renal failure without renal replacement therapy were not significantly associated with red cell alloimmunization (adjusted rr . , % ci . - . and adjusted rr . , % ci . - . , respectively). however, patients undergoing renal replacement therapy had a two-fold lower alloimmunization risk (adjusted rr . , % ci . - . ) as compared to transfusion recipients without renal failure, unrelated to type and duration of renal replacement therapy. summary/conclusions: these findings suggest that patients undergoing renal replacement therapy have strongly diminished red cell alloimmunization risks. further research should confirm these results and elucidate the underlying pathophysiological protective mechanism. background: the ability of allogeneic hematopoietic stem cell transplantation(allo-hsct) to prevent relapse depends partly on donor natural killer (nk) cell alloreactivity. nk effector function depends on specific killer-cell immunoglobulin-like receptors (kir) and hla interactions. thus, it is important to identify optimal combinations of kir-hla genotypes in donors and recipients that could improve hematopoietic transplantation outcome. aims: to obtain the optimal combinations of inhibitory kir and its ligand between donor and recipient which is helpful for the guidance of selecting donors and recipients in hsct. methods: the pcr-sbt method was used for kir dl , kir dl /kir dl , kir dl , kir dl and hla-a, -b, -c, -drb , -dqb genotyping. pairs of hla / identical donor/recipients matching samples were retrospectively analyzed. three different models of kir were established. there were kir-kir gene model, kirligand model and haploid model. in kir-ligand model, patients were divided into three groups: c /c homozygote group ( cases), c /c heterozygote group ( cases) and c /c homozygote group ( cases). according to the expression of dl , cases were dl positive and cases were dl negative. there were cases of bw /bw , cases of bw /bw and cases of bw /bw in the dl positive samples. according to the expression of a /a , they were divided into three groups: a /a negative group ( cases), a /a heterozygous group ( cases) and a / a homozygote ( cases). according to kir genotyping, kir haploidentical group ( cases) and kir haploid mismatched group ( cases) were divided. the clinical data on neutrophil and platelet remodeling time, gvhd and os of cases were statistically analyzed by the mann-whitney test or the kruskal-wallis test using graph-pad software v . . results: there was no significant difference in the time of neutrophil and platelet remodeling, the incidence of agvhd and the survival time after transplantation in the kir genotype model. in haplotype model, there was no significant difference in neutrophil and platelet remodeling time and survival time after transplantation. the incidence of agvhd was low when the kir haploid mismatched and kir dl was positive. it was conducive to neutrophil and platelet remodeling when bw /bw and a /a was heterozygosity. summary/conclusions: it is important to establish the three different models of kir genotypes, haplotypes and receptor-ligand mismatches for analyzing the effect on the prognosis of allo-hsct. kir-ligand model plays an important role in hla unre-background: transfusion of platelet concentrates (pcs) has been associated with adverse outcomes including transfusion-related acute lung injury (trali). the underlying mechanism of trali has been related to the accumulation of immunomodulatory mediators (e.g. lipids, cytokines/chemokines) present in pcs. current room temperature storage limits the shelf-life of conventional pcs to - days. alternative storage conditions, including cryopreservation, offers extended storage and a solution for blood banking logistics. cryopreservation of human pcs has been associated with higher concentrations of immunomodulatory mediators compared to room temperature stored pcs and it has been suggested that cryopreserved pcs may be immunomodulatory. to investigate the effects of cryopreserved pcs, a transfusion sheep model would be a beneficial approach. aims: to characterize immunomodulatory mediators in supernatants of sheep conventional and cryopreserved pc and to investigate whether storage duration and cryopreservation impacts the accumulation of these mediators. methods: buffy coat pooled sheep pcs (n = ), prepared in % plasma/ % ssp+ with minor modifications to standard human procedures, were stored room temperature (rt) for days (d) and sampled on d , d and d . cryopreserved sheep pcs (n = ), prepared by the addition of - % dimethyl sulfoxide, were stored at À °c and sampled pre-freeze and post-thaw. supernatant was prepared at each time point with double centrifugation and stored at À °c. concentrations of pro-inflammatory cytokines (interleukin (il)- , il- b, il- a), anti-inflammatory cytokine il- and chemokines (il- , monokine induced by gamma interferon (mig) and interferongamma induced protein (ip)- ) were measured using sheep specific in-house and commercial enzyme linked immunoassays (elisa). levels of non-polar lipid mediators, such as arachidonic acid (aa), -hete and -hete were assessed in the stored sheep pc-and cryo-pc supernatant using commercial elisa. results shown as mean ae standard deviation. the effect of storage was determined at p < . using paired t-test. results: in rt stored sheep pc supernatant il- , il- b, il- a, il- , il- , mig, ip- , -hete and -hete were detected at d , d and d . storage duration significantly increased accumulation of ip- at d ( . ae . pg/ml compared to . ae . pg/ml, p = . ) and further increased at d , and il- at d ( ae . pg/ml compared to ae . pg/ml, p = . ). cryopreserved sheep pc supernatant pre-freeze and post-thaw contained equivalent or higher concentrations of il- , il- b, il- a, il- , il- , mig, ip- , -hete and -hete than rt stored d pcs. however, cryopreservation did not impact levels of any of the platelet derived mediators. summary/conclusions: several platelet-derived cytokines/chemokines, including high concentration of il- with neutrophil priming activity, and non-polar lipids were found in stored sheep pc supernatant. these immunomodulatory mediators may contribute to adverse outcomes associated with pc transfusion. storage at rt, but not cryopreservation was associated with increased accumulation of immunomodulatory mediators in sheep pcs. most importantly, similar to human pcs, sheep cryopreserved pcs contained at least if not higher concentrations of majority of cytokines as pcs stored at rt, therefore making sheep a suitable model in which to investigate immunomodulatory effects of cryopreserved pc transfusion. background: transfusion, despite being a lifesaving therapy, has been associated with adverse transfusion outcomes. transfusion related acute lung injury (trali) remains one of the leading causes of transfusion-related mortality. accumulation of immunomodulatory mediators (e.g. lipids, cytokines/chemokines) present in blood products, including packed red blood cells (prbcs), have been implicated with the development of non-antibody mediated trali. however, how specific mediators contribute to the underlying mechanism is yet to be defined. during routine storage of human prbcs fewer than cytokines/chemokines and several biologically active lipids have been identified. a sheep model of trali has successfully been developed using human prbc supernatant, however transfusing sheep prbc has not been investigated. to support the use of sheep prbc in the trali model and to better understand the precise mechanism, characterization of the potential mediators in sheep prbc is required. aims: to characterize immunomodulatory mediators in sheep prbc supernatants and to investigate whether storage duration impacts the accumulation of these mediators. methods: sheep prbcs (n = ), prepared according to standard human procedures with minor modifications, were stored ( - °c, days (d) ) and sampled at d and d . supernatant was prepared by double centrifugation and stored at À °c. concentrations of pro-inflammatory cytokines (interleukin (il)- , il- b, il- a), antiinflammatory cytokine il- and chemokines (il- , monokine induced by gamma interferon (mig) and interferon-gamma induced protein (ip)- ) were measured using sheep specific in-house and commercial enzyme linked immunoassays (elisa). levels of potential non-polar lipid mediators (arachidonic acid (aa), -hydroxyeicosatetraenoic acid (hete) and -hete) were assessed in the sheep prbc supernatant using commercial elisa. paired t-test was used to compare fresh and stored prbc supernatant (p < . ). results are mean ae standard deviation. results: at day , aa ( , ae , pg/ml), -hete ( . ae . pg/ml), -hete ( . ae . pg/ml) and il- b ( . ae . pg/ml) were detectable in sheep prbcs supernatant. at day , storage duration significantly increased concentrations of aa ( , ae , pg/ml, p = . ) and -hete ( . ae . pg/ml, p = . ) in sheep prbcs supernatant. summary/conclusions: similar to reported findings of human prbcs, the predominant type of immunomodulatory mediators present in sheep prbcs were non-polar lipids. the concentration of these non-polar lipids increased during storage. these immunomodulatory mediators may contribute greatly to adverse outcomes associated with prbc transfusions. further investigation is required to determine whether stored sheep prbcs supernatant induce immunomodulation in sheep in vitro and in vivo transfusion models. background: dshtr incidence is reported as in , transfusions, presenting days to months after the transfusion. the published data addressing the correlation between the strength of the antibodies detected after a dshtr has taken place and the corresponding clinical symptoms as measured by laboratory parameters that assess the presence of hemolysis is limited. aims: the aim of this study is to evaluate the correlation between the results of the dat, automated and manual antibody reactivity strength with the corresponding clinical parameters of hemoglobin, lactate dehydrogenase (ldh), bilirubin, and haptoglobin. methods: a dshtr is defined as discovering a new antibody within days of a transfusion. for all positive antibody screens, a work-up is initiated consisting of identification panels, dats, antigen typing of the red cells transfused, and eluates at the discretion of the transfusion medicine physician. additional laboratory testing for hemolysis is requested when indicated. a retrospective review was conducted of patients who were identified as having a dshtr. levels of hemoglobin, ldh, and transfusion safety background: rhd immunoglobulin (rhdig) has been available for years in australia. since its introduction for routine antenatal and postpartum prophylaxis, alloimmunisation has decreased from % to . %, reducing the number of australian deaths from haemolytic disease of the newborn over a hundred-fold, to approximately . deaths per . blood matters serious transfusion incident reporting (stir) system has been collecting transfusion incidents and adverse events across four australian jurisdictions since . since january , rhdig administration errors have been reported. aims: to understand incidents relating to the administration of rhdig and increase safety and awareness of risks. methods: health services registered with stir (n = ) were notified of the inclusion of reporting rhdig incidents. when an incident is identified, the reporter sends an online notification to stir, prompting the appropriate investigation form to be sent for completion. the completed incident data are reviewed and validated by an expert group. data is de-identified and collated for reporting. results: during the period january -december , reports were received; reports were validated, with reports excluded (reactions rather than administration errors). reports were categorised as below: background: following the nice transfusion guidelines, recommending offering iron before and after surgery to patients with iron-deficiency anaemia (ida), we worked collaboratively with the anaesthetic and pre-operative team to implement a clear and robust anaemia pathway for pre-operative haemoglobin (hb) optimisation. oral iron was started, where appropriate, and our anaemia pro-forma was sent for review in a virtual clinic to assess eligibility for intravenous iron. we performed a retrospective evaluation of the patients who received iv iron during the anaemia pathway. aims: the aim of this retrospective evaluation was to look at the cohort of patients who had received iv iron in and assess the effect of iv iron on haemoglobin levels for different defined groups. methods: we classified patients, as described in munting and klein, , depending on their iron parameters as having either: -idaserum ferritin < mg/l -chronic inflammation with idaserum ferritin - mg/l with transferrin % of < %/crp > mg/l -anaemia of chronic inflammationserum ferritin > with transferrin % of < %/crp > mg/l patients were considered eligible for iv iron if the following criteria were met: . an inadequate response to oral iron, or were unable to tolerate oral iron or the interval between diagnosis and surgery was short . the anaemia pro-forma was completed . hb was ≤ g/l . they were classified as either having ida or chronic inflammation with ida or anaemia of chronic inflammation hb was measured prior and on average, days following the iv iron infusion. we excluded patients who had their post iv iron follow up blood tests done after surgery. results: this retrospective evaluation included patients. patients were classified as having ida and patients classified as having chronic inflammation with ida. those classified with ida had a mean hb of g/l ( - ), a mean mcv of . fl and a mean serum ferritin of lg/l. those with chronic inflammation with ida had a mean hb of g/l ( - ), a mean mcv of . and a mean serum ferritin of lg/l. follow-up hb was measured on average twenty days post iv iron infusion in both groups. the average hb post iv iron infusion in the ida group was g/l ( - ) with an average increment of g/l and in the group with chronic inflammation with ida the average post iv iron hb was g/l ( - ) with an average increment of g/l. summary/conclusions: in conclusion the group with ida had, on average, a lower starting hb that the group with chronic inflammation with ida and the average increment in hb days post iv iron infusion was greater in the group with ida. however, the group with chronic inflammation with ida cases also responded to iv iron and therefore we strongly consider the use of iv iron in both groups. further studies to evaluate the ongoing effect of iv iron would help assess whether the same level of increment seen with ida can also been seen for the group with chronic inflammation with ida over a longer period and how long the increment was sustained. background: the expansion of personalized genomic medicine has led to the development of targeted therapeutic approaches for patients. one example is sipuleucel-t, an autologous cellular immunotherapy product used to treat prostate cancer manufactured from the patient's white blood cells. this study describes our experience with incorporating autologous cellular immunotherapy products into the workflow of the blood bank. the policies supporting the workflow are outlined and compliance with them is assessed. aims: this study aims to evaluate the process and method used to dispense and track the infusion of sipuleucel-t. methods: this is a retrospective analysis of the dispensation and administration of sipuleucel-t from january -august , which was handled exclusively by the blood bank. standard operating procedures and hospital policies were reviewed and compliance with these policies evaluated. included were patients who had the sipuleucel-t product dispensed and administered. information collected included the total number of products dispensed, patient age, adverse reactions/incidents, premedication, and patient outcome. descriptive statistics were used for data analysis. results: there were products dispensed to patients. the recipients were male patients diagnosed with prostate cancer with a mean age of years. there were doses (a complete course) administered to / ( %) recipients and a partial course ( - doses) administered to / ( %) recipients, for a total of products. the blood bank workflow treated sipuleucel-t as a derivative in the computer system, listing the manufacturer (dendreon corporation) as the supplier. health care providers were instructed to follow the nursing policy for the administration of blood products and derivatives for the infusion of sipuleucel-t. this policy required documentation of the infusion in a transfusion nursing note and reporting adverse events to the blood bank as transfusion reactions. there were no adverse events reported to the blood bank, yet there were adverse events described in provider notes; of them necessitating transfer to the emergency department, and requiring hospital admission. of the infusions, infusions were documented in a chemotherapy note rather than a transfusion note ( %), and ( %) were documented as both a transfusion and a chemotherapy administration. there were additional deviations from the blood product administration policy: cases where the consent check was not performed, case where the product was infused with ringer's lactate rather than normal saline, and cases where the -person -way check erroneously indicated the product was irradiated. summary/conclusions: this study describes one approach to managing cellular therapy products as an extension to existing blood products dispensed by a blood bank. the findings demonstrate noncompliance with hospital policy with this new product as evidenced by failure to report adverse events and failure to follow hospital practices regarding administration. although sipuleucel-t is a product manufactured from an autologous blood product donation, the administration and perceptions of this product may be more similar to a pharmaceutical. as the field of immunotherapies derived from blood product donations continues to expand, these products may necessitate an entirely new approach to ensure proper management. abstract withdrawn. (ref ) . while the mnc procedure is fully automated, cmnc requires frequent interface checks to ensure the collection of the correct cell layer. at the rambam health care campus, a tertiary care center, solely the mnc procedure had been employed till , at which point, the cmnc has been introduced for the use in patients with a white blood cell (wbc) count of ≥ , /ll on the collection day. aims: the current study aimed to compare various parameters of peripheral blood stem cell (pbsc) collection, using the cmnc protocol in allogeneic donors and patients undergoing autologous stem cell (autosc) transplantation. additionally, data on autosc collection using mnc (n = ) and cmnc (n = ) procedures were compared. methods: data were retrospectively obtained from pbsc collection reports in consecutive cmnc procedures, including autologous and allogeneic donors. the following comparisons were made: cmnc results of allogeneic versus autologous donors, a sub-analysis of cmnc results for autologous donors with a pb cd + count ≥ /ll versus allogeneic donors as well as mnc versus cmnc results in autologous donors. the collection efficiency- (ce- ) was defined as the total cd + amount in the collection bag divided by the amount of cd + cells in the pb processed by the collection apparatus. results: in the cmnc, the following parameters significantly differed between autologous and allogeneic donors: mean collection time ( ae and ae min, respectively; p = . ), the total blood volume processed ( . ae . and . ae . , respectively; p = . ) and the final volume in the collection bag ( ae and ae ml; p = . ). the mean ce- in autologous versus allogeneic donors was ae and ae , respectively (p = . ). using cmnc, the collection was effective in % of allogeneic and % of autologous donors. in autologous donors, a significantly lower collection bag volume ( ae and ae , respectively; p < . ) and increased total wbc in the collection bag ( ae versus ae , respectively; p < . ) were obtained using cmnc compared to mnc protocol. thirteen patients were treated with plerixafor due to a low pb cd + count following g-csf therapy; of them achieved a cd count ≥ and their collection was considered effective. summary/conclusions: the cmnc protocol is highly effective in terms of the cell yield in both allogeneic and autologous donors with a pb cd + count ≥ /ll. significantly superior collection results are obtained in allogeneic donors versus autologous ones. cmnc provides a significantly higher wbc and a lower final collection volume than mnc. similar total cd + cell counts are obtained with both methods. . tbv processed ranged from - . tbv with mean of . , average was . tbv for females and . tbv for males mean pre-apheresis cd + count was . cells/ll (range . - . ). mean postapheresis cd + count was . cells/ll ( . - . ). mean cd + cells x / kg recipients body weight was . (range: . - . ). our target yield was ≥ cd + cells/kg body weight of the recipient and in only / ( %) cases, the yield was < . / ( . %) procedures were lvl and / ( . %) were svl. summary/conclusions: most of our pbsc were done for haematological indications ( . %) and the target dose was cells/ll in single leukapheresis. in cases ( %), target yield was achieved, only cases had < but > yield. in our study donors < years have shown to mobilize better than the older children. hematocrit (hct) and weight showed correlation with cd + cell yield but they cannot be taken absolute predictors. wbc count cannot be taken as a predictor for cd yield as high wbc count did not convert into high cd yield or vice versa. high prepheresis cd + count gave higher postpheresis cd + count. large volume leukapheresis (lvl), > tbv gave higher yield as compared to standard volume leukapheresis (svl). blood volume processed related to prepheresis cd + count and/or the weight difference between the donor and recipient. other parameters like hematocrit, wbc count, age etc did not show correlation to the volume processed. in our study, younger age and prepheresis cd + count were found as the most relevant predictors for stem cell yield. background: allogeneic hematopoietic stem cell transplantation is an established therapy for many hematologic disorders. since the discoveries of the potential of peripheral blood stem cells (pbsc) in the hematopoietic reconstitution mid s and early s pbsc gradually replaced bone marrow as the preferred source of stem cells. the introduction of hematopoietic cytokines that can mobilize large number of progenitors into circulation accelerated pbsc usage. aims: the aim of our study is to present our year experience with apheresis collecting of pbsc in donors. methods: this is a retrospective study performed in the institute for transfusion medicine of republic of macedonia and university hematology hospital for period background: obtaining unambiguous results of hla typing plays an important role in the transplantation of hematopoietic stem cells. appropriate selection of alleles in the level of hla between recipients and unrelated bone marrow donors reduces the risk of transplant rejection and graft-versus-host disease. new generation technology ensures the highest possible resolution and obtaining unambiguous genotyping results due to the high complexity of the hla system. currently, this is the selection method for obtaining hla test results at the high resolution level. aims: the aim of this study was to determine hla loci (hla-a, -b, -c, drb / / / , dqb , dpb , dpa , dqa ) in potential bone marrow donors from poland. the research included , potential bone marrow donors registered between and . a novelty of this paper was that the amplification of all hla loci was performed by using multiplex pcr primers in a single tube. that solution completely eliminated the need to pool amplicons. methods: the typing of the hla loci (hla-a, -b, -c, drb / / / , dqb , dpb , dpa , dqa ) of potential bone marrow donors was made by using the alltype tm ngs -loci amplification kit (one lambda). genomic dna was isolated from peripheral blood of , donors. hla genotypes were determined according to the manufacturer's protocol on the miseq illumina platform. the obtained sequencing data was evaluated by using the typestream tm visual ngs analysis software. results: the ngs method allowed to obtaining unambiguous results of genotyping of potential bone marrow donors, and also provided the identification of rare alleles, such as: c* : , c* : , c* : , c* : , drb * : , c* : , b* : , c* : , dqb * : , drb * : , drb * : . summary/conclusions: . new generation sequencing technology (ngs), which is based on pcr, ensures the highest possible resolution. . the ngs method allows to obtain more accurate sequencing results compared to the conventional methods. . the research has confirmed the superiority of the ngs method over conventional methods in obtaining unambiguous hla genotyping results at the high resolution level. background: the accurate results of hla typing are significant for ensuring the success rate of hematopoietic stem cell transplantation. currently, hla typing is mainly based on sanger sequencing, which has a high proportion of ambiguous combination results indicating potential errors for hla typing. it is necessary for finding a more accurate typing method to reduce the risk. next-generation sequencing (ngs) method could provide clonal sequencing of single molecules, which has been used for hla genotyping and improved the scope and precision of hla study. aims: to establish a full-length precision sequencing platform for hla-i gene (hla-a, -b, -c) based on ngs technology and be evaluated by classical sangersequencing method, which can effectively improve the accuracy of hla typing for donor and recipient in hematopoietic stem cell transplantation. methods: hla-i (hla-a, -b, -c) gene-specific primers were screened, and the amplification parameters were optimized to obtain full-length sequences of hla-i gene under the same condition. the sample library for the amplicon was prepared with transngs tn dna library prep kit and the sequencing step was carried out with illumina miseq platform according to the manufacturer' protocol. all the sequencing data in fastq format were analyzed by typestream visual software version . . (one lambda inc.)with the default setting. cord blood samples were collected for hla typing with the mentioned above next-generation sequencing method in our study. in parallel, all the sample were also tested with the sanger sequencing method according to the previous study in our laboratory. results: samples were successfully tested with two methods and the coincidence rate between two sequencing methods was %. with the next-generation sequencing method, the probability of ambiguous results among samples in our study is . %( / ) for hla-a, . % ( / ) for hla-b and % ( / )for hla-c. however, the probability of ambiguous results with the sanger sequencing method is . % for hla-a, . % for hla-b, % for hla-c. summary/conclusions: the full-length precision sequencing platform for hla-i gene (hla-a, -b, -c) based on ngs technology was established, which could greatly reduce the probability of ambiguous results and effectively improve the accuracy of existing hla typing techniques. key: cord- -t x gknd authors: nan title: abstract presentations from the aabb annual meeting san diego, ca ctober ‐ , date: - - journal: transfusion doi: . /trf. sha: doc_id: cord_uid: t x gknd nan background/case studies: zika virus (zikv) is associated with severe neurological consequences in fetuses and adults and potential for transfusion transmission (tt). rna persistence has been reported in whole blood (wb) long after clearance of viremia in plasma, raising concerns over the risk of tt with plasma based nucleic-acid amplification testing (nat). the dynamics of zikv persistence in asymptomatic infection are not well understood and are needed for understanding of the natural history of zikv infection. we sought to characterize the dynamics of infection through prospective enrollment of zikv rna blood donors. study design/method: donors identified through investigational zikv nat screening were enrolled into longitudinal follow up and assessed for viral and serological persistence and clinical outcomes. plasma and rbc were obtained from index donations and blood, urine, saliva and semen samples were collected prospectively at weeks , , , and following index donations from donors and detailed symptom questionnaires were administered at each study visit. blood compartments and body fluids were tested for zika rna by real time rt-pcr. plasma samples were tested for zika specific igm and igg antibodies results/finding: the percent of zikv rna samples, followed by the number of samples tested in parenthesis, for each sample type during each sampling interval is summarized in the table. plasma viremia declined rapidly after index donations whereas rbc-and wb-associated viral rna persisted for up to months and peripheral blood mononuclear cell (pbmc) associated virus was detected intermittently at low levels and waning by months. urine and saliva detection decreased significantly after weeks and was undetectable by months. of donors who were enrolled in the acute pre-seroconversion stage of infection % ( / ) developed multiple zikv related symptoms week post index donation, compared to only % ( / ) for donors detected post-seroconversion. conclusion: zikv rna persists in cellular blood compartments for several months following clearance from plasma and body fluids, with higher rates of symptoms than previously reported. the persistence of zika rna in rbcs has unknown implications for blood screening, which currently relies on plasma testing; infectivity studies are in progress. wb testing may be of value to extend detection of acute infection and for diagnostics and monitoring of pregnant women. iron status and novel risk factors for iron depletion in a diverse donor population bryan r. spencer* , yuelong guo , ritchard g. cable , joseph e. kiss , michael paul busch , grier page , stacy endres-dighe , steve kleinman , simone glynn , alan mast and for the nhlbi recipient epidemiology and donor evaluation study-iii (reds-iii) . american red cross, rti international, american red cross blood services, blood systems inc., blood systems research institute, university of british columbia, nih/ nhlbi, blood research institute, nhlbi background/case studies: blood centers and regulators in the united states (us) are evaluating strategies for minimizing iron depletion in blood donors. the logistics of donor management might differ across blood centers, but the optimal approach may also vary according to biological or behavioral differences across sub-populations of donors. studies donors have been conducted in predominantly caucasian populations, which may differ from racial/ethnic minority donors in iron metabolism and capacity to undergo repeat phlebotomy. study design/method: over , donors were enrolled from us blood centers for ferritin testing. the study population was enriched for racial minorities [ african-american (aa), asian (as), hispanic (hisp)] and for "super donors" ( , who had completed donations in two years without low hemoglobin deferral). the minority donors and the remaining non-hispanic white (nhw) donors were an unselected population with no specific eligibility criteria. subjects completed questionnaires on risk factors for iron depletion. logistic regression was used to identify demographic and behavioral predictors of absent iron stores (ais, ferritin < ng/ml) and low ferritin (lf, ferritin < ng/ml). results/findings: across all subjects, % had ais and % had lf, with a high degree of variability based on demographic factors and donation behavior. in models stratified by race, expected patterns common to all groups included a sharp increase in risk with increasing donation intensity, and a large decrement in risk for females > years old. in models including all subjects, race was an independent predictor of both ais and lf controlling for age, sex, body weight, donation frequency, and other factors (table) . aa and as donors showed % % decreased risk for ais compared to nhw, while hisp donors had % higher risk. daily use of exogenous iron reduced risk for lf and ais by to %, respectively, while the estimated benefit from less-than-daily use was lower ( to % protection). regular use of antacids was associated with a % or greater increment to risk. reported use of hormone supplements showed opposing effects in males and females. use of oral contraceptives or estrogen in females reduced risk by % - %, while males who reported current use of supplemental testosterone had twice the estimated risk for ais. conclusion: this large study confirms the high prevalence of lf and ais in us donors and the principal risk factors of age, sex, and donation frequency. the diverse population studied and the questionnaire data from donors identify additional demographic and behavioral risk factors of secondary importance. in developing iron mitigation strategies, practices based on age and gender could be further refined depending on a given blood center's operational context and donor population. data are reported as mean ( sd) *p < . compared to batf / , ul hod rbcs mfi, median fluorescence intensity background/case studies: during storage, red blood cells (rbcs) undergo multiple morphological, biochemical and molecular modifications, collectively called the storage lesion. the proportion of cleared rbcs is correlated with storage duration, which may be responsible for the rapid clearance of up to % of transfused rbcs, reducing transfusion yield. it has been shown, using imaging flow cytometry that a subpopulation of morphologically altered rbcs accumulates during storage. the reduced surface area of these small rbcs (srbcs) suggests their rapid elimination by the spleen in the hours following transfusion. this hypothesis remains to be clarified, since the physiological mechanisms of rbc clearance remain to be precisely identified. study design/method: murine "young" and "old" rbcs (respectively on d and d of storage) were transfused into different models including splenectomized or macrophage-depleted mice. flow cytometry was used to determine the kinetics of clearance, the transfusion yield and to quantify rbcs retention in organs. the accumulation, during storage, and the posttransfusion disappearance of srbcs were analyzed by imaging flow cytometry. results/finding: using a murine model of transfusion, we confirmed that the post-transfusion yield decreases with storage duration ( % on d vs % on d of storage). a clearance of the storage-damaged rbcs mediated by spleen and macrophages is shown by significant improvements in post-transfusion yield observed in the splenectomized ( %) and macrophage-depleted ( %) groups. as in humans, we observed the accumulation of a subpopulation of small rbcs (mouse small rbc: msrbc) of reduced projected surface area with altered morphology. these msrbcs disappear rapidly from the circulation in control or splenectomized mice with a decrease of more than % at h post-transfusion. in contrast, in macrophage-depleted mice, msrbcs are kept in circulation at h posttransfusion. at h, these msrbcs completely disappear in all models, suggesting the importance of their elimination and the presence of compensation clearance mechanisms. in control mice, storage-damaged rbcs are mostly retained in the spleen but also in the bone marrow (bm) . no retention is observed in the liver, kidney or lung. in macrophage-depleted mice, retention is decreased in the spleen and bm. conversely, elevated retention is observed in the bm of splenectomized mice, associated with a transient retention in the kidney and liver. conclusion: during storage of murine rbcs, damaged rbcs accumulate, and are eliminated following transfusion via spleen/macrophage-mediated mechanisms. they include, as observed in humans, a subpopulation of small rbcs which undergoes a rapid macrophage-mediated clearance. the increase in transfusion yield in the absence of spleen or macrophages suggests that the recipient's functional state is one of its determining factors. age dependent relapsing and remitting autoimmune hemolytic anemia in a murine model andrea sut ling wong* , amanda l richards and krystalyn e hudson . background/case studies: breakdown of tolerance to rbc antigens may result in development of pathogenic autoantibodies (autoab) and lead to autoimmune hemolytic anemia (aiha), a severe and sometimes fatal disease. aiha in humans has a number of known features, including increased frequency with age, and tendency to relapse and remit. however, the mechanisms behind such observations are not understood. to gain insight into tolerance (or loss thereof) to an rbc autoantigen, we utilized the hod mouse, which expresses an rbc-specific triple fusion protein consisting of hen egg lysosyme (hel), ovalbumin (ova), and, duffy (hod). hod mice were bred to a transgenic mouse that expresses a t cell receptor specific for an ova peptide in hod presented by mhcii (otii mice). thus, hod otii mice are predisposed to have autoreactive cd t cells. study design/method: four cohorts of hodxotii f mice ( - mice/ cohort) were bled monthly for months to assess for autoab production. peripheral rbcs were stained with anti-complement (c ) and mouse immunoglobulin ab. spleens were weighed and splenocytes were stained with anti-cd and ter to assess for the presence of rbc progenitors. statistical analysis between hod otii autoab vs. hod otii autoabvs. hod -otii was performed using kruskal-wallis test and corrected for multiple testing with dunn's test. results/finding: otii cd t cells were not deleted in the thymus of hod otii mice; rather, they matured to the periphery. despite these peripheral autoreactive t cells, no detectable autoab were observed in hod otii . however, as they aged, - % of hod otii were positive for rbc autoab by months. thereafter, $ % of the autoab mice stopped producing autoab within two months after onset and remained autoab free throughout the study. in of the cohorts, - % of autoab mice were female. hod otii autoab mice also had enlarged spleens compared to hod otii autoaband hod -otii mice ( . g vs. . g and . g, resp., p< . ). this may due to rbc consumption, extramedullary erythropoiesis, or both. consistent with increased erythropoiesis, elevated numbers of rbc progenitors (cd hi ter inter ) were observed in the spleens of hod otii autoab mice but not in hod otii autoaband hod -otii ( . % vs. . % and . % resp., p< . ). moreover, autoab and c deposition were found ( . - % and - %, resp.) on ter rbcs in all of the hod otii autoab mice analyzed. conclusion: several features known to exist in human aiha were observed, including age-dependant autoab production, relapsing of autoimmunity after onset, and an increased frequency in females. this model may serve as an experimental system to investigate the mechanisms of aiha. b -a a reduction in neutrophil numbers is a risk factor for rbc alloimmunization amanda l richards , christopher a tormey and krystalyn e hudson* . background/case studies: red blood cell (rbc) alloimmunization occurs in up to % of transfusion recipients (excluding abo and rhd). the underlying factors that influence alloimmunization are poorly understood; thus, there is currently no reliable way to predict who will make an alloantibody and who will not. patients who receive multiple rbc units or several separate transfusions are at higher risk of alloimmunization; likewise, certain disease states have higher rates of alloimmunization, such as myelodysplasctic syndrome (mds) and sickle cell disease patients. however, despite chronic transfusions, some patients never develop rbc alloantibodies. it has been recently reported that poly (i:c)-elicited inflammation leads to enhanced alloimmunization rates and is correlated with increased splenic neutrophil (pmn) numbers. additionally, rbc transfusion into an inflamed recipient leads to enhanced erythrophagocytosis by pmns. here, we test the hypothesis that pmns regulate rbc alloantibody generation. study design/method: mice: c bl/ (b ) mice were treated with pbs, or anti-ly g to deplete pmns, followed by poly (i:c) to elicit inflammation, and finally a transfusion of allogeneic dio-labeled rbcs expressing a synthetic antigen, hod (hel-ova-duffy). multiple splenic cellular subsets were evaluated for dio fluorescence, an indirect measure of rbc consumption, at - hours post-transfusion. anti-hod alloantibody generation was assessed days post-transfusion by flow cytometry. humans:retrospectively, mean white blood cell (wbc) and pmn counts were collected on chronically transfused mds patients at va connecticut healthcare. for alloimmunized patients (n ), wbc and pmn counts were assessed on the day of exposure to the alloimmunizing rbc unit, whereas counts were averaged for the entirety of rbc therapy for non-alloimmunized patients (n ). patients were matched for numbers of rbc transfusions. results/finding: mice: the mfi of anti-hod antibodies was significantly increased in pmn-depleted mice, compared to controls ( / experiments, p< . ). while many control mice made no alloantibody (non-responders), all pmn-depleted mice made detectable anti-hod. pmn depletion also led to a significant reduction in dio leukocytes, suggesting a lack of compensatory mechanism(s) for rbc consumption. absence of pmns also shifted rbc consumption from macrophages to immune-stimulating dendritic cell subsets. flow cytometric analysis revealed that pmns with internalized rbcs upregulated expression of co-inhibitory molecules (e.g. pd-l ), compared to pmns (without internalized rbcs) from the same mouse; thus, pmns may regulate alloimmunization through antigen presentation and/or inhibitory signals. humans:. alloimmunized mds patients had a significant decrease in pmns, compared to non-alloimmunized (p< . ); no significant differences were detected in mean wbc counts between the two arms. conclusion: these data demonstrate that in both murine and human settings, pmns may play a significant role in regulating rbc alloimmunization and may provide key insights into predicting which patients will become alloimmunized. b -a b cxcr pd and ccr expressions characterize responders to rbc immunization benoît vingert* , , , marie tamagne , , , sadaf pakdaman , , , anoosha habibi , , , philippe bierling , , , , , rachid djoudi and france pirenne , , , . efs ile de france, laboratory of excellence gr-ex, imrb u -eq , ap-hp, universit e paris est background/case studies: post-transfusion alloimmunization can induce life-threatening hemolytic transfusion reaction. in human, mechanisms responsible of rbc alloimmunization are not fully defined. cd t cells are major for antibodies production. we have already shown in responder patients that the majority of anti-rbc cd t cells have a th profile. in contrast, in whole blood of non-responder patients, there is an unexpected expression of circulating cd t cells with a cxcr pd phenotype. this phenotype is usually associated with the presence of tfh cells, specialized in the production of antibodies. it has been suggested that some of the activated circulating tfh could have a cxcr pd hi profile, with a differentiated expression of ccr . ccr is essential for t cells domiciliation in lymph nodes where the encounter t and b cells is major for b cell differentiation and antibody production. others chemokines receptor like ccr and cxcr can also differentiate circulating tfh subpopulations. in this study, we were interested in the phenotype and function of these cxcr pd lymphocytes which were paradoxically highly represented in non-responder patients. study design/method: the membrane and functional phenotype of the circulating cxcr pd cells were compared in groups of transfused sickle cell patients : alloimmunized (n ) and non-alloimmunized patients (n ). the analysis was also performed in non-transfused healthy controls (n ). all assays were performed on whole blood without separation procedures that are known to alter the expression of chemokine receptors results/finding: the cxcr pd hi subpopulation expression was identical between transfused groups and controls. ccr and cxcr expressions show no difference between the transfused groups or the controls. however, in non-responder patients, ccr expression was very strong independently of the expression of pd . in the aim to determine the help of the circulating cxcr pd cells in the production of antibodies, these cells were purified by flow cytometry and co-cultured for days with b cells, and in the presence of seb protein. the levels of antibodies after seb stimulation were identical with the cxcr pd subpopulations from transfused groups or controls. conclusion: the paradoxical presence of circulating cxcr pd cells in non-responder transfused patients do not appear to have any particular functions that can promote the absence of a humoral response. however, in responder patients, the high expression of ccr on circulating cxcr pd cells suggests remarkable migratory properties towards secondary lymphoid organs, and could facilitate allo-immune responses. in conclusion, the study of the cxcr pd profile and the ccr expression in these cells could help to differentiate responder and non-responder patients to rbc immunization. primed cd t cells to one rbc alloantigen can enhance subsequent alloimmunization seema r patel* , ashley bennett , kathryn girard-pierce , connie arthur , amanda mener , patty zerra , christopher a tormey , jeanne hendrickson and sean stowell . emory university, yale-new haven hospital, yale university, emory university school of medicine background/case studies: while red blood cell (rbc) alloantibodies can increase the probability of transfusion-related complications, not all patients become alloimmunized following transfusion. however, individuals that do generate alloantibodies appear to experience an increased rate of additional alloantibody formation following subsequent transfusion. however, how immunity to one rbc alloantigen primes immunization to a completely distinct alloantigen remains unknown. though cd t cell help classically occurs through direct recognition of a peptide that resides within a target b cell antigen, individuals who develop antibodies toward one rbc alloantigen experience increased rates of antibody formation against completely distinct rbc alloantigens. these observations suggest that cd t cells that respond to one alloantigen may directly facilitate immunity to a completely distinct rbc alloantigen. study design/method: b recipients were transfused with kel rbcs in the presence or absence of poly i:c (pic), followed by transfusion of hod rbcs, kel rbcs, rbcs expressing hod and kel (hod x kel), or a mixture of hod and kel rbcs (hod kel). to examine the role of cd t cells, pic/kel primed b recipients were cd t cell depleted prior to transfusion. in addition, b recipients were adoptively transferred with cd t cells from na€ ıve or pic/kel primed donors, followed by transfusion of hod rbcs or (hod x kel) rbcs. anti-hod and anti-kel alloantibody formation was evaluated using indirect immunofluorescence staining. results/findings: kel rbc transfusion in the presence of pic (pic/kel) not only enhanced anti-kel antibody production through a cd t celldependent process, but this same priming event directly facilitated anti-hod antibody formation following subsequent (hod x kel) rbc transfusion (p < . ); pic/kel primed recipients transfused with (hod kel) rbcs or hod rbcs alone failed to impact anti-hod antibody formation. the ability of immunity to kel to boost a humoral response to the hod antigen following (hod x kel) rbc transfusion required kel priming in the presence of pic. cd t cell depletion prevented pic/kel primed recipients from boosting an anti-hod antibody response (p < . ) and transfer of cd t cells from pic/kel primed recipients likewise directly facilitated anti-hod antibody formation following a (hod x kel) rbc transfusion (p < . ). conclusion: these results demonstrate that cd t cells primed to one rbc alloantigen can directly enhance the immune response to a completely distinct rbc alloantigen, suggesting a mechanism whereby alloantibody responders may exhibit an increased rate of additional alloantibody background/case studies: platelet refractoriness remains a significant clinical problem, yet the mechanisms by which it occurs are incompletely understood. immune-mediated platelet clearance by anti-platelet alloantibodies plays a significant role, and patients with detectable alloantibodies can be managed with transfusion of hla-matched platelets. still, many patients are refractory even after receiving hla-matched platelets. it was shown previously that cd t cells can play a direct role in platelet clearance, as allogeneic platelets are cleared within hours post transfusion in b celldeficient mmt recipient mice (ie in the absence of anti-platelet alloantibodies) and depletion of cd t cells prevents such clearance. since minor antigenic differences still exist between donor hla-matched platelets and a recipient, we hypothesized that minor antigens alone may mediate clearance of otherwise hla-matched platelets. study design/method: to test whether minor antigens can stimulate cd t cell-dependent platelet clearance we examined platelet refractoriness using mova and oti transgenic mice. leukoreduced donor platelets from mova mice, which express a membrane-bound form of chicken ovalbumin and thus present ovalbumin peptides complexed with murine mhc class i h kb, were labelled in vivowith the fluorescent dye cfse and transfused into wildtype (wt, c bl/ ) mice or oti mice, whose cd t cell receptors recognize a specific ovalbumin peptide in the context of mhc class i h kb. in some experiments oti mice were primed with mova or wt splenocytes one week prior to mova platelet transfusion, and in others wt mice were adoptively transferred with oti splenocytes hours before mova platelet transfusion. platelet recovery was measured immediately after transfusion as well as after , , , , and hours and on days - . results/finding: transfusion of mova platelets into oti mice results in significant platelet clearance as compared to transfusion with wt platelets. clearance kinetics demonstrate platelet loss starting after hours and peaking at hours, and are similar whether oti mice are na€ ıve or previously primed with mova splenocytes. specifically, mova platelet recovery in oti recipients is - % versus > % in wt recipients at hours (p< . ), whereas transfusion of wt platelets into either oti or wt recipients is approximately % at hours after transfusion. adoptive transfer of oti cd t cells into wt mice recapitulates the effect, with significant mova platelet clearance at hours compared to wt platelet clearance (p< . ). conclusion: this work extends the ability of cd t cells to mediate platelet clearance to a minor antigen, providing insight into the potential etiology of platelet refractoriness in patients receiving hla-matched products. this study also holds implications for the clinical management of any nonantibody-mediated platelet refractory patient, as therapies directed toward immunomodulation of t cell responses may prove beneficial. background/case studies: alloimmunization against major histocompatibility (mhc) antigens is a common complication of transfusion, and can negatively impact subsequent transfusions and transplants. we have previously demonstrated that pathogen reduction with riboflavin and uv light (uv r) is effective both at rapidly killing donor white blood cells (wbcs) and at blocking their ability to stimulate an allogeneic response in vitro. furthermore, uv r treatment of allogeneic platelet rich plasma (prp) prevents alloimmunization in mice, and provides partial antigen-specific tolerance to subsequent transfusions. as cells that die through different pathways can be either tolerizing or inflammatory, we sought to determine which cell death pathways are triggered by uv r, as well as evaluate the immunogenicity of prp containing wbcs killed by other methods. study design/method: wbc-rich prp was prepared from c bl/ mouse blood and treated with uv r, and wbcs prepared in parallel from the same blood were treated with known inducers of either apoptosis or necrosis. membrane integrity, phosphatidylserine exposure, caspase activity, and chromatin condensation were evaluated by flow cytometry. balb/c recipients were transfused with either uv r treated wbc-rich prp, or uv r treated wbc-poor prp either alone or with added untreated, apoptotic, or necrotic wbcs, all generated from allogeneic c bl/ donor blood. a second transfusion of untreated wbc-rich c bl/ prp was given weeks later, and alloresponses were compared against mice given no transfusion or only the second untreated transfusion. results/finding: uv r treated wbcs have a pattern of phosphatidylserine exposure and loss of membrane integrity consistent with early apoptosis, but fail to demonstrate significant caspase activity or clear chromatin condensation. alloantibody responses to transfusion were significantly higher in mice previously exposed to untreated (p< . ) or necrotic (p< . ) wbcs, but not those given uv r treated or apoptotic wbcs. ex vivo cytokine responses to stimulation with c bl/ wbcs were reduced in recipients of either uv r or apoptotic wbcs, and enhanced in recipients of untreated or necrotic wbcs. conclusion: the mechanism of wbc death following uv r treatment shares some membrane characteristics of early apoptosis, but is distinct from classic apoptosis. however, both uv r treated and apoptotic wbcs fail to trigger an alloresponse, and offer some protection against subsequent alloexposures. background/case studies: in mitochondria-less red blood cells (rbcs), oxygen is the main substrate for oxidative reactions and resulting oxidative damage is considered as one of the major causative factors in the development of rbc storage lesion. oxygen saturation (so ) of venous blood is generally assumed to be around - % as measured from a central venous line. however, a recent investigation of so levels in freshly prepared leukocyte-reduced red cell concentrates (lr-rccs) revealed unexpectedly wide so distribution (mean . % . % [yoshida et al. ; blood transfusion , ] . the present study was undertaken to determine the distribution of so in lr-rcc produced at a medium-size blood center using a novel non-invasive so probe. additionally, quantitative metabolomics were carried out to examine the redox status of the stored rbc under various so levels. study design/method: the so from units of lr-rcc were examined on five consecutive days representing % of the collected units during the period at a regional blood center where all the units were processed at room temperature within hours of blood collection. so was measured noninvasively through the pvc bag immediately prior to refrigeration by employing a resonance raman spectrometry (pendar microvascular oximeter a u ; pendar technologies, cambridge ma). in addition to so , process methods, rcc volumes, blood types, gender and process times were recorded for analyses. in a separate study, lr-rcc (n ) from human volunteers were stored in as- under normoxic, hyperoxic, or hypoxic conditions for up to days (so ranging from < to > %) prior to uhplc-ms metabolomics analyses in presence of c, n or deuterated internal stable-isotope labeled standards for absolute quantitation. results/finding: measurements of so carried out non-invasively at a blood center yielded a similar wide distribution as previous study from units of lr-rcc procured and sampled invasively within hours after blood collection [yoshida ibid]. the shape of the so distribution appeared near normal with the mean of . % . %, median . %, range < % to > % and inter-quartile range (iqr) of . %- . %. male donors showed higher so compared to female donors (p< . ). no correlations were observed between so levels and processing time, donor age or blood types. metabolomics workflow indicated that lower so levels ameliorate the energy and oxidative metabolic lesion. lower so levels yielded higher rate of gsh synthesis, higher nadph concentration, higher gsh / gssg and nadph/nadp ratios, lower supernatant urate consumption and lower purine oxidation. the surprisingly wide distribution of starting %so levels was observed from lr-rcc manufactured at a blood center using -hour room background/case studies: cellular prion protein (prp c ) is a gpi-anchored cell surface glycoprotein that is expressed mainly in the brain but also in peripheral organs including blood, bone marrow (bm), and lymphoid tissue. prp c can be converted post-translationally into scrapie-prp (prp sc ), which is involved in the pathogenesis of neurodegenerative diseases including creutzfeldt-jakob disease, kuru in humans, and scrapie and bovine spongiform encephalopathy in animals. however, biological functions of prp c have yet to be conclusively elucidated. study design/method: in this study, prp c knockout mice (ko) are utilized to investigate the role of prp c in the hematopoietic system with controls of age and sex-matched prp c transgenic mice harboring a slightly augmented prp c expression. peripheral blood was examined by hematology analyzer to establish counts. bone marrow, thymus, spleen, lymph nodes, and peripheral blood were harvested and analyzed by flow cytometry using a comprehensive panel of fluorochrome-conjugated antibodies specific for all hematologic cell precursors/ lineages. histology of bone marrow, spleen, thymus and lymph nodes were evaluated by light microscopy. results/finding: complete blood count (cbc) showed a significant increase of wbc in ko mice. closer analysis of wbc differential revealed that the elevated number of wbc in ko mice was due to lymphocytosis. specifically, ko mice had a -fold increase in the absolute lymphocyte count (ko . . x /l vs. wt . . x /l, p . ), as well as a higher lymphocyte percentage compared to controls. ko mice also had a trend toward higher hemoglobin, rbc, and hematocrit compared to wt mice. additionally, platelet count in ko mice was higher than control mice. of interest, the mean platelet volume indicating platelet size was significantly increased in ko mice compared to controls (ko . . fl vs. wt . . fl, p . ). a comprehensive flow cytometric analysis of all cell lineages revealed no significant differences in the numbers of rbc and megakaryocyte in bm, and of lymphocytes in the thymus, spleen and lymph nodes. histological analysis of bm, thymus, spleen and lymph node tissue from ko and wt animals failed to show morphological differences between the two groups. conclusion: absence of prp c resulted in significant leukocytosis and specifically higher absolute count and percentage of lymphocytes, as well as larger platelets in peripheral blood, but does not appear to affect hematopoiesis and lymphopoiesis. our findings indicate that prp c might be critical in the survival and trafficking of lymphocytes in peripheral blood. the molecular mechanisms underlying the observed changes in lymphocytes and platelets, and whether these involve functional changes in these cells will be subject of future studies. potential role of cd foxp regulatory t cells derived exosomes in their immune modulation yiming yang*, rufeng xie and jie yang. blood engineering laboratory, shanghai blood center background/case studies: exosomes are defined as one type of membrane vesicles secreted into extracellular space by most types of cells and are reported to involve in intercellular communications, mediate biological process. human periphery blood cd foxp tregs cells are reported as more stable regulatory cells with greater inhibition effects. however, cd foxp tregs derived exosomes and their functions involved in cd tregs mediated immune-modulation were seldom reported. study design/method: cd t cells were freshly purified from pbmcs, cultured with anti-cd /cd antibody packaged beads and il- , and then polarized with tgf-b and rapamycin into cd foxp treg cells. the harvest cells were co-cultured with cd /cd beads stimulating cd cd effector cells in the transwell plate. the supernatant derived from cd tregs was collected and ultrafiltrated by centrifugation and the remaining solution was precipitated with peg. the harvest precipitation was resuspended in pbs and exosomes were analyzed by sem and nta. exosome surface marker cd , cd , tsg and other proteins expression were evaluated by flow cytometry and western blot. microrna was isolated with mircute mirna kit and mir- , let- b, let- d were measured by qpcr. the precipitated exosomes were further purified by cd immunoaffinity capture and co-cultured with effector cells to investigate their function in immune modulation. results/finding: as compared with direct contact co-culture, separated cd treg cells could suppress the proliferation of effector cells with a small decline (p> . ), which means some non-contact factors involved in the cd treg mediated immune modulation. a total number of . . / cells exosomes were harvest. electron microscope analysis demonstrated a kind of round-shaped membrane vesicle - nm in diameter ( . . nm by nta). cd and cd were expressed on these background/case studies: regulatory t cells (tregs), containing cd and cd subtypes, play an essential role in immune regulation and autoimmune disease prevention which makes it a potential candidate for cell therapy on autoimmune disease (aids). unfortunately, due to the instability of natural cd foxp regulatory t cells (ntregs) in inflammation conditions (including instability of foxp , conversion to pro-inflammatory effector cells and was unable to modify established disease), thus, it is needed to investigate cd regulatory t cells stability both in vitro and in vivo. in our previous works, we found that cd treg has an effective therapeutic function on cia mice. in this study we aim to investigate the stability of induced polyclonal human cd regulatory t cells in inflammation and transfusion. study design/method: human cd tregs were induced with tgf-b and rapamycin from cd t lymphocytes in vitro. collagen-induced arthritis (cia) mice were induced with type-two collagen as an autoimmune disease model. in vitro the stability of cd tregs when encountering with inflammation were test by foxp expression, th and th cells conversion in inflammations conditions (il tgf-b il il and il tgf-b il b il ) on day , day and day . in vivo, cd tregs were transfused into cia mice and then their survival in mice and foxp express were evaluated to reveal the stability of cd tregs in an inflammation condition model. additionally, we also investigate the stability maintenance of cd tregs when induced factor tgf-b and rapamycin were removed by testing the foxp expression on day , day and day . results/finding: ex vivo induced human cd treg were foxp ( . . %) and did not secret il a (both in supernatant and % of cells). foxp express in cd tregs were maintained after induced factor tgf-b and rapamycin were removed on day , day and day . in vitro, foxp , il and ifn-c expression has no significant difference when compared with controlled tregs on day , day and day and did not secret il a when encounter with inflammation conditions (il tgf-b il il and il tgf-b il b il ). in vivo, cd treg cells were transfused into cia mice on the peak of disease onset ( days after the first collagen immunization, has inflammation condition in vivo) to test cd tregs survival. cd tregs were found in cia mice foot ( . . %), blood ( . . %) and spleen cells ( . . %) hours after transfusion and their % of foxp were remained. conclusion: the results revealed that ex-vivo induced and expanded human cd tregs are stable in inflammation and transfusion and can maintain foxp expression when induced factor were removed, these make cd treg a novel and stable cell for potential cell therapy on aids. this research can provide some instructive reference and improve the utilization of blood components. tolerogenic dendritic cells induced by mtor suppression and control inflammation in chs model through s k related proteins translation inhibition. li gao*. shanghai blood center background/case studies: tolerogenic dendritic cells (tdcs) adoptive cellular immunotherapy is a cutting edge strategy for treating hypersensitivity response disease, in which immune responses are directed against selfantigens, such as atopic dermatitis, systemic lupus erythematosus (sle), rheumatoid arthritis (ra),et al. however, the traditional strategy base on the tdcs was usually unstable and inconspicuous through cytokines inducing processing so that might be the limitations on tdc adaptive cell therapy in future clinical use. study design/method: human tdcs were derived from fresh purified monocytes from pbmncs isolated from buffy coat and induced by mtor inhibitors (rapamycin and temsirolimus) in safe concentrations confirmed by apoptosis assay when the cells were completely differentiated. the mature markers and endocytisis were detected by flow cytometry. the production of cytokines and chemokines was measured using elisa. mechanism investigation was analysis by real-time pcr and western blotting. contact hypersensitivity (chs) model, an atopic dermatitis animal model, was treated with tdcs induced via mtor suppression and analyzed by ear thickness and tissue leukocytes number calculating. results/finding: human tdcs treated with mtor inhibitors had a lower mature marker cd /cd /cd expression after tlr signaling activation, accompanied with a set of cytokines and chemokines remarkably downregulated in a concentration dependent manner but not the lps absent group. moreover, mtor suppression extremely reduced the capacity of lps treated human dcs to stimulate autogenic na€ ıve t cell proliferation, which is one of the most important characteristics of tdc. beyond expectation, the common signal transduction pathway, mapk and nf-jb pathway, were not the signal target so that it could hardly be the explanation for the tolerogenic performance of tdc when exposure to lps stimulation. however, the p s k and its downstreanm proteins, especially the protein s , which controls the protein translation, were shown in charge of the tolerogenic induction mechanism. the data were also supporting the suggestion that rare difference on mrna transcription of the related functional proteins in tdcs induced by mtor inhibitors when exposure to lps stimulation from the non-induced cells, although there was more transcription of ido induced by mtor inhibitors. more important, edema responses of ears were clearly weakened in the chs model and recruited less leukocytes to the tissue when co-sensitized with mtor inhibitors or with tdcs induced by mtor inhibitors suggested that the tdc induced by mtor suppression were able to control hypersensitivity inflammation response in vivo. conclusion: accordingly, tdc induced by mtor suppression is a potent adoptive cellular immunotherapy strategy for treating hypersensitivity response disease and the induction mechanism of it might be through suppressing systematically effective function proteins by mtor-s related protein translation inhibition. xiaoyun fu* , , mikayla anderson and james c zimring , . bloodworksnw research institute, university of washington school of medicine background/case studies: red blood cells (rbcs) undergo many changes when stored under blood banking conditions, collectively known as the storage lesion. bioactive lipids generated during rbc storage have been implicated in certain adverse outcomes. recently, we reported that bioactive lipids, especially polyunsaturated fatty acids (pufas) and their oxidized products (oxylipins) accumulate during rbc storage despite leukoreduction. to evaluate the extent of membrane lipid degradation and oxidation in stored rbc units among the donor population with different blood groups, we quantified pufas and lysophospholipids (lpls) in leukoreduced rbc units. study design/method: rbc units from different donors were acquired and processed on day (one day past their expiration). bioactive lipids including common fatty acids, oxylipins, and lpls were analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (lc-ms/ms-mrm). total fatty acid concentrations of selected units were also analyzed. a one-way anova test was used to determine significant difference of analytes amongst the different blood groups. results/finding: we observed a wide distribution in concentration of major pufas in stored rbc units. for example, arachidonic acid (aa) ranges from . - . mm, linoleic acid (la) ( . - mm), dihomo-c-linolenic acid (dgla) ( . - . mm), eicosapentaenoic acid (epa) ( . - . mm), docosahexaenoic acid (dha) ( . - . mm), and alpha-linolenic acid (ala) ( . - . mm). ten oxylipins including hetes, hodes, and dihomes, and lpls including lpcs, lpss, and lpes all showed a large variation in concentration among donors. of analytes quantified, showed a significant difference in concentration among different blood types by one-way anova testing (fdr< . ). the ab rh blood group consistently exhibited the lowest concentration of major pufas, while the o rh-blood group showed the highest, averaging a two-fold difference in concentration (o rh-/ab rh ). the fold increase of o rh-/o rh among pufas ranges from . to . , suggesting the rh blood group, independent of the abo blood group, correlates with donor to donor variation in lipid metabolism. conclusion: the wide distribution in the concentration of bioactive lipids among stored rbc units suggests that lipid degradation is highly donor-background/case studies: to ensure availability of biological products to hospitals, blood banks have developed and validated multiple storage conditions for each of their products to maximize shelf life and quality. in the case of labile products, their metabolism is known to remain active during storage, leading to storage lesions. micrornas (mirnas) levels are modulated by these storage-related damages, which makes mirnas ideal candidates as potential biomarkers of quality monitoring. lately, nanoparticles have been widely studied and used for biosensing applications. the objective of this work is to develop biocompatible gold nanosensors for sensitive, selective and direct detection of biomarkers to characterize and assess the quality of blood products delivered to hospitals. study design/method: gold nanoparticles (gnps) surrounded by a fluorescent silica shell were prepared using a wet chemistry method. mirna- was chosen as a potential target, since it is strongly expressed in platelet concentrates and its concentration fluctuates according to storage lesions. custom rna and dna molecular beacons were designed and used as a probe for the specific detection of mirna- targets in pbs and human plasma. these fluorescent transducer probes were conjugated at the surface of fluorescent silica shell-gnps using an edc/nhs cross-linking reaction. the hybridization reaction between the target and the probe initiates an energy transfer mechanism which can be recorded by fluorescence. results/finding: gnps ( nm) surrounded by a thick fluorescent silica shell ( nm) were prepared and used as nanosensors because of their optimal luminescence properties and long-term stability. conjugation of the probe onto the nanoparticles was confirmed by fluorescence spectroscopy and microscopy, as well as nanoparticle tracking analysis. the fluorescent response of the molecular beacons was studied and showed a reproducible and linear relationship (r rnaprobe . and r dnaprobe . ) with mirna- concentration, down to a -nm limit of detection. hybridization assays in % human plasma appear to demonstrate denaturation of rna probes and targets. conclusion: biocompatible fluorescent gnps were prepared and used as tools for blood product characterization. the conjugation of a molecular beacon at the surface of nanoparticles was achieved and characterized using spectroscopic and microscopic techniques. the functionalization of the probe is still being optimized. the fluorescence response of the molecular beacon was characterized for the detection of a model mirna target in pbs and in % human plasma. energy metabolism profile of erythrocytes during storage suping ren*, qun yu, yanbing wang, changlan li and yu wang. background/case studies: the moment the mature red blood cells (rbcs) leave the bone marrow, it is optimally adapted to perform the binding and transport of oxygen and its delivery to all tissues. red blood cells modulate oxygen transport, protect hemoglobin from oxidant-induced damage, and maintain the osmotic environment of the cell. glycolysis is the only energetic metabolic pathway for mature rbcs to obtain atp which is the energy for rbcs to maintain a number of vital cell functions. generally, the current methods used to measure rbcs glycolysis are not in living state in realtime, or are destructive to cells or require radioactivity.xf technology can be applied to different types of cells, in which the red blood cells are suspended and the cell shape and size are different from other cells, and more importantly, rbcs have no nucleus, mitochondria and other organelles, so application of the xf technology in erythrocytes and exploration of the assay conditions are necessary. . . a . . . . a . . . . a . . total atp,lm/ghb . . a . . . . a . . . . a . . extracellular lactate,mm a extracellular glucose,mm a a a extracellular na ,mm a extracellular k ,mm a a a a p< . , paired t-test b intercept blood system for red blood cells is not approved for commercial use. c this project has been funded in whole or in part with federal funds from the dhhs; aspr; barda; contract no. hhso c. background/case studies: pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide to make transfusion safer. in vitro studies have demonstrated the effects of pathogen inactivation on storage lesion, but little routine quality control data on blood banking outcomes have been reported. study design/method: swirling of distributed products was monitored one year before and one year after implementation of intercept pathogen inactivation. metabolic parameters like ph, glucose and lactic acid were determined in a random sample of expired pathogen inactivated products. furthermore, indicators of platelet storage lesion were measured in apheresis concentrates with premature low swirling and compared to controls with normal swirling. results/finding: in an experimental phase on a limited number of products (n ) to validate the intercept pathogen inactivation method, ph and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pooled buffy coat derived products. once pathogen inactivation was implemented, routine products showed glucose exhaustion more often when prepared by apheresis compared to buffy coat derived platelet concentrates despite more plasma carryover in the former. furthermore, the number of apheresis products with premature low swirling increased by % ( / , ) compared to the previous year without pathogen inactivation ( / , , p . , chisquare) . in contrast, the incidence of premature low swirling in platelet concentrates prepared by the buffy coat method decreased ( / , vs / , ). of note, apheresis concentrates with premature low swirling had a significantly higher median platelet count ( . x ) than unaffected controls ( . x ) and showed signs of increased storage lesion compared to controls expiring on day five without swirling defects. these signs included lower ph, higher lactic acid concentration, increased mean platelet volume, phosphatidylserine exposure and alpha-degranulation. conclusion: the risk of increased storage lesion rates following intercept pathogen inactivation is higher for apheresis than for buffy coat derived platelet concentrates, especially when platelet content is above . x . in vitro quality of single dose amotosalen/uva treated platelets in % plasma/ % pas- after days of storage crystal stanley , marguerite kelher , nero evero , melissa vongoetz , betsy donnelly and anna erickson* . belle bonfils memorial blood center, university of colorado, cerus corporation background/case studies: the interceptv r blood system for platelets is fda approved for the ex vivo preparation of pathogen-reduced amicus o apheresis platelet components (pc) in pas- to reduce the risk of tti, including sepsis, and to potentially reduce the risk of transfusion-associated gvhd. registration studies (clinicaltrials.gov nct ) are in progress to support approval of the trima o apheresis platform for collection of platelets components (pc) suspended in pas- and plasma. the objective of this study was to evaluate in vitro function of platelets suspended in % plasma/ % pas- , collected using the trima platform, after treatment with the intercept blood system for platelets. study design/method: double dose apheresis pc, . . platelets in ml, were collected on the trima apheresis platform in % plasma/ % pas- . a sample was taken from each donation prior to dividing the donation to produce intercept treated apheresis pc (t), using the small volume (sv) set, and an untreated control pc (c). input volumes for replicates, n , were ml (t) and ml (c) with doses of . . (t) and . . (c). all pc were stored under the same conditions and evaluated on day and day for physical/metabolic characteristics. results/finding: on days and all t and c pc had ph c ! . . the dose recovery for t was % %. on day , t had lower count, volume, dose, bicarbonate and glucose compared to c pc; however, parameters predictive of in vivo function (atp, morphology score, hsr, and esc) were equivalent between t and c (table ) . conclusion: trima pc in % plasma/ % pas- treated with the inter-cept blood system for platelets using the sv set and stored for days retained in vitro metabolic and functional properties consistent with in vivo functionality. induction of pluripotent stem cell-derived cardiomyocyte toxicity by supernatant of long term-stored red blood cells in vitro feng-yan fan , , yang yu , li-ping sun , shu-fang wang , rui wang , lei-ying zhang and deqing wang* . the department of blood transfusion, the pla general hospital, the department of blood transfusion, air force general hospital, pla background/case studies: recently, multi researches have reported that longer term-stored red blood cells(rbcs) units were associated with increased risks of clinically adverse events, especially in critically ill patients. however, other studies have concluded the negative results. whether rbcs storage duration was associated with increased risks of clinically adverse events is uncertain and had become a popular topic. to study the adverse effects of longer term-stored rbcs directly, we aim to look at the pluripotent stem cell-derived cardiomyocyte toxicity induced by supernatant of suspended red blood cells(ssrbcs), and study the possible mechanism. study design/methods: five doses of leuko-reduced rbcs were prepared, and supernatant was isolated by centrifugation on d , d and d . we looked at the cardiotoxicity of ssrbcs on human-induced pluripotent stem cell-derived cardiomyocytes (hips-cms). hips-cms were treated with ssrbcs in % final volume simulating the large volume blood transfusion. using real-time cellular analysis (rtca) technology the beating of hips-cms was recorded in real time in detail. levels of k and lactic acid (la) were tested using automatic biochemical analyzer. k and la solution with concentrations being consistent with ssrbcs were prepared and cocultured with hips-cms. we analyzed the cardiotoxicity of k and la solution on hips-cms. treated hips-cms with d ssrbcs, d k and cell culture media for h. the nuclear shape and integrity of filament and sarcomere was examined by immunofluorescence. total rna of hips-cms was isolated and mrna analysis microarray was implemented. screened for toxic effects related signaling pathways through bioinformatics analysis. results/findings: d ssrbcs had no obvious influence on beating state of hips-cms-hips-cms treated with d ssrbcs stop beating, but beating patterns restored at h. hips-cms treated with d ssrbcs stop beating, and beating patterns did not restored at h. levels of k and la in ssrbcs changed most obviously. only d k solution made hips-cms stop beating and can restore in h; d k, d k and la solution did not influence the beating pattern in at the end of the treatment for h, hips-cms treated with d ssrbcs show obvious shrinkage. at the end of the treatments for h, cells treated with d k and d ssrbcs both show obvious shrinkage, the shrinkage in d ssrbcs group was more serious. the immunofluorescence results show the integrity of filament and sarcomere was complete and no nuclear pyknosis was detected. gene expression array results show a total of genes were differentially expressed in d ssrbcs group compared with naive group. there was no consistent separation within the d k and naive group. fifteen differently expressed genes were selected with bioinformatics method which were likely to play an important role in the cytotoxic effect. under the condition of simulating the large volume blood transfusion, ssrbcs of long term-stored rbcs have toxic effect on myocardial cells. in addition to high potassium that induced cardiotoxicity, there must be other elements are involved in the toxic effects. further study should be applied to signal pathways on ssrbcs induced cytotoxicity. large volume transfusion of long term-stored rbcs may be a risk factor for adverse clinical outcomes, and clinical should pay attention to it. background/case studies: processing thawed, deglycerolized red cell concentrates (rcc) in a functionally closed system allows for a prolonged storage after thawing. thawed cells are better maintained in as- as compared to sagm. the presence of citrate in as- seems to be necessary to prevent hemolysis of thawed cells. during storage in as- , atp and , -dpg levels rapidly decline. recently developed additive solutions like pag m and as- have shown to better maintain , -dpg and atp levels during storage of normal, unfrozen, rcc. however, most probably due to the absence of citrate, these solutions are not suitable for storage of thawed cells. we therefore designed pag c in which the mannitol of pag m was replaced by citrate. the aim of this study was to investigate the in vitroquality of thawed, deglycerolized rbc during storage at - c in pag c. study design/method: leukoreduced rcc (n ) in pag c (phosphate, adenine, glucose, guanosine, gluconate, citrate) were stored at - c. on day , rccs were glycerolized using acp (haemonetics v r , braintree, ma) to a final concentration of % (w/v), frozen and stored for at least two weeks at - c. after thawing and deglycerolization using acp , rcc were resuspended in pag c. during storage at - c, stability (hemolysis), atp and , -dpg levels were determined. results were compared with thawed rcc (prefreeze storage in sagm, n ) resuspended in or sagm (n ). results/finding: pre-freeze storage in pag c resulted in increased , -dpg levels at day as compared to storage in sagm, resp. . . mmol/g hb and . . mmol/g hb. hemolysis during post-thaw storage in pag c remained below . % for days and was comparable with storage in as- . in sagm, hemolysis remained below . % for days. during the first weeks of post-thaw storage in pag c, both atp and , -dpg levels increased, followed by a gradual decline during prolonged storage. during the whole postthaw storage period, rccs in pag c showed significantly higher atp and , -dpg levels compared to as- or sagm. while in sagm and as- , , -dpg levels were undetectable after days post-thaw storage, in pag c, , -dpg levels only decreased to . lmol/g hb after days of storage. conclusion: pre-freeze storage in pag c resulted in increased , -dpg levels. as compared to as- , post-thaw storage in pag c showed comparable hemolysis while atp and , -dpg levels were much better maintained. based on a maximum allowed hemolysis of . % and an atp content of > . mmol/g hb, thawed rcc can be stored at - c for days in pag c. background/case studies: platelets (plts) are vital for effective treatment of hemorrhage. cold ( c, c) storage of plts in platelet additive solution (pas) is a promising alternative to conventional storage at room temperature (rt) due to a lower risk of bacterial concerns, preservation of plt function, and mitigation of plt activation. currently only apheresis (ap) and pas systems are fda-approved for use in the us: trima and isoplate-pas (iso; terumo) and amicus and intersol (int; fenwal) . the goal of this study was to assess the adhesive function of long-term cold-stored plts collected by fda-approved ap/pas methods. study design/method: plts were collected (n - ) in % iso using a trima or in % int using an amicus and stored for days at rt and c. samples were tested on day (baseline, bl), , , and of storage to assess plt adhesion under shear flow (bioflux). acd vacutainer tubes were collected from donors and centrifuged to obtain red blood cells (rbcs) for all bioflux runs. simulated whole blood was created by combining plts labeled with calcein-am with rbcs at % hct. labeled blood was perfused through microfluidic channels (fluxion) coated with ug/ml type- collagen at s - shear rate. images were acquired every sec for min using a fluorescent microscope and % surface coverage was reported. data were analyzed using two-way anova and posthoc tukey test with significance at p< . . results/finding: both rt-int and rt-iso plts showed significantly decreased adhesion by day of storage compared to bl (bl: . . %, rt: . . %; p< . ). c-int samples showed no difference in adhesion at any timepoint compared to bl-int but significantly enhanced adhesion compared to both rt-int and rt-iso. in contrast, c-iso plts showed significant enhancement of surface coverage compared to bl-iso by day (p . ) and compared to c-int by day (p< . ). conclusion: our work suggests that c storage of plts collected with a trima ap system in iso for up to days offers a significant enhancement in adhesive function compared to plts collected with an amicus system in int and stored at c. these results are surprising since both c-int and c-iso have been shown to express similar levels of cd p, pac- , and phosphatidylserine and may suggest differences in pas plt intracellular signaling. as expected, storage at c of plts collected on either platform demonstrated superior function to rt storage. a plt product with superior hemostatic function and a shelf-life x longer than the current standard-ofcare provides the potential for shipment of products to underserved areas and may bolster plt availability for trauma care in the us. table . comparisons of white blood cell counts and percentages of apoptotic cells in whole blood components after -week storage between unirradiated and irradiated groups (n ) tang, is an anti-inflammatory agents and has a good safety records in clinic. it could reduce the severity of experimental autoimmune encephalomyelitis (eae), asthma, colitis, systemic lupus erythematosus(sle) and other immune diseases.however,its potential in inducing transfusion tolerance remains to be explored.the aims of our study are to find if baicalin could inhibit red blood cell (rbc) immunization and to elucidate the possible mechanism of yin-chen-tang in preventing hdn. study design/method: we used human red blood cells with adjuvant lipopolysaccharide (lps) and transfused mice to induce antibodies, as an experimental system to study the effect of baicalin on rbc immunization. mice were divided into a normal control group, a human rbc transfused positive control group receiving human rbc and lps intravenously weekly for five weeks, a control group receiveing dexamethasone ( mg/kg/day) intraperitonealy daily for five weeks,a treatment group receiving baicalin ( mg/kg/day) intraperitonealy daily for five weeks. assessment of human rbc immunization was performed by measuring serum immunoglobulin g (igg) and immunoglobulin m (igm) against human rbc weekly. and the lymphocyte changes in spleen are also monitored by flow cytometry. results/finding: we found that baicalin treatment decreased serum igg but not igm production significantly since the second week, with a concomitant reduction in th cells and increase in cd regulatory t cells in both spleen and mesenteric lymph nodes. and there are no significant differences in the percentage of th ,th ,tfh and tfr cd subpopulation among all groups.in addition, baicalin treatment didn't decrease the size of spleen and the percentage of cd positive cells in spleen in baicalin treatment mouse but in dexamethasone treated mouse. our results indicate that baicalin could inhibit rbc immunization especially igg production without the damage to the function of spleen,while dexamethasone as a wildly used immune-suppressive drug in blood transfusion could damage the function of spleen.considering its good safety records in clinic, it may be exploited for suppressing transfusion immunization events. in addition, our results elucidate the inhibitory effect in antibody production of baicalin may be a possible mechanism for yin-chen-tang as a widely used chinese herbal medicines in preventing hdn. comparison of immucor's pak plus and pak lx assays for the detection of human platelet alloantibodies randy m schuller* , sarah kloss , sara crew and sandra j nance . american red cross, american red cross and american rare donor program background/case studies: alloantibodies directed against human platelet membrane glycoproteins (gp) ia, iia, iib, iiia, ib, ix, iv, and cd have been implicated in several clinically significant disorders such as fetal and neonatal alloimmune thrombocytopenia (fnait), post-transfusion purpura (ptp), refractoriness to platelet transfusions, and passive transfer of antibodies in donor plasma. polymorphic epitopes on these gps give rise to unique human platelet antigens (hpa). identification of the specific platelet alloantibody is crucial in diagnosing and treating these bleeding disorders. currently the only k fda approved test permits the identification of these hpa antibodies to the glycoprotein level. immucor has recently released pak lx, a research use only (ruo) assay in the united states that has the ability to identify hpa antibodies to a single nucleotide polymophism (snp). we compared the performance of pak lx to the fda approved immucor pakplus. study design/method: we compared pakplus and pak lx results from plasma and serum clinical specimens. group contained a single hpa alloantibody specificity with or without hla antibodies (n ). group included specimens with hla antibodies alone and group consisted of patient samples that were negative for both hpa and hla antibodies. pak lx utilizes a luminex bead based assay which allows the user to report antibodies to the platelet specific antigen (hpa- , hpa- , hpa- , hpa- , hpa- , gpiv) and hla class i. pakplus uses an elisa method and results can only be reported to the glycoprotein location (gpiib/iiia, gpia/ iia, gpib/ix, gpiv) along with hla class i. however, based upon the pattern of reactivity observed in the pakplus and pak lx assays it is possible to determine the most probable hpa antibody specificity to the hpa snp. results/finding: conclusion: when analyzing hpa antibody specificity, there is % concordance observed for hpa- a, hpa- b and hpa- b antibodies. the pak-plus assay had difficulty discriminating hpa- b from hpa- a antibody when hpa- a antibody was present ( false positive samples) although the pak-plus signal od to cutoff od ratio was significantly higher for hpa- a when compared to hpa- b in these samples. the discordant hla class i antibody results between the assays was isolated to very weakly positive antibody (within % of the cut-off for pakplus and < . adjusted ratio for pak lx). we conclude that pak lx is an easy to use platelet alloantibody screening method that has the ability to differentiate hpa antibodies to the allele level. histo-blood group antigen lewis y promotes cell migration via regulation of microtubule acetylation huijun zhu* and ping lu. shanghai blood center background/case studies: blood group antigens are critical for transfusion practices as antibodies raised against them can cause severe transfusion reaction. beside this, blood group antigens themselves are composed of sugar chains, proteins, lipids, etc, which may be involved in various biological processes. lewis y is a histo-blood group antigen belonging to abh family. ley consists of carbohydrate chains which may play important roles in cell recognition, adhesion as well as migration, which are all critical steps in tumor progression and thus attracts wide researches focusing on its relevance in tumor biology. ley is demonstrated to affect cell mirgration via various mechanisms. however. although changes in cytoskeleton organization is the basis for cell motility, little is known about the association between cytoskeleton and ley. as microtubule and its construction unit tubulin participate in various steps of cell migration, we aim to explore the role of ley in microtubule and cell migration using breast cancer cells, which may provide reference to clinical study of other histo-blood group antigens and change the way of thinking in transfusion practice. study design/methods: we first manipulate ley expression in breast cancer cells by overexpression or sirna knockdown of fucosyltransferases, and block ley activity in mda-mb cells using anti-ley antibody, to verify the effect of ley on cell migration. then, we detect acetyl-a-tubulin level change as microtubule acetylation is a sign for stability. to establish the role of ley in cell migration via microtubule modification, we use hdac specific (tubacin) and nonspecific (tsa) inhibitors to minimize deacetylation of acetyl-atubulin and test again the effect of fut overexpression on cell motility. results/findings: fut overexpression increases both ley expression and cell migration, while fut knockdown leads to the opposite. ley activity blockade by anti-ley antibody also significantly inhibits cell migration. western blot and immunostaining results show a-tubulin acetylation level is negatively related with ley expression. tubacin or tsa treatment increases the acetyl-a-tubulin level while inhibits cell migration; in the meantime, the significance of fut overexpression in promoting cell migration is eliminated. conclusion: it can be concluded from the results above that ley can promote cell migration via regulation of a-tubulin acetylation, wherein ley may have interaction with deacetylase hdac . as tumor promoter, hdac becomes the target of many anti-cancer drugs. we demonstrated the potential association of ley and hdac function in this study. many blood group antigens are also carbohydrate chains, which are not only critical in blood group determination, compatible transfusion and immunological reaction, but may also have an effect in the initiation and development of diseases as tumor, similar to ley; they can even be components in a network with other important molecules and contribute to the destiny of diseases. transfusion of blood products is frequently needed by tumor patients. most attention is focused on the search of compatible blood for reducing transfusion reaction. however, it may lower the chance for the disease to advance to take account background/case studies: reducing the risk of bacterial contamination in platelet (plt) products is of great concern since plt storage occurs at room temperature (rt). pathogen reduction technologies (prt) were developed to inactivate pathogens prior to transfusion; however, studies have shown that prt may damage plts over the course of extended storage at rt resulting in a greater loss of function than what is normally concomitant with platelet storage lesion. storage of plts in platelet additive solution (pas) at c helps to preserve plt function and reduces the risk of contamination. in this study, we established the impact of prt performed after long-term coldstorage of plts in pas, instead of before storage, on plt function, mitochondrial respiration, and cell death parameters. study design/method: plt units were collected in pas (n ) and stored at c for up to days. after this time period, the bag was treated using mirasol prt (riboflavin and uv). samples were obtained and tested on the day of collection (baseline, bl), pre-mirasol (pre), post-mirasol (post), and minutes post-mirasol . aggregometry (adp, collagen, trap), rotem, flow cytometry (cd p [p-selectin] , lactadherin [ps] , , and gpib), high-resolution respirometry (oroboros), and imaging flow cytometry (amnis) were used for analysis. data are reported as means sem, and paired student's t-tests were used to determine statistical significance (p< . ). results/finding: on day , p-selectin levels were significantly higher in pre than bl (p . ). mirasol treatment caused a significant increase in pac- expression compared to pre (pre: . . %, post: . . %; p . ), which remained after incubation. a significant drop in both collagen and trap aggregation was observed in post samples compared to pre, but adp aggregation response was preserved. no differences in p-selectin, gpib expression, and mitochondrial respiration were observed between pre and post samples. post- samples displayed significantly less function, higher activation levels, and lower mitochondrial respiration compared to pre and post. conclusion: prt treatment of plt units in pas after day storage at c presents a unique alternative to prt treatment of plts prior to rt storage. in addition to providing a lower risk of bacterial contamination, c-stored pas plts may provide better preservation of hemostatic function than standard-of-care rt plts, even after mirasol prt treatment. however, we show here that mirasol prt of day c-stored pas plts followed by incubation ( minutes or more) results in widespread cell damage and should be avoided. safety evaluation of lyophilized canine platelets in a model of coronary artery bypass graft (cabg) todd m. getz* , arthur p. bode , anne s hale , michael stanton , mark johnson and g. michael fitzpatrick . cellphire, bodevet, inc, cellphire, background/case studies: cellphire has completed a micro dose clinical safety trial using lyophilized human platelets. cellphire also evaluated the safety of lyophilized canine platelets (lcp) in comparison to liquid stored canine platelets, following intravenous administration in a model of on-pump coronary artery bypass graft (cabg) in the canine. this safety study was in support of a future phase ii human clinical trial in cardiac patients. study design/method: three groups of eight mixed breed hounds underwent cabg to create an anastomosis and were administered lcps equal to , , and . % of the total circulating platelet count (tcpc). one group of four animals served as the vehicle group which received lyophilization platelet-formulation buffer, and another group of four animals received control ( -day old liquid-stored platelets). safety was assessed through the collection of blood loss data, evaluation of blood flow through the bypass graft, evaluation of the development of acute thrombosis, and maintenance of patency through the graft over the hr evaluation period. full necropsies with complete tissue analysis were also performed. efficacy signals were evaluated through the collection of blood loss data and coagulation endpoints (pt, aptt, fibrinogen, and teg). the results demonstrated that administration of the test article at doses up to % of the tcpc was not associated with any unexpected mortality, adverse changes in hematology or coagulation parameters, development of thrombosis at the anastomosis sites, or evidence of adverse thrombosis formation either clinically or microscopically regardless of group. the mortality noted on study was considered to be related to the surgical model and not a result of test article administration. the results also demonstrated that administration of doses of % and % of the tcpc produced a significant decrease in blood loss. the lcps at % and % tcpc were as effective in mitigating blood loss as -day old liquid-stored platelets and trended towards being more effective. no appreciable differences in coagulation parameters were observed between groups. conclusion: the results of the study demonstrate that administration of lcp up to % of the tcpc was safe in a canine cabg model. the data also demonstrate that administration of lcp at doses of % and % of the tcpc reduced blood loss. these results suggest a starting dose above . % tcpc may be required to achieve an effective dose in future human phase ii trials in cardiac patients. although the study was not powered for efficacy, these data indicate a level of safety, as % tcpc had similar efficacy signals as % tcpc with no observable severe adverse events. the starting effective dose may vary depending on the clinical indication. future studies will be required. this study was funded under barda contract hhso c. the study on pcr-ssp technique for the genotyping of cd - del.ac mutation and the genetic polymorphism of cd - del.ac in chinese population lilan li* and guoguang wu. nan-ning institute of transfusion medicine background/case studies: cd (platelet glycoprotein iv, scarb ) is an important and characteristic platelet antigen implicated in immune-mediated thrombocytopenia in chinese population. except anti-hla, anti-cd is the most common antibody of clinically relevant platelet antibodies in chinese population, which is associated with the high frequency of cd deficiency in china. cd gene mutation is the main reason that leads to cd deficiency. cd - del.ac (frameshift at aa ) mutation is one of the cd mutations that causes cd deficiency. have had natural mumps, measles and rubella infections, resulting in lower antibody levels in their blood. the recommendations may thus be unfounded and outdated, and prevent valuable vaccination opportunities for children with frequent blood transfusions. this places an already highly vulnerable pediatric population at risk for acquiring preventable infections. the primary aim of this project was to determine mmr vaccination immunogenicity in patients chronically transfused with rbc. study design/method: medical charts were reviewed for vaccination and transfusion histories. mmr-specific antibodies were quantified in pediatric patients who received both doses of the mmr vaccine at and months of age while they were on a chronic rbc transfusion program for sickle cell disease, b-thalassemia major, diamond-blackfan anemia or pyruvate kinase deficiency. there was no formal control group; long-term immunity rates in the literature are ! % for all mmr components. results/finding: table shows immunogenicity to vaccine components. delays between vaccination and serology testing averaged . years ( . to . years). thirteen patients ( %) were chronically transfused at the time of serology. twenty-three patients ( %) seroconverted to at least one of the vaccine components. conclusion: to the best of our knowledge, this is the first study designed to measure the effect of rbc transfusions on mmr vaccine immunogenicity. although lower than the rates reported in the literature, the results suggest a high rate of immunogenicity to each component of the mmr vaccine in chronically transfused patients immunized prior to months posttransfusion. weighing the risks and benefits of disease prevention in a highly vulnerable population, and taking into account the aforementioned results, a reevaluation of immunization delays post rbc transfusions is called for in chronically transfused infants. post-vaccination serology should be considered. cold stored uncrossmatched whole blood can be safely administered to pediatric trauma patients christine m leeper , , franklyn cladis , richard saladino , darrell triulzi , barbara a gaines and mark yazer* . university of pittsburgh, children's hospital of pittsburgh of upmc, institute for transfusion medicine background/case studies: the use of uncrossmatched cold stored whole blood (wb) is becoming increasingly popular in the initial resuscitation of trauma patients without a current abo group. wb has advantages over conventional component therapy including greater platelet and factor concentrations, as well as less saline and additive solution compared to an equivalent volume of reconstituted whole blood. this report details the initial use of wb in pediatric trauma patients. study design/method: pediatric trauma patients ! years old and ! kg with evidence of hemorrhagic shock were eligible to receive up to cc/kg of cold stored, leukoreduced group o negative wb during their initial resuscitation. all wb units had a low titer of anti-a and -b (< ) to reduce the likelihood of hemolysis in non-group o recipients. biochemical markers of hemolysis were measured on the day of wb transfusion and the following two days. admission thromboelastograms were obtained and repeated as necessary during the resuscitation. after receipt of the maximum quantity of wb, conventional components were utilized. results/finding: in approximately months, trauma patients received wb: group o and group a recipients, % male, median (iqr) age was ( . - ) and % blunt trauma mechanism. patients were severelyinjured with a median (iqr) injury severity score of ( - ) and % mortality rate. the median (iqr) quantity of wb transfused to group o recipients was . ( . - . ) ml/kg versus . ( - ) ml/kg to non-group o recipients. no transfusion reactions were reported. the mean standard deviation haptoglobin concentrations for non-group o recipients was . . mg/dl on day , . . mg/dl on day , and . . mg/dl on day ; the corresponding haptoglobin concentrations for group o recipients were . . mg/dl, . . mg/dl, and . . mg/dl, respectively (p> . for all comparisons). similarly there were no significant differences in total bilirubin, ldh, creatinine, and potassium at any time point. regarding evaluation of cold platelet function, we compared the subset of patients who received wb but no warm platelets (n ) to a historical group of pediatric trauma patients who received conventional components including warm platelets (n ). the mean standard deviation platelet volume administered was cc for whole blood recipients versus cc for warm platelet recipients. when pre-and posttransfusion teg and platelet counts were analyzed, there was no difference in median platelet count or teg maximum amplitude (ma) between cold and warm platelet groups. conclusion: use of cold-stored uncrossmatched whole blood for the resuscitation of pediatric trauma patients is feasible, acceptable, and appears to be safe. receipt of low titer group o wb did not lead to detectable hemolysis amongst the non-group o recipients. given this finding, the maximum quantity of wb per patients will be increased to ml/kg. identification of red blood cell antibodies in human breast milk by novel adaptation of serological method philippe p pary*, alexis leonard, lauren hittson, naomi lc luban, deepika s darbari, yunchuan delores mo, cyril jacquot, valli criss and jennifer webb. children's national medical center background/case studies: human breast milk contains immunoglobulins that are present in maternal serum and secretions. data in mice has demonstrated the potential for kell antibodies to be absorbed enterally from breast milk and impact the survival of transfused kell positive cells; however, methods to test and titer human breast milk for red cell antibodies are lacking. a two week old infant with a history of rh-d hemolytic disease of the fetus and newborn (hdfn), previously treated with intravenous immunoglobulin and phototherapy, was referred for anemia and reticulocytosis. patient was o positive, positive direct antiglobulin test (dat) with anti-human igg only, and a positive antibody screen by gel method. antibody identification showed anti-d in both the plasma and eluate. patient was transfused o negative red cells and discharged. over several weeks, the patient returned twice for persistent anemia requiring additional transfusions. at eight weeks of age, evaluation showed a persistent dat igg reactivity concerning for continued antibody exposure. maternal breast milk was evaluated as a potential source. study design/method: based on similar properties of human breast milk and plasma, testing to identify igg antibodies using a stantard tube saline method was performed with a minute c incubation, followed by automated washes prior to the addition of anti-human igg reagent. as a control, breast milk from an o positive, antibody screen negative mother was used to assess for interference by milk proteins. antibody screens were performed on the plasma of the patient, the patient's mother and the control concurrently using the same method. antibody identification and titers were also performed when indicated. only freshly collected breast milk stored at room temperature for less than days was found suitable for this technique. results/finding: the patient's mother showed plasma anti-d with a titer and the breast milk showed anti-d with a titer between and . the patient had a consistent plasma anti-d titer of . the patient's mother chose to stop breast feeding after weeks, and the patient's hemoglobin was improved at and weeks of age. using this method, we identified two additional cases of breast milk induced hemolysis: another anti-d and an anti-jka. conclusion: testing showed that it is possible to identify red cell igg antibodies in human breast milk using a standard tube saline method. we identified implicated antibodies in the breast milk received by infants with persistent anemia due to hdfn. breast milk titers were generally lower than maternal serum titers, but titers varied depending upon the timing and frequency of breast feeding. cessation of breast feeding correlated with improved hemoglobin in affected infants. background/case studies: red blood cell (rbc) transfusion is lifesaving for patients with sickle cell disease (scd), but is commonly complicated by rbc alloimmunization. despite transfusion protocols serologically matching for c,e, and kell antigens, alloimmunization to rh antigens continues. scd patients often exhibit a hybrid rhd-ce-d gene which is often characterized by the production of a partial c antigen. it has been previously documented that % of c scd patients from the west indies and west and central africa are partial c and at ( %) risk for alloimmunization to the c antigen through transfusion of c rbcs. this study sought to determine the prevalence within a cohort of children with scd at a u.s comprehensive scd center. study design/method: rbc genotyping results performed on all scd patients using precisetype hea array (immucor, norcross, ga) at children's healthcare of atlanta were reviewed and compared to the serologic type for rh (c/c, e/e) antigens. the prevalence of c-antigen positive patients (serologically) was determined overall, and compared to the prevalence partial c antigen based on the detection of the rhce*ce( g, t) allele in the absence of an rhce gene encoding a conventional c antigen in trans, since this allele is commonly linked to the hybrid rhd*diiia-ce( - )-d gene which encodes the partial c antigen. review of the blood bank information system was performed to identify the number of c-antigen positive transfusion exposures and frequency of alloimmunization to the c antigen. results/finding: out of a total of patients with genotype/rh phenotype data available, ( . %) were c antigen positive serologically. the allele frequency of rhce*ce( g, t) was . . in total, ( . %) patients possessed rhce*ce( g, t) in the absence of conventional c gene in trans. of the c antigen positive patients, individuals ( . %) were predicted to be partial c based on four molecular profiles [rhce*ce( g, t)/rhce*ce: ; rhce*ce( g, t)/rh*ce: ; rhce*ce( g, t)/rh*ce( g): ; rhce*ce( g, t)/rh*ce( g, t): ]. in these partial c patients, no anti-c alloantibodies (or other rh antibodies) were detected after transfusion exposures ( c-antigen negative units; mean: , range: - ), likely from placement of a c-negative rbc restriction upon detection of the rhce*ce( g, t) allele. conclusion: this report confirms previous data of a high prevalence of the partial c antigen in scd patients historically typed as c-positive serologically, and demonstrates the benefits of rbc genotyping to prevent alloimmunization to a highly immunogenic rh antigen by identifying individuals who should receive c-negative blood. all patients with scd should have rbc genotyping performed for determination of their rbc phenotype, preferably prior to receiving transfusions. investigational detection of zika virus rna in us blood donors paula p sa a* , megan l nguyen , melanie c proctor , david e krysztof , gregory a foster , erin k sash , sandy s dickson , joua yang , jeffrey m linnen , kui gao , jaye p brodsky and susan l stramer . american red cross, grifols diagnostic solutions inc., grifols diagnostic solutions, inc, quality analytics, inc background/case studies: zika virus (zikv), an emerging flavivirus, is primarily transmitted by infected aedes aegypti mosquitoes, but recent outbreaks have revealed non-vector transmission routes including the unprecedented sexual transmission of an arbovirus. acute zikv infection is mainly asymptomatic or presents as a self-limited disease but also includes severe congenital defects and neurologic disorders. the large proportion of asymptomatic cases, high numbers of returning travelers from zikv-active areas, severe clinical consequences to developing fetuses, the detection of rna in asymptomatic donors during the french polynesia epidemic, and suspected cases of transfusion transmission in brazil led fda to release guidance documents to minimize the risk of zikv transmission via blood/ blood components. study design/method: investigational testing by mini-pool (mp)-nat using the procleix zika virus assay (tma) was implemented on collections from five presumed high-risk us states on / / (fl, ga, sc, ms, al). following revised guidance on / / , testing was extended to all blood donations; conversion from mp-nat to individual donation (id)-nat was implemented in phases and completed on / / . travel history questions were discontinued on / / . confirmatory testing included repeat tma; in addition, rt-pcr, serology and red cell (rbc) tma were performed. estimates of viral loads were performed by end-point tma on plasma and rbcs. results/finding: as of / / , , , donations were tested including , ( %) in , mps. no reactive donations were identified by mp-nat. of the , , id-nat donations, were initial reactive (ir) of which ( %) confirmed positive (cp) by subsequent testing (cp rate of : , ; positive predictive value of %; specificity of . %). five ( %) cp donations were id-nat repeat reactive (rr); ( %) donations were id-nat ir only, igm positive and rna positive in rbcs. cp donors resided in ma, tx, ca, ny, wv and in fl, of which were local transmissions. six donors had traveled to a zikv-active area returning to the us from to days prior to donation. two donors with a travel risk reported clinical symptoms; cp donors ( %) remained asymptomatic. zikv rna was detected in rbcs from all cp index donations with estimated levels varying from less than copies (c)/ml to about Ê c/ml. at the time of writing, the longest period of detection in rbcs was days vs. days in plasma from the same tma-rr donor. zikv rna levels in plasma were obtained from ir and all rr donors, ranging from to c/ml. study design/method: plasma from blood donors were screened by individual donation (id-nat) for the presence of zikv rna with the cobasv r zika test. id-nat samples were repeated in duplicate and further tested by a second nat to confirm infection and estimate vl, and for anti-zikv igm. simulated mps of were prepared by diluting nat plasma : and tested to discriminate id-nat only detectable donations. nat yield samples for which simulated mp and conclusive igm results were available (n ) were sorted into categories corresponding to sequential stages of acute zikv infection: igm-/low vl; igm-/high vl; igm /high vl; igm /low vl. results/finding: of , donations collected april -december , were reactive for zikv rna. igm-index donations had higher vls (mean . x vs . x iu/ml) and higher proportions of simulated mp-detectable results ( % vs %) than igm donations. the distribution by stage of infection was evaluated as the epidemic evolved. over the course of the epidemic, the rates of id-nat only detectible and igm donations increased (table ) . conclusion: this study demonstrates how the viral and immunological profiles of zikv infection in the index donations shifted through the course of the pr epidemic. categorization of index samples into stages of infection is important for blood safety considerations, since infectivity and utility of mp vs id-nat screening likely correlate with vl and serological stages of infection. staging of infections also has implications for diagnostic testing and understanding the durations of zikv viral and immunological markers in blood and persistence of zikv in body fluids and tissues. cobasv r zika is not commercially available for blood screening. data generated under the cobasv r zika ind is preliminary and has not been reviewed by fda. this project has been funded in whole or in part with federal funds detection of zika virus rna in united states blood donations using cobas v r zika on the cobas v r / systems lisa lee pate* , phillip c williamson , michael paul busch , susan rossmann , scott jones , ann butcher , john duncan , jean stanley and susan a galel . roche molecular systems, inc., creative testing solutions, blood systems research institute, gulf coast regional blood center -sugar land, qualtex laboratories background/case studies: in february , the us fda recommended that all blood donations in areas with active zika virus (zikv) transmission be tested with an fda approved nucleic acid test (nat) for zikv rna or treated with an fda approved pathogen reduction technology. the cobasv r zika test was approved under an investigational new drug application on march , and testing of puerto rico donations began on april , . as a precautionary measure some blood centers in the us states also began nat testing for zikv. in august , the fda recommended universal screening of all blood donations. the aim of this study is to describe the detection of zikv rna in blood donations collected in us states between april , -february , using the investigational cobasv r zika for use on the cobas v r / systems. study design/methods: donations were screened with cobasv r zika by individual donation testing. all initial reactive (ir) results were repeated in duplicate. supplemental testing included an alternative nat (altnat) assay which is less sensitive than cobasv r zika and serology testing for anti-zika igm and igg. reactive donors were invited to enroll in follow-up, which included cobasv r zika and serology testing. a donor was considered to be zika confirmed positive if at least one replicate of the repeat testing by cobasv r zika was reactive on index donation or follow-up, reactive by altnat on the index donation, or positive for anti-zika igm on index or follow-up. all ir donations were also retested at a : dilution to simulate mini-pool testing. results/findings: a total of , , blood donations were screened using cobasv r zika. of ir donations, were repeat reactive (rr), non-rr and had no repeat testing. of the rr donations, were positive by altnat; of these were igm positive. all altnat negative donors were igm positive. one donor was alt-nat equivocal and igm negative. of the rr donors that were not igm positive on index, enrolled in follow-up and all seroconverted. of non-rr donations, were altnat negative and is pending supplemental testing. / donors were igm positive on index. donors were igm negative on index; / enrolled in follow-up; remained igm negative and was gm inconclusive. of donations without repeat testing results, met criteria for positive ( was altnat positive, igm negative and altnat negative, igm positive). donation is pending additional testing. altogether, / ir donations met the criteria for true positive on the index donation. / ( %) true positive donations were reactive when retested in a simulated minipool. / were igm positive. conclusion: . % of the , , donations in us states screened for zikv rna were confirmed as true positives. cobas v r zika is not commercially available for blood screening use. using monte carlo simulation luiz amorim* , marc germain , gilles delage , maria esther lopes and yves gr egoire . hemorio, hemaquebec, h ema-qu ebec background/case studies: zika virus was implicated in very large and recent outbreaks, in french polynesia ( ) , and in brazil ( / ), which was followed by outbreaks in south america, central america and caribbean. four probable transfusion transmitted cases were reported in brazil; since % of zika cases are asymptomatic, the actual transfusion rates can be much higher than reported. in this study, we used a monte carlo simulation for risk estimation during the brazilian outbreak. study design/method: the data feeding the monte carlo simulation were collected from january st , through november, th , , from brazil (the whole country) and for rio de janeiro state, one of the outbreak epicenters. the data came from brazilian epidemiologic bulletins and from brazilian blood donation figures. the risk assessment was performed separately for whole blood (wb) donation and for apheresis platelets (ap). the model took into account the following parameters: zika incidence in brazil and in rio; lognormal distribution symptomatic viremia (period: days, with % of the values lower than days); % of infected donors with symptoms lasting days; . donation/donor/year for wb and . for ap. the formula for transfusion risk calculation was: incidence x infectious period x average donation number per donor per year (wb, x/y; aph, z/y) x ( -proportion of refused donors) x ( proportion of discarded donations due to post donation -pd -information). results/finding: the table below shows the results. the estimated risk of transfusion transmitted zika is very important in brazil and in rio de janeiro, where it can attain : , , for apheresis platelets. the severe consequences of zika in vulnerable populations -pregnant women and newborn -indicate that interventions to reduce this unfavorable outcome, such as donor testing and pathogen inactivation, should be considered in brazil dengue (denv) arboviruses in the population are not available in brazil. the objective of this study was to assess the contemporaneous incidence of these agents in donors at large geographically dispersed blood centers located in the southeast and northeast of brazil. study design/method: in the brazil public blood bank system, nat screening for hiv, hcv and hbv is performed on minipools (mp from donations). the residual volume of mp plasma, . - . ml, is routinely discarded. beginning in april each blood center saved $ mps/week for retrospective testing using the triplex zikv, chikv, denv transcription mediated amplification (tma) assay developed by grifols/hologic. mps were shipped to the usa and batch tested at grifols. in the first two weeks (april - ) mp were combined into pools of donations; thereafter mp were tested without additional pooling. to estimate the percent positive donors, the denominator was adjusted to account for the number of donations included in each pool each month and % confidence intervals (ci) calculated using the method developed by biggerstaff. results/finding: the triplex assay performance was shown to have very high sensitivity ( % limit of detection < copies/ml for zikv/chikv/ denvs) and to accurately discriminate each of the arboviruses. testing of the first months of samples is complete for , mp, comprised of , donations collected from april to october , . a total of pools were positive, with detected between april-june . the table summarizes the highest monthly estimated percent positive donors for each virus in each city. months with highest percent postive donors were april or may. at the peak over . % of donors in belo horizonte and rio were viremic for zikv, whereas zika was not evident in donors in recife, but over . % of donors in that city were viremic for chivk during the peak. conclusion: during the latter part of the arbovirus outbreak season in brazil in , zikv, chikv, and denv were being transmitted by mosquitoes to donors with asymptomatic donors donating, indicating that blood recipients in brazil were extensively exposed to viremic blood components. the use of donor mps for surveillance may be one of the most efficient approaches for public health monitoring of the onset and magnitude of arbovirus infections. universal zika screening for blood donors in singapore sally lam* , sze sze chua , mars stone , michael paul busch and ai leen ang . health sciences authority, blood services group, blood systems research institute background/case studies: singapore reported its first locally transmitted zika case on august . the numbers rose rapidly to cases by the end september, with eight clusters (hotspots) of cases island-wide. zika virus (zikv) shares the same mosquito vector, aedes aegypti, as the dengue viruses and can caused microcephaly in unborn fetuses of infected pregnant women and guillan-barr e syndrome, which hastened singapore's blood services group (bsg) to look into securing the safety of blood supply from the zika threat. we aimed to assess the assay performance of usa-fda investigational (ind) procleix zikv nucleic acid technology (nat) assay for universal blood donation screening in singapore to prevent transfusion-transmitted zika infection. study design/method: all blood donations were screened for zika with the procleix zikv nat assay since october . zika nat reactive samples were tested at blood system research institute (bsri) for zika rna in plasma and red cells by pcr and for zika and dengue igm and igg antibodies. a zika confirmed case was defined by the presence of zika rna by pcr and/or zika antibodies. the analytical sensitivity was evaluated using blinded frozen samples consisting of replicates of half log dilutions of the who international standard for zikv and replicates of negative controls prepared by bsri. probit analysis was performed to determine the % and % limits of detection (lod) . clinical performance of the procleix zikv assay was also assessed with local patient samples obtained from institute of infectious disease and epidemiology, singapore and a member blinded zikv reference panel from the usa-fda. results/finding: a total of , donations were screened from october to march , with false positive case and zika confirmed donation detected. alternative zikv pcr tested positive in both the plasma and red cells with an estimated plasma viral load of . x copies/ml. zika igm was negative in the index donation sample but present in the -day post-donation follow up sample.. the donor reported no clinical symptoms. the analytical sensitivity for the procleix zikv assay was determined to be . copies/ml at % lod and . copies/ml at % lod. the procleix zikv assay detected rna in out of patient samples and provided . % agreement to the results of the usa-fda zikv reference material. conclusion: the investigational procleix zikv assay showed good analytical sensitivity and clinical performance, suitable for blood screening of zika infection especially in asymptomatic donor populations. bsg commenced universal zika nat screening by individual donation testing following the zika outbreak with confirmed zika donation (high-titer and seronegative) interdicted, which translates to a risk incidence of in , donations in singapore. background/case studies: a cap/aabb work group suggested that steps be taken to phase in rhdgenotyping for patients with a serologic weak d phenotype. weak d types , and express all the major rhd epitopes and these patients can be managed as rhd-positive, which may lead to a reduction in unnecessary rh immunoglobulin (rhig) administration and conservation of rhd-negative rbcs. study design/method: rhd genotyping was performed on all patient samples with weaker than expected or discrepant rhd typing results, utilizing a commercially available genotyping kit manufactured by immucor (rhd beadchip). initially, testing was performed at a reference lab while the rhd beadchip was validated and implemented at this institution. a serologic weak d phenotype is defined as weak to reactivity on initial gel testing. if genotyping demonstrated weak d types , or , the intent was to manage the patient as rhd-positive. if weak d types , or were not detected, the patient is considered at risk for alloimmunization and treated as rhdnegative. while rhd genotyping results were pending, rhd-negative rbcs were used and if pregnant, the patient was eligible for rhig. results were generally available in to weeks. results/finding: rhdgenotyping was performed on patient samples over months. of these patient samples, ( %) were weak d types or . the remaining samples demonstrated a variety of alleles including known partial d variants (see table) . one patient identified as weak d type required multiple transfusions over the study period, and refused rhd-positive rbcs. the remaining weak d types and patients have not received transfusions at this institution since they were genotyped. four of obstetric weak d types and patients received rhig while genotyping was pending. conclusion: testing and management of patients with serologic weak d phenotypes is not standardized. rhd genotyping may lead to more consistent, personalized patient care and appropriate management of resources. in this month study period serologic weak d patients were identified who could be managed as rhd-positive, however this did not result in withholding any doses of rhig nor conservation of rhd-negative rbcs. genotyping results pertaining to the management of an obstetric patient were discussed with each obstetrician and it is possible this information may impact management of future pregnancies. these outcomes highlight the limitations of current genotyping processes, including long turn-around-time background/case studies: the rh blood group is highly immunogenic and the most clinically significant blood group secondary only to abo. currently, in the united states, blood donors who type rhd-negative by serology undergo weak-d testing to identify some weak and partial states of rhd expression. however, not all rhd expression can be detected serologically. it has been suggested that investigation of serologic rhd-negative blood donors using genotyping methods can more accurately identify units that may lead to alloimmunization in rhd-negative recipients. study design/method: rhd genotyping of all serologic rhd-negative blood donors presenting to our blood donor center was implemented to identify units with altered rhd alleles that should be characterized as rhdpositive. repeat donations were not tested. initial serologic testing of blood donors was performed using fda approved anti-d reagents. when reactivity with all reagents was negative, rhd genotyping was performed using a commercially available genotyping kit manufactured by immucor (rhd beadchip). this assay detects over rhd variant alleles and additional dna sequencing was performed in selected cases. to maximize efficiency samples were batched for testing; testing was generally performed once a month. if an rhd variant known or suspected to be associated with an increased risk of alloimmunization was detected, recipients of previous donations were investigated for evidence of alloimmunization, and all future donations were restricted to rhd-positive recipients. results/finding: over a period of months we tested rhd-negative blood donors. there were ( . %) partial-d, weak d ( . %), and ( . %) del donors. in one donor sample a novel rhd allele was identified through dna sequencing (rhd*ivs - _ deltctc). the phenotype associated with this allele variant is unknown. investigation of previous donations from these donors showed that rhd-negative recipients received rbcs from of these donors. five of these recipients underwent antibody screening after an average follow-up period of months; anti-d was not detected in any sample (see table) . conclusion: serologic testing occasionally fails to identify some rhdpositive donor units, which could place rhd-negative recipients at risk for alloimmunization. dna-based testing can be used to identify donors who have the potential to sensitize rhd-negative individuals. in this limited study period a small number of serologic rhd-negative donors, whose genotype indicated potential to sensitize recipients, were found. however, review of recipient transfusion records indicated that prior exposure to these donors' rbcs did not lead to detectable immunization to date. future potential sensitizing events will be avoided by restricting these units to rhd-positive recipients. grifols diagnostic solutions labs, grifols immunohematology center background/case studies: pregnant women with rhd variants may be candidates for rhig prophylaxis if molecular analysis reveals a genotype associated with possible anti-d formation. proposed testing algorithms advocate molecular characterization of weak d types but if a patient types as rhd-positive, no further action is proposed. women with partial d variants who may also be at risk of anti-d formation have not been included in algorithms proposed to date yet molecular testing may unmask this hidden subpopulation of women who type as d-positive but who may be candidates for rhig prophylaxis. our hospital is in an urban setting in which % of deliveries are to african-american patients. we initiated routine, full-gene rhdsequencing for obstetric patients whose serology demonstrated not only weak d, but also those who were categorized as "d " with reactivity to determine the prevalence of partial d patients in an ethnically-mixed population who may be at risk of anti-d formation. study design/methods: from october to march , we performed routine d typing (neo, immucor) on obstetric specimens followed by rhd sequencing on samples with either a serologic weak d phenotype or anti-d testing strength of using at least antibody. solid phase and manual testing used the series and series reagents. four additional anti-d reagents manufactured by grifols (dg gel anti-d), quotient (anti-d blend), biorad (anti-d (rh ) blend), and ortho (bioclone anti-d) were also used for supplemental testing. rhd sequencing was performed by sanger methodology using routine clinical protocols. results/findings: rhd polymorphisms or variations were identified in all samples. two of ( . %) were d with an rhd gene with only common, known intronic variants that is predicted to produce the "reference" rhd protein (ivs - c, rs ; ivs c, rs ; and ivs a, rs ). two ( . %) were d and heterozygous for two apparently new rhd coding variations which we are confirming by further testing. four ( . %) patients had rhd alleles with known potential to make anti-d (rhd*dol , rhd*dar . , and with weak d type . ). one had weak d type , which has uncertain susceptibility to alloimmunization and one was weak d type , which has not yet been associated with anti-d. interestingly, two ( . %) had variable d expression associated with apparently new alleles, pending ongoing confirmatory testing and cloning. one patient background/case studies: a weak d type is a variant of the rhd protein that comprises an amino acid substitution located in the th transmembrane segment and expresses a reduced amount of the d antigen. this variant is known to be associated with the missense mutation c. g>c which is the first nucleotide of the exon of the rhd gene and thus could be implicated in exon skipping when it is mutated. when performing ngs (next generation sequencing) analysis to fully genotype known patients, we identified an additional variant. study design/method: dna samples were studied by beadchip technology (immucor/bioarray solutions) and ngs using the sureselect human all exon v (agilent) and the nextseq platform. in silico analysis with different bioinformatic tools was used to predict splicing events. furthermore, a functional splicing assay was performed to determine the impact of the nucleotide variations on exon skipping of rhd gene. this study was completed by the comparative modeling between the wild type and the weak type rhd proteins. results/finding: by a targeted analysis of full exome sequencing, we have confirmed the blood group genotype of patients previously characterized by beadchip technology. interestingly, out of carry the c. - c>t intronic variation on the rhd gene, already described and associated with a del allele. among these last patients, one has been previously characterized as rhd weak type carrying the c. g>c (p.gly ala). independently, sanger sequencing on unrelated rhd weak type samples pinpoint to a linkage disequilibrium between c. g>c (exac, maf . ) and the c. - c>t (exac, maf . ). in silico analysis of both mutation located close to the splice acceptor site of the exon does not predict a significant reduction of its strength score. with minigene vectors harboring rhd wildtype exon , mutant rhd c. g>c, mutant rhd c. - c>t and double rhd mutants c. g>c plus c. - c>t, we showed no influence on skipping of exon due to these mutations. comparative modeling of rhd proteins pointed out an additional hydrophobic interaction on the rhd weak type between ala (transmembrane helix ) and val (transmembrane helix ) hampering membrane insertion. conclusion: the c. - c>t variation is always associated in cis with the missense mutation c. g>c on the allele rhd weak type . the c. - c>t can be found alone on the rhd gene as a neutral polymorphism. we assess that these two mutations isolated or combined do not lead to abnormal rhd transcripts. our results clearly demonstrate that the weak d antigen reactivity observed with rhd type red blood cells is due to the substitution of alanine at amino acid position to glycine. topology of jk-weak or jk-negative single-nucleotide missense variants in the kidd protein glenn ramsey*. northwestern university background/case studies: the human urea transporter-b (hut-b) protein carrying the kidd blood group has transmembrane (tm) and tilted ureapore a-helices, a long extracellular connector segment, and cytoplasmic segments at each end. numerous single-nucleotide missense variants (snmvs) weaken or abolish expression of jk a/b antigens determined at p. . we mapped all reported jk-weak or jk-negative (jk-neg) snmvs onto the hut-b structure to explore topological correlates of jk antigen expression. study design/methods: jk*a and jk*b snmvs affecting jk expression were compiled from dbrbc and isbt registries, literature searches and - aabb, isbt and british blood transfusion society meeting abstracts. snmv locations were correlated with the human homolog of the x-ray-crystallographic structure of mammalian ut-b derived for analysis of ut function (levin ej, ) . results/finding: seven snmvs located within amino acid (aa) from the exofacial or internal end of a tm helix are mostly weak variants (table) . all at the exofacial ends (p.a t, p.w r, p.v d) are jk-weak; the two jkneg exceptions p.g e and p.g e are at the internal end of the tm helix bearing jk a/b . four snmvs in the cytoplasmic n-terminal segment are mostly weak variants. in contrast, snmvs within membrane helices are mostly jk-neg variants. three jk-weak snmvs (p.v m, p.e k, p.v i) have been associated with allo-anti-jk a/b to the antigen on their alleles ("weak partial"). six of the jk-neg variants are within aa (p. -p. ) of jk a/b at p. . none of these snmvs are in the long extracellular connector region or the cytoplasmic c-terminal segment. jk-neg variants p.n s and p.s p are adjacent to p. f and p. l which line part of the urea transporter pore. conclusion: in the transporter-structured rhd and rhce proteins, snmvs with weak d, c, c, e or e expression are mostly within the rbc membrane, and non-canonical antigen-negative snmvs are unusual. in the structurally similar kidd hut-b, most jk-weak snmvs are at the ends of the tm helices or in the n-terminal cytoplasmic segment. among jk-neg snmvs, most are in membrane helices. however, whether a variant appears jk-weak or jk-neg may depend on the extent of testing. next-generation sequencing may provide more complete structure-antigen correlations. background/case studies: the kidd-null blood group is most often inherited as a recessive genetic trait due to biallelic mutations in the slc a gene, which encodes the urea transporter ut-b . the kidd-null phenotype is associated with transfusion risk and also is associated with abnormalities in the ability to concentrate urine. the cause of the identical kidd-null phenotype with dominant inheritance [in(jk)] has not yet been defined, though it was first described in . in contrast to recessively inherited kidd-null phenotype, this is not associated with mutations in the slc a gene. the aims of the studies was to identify and characterize the causative gene for dominant kidd-null red blood cell phenotype (injk). jk-weak (bold)/ jk-neg expression n within aa from tm a-helix end v i, a t, w r, w r, g e, g e, v d* cytoplasmic n-terminal v m, g s, e k, l p in membrane tm and urea-pore a-helices r w, r q, g d, i t, a v, l r, a t ‡, a a §, l f, n s, s p, t m / * second nucleotide variant in this allele is synonymous (p.p p). ‡reported as jk-neg but considered jk-weak by isbt. §near splice point. study design/method: we identified several families with dominant inheritance of the kidd-null phenotype in multiple kindreds in spain. we performed whole-genome linkage analysis, exome sequencing, expression (rt-pcr and western) analyses, and urea lysis using patients' cells. in addition, two probands underwent urine concentration tests. results/finding: using molecular approaches, we mapped the affected locus to a mbp region in q . - . with an lod score of . . using deep sequencing, we identified a potential deleterious mutation in the znf gene, which deletes bp resulting in loss of an entire zing finger domain. the identical del -znf mutation is present in all affected individuals, and is absent from all controls tested (n> ). in addition, two adult individuals who are homozygous for the entire haplotype including the deletion within the znf locus, thus completely lacking the common allele, were identified. we also obtained dna from an unrelated injk individual reported from japan. in this individual, there was a similar, though not identical, znf del . none of the other potential genetic variants identified in the spanish kindreds was present in the dna from the injk individual from japan. consistent with the fact that the kidd antigen, encoded by the slc a gene, is a urea transporter that has been associated with renal function, we found that people with the znf del in spain had an inability to concentrate their urine. conclusion: a predicted zinc finger deletion at znf , prevalent in southern spain due to a founder mutation, leads to ut-b dysfunction and underlies the dominantly inherited kidd-null blood phenotype. the phenotype associates subnormal urine concentrating ability. in background/case studies: di-( -ethylhexyl) phthalate (dehp) makes pvc film flexible and useful for blood products. during storage, dehp can leach from the bag film into solution and be metabolized. studies in rodents have suggested that exposure to dehp may be associated with adverse health effects, albeit at high dosages. attempts to find dehp alternatives for blood bags have been difficult due to the rbc membrane-stabilizing effect of dehp. bis( -ethylhexyl) terephthalate (deht) a non-ortho-phthalate is structurally and functionally similar to dehp, but distinct from a metabolic and toxicological standpoint. deht can undergo complete hydrolysis and has an excellent safety profile; it is not classified as a carcinogen, mutagen, reproductive toxicant or endocrine disruptor. the study objective was to evaluate the quality of fresh frozen plasma (ffp) stored in deht containers versus ffp stored in dehp containers at days and year. study design/methods: thirty-six wb units were collected into cpd solution, leukoreduced, centrifuged, and separated into rbc and plasma. abo identical plasma units were pooled together in groups of three. the pools included group a, group o and group ab. each plasma pool was weighed, mixed, sampled, divided into dehp and deht pairs, and frozen at less than - c within hours of collection. in vitro plasma testing (pt, aptt, factor v, factor viii, fibrinogen, protein c, and protein s) was done on day (pool), day , and year of storage. dehp and deht paired plasmas were thawed and tested at the same time. plasticizer concentrations were determined on day , day , and year of ffp storage. dehp and deht and their monoesters were analyzed by liquid chromatography-mass spectrometry. internal standards were deuterated-dehp, mehp, deht and meht. the lower limits of quantification (lloq) were: dehp . ppm; mehp . ppm; deht . ppm; and meht . ppm. results/findings: mean and standard deviation (sd) for key clotting factors and plasticizer results are summarized in the table. there was no statistical difference in any plasma parameter between dehp and deht bags at the same time period. factor viii retained greater than % of its initial value. plasma stored in deht bags had an average plasticizer content % lower than that of the dehp bags. background/case studies: plasma prevents dilutional coagulopathy in trauma victims by replacing coagulation factors and substrates during resuscitation with red blood cells (rbcs) and/or crystalloid solutions. spray-dried plasma (spdp) is lightweight and can be reconstituted in minutes making it ideal for use in combat and pre-hospital settings to rapidly provide plasma in situations where it is impractical to administer fresh frozen plasma (ffp). the spray-drying process preserves coagulation proteins, but high molecular weight multimers (hmwm) of von willebrand factor (vwf) are decreased. the objective of this study was to compare spdp and ffp in reconstituted whole blood (rwb) to test the hypothesis that spdp is not inferior to ffp in facilitating platelet adhesion and thrombus formation. study design/method: under an irb-approved protocol, whole blood from healthy volunteers was collected into sodium citrate and centrifuged at g to separate rbcs from platelet-rich plasma (prp). prp was diluted -fold in pipes-saline with . mm pge and centrifuged at g. the platelet pellet was resuspended in either spdp or ffp and recombined with the packed rbcs to create rwb with hematocrit of - % and , - , platelets/ml. in addition, two rwb pairs were reconstituted with spdp diluted : (spdp %) with plasma from a patient with type vw disease (t vwd). samples were fluorescently labeled with a gpiibiiia-specific antibody and the sample was flowed through a type i collagen-coated microchannel at a shear rate of s - for seconds. still images of adherent platelets and thrombi were captured in order to calculate surface area coverage (sa) along the length of the channel. ratio paired t-test was used to compare sa in samples reconstituted with spdp vs. ffp. the margin of noninferiority was % (spdp/ffp > . ). results/finding: six batches of spdp/ffp were evaluated using subjects. there was no statistical difference between the spdp/ffp pairs (p . ). the mean ratio of spdp/ffp was . with a % ci of . - . . comparing spdp vs. spdp %, there was no difference (median ratio . , range: . - . ) in sa. two-way anova demonstrated that batch did not significantly affect ratio of sa in spdp vs. ffp. conclusion: spdp, despite a decrease of vwf hmwm, was not inferior to ffp in ability to support platelet adhesion and thrombus formation. on average, sa in samples reconstituted with spdp was % greater than in samples reconstituted with ffp. the lower limit of the th % ci is a difference of %, which is less than the a priori determined margin of noninferiority of %. even with % dilution with t vwd plasma, there was no reduction in platelet adhesion and thrombus formation in the spdp rwb samples. these data support the development of in-human studies to evaluate the efficacy and safety of spdp in preventing and reversing trauma-related coagulopathy. spray-dried plasma deficient in high molecular weight multimers of von willebrand factor retains hemostatic properties michael a. meledeo* , qiyong peter liu , grantham c. peltier , ryan c. carney , ashley s. taylor , colby s. mcintosh , james a. bynum and andrew p cap . u.s. army institute of surgical research, velico medical inc background/case studies: restoring coagulation factors is key in acute resuscitation after traumatic hemorrhage, but blood products are frequently unavailable in emergency response due to shelf-life restrictions and storage needs. a single unit spray dried plasma (spdp) process has been developed that produces a long-lived and readily stored product that has a reduction in high molecular weight multimers of von willebrand factor (vwf) and an increase in low molecular weight multimers. vwf is critical in platelet adhesion and thrombus formation. following work demonstrating enhanced function with use of glycine-based reconstitution solutions for spdp, this study examines two different spdp pretreatment conditions. study design/method: the samples were: ( ) ffp; ( ) ffp with mm glycine; ( ) regular spdp without pretreatment (rspdp), rehydrated with glycine-hcl:glycine; ( ) spdp pretreated with glycine-hcl ( mm); and ( ) spdp pretreated with glycine-hcl:glycine ( mm: mm; both pretreated were rehydrated in water). six donor-matched plasmas of each type were tested. vwf activity was measured by ristocetin cofactor assay. fibrin polymerization kinetics were analyzed by turbidimetry. thrombin generation (tg) was observed by thrombogram. chemistry was evaluated by i-stat. residual cell material was quantified by flow cytometry. coagulation properties were measured by thromboelastography (teg) in plasma and reconstructed whole blood ( % hct with platelets/nl from typematched donors). platelet adhesion to collagen under shear was measured by bioflux. results/finding: pretreated spdp showed enhanced vwf activity over rspdp (p < . ). fibrin polymerization density was slightly diminished in rspdp vs. ffp ( . vs. . o.d., p < . ), but tg was unchanged. bicarbonate/base excess were lower in spdp samples vs. ffp (p < . ). residual cellular material (especially platelet-derived) was reduced threefold in rspdp vs. ffp (p < . ) and an additional twofold in pretreated spdps vs. rspdp (p < . ). teg results were unchanged in plasma-only samples; in reconstructed wb there was a reduction in amplitude (clot strength) in all spdp samples vs. ; p < . ). platelet adhesion was equivalent in pretreated spdps and ffp, while rspdp was improved vs. all other samples ( . % surface coverage vs. . - . %, p < . ). conclusion: spdp has a longer shelf life and easier storage requirements than ffp and was equivalent or superior to ffp in most of these in vitro assays. spdp pretreated with glycine solutions was similar to ffp in most assays and showed superior vwf activity and fewer residual cellular materials but inferior support for platelet adhesion to collagen while under flow compared with untreated spdp. clinical significance of these findings is unclear, but overall in vitro outcomes suggest clinical studies are warranted. the interaction between red blood cell transfusion and lung injury: the influence of blood component manufacturing methods mathijs wirtz* , anita tuip-de boer , ruqayyah almizraq , jason p. acker , philip j. norris , jennifer a muszynski and nicole juffermans . academic medical center, university of alberta, canadian blood services, blood systems research institute, nationwide children's hospital background/case studies: red blood cell (rbc) transfusion is associated with acute lung injury, in particular in patients on mechanical ventilation. the causative factor is not known but may include residual cells or extracellular vesicles (evs) . in this study we investigated the functional effect of different manufacturing methods of rbc products on the response of pulmonary cells in an in vitro model of mechanical ventilation. study design/methods: groups of rbc products (whole blood filtered [wbf] , red cell filtered [rcf] , apheresis derived [ad] and whole blood derived [wbd]) were manufactured from donors (blood type a or b). supernatants were prepared after - (fresh) and - days of storage (stored) for measurement of thrombin generation and ev analysis. a type ii alveolar cells were seeded onto flexible membranes and incubated with rbc supernatant. cells were subjected to % stretch using a cellstretcher. control cells were not stretched. after hours, il- and il- production were measured. results/findings: both fresh and stored supernatants from ad products significantly increased pulmonary cell il- and il- production compared to incubation with other rbc products and non-incubated controls, which was further exacerbated by cell stretching. ad products also had significantly increased thrombin generating ability compared to other rbc products, as well as a significantly increased number of rbc-derived evs compared to rcf and wbd products (p< . ) . incubation of stretched cells with stored wbf products resulted in higher il- production compared to other blood products and stretched controls. rcf products did not activate pulmonary cells, had an absence of tg and had low levels of evs compared to other products. conclusion: manufacturing methods markedly influence the interaction of rbc products with lung cells. ad products activate lung cells, which is further aggravated by cell stretching. this may in part be mediated by rbc-background/case studies: investigators previously demonstrated immunosuppressive effects of rbc supernatant on monocytes in vitro, with greater effects seen in response to older units. recent clinical data suggest that rbc manufacturing method may influence immunomodulatory potential, but this has not been directly measured. we used in vitro models to test the hypothesis that rbc supernatants obtained by different manufacturing methods will have differential effects on monocyte function. study design/method: rbc products were manufactured by different methods from individual donors, each: (whole blood filtration [wbf] , red cell filtration [rcf] , apheresis, and whole blood derived [wbd] ). rbc products were stored in sagm (wbf and rcf) or adsol-containing preservative solution (apheresis and wbd). supernatants were obtained after - days (fresh) and - days (expiry). monocytes were co-cultured in media plus % rbc supernatant or media only (control) followed by lps stimulation. experiments were performed in replicates, each with a distinct monocyte donor. comparisons between groups by anova with dunnett's post-test for multiple comparisons. data are mean sd of % of control values. results/finding: exposure to apheresis or wbd rbc supernatants suppressed monocyte lps-induced tnfa production capacity compared to controls (table ) . this was true for fresh units and those at expiry. for monocytes exposed to rbc supernatant alone without lps, interleukin- production was higher after exposure to fresh wbf ( % control, p . ) or wbd at expiry ( % control, p . ). conclusion: manufacturing method and/or storage solution significantly alters immunomodulatory effects of rbc supernatant on monocytes in vitro and may confound analyses of clinical effects of rbc storage duration, particularly within international multi-center studies. a magnetic levitation system to study the impact of donor gender, age and blood storage conditions on red blood cell density profile gozde durmus* , alessandro tocchio , anita howell , kaushik sridhar , jason p. acker and utkan demirci . stanford university, canadian blood services, centre for innovation, background/case studies: the amount of hemolysis in red blood cell units increases as the product ages and has been shown to be lower in female blood donors than in males. it is hypothesized that female donors possess, on average, a younger population of red cells, which results in the lower hemolysis that is observed in the pre-menopausal population. it is also hypothesized that the differences between donor populations are mitigated by lysis of older cells when whole blood units undergo processing steps to produce red cell concentrate (rcc) units. as red blood cells (rbcs) age in circulation, they undergo characteristic changes in density and membrane composition that allows for them to be separated from younger cells. study design/method: our aim is to study the effect of donor factors and method of manufacturing and storing conditions on the average rbc age and density of red cell units. we have recently developed a powerful yet simple and inexpensive magnetic levitation-based platform, which allows realtime, high-resolution imaging and monitoring of various cell populations. this label-free system allows density profiling for individual red blood cells, with an unprecedented resolution of - g/ml. first, to determine the effect of rcc storage on the density profile of rbcs, levitation and single-cell density profiles were measured at , , , , and days. in addition, to determine the effect of donor age and sex on the rbc density profile, blood samples from volunteers with four different age and sex categories (male, - years; male, > years; female, - years; female, > years) were profiled. results/finding: first, we observed that the levitation and density profiles as well as morphology of rbcs within rcc units change significantly during storage. in addition, rbc density was significantly different between young ( . g/ml) and older female donors ( . g/ml) (p < . ). moreover, rbcs from young males ( . g/ml) were significantly less dense compared to rbcs profiled from older female donors ( . g/ml) (p < . ). conclusion: we have developed a magnetic levitation system for the point-of-care, real-time evaluation of rbc and red cell concentrate (rcc) quality. we envision our results might inform decision makers about impact that donor deferral criteria may be having on the quality of red cell concentrates available in the blood banks, for the optimal clinical outcomes. cytokine production of pulmonary cells il- (pg/ml) il- (pg/ml) background/case studies: oxidation reduction potential (orp) or redox is the ratio of activity between oxidizers and reducers. redox imbalance caused by a higher production of reactive oxygen species (ros) and reactive nitrogen species or a decrease in endogenous protective antioxidants results in oxidative stress (os). while os can cause cellular injury and death, it is also important in the regulation of a healthy immune response to injury or disease. in the present study we investigated changes in hemoglobin, free heme, and orp as red blood cells (rbc) age and the effects of red blood cell age on icu patient morbidity and mortality. study design/method: icu patients were enrolled in this prospective observational trial investigating the effect of transfused rbc age on icu patient morbidity and mortality. all rbcs were pre-storage leukoreduced and abo identical. citrated blood samples were collected from each rbc unit prior to issue. the rbc supernatants were tested for free hemoglobin/ heme and orp. the patients were followed prospectively. results/finding: a total of rbc units were transfused. patients and rbc characteristics are shown in the table. significant reductions were detected in orp values over storage duration (p< . ). substantial correlations were also found between orp and free hemoglobin (p< . ) and orp and free heme (p< . ). interestingly, there was a statistically significant difference between the average orp values of the transfused rbc in patients who developed infection with higher orp values measured in rbc units given to patients who developed post-transfusion infections vs (p< . ). no significant differences were observed between orp and patient mortality, hospital/icu days, or thrombosis. also, no correlations were detected between free heme/hemoglobin or rbc age and infection development. conclusion: these data demonstrate that older blood has lower orp values as well as increased free heme/hemoglobin. there were no differences in orp values between the different blood groups once rbc age was controlled for and there were no statistically significant differences in patient mortality associated with orp, free heme/hemoglobin, or rbc age. the decreased orp values observed in the older blood are likely attributable to the "storage lesion". higher transfused rbc orp values were associated with subsequent development of infection, and younger rbcs were found to have higher orp values. thus, this data supports that young/fresher blood may predispose to subsequent development of infection in critically ill patients. further studies are needed. background/case studies: no randomized trials in humans have addressed whether only exposure to red blood cells (rbcs) that have been stored for a long time is associated with harm. we explore the effect on inhospital mortality of transfusing rbcs stored for more than days compared to rbcs stored for days or less. study design/method: data from a multi-national randomized controlled trial were used for this exploratory analysis. the patients were hospitalized adults who required transfusions and were randomly allocated to receive the freshest rbcs in inventory or the oldest (standard issue) rbcs providing a large cohort of patients receiving rbcs with storage durations along the entire rbc storage continuum of to days. using a time dependent variable patient exposure was defined by the maximum storage duration of rbcs received. this was then used to classify individuals on each day of hospitalization into one of three mutually exclusive exposure categories: freshest (exclusively exposed to rbcs less than or equal to days storage duration -reference group), medium age (at least rbc of - days storage), and oldest (at least rbc greater than days storage). the primary outcome was all-cause in-hospital mortality. cause-specific cox regression models of in-hospital death assessed the effect of exposure of rbcs in each category to exclusive exposure to rbcs stored for days or less. the effects of fixed and time-dependent confounders were dealt with through stratification and regression. sensitivity analyses were conducted with a) weekly partition with cut-points every days, and b) a finer partition using cut-points every days. results/finding: , patients receiving , rbcs were included in the analysis. exposure to rbcs stored for more than days was not associated with increased risk of in-hospital death compared with exposure exclusively to the freshest rbc units (stored for days or less) after adjusting for several fixed and time-dependent potential confounders (hr . ; % ci: . , . ; p . ). exposure to blood stored for at most - days yielded a similar hazard ratio (hr . ; % ci: . , . ; p . ). in the sensitivity analyses using weekly partitions, exposure to rbcs stored for greater than days compared to exclusive exposure to rbcs stored days or less was not significant (hr . ; % ci . , . ; p . ). the confidence intervals around the hazard ratios for the other -day intervals all include . similar findings were obtained with partitioning exposure data into day intervals where exposure to rbcs stored for - days was not associated with increased risk of death compared with exclusive exposure to rbcs stored for - days (hr . ; % ci . , . ; p . ). the confidence intervals around the hazard ratios for the other -day intervals all include . conclusion: individuals exposed to rbcs stored for more than days were not at increased risk of in-hospital death compared to individuals exposed exclusively to rbcs stored for days or less. transfusion of anaerobically stored red blood cells improves recovery in experimental rat hemorrhagic shock model alexander williams* , cynthia walser , tatsuro yoshida , andrew dunham and pedro cabrales . university of california san diego, new health sciences inc. background/case studies: hemorrhagic shock (hs) severely decreases oxygen (o ) delivery and induces cardiovascular collapse. in parallel to controlling the hemorrhage, clinicians respond by infusing large volumes of red blood cells (rbcs) to restore blood volume, o carrying capacity, and hemodynamic stability. the quality of the transfused rbcs determines the recovery from hs, and extent of clinical sequelae prompted by the hs. this study compares the ability to recover from hs with conventionally stored rbcs, anaerobically (o saturation < %) stored rbcs, or anaerobic/hypercapnic (o saturation < % and pco (@ c) $ mmhg) stored rbcs. study design/method: packed red blood cells (prbcs) stored in as- after leukorfiltration were created from donor sprague-dawley rats. prbc units were randomly stored under either ) conventional; ) anaerobic; or ) anaerobic/hypercapnic conditions. rats ( - g) were hemorrhaged to % of blood volume, held in hypovolemia for minutes, and resuscitated to recover blood pressure to % pre-hemorrhage with prbc stored for either or weeks. systemic hemodynamics, cardiac function, and blood gas parameters were monitored during shock and resuscitation; and vital organ inflammation, oxygenation, and function were evaluated post resuscitation. data were analyzed using two-way anova, followed by the appropriate post hoc analyses. ( %) neg patient showed short term response and ( %) patients showed progressive disease. at the neg group standard eval ( %) patient showed response and ( %) had progressive disease. ( %) neg patient had long term response compared to ( %) pos patients. at the pos short term eval ( %) patients showed response and ( %) patients had progressive disease. at the pos group standard eval, ( %) patients showed response and ( %) patients had progressive disease. overall, ( %) pos patients responded compared to ( %) neg. conclusion: there is a trend in lower response rate in patients with negative antibody screens compared to positive controls. these findings suggest that an anti-cd neutralizing substance could play a role in treatment response. alternatively, reduced cd expression may also contribute. the low response rates seen in both groups may result from biased selection. the need for repeat t&s and presumed repeat transfusions may be preselecting patients with more aggressive disease. also, only a small number of patients were suitable for review. a larger prospective study that controls for such variables is needed. a review of blood utilized during provider-activated and critical administration threshold-triggered massive transfusion events patrick ramos* and john hess. division of transfusion medicine, harborview medical center background/case studies: traditional definitions of massive transfusions -e.g., the transfusion of ten or more units of red blood cells (rbcs) in a -hour period -are limited in prospectively identifying patients requiring massive transfusions, excluded patients who may not survive long enough to meet criteria, or ignored the acuity of the event. to address these issues, a level i trauma center adopted the critical administration threshold (cat) as an additional indication for activating its massive transfusion protocol (mtp). this study reviewed blood utilized during massive transfusion events based upon whether the mtp was provider-activated versus cat-triggered. study design/method: all massive transfusion events between january and april were reviewed to identify the start time, termination time, number of components transfused, and the start time of each component transfused. the transfusion of three or more blood components in an hour defined cat. a massive transfusion was any event in which the concern for hemorrhagic shock either necessitated a provider to activate the mtp or blood components were transfused at a rate that met cat criteria. the massive transfusion start time is based on either the time the provider activated the mtp or the time the first blood component was transfused, whichever came first. unless the patient expired first, the termination of the massive transfusion event was determined by identifying the point in time in which the patient went three or more hours without the transfusion of any additional blood components. this information was tabulated to determine the monthly number of provider-activated mtps, cat-triggered mtps, and average blood component transfused per massive transfusion. conclusion: blood utilization is lower within the cat-triggered mtps even though it outnumbered provider-activated mtps. however, the mode for both groups suggests that most massive transfusion require less blood components than the average rate. using the mode provides an approximate % replacement of blood volume. this should be enough to counter the early signs and symptoms of hemorrhagic shock. though this study did not review the appropriateness of provider-activated mtps, using cat as an indicator ensures clinicians are prepared for a potential massive transfusion. further investigation is needed to determine the factors contributing to the downward trend of the average blood components transfused. the mode would suggest optimistically that patients are being stabilized faster and resuscitated more efficiently. if this is the case, defining massive transfusion should include the rate of components transfused in addition to the total volume transfused. the long term storage effect of . m dithiothreitol on red cell antigen integrity in reagent red blood cells heike carrel* , laurie sutor , , germ an leparc , marjorie doty and william crews . carter bloodcare, ut southwestern medical center, oneblood background/case studies: anti-cd drugs, such as daratumumab, pose a problem for the transfusion service. they may cause a number of false positives, including positive direct antiglobulin tests (dat), indirect antiglobulin tests (iat), and panreactivity in eluates. such results can prolong compatibility testing and delay delivery of blood products for patients. treating reagent red cells (rrbcs) with . m dithiothreitol (dtt) removes drug interference due to daratumumab and allows for the detection of underlying alloantibodies. this study aimed to investigate the effect of dtt-treatment on rrbc antigen integrity over a day period. study design/method: twelve aliquots of human plasma, each containing an antibody of a single, known specificity (anti-d, -c, -e, -c, -e, -m, -s, -s, -fy a , -fy b , -jk a , and -jk b ), were tested against untreated and . m dtttreated rrbcs (immucor panoscreen i, ii, iii; dtt from acros organics). dtt treatment of rrbcs was performed using the methodology described in the aabb technical manual ( th edition). each of the plasma aliquots was further separated into aliquots and stored at - c until day of use. fresh aliquots were thawed each day to avoid unintended antibody integrity degradation. a polyethylene glycol (immucor) enhancement technique was used and reactions were read at the iat phase. hemolysis, if present, was observed in the diluent each day prior to mixing the cell suspension and given a grade based on the haemonetics color comparator chart. serological antibody reaction strengths were observed and documented each day. ( ) a monthly breakdown for both groups also displayed a downward trend in the average use of blood components. results/finding: there was noticeably more hemolysis with the dtttreated cells over time compared to the untreated cells. red cell antigens remained serologically detectable on the dtt-treated cells throughout the study, despite a greater degree of observed hemolysis. there was minimal difference in reactivity strength between untreated and dtt-treated cells for antigens not affected by dtt. in most instances, the dtt-treated cells reacted slightly more strongly. none of the antibodies produced reactivity strengths of less than with the untreated or dtt-treated cells during the study. conclusion: long term storage of . m dtt-treated rrbcs does not compromise antigen integrity. advance dtt-treatment and storage of a large aliquot of rrbcs may serve to increase efficiency in the transfusion service. background/case studies: monocyte monolayer assay (mma) is a cellular bioassay used to evaluate the hemolytic significance of blood group antibodies and aid in the selection of rbcs for alloimmunized patients. the requirement for fresh auto/allogenic monocytes for mma is highly restrictive due to tedious processing of fresh peripheral blood (pb). our previous study described processing and cryopreservation of buffy-coat (bc) derived and fresh pb-monocytes for mma assay. the aim was to evaluate the functional properties of cryopreserved bc-monocytes as substitute for fresh pbmonocytes in mma in evaluation of previously reported clinically significant rbc alloantibodies. study design/methods: peripheral blood mononuclear cells (pbmcs) were isolated from buffy-coats (histopaque- ), pooled, suspended in cryopreservation media ( % dmso; : ) and stored in liquid nitrogen. pbmc membrane integrity post-thaw was determined by trypan blue exclusion. pbmcs were cultured on poly-l-lysine-treated coverslips ( c, % co , h) and monocyte monolayers incubated with fresh or cryopreserved antigen positive (o ) rbcs sensitized with either anti-d (positive control), anti-scianna- (sc ) or anti-anwj or lipopolysaccharide stimulated for h. aliquots of the sensitized rbcs were tested for opsonization by indirect antiglobulin test (iat). phagocytosis index (pi) was determined microscopically as the number of fully phagocytosed rbcs/ monocytes. supernatants were analyzed for cytokines using luminex technique. results/findings: cryopreserved pbmcs showed . % viability postthaw. we report no significant difference in phagocytosis of anti-d sensitized rbcs by cryopreserved monocytes vs fresh monocytes. we show a significant increase in tnf-a, il- b, il- , il- , mip-a (p < . ), mip-b and gro (p < . ) secretion from cryopreserved bc monocytes vs both fresh bc and pb-monocytes. sc -and anwj-sensitised rbcs resulted in a pi of . % and . . % respectively vs anti-d sensitized rbcs (pi: . %). a weak ( ) reactivity by iat was observed for anti-anwj sensitized rbcs while anti-d sensitized rbcs resulted in iat reactivity. these results correlated with previously reported results for clinical significance and mma when using freshly obtained autologous or healthy donor monocytes. conclusion: this study shows that cryopreservation preserved monocyte viability and phagocytosis function for mma. as previously reported with fresh monocytes mma assay, the two alloantibodies tested with cryopreserved bc monocytes were shown to have a phagocytic index of clinical significance (pi> %). the use of cryopreserved bc-monocytes has the ability we describe antigen typing discrepancies in patients, involving antigens (c, jk a , s), revealed when serologic results differed from the phenotype predicted by dna testing. all patients had - positive dat with anti-igg and warm autoantibodies identified in the plasma. investigation of the antigen typing discrepancies showed both false negative and false positive results using monoclonal reagents. study design/method: standard tube hemagglutination methods were used for antigen typing. rbcs were treated with edta glycine-acid (ega) using gamma ega kit. genomic dna was isolated from wbcs and hea precisetype performed. results/finding: the rbcs of patients and typed c-on initial testing with immucor gamma-clone anti-c, but were predicted c by hea precise-type. ega-treated rbcs gave reactions with the same anti-c reagent. patient rbcs gave variable reactivity (vw- ) with bio-rad seraclone and ortho bioclone anti-c. patient rbcs gave reactivity with all anti-c reagents when incubated for the maximum incubation time allowed. patient rbcs were jk(a ) with immucor gammaclone anti-jk a , which the manufacturer states is suitable for testing dat rbcs, but predicted jk(a-) by hea. ega-treated rbcs tested jk(a-) with the same reagent. rbcs from patients and tested s with bio-rad seraclone anti-s ( - ), but predicted s-by hea. further testing with immucor gammaclone anti-s showed rbcs from both patients were s-. ega-treated rbcs from both were non-reactive with both anti-s reagents. conclusion: commercial monoclonal reagents are valuable resources, especially when phenotyping dat rbcs but not all manufacturers include reagent limitations regarding testing of dat rbcs. we describe cases of false negative tests with monoclonal anti-c due to antigen blocking by igg, and cases with false positive tests with anti-s (n ) and anti-jk a (n ) typing. false positive tests would potentially be anticipated, but false negative results due to antigen blocking are unexpected. extended incubation as indicated in the reagent insert may reveal weak reactivity when antigen blocking is involved. results concordant with dna testing were obtained with ega-treated rbcs, but it is generally accepted that this is not necessary when using a direct-agglutinating monoclonal reagent. these cases caution the potential for both false negative and false positive results for samples with - positive dat and supports testing to dissociate igg from rbcs strongly dat before antigen typing. in addition, this report highlights the benefits of dna testing as part of the routine reference laboratory workup. background/case studies: sensitization to antigens expressed on transfused cells, by triggering premature antibody-mediated clearance, diminishes the therapeutic effectiveness of transfusion and may also lead to serious delayed hemolytic transfusion reactions. accepted us clinical practice, while providing that sensitized patients receive only cells lacking "offending" antigens, nevertheless ensures continued alloexposure, and thus possible sensitization, to additional antigens, thereby complicating patient management. to mitigate sensitization risk, especially in an era of increasingly cost-conscious procurement, a quantitative assessment of the immunogenicity of specific antigens will be desirable. giblett, long ago, introduced a relative scale relating the rbc antigen immunogenicities to (an assumed) immunogenicity of "k" (http://bit.ly/ opqfew ). here, we show that an absolute estimate of immunogenicities may be extracted directly from observed antibody counts provided these are properly normalized to the fraction of recipients at risk (namely those lacking a specific antigen) and the expected fraction of donors expressing that antigen. study design/method: we define immunogenicity, or sensitization risk, r, for any antigen ("ag") of interest, as the conditional probability of alloantibody ("ab") formation, given allo-exposure to ag, i.e. r : prob(ab|al-loexp), so that prob(ab) prob(ab|alloexp)*prob(alloexp) and r ; rewriting prob(alloexp) prob(recipient, "r", lacks ag)*prob(donor , "d" has ag); and estimating prob(ab) nab/nr, nab denoting the number of ab in nr recipients, we obtain: nab/(nr*prob(r lacks ag)) r * prob(d has ag), the left-hand side representing the observed sensitized fraction, u, i.e. the number of observed ab in relation to the number of recipients at risk. conclusion: several antigens, though corresponding antibodies may be rare (e.g. "jsa", "e", "u"), nevertheless are highly immunogenic, requiring only a single exposure (on average) for sensitization; in contrast, others (on average) will require many exposures and thus pose a relatively low risk. in conjunction with patient genotypes, our r -scale will facilitate the selection of patient-specific cells so as to minimize the risk of (proliferating) alloimmunization even when perfectly matched cells are not available. our approach may be readily extended to additional rbc antigens and other antigen systems. background/case studies: aabb and fda require a month deferral of donors with a tattoo applied using non-sterile needles or reusable ink. we review state regulations to ascertain if tattoo establishments are licensed and required to use sterile or single-use needles and single-use ink. we recently added two large states in which we collect blood to the acceptable states list (asl). we compared the rates of donors deferred before and after the addition of these states to determine potential donor gain with changes in state tattoo licensing regulations. study design/method: we analyzed allogeneic interview responses to the screening question, "in the past months have you had a tattoo?" and if 'yes', whether the tattoo was applied by a state regulated entity. blood centers in states were selected for the analysis before and after state tattoo regulation. in state a, a comparison period of similar months before ( / - / ) and months after ( / - / ) was selected; for state b, a similar months before ( / - / ) and months after ( / - / ) was selected. frequency and rate of responses were compared in before and after periods. among those who responded to having a tattoo in a regulated state, donations were reviewed for presence of infectious disease markers including hiv, hbv and hcv. results/finding: a higher proportion of donors presenting to give blood admitted to having a recent (< months) tattoo in the post period in both states. this increase occurred immediately following the addition of states a and b to the asl (data not shown). among those who responded yes to having a tattoo, in states a and b respectively, there was a -and -fold increase in accepted donors (table) . the absolute number of accepted donors with tattoos increased from to (state a) and to , (state b), which annualized, represents a potential gain of , (state a) and , (state b) additional donations. all donors who had a tattoo in regulated states (asl) tested negative for hiv, hbv and hcv. conclusion: to counter rising numbers of ineligible donors resulting from recently added deferrals, we considered recovery of donors deferred for tattoos as a way to enhance our donor base. the immediate rise in the number of donors reporting a tattoo following the addition of the states may reflect a decline in self-deferrals based on having had a recent tattoo. we demonstrated an increase in the potential number of donations without compromising safety. background/case studies: transgender donors represent a small fraction of blood donors. determining their eligibility to donate has been challenging for blood centers. to assess behavioral risk, the donor is required to answer gender specific questions. the same is true when assessing trali risk where the donor is asked about a history of prior pregnancies. prior to the implementation of the fda's final rule, blood centers asked donors for their birth gender and determined eligibility based on that gender. if the donor changed their gender they were asked to answer both the male and female questions. the final rule now allows blood centers to accept the donor's stated gender and to determine eligibility based on that gender. in order to assess the risk of failing to ask a transgender male donor (birth gender female) the pregnancy question, a review was done to determine the number of transgender males who were actively donating with a large blood center. and tracked. donors were contacted to resolve any descrepancies. donors who had changed their gender from female to male and who had answered yes to prior pregnancies were identified. hla antibody test results were reviewed for these donors to see if they had been tested and whether they had tested positive or negative. results/finding: from - , there were donors identified who had changed their gender from their birth gender; female donors changed their gender to male and male donors changed their gender to female. there were ( %) transgender male donors, birth gender female, who had answered yes to the pregnancy question at one of their donations. three of these donors were apheresis donors who had been tested for hla antibodies. one tested positive and the other two tested negative for hla antibodies. the four other donors were whole blood donors and had not been tested. an hla test was added to these donors' records so that the test could be performed the next time they presented to donate. conclusion: transgender male donors may have had prior pregnancies and are also choosing to become pregnant after having transitioned from female to male. six percent of transgender males that we identified reported a prior history of pregnancy. at our center, when a donor requests a gender change from female to male, an hla test is requested for the next donation. first time donors are qualified based on their stated gender so transgender donors with a history of pregnancy will not be identified unless they volunteer this information. consideration should be given to using educational materials to prompt the donor to reveal a history of pregnancy at the time of donation so that hla antibody testing can be performed. effect of variable volume scale introduction in a large multi-site blood center ralph r vassallo*, marjorie d bravo and hany kamel. blood systems, inc. background/case studies: regulations allow whole blood donation [wbd] of up to . ml/kg or % of estimated blood volume [ebv] . traditional measuring/mixing devices are set to halt blood flow at fixed volumes which, with testing samples, are consistently below the % limit. variable volume scales [vvs] can be programmed to vary unit volume (up to ml) by donor ebv. this maximizes transfusable rbcs and plasma and recovered plasma [rp] volume. rp from wbds is a small but important source of derivatives and blood center cost recovery. we report the effect of introducing the hemoflow vvs on donor reaction rates and rp volume in a large blood center. compared to previous fixed settings, variable collection volumes were expected to decrease by ml at ebvs < . l in donors ! yo, but increase by - ml for all others. study design/method: donor vasovagal reaction [vvr] rates (prefaints, prolonged/offsite reactions, and loss of consciousness [loc]) for successful wbds were obtained from the center's hemovigilance database for the mos. before a mo. phased implementation of the vvs, and the subsequent mos. multivariable analysis [mva] by -mo. periods was performed in a model incorporating donor sex, age, first-time [ftd] vs. repeat status, ebv and donation site. both the volume and number of units of plasma sent for fractionation were available for the same time periods from the blood center's data warehouse. results/finding: compared to the baseline period, a significant increase in prefaint reaction rates were noted in pre-implementation (impl) periods & , continued during impl and post-impl periods & , returning to the baseline rate in post-impl periods & (table) . more severe reactions showed an increasing trend that only became significant in post-impl periods & . the mva showed the vvs as independent factor contributing to the increased prefaint and more severe reactions. however, its contribution, as measured by odds ratios, was consistently lower than those exerted by known donor determinants of reaction rates: young age, low ebv, ftd status and collection site (not shown). plasma unit volume increased an average of . ml during post-impl periods & from the temporally matched baseline & pre-impl period . conclusion: following an initial increase in mild vvrs during and immediately after implementation of the vvs, vvr rates fell back to baseline, suggestive of transient staff distraction from usual donor care, or a minor effect of increased blood loss with a superimposed improvement trend. the subsequent increase in prolonged/offsite reactions and loc after prefaint reactions had already returned to baseline suggests that staff training, work load, donor compliance with mitigation strategies and other determinants of donor reactions have a far greater effect than the small additional blood loss due to the vvs. small but significant increments in rp volume improve derivative availability and offset the cost of the vvs. comparison of vasovagal and citrate reaction rates in donors according to type of apheresis procedure pierre robillard* and yves gr egoire . hema-quebec, h ema-qu ebec background/case studies: apheresis procedures expose donors to various volumes of citrate depending upon type and length of procedure and type of machine used. citrate reaction (cr) results from various degrees of hypocalcemia in donors. blood volumes taken from donors vary according to type of procedure and use of volume replacement. loss of blood volume is in part responsible for the occurrence of vasovagal reactions (vvr). this analysis was conducted to estimate the incidence of cr and vvr according to various types of apheresis procedures performed at our blood center. (yfv) were reported in some brazilian states -rio de janeiro, sao paulo, minas gerais and espírito santo, mainly. the vectors of those cases were mosquitoes from the haemagogus and sabethes genders, whose habitat is the tropical forests. since many brazilian urban areas are very close to rain forests, there is an outbreak risk in those areas, where the infection is transmitted by the aedes mosquitoes. in order to minimize this risk, rio de janeiro health authorities decided to promote a mass vaccination in late march, . the vaccine is produced with live and attenuated yfv, which can circulate for at least weeks after vaccination. in some individuals, the vaccine can elicit viscerotropic effects and sometimes severe diseases. due to that, brazilian blood regulation authority established a week deferral period after yfv vaccination. this action could dramatically affect the availability of blood donors. this study shows the measures taken by rio de janeiro blood center to circumvent this risk and attract more donors. study design/method: the strategy consisted in offering the population, at a single place -the blood center -the possibility to donate blood and, immediately after donation, to get vaccinated against yfv. there were no financial advantages to the donors, since yfv vaccine is completely free of charge for any brazilian citizen. the vaccine was administrated by trained nurses, in an office close to the donors session. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. the blood center annnounced just before the mass vacination campaign launching that it would vaccinate people who came to the blood center to donate blood. if, for any reason, the prospective donors were not able to donate, the vaccine was also offered to them, provide there were no contraindications. results/finding: during the five days of campaign, we received , blood donors candidates; from those, , were accepted as a blood donor, after medical interview. the deferral rate was . %. at the same period of the year , there were , prospective donors, and blood donations. the deferral rate was . %. the "get vaccinated against yfv . . .but give blood before" campaign was able to attract, in a five day period, , additional donors, compared to same dates. that represents a . % increase in the number of blood donations, without deferral rate increment. there was a slight increase in the proportion of first-time donors, from . % in to . % in . conclusion: the strategy was more than successful, and it allowed the blood center to build a blood inventory large enough to avoid risks of shortage due to mass vaccination against yfv. dose loss which must be accommodated when collecting plt donations to ensure the us plt dose of ! . x is met. currently, triple set kits for pr are only approved in europe. plt loss, and adjusted apheresis targeting parameters may impact split rate (sr) or products per apheresis procedure. inventory suitable for pr without impacting us blood center srs warrants evaluation and optimization. study design/method: , apheresis collections from centers with different srs were analyzed. a baseline sr for conventional pc was calculated assuming i) a minimum dose (allowing for production loss) of . x for single (s), . x for double (d), and . x for triple (t) conventional pcs, ii) concentration and volume requirements from apheresis device manufacturer were used. for each collection, dose, volume, and concentration were assessed for pr kit compatibility, based on storage medium (pas or % plasma) assuming i) a minimum dose (allowing for production loss) of . x for s and . x for d for pr units, ii) removing small quantities from units with excess volume or dose to meet pr specs., iii) if all or part of an out of parameter d or t collection could be divided into one or more kits for pr, eligible parts undergo pr, and the remainder treated conventionally, iv) collections unsuitable for pr specs. or would decrease sr if treated would be counted as conventional pcs. results/finding: conclusion: blood centers today can adopt pr for a significant percent of their current supply (as high as %) without affecting their sr. compatibly increases further by dividing t and large d donations. percent achievable depends on their current s, d, t proportion of collections and practices. changes to d and t collection parameters, optimized donation and counting accuracy, and volume reduction will improve pr compatibility further. individual analysis is warranted for each blood center. rbc rbc plt/p plt plt/rbc/p plt/rbc plt plt/rbc plt/p #donations citrate exposure (mls) - study design/method: a randomized ( : ), placebo-controlled, single blind, subject, single-site study of ascending microdoses of autologous (apheresis-derived) thrombosomes was conducted. subjects were divided into cohorts, receiving increasing doses, ranging from / , - / of the lowest effective dose found in the above rabbit model. cohorts and received the / th dose, but cohort received two / th doses one hour apart. the primary end points were safety and tolerability. subjects were monitored in-hospital for hrs post infusion and followed for up to days for adverse events, global neurological assessments, abbreviated physical exams, and laboratory tests. results/findings: there were no serious adverse events (saes) or subject discontinuation post-infusion due to a significant decrease in platelet count from baseline. there were a total of aes: were treatment emergent (teae), of which were treatment-related ( thrombosomes and control). all teaes were mild or moderate in severity. in cohorts and , / thrombosomes subjects had treatment related adverse events. one cohort subject developed an upper respiratory infection and elevated wbcs within hours post infusion, which resolved by hours, and an elevated d-dimer at hours post infusion, which resolved by day . this subject also had an elevation of prothrombin fragment at baseline, which increased post transfusion and peaked at hours with resolution by day . one cohort subject developed non-specific t-wave changes at and hours following her nd infusion that resolved by day without clinical symptoms. troponin levels and echo stress tests were normal. ekgs were considered possibly a normal variant or related to placement of the ekg leads. another cohort subject developed an igg platelet autoantibody on days - , which was undetectable on days - ; there was no change in platelet counts. the thrombosomes autoantibody assay was positive at baseline, days - , and negative on days - . background/case studies: cryopreservation of platelets (plts) could extend the shelf life from - days to over two years. cryopreserved plts (cryoplts) appear to have a greater in vivo hemostatic effect than liquidstored plts. plts have been shown to require protein synthesis capabilities for certain functions such as clot signaling and immune responses. this study was designed to assess whether reconstituted cryo-plts carry out protein synthesis upon thawing and short term storage. study design/methods: apheresis plts were cryopreserved with % dmso and stored at c. after thawing, the unit was reconstituted in thawed ffp spiked with either lm puromycin (pm) or nm biotinlabeled pm. plts were stored at room-temperature with agitation. samples were drawn immediately after reconstitution as well as after , and hours to assess pm incorporation as a measure of protein synthesis, and for in vitro assays to determine platelet activation by cd p binding, phosphatidylserine exposure by annexin-v binding and microvesicle count in the supernatant. plt microvesicles (pmv) were prepared from the supernatant by ultracentrifugation. plts and pmv were lysed in a triton x- containing buffer and qualitative proteomics was performed on samples following affinity-purification with streptavidin beads. results/findings: in vitro parameters of reconstituted and subsequently stored platelets were in line with previously published results, with high surface levels of cd p and phosphatidylserine. pmvs were generated during cryopreservation and the count increased by -fold during hour storage. immunoblot analyses of the plts showed a -and -fold increase in pm incorporation after and hours of storage, respectively. massspectrometry revealed unique proteins that were synthesized after hours of storage, which was confirmed for gtpase and gtpase-regulatory proteins rac , rap and rhogdi by immunoblot analyses. analyses of the pmv translatome also revealed the presence of synthesized proteins; however, these did not change throughout storage. this finding suggests that a defined panel of proteins is packaged into pmvs upon freezing and thawing. additionally, the pmv translatome profile comprised a smaller subset of synthesized proteins compared to the cryo-plt translatome, including the proteins rac , rap and rhogdi. conclusion: this study has demonstrated that cryo-plts can synthesize proteins upon reconstitution in ffp and subsequent storage. discovery of a subset of these proteins in the pmv suggests their encapsulation, possibly in a selective manner. this observation provides novel insights into the capacity for protein synthesis in cryo-plts and the potential regulation of protein packaging into pmv. background/case studies: in , the authors' hospital-based blood bank received variances from the fda and aabb for the use of cold stored platelets (csps) with a shelf life of days. these group a csps, stored in a refrigerator in the emergency department, were used to support the trauma program for use in massively bleeding patients. the placement of the csps on the air ambulances, stored in coolers, was the next logical step in providing platelet therapy sooner to these patients. study design/methods: eight double unit csps were collected using the trima accelv r . two double csps were pathogen reduced using the inter-ceptv r pathogen reduction system. half of the csp pairs were subjected to flat storage in a refrigerator; the other half were loaded into a credov r - cooler with units of ffp, units of rbcs, and unit of whole blood. three to ml of platelets were collected via syringe from each unit at min (before storage in cooler or refrigerator) and after . , , , , and hours of storage for functional validation of platelets. the platelet count, agonists (thrombin receptor agonist peptide (trap), adenosine diphosphate (adp) and collagen stimulated platelets aggregation), non-activated and agonists activated platelet surface expression of phosphatidylserine (ps, annexin-v binding), p-selectin, fibrinogen receptor (pac- binding) were measured by coulter counter, channel aggregometer, and digital flow cytometer. paired wilcoxon rank sum tests were used to analyze differences in degradation rates with p< . deemed significant. conclusion: platelets, including pathogen reduced, stored in an oxygendeprived environment, (cooler), do not lose functional capabilities when compared to those platelets stored in a refrigerator with adequate oxygen for hours. therefore, cold stored platelets transported in a cooler are a viable option for providing timelier platelet intervention for severely injured patients prior to hospital arrival. c -a h molecular sieving: beyond genotyping ghazala hashmi , reinhard klemm and michael seul* , . biomolecular analytics, immunoinformatica background/case studies: more than a decade after its commercial introduction (hashmi http://bit.ly/ ohlehe), blood group genotyping, though available in several formats, has remained a tool for special tasks, e.g. the profiling of difficult patient samples or the identification of rare antigen combinations, while serology has remained the tool of choice for routine antigen typing. here, we introduce molecular sieving as an alternative to the current approach of managing special donor unit inventories. this novel process for dna analysis combines the "multiplexing" of markers offered by existing genotyping methods with the pooling of multiple samples in manner permitting the step -wise refinement of candidate sets by molecular attribute patterns. study design/method: molecular sieving is a special format of leansequencing, a proprietary process that permits the simultaneous analysis of up to four samples for alleles encoding rbc antigens in mns,rhce, lu,kel,fy,jk, di,yt,do and co, including the identification of rhce alleles. molecular sieving extends these capabilities to the analysis of pools to attain large scale. thus, in one format of the process, * * samples are accommodated in a single run. following the completion of the sieving step, candidates may be directly assigned to requests, or may be selected to enrich a subsequent profiling step for samples with rare or otherwise desirable attributes. here, molecularsieving was used to identify suitable donor units for sensitized sickle cell anemia ("sca") patients (tb in cas-tro , http://bit.ly/ oplxhr, excluding le and e(variant) and assuming request per patient), presenting with up to allo-antibodies ("ab") in multiple combinations. proprietary "greedy" algorithms were invoked to optimally pair candidate units with requests. results/finding: sieving of only = plate holding * candidate units from actual black donors, followed by profiling of samples selected to enrich for "e neg" and "c neg" and "c-e-k-fya neg", produced assignments for of requests ( . %), as indicated by colors, and shown in the row "assigned" below: thus, the number of assignments substantially exceeded the number of wells processed. moreover, the remaining pooled samples produced additional assignments to a second set of requests, for a total of assignments from only wells. in another scenario, sieving of a full plate of * samples, produced $ assignments for two successive batches of requests from sca patients, a yield exceeding . x. sieving alone typically fills - % of requests of moderate complexity ( ab). conclusion: molecularsieving, by widening the "funnel" while focusing the search for candidate donor units, attains a new level of efficiency in procuring suitable units for patients with hemoglobinopathies. molecular sieving for identifying red blood cells with special phenotype attributes kristopher fernandez , monica kalvelage , ghazala hashmi* and michael seul . biomolecular analytics, lifeshare blood centers background/case studies: providing transfusion support to patients with sickle cell anemia and other hemoglobinopathies remains a challenging logistical task that must accommodate pre-existing allo-antibodies in multiple combinations preferably while minimizing the risk of (continued) transfusion-related sensitization. the allelic diversity of the predominantly black patient population, especially at the rh locus which encodes a variety of "partial" phenotypes further complicates the problem (chou http://bit. ly/ ppvfeq ). study design/method: molecular sieving is a proprietary new process that, in order to rapidly probe candidate donor units in large numbers for multiple phenotype patterns, permits the analysis of pools and pools of pools of samples for a multiplicity of alleles (including at the rhce locus) that encode mns, rh, lu, kel, fy, jk, di, yt, do and co antigens. based on sieving, samples may be grouped by molecular attribute patterns ranging from single "ag " (e.g. e ,c ,e ,c ) to specific combinations of "ag " (e.g. c e k fya and c e jsa ) or combinations of alleles such as those encoding partial rh phenotypes. sieving, optionally, may be followed by profiling of samples selected for desirable attribute patterns. genomic dna from (predominantly) black donors, independently genotyped by one of two commercial methods were provided by lbc. at bmx, pools were prepared prior to amplification, and analyzed by a novel leansequencing method. results/finding: all pool genotypes were consistent with available individual sample genotypes. antigen patterns of particular interest included two groups, namely: several for which pools were homozygous and certain others t for which pools were heterozygous. illustrative of the former pattern type are these: appropriate pool queries revealed that sieving alone identified, among the c samples, that were also v and vs and, among the e samples, that were also negative for any partial_e phenotype. illustrative of the latter pattern type are pools identified as heterozygous ("het") for alleles encoding antigens of high or low prevalence. by segregating het pools into subpopulations, we were able to select specific "ee" pools of which were demonstrated (in subsequent profiling) to contain an e-sample. we also identified pools "het" for alleles indicating the possible presence of a rare donor, for example yta|b ( pools), co a|b ( ) and others. conclusion: molecularsieving of a single -well plate identified many desirable "antigen-negative" phenotypes and permitted selection of pools for combinations of "ag-neg" patterns including "partial:" rh phenotypes and combinations of c , e and jsa . these samples are thus confirmed "ag neg" and available for assignment. sieving also facilitated the enrichment of subsequent refinement of molecular attribute profiles in accordance with pending or anticipated demand. "antigen-neg" pattern partial_c partial _c, _e samples available after sieving background/case studies: sequence information generated from next generation sequencing (ngs) is often computationally phased using haplotype-phasing algorithms. utilizing experimentally derived haplotype information improves this prediction, as routinely used in hla typing. among the blood group systems, however, experimentally derived haplotypes are known for short genes only, such as icam (landsteiner-wiener) and ackr (duffy). for longer genes, such as abo of > kb, most haplotypes are only statistically derived. we recently established a large dataset of long ermap haplotypes, which code for the scianna blood group system. study design/methods: the nucleotide sequence of > kb each was used for all physically confirmed ermap alleles that we previously published. full-length sequences were aligned and variant sites were extracted manually. the bayesian coalescent algorithm implemented in beast v . . was used to estimate a coalescent phylogeny for these variants and the allelic ancestral states at the internal nodes of the phylogeny. results/findings: we found at least clades representing clusters of to alleles. for each clade, one observed allele was identified as the ancestral allele for its cluster of alleles. using the alleles, we were able to predict alleles with high posterior probability, which were ancestral to the observed alleles and, while not yet observed, may be extant. conclusion: we explored the phylogenetic structure and evolutionary events underlying the origin of different ermap alleles and predict ancestral alleles. in the present study, we show means to predict alleles and to calculate the distinct probabilities of correctness for such predicted alleles. the probabilities can be instrumental in defining a cut-off value to determine which computationally predicted alleles are worth confirming by physical evidence. the alleles identified by studies like ours may be utilized in designing of microarray technologies, imputing of genotypes and mapping of ngs data. the new alleles with nucleotide insertions would be predicted to cause complete loss of expression of the corresponding antigen from a bioinformatics perspective and to encode group o. rather very weak expression of the respective antigen and lack of the corresponding antibody in the plasma was found, confirming these represent subgroups of a and b and suggesting that transcriptional slippage, which has been observed before, is responsible for low level antigen expression. abo genotyping is powerful when both serology and molecular results are evaluated together, and these studies are needed to inform development of bioinformatics tools to accurately associate abo genotypes with phenotypes. background/case studies: evolutionarily related abo and gbgt genes encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the biosynthesis of a and b, and forssman (fors ) oligosaccharide antigens responsible for the abo and fors blood group systems, respectively. human at and bt possess leuglygly and metglyala, respectively, at codons - , and these tripeptides are important in determining the sugar specificity of enzymes, n-acetyl-d-galactosamine (galnac) for at and galactose for bt. functional fss possess gly-glyala at the corresponding codons, and exhibit galnac specificity. it has been recently shown that human at gained weak fs activity when the leu-glygly was substituted by glyglyala, suggesting that the tripeptide is involved in the recognition/binding of acceptor substrates, in addition to donor nucleotide-sugar substrates. study design/methods: we have searched for additional mechanisms that might enable human at to express fors . a variety of amino acid substitution constructs of human at were prepared. additionally, exon deletion constructs of at mrna transcripts were also prepared. dna from those expression constructs was transfected into cos (b galnt ) cells, and cell-surface expression of fors antigen was immunologically monitored with a monoclonal anti-fors antibody. results/findings: we found that met thr/ser substitutions also conferred human at with weak fs activity. we also found that the deletion of exon or of human at transcripts bestowed weak fs activity. because altered rna splicing is frequent in cancer, this mechanism may explain, at least partially, the appearance of fors antigen on certain cancer cells and tumors in forssman antigen-negative human species. furthermore, the co-introduction of one of those changes together with the glyglyala substitution synergistically conferred strong fs activity, in addition to strong at and bt activities. conclusion: the substitution of the glyglyala tripeptide codon in the catalytic domain may modify the acceptor specificity of the enzyme. met thr/ ser or exon / deletion may alter the intra-glogi localization of the enzyme. and those mechanisms function in synergy. the overlapping usage of acceptors by glycosyltransferases encoded by abo and gbgt genes is reminiscent of common ancestral origin of alpha , -gal(nac) transferase genes. the finding that at can synthesize fors implicates that the boundary between abo and fors systems may not be as strict as was previously delineated due to the crosstalk in-between. rh typing is required by the fda and fact/aabb for identity testing. since most antibodies in cb plasma are maternal in origin, the abo/rh phenotype relies only on the red cell typing. a and b antigens are not fully developed at birth, presenting about one third of a or b antigen expression levels compared to adult cells. this can result in indeterminate abo results for some cb products. we evaluated the use of dna-based methods for abo typing to aid the resolution of inconclusive ("indeterminate") or discrepant serologic typing results. study design/methods: a total of , cb units (cbu) were typed for abo/rh (beckman coulter pk system blood grouping and phenotyping) during the period / / - / / . abo genotyping targeting specific snps for groups a, a , b, o , and o and, if needed, gene sequencing was conducted in cases with indeterminate results, and in cbu that were provided for transplantation with abo discrepancy found at the transplant center. results/findings: sixty-two ( . %) cb samples had no reportable abo/ rh phenotype on initial testing, and therefore the cbu could not be used clinically. molecular abo/rh typing resolved all but one. all cases were heterozygous (a/o, b/o, or a/b); in % the predicted abo phenotype was a rh neg (table a ). the predominant donor race was caucasian ( %). four cbu with abo discrepancy were also evaluated by genotyping (table b) . in of those, abo typing performed at the hospital on the day of transplant differed from that reported by the cb bank; the fourth was identified by posttransplant abo typing of the recipient. molecular genotyping resolved the discrepancies. cbu identity was always verified by confirmatory hla typing. conclusion: there is currently no fda approved dna-based abo assay. however, abo genotyping is a useful method for samples where antibody tests alone cannot be conclusive, and can "rescue" cbu that could not be used otherwise. further, genotyping can help resolve abo discrepancies. abstract cobas v r hev for use on the cobasv r / systems is a qualitative pcr test for the detection of hev rna in human plasma. the purpose of this study was to evaluate the prevalence of hev rna among us blood donations collected in the midwest, a region reported to have a higher prevalence of hev infection, and the eastern us. study design/methods: , fresh and , frozen edta plasma samples from american red cross donors, collected from february - , were de-identified and screened by individual donation testing (id-nat) using cobasv r hev for use on the cobasv r system under a research protocol. samples were primarily from midwestern and eastern regions of the us. samples reactive on cobasv r hev were further tested by an alternate hev nat, hev rna quantitation, hev genotyping, and for hev antibodies. results/findings: of , valid results, a total of donations were reactive on cobasv r hev and all were confirmed positive. the confirmed donations were from a -year old male in indiana, a -year old male in california, and a -year old female in kentucky. all donations were positive by hemi-nested pcr and alternative hev nat; however, only the kentucky donation had a high level of hev rna ( iu/ml), and was strongly positive for both igm and igg hev antibodies. the indiana donation was genotyped as a, the california donation genotype b, and no genotype determined for the kentucky donation (see table) . the clinical specificity for the cobasv r hev test in id-nat was % ( % exact ci: . % to %). conclusion: based on the confirmed-positive donations of , tested, the hev prevalence was . % ( % exact ci: . % to . %) with a detection rate of : , ( % ci, : - : , ). to date, no cases of tt-hev have been documented in the us. however, based on the prevalence observed, immunosuppressed transfusion recipients may be at increased risk for transfusion-transmitted hev. background/case studies: monitoring the epidemiology of ttis within the donor population is critical to provide an ongoing assessment of infection risks associated with fda policy changes such as the msm deferral criteria. ttims is a multi-center, federally-funded program intended to derive hbv, hcv and hiv prevalence, incidence, viral genotypes, and donor risk factors for greater than % of blood collected in the us. ttims is supported by two distinct coordinating centers (laboratory and risk factor, lrcc, and donation database, ddcc). here we report months of prevalence along with demographic trends from the ddcc. study design/methods: four blood providers and their respective testing laboratories participated. standardized consensus-positive (cp) monitoring definitions were established for donor test results for hbv, hcv and hiv. these results, along with demographics for each donor and donation status (first-time vs repeat) were assembled into a single data set. rates of nucleic acid test (nat) yield (seronegative) and concordant positives (serologic plus nat positives) were combined to comprise cps, were computed overall for donors and donations and by demographic, geographic and temporal characteristics. where appropriate, rates were compared for differences using % confidence intervals. this analysis contains data from / / - / / . results/findings: among , , donations reported ( . % from firsttime and . % from repeat donors), there were respectively , and cp results for hbv, hcv and hiv with corresponding rates of . , . and . per , (pht) donations. prevalence among firsttime donors was, as expected, higher than among donations from repeat donors with ratios of : , : and . : for hbv, hcv and hiv. rates (pht) among males were higher than among females for all markers (hbv . vs . ; hcv . vs . ; hiv . vs . ). in general, higher rates for all markers were seen among minority donors, those in the - -year age group (also - year for hiv), and those from the southeast (and south central for hiv and hcv, and southwest for hbv). no trends were noted over time when -month periods were compared. conclusion: data from major us blood systems were successfully combined and are a baseline for monitoring purposes. demographic trends are similar to those observed in other donor studies and generally agree with community trends. changes in rates will require analyses in the context of potential changes in the demographic structure of the donor population. screening donated blood from babesia endemic regions of the united states using a transcription-mediated amplification assay on a fully automated system vanessa bres* , melanie c proctor , deanna self , monique portugal , adrian gurrola , laura tonnetti , sonia bakkour , cheryl lobo , michael paul busch , susan l stramer and jeffrey m linnen . grifols diagnostic solutions inc., american red cross, blood systems research institute, new york blood center background/case studies: the procleix v r babesia assay on the procleix panther v r system is a qualitative in vitro nucleic acid test currently under development. the assay, which is based on transcription-mediated amplification (tma), detects four clinically relevant babesia species (b. microti, b. divergens, b. duncani, and b. venatorum) in human whole blood specimens. this test is intended to screen blood donations individually and in pools of up to donations. whole blood samples are lysed and then pooled on the automated procleix xpress v r system prior to testing on the procleix panther system. these studies evaluated the preliminary analytical and clinical performance of the procleix babesia assay on the panther system. study design/method: analytical sensitivity was determined by diluting in vitro synthesized rna transcripts for the four babesia species. fresh b. microti-infected hamster whole blood, cryopreserved b. duncani-infected hamster whole blood and fresh b. divergens-infected human erythrocytes were tested to determine the limit of detection (lod) of parasites/ml (p/ml) by probit analysis. clinical sensitivity and specificity were determined by screening , unlinked whole blood donations collected from august th to april th in the northeastern united states. initial reactive donations were confirmed by repeat testing, pcr, and/or igg immunofluorescence assay (ifa). reactive individual donor lysates were tested in pools of . results/finding: the procleix babesia assay detected all four babesia species with a % lod ranging from . - . copies/ml. the preliminary % lod in parasites/ml ranged from . - . p/ml for b. microti (n ), from . - . p/ml for b. duncani (n ), and from . - . p/ml for b. divergens (n ). of the , donations screened, initial reactive and confirmed positive donations were identified for specificity of . % ( %ci: . - . %). of the confirmed positive specimens, were reactive by both ifa and pcr, by ifa only and by pcr only. all confirmed positive samples were reactive in lysate pools of . donors of reactive donations resided in ct ( ), nj ( ), nh ( ) and me ( ) for an overall incidence of : , , and : , in ct. conclusion: the procleix babesia assay on the procleix panther system demonstrated high clinical specificity and sensitivity and detected all four babesia species with similar sensitivity. all confirmed positive donations were also detected in pools of thus demonstrating the effectiveness of pooled lysate screening. conclusion: use of the lag avidity assay shows that in both first-time and repeat hiv-positive us blood donors, newly-acquired (i.e., incident) hiv infections are more frequent in younger donors. the use of this approach provides an additional monitoring tool to assess changes in characteristics of donors whose risk exposure was proximate to the date of donation and will also complement traditional incidence methods by allowing derivation of incidence by donor type. epidemiology of hepatitis b virus, hepatitis c virus and human immunodeficiency virus in united states blood donors lauren a crowder* , whitney r steele , ed p notari , james haynes , roger y dodd and susan l stramer . american red cross, american red cross (retired) background/case studies: from - , the prevalence of hbv and hcv in us blood donors decreased, while hiv rates remained constant. however, incidence has not been recently calculated. here we report the prevalence, incidence and residual risk (rr) of hbv, hcv, and hiv in a large us blood system from - . study design/methods: prevalence was calculated in -year intervals. incidence was measured as the number of positives among repeat donors divided by the total time at risk, in person-years (py). rr was calculated using the window periods of . , . and . days for hbv, hcv and hiv, respectively. linear regressions were calculated with p< . (*) as significant. results/findings: from / / - / / , there were more than million donations from , , donors ( . % female, % first-time (ft), . % caucasian). there were significant decreases in donation prevalence for hbv and hcv (p . and . ), but no significant decrease in hiv during the years (see table for f and r values). a significant decrease was seen in ft donor prevalence for hbv and hcv (p . and . ). prevalent ft donors were significantly more likely to be male ( . % -hbv, . % -hcv, . % -hiv; p< . ). incidence for all agents declined (significant only for hbv; p . ). the decrease in hcv incidence was not significant, but there were fewer incident donors in the last -year period ( in - vs. in - ) . hcv incident donors in - were more likely to be male ( . % vs . % in - , p< . ) and were younger ( . % vs. . % in - < years, p . ). overall, incident donors were more likely to be caucasian males (p< . ). rrs for all agents decreased over time with rrs in - of in , , ; in , , ; and in , , for hbv, hcv and hiv, respectively. conclusion: prevalence, incidence and rr of hbv, hcv and hiv have generally decreased within this blood system over the -year time frame. as donor screening and deferral regulations evolve, it is important to monitor these risks. it is critical to note that even in a large population, small changes to the number of positives can have a significant impact on prevalence and incidence rates. furthermore, in , mayv was isolated from a patient in haiti, suggesting the virus is already circulating in the caribbean. the extent of mayv transmission could be underestimated due to limited surveillance and diagnostic capabilities; therefore, it is necessary to be prepared for mayv emergence and the potential risk for the blood supply in case it can be transmitted through blood transfusion. study design/method: platelet components (pc) prepared in pas were spiked with mayv and treated with amotosalen and uva illumination. samples were collected pre-uva and post-uva illumination for infectious titer determination. as- rbcs were spiked with mayv, mixed with glutathione (gsh)/processing solution, dosed with lm amustaline, and incubated for hrs at room temperature. samples were collected prior to the addition of amustaline (pre-treatment) and following the hr incubation (post-treatment) to determine infectious titers. infectious titers for all samples were determined by plaque assay on vero cells. the extent of inactivation was determined by comparing the infectious titers (plaque forming units (pfu)/ml) in pre-vs. post-treatment samples. results/finding: mayv was inactivated to the limit of detection in both pc and rbcs. in platelets, > . log , or > . log pfu/ml, inactivation of mayv was achieved. in rbcs, inactivation of mayv was > . log , or > . log pfu/ml. conclusion: this study demonstrates robust inactivation of mayv by both amotosalen/uva treatment in pc and amustaline/gsh treatment in rbcs. these systems are efficient at inactivating alphaviruses that have demonstrated or have the potential for transfusion-transmission, including mayv, chikv and rrv. prt offers potential as a mitigation strategy for maintaining blood component availability in areas where multiple alphaviruses are epidemic or endemic, and testing is not feasible. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). thrombotic thrombocytopenic purpura with high adamts- inhibitor may represent a distinct disease subset in response to therapy based on immature platelet count (a-ipc) dynamics hamza n gokozan* , , hollie m reeves , and robert w maitta , . case western reserve university school of medicine, university hospitals cleveland medical center background/case studies: thrombotic thrombocytopenic purpura is a lifethreating consumptive thrombocytopenia and microangiopathic hemolytic anemia causing diffuse ischemic damage to tissues. early therapeutic plasma exchange (tpe) initiation has improved survival. absolute immature platelet count (a-ipc) has been found to aid in diagnosis and follow-up of ttp patients. a-ipc changes in response to therapy in patients with low adamts activity and high inhibitor have not been analyzed in a patient cohort. we analyzed a-ipc response to therapy in five patients with adamts deficiency and high inhibitor at a large tertiary academic medical center. study design/method: patients had adamts activity of < % and high inhibitor ( . - ). mean age of cohort . years (range - ). four patients were female and one was male. patients presented with microangiopathic hemolytic anemia, thrombocytopenia (mean . x /l, range - x /l) and low a-ipc (mean . x /l, range . - . x /l). patients were initiated on daily tpe and prednisone; additional immunosuppression during hospital stay for cohort consisted of rituximab mg/m ( patients) and cyclophosphamide mg/m (one patient). tpe continued until platelet count reached x /l for at least two consecutive days. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: patients responded rapidly to daily tpe (mean of . days [range - days]) when they achieved a three-fold increase in a-ipc from baseline (mean . x /l, range . - . x /l) and a rapid improvement in platelet count. however, this improvement in platelet count was not accompanied by expected decreases in a-ipc, suggestive of recovery from disease. all patients experienced platelet (mean . x /l, range - x /l) and a-ipc (mean . x /l, range - . x /l) decreases that occurred concurrently while receiving daily tpe so that after a mean of . days (range - days) mean platelet count was . x /l (range x /l) and mean a-ipc . x /l (range . - . x /l). patients were initiated in either rituximab or cyclophosphamide therapy in conjunction with tpe after a mean of . days of a-ipc and platelet count instability. a-ipc trended to levels indicative of restoration of a negative feedback after this time. conclusion: rapid decreases in platelet counts after a good response in ttp patients may raise suspicion for presence of high adamts inhibitor. patients with a high inhibitor have similar a-ipc dynamics during which initial high a-ipc production is followed by unexpected decreases in a-ipc concurrent with platelet counts. recovery occurs once negative feedback between platelet and a-ipc production is re-established. patients with a high inhibitor may represent a distinct subset of ttp as suggested by a-ipc responses. benchmarking the centralized urgent plasma exchange service for patients admitted with a diagnosis of thrombotic thrombocytopenic purpura at a multi-hospital healthcare system jansen n seheult* , michelle n stram , joan sevcik , alesia kaplan , and joseph e. kiss , . department of pathology, university of pittsburgh medical center, blood systems inc., university of pittsburgh background/case studies: consensus guidelines recommend that therapeutic plasma exchange (tpe) must be started as early as possible and within - hours after the diagnosis of thrombotic thrombocytopenic purpura (ttp) has been made; however, there are limited data documenting actual practice. there are several operational facets of delivering a centralized urgent tpe program in a multi-hospital healthcare system, including: central venous (cv) access, ordering, release and delivery of thawed plasma, and transportation of personnel and equipment to perform the procedure. this study analyzes the time elapsed between major steps from diagnosis to initiation of tpe in patients admitted with ttp. study design/method: a retrospective review of the electronic medical record and laboratory information systems from january , to november , was conducted to identify all ttp patients undergoing urgent tpe. demographics, comorbidities, and other pertinent laboratory tests (such as adamts- activity levels, complete blood count, biochemical markers of hemolysis and coagulation studies) were reviewed on all identified patients. temporal data for tpe request, cv access placement, plasma product release (which usually happens after cv access), arrival of tpe team and initiation of the procedure were extracted from procedure notes and the blood bank information system. descriptive and summary statistics were generated using stata version (statacorp, tx). group comparisons were made based on hospital location, level of care and history of ttp using a wilcoxon rank-sum test. results/finding: of the ttp patients identified, were excluded due to missing temporal data for important variables. the majority ( %) of patients were treated at central academic centers, with the remainder being treated at peripheral sites. fifteen patients ( %) had a prior history of ttp and % had severe adamts deficiency on admission. the median time from tpe request to initiation was . hours (interquartile range: . - . hours). there were non-significant trends to shorter time intervals from request to cv access and request to tpe initiation in patients admitted to the intensive care unit (icu) versus non-icu patients (table ) . treatment was not started within an -hour window in patients; the median time to cv access was significantly longer in these patients ( . vs . hours, p< . ). two of these patients had a prior history of ttp and only four patients had severe adamts- deficiency. the majority (more than %) of the time interval between tpe request and tpe initiation was spent obtaining cv access and plasma products. there were no significant differences in time intervals comparing patients with a new diagnosis of ttp versus patients with recurrent/ relapsed disease (table ) or between patients treated at a central academic center versus a peripheral hospital. conclusion: the consensus - hour target window from tpe request to initiation appears feasible for a centralized tpe program servicing a multi- a transfusion vol. supplement s hospital healthcare system. addressing limitations in availability of cv access would likely yield the greatest improvement in timeliness of urgent tpe. cytoreductive therapy for cellular hyperviscosity: utility of cytapheresis treatment for chronic myelogenous leukemia and essential thrombocythemia. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: several retrospective, case series have suggested that cytoreductive therapy to treat cellular hyperviscosity and prevent thrombotic events in patients (pts) with chronic myelogenous leukemia with accelerated transformation (cml-at) or essential thrombocythemia (et) may improve short-term outcomes. however, no randomized controlled trial (rct) assessing the efficacy of cytapheresis treatment in this group of pts has been performed. study design/method: from january, through january, , we performed cytapheresis (cy) treatments (txs) for pts with either cml-at or et, and clinical and/or laboratory evidence of cellular hyperviscosity. pts ( %) had cml-at and received leukapheresis (lp) txs; pts ( %) had et and received thrombocytapheresis (tc) txs. cml-at pts presented with median wbc x /l (range - x /l), of which % had blast percent > % or blast count > x /l. median age was years ( - years); % were male. cns symptoms (sxs) of leukostasis (lks) were defined as: headache, cognitive decline, confusion, somnolence, visual abnormalities, or seizure; pulmonary (pulm) sxs of lks were defined as: dyspnea, hypoxia, or bilateral chest infiltrates. % of cml-at pts had no sxs of lks; % pts had sxs of either cns or pulm lks ( sxs), and % pts had sxs of both cns and pulm lks ( sxs). et pts presented with median platelet (plt) count of: x /l ( - x /l)and % pts had sxs of thrombosis (evidence of cva or tia, mi, or dvt). median age was years ( - years); % pts were male. results/finding: all pts received a course of cy tx with following objectives: ) decreasing the risk of thrombotic/ hemorrhagic complications related to hyperviscosity, and ) stabilizing cml-at pts for induction chemotherapy (ind chemo). wbc (or plt ct) tx goals were: wbc count (ct) < x /l for cml-at pts, and plt ct < x /l for symptomatic et pts and < x /l for asymptomatic et pts. cml-at pts received median of lp txs (mean . txs/pt; range - txs). et pts underwent median of tc txs (mean . txs/pt; - txs). outcomes were evaluated by percentage of pts who: ) reached wbc (or plt ct) tx goal, and ) received ind chemo. "improved" outcome was defined as pts who reached their wbc (or plt ct) tx goal during cy tx; "stabilized" were pts who achieved > % reduction in wbc (or plt ct) without reaching goal; and "unchanged" were pts who achieved neither. in cml-at cohort, % pts improved, % pts stabilized; and % pts worsened. in et cohort, % improved, % stabilized, and % were unchanged. for cml-at pts, median final wbc ct x /l (range - x /l); % pts received ind chemo. for et pts, median final plt ct x /l ( - x /l); % pts had resolution of thrombotic a transfusion vol. supplement s symptoms. % of cml-at pts and % of et pts expired within - days after course of cy tx. of expired pts, pts had both blast crisis and sxs of cns/ pulm lks; pt had intracranial hemorrhage or cva; and pts were hypotensive, intubated, or unable to tolerate ind chemo. conclusion: pts with cml-at or et and evidence of impending thrombosis may benefit from cytoreductive therapy. a limited number of cytapheresis treatments (median - txs) can enable a high percentage of pts to receive definitive treatment and may improve short-term clinical outcomes. a rct to assess efficacy of cytapheresis treatment versus induction chemotherapy (or platelet inhibitor tx) alone in this subset of pts would be very useful. background/case studies: partial normal saline replacement during plasma exchange procedures is common practice. benefits of using normal saline as a replacement fluid include reduced procedure costs and possible reduction of the hypothetical hyper-oncotic effects of standard albumin formulations. however, the use of normal saline may increase the risk of undesired, and potentially costly, adverse events, such as hypotension and citrate reactions. the goal of this study was to compare the frequency of reported adverse outcomes for patients that received all albumin versus albumin/ saline as replacement fluid for plasma exchange at our institution. study design/method: a four year retrospective chart review was done of all therapeutic apheresis procedures performed by our apheresis service that used % albumin or % albumin- % normal saline ( / ) as replacement. patients who received plasma entirely or partially as replacement were excluded. the procedure type ordered ( % albumin vs / ), the percent of normal saline actually used during the procedure, age, gender, and any noted adverse events during the procedure were recorded in all cases. repeated procedures were modeled using a generalized linear mixed model to examine the risk of having hypotension and/or citrate toxicity where % albumin was used versus those that used / . covariates included were fluid types, age and gender. odds ratios (or) and % confidence intervals (ci) were used as a measure of risk. we used the term significant for a two-sided p-value < . . results/finding: during the study period, procedures were documented for subjects ( % female), age range - years, of which , ( . %) received / . the type of fluid used as replacement had a significant effect on the risk of having either hypotension or citrate toxicity. replacement with % albumin had a significantly lower risk of having either event than by using / , [p . , or (ci): . ( . , . )] , and also had a significantly lower risk of causing hypotension [p . , or (ci): . ( . , . )] in addition to a lower risk of causing citrate toxicity [p . , or (ci): . ( . , . )]. age had a significant effect on having a hypotensive event [p . , or (ci): . ( . , . )] but no effect on citrate toxicity or the combined outcome. gender had no effect on frequency of any event. conclusion: partial saline use as a replacement fluid with albumin during plasma exchange significantly increases the risk of hypotension and citrate toxicity during the procedure. age also increases the risk of hypotension. use of saline as replacement fluid during plasma exchanges should be minimized to maximize patient safety especially in older patients. background/case studies: therapeutic apheresis (ta) is a complex procedure that is mostly well-tolerated and rarely associated with adverse events (aes). there are few studies published on aes associated with ta but they lack uniformity of data. moreover, there is no common database in the united states (us) to report ta-associated aes. we evaluated the annual incidence rates of aes associated with ta at a large tertiary academic medical center over a year period and compared it to published literature. study design/method: we conducted a -year retrospective study of ta procedures performed and aes were classified according to criteria described in table . during the study period, ta were performed using cobe spectra (software versions . and . ) and since the spectra optia apheresis system (version . ). literature search was conducted for data published on aes associated with ta. four studies from us and non-us studies (canada, europe and japan) were analyzed. trend for ae rates from - was also analyzed. statistical analysis was performed using chi square and spearman rho tests. results/finding: the overall ae incidence was . % ( of , procedures) during year period. frequency of aes associated with therapeutic plasma exchange (tpe) was significantly higher ( . %, p< . ) compared to other ta procedures. we found significant correlation between number of tpe and aes (spearman rho . , p . ) over the years and significant down trend of moderate and severe aes with a spearman rho of - . (p . ) and - . (p . ) respectively. there were no fatalities during the study period. majority of aes were grade i ( %) and grade ii ( %): / ( . %) procedures were not completed due to aes. comparison of aes [ . % ( / , )] to both european [ . % (n , , / , ) ] and other us studies [ . % (n , / , )] showed a statistically significant difference (p< . ). conclusion: overall incidence of aes was significantly lower than current published literature. incidence of aes published in other countries is significantly lower than rates published in us. differences in incidence of aes in literature emphasizes need for uniform reporting and stratification of aes and development of a common database to report ta-associated aes. we propose a grading rationale in order to standardize reporting of ae (table ) . variations in biochemical markers of bone metabolism during plateletpheresis: impact of socio-demographic and lifestyle factors? markus dettke*. akh vienna university hospital background/case studies: plateletpheresis is associated with short-term variations in biochemical markers of bone turnover. socio-demographic factors and lifestyle behaviors are recognized factors which influence mineral metabolism and bone health. in the present study we analyzed the influence of demographic and lifestyle factors on the observed changes in bone markers in a large cohort of routine platelet donors. study design/method: altogether platelet donors with a donation activity of up to platelet donations participated in the study. after a detailed anamnesis all participants underwent a standardized questioner asking for several lifestyle factors known to affect bone metabolism. blood was sampled before and after plateletpheresis and was analyzed for the bone formation marker osteocalcin (oc) and the bone resorption marker cross-linked telopeptides of type i collagen (ctx), among other parameters. the effect of calcium supplementation on bone metabolism was tested in a placebocontrolled crossover study involving ten donors. results/finding: plateletpheresis resulted in an increase in the serum levels of the bone resorption marker ctx and the bone formation marker oc. both parameters returned to base levels within hours after the end of the collection. multiple regression analysis including the parameters sex, age , positive family history of bone disease, but also individual factors like hormonal contraception, smoking, regular alcohol consumption or sportive activity revealed no influence of socio-demographic or lifestyle factors on the observed variation in ctx or oc. there was no association between individual donor career or the number of previous donation and the observed increase in bone turnover. the only predictive parameter we could identify was the amount of citrate exposure during plateletpheresis. increase in serum ctx, showed an inverse correlation to changes of serum ionized calcium. continuous iv supplementation of calcium-gluconate throughout plateletpheresis reduced the variations in bone markers, although this effect was more pronounced for ctx compared to oc. conclusion: the amount of citrate infused during routine plateletpheresis is a predictive parameter for the transient increase in serum markers of bone metabolism. known risk factors for bone diseases, including sex, age, smoking or alcohol consumption, seems to have a low impact on the observed citrate-related variations in serological biomarkers of bone turnover. transfusion with optimized blood products versus transfusion with standard products in a trauma-transfusion rat model mathijs wirtz* , jordy jurgens , jacoline buchner-doeven , joris roelofs , philip spinella , jennifer a muszynski , carel goslings and nicole juffermans . academic medical center, washington university school of medicine, nationwide children's hospital background/case studies: transfusion is associated with nosocomial infection and organ dysfunction in trauma patients, which may be mediated by soluble bioactive substances in blood products. we hypothesized that removing these bioactive substances improves host immune response and reduces organ dysfunction. study design/methods: blood products were prepared from syngeneic rat blood according to blood bank standards. soluble mediators were removed from red blood cells ( days old) and platelets ( days old) by washing. plasma was filtered through a . um filter. rats ($ grams) were poly-traumatized by crush injury to the small intestines, the liver lobes, and by fracture of the right femur and hemorrhaged $ % of their estimated blood volume, which was calculated to be ml/kg. hemorrhage continued until a mean arterial pressure of mmhg was reached. rats were randomized to resuscitation with standard blood products, washed/filtered blood products or sham. blood samples were taken up to h after trauma to assess biochemistry and coagulation status. ex vivo whole blood stimulation tests with lps were performed after sacrifice, and organ damage was assessed by histopathology. blood products were sampled to assess for biochemical changes. comparisons between groups was done by anova and dunnett's post-test for multiple comparisons. results/findings: filtering or washing of blood products significantly stabilized ph, sodium and potassium concentrations and decreased lactate levels in the products compared to standard products. both resuscitation groups received an average of ml/kg of blood products in a : : ratio. however, use of washed/filtered products did not improve organ failure, as assessed by histopathologic score and levels of creatinine, asat and alat. the coagulation status as assessed by thromboelastometry was deranged in all groups and normalized during transfusion, showing no significant differences between washed/filtered products and standard care. immune response to lps was decreased following trauma compared to healthy controls but did not differ between groups. conclusion: filtering or washing of blood products reduces some aspects of storage lesion of blood products, without affecting the hemostatic capacity of the products, but does not improve organ injury in a rat trauma and transfusion model, nor does it improve the immunosuppressive host response. these results suggest that washing or filtering of blood products may have no relevant clinical effects in a rat polytrauma model. safety and efficacy of tranexamic acid during cardiovascular surgery: a single center before-and-after study takuma maeda* and shigeki miyata. national cerebral and cardiovascular center background/case studies: tranexamic acid (txa), an antifibrinolytic agent, has been widely used in cardiovascular surgery, since several studies have shown that prophylactic use of txa is effective in reducing blood loss after cardiovascular surgery. however, there is concern about the risk of thromboembolic events and adverse neurological effects such as seizures, which might worsen patient outcomes. consequently, we stopped using txa in april , which enabled us to conduct a before-and-after study. the present study aimed to examine the association between txa and adverse effects (seizures, thromboembolism, and renal dysfunction) in patients undergoing cardiovascular surgery using a propensity score matching model. we also assessed the association between txa and other clinical outcomes (reoperation for bleeding, transfusion volume, blood loss, ventilation time, intensive care unit stay, and -day mortality). study design/method: this single center retrospective cohort study involved patients who underwent cardiovascular surgery with cardiopulmonary bypass or offpump coronary artery bypass grafting between january and july (n ). because of missing data on patient characteristics, patients were excluded. the incidence of adverse effects associated with txa and other clinical outcomes were evaluated before (january to march , n ) and after (april to july , n ) using a propensity score model. we estimated propensity scores using a logistic regression model for txa use as a function of baseline variable, generating pairs of patients who received or did not receive txa. we also evaluated the adverse effects of txa using segmental regression analysis. results/finding: propensity-matched analysis showed that seizures were more common ( . % vs . %, p< . ) and ventilation time was longer ( h vs h, p . ) significantly in the txa group than in the non-txa group. in contrast, transfusion volume and blood loss were significantly lower in the txa group than in the non-txa group ( ml vs ml, p . ; and ml vs ml, p< . , respectively). however, -day mortality was not statistically different between the groups ( . % vs . %, p . ). none of the other outcomes were significantly different. segmental regression analysis yielded similar results. conclusion: even though txa may be associated with an increased rate of seizures and longer ventilator time, it does not increase mortality. the use of txa is significantly associated with decreased blood loss and transfusion volume, providing social benefit by reducing the need for blood transfusion because the supply of blood components will be limited with the aging of japanese society. it seems to be advantageous to use txa because decreased blood loss and transfusion volume and the associated social benefit outweigh the disadvantages of an increased rate of seizures and longer ventilator time. sustained impact of blood management strategies in orthopedics: continuous quality improvement linda levinus* and michele deeney. new england baptist hospital background/case studies: transfusions are one of the most over-utilized treatments performed in any hospital setting (choosing wisely campaign, april , www.choosingwisely.org/societies/american-association-of-bloodbanks). costs and risks associated with transfusions are high and may have a significant impact on patient safety. in our institution we perform over , joint replacements and spine surgeries per year, making transfusion-associated costs very high. since our last formal evaluation of the metrics used post implementation of patient blood management (pbm) strategies, questions regarding the feasibility of continued transfusion reduction and sustainability of the program were raised by administration and key stakeholder physicians. the objective of this study is to determine what, if any, sustainable improvement to our blood utilization dashboard table ). the data collected show that there has continued to be a reduction in transfusion rate, and blood expenditures through fy . length of stay has also shown a continued reduction, which is an indicator that the pbm strategies implemented have not compromised quality outcomes. further, continued review and monitoring of the chosen metrics, evaluating changes to policy and practice related to transfusion medicine, and communication of findings to providers/administration upon immediate restrospective analysis, are integral to the continued success and sustainability of our pbm program. going forward, these practices, along with investigating use of additional pbm strategies, will provide the basis for an effective continuous quality improvement program in transfusion medicine for orthopedics. safety and efficacy of -factor prothrombin complex concentrate: a retrospective review of outcomes at an academic hospital stephanie jalaba*, hollie benson, nan zhang, jill adamski and theresa kinard. mayo clinic arizona background/case studies: -factor prothrombin complex concentrate (pcc) contains factors ii, vii, ix, x, proteins c and s and is used for reversal of vitamin k antagonists in acute major bleeding or urgent, invasive procedures. occasionally, it is used off-label when plasma is not optimal for achieving hemostasis. this study compares the efficacy of on-label and off-label use of pcc in correcting coagulation parameters and reducing allogeneic blood transfusion. study design/methods: a retrospective chart review was performed for pcc use at our institution in . marginal modeling (gee method) was used to account for within patient correlation and assess changes in lab values and products transfused. logistic regression (gee method) was used to evaluate potential risk factors for unsuccessful hemostasis (uh rate of transfusion after pcc ! rate before pcc) or thrombotic complications. results/findings: the reduction in pt (p . ) and ptt (p . ) was significantly greater in on-label than off-label use. interestingly, transfusion reduction in rbc (p . ) and plasma (p . ) after off-label use was significantly greater than on-label use. cases, both on-label and off-label, with uh were associated with cell saver, acute normovolemic hemodilution (anh), or cardiopulmonary bypass (cpb). the odds of having uh were . times (p . ) more with cell saver or anh, and . (p . ) times more with cpb. post-pcc thromboses were identified in cases, but no association was found with potential risk factors: use of antifibrinolytics, vitamin k, factor viia, or extracorporeal support. background/case studies: when a pregnant woman with high risk pregnancy (diagnoses such as abnormal placentation, multiple gestation) is admitted to inpatient bedrest the obstetrical team would like to assure ability to crossmatch red blood cells (rbc) at all times by always having an in-date type and screen specimen. per current aabb standards, this necessitates a new sample every days. this can lead to excessive iatrogenic blood loss and increasing difficulty with obtaining intravenous access in the patient, to the point that an invasive catheter such as a picc line may be placed. in order to mitigate these issues, we chose to extend the type and screen specimen to expire after days in patients without rbc alloantibodies other than passively acquired anti-d due to rh immune globulin administration. study design/method: patients expected to have an antenatal hospitalization of at least days with high risk for transfusion need are identified by the obstetrical service, which submits a request to the transfusion service for extension of pre-transfusion specimens to days. the transfusion service medical director reviews the case and gives final approval. we observed only patient did not have an in-date specimen when the extended out-dating was requested. thirty-eight ( ) patients were in-patients continuously until delivery. five patients were discharged prior to delivery- moved to another state, was admitted later at another local hospital, and three were readmitted for later deliveries. the mean interval from approval to delivery was days (range - ). six ( ) patients delivered within days of approval. after approval, the mean number of additional specimens per patient was . (range, - ). no patient required transfusion prior to delivery. five patients received transfusion of at least rbc at the time of delivery, and none had evidence of transfusion reaction. conclusion: since no new antibodies were identified prior to discharge or delivery and no transfusion reactions were observed, the process appears safe. with only patients delivering within days of approval for extended specimens, patients avoided collection of at least specimen each, and patients avoided at least collections each. since new antibodies are not detectable for at least days after immunization, even longer extension of pre-transfusion specimen out-date may be considered. although this requires further study, we believe our practice of extending the pre-transfusion testing sample expiration date to days is safe and is justified, when weighed against the risk of excess iatrogenic blood loss and placing an invasive line for blood sampling in a pregnant patient. iron metabolism in critically ill patients developing anemia of inflammation margit boshuizen* , , jan m. binnekade , benjamin nota , pieter r tuinman , kirsten van de groep , olaf l cremer , janneke horn , marcus j schultz , robin van bruggen and nicole p juffermans . academic medical center, sanquin research and landsteiner laboratory, vu university medical center, university medical center utrecht background/case studies: anemia due to inflammatory processes (anemia of inflammation, ai) frequently occurs in critically ill patients. in ai, inflammation-induced hepcidin decreases iron availability, a process that is thought to be regulated by erythroferrone, which impact erythropoiesis. knowledge on changes in iron metabolism during the course of ai is limited, hampering the development of strategies to counteract ai. this study aimed to investigate the dynamics of parameters of iron metabolism during the development of ai in critically ill patients. study design/methods: a case control study was performed in tertiary icus in the netherlands comparing patients who developed ai during icu stay with control groups: non-anemic patients with sepsis, non-anemic patients without sepsis, and patients with anemia due to acute blood loss. patients were matched on age and sex. a linear mixed model was used to assess differences in parameters of iron metabolism between groups and over time. results/findings: in patients with ai, levels of iron, transferrin and transferrin saturation decreased already prior to the development of anemia, with lower levels compared to controls (table) . ferritin and hepcidin were increased in ai compared to controls. in the course of ai development, erythroferrone decreased. differences in iron metabolism between groups were not influenced by disease severity. patients with ai differed from patients with anemia due to acute blood loss, the latter was characterized by high iron ( . vs. . mmol/l, p< . ) and transferrin saturation ( vs. %, p< . ), and low ferritin ( vs. mg/l, p< . ). conclusion: in critically ill patients with ai, iron metabolism is already altered prior to the development of anemia, suggesting a potential window of opportunity for therapy. iron metabolism in ai is more disturbed than in non-anemic septic controls, irrespective of disease severity, indicating that ai is not solely determined by severity of inflammation. iron metabolism in ai patients differs from patients with acute blood loss, suggesting that efforts to modulate iron metabolism in anemic icu patients should take the cause of anemia into account. clinical oral abstract session: novel approaches to processing and assessing cell therapy products a paradigm shift in stem cell isolation and storage jeffrey drew*. cells life group llp background/case studies: widespread use of umbilical cord blood is limited by processing yield and post-thaw recovery of viable nucleated cells. the recommended therapeutic cell dose is approximately . x cells per kg body weight indicating that a single cord unit may be insufficent to treat larger individuals. cell isolation methods were developed to remove erythrocytes whilst recovering the white cell fraction (wcf). however, all current methods result in significant loss of the wcf, some up to %, whilst leaving % of the starting volume of erythrocytes. additionally, there is an almost total loss of potentially important, low abundance cellular subsets. the use of cord blood for hematopoietic reconsititution and in regenerative medicine would be widened if processing methods improved postprocessing and post-thaw viable cell recovery. study design/methods: we have developed a solution consisting of a defined concentration of reagents routinely used in blood therapy. on combination with blood, this solution results in the selective sedimentation of erythrocytes by gravity within minutes. the wcf remains in solution and can be easily separated from the erythrocyte sediment. the wcf can then be concentrated by gentle centrifugation into a small volume containing less than % of the original erythrocyte content. the addition of dmso for cryogenic storage and controlled freezing using standard procedures then completes this simple process. results/findings: we have clearly demonstrated that this method allows almost the entire wcf to be isolated and/or concentrated with only modest loss of any of the cellular sub-sets thus far examined. in addition to improving pre-freeze yields, post-thaw recoveries of viable cells are markedly increased, with a yield of approximately % of the cd fraction post separation and freeze thaw (table ) . possibly more important, the cfu assay results reproducibly yield higher counts of cfu-gm, cfu-gemm and bfu colonies (table ) which is a strong indicator that this method will improve patient outcomes. in addition, our separation method isolates and preserves the megakaryocyte-like cells (cd cd ) and early projenitor cells expressing oct and nanog (markers for vsels) which are two examples of cellular subsets usually lost using current separation techniques. conclusion: these results demonstrate that our method achieves: . routine recovery of the wcf at levels higher than current methods, independent of volume. . higher percentage recoveries of all cell types tested than can be achieved with existing methods. . markedly higher post-thaw recovery of viable nucleated cells than any current methodology. . almost complete removal of hematocrit. as a result units of cord blood separated using this new method will contain cell yields that could only otherwise be achieved through pooling multiple separate units. therefore, this new method has the potential to increase the demand for cord blood in therapy, expanding to larger individuals and adults, where up until now, it has been suppressed due to limited cell yields delivered by existing methods. effects of implementation of an absolute lymphocyte count target, in addition to cd target, for hematopoietic progenitor cell collection edwin a burgstaler*, luis f porrata, dennis a gastineau, eapen k jacob and jeffrey l winters. mayo clinic background/case studies: lymphoma patients receiving > . x lymphocytes(lymph)/kg during peripheral blood stem cell transplant have superior survival. in addition to a cd cell target of . x /kg, a lymph target was also implemented. fifty patients before (no alc) and after (alc) implementation were retrospectively evaluated. study design/method: lymph and cd yields, number of collections, lymph target reached, and days to engraftment were examined. mobilization was g-csf (g) or g-csf plerixafor (g pl). consecutive no alc and alc procedures were examined. the mann-whitney and chi square tests were used for statistical comparison, p< . considered significant. results/finding: no alc and alc collections occurred among the patients. fenwal amicus was used for % of the no alc and % of the alc collections (terumobct spectra optia cmnc used for remaining). diagnosis was hodgkin's and non-hodgkin's lymphoma (no alc); hodgkin's and non-hodgkin's lymphoma (alc). pre procedure wbc and lymph counts were significantly higher for no alc (wbc . , lymph . x /l) than alc (wbc . , lymph . x /l). equivalent whole blood (corrected for ac) was processed for no alc ( . l) and alc ( . l). for alc group, extra collections beyond cd target were: days: %, day: %, days: %, days: %, and days: %. significantly more patients were mobilized with g pl in no alc group (n ) than alc group (n ) and collections in alc group had mobilization discontinued after cd cell target reached. there was no significant difference in g ( . x lymph) compared to g pl mobilized collections ( . x lymph); both were significantly higher than the collections where mobilization had been discontinued ( . x lymph). days to wbc engraftment ( . no alc vs . alc) and platelet engraftment ( . no alc vs . alc) were not significantly different. median number of collections for no alc ( ) and alc ( ) were not significantly different. data (medians) in the table. conclusion: not all patients achieved the . x lymph/kg or even the . x lymph/kg targets. implementation of a lymph target increased patients obtaining . x lymph/kg from % to %. only % had < . x lymph/ kg. discontinuation of mobilization once cd cell target was reached significantly reduced lymph yield. the median increase of one collection per patient following implementation was less than had been expected. extended preprocessing storage impairs cord blood hematopoietic stem cell activity suria jahan* , and nicolas pineault , . canadian blood services, university ottawa, canadian blood services, centre for innovation background/case studies: large distances between collection and processing sites combined with staff availability can result in long processing delays of umbilical cord blood (ucb) unit. current net-cord-fact standards specify that units can be stored for almost hours at room temperature (rt) as long as units are cryopreserved by -hours post-collection. the impact of such delay on hematopoietic stem cell (hsc) function is unclear since most studies have not used transplantation assays that measure hsc key properties and activities. we hypothesized that such processing delay reduces the engraftment activities of ucb units. we set out to measure the loss in engraftment activities associated with preprocessing storage. study design/method: ucb units (n ) were split with one half processed immediately (baseline - hours) and the second after hours storage at rt. ucb were then processed with hetastarch and buffy coat maintained cryopreserved in liquid nitrogen until use. viability was assessed post-thaw, and thawed ucb buffy coat cells were transplanted into nsg mice. serial transplantation was used to test the self-renewal and differentiation activities of hsc, while limiting dilution (ld) assay and poisson statistic were used to estimate the frequency of scid repopulating cells (src) in thawed units. results/finding: storage before processing had no significant impact on the recovery of viable post-thaw cd cells and cd cell (n ). primary nsg mice were transplanted with a ucb cell dose that contained a total of , annexinv neg viable cd cells. the latter was done to avoid any bias towards one group or another. short term platelets ( vs. hplt/ml, p . ) and leucocytes ( . % vs. . % hcd , p< . ) engraftment at -weeks were significantly reduced in stored mice vs. baseline (n ), and similar results were observed long-term at -weeks. long-term human bone marrow (bm) engraftment was also reduced in primary transplants from stored samples ( myeloid engraftment was however confirmed in both groups. bm cells from primary mice were transplanted into secondary recipients and human engraftment investigated months post-transplant. strikingly, the frequency of human cd bm cells was -fold greater in baseline vs. stored mice (p< . , n ). hence, storage at rt of ucb units is associated with a deficit in engraftment activity likely due to a loss in hsc activity and/or numbers. to distinct between both possibilities, the net number of src in baseline and stored samples for two units were calculated by ld transplantation assay. the net number of src measured -weeks post-transplants were reduced by % in unit , and by % in unit . conclusion: prolonged preprocessing rt storage significantly impairs the engraftment activities of ucb units. the reduced engraftment in secondary transplants coupled with the results from the ld assays suggest that this engraftment deficit origins from loss of hsc numbers. our results stress the importance of rapid ucb processing to avoid loss of engraftment activity. acoustic microfluidic separation of blood components charles lissandrello, ryan dubay, kenneth kotz and jason fiering*. draper background/case studies: new cell therapies require efficient and automated methods for purification of target cells prior to subsequent processing. while apheresis, density gradient centrifugation, and magnetic separation achieve some of the requirements, no method is currently available that fully meets clinical needs for a closed, automated, and scalable process. continuous acoustic separation in microchannels is emerging as a versatile method for sorting, separating, and concentrating cells from blood. it has advantages over centrifugation because it is scalable to small or large quantities and can discriminate cells by size as well as density. meanwhile, unlike magnetic methods, acoustophoresis is "label free" and adds no reagents to the therapeutic cells. it has been shown previously that acoustic separation can separate blood components including purification of lymphocytes. however, these studies used devices that were constructed from silicon or glass and have limited potential for scale-up or production as disposable cartridges. in contrast, we report the first ever demonstration of acoustic lymphocyte enrichment along with rbc and platelet depletion in a disposable plastic chip, and we present a cartridge concept that enables clinical scale throughput by linking microchannels in parallel. study design/method: acoustophoresis uses ultrasonic waves to oscillate a rectangular microchannel having a cross section on the scale of the ultrasonic wavelength ($ mm). this results in an acoustic force across the channel that drives cells toward the axial center stream. because the force increases with a cell's size and density, lymphocytes experience a weaker force than rbcs and other classes of wbcs. thus, as blood product flows through the device, the lymphocyte population is enriched at the sides of the channel and can be captured in a branching outlet. likewise, platelets can by separated from lymphocytes. initial and output cell counts are measured by a standard hematology analyzer. results/finding: in our acoustic system, lymphocyte purity (% of total wbcs) was enriched up to %, using leukapheresis product as the starting material. this enrichment was achieved in a single pass through the device (residence time of sec). total lymphocyte recovery was % and monocyte concentration was reduced %. furthermore, in a two-pass process platelets were reduced by %. in a -fold parallel system we tested rbc separation from plasma and achieved % separation at ml/hr. conclusion: acoustic lymphocyte enrichment along with platelet depletion from standard blood product was demonstrated for the first time in plastic microchannels. such disposable devices are suitable for scale up to clinical bioprocessing systems. lymphocyte purity is comparable to existing methods with the advantage of monocyte and platelet depletion and potential for an automated instrument. background/case studies: the use of natural killer (nk) cells as a cellular immunotherapy has increased over the past several years, specifically their use in patients with hematologic malignancies. nk cells have been used at our institution for the past years. most patients have a reaction with nk cell infusion with some reactions being quite severe. we retrospectively analyzed the reactions associated with nk cell infusions to help address why some patients have more severe reactions than others. study design/method: retrospective chart review of nk cell infusions performed at our institution from clinical protocols from - . an infusion reaction was defined as any symptom from the time of nk cell infusion up to hours afterwards. a severe reaction was defined as any symptom with grade or higher severity (graded on common terminology criteria for adverse events-ctcae). preliminary data was analyzed using r . . . two major endpoints of interest were: ) infusion reaction with any symptom and ) severe infusion reaction. to numerically summarize the association of continuous variables with our endpoints, the median, (range) and interquartile range (iqr) were used. a wilcoxon test was performed to test the association between the continuous variables and our end points. a chi-square test was used to test the association between categorical variables and our endpoints of interest. results/finding: there were a total of nk cell infusions. there were ( %) patients with an infusion reaction of any symptom and there were ( %) patients with a severe reaction. infusion rate (ml/min) was similar among those with any reaction (median . , p . ) and those with severe reaction (median . , p . ). infusion rate (ml/min/kg) was also similar among those with any reaction (median . , p . ) and those with severe reaction (median . , p . respectively). incubation of nk cell product overnight in il- vs il- had similar reaction rates for those with any symptom ( % had reaction with il- , % had reaction with il- , p . ) and those with severe reaction ( % had severe reaction with il- , % had severe reaction with il- , p . ). patients with severe reaction had a higher calculated monocyte dose (monocytes/kg) in the nk cell product (median . x ) versus those without (median . x , p . ). conclusion: our preliminary data analysis reveals that a higher number of monocytes in the nk cell product may contribute to severe infusion reactions, causing patients to have a grade or higher symptom. limitations to this study include this was a retrospective review at a single institution. a streamlined mixed lymphocyte reaction (mlr) assay for evaluation of human mesenchymal stem cell immunomodulation activity christopher p delavan , maryanne c herzig* , barbara a christy , james a. bynum and andrew p cap . us army institute of surgical research, u.s. army institute of surgical research background/case studies: mesenchymal stem cells (msc) have been investigated for treatment of acute respiratory distress syndrome (ards), graft versus host disease (gvhd), wound healing and trauma. a consensus is building that the immunomodulation by mscs is key to their therapeutic potential. mscs suppress peripheral blood mononuclear cells (pbmc) proliferation in vitro, suggesting a correlation for suppressing pbmc inflammatory responses in vivo. current mixed lymphocyte reaction (mlr) assays generally rely on either direct co-culture or indirect culture using transwell systems for monitoring the proliferation of isolated pbmcs in the presence of mitotically inactive mscs. in the study detailed here, mscs are analyzed in a direct co-culture with pbmcs using a luminescent atp assay. study design/method: blood was obtained from an in house blood bank and pbmcs were separated by centrifugation over ficoll-paque in leuco-sep tubes as specified by the manufacturer. the pooled donor pbmcs were stored at - . mscs derived from bone marrow, adipose tissue or umbilical cord (bm-msc, ad-msc, uc-msc, respectively) or human umbilical cord endothelial cells (huvec) were serially diluted starting at - , cells/ well and cultured in well plates for - h in their respective medias. on day , mscs were washed, resuspended in pbmc media and incubated with or without , freshly thawed pbmcs/well, in the presence or absence of phytohemagglutinin a (pha, - lg/ml). proliferation of both mscs and pbmcs was assessed in triplicate wells by quantitation of atp levels using the bioluminescent reagent cell titer-glo (promega). results/finding: pbmc proliferation in response to pha gave a robust atp signal by h, with > fold increase over control pbmcs. no increase in atp response or proliferation was seen in the absence of pha. co-culture with mscs inhibited pbmc proliferation dependent upon msc passage, source, msc media additive. intra-assay variance of triplicate samples was . %. inter-assay variation of msc preps run under identical conditions was . %. inhibition of pbmc proliferation was graded from - % over the range msc concentrations therefore an ec of msc cell number resulting in % suppression of pbmc could be determined for each msc prep. this ec however was dependent upon pbmc donor pool. conclusion: direct co-culture of live mscs with freshly thawed pbmcs give a robust determination of immunosuppression by mscs. graded responses can be determined, allowing comparison of potency between msc preparations. this streamlined assay can be performed within h, without irradiating cells and with minimal equipment outlay. background/case studies: a high prevalence of iron depletion (id) in blood donors has been documented by recent studies, but none targeted high school aged donors, who consistently contribute % or more of the us blood supply. differences between donors - years old (yo) and adults in baseline and donation-altered iron status are important to understand because teenagers need increased iron for physiological growth and development and may be more susceptible to harm from iron depletion. study design/method: donors aged - were eligible for ferritin testing if they donated at a high school (hs) blood drive at the start of the / academic year at two blood centers. samples from return donations over the remainder of the school year were also tested. the prevalence of absent iron stores (ais, ferritin < ng/ml) and low ferritin (lf, ferritin < ng/ml) were estimated for , , and - yo groups separately for both genders. linkage to operational databases established first-time (ft) vs repeat (rpt) donor status. linear regression analysis tested for differences in natural log of enrollment ferritin values by age. multiple logistic regression assessed whether young age independently predicts iron depletion controlling for donation frequency and other factors. results/finding: a total of donors contributed donations. donors were evenly split by gender, % were ft donors, and % were - yo. ft and rpt - yo donors had on average lower ferritin values at enrollment (p<. ), and a greater percentage were iron-depleted than donors - yo (table) . in repeated measures logistic regression analysis using data from all visits, female sex, greater number of previous donations, shorter interval since last donation, and lower body weight were risk factors for both ais and lf. controlling for these covariates, donors aged - have sharply higher risk for iron depletion than donors - yo. odds for lf were to times greater in the younger donors, and for ais were -to fold higher. preliminary statistical models indicate yo donors may have greater risk for lf than or yo by to percentage points, controlling for other factors (p . ). conclusion: the prevalence of iron depletion varies markedly by age, sex, and donation frequency, but was considerably higher in - yo donors than in adult controls. logistic regression analysis confirms lower age as an independent risk factor for iron depletion. blood centers should implement measures to mitigate higher risk for iron depletion and the potential adverse consequences for this population of vulnerable donors. mitigation of iron deficiency in young donors -a preliminary report ralph r vassallo*, marjorie d bravo, mary townsend and hany kamel. blood systems, inc. background/case studies: iron deficiency is observed in blood donors who meet regulatory hemoglobin (hb) requirements for blood donation. frequent donations result in negative iron balance and eventually lead to anemia. young donors may be at risk for adverse health consequences (cognitive dysfunction, pregnancy-related complications, fatigue, decreased exercise endurance and pica) even before anemia occurs. study design/method: serum ferritin testing was implemented on / / by a large blood collector. testing was performed on successful - y/o whole blood and apheresis donations. low ferritin (lf) was defined as a value < ng/ml in females (f) and < ng/ml in males (m). donors with low ferritin were notified of deferral from red blood cell (rbc) donations ( months for f and months for m) and counseled to take - mg of elemental iron daily for days. for m and f, a ferritin < ng/ml indicated absent iron stores (ais) and < ng/ml indicated iron deficient erythropoiesis (ide). ferritin levels ! ng/ml in f and ! ng/ml in m were considered as indicating an iron-replete state. conclusion: ferritin testing of young donors identified individuals with lf who would benefit from risk mitigation, e.g., delaying subsequent rbc donations and/or taking iron supplements. lf is more common in f than in m donors. lf is more prevalent in m and f donors with any rbc donations in the prior months. an appreciable number of donors with no rbc donations in the prior months presented with lf. these data may be useful in conducting a riskbased decision making exercise to establish recommendations for risk mitigation which could be different for m than for f, e.g., universal iron replacement in teen male donors may not be warranted above a certain hb value. ferritin blood screening in minor or young adult donors jennifer l ritter* , joan williams , michelle humphries , nancy haubert , ben reynolds , michael phillips , randall spizman , ralph r vassallo , hany kamel , sally caglioti , german leparc , and phillip c williamson . abstract completely investigated. the adolescent growth spurt, poor nutrition and onset of menses increase the risks of iron depletion in young donors. new studies show that teenage donors who give blood frequently may be more susceptible to becoming iron deficient than older repeat donors. study design/methods: over , serum samples from donors aged , and years were analyzed for ferritin levels using the beckman coulter au instrument and reagent kit. the anti-ferritin reagent is a suspension of polystyrene latex particles, of uniform size, coated with polyclonal rabbit anti-ferritin antibody. immune complexes formed in solution scatter light in proportion to their size, shape and concentration. the decrease in light intensity is measured spectrophotometrically. results/findings: background/case studies: the risk of cardiovascular (cv) disease in adults can often be identified during adolescent years. the presence of even borderline levels of multiple risk factors increases the likelihood of a cv event. our blood program routinely provides a total non-fasting cholesterol (tc) and blood pressure (bp) measurement for all blood donors. we added glycated hemoglobin (hba c) determination and performed analyses of the prevalence of abnormal (borderline or elevated) levels of multiple risk factors among , adolescents (ages - ; . % female) who donated blood from to . study design/method: abnormal risk factor levels were defined as hba c ! . %, sbp/dbp ! / mm hg and tc ! mg/dl, as suggested by the american heart association for adolescents. the presence of isolated risk factors was defined as one single abnormal risk factor per individual. clustering of risk factors was defined as the presence of or more abnormal risk factors in the same individual. donor sex was recorded at the time of donation. results/finding: table shows the prevalence of isolated abnormal risk factors and the prevalence of abnormal risk factor clustering in the study cohort. overall, , ( . %) adolescents had at least one abnormal risk factor ( . % of males, . % of females). of these, , adolescents had isolated abnormal risk factors, and , adolescents had clustering risk factors. higher proportions of males were in the abnormal bp alone, background/case studies: pre-donation determination of hemoglobin (hb) level in candidate blood donors is a pre-requisite in the majority of blood services and is used to ensure donor safety and blood product quality. however, a variety of hb testing strategies are used across blood services to satisfy this selection criterion. this study aimed to identify how hb screening practices vary across blood donation services and to what extent they influence deferral rates for low hb. study design/method: an online survey was performed among members of the biomedical excellence for safer transfusion (best) collaborative. additionally, data from literature were used to extend the dataset. the survey involved a detailed assessment of hb screening practices, numbers of donations and low hb deferrals for male and female donors separately. multivariable negative-binomial regression models were built to estimate the adjusted effects of minimum donation intervals, hb cutoffs (high/low with high defined as ! . g/dl for men and ! . g/dl for women), iron monitoring (y/n), iron supplements (y/n providing or prescribing), and geographical location on deferral rates due to low hb. results/finding: data were included from blood services worldwide and complete data were available for blood services. deferral percentages for low hb varied from . % to . % among male donors and . % to . % among female donors. hb deferral rates were notably higher in asian blood services. overall, iron monitoring was associated with % lower hb deferral rates in men ( % confidence interval [ci] % to %) and % lower rates in women ( %ci % to %). iron supplementation was associated to % lower hb deferral rates among women ( %ci % to %) but there was no evidence of such an effect among men (p . ). each one-week increase in minimum donation intervals resulted in % lower hb deferral rates among women ( %ci % to %) but not among men (p . ). at the % level of significance, higher hb cutoffs do not appear to have an effect among men or women. conclusion: the variation in hb deferral rates across blood donation services can be, particularly in female donors, explained by differences in hb screening and deferral practices. mitigation strategies should consider the variable response among men and women. these insights can help improve both blood service efficiency and donor care. were: characteristics of donors (age, sex, size, weight, region); hb levels, date and volume of donation for index application and previous donation; and number of previous donations (in the previous years and the lifetime). data were analyzed using logistic regression stratified by sex. results/finding: . % of all candidates for wb donation were deferred in continental france in . deferral was significantly more frequent in women ( . %) than in men ( . %), due to anemia in . % of deferred women and . % of deferred men. plotting mean hb recovery against time showed mean recovery times ranging from to weeks. analysis (table) identified main factors associated with a higher likelihood of hb recovery: higher logarithm of time since previous donation, lower levels of hb at previous donation, higher number of blood donations in the previous years. conclusion: the main factors associated with higher likelihood of hb recovery after wb donation are probably linked with hematopoiesis stimulation and selection bias among high-frequency donors. mean times required for hb recovery were long enough to require further studies to assess interdonation intervals in france. background/case studies: red blood cell (rbc) transfusion has been related to thrombo-embolic events. microvesicles in the rbc product may support coagulation, which in part may depend on storage time because microvesicles have procoagulant effects in vitro and the amount of microvesicles increase with storage duration. study design/method: we investigated whether transfusion of rbcs containing microvesicles promotes coagulation in human recipients. as transfusion is mostly administered to ill patients, we used a model of mild endotoxemia. eighteen healthy volunteers were randomized to receive either saline, days stored or days stored autologous rbc transfusion two hours after infusion of lipopolysaccharide (lps, from e.coli, ng/kg). blood was sampled every hours up to hours after lps infusion. results/finding: lps resulted in a mild increase in thrombin generation. during storage, the total number of microvesicles increased from . e (iqr . e - . e ) /ml in the fresh product to . e (iqr . e - . e /ml; p< . ) in the stored product (p < . ), which were mostly rbc derived vesicles. after transfusion, microvesicles from stored rbc products, but not from fresh products, could be detected in the circulation of healthy volunteers and were cleared within hours. however, infusion of stored rbc microvesicles did not augment thrombin generation. levels of d-dimer and thrombin-antithrombin complex were also unaffected. conclusion: transfusion of autologous rbcs containing high levels of microvesicles does not enhance coagulation in human volunteers with mild endotoxemia. background/case studies: transfusion-associated circulatory overload (taco) is characterized by hydrostatic pulmonary edema related to blood transfusion. we sought to examine contemporary risk factors and outcomes for taco during a period where patient blood manaement has led to declines in blood utilization. study design/methods: at four academic hospitals, cases of taco were detected by active surveillance of all adult hospitalized patients who received a blood transfusion, and transfused controls were matched to cases by transfusion intensity. taco incidence was calculated, and clinical characteristics were compared with control patients. odds ratios (or) were calculated using multivariable logistic regression. hospital mortality and length of stay were modeled using cumulative incidence functions in proportional hazards regression. results/findings: cases of taco and matched controls were enrolled from , transfused patients who received , blood components from may until july . taco incidence was case per patients transfused. in addition to well described cardiac and renal comorbidities, multivariable analysis identified the following independent predictors of taco: number of plasma units, emergency surgery, pre-transfusion diuretic use, and higher post-transfusion hemoglobin levels (see table) . compared to controls, taco cases were more likely to require mechanical ventilation ( % vs. %; p < . ), experienced longer intensive care ( vs. days; p . ) and hospital length of stay following transfusion ( vs. days; p< . ), and had higher mortality ( % vs. %; p . ). conclusion: the incidence of taco was lower than what has been reported by prior active surveillance studies. despite declines in its incidence and the number of blood components transfused per case, taco remains a complication of transfusion with significant associated morbidity and mortality. in addition to risk factors for cardiovascular and kidney disease, plasma transfusion and higher post-transfusion hemoglobin levels were associated with taco after controlling for other covariates in the model. additional research is needed to examine the utility of these risk factors in the development of real-time predictive algorithms and the benefit of reduced erythrocyte or plasma exposure in patients at high risk for taco. background/case studies: the residual risk of bacterial contamination of single-donor apheresis platelets (ap) was recently addressed by the march fda draft guidance to enhance the safety of platelet transfusion. this document also describes an existing pathway for ap outdate extension from to days using an fda cleared rapid test (rt). our hospital based transfusion service has used this rt to enhance the safety of ap transfusion since july and to routinely extend ap outdate to day since february . this study reports a month experience of secondary screening of ap using a rt. study design/methods: all ap were obtained from our hospital-based donor center or one of four external suppliers. ap were screened by culture based methods post-collection and prior to entry into our inventory. from july -january , ap underwent rt on day . day and units were transfused with physician approval when deemed medically necessary. any units remaining in inventory on day had a second rt performed. from february -january , ap underwent rt on day with routine outdate extension to days by performing a second rt on day and a third rt on day , as per manufacturer instructions. any positive rts were repeated in triplicate. repeat rt positive units were quarantined and cultured to identify true positives. false positives (fp) were defined as repeat rt negative (type ) or repeat rt positive with negative confirmatory culture (type ). all rt results were reviewed during both study periods. ap transfusion and outdate rates were also summarized. results/findings: since july , , ap were entered into inventory. of these, , ( %) were transfused prior to rt testing. the remaining ( %) underwent rt on day or day . of these ( . %) were rt positive ( type fp, returned to inventory; type fp, discarded), leaving a total available inventory of units tested by rt. of these, ( % of original inventory) were transfused before the end of day and the remaining ( % of original inventory) reached a day outdate. a total of ( % of original inventory) were transfused on day or day . of these, underwent a second rt on day ( rt positives; fp type one and fp type ) and underwent a third rt on day (no positive results). a total of ( % of original inventory) outdated on day . of these, underwent a second rt on day (no positive results). conclusion: to date we have performed rts on ap at our hospital. no true positives have been identified. use of rt over the study period decreased our outdate rate from a predicted % to only %. a total of ap have been tested twice by rt ( on day and ; on day and ) with ( . %) positive results, both of which were deemed fp by repeat testing or culture. a total of units have been tested times (day , day and day ) with no additional positives identified. we have not yet identified any units with an initial negative rt result that subsequently converted to a true positive. there is a low fp rate which should also be expected when performing repeat testing on the same unit. these data suggest that the yield for repeating the rt every hours, as currently specified by the manufacturer instructions, is quite low. additional studies are needed to clarify how rt can optimally be used to enhance detection of ap bacterial contamination. survival of trypanosoma cruzi in human blood components laura tonnetti*, aaron thorp and susan l stramer. american red cross background/case studies: trypanosoma cruzi, the agent of chagas disease, is associated with to million infections worldwide, mostly in latin america. despite the extensive immigration from endemic areas, only cases of transfusion-transmission (tt) t. cruzi have been reported in the us, before blood donor screening was implemented in . contributing factors to the low number of tt cases are a possible association between parasite lineage and tt, and high numbers of unreported cases. platelets are almost exclusively involved in t. cruzi tt cases; however, during preparation of components a large fraction of the parasites can be found in red blood cells (rbcs). we investigated if blood component preparation and storage time affect the survival of the parasite and thus play a role in tt of t. cruzi. study design/method: whole blood (wb) units were spiked with t. cruzi trypomastigotes to a final concentration between - , parasites/ml. each parasite concentration in wb was tested x . an aliquot of contaminated wb was used to prepare hemocultures to detect live parasites before preparation of components. rbcs were separated and half of the components leukoreduced (lr) by filtration. platelets and plasma were separated, along with one aliquot of plasma collected before lr. rbcs were stored at c for up to days; platelets were stored at c (rt) under agitation for days and plasma was frozen at - c. aliquots for culture were removed weekly from rbcs, daily from platelets and after days from frozen plasma. all samples were cultured in liver infusion tryptose (lit) media at c for detection of live parasites for up to weeks. results/finding: hemocultures from spiked-wb were positive at all concentration of parasites. lr'd and non-lr'd rbcs cultured before storage were positive at all concentrations. after storage at c, rbcs from all units spiked with , parasites/ml were positive for up to days; all further times yielded negative results. at lower concentrations, only non-lr'd rbcs spiked with parasites/ml were positive for up to days. plasma samples cultured before freezing were positive at the highest concentration in one non-lr'd sample, while all others were negative. platelets obtained from wb spiked with , and parasites/ml were positive up to days at rt. no parasites were observed in plasma or platelets prior to storage at lower concentrations. molecular analysis to determine the presence of parasite dna in each component is on-going. conclusion: platelet storage conditions offer a suitable environment for t. cruzi survival; however, high concentrations of parasites also survived in rbcs at c for up to weeks. leukoreduction offers partial protection, while freezing conditions appears unsuitable for t. cruzi survival. hemovigilance monitoring of platelet septic transfusion reactions (str) after treatment with intercept tm pathogen reduction or large volume, delayed bact/alert tm bacterial culture screening richard benjamin* , marion lanteri and larry corash . cerus corporation, scientific affairs department, cerus corporation background/case studies: amotosalen/ultraviolet a (uva) light (inter-cept tm blood system, cerus corporation) pathogen reduction (pr) and delayed, large volume, bacterial culture with the bact/alert tm system (dlvbc) (biomerieux, inc) represent respective best-in-class systems to reduce the risk of str associated with platelet concentrates (pc). where implemented, hemoviligance (hv) programs continue to receive reports of suspected str, most of which have low imputability as other causes are more likely or insufficient information is available to impute system failure. study design/methods: united kingdom ( - ), french ( - , swiss ( - ), and belgium( - hv reports, and cerus corporation's adverse event records were reviewed to assess the residual risk and imputability of str with amotosalen/uva-treated or dlvbc-screened pc. results/findings: approximately . million dlvbc-screened were issued with a day outdate after release into inventory days after collection, and $ . million amotosalen/uva-treated pc were released into inventory on day or , with a to day shelf-life. no septic fatalities were reported with either technology. the french, belgium and swiss hv programs monitored > . million conventional, non-dlvbc-screened pc and recorded str and fatalities. concurrently, zero definite and possible str were reported with , amotosalen/uva-treated pc, significantly fewer than with conventional pc (table ) ( . str per million vs. . per million, p< . ). one definite, possible, undetermined/indeterminate non-fatal str and contaminated "near miss" pc were reported with . million dlvbc-screened pc between and , for a reduced falsenegative rate compared with the prior five years ( . str per million vs. . per million, p < . ). hv programs highlight a major weakness when reporting str. stringent criteria are used to determine definite imputability, including evidence of patient infection, pc contamination and irrefutable evidence of a donor source, with confirmation of strain identity. reports with incomplete investigations are considered undetermined or indeterminate, or possible sepsis. some of these cases are almost certainly due to bacterial contamination of pc, suggesting that the actual rates of sepsis are considerably higher than that reported by hv programs. conclusion: best-in-class pathogen reduction and bacterial culture systems reduce str risk, although underreporting and inadequate clinical data may result in underestimation of the true rates. pathogen reduction of background/case studies: despite extant mitigation measures (e.g. diversion pouches and primary platelet culture at the collection facility), bacterial contamination of platelets and associated septic transfusion reactions remains a leading cause of transfusion-associated fatalities in the united states (us). consequently, the us food and drug administration has recommended adoption of additional measures such as point of release testing (port) and/or pathogen reduction to safeguard against transfusionassociated sepsis. however, port poses logistical challenges, particularly in institutions with high-volume platelet utilization, while pathogen reduction is a high cost intervention. we evaluated a second bacterial culture to contend with residual risk. study design/method: phased implementation of secondary bacterial culture testing (bact/alert tm ,biomerieux, inc., durham, nc) was initiated in october for all platelets received at our institution. at time of receipt at the blood bank (day post collection), products were sampled using a sterile connection device (tscd tm , terumo, elkton, md) and a sampling kit (sam-plok tm sampling kit, ml, itl biomedical, malaysia). five mls of product was transferred aseptically to bact/alert bpa (aerobic) culture bottles using the same sampling device. inoculated culture bottles were loaded into the bact/alert incubator modules and incubated at c for three days. results/finding: a total of / , ( . %) platelet products were successfully cultured ( / [ . %] and / [ . %] in october and march respectively). over the -month period, two true positive cultures were obtained (incidence of in platelet products). the cultures grew acinetobacter species (case a) and coagulase negative staphylococcus species (case b); both positive results were obtained four days following collection. repeat testing of cases a and b grew the same organisms identified in the initial cultures. there was a co-component in our inventory (case a) with negative initial and repeat cultures. none of the products were released for transfusion. the initial post-collection product cultures remained negative at the collection facility. over the same time period, no false positives were detected. implementation required hiring one additional dedicated fte; the total cost (technologist time, equipment and related supplies) was calculated to be $us . per product tested. the cost per averted case was $us , . conclusion: we demonstrate the feasibility of implementation of a secondary bacterial culture test of apheresis platelets to interdict bacterially contaminated units and prevent septic transfusion reactions. this presents a low-cost strategy (as compared to pathogen reduction) to mitigate risk of septic transfusion reactions. importantly, it offers a viable alternative to port in high volume institutions where logistic (e.g. time and personnel) constraints impede practical adoption of port. an increase in cases of blood culture positive transfusion reactions (bcptr) was noted at our hospital; bcptr was defined as bacterial culture positivity in the transfusion recipient and/or associated transfused blood product during investigation of a transfusion reaction. we sought to characterize the risk and clinical presentation of bcptr at our institution. study design/method: an analysis was conducted of all reported transfusion reactions at johns hopkins hospital (jhh) between january and december . the data, extracted from hemovigilance records, were evaluated to determine the incidence of bcptr; the severity and symptoms were evaluated in concordance with recipient data, including patient diagnosis, medications and clinical manifestations of the reaction. bacterial culture results were evaluated for both patients and associated blood products (i.e. partially transfused or residual product in blood bag). results/finding: in the -year study period, a total of transfusions reactions were reported, of which were bcptr ( . % of transfusion reactions). of the bcptr, ( %) were associated with apheresis platelets, ( %) with red blood cells, and ( %) with plasma. recipient diagnoses spanned hematologic/oncology (n ), renal (n ), cardiac (n ), autoimmune (n ), and obstetrics (n ). an organism was identified in both the blood product and recipient in ( %) cases; in ( %) cases an organism was grown in the blood product but not the recipient; and in ( %) cases an organism was isolated from the recipient only, due to inability to culture the product. the transfusion recipients in of the cases that did not isolate organisms in the recipients were on broad-spectrum antibiotics at the time of transfusion. symptoms of bcptrs included fever ( %), chills ( %), nausea and vomiting ( %), pain ( %) and dyspnea ( %). blood pressure (bp) decreased in %, increased in %; % of reported bcptrs had no change in bp. conclusion: the signs and symptoms of bcptrs are not specific and overlap both with underlying disease as well as other types of adverse transfusion associated events, thus contributing to delayed diagnosis and under-reporting. furthermore, high rates of antibiotic use in transfusion recipients can mask symptoms of true septic transfusion reactions. hospitals should consider expanding the clinical indications for culturing blood components that are implicated in transfusion reactions. furthermore, excessively stringent criteria (cdc/nhsn blood safety surveillance) for transfusion-transmitted infection, may contribute to misclassification of septic events in some recipients, particularly if on antibiotics. clinical oral abstract session: immonohematology and genetics --sickle cell disease and beyond blindspots and cross-reactivities of anti-human globulin specific for igg subtypes heather howie , jenna lebedev , linda kapp , xiaohong wang , meghan delaney , lay see er and james c zimring* . bloodworksnw research institute, bloodworks nw, university of washington school of medicine background/case studies: there are four different subclasses of human igg (igg -igg ), each with different effector function. essentially all existing data on the effect of igg subclass on hemolytic transfusion reactions and hdfn, were generated using ahg specific for igg subclasses. in recent decades, it has become appreciated that there are at least natural human variants of igg. in this study, the reactivity of igg specific ahg was tested against all known variants. study design/methods: the heavy and light chain variable regions of an anti-k monoclonal antibody were sequenced and cloned into expression plasmids that fused variable regions (in frame) with each of the known igg variants. plasmids were expressed by co-transfection into cho cells. the resulting panel of antibodies were pre-incubated with k rbcs and were then subjected to testing with currently available igg subtype specific ahg (monoclonal ahgs from southern biotech and sanquin, polyclonal ahgs from sanquin and the bindingsite). all testing was carried out by flow cytometry. results/findings: polyclonal reagents against igg , igg , and igg had cross-reactivity with variants found in other igg subclasses, and specific amino acids responsible were identified by site directed mutagenesis (table ). titrations of the ahgs did not identify a dilution at which crossreactivities were lost, but authentic targets were still detected. however, cross-reactivity could be neutralized by pre-incubating ahg with the crossrecognized igg forms (against a third party antigen); the remaining reactivity recognized the intended igg subtype without detectable cross-reactivity. no cross-reactivity was detected for polyclonal anti-igg or for any of the monoclonal ahgs tested. monoclonal anti-igg had a blindspot for igg - , due to the shorter hinge region on igg - . no blindspots were detected in other monoclonal or polyclonal ahg. conclusion: the relative quantitation of different igg subtypes has been studied in multiple immune settings, and plays important roles in diagnosis and research of human disease, including immunohematology. herein, we demonstrate that the reagents used to generate this body of knowledge suffer problems of cross-reactivities and blindspots. as such, the existing data regarding igg subtype biology may have some inaccuracies as a result of these defects in igg specific ahg. genotype matching for pediatric sickle cell disease patients nancy robitaille* , yves dominique pastore and maryse st-louis . chu sainte-justine, hema-quebec background/case studies: among the different treatment modalities available for sickle cell disease (scd), blood transfusion is frequently used. however, alloimmunisation remains a significant problem, even if prophylactic antigen matching is performed for c, e and kell antigens. this is partly explained by different antigen frequency among caucasian blood donors and african-american recipients, and by variants in the rh blood group of people of african-descent. blood group genotyping has been proposed as a potential way to alleviate this problem. the scd cohort of a pediatric academic hospital was genotyped for rhd, rhce and fy genes. the primary objective of our study was to evaluate whether compatible genotyped blood donors presenting similar rh variants could be identified. study design/methods: since , our local blood provider intensified recruitment of african-descent blood donors. these donors were phenotyped and genotyped for clinically relevant antigens by different means: genomelab snp stream, laboratory-developped assays and idcorext. as of , scd children were genotyped by sequencing rhd, rhce and fy cdnas after obtaining informed consent. extended red blood cell phenotypes were done at diagnosis at the hospital. patients' genotypes were compared to h ema-qu ebec's donor database to attribute blood donors to specific patients. results/findings: from diagnosis until september , ( %) patients had been transfused and had antibodies with known blood group antigen specificity: anti-c, anti-e ( ), anti-hrb, anti-fya, anti-jka, anti-jkb ( ), anti-s, anti-m, anti-sc , anti-leb ( ). seventeen patients ( . %) were either d or partial d. rhce results showed that patients expressed a normal c antigen and expressed partial c. as for e antigen, had a normal antigen, bore a partial antigen and were weakly expressed. fy(a b ) phenotype was found in ( %) patients. a total of genotyped blood donors of african-descent were available. the table below indicates the compatibility with these donors. conclusion: this study shows that several patients have rhce variants difficult to match, even with available genotyped blood donors from their community. although this measure is probably beneficial to decrease alloimmunisation, a larger donor pool is still needed to fulfill the patients' needs. the continued effort put towards recruitment and pheno/genotyping should improve the situation. using genetic markers to select responders and non-responders sickle cell disease (scd) patients for transfusion with rh haplotype matching red blood cell (rbc) units tamires delfino dos santos , emilia sippert , mayra dorigan de macedo , sheila fatima perecin menegati and lilian castilho* , . hemocentro unicamp, university of campinas background/case studies: rbc alloimmunization has been associated with several factors and with individual characteristics of each patient. we recently found that tnfa- a, il b- t cytokine polymorphisms, rhag g>a and hla-drb * alleles may predict a good responder phenotype (sippert et al, transfusion ) and that rhag a and hla-drb* alleles are closely linked to rh alloimmunization. based on this and considering the challenge to fulfill the transfusion needs of the patients with rh variants, we used these genetic markers to select responders and nonresponders scd patients for transfusion with rh haplotype matching rbc units and evaluated the risk of alloimmunization. study design/method: our study included non-alloimmunized patients with scd, homozygous for hbs, receiving a range of - rbc units. rbc antigen phenotypes of each patient and history of rbc antibodies were obtained from the medical records and transfusion service computerized database. rbc genotyping was performed using whea, wrhd and wrhce beadchip arrays (bioarray solutions, immucor) in accordance with the manufacturer's instructions. cytokine gene polymorphisms (tnfa- g>a, il b- c>t) and the rhag g>a gene polymorphism were analysed by pcr-rflp and taqman assays. hla class ii genotyping was performed using pcr-sso. results/finding: among non-alloimmunized patients, were homozygous or compound heterozygous for rh variant alleles. from those, had rhag a and/or hla-drb* alleles and at least one cytokine polymorphism (tnfa- a or ilb - t) associated with risk of alloimmunization and were transfused with extended and rh haplotype matching rbc units. the other patients with no risk factors associated with rbc alloimmunization were considered non-responders and were not transfused with extended and rh matching units. all patients were followed for one year and did not develop rbc antibodies. conclusion: these findings contributed to the development of a transfusion strategy for non-alloimmunized scd patients as typing for these polymorphisms could potentially help in the classification of responder and nonresponder scd patients, allowing blood with high level of compatibility to be five discrepant samples required sequencing. id core xt identified three rhce*cear samples encoding a partial c, and a partial e (predicted phenotype: vweak, vs-) and were confirmed by sequencing. the third sample was found to be rhce*cevs. ,rhce*cebi on sequencing (predicted phenotype v ,vs ). the samples were typed as v (or ce s ) and vs (or e s ) by hea. in addition, id core xt accurately identified rhce*ce[ g]in samples. this snp has been linked to various allelic variants affecting c and e antigenic expression. both samples were predicted to be c by hea. conclusion: blood group genotyping platforms vary depending on the specific snps that are included in each assay. such variations may be clinically significant when genotyping is used as a tool for providing matched blood. discrepancies leading to differences in the predicted phenotype could affect unit selection. despite the discrepancies between the methods, the high concordance rate and the limitations of serology warrant further reconsideration for the need for serologic confirmation of extended phenotypes. background/case studies: over three decades ago, two independent groups published work suggesting a novel categorization of warm autoimmune hemolytic anemia (waiha) on the basis of dat scores of agefractionated rbcs: type i waiha, comprising % of patients, showed increased binding of autoantibodies to aged rbc, whereas type ii waiha autoantibodies ( % of patients) bound young and old rbcs with no apparent prejudice. band- is a ubiquitously expressed rbc transmembrane protein which plays a vital role in maintenance of rbc structural integrity, cellular hemostasis, and regulation of senescence; and, has been suggested to be targeted by autoantibodies from patients with waiha. band- is regulated through phosphorylation of key residues; its hyperphosphorylation is a hallmark of normal rbc senescence, which causes band- to disengage from the cytoskeleton, increasing its lateral diffusion, thereby permitting the formation of band- aggregates forming new epitopes which are recognized by natural igg autoantibodies causing phagocytosis and destruction of senescent rbcs. type i waiha has been postulated to be caused by an exacerbation of normal rbc senescence. study design/methods: in an effort to confirm and characterize the two waiha subtypes we age-fractionated whole blood samples from patients with waiha on discontinuous percollv r gradients and looked for differences in dat results between less (young rbcs) and more dense (aged rbcs) fractions, fractionation patterns and band- tyrosine phosphorylation. results/findings: we confirm that two distinct types of waiha can be identified based on autoantibody reactivity with the youngest and oldest autologous rbcs. further, comparing type i and type ii patients, we found that type i is characterized by percollv r fractions (similar to healthy storage-matched controls) but increased band- tyrosine phosphorylation compared to healthy storage-matched controls, with phosphorylation occurring during younger stages of rbc development. type ii patients were characterized by - percollv r fractions, lacking the fraction containing the oldest rbcs, and showed a complete lack of, or dramatic decrease in, band- tyrosine phosphorylation compared to healthy storage-matched controls. conclusion: these results confirm the two distinct types of waiha. in type i waiha, the increased binding of autoantibodies to older rbcs coupled with increased tyrosine phosphorylation of band- suggests that rbcs from type i patients are aging faster than rbcs from normal healthy controls; this may represent an accelerated and pathogenic form of normal rbc senescence. in contrast, type ii waiha where autoantibodies bind strongly to either young or old rbcs coupled with a lack of fractionated bands that represent the oldest rbcs and a dramatic diminution in tyrosine phosphorylation of band suggests faster destruction of rbcs, consistent with the early published data, and metabolic changes that could affect rbc function. microbial pathogen primary sequence correlates with blood group antigen immunogenicity ian baine* , burak bahar , jeanne hendrickson , krystalyn e hudson and christopher a tormey . yale-new haven hospital, yale university, background/case studies: it is known that specific groups of patients immunologically respond more readily than others to rbc antigens. while rbc antigenic differences between donors and recipients are required for humoral immune responsiveness, other variables are also involved. studies have shown that there is significant primary sequence identity between common rbc antigens and microbes, and that cross-reactivity is possible between antigens in experimental models. we hypothesize that responder populations may be immunologically primed to form rbc alloantibodies via environmental exposure to cross-reactive microbial antigens, and that such a correlation may be linked to observed blood group antigen immunogenicity. study design/method: we performed peptide homology searches of the most immunogenic rbc antigens, based on previously published antigenicity findings. thirteen amino acid peptides containing the polymorphic residues of k, jk a , lu a , e, c, m, c, fy a , and s antigens were queried for identity with microbial peptides using the blast database (blastp, pam abstract algorithm, e value x - , word size , gap costs: existence exten-sion ). search results were restricted to bacteria and fungi, with a selective threshold of > % identity set for inclusion criteria. to corroborate with observed patient data, we also examined preceding cultures from alloimmunized patients to explore agreement between specific pathogens and rbc alloantibodies. results/finding: significant peptide identity was found between rbc antigens and pathogenic organisms including b. fragilis, p. aeruginosa, candida spp. among others. linear regression analysis of the number of genuses in microbial kingdoms meeting inclusion criteria showed a statistically significant inverse trend in predicting the degree of immunogenicity when fy a (an outlier) was removed (b - . , r . & p . ); that is, lower immunogenicity antigens were associated with larger number of kingdoms. k-medoids cluster analysis comparing immunogenicity and kingdoms showed that antigens clustered to low (c), moderate (e, c, s, m) and high (k, jk a , lu a , fy a ) immunogenicity groups, suggesting that an antibody response is inversely associated with environmental antigenic prevalence. of alloimmunized patients reviewed, were culture-positive. of these, % of the anti-c/c group ( of patients) and % of the anti-k group ( of patients) had microbe-antibody agreement. remaining microbe-rbc antibody agreements ranged from - . %. overall, . % ( of patients) demonstrated agreement. interestingly, we observed a particularly strong agreement between infection with klebsiella species and anti-k, despite the lack of > % sequence identity. while . % ( of ) patients reviewed had positive cultures for klebsiella species, . % of these ( of patients) demonstrated an anti-k. conclusion: our study highlights the potential connection between microbial infection and rbc alloimmunization, based on shared epitopes. we speculate that low-level antigenic exposure to highly prevalent microbial antigens such as commensals may promote immunotolerance, providing a model for the inverse relationship between rbc antigen immunogenicity and prevalence of microorganisms. longitudinal studies of microbial carriage (or acute microbial infection) and rbc alloimmune responses in larger patient cohorts may be informative. background/case studies: thromboelastogram (teg) has been incorporated into many hospital armories to manage transfusions during cardiovascular (cv) surgeries. some institutions use well-defined protocols for teg utilization at different stages of surgery (baseline, rewarming, postprotamine, and post-operative). on the other hand, at some institutions teg utilization is driven mainly by clinical judgment. when teg is ordered based on clinical judgment (clinical bleeding in most cases), some patients receive blood transfusions before teg is performed. there is no published literature on how pre-teg transfusions impact teg results and guide further transfusion requirements during cv surgeries. in this study, we have tried to address this issue. study design/method: we retrospectively reviewed tegs performed on patients undergoing cv surgeries at our institution from jan to dec , . no specific teg protocol was used to direct transfusions (plasma, platelets, and cryoprecipitate) during that period. only the first teg performed during surgery was included in the analysis. we excluded the patients that received only red blood cell (rbc) transfusions during the surgery because rbc transfusions are usually not based on teg results. for the tegs analyzed, teg results were divided into three categories: "normal" (reaction time (r), kinetics (k), angle (a), maximum amplitude (ma), and lysis at minutes (min) all within reference range), "hypocoagulable" (r> min, k> min, a< degrees, ma< mm) and "hypercoagulable" (r< min, k< min, a> degrees, ma> mm). fisher's exact tests and z-scores for two population proportions were used to identify statistically significant differences in teg results and blood product utilization. results/finding: out of tegs analyzed, patients ( %) received pre-teg transfusions. we found significantly fewer hypocoagulable teg results in pre-teg transfused patients than nontransfused patients ( % vs. %, p . ). the data also reflected a trend suggesting that there may be more normal teg results in pre-teg transfused patients compared with nontransfused ( % vs. %, p . ). there was no statistically significant difference in transfusions after obtaining teg results in both groups. however, there was a trend suggesting that hypocoagulable state was more likely to be corrected by transfusion in patients who were already transfused pre-teg compared to nontransfused ( % versus %, p . ). conclusion: pre-teg transfusions impact teg results (transfusions correct/normalize coagulopathy) but do not significantly impact further blood product utilization during cv surgeries. the decreased threshold (more transfusions) for correcting hypocoagulable state in patients who already received pre-teg transfusions may be due to more clinical significant bleeding in these patients to begin with. background/case studies: orthotopic liver transplantation (olt) is associated with significant blood loss, due to the complexity of the procedure and extensive liver vascularity, demanding blood transfusion. in this setting, cell salvage autotransfusion (cs) is been used as an alternative to decrease allogeneic red blood cell transfusion. however, as long as some studies have shown that cs in olt decreases allogeneic blood transfusion, others reported that cs presented little benefit or might have been associated with increased blood loss through fibrinolysis. in this study, we evaluate cs efficacy in reducing allogeneic blood transfusion in the intraoperative period. study design/method: we retrospectively evaluated data from liver transplants, performed from to in a single-center. patients were divided in two groups: one with cell salvage (cs) and another without cs (ncs). study endpoint included the requirement of allogeneic blood components transfusion during intraoperative period in both groups. cs was used in all liver transplant recipients but patients with malignancy and sepsis. blood transfusions were indicated based on clinical and hemodynamic criteria. clinical data included age, gender, diagnosis, body weight, height, warm and cold ischemic time and model for end-stage liver disease (meld) score. statistical analyses were performed using t-test, chi-square test, mann whitney test. results/finding: in this study period, olts were performed. a total of patients was submitted to cs. the median age was years (range - yo). cirrhosis caused by chronic hepatitis c virus infection was the main etiology of liver disease. hepatocellular carcinoma (hcc) was found in , % of the patients. the average meld score was , , and it was slightly higher in the cs group ( , vs , , p< , ) . there was no statistically significant difference in other variables such as body weight, height and cold ischemic time. the mean salvaged blood volume was ml and mean reinfused blood volume was ml. allogeneic blood transfusion was required in , % patients in the cs group, compared to , % patients in the ncs group. however, average red blood cells (rbc) and fresh frozen plasma (ffp) units transfused were lower in the cs group. the threshold for rbc transfusion was significantly lower in the cs group ( , units vs , units, p< , background/case studies: hemorrhage is a leading cause of mortality in trauma patients and morbidity in non-trauma patientsaddin en.cite.data. massive transfusion protocols (mtp) reduce mortality in trauma and nontrauma settings; however, this may be at the cost of blood product wasta-geaddin en.cite.data. blood product wastage benchmarks are loosely established, and data on wastage associated with mtps especially sparse. with a redesign of mtp and obstetric massive transfusion protocols (obp) which have different blood product preparation schedules, we assessed wastage, delivery method, and product utilization to identify differences in wastage during these protocols. study design/method: following institutional review board approval, a retrospective study on blood product wastage associated with the mtp and obp between july -december was performed. data on numbers of products dispensed and wasted were manually collected from transfusion service paper and electronic records and an automated data report from the electronic medical record. results/finding: the mtp resulted in higher total number of wasted products than the obp ( and products, respectively) however, obp wastage occurred more frequently in the month period. this reflects automatic thawing of cryoprecipitate in the first round of deployed products in the opb. mtp-trauma activations contributed higher wastage than non-trauma activations ( versus products). this is skewed by one month when products were wasted due to expiration of product on the floor. cooler-related issues ( ) and products dwelling too long out of a controlled environment ( ) were common reasons reported for wastage. the overall product wastage rates for mtp: trauma, mtp: nontrauma, and obp were . %, . %, and . %, respectively, with a total exsanguination protocol waste rate of . %. the difference between the overall proportion of waste between the mtp and obp protocols was insignificant (p . ). conclusion: wastage associated with both protocols was low and there is no statistical difference between mtp versus obp wastage. coolerrelated issues accounted for most product wastage, allowing for targeted waste reduction strategies including educational outreach and improved product delivery methods. better documentation of waste events identifies wastage trends for further product utilization optimization during these protocols. a year old female with multiple gun shots was admitted to a level one trauma center and received uncrossmatched group o, rh negative (d-) red blood cells (rbcs) through a rapid infuser during resuscitation. transfusion of uncrossmatched products before sample collection can lead to errors and confusion in blood typing, as can the venipuncture site used for collecting the patient's blood sample. the current fda guidance and aabb standard of two samples for determination of blood type to prevent cases wrong blood in tube (wbit) or electronic identification systems do not always catch or clarify these errors. study design/methods: patient was tested by manual tube method. two different technologists using two different reagent racks performed initial testing with matching results. results/findings: two samples were collected during resuscitation from the patient and typed as o d-. patient was transfused with units of o d-rbcs before stabilizing. two days later another sample was collected and typed as o rh positive (d ) with mixed field being seen on the anti-d. a weak d testing was performed to see if the negative result with anti-d could be strengthened through incubation. both original samples still resulted as d- (table a) . after consulting the patient care team it was discovered the samples were collected above the iv site after one unit had been completed and while the second unit was being transfused. it was also discovered all other clinical laboratory samples were rejected due to possible line contamination when results for the sodium, potassium, and glucose appeared inaccurate. the transfusion service laboratory is in a different area of the hospital and was unaware those samples had been rejected. conclusion: the initial samples were collected above the iv site and were contaminated with the d-blood product being rapidly transfused during resuscitation. the samples collected during the initial trauma response should have been rejected and a request made for samples drawn below the iv site. because both samples were collected while the unit was being transfused, contamination was in both. use of a handheld barcode system would not have caught this error because the patient had been correctly identified. future prevention of the above anomaly would be the education of transfusion testing staff to recognize an abnormal high hematocrit: secondly reminding the staff collecting samples to be aware of the proper collection procedures for laboratory testing, which would include type and screen. facilities also should strive to perform collection of the confirmatory sample from a completely different venipuncture site. impact of cell saver usage during solid organ transplants at a major institution holly ross* , edward smith , thomas brown , foeks jeremy , metcalf suzanne , james johnson , peter davis , karafa sw badjie and abba zubair . department of laboratory medicine and pathology, transfusion medicine, mayo clinic, department of anesthesia, mayo clinic background/case studies: our institution performs an average of solid organ transplants (sots) yearly. transfusion support for transplants can be tremendous, accounting for a large percentage of red blood cell (rbc) transfusions annually. even the best practices for allogeneic transfusion are not without risk. transmission of pathogens is possible with even the strictest screening methods, and each transfusion increases the risk of alloimmunization. the advent of intraoperative blood recovery has reduced the need for allogeneic donor rbcs during surgeries expected to bleed heavily. with the cell saverv r (haemoneticsv r , braintree, ma), patients' own blood shed during surgery is collected, washed, concentrated, and reinfused, lessening the need for transfusion support. this study sought to examine the amount of allogeneic donor rbc units saved during sots through the use of the cell saver for intraoperative blood recovery. study design/methods: data was collected for sots which utilized the cell saver. these included liver, liver/kidney combination, lung, and heart transplants. data a y.o. female was admitted to the trauma department after a motor vehicle collision (mvc) and transfused o( ) rbc units from the kiosk. her blood type was determined as o(-) with a negative rbc antibody screen (as). she was transfused more units of o(-) rbc. two months later, a repeat as identified two new rbc alloantibodies, anti-d and anti-e. the anti-d formation resulted from the o( ) rbc transfused from the kiosk, but the source of the anti-e was undetermined since e antigen is expressed in % of rh(-) individuals. the trauma department staff was notified of delayed serologic transfusion reaction and asked to investigate further since a y.o. female patient should not have received o( ) rbcs. study design/method: an investigative plan was developed by the trauma staff involving a patient census, review of the chart and kiosk inventory, obtaining feedback from clinical providers, and review of information provided by emergency services (ems). results/finding: the trauma unit was busy with admissions during the hours preceding the patient's arrival. the chart review found the following physical attributes; patient was overweight ( kg) with obvious facial deformities from the mvc, that compromised age assessment. it was determined that the kiosk was fully stocked with both o(-) and o( ) rbc units. one clinical provider recalls that the patient identification (id) might have been unknown. review of the ems communication states "patient is a y.o. female." conclusion: use of visual examination to determine age was significant in the selection of o( ) rbc for this patient. the trauma staff proposed and implemented a change in policy to prevent future incidents. any female patient that arrives without id or written confirmation of age will be transfused o(-) uncrossmatched rbc until a blood type can be determined. after being notified of the incident, the trauma staff took the lead in investigating and providing a process improvement resolution. this is credited to the excellent collaborative relationship between the transfusion service and trauma department on ensuring patient safety during emergent, uncrossmatched rbc transfusions. rate of abo/rh confirmation in outpatient pelvic organ prolapse surgery alexis r peedin*, taylor brueseke, yara park and jay s raval. university of north carolina background/case studies: approximately , surgeries for urinary incontinence or pelvic organ prolapse (pop) are performed annually. for abdominal pelvic floor disorder (pfd) surgeries, transfusion rates historically range from - %, whereas transfusion rates for vaginal and robotic pfd surgeries range from . - . % and . - . %, respectively. since the implementation of college of american pathologists (cap) requirements for abo/ rh confirmation, approximately % of patients who receive a transfusion in our hospital required a second abo/rh specimen to be drawn; however, limited data are available regarding the impact of this new requirement on patients preparing to undergo outpatient surgery that currently require preoperative type & screen (t&s). the primary objective of our study was to assess the rate of abo/rh confirmation in women who underwent outpatient pop surgery. study design/method: this was a planned secondary analysis of a retrospective cohort study of consecutive patients undergoing pop surgical repair from may -may in our academic tertiary care institution. among this sample, patients were excluded if their first t&s was drawn before our institution implemented the abo/rh confirmation requirement. fisher's exact test was used, and statistical significance was defined as p< . . results/finding: we identified patients for analysis, of whom ( . %) had a preoperative t&s ordered. two ( . %) of these patients had positive antibody screens; one patient had an anti-k and one had a warm-reacting autoantibody. fifty-nine ( . %) of the patients required a second abo/rh specimen per hospital protocol; ( . %) of these actually had a second specimen drawn. in patients for whom abo/rh confirmation was indicated, there were no differences between those who did and did not have abo/rh confirmed when comparing age, body mass index (bmi), pre-operative hemoglobin (hgb), or surgical approach (table ) . no abo/rh discrepancies were identified. one patient received unit of red cells after abdominal pop surgery. conclusion: the rate of requiring abo/rh confirmation before pop surgery was markedly higher than that seen in all patients receiving transfusions at our institution ( . % vs. %, respectively). because the vast majority of women undergoing vaginal or robotic pop surgery are not transfused perioperatively, hospital transfusion services should consider eliminating routine pre-operative t&s for this low-risk population in the maximum surgical blood ordering schedule, avoiding this unneeded test and subsequent abo/rh confirmation. volume reduction of red cells to reduce transfusion-associated adverse events related to hyperkalemia maressa t pollen*, laura knicks, linda van tol and c. michael knudson. background/case studies: one attribute of older blood is an increase in supernatant potassium level which can contribute to transient hyperkalemia. this problem is exacerbated in conditions of massive transfusion and in patients with renal failure. washing rbcs can effectively remove free potassium but is time consuming and can often only be performed on one unit at a time. here, we estimate the amount of potassium that is removed by volume reduction of red cell units. we also examined whether this technique would be feasible in the setting of massive transfusion in a patient with hyperkalemia. study design/method: expired or over temperature units (n ) that had been removed from inventory were utilized for these studies. each unit was weighed and a volume reduction procedure was performed. the supernatant was weighed and the potassium of the supernatant was measured using routine laboratory assays. for all formulas, weight was converted to volume using a specific gravity of . g/ml. the hematocrit (hct) of the volume reduced rbc was measured using a sysmex xs- i instrument. the percentage of supernatant removed was calculated by dividing the residual supernatant in the volume reduced unit (rbc hct x rbc volume) by the total supernatant prior to the procedure (residual supernatant removed supernatant). the remaining free potassium (meq) was calculated as the (concentration of potassium in the supernatant (mmol/l) x the estimated red blood cell residual supernatant volume. to simulate the process that would occur in the setting of a massive transfusion protocol (mtp), units were subjected to the volume reduction while recording the time needed to process all units. this was performed twice for a total of units processed in this manner. results/finding: the volume reduction procedure reduced the supernatant volume by an average of % (range %- %). in units between and days (n ), the estimated mean residual k was . meq (range . to . ). in the two mock mtp trials, the time to complete the procedure was approximately minutes and we estimate an additional - minutes would be required to modify and issue the units in our lis/emr. conclusion: a manual volume reduction protocol in red cell units significantly reduces the amount of potassium administered in a unit of red cells. this procedure may be useful when only older red cell units are available for a patient at risk for hyperkalemia. the procedure can be performed in less than one hour and may be useful under the conditions of massive transfusion. processing, cryopreservation, and non-specialized hospital collection. preliminary studies of three shipping conditions after collection were tested using sterile containers with sterile normal saline (ns) alone, ns plus antibiotic/antimycotic (ab/am) and a dry container. prolonged exposure to ab/am solution retarded outgrowth of mscs, but control of microbial growth in cultured tissue samples was needed. these findings were used to construct a validation study. study design/methods: a validation study designed to test procedures to collect, transport, process, and store umbilical cord tissue was measured by post-thaw outgrowth. collected uc tissue from consenting mothers was transported to the distant lab in validated shipping containers in a dry, sterile cup from vaginal ( ) and caesarian ( ) births. uc collections were divided into segments to test conditions. segment explants were placed on . % gelatin-coated gridded tissue culture plates ( explants per plate) in enriched medium specified for msc outgrowth containing antibiotic only with an endpoint of days. growth was scored as the number of squares with explants exhibiting outgrowth compared to the total planted explants. one segment (fresh control) was dissected and planted without further processing. the remaining tissue segments were soaked in (ab/am) saline solution for hr and hrs at c, respectively. tissue segments were frozen in cryo bags with a proprietary % dmso/large molecular weight sugar solution. background/case studies: it has been the practice in our institution to process or times the total blood volume (bv) of the patient, up to a maximum of liters (l) per procedure, to obtain peripheral blood cd stem cells. as a consequence, a patient often would need to spend hours or more on the machine. it would be desirable to be able to specify the exact volume of blood to process to achieve the desired cd cell yield, thus minimizing the patient's time on the machine, the nurse's time performing the procedure, and the number of bags that have to be submitted for cryopreservation and storage. study design/methods: our institution recently implemented the new spectra optia cmnc collection protocol, a continuous flow and continuous collection procedure that uses the automated interface management (aim) system to precisely manage the separation interface. an analysis of our collection data suggested a highly reliable collection process, so a prediction algorithm (pa) based on the linear regression between the patient's cd pre-count and cd yield, normalized per liter of blood processed, was derived utilizing the patient's cd pre-count, the patient's weight in kilograms (kg), and the target cd dose/kg. this pa calculated the exact volume of whole blood to be processed to achieve the requested dose of peripheral cd stem cells. the initial equation was modified to add an additional % to the predicted volume, to account for the natural variability of the process. this pa was then tested prospectively in the clinical setting. results/findings: in patients, representing both allogeneic and autologous donors, the average blood volume processed was . l. the range was . l - . l. the target dose was achieved in all patients. our previous practice for these patients would have required, assuming a standard bv procedure, processing an average of up to l per patient, with a range of - l. to quantify how well the new pa works, it was decided to evaluate the ratio between actual and predicted volume vs. the ratio between the actual and expected cd yield. the result was a high correlation between these two ratios (r . ), indicating that the algorithm produces very consistent results. conclusion: the predictability of our collection process during the time period analyzed was a robust r . , confirming the findings in the first data analysis. the blood volumes processed and patient time on the machine decreased substantially, with some patients only needing hours or less to achieve their target dose. nurses and lab medical technologists have seen a dramatic change in their workflow. the number of bags to process has dropped for the lab, with the consequent freezer space savings and the shorter collection times allowing the lab medical technologists to finish with their work earlier in the day. all in all, implementation of this pa has produced huge increases in patient and provider satisfaction. important factors that likely contributed to the success of the protocol included the precision and consistency of the aim system of the apheresis device, as well as the small number of nurses ( - ) who performed the procedures, resulting in less variability. the economic impact of this pa has not been quantified, but might be an interesting area for future studies. background/case studies: zarziov r , a biosimilar granulocyte colonystimulating factor (g-csf) has recently been introduced into clinical practice. its use has stimulated a certain debate regarding their possible less efficacy and security on cd mobilization. the aim of this study is to evaluate if there are differences between good and bad mobilizers and assess the need for plerixafor when a biosimilar as g-csf is used. study design/method: we retrospectively evaluated autologous mobilization processes performed between june and march . patients (n ) evaluated were diagnosed with malignant lymphoma (n ), multiple myeloma (n ) and primary amyloidosis (n ) and were mobilized according to standard protocols. collection cd cellularity target was established ! x e /kg. two groups, good and bad mobilizers, have been determined. predictors of unsuccessful mobilization were defined by > years old, previous fludarabine, lenalidomide, or bendamustine treatments or ! previous regimens, present peripheral cytopenias, active disease and previous mobilization failure. mann-whitney u test was used to compare means and comparisons of medians were performed by the median test. cd count was performed according ishage protocol. adverse events (ae) were analysed according to ctcae v . . results/finding: the media (range) general collection parameters were: cd (day ) . /ml ( . - . /ml), blood volume processed ml ( - ml) and . ( - . ) exchanged volemias. seventeen patients were considered bad mobilizers, needed plerixafor and had to undergone a collection procedure twice. there were statistically significant differences between both groups on mobilization characteristics and product cellularity [mean (sd) ); p . ]. there were no significant differences on mobilization characteristics and product cellularity between both groups. five mobilization ae were observed [muscle pain (n ), fever (n ) and flu syndrome; all grade ]. two patients could not undergo hematopoietic stem cell transplantation due low cd cellularity. conclusion: there are differences between products collected from the good mobilizer (rich in gm and cd ) versus poor mobilizer (with plerixafor) rich in cn and cmn. the mobilization with zarziov r could be smaller than expected since there are no significant differences if we compare the good mobilizers versus the bad mobilizers although the number of cases studied can be limiting. background/case studies: mesenchymal stem cells (mscs) have been widely studied and have shown beneficial effects on tissue regeneration, immunomodulation, and improvement of multiple organ failure caused by infection, sepsis, and trauma. however, mscs express tissue factor, which may be a risk factor for thrombosis especially if administrated systemically following trauma when coagulopathies are common. before applying mscs in a preclinical animal model, we sought to determine the procoagulant properties of rat mscs in vitro. study design/methods: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured and passaged using dmem medium with % fetal bovine serum. bmsc and amsc at passage - were used in this study. the tissue factor expression of mscs was determined by immunohistochemistry. citrated whole blood collected from normal rats was treated with rat bmscs and amscs at low, medium and high doses ( . /ml, /ml and . /ml respectively). the prothrombin time (pt), coagulation properties and platelet aggregation (response to adp, collagen and par ) were measured by hemostasis analyzer, rotational thromboelastometry (rotem) and impedance aggregometry (multiplate) respectively within min and hr after incubation. results/finding: tissue factor was significantly expressed among both bmsc and amsc at all passages in vitro. bmsc and amsc at any dose and time of treatment neither shortened nor elongated pt in whole blood. however, both bmsc and amsc significantly shortened the clotting time (ct) (none: seconds, versus low, medium and high doses of amsc ( , , and seconds), and bmsc ( , , . seconds), p< . ), clot formation time (cft, p< . ) and increased alpha angle (p< . ) by natem measurement, but did not significantly affect the ct, cft and alpha angle by extem. maximum clot firmness (mcf) and fibrinolytic index were not affected by mscs. there was no significant impact of both bmsc and amsc on platelet aggregation simulated by adp, collagen and par . no significant differences of hemostatic and platelet function were found between the treatments of bmsc and amsc. conclusion: consistent with reports from human derived msc, both rat bmsc and amsc significantly expressed tissue factor in both early and late passages, which led to a significant decrease in clotting time at various dose and time of treatment. however, mscs had no direct impact on platelet aggregation in vitro. as considering the procoagulant capability of mscs, future study will be necessary to determine the optimal dose and safety of using mscs for systemic application in vivo. comparison of the terumo bct mnc and cmnc protocols for peripheral blood stem cell collections lindsey westbrook* , neil bagamasbad , reynold dilag , melissa nasser , nicole bauer , jennifer wheeler and mary berg . department of pathology, university of colorado -anschutz medical campus, department of medicine, division of hematology, university of colorado hospital, scientific support, terumo bct background/case studies: terumo bct recently offered a new method of peripheral blood stem cell (pbsc) collection using the spectra optia, an apheresis instrument. the new protocol, continuous mononuclear cell collection (cmnc) collects cells continuously as opposed to the older protocol, the mononuclear cell collection (mnc) protocol, which is batch collection or dual stage collection, involving an additional step where platelets are separated from mnc within a cell separation chamber. our institution has used both protocols and the purpose of this study was to compare pbsc product characteristics and run times between the cmnc and the mnc protocols. study design/method: a retrospective review and comparison of parameters from collection procedures using the mnc protocol and collection procedures using the cmnc protocol was done using the t-test. data from patients/donors (including allogeneic donors) as well as procedure details including run time, flow cytometry marker for stem cells (cd )-positive (cd ) throughput, cd collection efficiency (ce%), platelet loss a transfusion per total blood volume processed (plt loss/tbv), and collection product characteristics were included in the analysis. results/finding: numerical results are summarized in the table. the mnc and cmnc donor groups included and allogeneic donors, respectively. donor weight was not significantly different between the two groups. pre-procedure wbc values were also similar between the two groups. run time was found to be significantly shorter using the cmnc protocol compared to the mnc protocol. product volume was also significantly lower in the cmnc group compared to the mnc group. although the volume was lower, the cmnc product had significantly higher percentages of mononuclear cells (mono%) and lymphocytes (lymph%) collected when compared to the mnc product. the cd throughput was significantly higher in the cmnc group than the mnc group. the cd ce% was found to be slightly increased in the cmnc group, though not significantly. the platelet loss was not significantly different between the protocols when normalized for total blood volume. product hematocrit (hct%) was significantly higher using the cmnc protocol; however, the red blood cell volume never exceeded ml due to the lower product volume with the cmnc protocol. the cmnc protocol collects a smaller volume of a purer product when compared to the mnc protocol with comparable platelet and red blood cell loss. staff members who perform apheresis procedures are pleased by the shorter run time. background/case studies: hematopoietic stem cell (hsc) donors and their recipients need not have a matching blood type. eventually, the hsc recipient will become the blood type of the hsc donor. this scenario can become quite a conundrum if the hsc recipient becomes a patient in need of an organ transplant. in order for a patient to receive a donor organ, the patient and donor's blood type and hla typing must be compatible. study design/methods: blood type was determined using gel test cards. hla typing was determined by using sequence-specific oligonucleotide (sso), sequence-specific primer (ssp), and sequence based typing (sbt) technologies. hsc sources were bone marrow and umbilical cord blood. results/findings: patient # , originally typed as an a , had bone marrow donor and cord blood transplants. one of the cord blood transplants successfully engrafted. the engrafted unit was from a type o donor. patient # is now typing as type o. patient # was originally typed as a and received a bone marrow transplant from a type b donor. patient # is now front-typing as a b and backtyping as an ab. since the patient's abo front and back-type do not match, a note must be made, that when confirming abo during crossmatch, the abo will not match. the patient now has an hla and abo identical kidney match (his father who is a type b). previously, the patient and his father were abo incompatible. the abo and hla results on both patient # and patient # indicate that the hsc transplants have engrafted. results also indicate that the abo and hla now match that of the donor and differ from the recipient's original abo and hla type. due to various reasons, for example, a side effect of the immunosuppression, both patients now need a kidney transplant. both patients will be entered into the unet system according to their "new" abo and hla types, as unos regulations require patients to be listed as per the results of two separate abo typing tests. the patients' antibodies will be monitored as per lab policy and communication with the transplant centers and blood banks is crucial. background/case studies: mesenchymal stem cells (msc) are beneficial for tissue regeneration, immunomodulation and improvement of multiple organ failure caused by infection, sepsis, and trauma. mscs express tissue factor (tf) that activate the clotting cascade and interfere hemostasis. hypoxia is a condition that occurs after trauma globally during shock or at the site of injury, and is known to change or influence the phenotypes of cells, including mscs. in this study, we want to determine if hypoxia changes the expression of tissue factor and the pro-coagulant properties of rat msc in vitro. study design/method: bone marrow and adipose derived mscs (bmsc and amsc) were isolated from bones (femur and tibia) and visceral fat tissue in normal young sprague dawley rats respectively. both bmscs and amscs were cultured using dmem medium with % fetal bovine serum under either normoxia ( % o ) or hypoxia ( . % o ). msc growth curves were measured by cell counter. the tf expression was determined by immunohistochemistry. cd /cd and cd were measured as positive and negative markers of msc respectively by flow cytometry. the citrated rat whole blood was treated with msc ( . /ml) either from normoxia or hypoxia. the coagulation properties were measured by hemostasis analyzer and rotational thromboelastometry (rotem). results/finding: hypoxia potentiated the growth of bmsc by %, but depressed the growth of amsc by % at day in comparison to normoxia. both bmsc and amsc equally expressed cd and cd but not cd under any culture condition. tissue factor was significantly expressed among bmscs and amscs from both normoxia and hypoxia. whole blood treated with bmscs and amscs from normoxia significantly shortened the clotting time (ct: (control), versus (bmsc), and (amsc) seconds) by natem. hypoxia also significantly shortened ct ( (bmsc), (amsc) seconds, p< . as compared to control), but the changes in ct were not significantly different between bmscs and amscs. maximum clot firmness (mcf) and fibrinolytic index did not change after treatment with bmsc and amsc regardless of the normoxia or hypoxia conditions. conclusion: tissue factor is constitutively expressed in rat bmscs and amscs. adjustment of the msc culture condition to hypoxia did not affect tissue factor expression or the procoagulant properties of msc (bmsc and amsc). this study also suggests that the procoagulant properties will not be affected if mscs are recruited into injured tissues with hypoxic environments. future study will be necessary to determine the optimal dose msc and whether it is safe to use mscs for systemic application in trauma. effect of double-end cryopreservation on gene-transduced human hematopoietic stem and progenitor cells sandeep k srivastava*, jiaqiang ren, steven highfill, narda theobald, suksee deravin, andre larochelle, david f stroncek and sandhya r panch. national institutes of health background/case studies: current early-phase clinical gene therapy trials use freshly collected or cryopreserved cd cells as the starting fraction prior to gene manipulation. following gene-transduction and culture, the end product is infused fresh into recipients. for wider applicability and scale-up, gene therapy manufacturing protocols would benefit from double-end cryopreservation (dec) of cd cells during manufacture (i.e. immediately post-collection and again, post-gene modification). dec helps delink patients' preparative conditioning phase from cell manufacture, eases logistics of inter-facility cell transportation, and ensures fulfillment of regulatory product release criteria before infusion. our objective was to study the effects of dec on gene transduced mobilized peripheral blood (mpb) cd cells. study design/method: cryopreserved cd cells from healthy adult donors were thawed and transduced (tr) in retronectin coated tissue culture bags with an ef -alpha-yfp lentivirus ( . % concentration) and media (x-vivo- , human serum albumin(hsa), ng/ml each of cytokines (scf, tpo and flt -l) over days. untransduced (utr) cells were cultured as controls. tr and utr fractions were re-cryopreserved. a standard freeze-mix of % dmso, % pentastarch, hsa, plasma-lyte a was used for cryopreservation. viability, hematopoietic stem cell (hsc) (cd cd -cd ra -cd cd f cells) phenotyping and cfu assays were done following first thaw (pt ), post-transduction (ptxn) and second cryopreservation-thaw (pt ). results/finding: tnc recovery decreased gradually in the donor samples at each step. transduction efficiency, cd %, cfus were similar before and after pt . hscs ranged from to cells/ cd cells in the pt -tr arm compared to a range of to / cd cells after pt . viability, % cd and cfus were lower in the tr compared to the utr arm. this difference was not altered after pt (table) . conclusion: dec of mpb human cd cells decreases tnc recovery, but has minimal effects on cd cell phenotype, transduction efficiency and cell function. hsc numbers were within acceptable range after recryopreservation. lower viability and cd % in the tr arm compared to the utr arm is likely due to vector toxicity. this was unaffected by recryopreservation. additional studies to assess dec mediated changes on cd cell early apoptotic markers, telomere lengths, gene expression and engraftment potential in nod/scid mice will inform clinical trials. background/case studies: autologous peripheral blood stem cell (pbsc) transplantation has been used as a powerful resource during the treatment of some hematological malignancies. cryopreservation of these cells is routinely performed to allow for patient adequate conditioning and chemotherapy. in some cases, pbsc are harvested as a backup option and remain stored for several years, although effect of storage lesion in this product is still controversial. our work presents retrospective data on pbsc infusion after long-term storage. study design/method: all products were harvested after patient mobilization with g-csf by apheresis with cobe spectra v r . flow cytometry analysis of cd cells was performed prior to cryopreservation. the cryoprotective solution was freshly prepared by addition of % hydroxyethyl starch, % human serum albumin and % dmso at final concentration. pbsc were cryopreserved by direct immersion on - c mechanical freezer (dump freeze) and stored until transplantation. post-thaw viability was determined from stored cryotube samples by trypan blue exclusion minutes prior to infusion. cells were thawed and infused on bedside. engraftment was defined as the first day of consecutive days of neutrophil count > . x /l and platelet count > x /l after days. with g-csf for four days and patients with g-csf for five days with use of mozobil when cd was below x cells/l on the fourth day. hpc collection was performed on the fourth day of mobilization for healthy donors and on the fifth day for patients. all procedures were realized based on a prediction algorithm using pre-cd on the day of the collection and estimating wbc liters to be processed to obtain sufficient stem cells for the transplant. this algorithm was designed using linear regression of peripheral blood cd on the day of the collection versus collected cd per liter of blood processed. there was no distinction between patients and donors, once the efficiency coefficient was used for both. collected material was sent to analysis and total cd was calculated. final laboratory count of cd per kilogram was compared with the number predicted by the algorithm with spearman's correlation to evaluate whether the formula is effective. calculations were made using ibm spss software. results/findings: among patients collecting hpc for autologous transplantation, , % needed only one day of hpc harvesting, while , % needed two days and , % needed three or more days. our collection efficiency (ce) and standard error of the mean (sem) was - , %. after comparing predicted values with cd collected in the final product, we found a very strong correlation of . (p< . ) for patients and a strong correlation . for healthy donors (p< . ). conclusion: the use of a mathematical model with a prediction algorithm is safe, has low cost and provides a good tool to estimate wb liters to process and avoid unnecessary procedures in both patients and healthy donors. this study evaluated the phenotypic characteristics of uc-mscs derived from fresh and cryopreserved cord tissues (ct), as described in isct's position paper on minimal characteristics of mesenchymal stem cells (plastic adherent; ! % cd , cd , cd and % cd , cd , cd , cd , hla-dr) study design/method: umbilical cord tissue (n ) was washed, blood vessels removed, cut into . - mm pieces, and washed twice in saline. fresh tissue was immersed in . % saline for same day culture, while frozen tissue was cryopreserved for at least hours prior to culture. for colony forming unit (cfu) testing tissue was plated directly in a cm tissue culture flask following a wash in pbs with antibiotic/antimycotic. the tissue was allowed to adhere for minutes prior to the addition of cell culture media. media was changed several times a week. cells were passed when robust colony growth was observed and in subsequent cultures > % confluence. all cells were tested on an msc flow panel at passage just prior to confluence. results/finding: both fresh and cryopreserved tissue showed excellent colony forming capabilities. average time for cellular emergence of days (fresh . , frozen . ), and days (total) for the msc's to reach passage (fresh . , frozen . ). all cells were ready for flow analysis in approximately weeks time. there was no statistical difference between fresh and frozen tissue in their colony emergence (p . ), or their growth rates (p > . for all). flow cytometry showed average ! % for positive markers and % negative markers. there was no statistical difference between fresh and frozen flow result (p > . ). conclusion: uc-msc's show excellent adherence to plastic in both fresh and frozen explant cultures, with a consistent fibroblast-like morphology. flow cytometry analysis showed strong msc phenotype in both fresh and frozen samples. the data show that the cryogenic process does not appear to have any detrimental effects on the ability to obtain msc colonies. studies have shown that hsct improves survival and disease-free survival rates when compared to conventional chemotherapy treatments. the increase in the number of hscts over the last years has demanded quality and safety improvements of cell processing and cryopreservation services. cell recovery and viability are crucial parameters to assess ucb quality as a viable hsct graft source. study design/method: twenty-five ucb units cryopreserved for periods of up to years ( to ) were analyzed. units underwent red cell and plasma depletion and then subjected to controlled rate freezing and subsequent cryopreservation using dmso (dimethylsulfoxide) cryoprotectant with % concentration. informed consent and the unit discard terms for all units were obtained. units were thawed in a c water bath and . ml aliquots were diluted at a : proportion with % human albumin solution and plasmin were prepared, enabling dmso stabilization and concentration reduction. the following analysis were performed: nucleated cell count (tnc) in an automated hematologic counter and cell viability using flow citometry. post-processing (pre-cryopreservation) cell viability was tested using trypan blue as exclusion dye, while post-thaw cell viability was assessed using -aad marker through flow cytometric analysis. results/finding: ucb storage period was . years (mean) and cell recovery was . % (mean). there was no statistically significant correlation between storage period and post-processing cell recovery (p . ). post-thaw cell viability of . % (mean) showed no statistically significant correlation with unit storage period (p . ). post thaw cell viability results are within parameters defined in other studies. background/case studies: umbilical cord (uc) tissue is a rich source of mesenchymal stem cells (mscs) that can be collected noninvasively at birth and stored for potential future use. as such, a growing number of stem cell banks have established uc storage programs based on mounting preclinical evidence of its therapeutic potential. however, little has been reported on the ability to isolate msc-like cells from uc tissue after extended periods of cryopreservation. this work describes and characterizes the isolation of mscs from uc tissue cryopreserved as a composite material at a family stem cell bank for years. study design/method: donated uc units from consenting mothers were evaluated. units had been cryopreserved as composite tissue pieces in ln vapor in a dmso-based cryoprotectant for yrs. ( . . ; n ). units were rapidly thawed and rinsed in dpbs, then pieces were excised from each using a biopsy punch. pieces from each unit were explanted in a x grid pattern in msc-supportive medium and incubated for days, after which the tissue was discarded and media exchanged. cells were isolated on the th day, counted, and subcultured for two passages. at the end of each passage, cells were collected, counted and population doubling time was calculated. isolated cells from each unit were also evaluated for msc immunomarkers. results/finding: small, proliferative cells with fibroblastic morphology were obtained from all explants, yielding a % success rate. cells were positive for the msc markers cd , cd , and cd ( . . %, . . %, and . . %, respectively) and negative for the hematopoietic markers cd / ( . . %). passage and passage doubling times were . . days and . . days, respectively, which are in line with values reported for mscs isolated from fresh uc tissue. conclusion: due to their immature status, ease of collection, and potential therapeutic value, uc mscs are an appealing candidate for future clinical a transfusion vol. supplement s research and treatment. the present work demonstrates that the long-term cryopreservation of uc tissue does not disrupt the ability to isolate functional mscs from the tissue at a later date. importantly, growth characteristics of isolated mscs appear to be comparable to those reported for mscs from fresh uc tissue. based on the consistent isolation and lack of apparent impact on proliferation kinetics, it is reasonable to expect cell yields in the range anticipated for therapeutic requirements and more than sufficient for moving to clinical grade bioreactors for expansion. these results support the feasibility of storage of uc as a composite material for future potential cell isolation and expansion to clinically relevant doses. large volume leukapheresis with spectra optia cmnc protocol in adult and pediatric patients: performance and determination of cd yield prediction algorithm ines bojanic* , nelly besson , ivana vidovic and branka golubic cepulic . department of transfusion medicine and transplantation biology, university hospital centre zagreb, terumo bct background/case studies: large volume leukapheresis (lvl) have shown to enhance cd cell yield collected. this study evaluated performance and safety of the spectra optia cmnc protocol (version ) in adult and pediatric lvl. a prediction algorithm for cd cell yield was also tested. study design/method: we evaluated retrospectively lvl performed in adult patients, and lvl in pediatric patients treated in uhc zagreb from march till september . mobilization regimen combined chemotherapy and filgrastim; poor mobilizes received plerixafor additionally. a combination of acd-a and heparin was used as anticoagulant (acd-a:whole blood ratio : ). in patients weighting kg (n ), a rbc prime was performed. cd , lymphocyte(ly) and monocyte(mo) collection efficiencies (ces) were calculated. a customized prediction algorithm was determined on linear regression between pre-cd cell count and cd cells collected / blood volume processed. prediction accuracy was evaluated by comparing predicted cd values to real cd yield. results are presented as median (iqr). results/finding: in both groups, cd , ly and mo ces were high. target cd dose was successfully reached in procedure in ( , %)adults and in ( . %) children. all procedures were well tolerated: adverse reactions were restricted to mild citrate toxicity symptoms in ( . %) adults, while all pediatric apheresis went uneventful. no bleeding episodes occurred, and no transfusion was needed. product and procedure characteristics* a high correlation between precd cells and cd cells collected/ blood volume was observed in both groups (r . and . in adults and children respectively, p< . ) suggesting cd yield could be predicted based on precd cells and blood volume to process. linear regression equations served as prediction algorithm. the high correlation between predicted cd yield and observed cd yield (r . and . in adults and children respectively, p< . ) showed accuracy of the algorithm. implementation of the algorithm could have allowed sparing a median of . ( . - . )l of blood in adult procedures, and . ( . - . )l in pediatric procedures. conclusion: lvl performed using spectra optia cmnc protocol is safe and efficient in adults and in low body weight children. high cd , ly and mo ce were observed in both groups. implementation of a predictive algorithm can reliably minimize blood volume processed, shorten procedure duration, reduce anticoagulant volumes infused, and improve patient comfort. mesenchymal stem cell therapy in steroid refractory graft-versus-host disease (gvhd) emese molnar* , aniko barta , arpad batai , zoltan csukly , zita farkas , laszlo gopcsa , gabor tatai background/case studies: steroid refractory acute graft-versus-host disease (gvhd) is a serious complication of allogeneic hematopoietic stem cell transplantation (hsct). more experience accumulates in the immunomodulatory effect of mesenchymal stem cell (msc) infusion in numerous immunopathological disorders -such as gvhd -and signals. mscs have a hlarestrictive and non-immunogenic nature. study design/method: we have evaluated the efficacy of msc transfusions in cases of acute gvhd refractory to conventional immunosuppressive treatment. the patients with steroid-resistant gvhd had received third-party mscs (derived from wharton's jelly and bone marrow) times per case weekly at a dose of million cells/kg. clinical response was assessed days after administering the first dose. complete remission was defined as the complete disappearance of symptoms. partial remission was assessed by the significant relief of symptomsand by the general improvement of the patient's condition. results/finding: in all patients had received cycles of msctreatment ( dose per cycle). the median age was years old ( - ) with a male/female ratio of : . distribution of the original malignancies (n): acute myeloid leukemia: ; acute lymphoblastic leukemia: ; myelofibrosis: ; myelodysplastic syndrome: ; multiple myeloma: ; t-cell lymphoma: . nine patients had undergone allogeneic hsct with matched unrelated donors, the other three had stem cells derived from hla-identic relatives. the first episode of gvhd after hsct was started on the median rd day ( - ). the involved organs were skin ( ), gut ( ), skin and gut combined ( ) and even lung in cases. the median time of msc's first infusion was days after the stem cell transplantation (hsct) and ( - ) days after the first episode of gvhd. of the cycles of msc-treatment led to complete remission ( . %) and resulted inpartial remission ( . %). conclusion: we have evaluated msc-therapy as an effective treatment of gvhd in the majority of the observed cases with % overall cumulative response rate. the application of third-party mscs offers a promising alternative in the therapy of gvhd and other gvhd-associated complications after hsct. further research is needed to determine the optimal start of the treatment, along with the issue of long-term safety. background/case studies: stem cell collection by leukapheresis for transplantation is a significant endeavor for the patient and the clinical team. whether the collection is allogenic or autologous, the patient undergoing the collection and the physicians caring for the patient are always concerned whether they will be able to harvest enough cells for transplantation and engraftment. a typical goal for most adult procedures is million cd cells/kg. if a patient does not reach this goal on the day of the procedure, they will likely have to return the following day to undergo a second procedure to reach the desired goal. given the logistical challenges in planning transplantation, it is reasonable to attempt to optimize the number of cells collected while minimizing the number of collections. measuring a patient's cd cells/ml in their peripheral blood before the leukapheresis procedure has been used to predict if the collection will successfully reach the million cells/kg goal. the ideal minimum cd cells/ml that will lead to successful harvest has not been conclusively identified. study design/methods: we analyzed the collection data from patients to evaluate the predictive value of the cd cells/ml level. data was collected over months from every patient who underwent a stem cell collection. four patients were allogenic donors and were autologous donors. the patients' weight, diagnosis, and pre-procedure cd cells/ml level were all collected. the run time, amount of volume processed, and the absolute viable cd cells collected were recorded. the collection efficiency and the cd cells/kg were calculated for each patient. results/findings: our data showed a strong linear correlation between pre-procedure cd cells/ml and post-procedure cd cells/kg (r . ). any patient who had a pre-procedure cd cells/ml count of or greater had a collection of at least million cells/kg. any patient who had a pre-procedure cd cells/ml count of or less collected less than conclusion: the pre-procedure cd cells/ml level in the peripheral blood has a very strong predictive value for the post-procedure cd cells/kg level. to confidently know that a patient will be able to produce the desired million cells/kg, a pre-procedure cd cells/ml count of at least should be obtained. for any patient with a count below , they should be counseled that their collection is likely to take at least a second day and a second procedure. further studies, including potentially lengthening the run time and the volume processed, to evaluate how to handle the patients who fall between and cd cells/ml should be conducted. heidi elmoazzen , antonio giulivi , michael halpenny* , lisa martin , donna perron , chris bredeson , lin yang , locksley mcgann , paul birch and jason p. acker . canadian blood services, ottawa hospital background/case studies: a critical aspect of hematopoietic progenitor cell processing is the cryopreservation method. our program uses a "dump" freeze method consisting of product placement directly into liquid nitrogen vapour after addition of a cryopreservation solution containing dmso ( % final concentration) and hes (hydroxyethyl starch). pentastarch (hes source) a critical component of the cryoprotectant formulation was discontinued by the commercial vendor. this required that an alternative cryoprotectant formulation be validated to minimize the risk to patient safety without compromising engraftment quality. study design/method: the validation study consisted of phases; firstevaluation of the efficacy of four different cryoprotectant formulations, second -evaluation of full scale production and crypreservation and third -a concurrent validation for clinical transplant. phase i -samples from four different cryoprotectant formulations were tested for tnc, cd , viability and cfu at three points during manufacturing (fresh, post processing and post thaw). phase ii -mock hpc, apheresis units were used for a side-by-side comparison of freezing curves for the control and replacement formulations. phase iii -five clinical transplants were performed with hpc, apheresis products cryopreserved using the recommended replacement (hetastarch). results/finding: phase i -results indicate that aliquots cryopreserved in % dmso and . % hes (hetastarch) did not behave significantly different than cells cryopreserved in the control in terms of cell recovery, viability or cell proliferation assay (cfu). phase ii -the majority of freezing profiles displayed typical or expected bulk freezing profiles for both formulations. phase iii -transplants performed resulted in a mean engraftment time of . days for anc with no adverse patient reactions observed. engraftment times using the new hetastarch formula were compared to the previous engraftment times with no significant difference. conclusion: a change in the formulation of a cryoprotectant solution represents a major change that could have a significant impact on quality. in addition, maintaining the current % dmso final concentration was critical as post thaw washing is not performed at the clinical site, history demonstrating a very low toxicity rate with the existing formulation. this study demonstrated the acceptability of the hetastarch formulation using % dmso and . % hetastarch to replace pentastarch in the cryoprotectant formulation used for cryopreservation of hpc, apheresis products. background/case studies: autologous stem cell transplantation is usually performed with mobilized peripheral blood stem cells (pbscs). traditional mobilization regimens include granulocyte colony stimulating factor (g-csf) with or without chemotherapy, but have failure rates ranging from % to %. plerixafor is an adjunct agent used to improve mobilization in many clinical settings. however, its high cost is a significant concern. the manufacturer-recommended dose is . mg/kg, therefore patients weighing > kg would require a second vial, thus doubling the drug cost. in we implemented a policy of capping plerixafor at mg for patients weighing > kg. this retrospective study compares the mobilization of patients > kg who received capped doses ( ) ( ) ( ) ( ) , with historical control patients ( - ) who received full or uncapped doses. study design/method: patients weighing > kg with crcl > ml/min who received capped and full doses of plerixafor were identified in the pharmacy database. electronic medical records were used to collect baseline characteristics and cell collection data. results/findings: a total of and consecutive patients were included in the capped and full dosing groups, respectively. they showed comparable baseline distributions of age, weight, gender and diagnoses. plerixafor was given upfront, or as a rescue agent due to suboptimal mobilization in both groups. in the capped dosing group, fewer patients received chemomobilization or plerixafor upfront. when compared to historical controls, they used half of the number of vials of plerixafor, but collected similar numbers of cd /cells kg and achieved a comparable collection success rate. the strategy dose capping plerixafor at mg for patients > kg is cost-effective and achieves comparable mobilization outcomes while decreasing the drug cost by half. mean and range of %cd in peripheral blood were calculated. the data show that in the non-hispanic group, the youngest donors (< yrs) have a higher pre-apheresis %cd level than any of the other groups, reaching statistical significance when comparing the %cd pre-apheresis between the youngest group (< yrs) and the oldest group (> yrs). hispanic donors show statistically similar %cd pre-apheresis levels over all age groups. moreover, the hispanic older age group (> yrs) had a statistically higher %cd pre-apheresis level than the non-hispanic older age group. conclusion: in this analysis of sequential unrelated pbsc donors, hispanic donors maintain a similar pre-apheresis %cd level even as the donor ages, while non-hispanic donors show a decreasing pre-apheresis %cd level as they age. if proven, this data would suggest there are genetic factors that modulate a person's ability to mobilize stem cells as they age and that these genetic factors differ between ethnic groups. this small data set would suggest that people of hispanic ethnicity maintain a more robust and quickly responsive stem cell pool, even as they age. further studies of larger cohorts are needed to validate this observation. if proven, this has far reaching implications within the stem cell research and therapy arena. background/case studies: an update in hpc apheresis collection software led to higher collection volume in the organization's human progenitor cell (hpc) products without a corresponding increase in total cellular counts. incorporation of a volume reduction step was therefore warranted as larger product volumes require additional time to transfuse and lead to a larger dmso load to the recipient, often resulting in the need to transfuse over several days. the objectives of this study were to develop suitable mock hpc (mhpc) products and evaluate the effectiveness of the biosafe pericell volume reduction technology on white blood cell (wbc) recovery and viability. study design/method: hpc products are not readily available for development. mhpc were created from whole blood buffy coats (bcs). fresh abo compatible bcs were pooled and concentrated using centrifugation and manual extraction of supernatant and red cells. the mhpc products were then diluted in plasma to produce an appropriate concentration and volume. hpc collection data from last years was analyzed to determine the th percentile, median and th percentile values for both hpc volume and wbc concentration. six mhpc products were tested; three high wbc ( x cell / ml) and three low wbc ( x cells / ml) concentrations, each at high ( ml), low ( ml) and median ( ml) volumes. each unit was processed sequentially from high, median and low volumes. hence, the highest mhpc volume was processed for volume reduction first with a sepax (pericell protocol, cs. . kits), analyzed and then reconstituted and volume adjusted to the next volume target before being volume reduced again, and so forth. one additional mock product was prepared for a reproducibility study and was volume reduced three times. wbc concentration and -aad viability was determined before and after each volume reduction. a control sample was removed from the product prior to processing and sat on the bench top until the end of the protocol to assess the change in cell concentration and viability over time. results/finding: mock hpc products had a mean starting -aad viability of % [range - ]% and a hematocrit of % [ - ] which is well below the maximum allowable limit of the pericell. no significant differences in wbc recovery or change in viability were seen between the mhpc products. aggregate data showed that the mean wbc recovery of the volume reduction process was % [ - ] with a % [- - ] change in viability. the recovery protocol used to salvage product after each volume reduction gave a recovery of [ , ] % and a change in -aad viability of [ , ] % from the input product. the method was found to have a cv of . %. the change in wbc concentration and wbc viability of the test products was not significantly different from the unprocessed control samples. conclusion: mock bc products are a suitable alternative where hpc products are not available for development and are a good use of product otherwise directed for rejection and disposal. the volume reduction protocol evaluated had minimal impact on the wbc concentration and wbc viability in the mock products and was found to be highly reproducible, giving confidence that it will be a valuable processing step with hpcs and will facilitate transfusion of hpc products into the recipient. the protocol is now in use with patient hpc products and engraftment kinetics will be tracked in a postimplementation study. validating a transfusion clinical assessment. in the first phase, cryopreserved pbsc products were tested. two aliquots were thawed simultaneously for each product: one was passed through a pre-set infusion pump and a second control aliquot was drained by gravity. each aliquot was tested for baseline total nucleated cell (tnc) count and viability, and for final tnc recovery, trypan blue (tb) viability, cd -aad viability, and potency (cfu). the effect of longterm exposure to dmso was assessed by visually inspecting the product for aggregates and measuring viability up to hours post thaw. the second in vivo phase included use of an infusion pump for consecutive autologous patients, with comparison of infusion and transplant outcomes to previous infusions by gravity drip. comparison variables included infusion rate, adverse events (ae), and engraftment time. results/finding: no significant differences were observed between infusion pump and drip for the products tested in vitro, including tnc recovery, cell viabilities, and potency. for both methods tnc tb viability decreased by more than % within hour, while cd cell viability remained stable up to hours post thaw. small aggregates appeared after hour for both methods and increased by a similar rate over time. comparison of infusion and transplant outcomes between drip and infusion pump patients showed no significant differences for all measured variables. engraftment time was similar for both groups. anc days to engraftment for pump and drip were . . and . . , respectively (p-value . ). platelet days to engraftment for pump and drip were . . and . . , respectively (p-value . ). infusion rates were slightly higher for the pump group. for control patients, required transfer of products to syringes due to slow infusion rate and others experienced allergic and hypotension infusion adverse events. conclusion: no significant in vitro or clinical differences were observed between thawed pbscs infused by gravity or an infusion pump. these results demonstrate that the use of a pump for pbsc infusion is safe, provides consistent infusion rates, eliminates the need to transfer products to syringes, and results in comparable engraftment times. donor racial distribution among the zikv ineligible cbus was: caucasian %, asian %, black/aa %, and multi-race %. racial distribution of all clinical cbu donors was caucasian %, asian %, black/aa %, and multi-race %, suggesting there is no race correlation for this risk factor driven by cultural habits such as family travel. there were no cases in which onlythe sexual partner's potential exposure determined donor's ineligibility. conclusion: our study indicates that currently the leading risk factor for ineligible cb donors is potential exposure to zikv: % of all ineligible cbus and % of all banked cbus in the study period. we anticipate the number of cases to decrease following maternal education and travel warnings. recognizing the importance of zikv in public health, and its potential transmission via hct/p products, an fda approved screening test for hct/ p donors becomes a timely necessity. acknowledgments: funded by zimmer biomet, a zimmer biomet company, ibgrl red cell reference and nhsbt reagents background/case studies: during storage, red blood cells (rbcs) become less deformable, deplete , -diphosphoglycerate ( , -dpg) and adenosine triphosphate (atp), release pro-coagulation phospholipids, accumulate pro-inflammatory molecules, free iron and haemoglobin and increase their potential for adhesion to a recipient's vascular endothelium. longer rbc storage may impair transfusion outcome due to impaired oxygen delivery, promotion of oxidative stress, increased pro-inflammatory state and coagulation. a sterile, non-pyrogenic rejuvenation solution, containing pyruvate, inosine, phosphate, and adenine (citra labs, llc, braintree, ma), is approved by the u.s. food and drug administration for the rejuvenation of stored rbcs. the solution acts by restoring , -dpg and atp in stored rbcs to levels equivalent to those in the circulation. the aim of the study was to investigate the effect treatment with this rejuvenation solution had on the crossmatch reaction profile and phenotypic state of stored rbcs. study design/method: a ml aliquot was removed from abo/rh grouped, leucocyte depleted rbc units (n ), which were stored in sagm for days, to act as untreated controls. the remainder of each unit ($ ml) underwent treatment with the rejuvenation solution ( ml, minutes at o c), followed by cell washing twice in sagm ('manual' centrifuge-based process). to represent current transfusion laboratory practice, units were crossmatched against plasma from random donors, using both diamed gel column and glass tube technique. phenotype investigation with commercial antisera was performed to identify the effect the rejuvenation solution treatment exerted on rbc surface antigens (a, b, d, c, c, e, e, k, m, n, s, s, p , lu a , k, kp a , kp b , le a , le b , fy a , fy b , jk a , and jk b ), including whether it exposed crypt antigens (t, tn, tk*, th, tx*, and cad). crossmatch and phenotype agglutination scores observed for the untreated and treated rbcs were then compared. results/finding: crossmatch findings were defined as compatible, suitable, and incompatible. the study identified no difference between the crossmatch reaction profiles of untreated and treated rbcs. furthermore, no difference was observed in the phenotypic state between untreated and treated rbcs. conclusion: treatment of day old stored rbcs with the rejuvenation solution had no effect on crossmatch reaction profiles or phenotypic state when compared to matched untreated samples. background/case studies: cryopreserved platelet production is burgeoning worldwide. currently, there are no automated platelet cryopreservation methods. by contrast, red blood cell cryopreservation using the acp (haemonetics corp., baintree, ma) has automated the processing within a closed system, increased labour productivity and provided high quality blood components. purpose: to automate platelet cryopreservation procedure. study design/method: apheresis platelet concentrates (pc) were collected on the trima accel system. platelet counts were performed using an abx micros . pc were centrifuged at g in a sorvall rc c centrifuge (sorvall, usa) for min. the combination cryoprotectant dmso dextran (cryosure dex , germany) was used for pc cryopreservation. cryopreserved pc (cpc) were frozen and stored in a kelvinator chest freezer. cpc were thawed at degrees c (barkey plasmatherm) for min. cpc osmolality was measured with an osmomat osmometer. results/finding: staged platelet cryopreservation technology has been developed. platelets were cryopreserved in a closed system (patent no.: ru u ). during the first stage, cpc were spun to separate a plateletrich plasma (prp) fraction from platelet-poor plasma (ppp). the second step was to resuspend the prp by adding a combination of dmso dextran (cryosure dex ) , as a cryoprotectant, to obtain a final concentration of % dmso in the platelet suspension. the injectomat mc agilia and npbi compomixer m were instrumental in automating that phase. pc to be frozen had an osmolality of no less than mosm/l. prp and ppp were frozen at a cooling rate of - c/min and stored at - in the chest freezer for up to months. pre-transfusion defrosted platelets were also processed in a closed system (patent no.: ru u ). our transfer set made it possible to automate platelet resuspension in plasma through the agency of the exadrop v r . post-thaw prp was resuspended in plasma, which lowered the osmolality to mosm/l. freeze-thaw recovery of platelets was % or more of the original population. defrosted pc were stored at - with continuous gentle stirring from a helmer platelet agitator for no longer than hours before transfusion. it took no more than min to cryopreserve pc and process pre-transfusion thawed platelets. the automated processing accounted for the bulk of the time (over min). conclusion: the automated technique developed reduced the workload while offering reproducibility of the procedure and high cpc quality. the use of closed systems ruled out bacterial contamination. employing the infusion pump, platelet stirrer and precision flow regulator enabled adequate osmolality monitoring. bacterial detection in leukoreduced apheresis platelets on day and day evelyn c. oyler*. suncoast blood bank background/case studies: the recently published fda draft guidance describing bacterial testing to enhance the safety and availability of platelets outlined the steps for blood collection establishments and transfusion services to extend apheresis platelets dating for up to days. this evaluation will compare culture based and rapid based test methods for detecting bacterial contamination in apheresis platelets. study design/method: a large community blood center and transfusion service collects leukoreduced apheresis platelets (lrap) using amicus separator system (fenwal, lake zurich, il) and trima accel system (terumo bct, lakewood, co). previously-cultured lrap units were sampled on day for secondary culture using bact/alert (biomerieux, durham, nc) and rapid bacterial tests using bactx (immunetics, boston, ma) and pgd (verax, marlborough, ma). if lrap unit is still available, it is also sampled and tested for rapid testing on day . a total of lrap units were tested over a -month period: were cultured and rapid tested on day ; were rapid tested on day . the rapid test methods were also evaluated based on cost, ease of use, incubation time and indication for use. results/finding: of the lrap units evaluated for this study, there were true negatives (tn) and false positive (fp) on day when tested by bact/alert, with tns on day . bactx testing results showed tns on day and tns on day . testing using the pgd kit showed tns on day ; and tns and fps on day . fp results were confirmed by performing a secondary culture, which were found to be negative. bactx requires a specific analyzer and minutes are required for result interpretation. there is no instrument requirement for pgd and reactions can be read within minutes. conclusion: the results of this evaluation makes pgd the best fit for this blood center based transfusion service. pgd offers a shorter time for reading of results, does not need an initial investment for an analyzer and is indicated for lrap in % plasma and lrap in pas/plasma. its ease of use allows for testing of lrap on day and day during the night shift to be accomplished without additional staffing and allows to extend outdate to day storage of lrap. change in growth factor content of human serum for use as eye drops during frozen storage for year jos lorinser , pieter f van der meer , hans van der heiden and dirk de korte* . department of product and process development, sanquin blood bank, mu-drop background/case studies: growth factors are thought to be among the active components in serum used for treatment of dry-eye syndrome. stability of growth factors during frozen storage in mini containers ( ml) is unknown. if these products can be stored at - c it will be feasible to store this product in -star household freezers, making the product available for patients in need of serum eye drops. the purpose of this study is to demonstrate stability of growth factor content in human serum during longtime storage at - c or <- to - c packed in a new micro dose device for single use as eye drops. study design/method: serum produced from ml whole blood donations from non-remunerated healthy donors was quickly frozen. after frozen storage at <- c for - months and controlled thawing, six different sera were used to fill a large number of mini ( ll) containers, which were refrozen and stored at either - c or <- c. during storage at months intervals, samples were tested for several growth factors, using magpixv r luminex multiplex assays and compared to control samples stored at <- c. growth factors tested were pdgf-aa&ab/bb, tgf-ß / / , vegf, a transfusion vol. supplement s egf, fgf . the study was a fact-finding study, without preset acceptance criteria. results/finding: pdgf-ab/bb and tgf-ß were the most abundant growth factors, on average , resp. ng/ml. also pdgf-aa was detected at relatively high concentration in human serum, on average ng/ml. tgf-ß , egf and vegf were detected at relatively low values, resp. ng/ml, . ng/ml and . ng/ml. average levels of fgf and tgf-ß were close to detection limit (< . ng/ml). the controls stored at <- c showed for all growth factors close to % of the initial values in samples at t (moment of filling mini containers). for serum stored at <- c for up to months, most factors showed less than % decrease, except for pdgf-aa and tgf-ß , showing % resp. % lower values. for serum stored at - c the values for tgf-ß , egf and vegf were stable, whereas pdgf-ab/bb, pdgf-aa and tgf-ß showed a decrease of resp. , and %. conclusion: human serum eye drops can be stored in the new micro dose device at - c ( -star household freezers) or <- c (professional freezers) for at least one year after preparation without large decreases in growth factor content. the maximum decrease was found for pdgf-aa in serum stored at - c. it is yet unknown if the tested components add to the in vivo effectiveness of serum eye drops and what the minimal concentration is to ensure in vivo effectiveness. further stability testing in combination with in vitro and in vivo application is required to extend the shelf-life beyond year. ruqayyah almizraq* , heather inglis , phillip norris , , jennifer a muszynski , nicole juffermans , jelena holovati and jason p. acker , . university of alberta, blood systems research institute, university of california, san francisco, nationwide children's hospital, academic medical center, canadian blood services background/case studies: different blood manufacturing methods can influence residual cell numbers and membrane vesiculation, which may affect quality and safety of blood components. the aim was to identify, quantify and characterize residual cells and extracellular vesicles (evs) in stored rbc products produced by different blood manufacturing methods. study design/methods: thirty-two rbc units produced using whole blood filtration (wbf), red cell filtration (rcf), apheresis, and whole blood derived (wbd) methods were examined (n per method). residual platelets and white blood cells (wbcs) were measured on day using flow cytometer (fc). on storage day and , number and cell of origin/surface markers of evs were assessed with fc, and concentration and size-profile of evs were examined using tunable resistive plus sensing (trps). results/findings: on day , apheresis and wbd units had significantly greater residual platelets in comparisons to rcf (vs: apheresis p< . , wbd p< . ) and wbf (vs: apheresis p< . , wbd p< . ) methods. while rcf units yielded the lowest count of platelet-evs (cd a ) on day and , the highest number of platelet-evs were in apheresis (day ) and in wbd (day ). similarly, there was significant difference among methods in the number of wbc-evs (cd , cd , cd , cd , cd b ) and rcf contained the smallest concentration. moreover, both trps and fc showed an increase in the total number of evs on day vs day in all of the processing methods. noteworthy, trps showed that the number of small evs/exosomes (< nm) was greater than large evs (! nm) in all of the products on day and , and the highest level of evs < nm were in apheresis units. trps results also showed a significant difference in the evs size-profile amongst all rbc products (p< . ). conclusion: this study shows that the method of manufacturing significantly affects rbc and non-rbcs evs characteristics throughout storage, which has the potential to impact quality and safety of rbc products. the differences in the evs cell-of-origin, concentration, and size-profile observed between manufacturing methods, warrants further examination of their potential immunomodulatory effects and clinical consequences. coagulation and complement assays in whole blood stored at centigrade maryanne c herzig* , crystal lafleur , chriselda g fedyk , sherrill j. slichter and andrew p cap . us army institute of surgical research, u.s. army institute of surgical research, university of washington background/case studies: whole blood has been demonstrated to retain hemostatic activity, including platelet aggregation function, over at least weeks of storage at c without agitation. it may be possible to extend the preservation of platelet function by agitating wb. in order to more fully characterize the quality of wb stored at c with or without agitation, we evaluated complement activation as a marker of inflammatory potential. study design/method: subjects donated one unit of wb collected in cpd-a (citrate phosphate dextrose anticoagulant with adenine). the wb was not leukoreduced nor was it separated into components. units were stored under refrigerated conditions for , , , or days after collection. units were stored for days without agitation. units stored for , or days were agitated during storage with a model hybridization incubator at c set for end over end rotation at - rpms. at the appropriate time point, platelet free plasma was obtained from the wb sample and stored at - c. the frozen plasma was analyzed by elisa assays to determine: thrombinantithrombin complex (tat) as a marker of coagulation; soluble cd l as a measure of platelet activation and granule release; plasmin anti-plasmin complex (pap) as a marker of fibrinolysis; plasminogen activator inhibitor (pai- ) as another fibrinolytic measure; and complement activation markers c a, c d, c a and c b- . data was analyzed by one way repeated measure anova. results/finding: only % of the platelets were recovered in units stored for days without agitation. these levels did not meet fda requirements of . x platelets per wb unit. subsequently, wb was agitated and platelet recovery was - %. no difference was seen in elisa analysis for agitated or non-agitated samples. no change was seen in tat or pap levels between t (day of collection) and t , , , or measurements. significant elevations of pai- and scd l indicate activation of platelets and inhibition of fibrinolysis (p< . ). activated complement peptides c a, c a, and c d were all elevated over time (p< . ) while sc d- was not. however, only c a and c d levels at t were above normal reference ranges at . and . times maximum reference, respectively. conclusion: whole blood agitation appeared necessary to recover platelets at or above fda requirements. whole blood stored at c for - days did show some activation of complement proteins. in contrast to studies in stored red blood cells with elevations of sc d- reported, wb showed elevation of c a, a and c d and not sc d- . complement was gradually and modestly activated with most levels remaining within reference ranges over whole blood shelf life. meredith lummer* and christian todd . cerus corporation, community blood serivces background/case studies: the interceptv r blood system for platelets (cerus, concord ca) is used for the pathogen reduction (pr) of platelet collections, and replaces irradiation, cmv testing, bacterial culture and point of issue bacterial testing. to better understand pr compatibility and impact to split rate, data were analyzed from a mid-size blood center with roughly . x . x . x . x rcf . x . x . x . x apheresis . x . x . x . x wbd . x . x . x . x platelet collections must meet specific volume, concentration, and dose ranges to qualify for intercept pr. changes made to apheresis devices included adding the following collection targets: . x in ml, . x in ml, . x in ml, and . x in ml. study design/methods: four months of collections were retrospectively analyzed. platelet collections were evaluated to determine eligibility for pr treatment, and all products meeting pr processing specifications (unless intended for an hla matched recipient at a hospital not able to accept pr products) underwent pr treatment regardless of potential impact to split rate. a minimum post-treatment dose of . x or . x was required to classify collections as singles or doubles respectively. volume/dose mitigation (removal of volume to increase the number of products eligible for pr) was not utilized during this study. thus units were treated conventionally if volume, dose, and/or concentration did not meet pr specifications without further manipulation. results/findings: % of all single and double collections were eligible for and underwent pr treatment. split rate for single and double collections was . . conclusion: it is possible to treat % of single and double platelet donations with intercept pr at the blood center's current state with only a slight impact to split rate if centers are willing to make alterations to their targeting practices. platelet collections that fall outside of the specifications for pr are processed and distributed as conventional products. strategies to increase eligibility toward % while minimizing impact to split rate are being investigated, including incorporating new collection settings, splitting triples, and volume/dose mitigation. further evaluation is needed to determine the additional quantity of pr eligible products resulting from such changes. monique p gelderman* , andrey skripchenko , fei xu , ying li , stephen j wagner , pamela h whitley and jaroslav g vostal . fda/cber/ obrr/dbcd/lch, american red cross holland laboratory, american red cross mid-atlantic research facility background/case studies: platelets (plts) stored at room temperature (rt) can support bacterial proliferation in contaminated units and therefore septic transfusion reactions may occur. storing plts at cold temperature ( - o c [ct]) limits bacterial growth but results in rapid clearance upon transfusion. the development of alternate storage conditions usually involves costly radiolabeling human studies but success in these studies is difficult to predict based on in vitro studies. thus, an animal model of plt circulation that could predict performance of human plts in human volunteers would positively impact the development of alternate storage conditions. study design/method: we designed an immunodeficient (scid) mouse model to evaluate recovery of human plts and compared this side by side to a radiolabeling study in human volunteers that was conducted for evaluating a new plt storage condition: thermocycling plts ( hrs ct: hr o c [tc]). autologous apheresis plts stored for -days at rt, tc and ct were radiolabeled and infused into healthy human volunteers (n ) and the same non-labeled plts were also infused into mice (n ). blood samples from humans and mice were collected over time to generate survival and clearance curves of the plts in circulation. flow cytometry was used to detect and analyze the human plts in the mouse samples to generate such curves; counts < % were considered background. results/finding: the mean recoveries of infused plts were . . % for rt, . . % for tc and . . % for ct in humans. in mice, mean recoveries of the same plts were . . % for rt, . . % for ct and . . for ct (mean sd). to compare performance of the plts in humans and mice we expressed all recoveries as a percentage of the rt recoveries. in humans tc was $ % and ct was $ % of rt. in mice tc was $ % and ct was $ % of rt. the area under the survival curve (auc) was calculated for the individual mouse study and human trial data sets. the results of both auc were normalized to % for rt plts. human tc plts had % auc while ct plts had % auc compared to rt plts in humans. in comparison, the same tc plts had % auc and ct plts had % auc of the rt auc in the mice. the calculated ratios of the auc between the tc plts and ct plts of the human data set and mouse model data set are . and . , respectively. conclusion: the scid mouse model differentiates between rt plts and ct plts similar to humans based on auc and plt recovery data. however, the mouse model cannot differentiate between ct plts and tc plts as occurs in humans. even though the mouse model cannot differentiate between ct plts and tc plts, it may still be a useful tool to screen other novel storage conditions for human plts. converting the component manufacturing from a manual process to automation nicole peters* and geeta paranjape , . coastal bend blood center, carter blood care background/case studies: initiatives focused on improvements to donor collection processes drove us to investigate opportunities in our component manufacturing processes. our goal was to maintain blood quality while streamlining manufacturing and automating the in-process documentation. the compomat g was evaluated using a multi-team approach including component manufacturing staff, equipment management, qa, regulatory affairs and it. study design/method: after a comprehensive evaluation, the team decided to purchase the compomat g with the compomaster net software for data management. implementation was planned for a november go-live. . to centralize processing, new work counters were installed. fresenius kabi installed the compomat g s and compomaster in june . training and validations were successfully completed and a full launch occurred mid-march . device and sop training was performed. training qualification checklists were completed for each technician with a required number of successful units processed and completed december . validation was completed and signed off in march of . manufacturing data was collected using the compomaster net data management system and our quality control software for platelet (plt) parameters, including plt count, plt weight, and plt yield from before implementation (bi) and after implementing (ai) of the compomat g system. data points were collected from units bi and units ai. results/finding: upon initial implementation, staff training and use, the compomat g was found to be easy. plt weight spread was reduced from an average of gm to an average of gm. actual plt weights were reduced from an average of gm to gm, resulting in an average increase in recovered plasma of . ml per unit. plt count on average increased from a count of to ( /mm ) with a negligible change in plt yield. conclusion: plt weight spread was reduced by . % after implementation of the compomat g and our plt concentrations increased on average by %. we were able to consistently produce a smaller volume plt (average gm), which gave us . ml more plasma per unit for recovered plasma. the team intends to review a dryer cryo as a next step for potential additional plasma yields for recovered plasma. deglycerolization of manually glycerolized, frozen rccs using a closed system cell processor anita howell , angela hill , brandie dennis and jason p. acker* , . canadian blood services, centre for innovation, canadian blood services, university of alberta background/case studies: upon implementation of a closed system cell processor for glycerolization and deglycerolization of red cell concentrates (rccs), many rare rccs frozen using the current manual, open system glycerolization method will remain in the organization's frozen inventory. a study was undertaken to assess the feasibility of deglycerolizing this existing inventory on the closed cell processor and to evaluate how the change may impact post-thaw red blood cell (rbc) in vitro quality. as the closed cell processor uses a fixed centrifuge bowl for deglycerolization and rbc resuspension, both large and small units were assessed to determine the impact of cellular loss and variability in hematocrit on the post-thaw product. study design/methods: abo/rh matched lr sagm rccs were pooled and split to produce large ( ml) and small ( ml) rccs. the rccs were stored to d and glycerolized manually by mixing ml of glycerol with the rcc in a ml freezing bag. units were frozen at - c for ! h before being removed from frozen storage and thawed in a c water bath. large rccs and small rccs were deglycerolized using the organization's current procedure on the cobe cell processor prior to re-suspension in . % saline, . % dextrose. the remaining rccs were transferred into a l bag, spun to allow removal of excess glycerol by manual extraction to achieve a hematocrit of %, and deglycerolized in a ml centrifuge bowl on the acp- with re-suspension in as- . rbc quality was tested at h post-deglycerolization. results/findings: large rccs had significantly higher hemoglobin per unit (cobe: p . , acp : p . ) and lower cell recovery (cobe: p . , acp : p< . ) post-deglycerolization than smaller rccs on both cell processors. large rccs deglycerolized on the cobe had higher hemolysis (p< . ) and supernatant potassium (p . ) than did small volume rccs. large cobe rccs had higher hematocrits (p . ), hemoglobin (p . ), and recovery (p . ) than did large acp- rccs. however, all cobe rccs had higher (p< . ) hemolysis ( . . %) levels than did acp- rccs ( . . %). cobe rccs failed to meet regulatory hemolysis standards of . %. conclusion: addition of a ml bolus dose of glycerol to rccs of different volumes results in different concentrations of glycerol in the frozen rcc product and may lead to differences in frozen rcc quality. additionally, the size of the rcc impacts quality for rccs processed on the closed cell processor due to centrifuge bowl volume limitations which result in lower recovery, hemoglobin, and hematocrits. use of the closed cell processor with resuspension in as- and storage for h, met in vitro quality standards for recovery, hemoglobin, and hematocrit, and drastically reduced hemolysis levels in rccs glycerolized manually. the acp- cell processor can therefore be used to deglycerolize rccs glycerolized using a manual, open system glycerolization method. background/case studies: washed platelets may be indicated for thrombocytopenic patients who experience severe allergic/anaphylactic or febrile reactions to conventional platelet transfusions. platelet washing process is time-consuming which may delay transfusion. this study was conducted to evaluate the manual platelet washing method (mm) using . % saline and centrifugation and the semi-automated washing method (sam) using the cobe blood cell processor. study design/method: in this study, units of single donor platelets were evaluated ( washed using the mm and washed using the sam. the collected data included product weights (pre-and post-wash), platelet counts (pre-and post-wash), total plasma protein (pre-and post-wash), presence/absence of platelet clumps, calculated % protein removal, and calculated % platelet recovery rate. the platelet counts were measured on the sysmex exn and the total plasma protein samples were measured on the roche cobas . results/finding: table shows that the average platelet recovery for the sam ( %) was significantly higher compared to the mm ( %). the mm had a slightly higher average protein removal compared to the sam. no platelet clumps were observed in either the mm or the sam. it was observed that the hands-on time for the mm took - minutes longer than the sam. background/case studies: the interceptv r blood system for platelets is currently licensed for pathogen reduction (pr) of amicus platelets in inter-sol (pas- ) for input platelet doses of . to . platelets in to ml of to % plasma and - % pas. a new platelet processing set was designed with three storage containers (ts) to process apheresis platelet components in pas- containing doses of . to . platelets in a volume of to ml. study design/methods: apheresis pcs (amicus v r ) were collected in % plasma and % pas- . one study was performed at the nominal dose ( . - . x platelets), volume ( - ml) in % pas/ % plasma using single donor apheresis collections. two studies were performed to evaluate the high dose and high volume condition ( . - . x platelets in - ml) using either single or pooled donations. input pcs (n ) were treated with the intercept ts set by the end of day post collection; the incubation time in the compound adsorption device (cad) container ranged from to hours and the intercept treated pcs were stored in containers (n ). day and post-donation pcs were evaluated using a panel of in vitro platelet function assays results/findings: in vitro function data for apheresis pcs in pas- treated in the intercept ts set demonstrated acceptable in vitro function (table ). all intercept treated pcs had ph( c) ! . . platelet dose and volume recovery post-treatment ranged from % to % and % to %, respectively. conclusion: pathogen reduced platelet components processed using the intercept ts set from either single or pooled apheresis donations maintained acceptable in vitro quality through days of storage. intercept blood system for platelets ts set is currently not approved for use in the us. background/case studies: the possibility of transmitting infectious organisms via blood products, plasma and their derivatives is a major public health concern. while current screening measures have considerably improved transfusion safety by reducing the risks associated with known pathogens, they cannot protect from emerging infectious threats. the pathogen reduction technology (prt) represents a proactive strategy to further reduce transfusion-transmitted infectious risk. however, the scientific community broadly agrees over the fact that prt has negative impacts on the product's quality markers. this study aims at evaluating the impacts of the mirasol prt on platelet (plt) quality and plt processing. study design/method: two abo-compatible platelet concentrates (pcs) containing % plasma obtained from either apheresis or sagm whole blood (wb)-derived processing were paired, pooled and then split into two equal units. one unit was used as a non-treated control (ctrl) (n ). riboflavin was added to the other pc unit and then exposed to uv light according to the manufacturer's instructions for the mirasol prt (teru-mobct) (test) (n ). numerous in-vitro quality markers (plt concentration, atp, po , pco , ph, glucose, lactate, sodium, and potassium) were measured for both mirasol-treated and non-treated pcs on days , , and for apheresis pcs, and on days , , and for wb-derived pcs. two flow cytometry assays were used to evaluate cd p expression with and without thrombin activation, and to measure the percent annexin vpositive plt. transfusion vol. supplement s results/finding: platelet recovery was % and % for apheresis and wb-derived pcs, respectively. mirasol-treated pcs showed higher levels of annexin v-positive cells ( % (test), vs. . % . (ctl) on day ) and a higher rate of cd p expression than control pc units ( % (test), vs. % (ctl)) on day ). the mirasol treatment generates changes in ph, glucose and lactate for pcs during storage. conclusion: the mirasol treatment induces a loss in the net number of plts/unit and elevated platelet activation. changes in ph, glucose and lactate suggest that prt affects plt metabolism. finally, prt has numerous impacts on logistic, storage and processing time constraints of blood bank operations. nevertheless, the mirasol prt is routinely used in europe with acceptable clinical outcomes. evaluation of a test method to detect bacterial contamination in platelets; bactx tm assay ji hye park sexton* , lorraine blagg , christi e marshall , herman woodson , sean erony , krishna patel and eric gehrie . the johns hopkins hospital, johns hopkins hospital transfusion medicine dept, johns hopkins university school of medicine background/case studies: bacterial contamination of platelets (plts) is the leading infectious risk of platelet transfusion therapy and it is the most significant infectious cause of transmission-associated morbidity and mortality. therefore, detecting various potential bacterial contaminants in platelets in a timely manner is critical. the bactx assay is a rapid colorimetric assay that detects peptidoglycan, a cell wall component of both gram-positive and gram-negative bacteria. here, we report an analysis of the bactx assay at our hospital. study design/method: we aimed to determine the sensitivity and specificity of the bactx assay. intact leukoreduced apheresis plt (lrap) units were tested by bactx at storage day . as a control, each intact lrap was also cultured by an automated bacterial detection system (bact culture) on storage day . the results of the bactx test were compared to the results of the bact culture system. results/finding: a total of lrap were tested. lraps initially tested negative by bactx, while lraps initially tested positive by bactx. all initial positive bactx tests were negative when subjected to repeat testing. in contrast, all lraps tested negative with the bact culture system. the specificity of the bactx test was . %. we did not have any true positive test results; therefore, the sensitivity of the bactx could not be determined. conclusion: this is a small study of only platelet units. the expected rate of bacterial contamination of platelets is less than per units. the . % initial positive rate was therefore higher than expected, but given the small sample size, it is clear that further study is needed to more rigorously assess the true sensitivity and specificity of the bactx assay. in vitro quality of rejuvenated and washed cpd/as- and cp d/as- rbc alan d. gray* , matt landrigan , pamela whitley , michael wellington , sherrie sawyer , shalene hanley , emily rondeau , louise herschel , neeta rugg , patricia a.r. brunker , shawnagay nestheide , jose cancelas-perez , larry dumont and zbigniew m. szczepiorkowski . and , -dpg to fresh levels. the objective was to demonstrate that in vitro quality measures are maintained for rbc when stored for > hours after treatment with an fda approved rejuvenation solution. study design/method: whole blood ( - ml) was collected and processed at sites into leukocyte-reduced rbc (a total of n cpd/ as- and n cp d/as- ). ml of rejuvenation solution (citra labs) was added to each rbc on day (d- ), incubated for minutes with agitation at c water bath (helmer dh ), washed (haemonetics acp ), and stored in as- at - oc for days (d- through d- ). in vitro recovery (%) was calculated and hemolysis, atp, and , -dpg were determined on day , d- , d- after rejuvenation and washing (postrjv), d- , d- , d- , and d- . all units were cultured on d- postrjv and on d- , and then concentrated by centrifugation on d- . results/finding: in vitro rbc recoveries were . % and . % (as- and as- , respectively) and no bacterial growth was observed. hemolysis on d- was maintained < % in / ( %) as- units and / ( . %) as- units. all as- and as- units ( %) had hemolysis < % following concentration by centrifugation. morphology score was reduced to % (as- ) and % (as- ) by d- , restored after rejuvenation ( %, %, respectively) and maintained through d- (> %). atp was restored and maintained above fresh levels after rejuvenation. , -dpg was restored above fresh levels and was maintained ! % of fresh levels through d- . all values were significantly different compared to d- except as noted (p< . , paired ttest) ( table ) . conclusion: rejuvenation of stored rbc restores atp and , -dpg above fresh values and morphology to near fresh levels while maintaining improved in vitro rbc quality measures through d- when compared to nonrejuvenated rbc on d- . this study is funded by zimmer biomet. storage > hours is not fda approved for use at the time of this publication. liposomes and rejuvenation: new approach for improving quality of stored red blood cells luciana da silveira cavalcante , jason p. acker* , and jelena holovati . background/case studies: liposomes have been shown to minimize rbc membrane damage occurring during -day hypothermic storage (hs), while rejuvenation solutions have been shown to restore rbc metabolism by maintaining atp and , -dpg levels. this study aimed to evaluate the effect of combining liposomes and rejuvenation on the quality of stored rbcs. study design/methods: five leukoreduced packed rbc units obtained were pooled and split. the units produced were segregated into four experimental groups: sham control (s), liposome-treated (l), rejuvesol-treated (r) and liposome rejuvesol-treated (l r). the prbcs were incubated for h at c with hepes-nacl (sham), liposomes (dopc:chol, : mol%, mm lipid), rejuvesol or liposomes plus rejuvesol. the in vitro quality was accessed by hemolysis, deformability, aggregation, atp and , -dpg at day hs. results/findings: hemolysis was significantly decreased in all treatments compared to sham control ( . . %): l ( . . %, p . ), r ( . . %, p . ), l r ( . . %, p . ). ektacytometry analysis showed an increase in maximum elongation (ei max ) in r ( . . , p . ) and l r ( . . , p . ) treatments compared to s ( . . ) but not l ( . . , p . ). rbc rigidity (kei) increased in all treatments compared to sham ( . . ): l ( . . , p . ), r ( . . , p . ) and r l ( . . , p . ). aggregation amplitude was significantly increased by r treatment only ( . . au vs. . . au, p . ). atp levels were significantly higher in all treatments compared to sham ( . . mmol/g hb): l ( . . mmol/g hb, p . ), r ( . . mmol/g hb, p . ), l r ( . . mmol/g hb, p . ). the levels of , -dpg were no longer detectable in s and l treatments at day . the combined treatment was comparable to r ( . . mmol/g hb vs. . . mmol/g hb, p . ). conclusion: both rejuvenation and liposome treatments improved the quality of stored rbcs compared to sham control. the combined treatment (l r) did not have a greater impact in improving in vitro quality of stored rbcs compared to rejuvenation alone. step toward a unique and adaptable thermoregulation system lucie boyer , eric ducas , patricia landry , nathalie dussault , jacques bernier , danny brouard* and anne maltais . h ema-qu ebec, institut de technologie des emballages et du g enie alimentaire background/case studies: h ema-quebec (hq) is facing major logistic challenges in the transportation and distribution of blood components over a large geographic area. in collaboration with the institut de technologie des emballages et du genie alimentaire, our applied research group is working on the development and optimization of a transport packaging for the -ml whole blood leukotrap rc system (haemonetics corp.). the objective is to design a packaging system for the rapid cooling (t < c) of one to six -ml whole blood units (wbu) within h from collection. moreover, the insulating and thermoregulation system must maintain the internal temperature of wbu between c and c for h under extreme external conditions (- c to c), including the initial blood cooling period. study design/method: the proposed packaging design is based on an external coroplast box containing six vacuum insulated panels (vip) for increased insulating efficiency. preservation of the initial cooling period and extended thermoregulation properties were ensured by an assembly of preconditioned c phase change material (pcm). the number of pcm, their position and conditioning were optimized and tested in order to meet the expected performance criteria. preconditioned pcm were stored into vip boxes for h at - c before each test to mimic a worst-case scenario for remote blood drives. for the experimental testing, -ml wb bags were filled with ml saline . % at t c to mimic freshly collected wb. probes were positioned inside the saline-filled bags to monitor temperature profiles of wbu under extreme winter (- c) and summer ( c) conditions. shipping boxes were filled with either one or six bags (n ). results/finding: the results showed that the thermoregulation box prototype is able to cool wbu bags under c in . . h and maintain their internal temperature between c and c for h with final values ranging between . c and . c for the extreme summer scenario. similar results were obtained for the extreme winter scenario; units reached the c threshold value in . . h and the bags' internal temperatures were within the acceptable range for h. conclusion: the insulating and thermoregulation system met hq performance criteria. preliminary results showed that pcm could be conditioned at temperatures higher than - c without any significant impact on the system performances. hq is currently validating the shipping box prototype performances. additionally, we are working on reducing the pcm conditioning time to optimize logistic operations. as this packaging has many advantages in terms of durability, price and convenience, hq intends to evaluate this system for the packaging and transport of other lines of blood products. stuart weisberg* , christopher c. c silliman , beth shaz , marguerite kelher and claudia s. cohn . new york blood center, bonfils blood center, department of laboratory medicine and pathology, university of minnesota background/case studies: platelets collected and stored in platelet additive solution (pas) reduce recipient exposure to donor plasma components. to better define the effects of pas on platelet supernatant composition, we compared total protein, isohemagglutinin titers, hla antibodies and in vitro neutrophil (pmn) priming activity in supernatants of pas-c platelets to plasma platelets. study design/methods: apheresis platelets from group o blood donors were collected into either % donor plasma (n ) or % pas- / % donor plasma (n ). within hours of collection, samples of the product supernatant were frozen, assayed for total protein concentration, anti-a and anti-b titer, and pmn priming activity within the total and lipid extractable fractions. all samples were screened for hla antibodies. screen-positive samples were tested using luminex single bead assays for antibody strength and specificity. soluble cd ligand (scd l) was measured using solid-phase elisa. results/findings: supernatants of pas-c platelets had significantly lower total protein concentration, anti-a and anti-b titers compared to plasma platelets. there was no significant difference in the number of hla-antibody screen positive pas-c ( / products) compared to plasma platelets ( / products); however, the hla-antibody screen-positive supernatants of pas- a transfusion vol. supplement s abstract c platelets had fewer hla specificities ( specificities) compared to those of the plasma platelets ( specificities). pmn priming activity was significantly increased in the supernatant of pas-c platelets. the lipid extractable fraction was not affected; however scd l levels were increased in the supernatant of pas-c compared to plasma platelets (table ) . conclusion: decreased plasma proteins likely underlie lower rates of allergic and febrile non-hemolytic transfusion reactions seen with use of pas-c platelets. decreased anti-a and anti-b titers may prevent hemolysis from minor abo mismatch. lower hla-antibody specificities may mitigate transfusion related acute lung injury (trali). increased pmn priming by pas-c platelets is likely due to platelet membrane release of scd l and not bioactive lipids. although scd l has been associated with trali, only pmn priming with lipid -not cytokine -agents has been causally linked with trali. the mechanism and clinical impact of increased scd l in pas-c platelets remain to be elucidated. background/case studies: current guidelines require a reduction of residual white blood cells (rwbc) below x wbc in us and x wbc in europe, per unit. the established reference method for testing rwbc in platelet (plt) and red blood cell (rbc) products is flow cytometry. alternative technologies have been developed including hemocytometry and microfluorometry. study design/methods: this study compared performance and workflow efficiency of the facsvia, a flow cytometer with a simplified workflow and automated loader to the adam automatic microscopic cell counter based on imaging technology. nonfiltered whole blood (wb) samples, apheresis platelet units (n ) and leukoreduced (lr) rbc units (n ) were used to generate spiked samples. apheresis platelets and lr rbc were filtered to deplete wbcs and were used as a diluent. nonfiltered wb samples were the source of wbcs to prepare a sample of wbc/ul. the spiked samples of , . , , , and wbc/ul were prepared from the source sample of wbc/ul and filtered platelet and rbc units. to evaluate linearity, wbc concentrations ( , . , , , , wbc/ul) were measured using adam and facsvia. samples were stained and run in triplicate on each analyzer. data was analyzed using linear regression. the results were proportional to the wbc concentration in the spiked samples. reproducibility of the two systems was measured by running spiked samples ( , , , wbc/ul). tubes of each sample were stained and run per system. the %cv and %diff were calculated. a batch of samples (plt and rbc) were run on both analyzers, repeated for days. workflow efficiency was assessed observationally by measuring the time of tasks performed. tasks recorded were instrument qc, assay controls and sample testing and analysis. results/findings: the wbc concentration results for plt and rbc samples on facsvia correlated well with adam (r-plt . , slope . ), (r-rbc . , slope . ). the %diff-plt at , , wbc/ul were . , . and , respectively. the %diff-rbc at , , wbc/ul were . , . and . , respectively. the average total testing time was similar on both instruments; min for the facsvia and min for the adam. of the total testing time, adam required continuous hands-on time, while facsvia demonstrated % ( of min) hands-off time. conclusion: both instruments showed comparable precision, linearity and accuracy. while the average total testing time was similar on both instruments, facsvia offered a significant workflow efficiency advantage. users saved an average hands-on time of minutes that could be used on other tasks. platelet rich plasma and quality control: is there a role for the blood bank? claudia s. cohn* and mickey koh . department of laboratory medicine and pathology, university of minnesota, st george's hospital and medical school background/case studies: autologous platelet-rich plasma (aprp) is a poorly regulated blood component often produced at the patient's bedside and used for indications such as chronic and acute orthopedic injuries, wound and incision-healing and rheumatologic diseases. prp isolation can be done by apheresis, which yields a consistent, platelet-rich fraction; however, most aprp is made using small bench-top centrifuges with cartridges that deliver uneven platelet enrichment. thus, the consistency and quality of aprp is questionable and the lower yielding prp may have decreased efficacy. study design/methods: a survey was designed to assess aprp manufacture, usage and quality control (qc) measures taken prior to its use. a survey was developed with input from content experts. the survey was sent to members of best and isbt. survey respondents were encouraged to forward the survey to colleagues, thus a true denominator is unknown. a total of completed and partially completed surveys were received. results/findings: responses came from countries, but the majority of responses came from the united states (us). of the respondents, % reported aprp use in their hospital. aprp was used predominantly for outpatients, though > % of hospitals also used aprp in the in-patient setting. in most hospitals, aprp was used by - mds; however, hospitals had > mds using aprp. the aprp was used for orthopedics, wound/incision repair, rheumatology and other indications. in the us the aprp was manufactured outside of the blood bank, while outside the us aprp was isolated by blood bank personnel. nearly all the aprp manufacturing was done with no quality control (qc) measures ( %); however, respondents assessed the final product prior to release. these qc measures included a platelet count to measure the enrichment of the platelet fraction, culturing the product and infectious serology testing. in some cases, if the aprp failed qc it could still be used, pending an md's approval. in the hospitals conducting qc on the final aprp, the testing was done by the blood bank. a subset of respondents from african nations also used allogeneic prp (allprp). in contrast to the patterns of use with aprp, allprp was used primarily for inpatients for indications including orthopedics, wound/incision repair and 'other'. the allprp was manufactured in the blood bank or the donor center with no qc other than a regular check of the centrifuge used to isolate the prp fraction. conclusion: prp is used in hospitals throughout the world for a wide variety of indications. the blood bank is involved in its manufacture in some countries, but in the us aprp is made outside of the blood bank. quality control of aprp production and the final product is not done in most hospitals. to improve the consistency and efficacy of prp, more stringent qc measures need to be in place. background/case studies: the morphology of donated red blood cells (rbc) change with storage, along with a loss of deformability, increased surface exposure of phosphatidylserine (ps), and decreased intracellular atp. these changes have been associated with increased rbc clearance within hours of transfusion. analysis of morphological alterations of stored rbc with imaging flow cytometry (ifc) has identified a subpopulation of small rbc that accumulates upon storage. this rbc subpopulation has a reduced projected surface area and undergoes a spherocytic shift which is expected to induce their retention in the spleen (roussel, dussiot et al, ) . some of the storage alterations are reversible when the rbc metabolism is reestablished. as such, treatment with a rejuvenation solution (citra labs) before transfusion is expected to restore some of the rbc properties and thus potentially increase their capacity to stay in circulation and operate effective tissue oxygenation following transfusion. study design/methods: a multi-parametric analysis of rbc alterations was performed to evaluate the effect of rejuvenation on rbcs stored in sagm (n ) under blood bank conditions at day (d ), at day (d ), after rejuvenation (r), and after rejuvenation and washing (rw). morphological alterations of stored rbcs were evaluated with ifc (imagestream x mark ii, amnis v r ). results/finding: rejuvenation increased the level of intracellular atp, confirming the metabolic effect of this process. population distribution as per rbc projected surface area measured by ifc depicted a well-demarcated subpopulation of small rbc that increased with storage from . - . % at d to . - . % at d . rejuvenation markedly reduced this storage-induced spherocytic shift ( . - . %) and partially restored rbc morphology, an effect confirmed by differential interference contrast microscopy. the restoration effect of the rejuvenation process did not correct the storage-related loss of rbc elongation but was associated with a decrease in ps exposure (table) . conclusion: our multi-parametric analysis shows that some but not all storage-related alterations are therefore corrected by metabolic rejuvenation. the impact of these effects while generally positive at the cellular scale requires further analysis by specific clinical studies assessing transfusion yield and tissue oxygenation. red cell concentrate volume and manufacturing method impact post-thaw quality in cryopreserved products processed using a closed cell processor anita howell , angela hill , tracey turner , april xu , brandie dennis and jason p. acker* , . canadian blood services, centre for innovation, canadian blood services, university of alberta background/case studies: the blood service uses both top/top with whole blood filtration (wbf) and top/bottom with red cell filtration (rcf) methods to prepare cpd/sagm lr red cell concentrates (rccs). mean volume (ml) is higher in wbf units ( ) than in rcf units ( ), with similar hematocrits. a closed system cell processor is currently being implemented for cryopreservation of rccs. post-deglycerolization re-suspension in as- additive solution is performed on-instrument to a defined total end volume, as dictated by the centrifuge bowl size. the impact of the resulting variation in hematocrit on post-thaw in vitrorbc quality was evaluated to ensure that regulatory standards can still be met for rccs at the extreme edge of the input volume range. study design/methods: small rcf ( - ml) and large wbf ( - ml) rccs were stored for d before being glycerolized and frozen at - c for ! h. large rccs whose red cell mass exceeded the capacity of the ml deglycerolization centrifuge bowl were volume reduced prior to glycerolization. rccs were thawed in a c water bath, deglycerolized and re-suspended in as- . rccs were stored d and then tested for in vitrorbc quality. results/finding: small rcf rccs had lower (p< . ) hematocrit, specific gravity, hemoglobin per unit, supernatant k and na concentration, deformability (ei max ), and higher (p< . ) recovery than did large wbf units. no significant differences in hemolysis, atp, , -dpg, p , rbc indices, rbc morphology, or residual glycerol were seen between groups. the majority of units met acceptance criteria (table ) , however of large wbf units had rbc recoveries < % due to pre-glycerolization volume reduction, and of the small rcf units had hemoglobin values < g per unit. when the recovery and hemoglobin failure rates are analyzed against the organization's rcc production volume distribution, the mean recovery is projected to be well above % and the hemoglobin failure rate would be below % of units tested; compliant with regulatory standards. conclusion: the differences between groups in the cryopreserved rcc physical characteristics were expected due to the re-suspension method and differences in the input product red cell mass. the lack of significant metabolic differences between groups indicates that the differences in postdeglycerolization hematocrits are not adversely affecting product quality. . . . . . . . . elongation index ( pa) . . . . . . . . this study is funded by zimmer biomet. (hasan ) . the objective was to determine the effect of rbc rejuvenation on rbc oxygen release capacity (orc) and estimated oxygen consumption (vo ) after simulating a single unit transfusion of either standard or rejuvenated rbc stored for days. study design/method: oxygen dissociation curves (odc) (hemox analyzer, tcs scientific) were generated from fifty-two ( ) rbc units (leukocyte-reduced), cpd/as- or cp d/as- , on day , day , and after rejuvenation and washing (pw). the odc for each sample was used to determine orc (ml o /g hb) and total releasable oxygen (tro) of the unit (ml o ). orc was determined by assessing the change in % o saturation from mm hg po (e.g., lung) to mm hg po (e.g., venous blood) multiplied by . ml o /g hb (li ). a simulated baseline pretransfusion vo of ml o /min was estimated using the day orc and assuming a g/dl transfusion trigger with a cardiac output of l/min and l blood volume. paired student's t tests were used for comparative statistical analyses. results/finding: rbc rejuvenation on day restored orc and tro to levels greater than day ( table ) . orc of the rejuvenated unit was . . times and . . times greater than rbc on day and day , respectively (p< . ). vo increased after a simulated single unit transfusion of rbc (day , day , and pw) by . %, . %, and . % over the pre transfusion vo , respectively (p< . ). conclusion: these results suggest a transfusion with rejuvenated rbcs has the potential to release . times the volume of o compared to standard, untreated rbcs stored for days. inferior oxygen delivery to tissues (vo max) has been observed during exercise in healthy human volunteers after transfusion of two autologous rbc units stored for days vs days which seem dependent on genetic variability and storage time (bennett-guerrero ). therefore, transfusion practices to correct anemia may be less effective than intended due to the variable orc of standard stored rbc units. transfusion strategies should consider whether the use of rbc with increased orc may be physiologically advantageous. disclosure: this study was funded by zimmer biomet. rejuvenation solution as an adjunct storage solution maintains physiological hemoglobin oxygen affinity during rbc unit storage andrea ansari* , jay srinivasan , gustaaf de ridder , alan d. gray , matt landrigan , keaton charles stoner , angela crabtree , jessica poisson and ian welsby . duke university school of medicine, duke health pathology, citra labs, a zimmer biomet company, zimmer biomet, duke university, department of pathology, durham veterans affairs medical center, duke university hospital, duke university medical center background/case studies: deleterious changes develop during the storage of packed red blood cells (rbcs) collectively called the "storage lesion". these include altered membrane composition and decreased deformability, increased in-bag and post-transfusion hemolysis, loss of atp, snitrosohemoglobin, vasodilatory capacity, and cell surface ps expression, and depleted , -diphosphoglycerate ( , -dpg). the loss of , -dpg increases the oxygen affinity of hemoglobin, resulting in lower p (partial pressure of oxygen at % hemoglobin saturation). decreased p may negatively impact the ability for transfused rbcs to release oxygen to peripheral tissues. an fda-approved rejuvenation solution (citra labs) can restore normal levels of atp and , -dpg, normalizing membrane function and oxygen affinity, respectively. this process requires incubation at c for an hour, an impractical step in time-sensitive situations, followed by washing of the rbcs. we tested the hypothesis that rejuvenation without the incubation step ("cold rejuvenation") could prevent or reverse changes in oxygen affinity, deformability, and susceptibility to hemolysis of rbcs. study design/method: eight units of group a , leukoreduced prbc stored in as- were obtained from our local blood center. after days of storage, units were divided into separate aliquots: control (ctl), wash (w), standard rejuvenation (sr), and cold rejuvenation (cr). the rejuvenation solution ( ml) was added to the cr group, and all groups were then stored for another days at - c. on day of storage, the sr group was incubated for hour at c with rejuvenation solution, after which the w, sr, and cr groups were separately washed on a c.a.t.s v r (fresenius kabi) using the high quality wash setting. hemoglobin p was measured by tonometry using a hemox analyzer (tcs scientific). deformability (elongation index or ei) was measured by ektacytometry (lorrca mechatronics). supernatant plasma free hemoglobin (pfhb) was measured using visiblelight spectrophotometry. cell surface ps expression (ps ) was measured by annexin v flow cytometry. all group results were compared using nonparametric wilcoxon signed-rank tests with a . . results/finding: significant differences in p were noticed between all groups (table ) . ei, ps , and pfhb did not differ between groups. conclusion: cold rejuvenation prevents the increased oxygen affinity (lower p ) seen over days of rbc storage without adverse effects on deformability or hemolysis. this offers an alternative to incubated rejuvenation to provide clinicians with ready access to rbcs with a high/normal p that may better release oxygen to the tissues. cause transient and potentially fatal cardiac arrhythmias upon transfusion, particularly in infants, and massively-transfused patients, and those with compromised renal function. reactive antibodies and other inflammatory agents in rbcs can also elicit life-threatening reactions, potentially causing high fever, transfusion-related acute lung injury (trali), anaphylaxis, and even death. in this study, a multifunctional bead-based filter was evaluated for removal of k , along with free hemoglobin (hb) and other prbc contaminants that can contribute to transfusion related adverse events. study design/method: ten leukocyte-reduced prbc ( ml) units stored in as- , obtained from a regional blood donor center at expiration ( days), were passed by gravity through sorbent-devices containing ml of multifunctional polymer bead, at a flow rate of ml/min. supernatants were analyzed for k removal as well as free hb, antibodies and cytokines ( -plex, biorad). rbcs were analyzed for viability and integrity via flow cytometry and osmotic fragility assay, respectively. results/finding: filtration of the aged prbc units through the sorbent device reduced [k ] from . . to . . meq/l; equivalent to an . % reduction. free hb was reduced by . % from . . to . . mg/ml. antibodies, specifically igg, iga, and igm decreased from . . to . . mg/ml ( . %), . . to . . mg/ml ( . %), and . . to . . mg/ml ( . %), respectively. inflammatory cytokines were significantly reduced, specifically: ip- from . . to . . pg/ml ( . %), mip- b from . . to . . pg/ml ( . %), and pdgf from . . to . pg/ml ( . %). filtration had no significant impact on cell surface markers of rbc viability (< . % decrease) or sensitivity to osmotic changes. values listed represent mean sem (p < . for all analytes tested). a paired ttest was used to assess significance. conclusion: the sorbent filter was highly effective in reducing the levels of extracellular k as well as free hb, antibodies, and cytokines from prbcs without impact on rbc viability or integrity. this study demonstrates the viability of a multifunctional sorbent filter for removal of k along with other detrimental components from stored prbcs that can readily be incorporated into transfusion practices to minimize adverse effects. background/case studies: platelets carry no rh antigens, but residual red blood cell (rbc) in platelet products can immunize d negative recipients if the donor is d positive. current recommendation is to give rh immunoglobulin (rhig) to rh negative patient if they receive rh positive platelet unit to avoid potential alloimmunization to d antigen. a recent study has shown a very low frequency ( . %) of d alloimmunization when a rh mismatch platelet is transfused. restricting d negative patients to receive only d negative platelets could create shortage and cause inventory challenges. higher yields of platelets with minimum to none residual rbcs are obtained with new generations of apheresis machines. as a consequence, the need for prophylactic rh immunoglobulin (rhig) may be unnecessary with the use of apheresis derived platelets. the accurate determination of residual rbc in a platelet unit is important for patient safety to prevent rh alloimmunization. hemocytometer is considered the gold standard for cell counting. however, the rapidity and convenience offered by automated methods resulted in widespread use of automated hematology analyzers. currently there are no standardization and/or guidelines to advise what system to use for rbc quantification in platelet products. study design/method: we designed this study to quantify the residual rbc in apheresis platelets and whole blood derived platelets comparing hemocytometer and automated methods. we measured the amount of red blood cells per microliter in apheresis and whole blood derived platelet units using hemocytometer and two different automated hematology analyzers, namely, sysmex (sysmex america, lincolnshire, il) and advia (siemens healthcare diagnostics, tarrytown ny). the whole blood derived platelet units were produced using acrodose tm system technology. we conducted non-parametric permutation test based on permutations to compare sysmex and advia between apheresis and whole blood derived groups. abstract collection) rbcs to rbcs stored for days and after treatment with an fda approved rejuvenation solution. study design/method: the addition of a rejuvenation solution to stored red blood cells (rbcs) has been shown to increase atp and , -dpg profiles to fresh levels. the objective was to compare % hemoglobin-oxygen saturation (p ) and morphology profiles of fresh(day of collection) rbcs to rbcs stored for days and after treatment with an fda approved rejuvenation solution. results/finding: in vitro rbc recovery (overall) was . . %. hemolysis (%) was similar on day before and after dry-air incubation with the rejuvenation solution ( . . % vs . . %). percent hemolysis (%) decreased after washing ( . . %) and was maintained below < % for all units during storage for hr ( . . %). average atp and , -dpg were restored above the average fresh values. the morphology score decreased $ % by day , which was restored to near fresh values following rejuvenation and washing and storage hr ( . % and . %, respectively). rbc oxygen affinity, as assessed by p , was restored above fresh values. all values were significantly different compared to day (p< . , paired t-test) ( table ) . conclusion: rbc morphology was restored to near fresh and average atp, , -dpg, and p were restored above fresh values when incubated with a rejuvenation solution using the dry-air incubation process. rbc morphology, atp and , -dpg were maintained during storage hr. rejuvenation of refrigerated rbcs may offer avenues to improve rbc quality prior to transfusion. vandi ly*, dimath alyemni, warren r korn, matthew j brune and julie katz karp. thomas jefferson university hospital background/case studies: blood donors are screened with a donor history questionnaire that includes questions regarding behavioral risk factors, but none that specifically screen for the use of marijuana. therefore, there is the theoretical possibility of transfer of active cannabis metabolites through transfusion. donor plasma collected at an urban, hospital-based blood donor center was examined for the presence of active cannabis metabolites, d tetrahydrocannabinol (thc) and -oh-d -tetrahydrocannabinol ( -oh-thc). study design/method: de-identified donor plasma segments were sequestered and stored frozen until time of testing. testing for thc and -oh-thc was performed by liquid chromatography-tandem mass spectrometry (lc-ms/ms) based on a method modified from lacroix and saussereau. in summary, this method used dabsyl chloride derivatization of thc and -oh-thc to produce samples for lc-ms/ms analysis. lc used a c column. post-column detection by ms/ms used positive ion electrospray with q :q ion pairs of m/z . : . (internal standard (is), d -thc), m/ z . : . (thc), and m/z . : . ( -oh-thc). quantitative results for thc and -oh-thc were obtained from a standard curve (ratio of analyte integrals to integrals of internal standard) ranging from - ng/ ml for both thc and -oh-thc. limits of quantitation, defined as standard deviations above background, were . ng/ml for thc and ng/ml for -oh-thc. results/finding: a total of donor plasma samples were tested for thc and -oh-thc. no samples tested positive for either thc or -oh-thc. theoretical calculations according to statistics of a poisson distribution indicated that there would be a % probability of one or more positives at a prevalence of . % positive samples, and a % probability of one or more positives at a prevalence of . % positive samples. results thus indicated a boundary of prevalence of the presence of active thc-metabolites in plasma samples to be less than % among this donor population. standard pharmacokinetics of cannabis metabolism in previous studies indicate a likely time window of less than hours for post-exposure detection of thc and/or -oh-thc in plasma. conclusion: testing of donor plasma samples for active metabolites of cannabis at one urban, hospital-based blood donor center produced no testpositives. statistically, results indicated that prevalence of positivity, if greater than zero, is at most less than %. probability of occurrence of cannabis metabolites in blood donor samples is likely to be highly variable across donor centers and is largely dependent on blood donor demographics. elisabeth maurer-spurej* , ruqayyah almizraq , daniel millar and jason p. acker . university of british columbia, university of alberta, lightintegra technology inc., canadian blood services background/case studies: the controversy around the quality and clinical impact of aged red blood cell concentrates (rcc) is ongoing. current studies are limited by the lack of quality measures suitable for routine screening of rcc. based on evidence that fragments called microparticles (mp) or extracellular vesicles are markers of cellular activation or degradation, this study investigated the utility of mp screening to characterize the effect of rcc production methods and storage. study design/method: red blood cell concentrates were prepared by whole blood filtration (wbf; top/top) or red cell filtration (rcf; top/bottom) methods, centrifuged to prepare a supernatant and tested for mp content (as measured with dynamic light scattering or a tunable resistive pulse sensing technique), hemolysis, atp and red cell deformability on days , , and of storage. one rcf rcc was tested on days , , , , and and six ml aliquots were stored in parallel and tested on days , , and . all samples were tested for mp content and compared to the other quality indicators. results/finding: mp content showed a linear increase with storage time with statistically significant differences between days , and (p< . ) and correlated with supernatant hemoglobin, and inversely with atp or rbc elasticity. both mp testing methods agreed with respect to total mp content. starting levels of the quality indicators varied between donations, preparation methods (wbf rcc contained much higher levels of mp), and storage time. mp content in the aliquots were consistent at each time point but statistically higher than in the original rcc on and after day of storage. conclusion: mp content correlates with measures of hemolysis and other rbc quality indicators and could be implemented as a routine screening tool. differences in mp content between donors, processes and age could be monitored and used to inform component production decisions. measuring mp content would allow % screening of rcc products in studies and pragmatic qc initiatives which are needed to settle the controversy about the clinical effect of rcc age. single donor spray-dried plasma: the future of plasma therapy? qiyong peter liu*, jihae sohn, ryan c. carney, sruthi sundaram and mark a popovsky. velico medical inc background/case studies: frozen plasma is integral to hemostasis management in many situations but logistically cumbersome because of frozen storage and long thawing time. spray-dried plasma (odp, on demand plasma) is potentially superior because it may be stored under refrigeration near the patient and reconstituted in minutes at the point-of-care. the objective of this study is to determine if odp can be consistently manufactured at a blood center with key proteins and coagulation function comparable to ffp. study design/methods: units of never frozen plasma collected at a blood center were processed on-site at a fixed volume into odp using velico's spray dryer. odp (n ) and paired ffp aliquots were stored for - days at - c and - c, respectively, reconstituted with a fixed volume of rehydration fluid (sterile water for injection), and extensively characterized with respect to the levels of hemostatic proteins, coagulation and complement activation markers, and clotting performance. the volumes of processed plasma and rehydration fluid were pre-determined ensuring similar total protein concentration in reconstituted odp and ffp for direct comparison. results/findings: compared to ffp, odp had ! % levels of functional clotting factors (fibrinogen, factors ii, v, vii, viii, ix, x, xi and xii), plasminogen, and protease inhibitors (antithrombin iii, protein c, protein s; plasmin, c esterase and alpha -proteniase inhibitors). the level of factor xiii in odp was slightly lower, about % of ffp by both activity and antigen assays. odp was identical to ffp in the levels of albumin, immunoglobulins (iga, igg and igm), lipoproteins, calcium, citrate, and coagulation proteins evaluated by antigen assays except for factor xiii. the levels of the markers for coagulation (thrombin-antithrombin, prothrombin fragments i ii and ddimer) and complement (c a and c a) activation in odp remained similar to ffp. odp was equivalent to ffp when assessed by aptt, pt and thrombelastography. transfusion vol. supplement s abstract spray-drying fragmented a substantial number of high molecular weight von willebrand factor (vwf) multimers into smaller ones, leading to a net increase of vwf multimers in odp. the size re-distribution reduced the vwf ristocetin cofactor activity (vwf:rco) to % in odp relative to ffp, but had no impact on vwf antigen and factor viii function (stabilized by vwf). vwf-specific studies have shown that odp retains hemostatic function in supporting platelet adhesion and aggregation (see abstracts by meledeo et al/us army institute of surgical research and bercovitz et al/blood center of wisconsin). conclusion: odp can be manufactured at a blood center with a quality comparable to that of ffp. future studies will determine if the product is bioequivalent to ffp and comparable in safety and efficacy. background/case studies: the collection time of whole blood is, according to european guidelines, limited to minutes. in addition, donations with collection times between and minutes should not be used for preparation of platelet (plt) concentrates (pc) because of the chance of too much activation of plt. it seems justified to re-evaluate the quality of plt from these donations because new generations collection systems and mixers were introduced, including a more efficient needle. the aim of this study was to investigate the in vitro quality of pc prepared from - minutes buffy coats (bc) with the aim to prevent unnecessary discarding of bc and to simplify the total blood bank process. study design/method: single-donor pc (spc, n ) were prepared from one - minutes bc and ml of autologous plasma in a ml pvc-dehp container. as a reference, spc from donations with collection times of < minutes were prepared (n ). in addition, pc were prepared from bc, of which at least bc were from - minutes donations (n ). after pooling of the bc, ml of pas-e was added and a standard pooling set with a pvc-bthc storage container was used for storage of pc. all pc were stored for days at c and sampled at regular intervals for determination of the in vitro quality. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg), using kaolin as an activator, was applied for assessment of the overall clotting capacity. values are expressed as mean sd. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or non-normal distributed data respectively. results/finding: volume ( vs. ml) and platelet content ( vs. x ) were similar in both groups. at the end of storage, both groups showed comparable in vitro quality (day , ph( c): . . vs . . , other data not shown). no differences in aggregation response after stimulation with arachidonic acid, adp or collagen were measured. teg parameters in both groups were also comparable. the five-donor pc fulfilled all requirements of european guidelines, aside from occurrence of small aggregates at day and/or in / pc (possibly because sometimes ab incompatibility was accepted). on day , plt showed low cd p expression ( . . %) and phosphatidylserine exposure (annexin v binding, . . %). hypotonic shock response of platelets was comparable with historical data. conclusion: single-pc in plasma as well as five-donor pc in pas-e, prepared from - minutes whole blood donations had a normal composition and showed good in vitro quality during day storage. to substantiate that the exclusion of - minutes donations for pc preparation could be stopped, further studies will be performed. the effects of a pneumatic tube system on red blood cell units amy mata* , jessie miller , ranee marie wannarka-farlinger , sandra bryant , scott a hammel , sherry stern and camille van buskirk . mayo clinic, mayo clinic rochester background/case studies: the use of pneumatic tube systems (pts) has become commonplace in many healthcare facilities throughout the world. the purpose of these systems is to transport products and specimens, resulting in reduced turnaround time for laboratory testing and to aid in the timely delivery of patient care. a downfall of ptss is that they have the potential to play a role in increased hemolysis. while several studies have been published on the effects of ptss on blood specimens, there are very few that address the effects on blood products, specifically red blood cells (rbc). the objective of this study was twofold: to determine if the pts that is in use at our facility contributes to an increase in hemolysis of rbc units and to evaluate how the pts system affects red cell microparticle (rmp) levels. study design/method: forty-one units of as- rbcs, irradiated and non-irradiated, were selected for the study. the units varied in age, ranging from to days old. specimens were obtained from each unit both prior to and after being transported through the pts, which runs underground and spans the length of a mile and a half. specimens were spun down and the plasma supernatant was removed. all specimens were evaluated for plasma hemoglobin (hgb), potassium (k), hemolysis index (hi), and rmps. the wilcoxon signed-rank test and p value were used to compare the pre and post values. additional statistical analysis was performed to compare the values after adjusting for age and irradiation. results/finding: after sending the rbc units through the pts, hgb, hi, and rmps were statistically (p< . ) higher than before. when adjusted for irradiation, the same analytes remained statistically higher, however when adjusted for age, the p-value was only significant for hgb and hi. the k values did not significantly change. rmps significantly increased, but only if the units were irradiated (p . ). (table) conclusion: the use of a pts provides an effective means to transport blood products; however, it can contribute to biological changes within rbc units. it is uncertain at this time how those changes can affect the outcome of patients who receive these products. each pts system is different in its specifications and should be validated prior to being used to transport blood products. validation of factor viii levels of thawed fresh frozen plasma after days of storage pei lun karen lim* , erma sofia sumardi , isamar eduardo ancheta , susan lim , christina yip , lip kun tan and shir ying lee . national university hospital singapore, national university hospital, national university hospital, singapore background/case studies: plasma transfusion is indicated in patients with coagulation factor deficiencies and active bleeding, or who are about to undergo an invasive procedure. fresh frozen plasma (ffp) has to be placed in the freezer within hours of processing and stored at - c or colder in order to preserve its coagulation factors. thawed ffp has an expiration period of hours hence to reduce wastage, this study aims to investigate factor viii (fviii) activity in thawed plasma stored for days and kept at to c. fviii was chosen as it is an important coagulation factor in correcting coagulopathies. arbitrary fviii level acceptance limit was set as not less than iu/dl. study design/method: randomly selected units of ffp (n ) were measured for fviii concentration based on clotting assay (stav r -deficient a transfusion vol. supplement s viii diagnostica stago). fviii levels were measured at five time points: prefreezing, , , and hours post-thawing. ffp were thawed using helmer plasma thawer (helmer scientific) at to c for minutes. an aliquot of thawed ffp from each unit was removed and measured for fviii before refrigeration ( hours post-thaw). thawed plasma (tp) units were kept in a refrigerator at to c for days for subsequent testing. results/finding: results obtained were listed in table . units to were not tested for fviii at post thaw- hour due to operational issues. the overall fviii concentration decreased at an average of % from pre-freezing to post thaw hour. after further storage of tp post thaw- hour and - hour, residual fviii level remain to be above iu/dl except unit which had a lower initial fviii concentration. at post thaw- hour, out of units tested had residual fviii activity within the pre-set standard of iu/ dl. the average decline from -hour post-thaw to -hour, -hour and hour post-thaw was . %, . % and . % respectively. there was no observed trend of any blood group having higher or lower pre-freezing fviii and this is likely due to small sample size. conclusion: decrease of coagulation factor such as fviii in ffp is expected due to its diminishing stability. nevertheless, our data showed that majority of the tp retained at least iu/dl of fviii. typically patients with factor levels below iu/dl may start to show abnormal coagulation profile. while tp is not used for specific factor replacement therapy, it may be indicated for patients with general coagulopathies and active bleeding. further study extending to measurement of other labile factor such as fv may add value to the validation study. validation of the pathogen reduction method using amotosalen/ uva: comparing pathogen-reduced pooled prp-platelets and conventional single prp platelets for quality and bacterial inactivation efficacy lubna ahmed almenawi , ayman mohamad sabri , ali abdullah alajeafi , ashwaq hasan alhekri , saleem bin mahfouz , ali hasan alkhodari , rawya saeed shealy , marcus picard-maureau* and hussain bana almalki . king abdulaziz hospital and oncology center, cerus europe bv background/case studies: the growing number of transfusiontransmitted infectious (tti) risks, including emerging and endemic pathogens, is a constant challenge for blood centers in saudi arabia. while for a limited number of these pathogens tti risk can be reduced using blood screening assays, alternative solutions are anticipated. pathogen reduction (pr) technology was identified as a potential solution. validation of amotosalen/uva photochemical treatment in our blood center was performed by comparing the platelet component (pc) quality of the standard "control" single-donor prp-concentrate in % plasma over a day storage period and the new "test" pathogen-reduced, pooled (pools of ) prp pc in % plasma over a day storage period. the efficacy of the bacterial inactivation was also assessed in our setting. study design/method: the quality parameters of leucoreduced test pcs were assessed at day of storage and compared to leucoreduced control pc at day of storage. the test pcs were pathogen-reduced with the intercept blood system (cerus corporation, concord, u.s.a.) at day ; the process was completed by day post-collection. samples were taken daily for quality analysis from test and control pc until day and day , respectively. for bacterial spiking, additional pc were spiked with each receiving ml of mcfarland ($ . x cfu) s. aureus, s. epidermidis, e. coli, p. aeruginosa or s. viridans, respectively, to challenge pr efficacy. results/finding: the average platelet loss in the test pc post pr treatment was . % . , the total average platelet loss at day was . % . . the average platelet loss in the control units at day was . % . . the average ph of the test units at day was . . and in the same range as the control pc, ph . . . glucose concentration in test pc at day ( . . mmol/l) was lower than in the day control units ( . . mmol/l). lactate levels increased during the course of storage; lactate levels at days and were outside the range of the assay (> mmol/l). cultures inoculated with pathogen reduced, bacterially spiked units were negative after days of incubation, in contrast to those inoculated with nonpathogen reduced samples from the control units, which were positive for bacterial growth. conclusion: the quality parameters of the pathogen reduced test pc were within specifications and comparable to the conventional control pc. the high efficacy of bacterial inactivation together with comparable quality parameter values suggests the use of amotosalen/uva pathogen reduction is safe and efficient to enhance pc transfusion safety. keaton charles stoner* , jay srinivasan , jessica poisson and ian welsby . duke university, duke university school of medicine, duke university hospital, duke university medical center background/case studies: the coagulation cascade relies on a complex interaction between proteins known as clotting factors. cryoprecipitate (cryo) is a plasma-derived blood product that contains several of the proteins central to the clotting cascade and is typically used as a fibrinogen replacement in bleeding patients. however, cryo contents tend to be variable, and little quantitative evidence exists regarding the exact therapeutic effect of cryo on coagulation. my study aimed to better characterize cryo for consistency across and within sources in terms of its functional effect on in vitroclot formation. study design/method: the duke proteomics core conducted a semiquantitative liquid chromatography-mass spectrometry/mass spectrometry transfusion developed an in vitromodel for a coagulopathic patient using serial dilutions of pooled normal plasma with saline and then added the equivalent of one, two, and three cryoprecipitate doses. a tissue factor-activated test on the rotemv r delta hemostasis analyzer (extem) was performed on each condition. for each source, dose-response curves for clotting time (ct), alpha angle, and maximum clot formation (mcf) were generated using linear regression models. inter-source unit variability was determined by anova and tukey's hsd post-hoc analysis (rstudio inc.). results/finding: lc-ms/ms identified proteins in cryo; of the most abundant, only fibrinogen was relevant to coagulation. notably, the american red cross (arc) single donor source had the steepest slope for mcf ( . mm/dose), indicating a greater per dose potency than the other sources. the arc single donor source had the highest mean mcf across all dosing levels, but also the highest standard deviations and response variability. the arc single donor source was significantly more potent than the australian source. conclusion: paired with our estimates regarding the variability of clot formation responses to cryo, the quantitative dose-response curves provided in this study for ct, mcf, and alpha angle can provide physicians with more information regarding cryo dosing. future studies that evaluate the therapeutic effect of cryoprecipitate versus fresh frozen plasma or fibrinogen concentrate would be of clinical importance and give us further insight into the relative utility of and dose requirements for cryo to correct dilutional coagulopathy. viral inactivation and enrichment of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers from fresh frozen plasma (ffp)using, "vips plasma, virus inactivation treatment system". background/case studies: the solvent/detergent (sd) process used for plasma can safely inactivate all lipid-enveloped viruses. the method proved effective in the processing of coagulation factor concentrates by disrupting the membranes of lipid-enveloped viruses, cells and most protozoa, while leaving the labile coagulation factors intact. this study is done to assess viral inactivation and, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers enrichment capacity of, "vips plasma, virus inactivation treatment system". study design/method: "vips plasma, virus inactivation treatment system" comprise of interconnected bag system where the s/d reagents are removed by filtration and the final products subjected to bacterial ( Á lm) filtration. cryoprecipitate mini-pools ( ml) were subjected to doublestage s/d viral inactivation, followed by one oil extraction and a filtration on a s/d and phthalate [di( -ethylhexyl) phthalate (dehp)] adsorption device and a Á lm filter. the initial and the final products were compared for visual appearance, blood cell count, factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers. initial and final products were also checked for hiv, hbv, hcv, dengue, malaria and bacterial contaminations. results/finding: our analysis showed that the treated cryoprecipitate were very clear, with negative blood count and the protein content of factor viii, factor xiii, fibrinogen and von willebrand factor (vwf) multimers were well conserved (table ) . kit ensured bacterial sterility (table ) and most importantly, final product was free of hbv, hcv and hiv (table ) . conclusion: it's the first time, "vips plasma, virus inactivation treatment system", is used in south asia for product enrichment and viral inactivation. results showed effective product enrichment and viral inactivation in our conditions. but further investigation is needed to characterize functional activity of the enrich component. irrespective of that the process may offer one additional option to blood establishments for the production of virally inactivated plasma components especially in low income countries. background/case studies: buffy coats (bc) from donors who used pain medication like aspirin and ibuprofen up to days prior to the donation are discarded, because a known side effect of these non-steroidal anti-inflammatory drugs (nsaids) is inhibition of platelet (plt) aggregation. these nsaids inhibit the enzyme cyclooxygenase- , thereby blocking synthesis of thromboxane a from arachidonic acid. however, the quality of platelet concentrates (pc), prepared from this bc is not known. the aim of the study was to investigate the in vitro quality of pc prepared from nsaid-bc and autologous plasma during storage. study design/method: single-donor pc (spc, n ) were prepared from a nsaid-bc and ml of autologous plasma. information about the type of pain medication was extracted from the anamneses form. the spc were stored for days at c and sampled at regular intervals. aggregation tests were performed with chronolog (adp or collagen) and multiplate (arachidonic acid) aggregometers. thromboelastography (teg, kaolin) was applied for assessment of the overall clotting capacity. spc in plasma from normal controls (n ) were investigated as a reference. values are expressed as mean sd or as median & iqr. a non-paired t-test or a mann-whitney u test was applied for statistical analyses of normal or nonnormal distributed data respectively. results/finding: volume ( vs. ml) and plt content ( vs. x ) were similar in both groups. on day , both groups showed comparable ph and changes in plt content (data not shown). phosphatidylserine exposure on day was significant higher in a subset of donors who had used ibuprofen (n ). aggregation tests with arachidonic acid revealed in general a low or absent response for spc with aspirin ( , - , p< . ), diclofenac ( , - ) and naproxen ( , - , p< . ), compared to normal controls ( , . no differences were detected in aggregation with adp or collagen. with teg, slightly longer r-times (initiation phase) were measured on day in spc with aspirin, diclofenac and naproxen, compared to the normal controls (only significant for naproxen). these differences disappeared during storage. conclusion: storage properties of spc prepared from nsaid-bc were comparable with spc from normal controls. main differences were observed in aggregation and coagulation properties for donors who used aspirin, diclofenac or naproxen. plt from donors who used ibuprofen showed little or no deviations. this is most likely caused by the fast (< hour) disappearance of ibuprofen from the blood circulation and the reversible binding to plt. the use of bc from donors who used ibuprofen will be further investigated in a 'worst case' (pc in plasma) and 'best case' (pc in additive solution) scenario. the effects of ibuprofen on aggregation and coagulation properties will be further investigated in a dose-response study design adding different levels of ibuprofen to plt. background/case studies: previously it was shown that donors could be classified as having platelets (plt) with good, average or poor storage properties [bontekoe, transfusion, ] . a main difference between 'good' and 'poor' storage properties involved metabolic activity, resulting in a faster decline of ph during storage of 'poor' plt concentrates (pc). this might be caused by a different functionality of the plt mitochondria and there are indications that donors with a history of 'poor' pcs are more likely to have health issues, pointing towards metabolic syndrome and type diabetes (t d). because of the strong rise of people with t d in the dutch population, the aim of this study was to characterize plt from whole blood donors diagnosed for t d, but accepted as donor. study design/method: twelve whole blood donors with t d, not using insulin, were selected and buffy coat (bc) and plasma were, after overnight hold, used for preparation of a single-donor pc (spc). an equivalent number of spc was prepared from age and sex matched control donors, derived from the same collection sessions. spc were stored for days at c and sampled on day , or and . the diabetic marker hba c was determined in red cells and cholesterol and triglyceride levels in plasma. from both groups 'good' (ph day > . ) and 'poor' (ph day < . ) storing spc were selected and analysed in more detail. results/finding: donors were of age year and primarily men ( %). donors with t d had a higher mean bmi ( . . vs. . . kg/m ) and higher hba c than controls. the spc of both groups had the same volume ( vs ml) and plt content ( vs x ) but on day glucose concentration was higher in the diabetic group ( . . vs . . mm, p< . ). on day , the average in vitro quality was comparable in both groups (data not shown). when combining a transfusion vol. supplement s the selected 'good' and 'poor' storing plt from both groups, a large difference in lactate production was observed ( . . vs . . mmol/ day/ plt). the 'poor' plt showed a faster decline of the mitochondrial membrane potential (as measured with jc- ) during storage than 'good' plt. remarkably, a difference in triglyceride levels was detected on day ('poor': . . vs 'good': . . , p< . ). conclusion: bc from donors with t d who did not use insulin and fulfilled all donor criteria, were comparable with bc from age and sex matched controls, and seem suitable for preparation of pc. when selecting the 'good' and 'poor' storing plt from the combined groups, the results of our previous study were confirmed, with significant differences in glycolysis rate and functionality of mitochondria. metabolic syndrome and t d are still suspected as health issues involved in 'poor' storage of plt because donors were of high mean age and because of the observed differences in triglyceride levels between 'good' and 'poor' stored pcs. whole blood leukoreduction failures --following manufacturer's instructions may not be enough karen klinker*, nancy m. dunbar and zbigniew m. szczepiorkowski. background/case studies: our hospital based blood donor program uses a blood collection system which leukoreduces the unit at room temperature prior to centrifugation. the manufacturer recommends minimum wait time of minutes prior to filtration. anecdotally, the vendor states waiting an hour improves the leukoreduction. we experienced leukoreduction failures in january and february of detected by our routine qc. we initiated an investigation as to the cause of these unexpected failures. study design/method: for each of the leukoreduction failures, the following factors were analyzed: collection time, length of filtration, length of wait time prior to filtration, platelet count, staff performing the process, the lot number of the collection system bag, and whether or not units collected from the same donor failed leukoreduction in the past. hemoglobin s determinations were not sought out as no repeat donor failures were noted and our donor population would suggest a minimal number of donors would be found to be hemoglobin s positive. results/finding: a relationship was established between the length of time the product rested or waited prior to filtration and leukoreduction failure. we found that shorter wait times increased the percentage of leukoreduction failures (see table ). all units that failed had wait times less than one hour. a similar trend was noticed for the previous year. the investigation showed no relationship between length of collection time, or the length of filtration time and leukoreduction failure. staff performing the filtration was ruled out as possible cause as the failures were spread out among numerous personnel and observation of their technique displayed no sample collection issues. platelet counts on the donors involved were available and none were outside of the normal range. various lot numbers of the collection sets were involved, and no donors were repeat failures. conclusion: in our small study, we found that following manufacturer's recommendations for the resting or wait time prior to filtration was insufficient to avoid excessive leukoreduction failures. we extended our minimum wait time to minutes based on our data. we have not experienced any leukoreduction failures after this change. absolute immature platelet count in diagnostic algorithm and management of pediatric thrombotic microangiopathy hamza n gokozan* , , katharine a downes , , hollie m reeves , and robert w maitta , . case western reserve university school of medicine, university hospitals cleveland medical center background/case studies: prior studies highlighted the utility of absolute immature platelet count (a-ipc) and a-ipc ratio once therapeutic plasma exchange (tpe) is initiated to differentiate thrombotic thrombocytopenic purpura (ttp) from other thrombotic microangiopathies. this can be helpful to determine those who may benefit from prompt initiation of tpe when tests such as adamts are not readily available. we report a young pediatric patient presenting with diarrhea in the setting of laboratory results suggestive of a microangiopathic thrombocytopenia suspicious for ttp in which a-ipc measurement was clinically useful. study design/methods: previously healthy month old unvaccinated girl presented with history of diarrhea for days which was bloody at onset, accompanied by fever and dehydration. laboratory results showed: white blood cell count: x /l, platelets: x /l, bun: mg/dl, creatinine: . mg/dl, lactate dehydrogenase u/l. hospital course was complicated by tonicclonic seizure episodes that stopped with anti-convulsants and acute kidney injury requiring hemodialysis. peripheral blood smear revealed schistocytes. on third day of hospitalization, platelet count decreased to x /l, adamts sample was sent out and tpe was initiated for clinical suspicion of ttp versus hemolytic uremic syndrome, atypical versus shiga-toxin mediated. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/findings: platelet count began to increase prior to tpe initiation ( x /l and a-ipc of . x /l). two consecutive tpe were completed which resulted in a platelet count decrease to x /l and a-ipc of . x /l. a-ipc ratio was . below the ratio of which has been reported for ttp patients. similarly a-ipc count was not below x /l threshold reported in setting of ttp with severe adamts deficiency. at this time stool culture obtained prior to start of tpe came back positive for e. coli o :h toxin. testing of c , c , factor h, factor h autoantibody, factor i and factor b were normal. adamts activity was %. patient was treated for the infection and platelet count improved within days to x /l, with resolution of her renal failure: bun: mg/dl, creatinine: . mg/dl. no additional seizures were observed during follow-up. conclusion: measurement of a-ipc can be used to aid clinical decisions in pediatric patients suspected of ttp especially when adamts testing and those for other etiologies are still pending. tpe did not seem to have a significant effect in a-ipc but decreased platelet counts in this patient. a-ipc is rapid to obtain and can provide helpful information in the setting of potentially overlapping etiologies in the setting of other testing with longer turnaround time. background/case studies: thrombotic thrombocytopenic purpura (ttp) is a thrombotic microangiopathy characterized by low adamts activity. many patients with severe autoantibody-mediated adamts deficiency at initial disease presentation may suffer from one or more recurrent episodes over the following months or years. it is unclear if disease course and characteristics of recurrent/relapsed ttp may be different from that seen at initial presentation. since absolute immature platelet counts (a-ipc) have been shown to be useful in the diagnosis and to follow response to therapy of ttp patients, we proceeded to evaluate if a-ipc pattern was different in relapsed verse initial presentation. study design/methods: our study cohort consisted of three patients (two female and one male) with acquired ttp (adamts activity < %) who underwent daily therapeutic plasma exchange (tpe). clinical course and laboratory values were reviewed. platelet count (plt), immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were analyzed during treatment course. a-ipc values at presentation and peak, a-ipc peak time (days), and plt count recovery time (days) were compared between initial onset and relapse episode for each patient. a-ipc percent change in relapse episodes compared to initial presentation was calculated. results/findings: all patients had an increased %-ipf, and decreased a-ipc and plt count at presentation in both initial and recurrent episodes. once tpe treatment was initiated, a-ipc rapidly increased and reached a peak value - days prior to plt count recovery, consistent with that previously described in ttp patients. however, compared to first onset, recurrent episodes featured lower a-ipc at presentation (results shown as percent decrease, column ), increased peak a-ipc value (results shown as percent increase, column ), delayed a-ipc peak, and delayed plt recovery (table ) . moreover, recurrent episodes required more procedures compared to initial presentation (table ) . conclusion: recurrent/relapsed ttp demonstrate lower a-ipc at presentation and a delayed and increased a-ipc peak value in response to tpe compared to initial presentation. a longer treatment course was observed in recurrent patients. future studies of more relapsed ttp patients are needed. donors undergoing frequent plateletpheresis and its effect on the hematological parameters sweta nayak*, poonam coshic and r.m pandey. all india institute of medical sciences background/case studies: frequent plateletpheresis donors are assets for the blood banks. the well-being of these donors has been a matter of concern. in our study we intend to analyze the effect of plateletpheresis on the hematological parameters of these donors assessed prior to each subsequent procedure. we also try to compare the effect cell separators used for plateletpheresis on the post donation hematological parameters. study design/method: the study was conducted during february to march on all the repeat plateletpheresis donors coming to the department of transfusion medicine for the nd time within a month of the first plateletpheresis. the values of the hematological parameters including red cell and platelet indices tested prior to each plateletpheresis were entered into the excel sheet and gap between each donations were calculated. the plateletpheresis were done either on hemonetics mcs separator (hemonetics corporation, braintree, massachusetts, usa), fresinius separator (com.tec), dn (fresinius hemocare gmbh, bad homburg v.d.h, germany) and gambro trima accel, software version . after taking consent from the donors. the target collection of each procedure was a dose of x platelets in - ml of plasma. to compare the effect of the cell separators on the hematological parameters due to the plateletpheresis, parameters at consecutive donations within days were considered. data was analyzed by stata . within change in the continuous variables were assessed by paired t-test and between two groups comparison was done by independent t-test or wilcoxon rank sum test. the comparison among the cell separators was done by kruskal-wallis test or one way anova. results/finding: of the donors, repeated the plateletpheresis within a week (group i) and underwent nd plateletpheresis within - days (group ii). no significant alteration was found in the red cell or the platelet indices within either group but a significant difference in the variation of platelet counts of the groups (p . ). though above the eligibility cutoff of . lakhs/ml, platelet counts were lower than baseline in group i donors whereas it was higher at nd plateletpheresis in group ii donors. there were donors who presented to us for the rd time for plateletpheresis with a mean gap between st and rd plateletpheresis being days. no significant difference in the parameters assessed prior to any of the plateletpheresis was found except the platelet distribution width (p . ). plateletpheresis through all the cell separators had similar effects on the hematological parameters. conclusion: there was no significant change in the hematological parameters in the plateletpheresis donors who underwent frequent plateletpheresis. post donation follow-up hematological parameters were not affected by the cell separators used for plateletpheresis. efficacy of therapeutic plasma exchange on angiotensin ii type receptor antibodies in two kidney transplant recipients chisa yamada*, silas p. norman, milagros samaniego and laura cooling. background/case studies: some kidney transplant recipients develop antibody mediated rejection (amr) without detected hla donor specific antibodies (dsas) in sera. in recent years, angiotensin ii type- receptor antibody (at rab) has been reported to cause amr, especially refractory amr, possibly by contraction of renal arteries. at our institution, therapeutic plasma exchange (tpe) followed by ivig every other day has been applied to reduce at rabs in kidney transplant recipients, and we here report efficacy of tpe treatments in two cases. study design/methods: two kidney transplant recipients who received tpe treatment followed by ivig to decreased at r ab are reviewed. results/findings: case : the patient is a currently -year-old female with focal segmental glomerulosclerosis who received her first kidney transplant from a living related donor at age , and a second deceased donor transplant due to a rejection of the transplanted kidney at age . three years post-transplant, her creatinine (cr) started to rise from . to . mg/dl and a biopsy showed banff criteria grade amr, grade a t-cell mediated rejection (tcmr) and grade interstitial fibrosis and tubular atrophy. hla dsa had been negative in serum, but high level at rab was identified at > u/ ml (high: > u/ml, intermediate: - u/ml, negative: < u/ml). she received tpe treatments every other day and started losartan. after a course of tpe, at rab decreased to u/ml and histology showed improvement of amr and tcmr, however, cr kept increasing slowly to . ml/dl. in one month, her at rab increased again to > u/ml, therefore, she received more tpe treatments with a decrease in her at rab to u/ml. although at rab level increased slightly to u/ml after months, her cr has been stable at . - . ml/dl. case : the patient is a -year-old mean /-se - . /- . % * . % /- . %* * p< . a female with malignant hypertension who received a deceased donor kidney transplant at age . her cr started to rise weeks post-transplant from . to . mg/dl without detectable hla dsa. although biopsy showed no amr or tcmr, there was focally severe arteriopathy. she was found to have high at rab level at u/ml. she received tpe procedures every other day and at rab decreased to u/ml with a decrease of cr to . mg/dl and improved arteriopathy in histology. because her at rab level slightly increased to u/ml over the next weeks, she started weekly tpe treatment. after weekly tpe, tpe treatment was stopped because her at rab level remained relatively unchanged. her cr has been stable at around . ml/dl to date. conclusion: we present kidney transplant recipients who received tpe treatments for high at rab levels. a course of tpe procedures followed by ivig every other day was effective to decrease at rab levels; however, weekly tpe had no effect on reducing at rabs. tpe treatment may be also beneficial to improve histological amr and clinical kidney function. experience in management of thyroid storm by plasmapheresis tatiana belousova*, vanya jaitly, brian castillo, hlaing tint, kimberly klein and yu bai. university of texas health sciences center at houston background/case studies: thyroid storm (ts) is an extreme manifestation of thyrotoxicosis that is a serious complication occurring primarily in patients with graves' disease. clinically they may present with a wide range of hypermetabolic symptoms which may be fatal if not managed appropriately. we report two cases where ts with severe cardiac complications was managed by plasmapheresis (plex) with excellent effect. study design/method: a year old man (patient a) with a medical history of hyperthyroidism present with ts complicated with cardiogenic shock [ejection fraction (ef) < %], renal and hepatic dysfunction as well as coagulopathy. patient was persistent tachycardic while being intubated, sedated and requiring tandem heart support. a year old man (patient b) with a medical history of hypothyroidism (on synthroid for years), end stage renal disease and non-ischemic cardiomyopathy (ef of - %) presented for evaluation of dual kidney-heart transplant. he subsequently developed ts with multiorgan failure. standard steroid medication treatment showed little response. results/finding: both patients underwent urgent plex along with standard medication administration as soon as the clinical suspicion of thyroid storm was raised. a - . plasma volume, iso-volumic procedure using fresh frozen plasma as replacement was performed in the intensive care unit where the procedure associated hemodynamic impact could be easily managed. both patients showed significant clinical improvement within hours of the procedure completion. their total t , t and free t levels trended to normal or near normal range within hours (table) . in addition, the plex effect on hormone and the associated antibody removal seemed remained and no "rebound" phenomenon was observed in both cases, making repeated plex unnecessary. both patients had total thyroidectomy - weeks after the event with great clinical outcome. conclusion: our cases demonstrate that plex is a safe, effective treatment option in managing ts patient with severe cardiac dysfunction. the procedure can not only lead rapid decrease in thyroid hormone and its associated antibody levels, but also lessen the severity of tissue injury by moderating the inflammatory process and correcting complications. extracorporeal photopheresis in s ezary syndrome treatment: hospital-based blood bank experience sandra ortega s anchez* , laura martínez molina , cristina muniesa montserrat , octavio servitje bedate , silvia cosano navarro and maria isabel gonz alez medina . banc de sang i teixits, dermatology service. background/case studies: extracorporeal photopheresis (ecp) is an immunomodulatory therapy widely used since years in cutaneous t cell lymphoma, several autoimmune diseases and organ transplant rejection, and in the last years, also used in graft versus host disease treatment. the use of ecp in cutaneous t cell lymphoma (ctcl), mycosis fungoides (mf) and s ezary syndrome (ss) in their erytrodermic form are recently categorized by the american society for a pheresis (asfa) , as first line treatment alone or in combination with other therapies, with a strong recommendation: grade ib, category . since mf and ss are incurable diseases current therapies are focus in controlling skin symptoms and minimizing immunosuppression. the objective of this observational study is to assess outcomes of patients diagnosed with ss and compare them in their first evaluation once the th procedure is been performed. study design/method: ecp is a leukapheresis-based therapy, ex vivo exposition to a photosensitizer drug ( -methoxypsoralen, -mop) and uva light, and subsequent reinfusion of the treated cells which are now induced to apoptosis. volume treated varies from . to total body volume (tbv) and the schedule for ss disease is one cycle (two daily ecp procedures) twice per month. the venous access was peripheral in all cases except in where central catheter was needed. the procedures were performed with optia or amicus devices for the aphaeresis and external uva irradiation for off-line system (in / patients) and with online system (therakos) just in . main parameters for evaluation were cutaneous response rate, number of s ezary cells, previous treatments, duration of the response and possible complications during ecp treatment. results/finding: global response rate is ' % (partial remission . % and complete remission . % with maintained response). no severe side effects related with the procedure were found. the patient outcomes analyzed are similar to results in published literature. conclusion: cases treated in our hospital confirm the efficacy of ecp in ss treatment, with a good safety profile. another great advantage of ecp is the relative lack of immune suppression. many questions remain still unanswered about ecp: which schedule is the most suitable one, how we must continue or stop when partial or complete remission is achieved; and the number of leukocytes to be treated, as techniques as mini-photopheresis are also getting good results. all these questions and more make prospective studies necessary to be performed. : u/l) requiring transfusions, mild thrombocytopenia ( x /l), acute kidney injury (bun mg/dl, creatinine . mg/dl). by the third hospitalization day hgb improved to g/dl, however with worsening thrombocytopenia ( x /l) that led to clinical concern for ttp. peripheral smear showed many red cell fragments. patient was transfused with platelets day prior to first tpe. immature platelet fraction (%-ipf) and a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. results/finding: four tpe in five days were performed (hospital days - ). platelet count and a-ipc improved to x /l and . x /l respectively just prior to first tpe. response to four tpe led to a decrease in both platelet count ( x /l) and a-ipc . x /l. these dynamics did not resemble those which had been described for ttp patients with adamts deficiency. adamts obtained prior to tpe initiation was resulted at this time and was %. no causative organism or toxin was identified after urine, blood, and stool examination and culture. based on these results, tpe was discontinued which led to an immediate increase in a-ipc ( . x /l) that preceded platelet count increase to x /l three days later when patient was discharged. other laboratory values at this time were ldh of u/l, hgb: . g/dl in the setting of recovery of renal function. conclusion: timely diagnosis of ttp is essential to start of tpe. a-ipc dynamics differ in ttp compared to other thrombotic microangiopathies. in our patient a-ipc failed to improve despite tpe and improved once procedures were discontinued and were followed by increases in platelet counts three days later. when ttp is not the causative etiology, a-ipc can help adjust therapy and lead to clinical improvement. further research is needed to characterize immature platelet dynamics in non-ttp microangiopathies. infection and its role in the clinical course of idiopathic thrombotic thrombocytopenic purpura associated with severe adamts deficiency eiman hussein* and jun teruya . department of clinical pathology, cairo university, texas children's hospital background/case studies: ttp is a life threatening disease, defined by microangiopathic hemolytic anemia, thrombocytopenia and severely deficient adamts . since the introduction of therapeutic plasma exchange (tpe) as a treatment modality for ttp, its prognosis has improved dramatically. nonetheless, some patients may develop relapse or refractoriness, with potentially fatal outcomes. despite the notable progress that has been made with studies that emphasized the pivotal role of adamts , the epidemiology of ttp remains uncertain. previous studies have suggested that many factors appear to influence its pathogenesis. some studies point toward infection as a possible trigger which may contribute to the development and can ultimately influence its clinical course. one of the theories to explain this association is the possible cross reactivity between antibodies targeting infectious pathogens and those directed against adamts . the aim of this study was to prospectively examine the potential association between infection and the clinical outcome in a cohort of patients with idiopathic ttp. study design/method: patients with idiopathic ttp who underwent tpe from january through march were studied. sessions were performed daily until platelets and reticulocytes had been normal, then sessions were gradually tapered. we only included patients with adamts activity of less than %. data on infections that occurred at or within a week prior to the development of ttp were analyzed. results/finding: thirty-two patients were categorized as idiopathic ttp with severe adamts deficiency. eight patients ( %) were associated with suspected bacterial infection. four of the patients ( %) showed acute relapse coincident with bacterial infections. central line associated staphylococcus aureus infections occurred in three patients and acinetobacter urinary tract infection was reported in one patient. one patient had symptoms of respiratory infection before the development of ttp, on his initial as well as his relapsing episode. refractoriness to treatment was demonstrated in patients. it was associated with dental abscess in one patient. the other two were associated with mycoplasma pneumonia. tpe sessions were continued in all refractory patients until their death. conclusion: in patients with idiopathic ttp refractory to conventional treatment, a serious consideration should be given to non-idiopathic causes, particularly the presence of a remote source of infection, which can be an additional triggering factor for their initial and / or recurrent episodes. sandra satoe kayano*, marcos paulo colella, rafaela guerra maciel, ingrid priscila ribeiro paes ferraz and rafael colella. a c camargo cancer center background/case studies: therapeutic leukapheresis (tl) has become an ordinary procedure in low body weight children with cancer, and its use over the time has been replacing exchange transfusion. leukodepletion preceding chemotherapy helps preventing leukostasis and hiperviscosity, and aims to reduce metabolic and renal complications associated with cell lysis. the objective of this study is to evaluate the efficacy and safety of leukapheresis procedure in pediatric patients with less than kilograms using a single apheresis procedure. study design/method: in october and june , two children with possible leukemia were submitted to tl procedure. they were and months old, and weighted , and , kilograms. central venous catheters were placed, and apheresis were performed using a continuous flow apheresis system. the device was primed with ml of abo, rh and kell compatible, leukocyte-reduced, irradiated, % hematocrit packed rbcs, and the anticoagulant used was acd-a plus heparin ( ml of acd-a and , units of heparin), at a blood to anticoagulant ratio of : . a complete blood count was determined before and after apheresis. the room was heated to avoid hypothermia, and ionized calcium was measured every minutes to prevent hypocalcemia. during the collection, changes in blood pressure, oxygen saturation and heart rate were observed. net fluid balance was calculated as the sum of the volume of anticoagulant, cation and nondiverted apheresis prime solutions minus the product volume. when the procedure was completed, the blood that filled the apheresis tubing was discarded. the patients were in the intensive care unit (icu) under the supervision of a pediatric physician and icu nurse who were aware of potential adverse events, and the procedure were performed by two hematology physicians and the nurse practitioner. results/finding: the white blood cell (wbc) in blood was counted immediately before apheresis in both subjects, and were . and . / mm . the formula "collection pump flow , x inlet flow x preapheresis wbc count" was used with the goal of removing up to x leukocytes/ml. a single leukapheresis procedure was performed with total blood volume processed per patient. immediately after the -hour procedures, wbc count were . and . wbc/mm , and -hour post tl, wbc count were respectively . and . /mm . net fluid balance was zero in both procedures, and the patients required no transfusion. conclusion: tl was safe and efficient. experience with leukodepletion in infants is limited, and a procedure in children weighing kg or less needs forethought and a multidisciplinary effort, hence operators need to customize procedures for safe collection. however, despite the potential complications that may occur (placement of adequate vascular access, management of low extracorporeal blood volume, anticoagulant-related toxicity with metabolic and hematologic issues), remains an excellent source for leukoreduction in hematologic malignant diseases. background/case studies: nationwide apheresis registry can give us information on the current status and trend regarding apheresis procedures. data can be compared with other regions to find and understand differences in perspectives, indications, technology, and clinical practice. the korean society for apheresis (ksfa) has launched an online web based registry system for apheresis procedures since . we report the data from the year . study design/method: the registry is consisted of two sub-registries. one addresses the overall aspects of apheresis procedures performed at each institute, and the other is focused on therapeutic plasmapheresis procedures. data is registered by voluntarily participating hospitals in korea. results/finding: a total of , apheresis procedures were performed at hospitals. therapeutic plasmapheresis was the most frequent procedure ( . %) followed by autologous peripheral blood stem cell (pbsc) collection ( . %), allogeneic pbsc collection ( . %), donor leukapheresis ( . %), and therapeutic leukapheresis ( . %). cobe spectra ( . %) and amicus ( . %) were the most widely distributed instruments. centrifugation was the dominant technique ( . %) for therapeutic plasmapheresis. detailed information was given for , therapeutic plasmapheresis procedures performed on patients (some items were not completely filled out). spectra optia ( . %) and cobe spectra ( . %) were the most frequently used instruments for therapeutic plasmapheresis. fresh frozen plasma (ffp) was used most frequently ( . %) as the replacement fluid followed by % albumin ( . %), % albumin ( . %), and % albumin ffp ( . %). most of the procedures were performed for plasma volume ( . %). acd ( . %) and heparin ( . %) were used for anticoagulation. central venous catheter ( . %) was the dominant type of vascular access. major clinical indications were desensitization for abo incompatible renal transplantation ( . %), antibody mediated rejection in renal transplantation ( . %), thrombotic microangiopathy ( . %), desensitization for abo compatible renal transplantation ( . %), neuromyelitis optica spectrum disorders ( . %), and hyperviscosity in monoclonal gammopathies ( . %). adverse reactions were observed in . % of the procedures. allergic reaction ( . %), hypocalcemic symptom ( . %), and hypotension ( . %) were frequently reported. therapeutic effect was achieved in . % of the patients. our apheresis registry has been well run for years. recent data reflects the increase of abo incompatible transplantation in korea. revision and update of the registry planned this year will help us achieve better understanding on the apheresis status of our region. plasma exchange may not always be necessary in patients with severe hypertriglyceridemia and acute pancreatitis. jan c hofmann* and dobri d kiprov. california pacific medical center background/case studies: hypertriglyceridemic pancreatitis (hp) is characterized by severe hypertriglyceridemia (shtg: triglyceride > - mg/dl), acute pancreatitis (ap), and absence of other causes. hp is a potentially fatal complication of acute pancreatitis with an incidence of $ deaths/ , cases/year. complications of shtg include: abdominal pain (nausea/vomiting), acute pancreatitis, hepatosplenomegaly, eruptive xanthomas, lipemia retinalis, memory loss, dementia, and peripheral neuropathy. we report on the effective use of plasma exchange (pe) to treat patients (pts) with hp refractory to conventional medical therapy (lipid-free diet plus pharmaceutical interventions). study design/method: we reviewed the medical records of pts who were diagnosed with hp from january, through january, , and referred for immunotherapy evaluation. / ( %) pts received conventional therapy (ct) and pe (pe group), and / ( %) pts received ct alone (ct group). mean age was years (range - ), and % were female. baseline mean triglyceride level (normal < mg/dl) for pe group was , mg/dl ( , - , ) versus , mg/dl ( , - , ) for ct group. baseline mean lipase level (normal < u/l) for pe group was , u/l ( - , ) versus u/l ( - , ) for ct group. results/finding: all pts were treated with dietary restriction (lipid-free diet, or nothing by mouth) and aggressive lipid lowering protocols involving - medications. / ( %) of pe group and / ( %) of ct group received insulin therapy to manage symptoms (sxs) of hyperglycemia and/or diabetic ketoacidosis. / ( %) of pe group and / ( %) of ct group received heparin therapy to stimulate lipoprotein lipase release. the pe group underwent an average of . pe treatments (txs) (median of , range - daily txs) using % albumin; / ( %) required ffp to treat dilutional coagulopathy. in most cases, we did not perform pe txs when baseline triglyceride levels were < - mg/dl and lipase < - u/l ( . - . x upper limit of normal). mean triglyceride levels after pe txs were , mg/dl ( - , ) for pe group (mean decrease %); mean triglyceride levels after additional hours of ongoing ct were , mg/dl ( - , ) for ct group (mean decrease %). while the pe group achieved a greater mean decrease in triglyceride levels after pe txs (compared to the ct group after hours of ct), both groups experienced marked improvement in clinical sxs of pancreatitis and hyperglycemia (p> . ). limitations of the retrospective cohort study include lack of long-term follow-up. conclusion: this small study adds to the literature which demonstrates that plasma exchange is very effective in rapidly lowering triglyceride levels in pts with acute pancreatitis and hypertriglyceridemia. it suggests that there may be a threshold (or range) of triglyceride and lipase levels below which conventional therapy may be nearly as effective in achieving clinical resolution of symptoms. randomized controlled trials would further elucidate the appropriate use of adjunctive plasma exchange in the setting of hypertriglyceridemic pancreatitis. role of plasma replacement in therapeutic plasma exchange for hypertriglyceridemia: a single patient study geoffrey wool* and angela treml. university of chicago background/case studies: our apheresis service performs chronic therapeutic plasma exchanges (tpe) for a -year-old man with a chronic history of hypertriglyceridemia > mg/dl, diabetes mellitus type ii, and chronic abdominal pain. his abdominal pain is severe and persistent, but there is not overt evidence of chronic pancreatitis on imaging or fecal elastase testing. targeted sequencing has not revealed a pathogenic mutation to explain the patient's hypertriglyceridemia. hypertriglyceridemic pancreatitis is a category iii indication for tpe by asfa guidelines, in a patient unresponsive to optimal medical management. asfa guidelines for this disorder state that "some have used plasma as it contains lipoprotein lipase and could enhance triglyceride (tg) removal. no direct comparisons of replacement fluids have been reported". there are three apheresis physicians on our service and use of partial plasma replacement has been variable. we undertook a retrospective study of the efficacy of partial plasma replacement in this patient. study design/method: we have performed tpe on this patient. we performed a chart review to capture replacement fluid use and pre-and post-tg levels, if drawn. tpe was performed using spectra optia (terumo, lakewood, co) exchanging approximately one plasma volume, using entirely % albumin for exchange fluid ( % albumin procedures) or partial plasma replacement ( - units of thawed plasma). twenty-six tpe had pre-and post-procedure tg values available. we determined the percent tg reduction achieved by the tpe. we also determined the daily rate of tg increase until the next tpe appointment (to assess any long-term effects of plasma preventing tg rebound). significance was assessed by student's t-test (one-tailed, heteroscedastic). results/finding: twelve tpe were performed with partial plasma replacement, while were performed with % albumin replacement. table shows that partial plasma replacement was associated with significantly greater % tg reduction. the rate of subsequent daily tg increase was also lower with partial plasma replacement, but this did not meet significance. one mild allergic reaction has occurred during partial plasma replacement which responded quickly to additional iv diphenhydramine. conclusion: we have performed an ad hoc cross-over study on the efficacy of partial plasma replacement in tpe for hypertriglyceridemia. in this patient without lipoprotein lipase mutations, plasma was significantly associated with improved % tg reduction, but not with prevention of post-tpe tg rebound. safety and efficacy of local albumin replacement for therapeutic plasma exchange phandee watanaboonyongcharoen* , , metha apiwattanakul , sompis santipong , jutaluk jaipian , jettawan siriaksorn and ponlapat rojnuckarin . chulalongkorn university, king chulalongkorn memorial hospital, prasat neurological institute background/case studies: therapeutic plasma exchange (tpe) with albumin replacement has been used to treat a variety of diseases. however, there had been rising cost and supply shortage of imported albumin in our country. to solve the problem, our national blood centre had established a plasma fractionation plant to manufacture plasma derivatives including albumin. the objective of the study was to evaluate the safety and efficacy of local albumin as a replacement for tpe. study design/method: all tpes using local albumin as a replacement from two tertiary care hospitals performed from june through february were included. complete blood count and serum calcium were tested before tpe. serum albumin was tested before and after tpe. local albumin is available as a % solution. before using, it was diluted to a % albumin concentration with normal saline. all the patients were hospitalized and received oral calcium before tpe to prevent hypocalcemia. the adverse effects were recorded. results/finding: the total of tpes in patients were included as shown in the table. neurologic disorders were the most common indication for tpe, followed by autoimmune diseases. the median total plasma volume was , (range , - , ) ml. although the corrected calcium level was low (< mg/dl) in . % ( / ) before the procedure, no clinical manifestation of hypocalcemia was detected. adverse effects were observed during the tpe procedure in patients. the first patient had events of mild symptomatic hypotension. he previously took angiotensin converting enzyme inhibitor. the second patient complained nausea after finishing tpe. all reactions were mild. the incidence of adverse effects was . % ( / ). in , the incidence of tpe adverse effects was . % ( / ) when commercial albumin was used. the difference was not statistically different (p . ). median serum albumin levels pre-tpe and post-tpe were . ( . - . ) and . ( . - . ) g/dl. the increase in serum albumin after tpe was statistically significant (p< . ). eighty-two percent of pre-tpe serum albumin levels were lower than . g/dl explaining the rises of albumin after the procedures. we demonstrated that local albumin was safe and effective in maintaining albumin levels in patients undergoing tpes. safety, efficancy and cost-effectiveness of mononuclear cell collections for autologous immunotherapies: experience from a private outpatient collection facility within the eu markus dettke*. akh vienna university hospital, cyto-care.eu background/case studies: within the eu the collection of mononuclear cells (mnc) as starting source for the manufacturing of autologous cell therapies are mainly performed in hospitals or hospital-associated apheresis centers. we report about the challenges to perform the leukapheresis procedure (la) at a private held medical practice, with specific emphases on safety, cell collection efficiency, and cost-effectiveness. study design/method: we reviewed the records of altogether outpatients who underwent a total of la procedure at cyto-care, a private held medical practice/ certified cell collection facility located in vienna, austria. all patients participated in various industry-sponsored clinical p i-iii trials; the study sponsors were responsible for the manufacturing of the active cell product. disease entities were mainly prostatic cancer ( %) and ovarian cancer ( %). based on differences in the study protocols la was performed either one-time ( %), two-times ( %) or three-times ( %), with an interval of at least weeks between repeated collections. results/finding: all patients successfully completed the apheresis course. because of poor venous access, out of patients ( %) required a shortterm femoral catheter insertion. there were no serious side effects in patients who required a femoral catheter, or in patients with repeated la procedures. side effects of the la procedure mainly consisted on mild hypocalcaemia-related symptoms in % of patients. a follow-up survey one week after completion of the la revealed no infectious complications, and no patient required hospitalization. median cell yield collected per single apheresis was . x wbc consisting of . x mnc. mnc cell yields remained stable even in repeated la collections. all cell products were successful transformed into an active cellular product. analysis of the cost structure showed that the total cost of care was % lower in the setting of a private collection center compared to hospital-based apheresis centers. conclusion: leukapheresis performed in a private medical practice/ certified cell collection facility is safe and effective, with low rates of complications and high levels of patient satisfaction. this service model is costeffective and can help to reduce the cost of manufactured goods in the production of innovative cellular products. although typically associated with monoclonal gammopathies (e.g. waldenstrom's macroglobulinemia and multiple myeloma), hvs has rarely been reported in patients with disorders of immune system such as rheumatoid disease, sjogren's syndrome, hiv and igg -related diseases. therapeutic plasma exchange (tpe) is indicated in hvs due to monoclonal gammopathy (asfa category indication). however, there are limited data for the utility of tpe in hvs due to polyclonal gammopathy. study design/methods: a year old female patient with a medical history significant for seropositive erosive rheumatoid arthritis, hypertension, diabetes mellitus, cutaneous lupus and diffuse parenchymal lung disease, presented to our institution with complaints of progressive fatigue, muscle weakness, poor appetite, headache and epistaxis for a few months. fundoscopic examination showed dilated and tortuous vasculature as well as bilateral retinal hemorrhages (mixed flame-shaped and dot-blot patterns). pertinent laboratory findings included a positive anti-nuclear antibody screen with anti-histone antibodies and anti-ro antibodies. serum rheumatoid factor was markedly elevated to , iu/mls (ref. range < ) and anti-cyclic citrulline peptide antibody was elevated to , units (ref. range < ) . serum protein electrophoresis and immunofixation demonstrated a polyclonal hypergammaglobulinemia; protein precipitates were noted at the point of application, suggestive of circulating immune complexes. serum igg, igm and iga were , and mg/dl respectively. a cryoglobulin screen was negative. serum free kappa to lambda ratio was . . peripheral blood flow cytometry did not identify any monoclonal bcell population. plasma viscosity was noted to be . centipoise (cp) at admission (ref. range . - . ). pet-ct imaging was negative. the patient was treated with high dose steroids; a single tpe procedure was performed using the following parameters: volume treated - total plasma volume; replacement fluid - % albumin and normal saline in a : ratio; replacement fluid volume: % of the total volume processed. the procedure was tolerated without complication. results/findings: immediately post-tpe her plasma viscosity level dropped to . cp. serum igg, igm and iga levels decreased to , and mg/dl respectively. her rf had decreased to , iu/ml. the patient reported subjective improvement in strength. she subsequently received two infusions of rituximab separated by two weeks. her plasma viscosity has remained less than cp since tpe. conclusion: polycolonal gammomathy (e.g. secondary to ra) is a rare cause of hvs. tpe can provide transient relief of symptoms in unusual cases of hvs and may facilitate therapy to prevent recurrent hvs episodes. therapeutic plasma exchange in neuromyelitis optica spectrum disorders -experience from tertiary care centre in north india ratti ram sharma*, rekha hans, satya prakash, naveen sankhyan and neelam marwaha. postgraduate institute of medical education and research background/case studies: neuromyelitis optica spectrum disorder (nmosd) is an idiopathic inflammatory demyelinating disorder of central nervous system preferentially involving optic nerve and upper segments of the spinal cord leading to optic neuritis and myelitis. tpe is indicated in acute phase or as a maintenance therapy to treat or prevent relapses in chronic phase. study design/method: to assess the efficacy of plasma exchange in patients of nmosd not responding to high dose intravenous steroids. we did a retrospective review of tpe records for patients with nmosd over a period of three years (jan -dec ). tpe was done using, cobe spectra (terumo bct, lakewood co. usa), replacing one to one and half patient plasma volume with % human serum albumin or fresh frozen plasma on alternate days. the improvement in clinical signs and symptoms was recorded after each tpe procedure and at the end of the therapy. adverse reactions if any were also recorded results/finding: eleven patients of nmosd between to years age (m: f; : ) underwent tpe procedures with an average of . per patient. all the patients were on high dose immunosuppressant therapy without much clinical improvement. three ( %) patients had only visual symptoms, ( %) had both visual as well as muscular symptoms whereas ( %) patients had muscular symptoms only. three ( %) out of the seven tested, were positive for aqp -igg. all the patients showed significant improvement in their visual symptoms post exchange, from no vision/light perception to finger counting in two patients, recovery of colour vision and diplopia in six patients. post exchange recovery in the muscle power was observed in patients with grade- , in patient, and by grade- , in seven. adverse events were observed in % ( / ) of the procedures with allergic reactions to replacement fluid as most common event (n- ) followed by hypotension (n- ). follow up was available in % ( / ) of patients and are doing well on immunosuppressive therapy. one patient died due to respiratory failure after months and another had relapse for which he underwent second tpe cycle and continue to do well. conclusion: tpe is a safe and effective adjunct therapy to high dose immunosuppression in nmosd. trima accel software upgrade from . to . for platelet collections rachel m beck*, kimberly j duffy, sandra bryant, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: terumobct released trima accel software version . as an enhancement to allow for the collection of platelets (plt) with platelet additive solution (pas) and provide additional improvements to increase overall reliability. additionally, the manufacturer identified a slower centrifuge speed at low draw flow rates. this software was expected to function similarly to version . . the objective of this retrospective study is to identify any variances with the software upgrade influenced the plt products collection process or products collected. study design/methods: prior to / / , plt collections were performed on nine trima accel machines operating with version . . upgrading and validating all nine machines to version . occurred from / / to / / . the trimas were programmed with the same plt configurations both before and after software update. platelet collection data from version . ( / / to / / ) was compared to version . ( / / to / / ). incomplete collections, runs identified as having possible leukocyte contamination, duration of collection, and plt split rate were evaluated for each time period. generalized estimating equations (gee) were used to assess differences between plt collections with version . and . , adjusting for multiple visits per donor, with significance defined as p-value < . . results/findings: following the upgrade to version . , staff observed a number of changes including an increased centrifuge recovery time on a donor with a low flow and a notable increase in possible leukocyte contamination products. version . of the trima accel showed a statistically significant increase in possible leukocyte contamination from % to % of collections as compared with version . . both the duration of collections and the plt split rate remained constant even with centrifuge speed adjustments in version . . conclusion: due to fda limitations not allowing for the implementation of trima accel pas plts with the currently available pathogen reduction system, the institution decided to implement only the pathogen reduction system at this time. subsequently, the version . software is no longer required. with the noted slight increase in possible leukocyte contamination as well as the lack of enhancements for plt collection, the upgrade to version . currently does not provide added value over version . for plt collection. pulmonary and neurologic symptoms due to leukostasis. therapeutic leukocytaphersis (tl) is used as an adjuvant therapeutic modality in these patients with symptoms suggestive of leukostasis. tl procedures are performed using cell separators where anticoagulated blood is subjected to centrifugal force resulting in separate layers of cells and plasma depending on their density. there are two programs in the cell separator, a mononuclear (mnc)program which has greater centrifuge speed and efficiency for the collection of mncs and a polymorphonuclear (pmn)cell program with lower centrifuge speed for the collection of pmns. hydroxyethyl starch(hes) is preferred for the collections of granulocytes for transfusion from healthy donors. use of hes facilitates the sedimentation of the granulocyte layer and increases the efficiency of collection. though use of hes in tl was not associated with adverse events with its use as a volume expander (pagano) its use in tl varies and no reports are available on the efficiency of leukodepletion using hes for tl. study design/method: we received a request for leukoreduction in yearold lady with chronic myelogenous leukemia (cml) who had a good response to imatinib. she is weeks pregnant with an increased wbc count due to the discontinuation of imatinib. we performed tl with the cobe spectra using a replacement fluid of ml % albumin. wbc counts were monitored pre and post tl in the patient and in the collected product. we modified the collection based on these results using the mnc program with acd-a or the pmn program with acd-a . as leukodepetion was not adequate with these programs we elected to use hes after discussion with the patient and her physician. tl was performed using ml of hes with citrate and the pmn program. wbc pre procedure, immediate post procedure and the product was obtained and the efficiency of leukodepletion with the different programs was calculated. results/finding: the efficiency of % wbc depletion was calculated by product wbc to patient wbc based on blood volume and also pre to post wbc the patient tolerated the procedures well and there were no adverse reactions in the patient and in fetal monitoring during the procedures conclusion: therapeutic leukocytapheresis in cml patients is safe and more effective in reducing the wbc count with the use of ml of hydroxyethyl starch with anticoagulant. post procedure patient wbc counts sometimes may not provide the data on the efficiency of leucodepletion. background/case studies: early recognition of hypertriglyceridemia (htg) in the setting of acute pancreatitis (ap) is critical to initiate effective therapy. the role of plasmapheresis as an early/adjuvant approach in acute htg-induced pancreatitis is controversial. currently, there are no consensus guidelines in optimal therapy and is asfa category iii. reported here is a case where the tg level as well as clinical symptoms improved after one therapeutic plasma exchange (tpe). study design/method: a years old male with history of hypertension, htg, and diabetes mellitus (dm) presented to our emergency department with excruciating abdominal pain. the patient was diagnosed with htg at years old. he was treated initially with diet and lifestyle modification. however, his clinical course has been compromised after developing pancreatitis with acute episodes requiring prolong hospital admission of approximately months each which were successfully treated medically. however, the recurrent episodes resulted in chronic pancreatitis which was complicated with pancreatic pseudocyst and pancreatic insufficiency. since the first episode of pancreatitis, he was then medically managed with fenofibrate, lovaza, lisinopril, levemir and novolog. during evaluation on current admission, he was found to have a tg level of mg/dl, lipase u/l, glucose mg/dl, bicarbonate mmol/l, anion gap . ct findings were consistent with ap without evidence of necrosis and stable pancreatic pseudocyst. medical therapy was started with omega fatty acid, fibrate, statin, hydration as well as pain control. statin therapy was suspended on day of hospitalization, because he was noted to have elevated liver function tests (lft) and tpe was requested and started on day after admission. results/finding: the patient tg decreased by % ( mg/dl) with medical therapy, followed by additional % ( mg/dl) after one volume of tpe. his symptoms significantly improved and was discharged with medical treatment on day after admission. compared to previous episodes, his hospital stay was significantly decreased. tg levels remained below mg/dl at days follow up after discharge. conclusion: early tpe may be of value in treating patients with elevated tg associated with recurrent pancreatitis. plasmapheresis might be an effective early adjuvant therapy to mitigate length of hospital stay, improve cost-effectiveness and patient safety. background/case studies: from to , a national blood donor center in southeast asia conducted a program to monitor the ferritin levels of platelet blood donors. the aim of this study was to explore the trend of changes in ferritin. study design/method: in this study, we collected , cases whose ferritin levels have been monitored more than twice with an interval of detection in - days. the collected plasma samples were tested for ferritin by chemiluminescence using a commercial assay. inclusion criteria included apheresis platelet blood donors with over two results of ferritin, and first time ferritin test result was over lg/l. and the upper limit was set to be lg/ l in male and lg/l in female as described in manufactures insert. the impact on ferritin from gender, age, and the blood donation frequency were examined with anova test. the blood donations frequency was categorized into five groups: times, to times, to times, to times and more than times. the high frequency (more than times group) blood donors were analyzed ferritin changes in longitudinal data. results/finding: there were , donors included in the study, of which , were male ( . %) and were female ( . %). the mean ferritin was . lg/l in male ( % ci: . - . lg/l) and . lg/l in female ( % ci: . - . lg/l). the result of anova indicates that the group with the highest frequency (more than times) has the significant lowest ferritin level (p< . ). the average change of ferritin if donation over times would up to . and . lg/l in younger and elder y/o male and and lg/l in female. and then for high frequency (half a year more than times the group of blood donors) for longitudinal analysis and found that the long-term sustained high frequency of blood donation caused a significant decline in ferritin. the average change about ferritin in high frequencies donors (over times in $ days) was reduced from . lg/l in the first period to . lg/l in the third period ( period $ days). along with the more and more period, the decline of ferritin decreased. conclusion: this analysis revealed that frequent apheresis platelet donation would decrease ferritin of donors. but the high frequency of platelet blood donors who continue to donate after a year, the decline of ferritin slowed down. a rare case of blood donation precipitating acute delirium joseph griggs* , mary townsend and lizabeth rosenbaum . university of new mexico hospital, blood systems, inc., blood systems inc. background/case studies: we report a case of whole blood (wb) donation that precipitated a transient agitated delirium. a year-old first time male donor presented to the local blood center, completed the donor health questionnaire, mini-physical exam, and hemoglobin check, and was deemed eligible for blood donation. approximately minutes after an uncomplicated wb donation, the donor had an observed, brief loss of consciousness in the post-donation area. no fall or injury was seen. shortly after regaining consciousness, the donor became agitated, confused, and was not oriented to month or year; was unable to remember the names of friends and family members; was unable to read an analog clock; and had difficulty with word finding. the donor was transported to the local university hospital where he was noted to be combatively delirious and had altered mental status; he had to be forcibly restrained. he ultimately was sedated and intubated, and transferred to the intensive care unit. study design/method: an extensive laboratory investigation was performed including standard hematologic and chemistry panels; serologic and pcr-based studies for multiple organisms including west nile, herpes, hiv, varicella zoster, and syphilis; aerobic and anaerobic blood cultures; and a urine drug screen for multiple drugs of abuse. radiographic imaging was performed including a chest x-ray, and a ct and mri of head and spine. in addition, an eeg was performed. the inpatient neurology and psychiatry services were consulted for this patient. results/finding: after the sedation was discontinued, the patient was successfully extubated and rapidly improved. he completely returned to baseline within hours of onset of the event. laboratory investigation revealed no signs of infectious organisms or evidence of drugs of abuse. radiographic imaging and eeg studies showed no abnormalities. in addition, infectious disease marker testing performed by the blood center laboratory was negative. investigation revealed that the donor was experiencing high levels of stress at school, had an aversion to the sight of blood, and was coerced into donating by his girlfriend and peers. a week following hospital discharge, the blood center medical director contacted the donor by phone; the donor had resumed his normal routine and was attending his graduate level classes. conclusion: to our knowledge, this is the first report of blood donation precipitating a transient acute delirium. at the time of donation, the health status of all potential blood donors is assessed to help ensure the safety of the donor and the recipient. the health questionnaire, physical exam, vital signs, hemoglobin level, and infectious disease testing help to identify overt signs of medical illness that may disqualify a donor. however, routine donor screening does not explicitly evaluate mental health issues, both diagnosed and undiagnosed. although exceedingly rare, this case highlights the limitations of donor screening to identify donors who may be at risk for mental health adverse reactions when donating blood. a targeted approach to increasing the african american blood donor pool arnethea sutton* , william korzun , teresa nadder , susan roseff and elizabeth ripley . virginia commonwealth university, virginia commonwealth university medical center background/case studies: a continuous need for blood products for those who require frequent transfusions, such as individuals with sickle cell disease who could benefit from products collected from african american donors, warrants the need for targeted interventions to increase blood donations from underrepresented populations. one population in particular, african americans, only account for % of blood donors in the united states. literature indicates numerous reasons why this population is underrepresented amongst donors, including fear, lack of knowledge about the blood donation, and specific to this population, lack of trust in the medical community. study design/method: african americans in richmond and norfolk, virginia were recruited through churches and local universities. the study's aims were to develop, implement, and assess a targeted educational approach incorporating the theory of planned behavior and various teaching methods, to develop and implement a survey to evaluate participants' feelings, attitudes, and intent to donate, and to motivate african americans non-donors to attempt to donate blood. participants attended a -hour educational session where they were educated on the importance of red blood cell donations from african americans. participants completed three surveys -one before the session, one directly after the session and one, two months after the session. a two-proportion z-test was used to compare the known proportion of african americans who present to donate in the study areas to those who presented to donate in this study, while regression analysis was used to estimate the relationships among survey variables. results/finding: a total of subjects were included in the data analysis. sixteen percent of the study participants presented to donate as a result of attending the educational session. this resulted in a statistically significantly higher proportion of african americans presenting to donate than the current proportion in the areas of the state where this study was conducted. results from the first two surveys indicated that subjective norm and attitude were significant predictors of one's intent to donate blood, while perceived behavioral control was not a factor. the educational session increased survey scores related to intent to donate in comparison to scores obtained prior to the session. conclusion: this study shows that a targeted educational program can change attitudes toward blood donations in african americans resulting in an increase in new blood donors. additional studies are needed to see if this behavior will continue and whether african americans can influence their community to increase awareness and motivation for life-long blood donation. were from female basic trainees conclusion: the significant increase in hemoglobin deferrals at basic training site a from to could be a result of a change in the blood drive timing of the training schedule of that location. in , basic trainees at site a were scheduled at day of . in january , the blood drive date changed to day of . the extra three days in the basic training atmosphere, and its associated diet changes and increased physical activity may have had an effect on the hemoglobin levels in that population. at basic training site b, the significant increase from to of hemoglobin deferrals can be attributed to a larger male population presenting at this site for basic training. additionally, the percentage of female recruits donating at the blood drives decreased in . these observations support the hypothesis that the increase in hemoglobin deferrals in resulted from the implementation of the male hemoglobin standard change from . to . g/dl at basic training site b. when planning for blood drives at basic training site b, screening of an additional % of recruits must be considered when performing these blood drives, in order to meet the same collection goals set prior the implementation of the change in the male hemoglobin standard. blood donation in the donor with spinal cord injury joan-ramon grífols* , eva alonso , oscar bascuñana , monica romero , teresa vich , elena castaño , laura carbonell , eva palomas , saray almerge , francesc carpio and xavier curia . banc de sang i teixits, institut guttmann background/case studies: donation of blood components (bc) in donors with spinal cord injuries (sci) is poorly studied. paralysis is a state, not a disease, after a reasonable time since its acquisition these people should not be differentiated from the rest of the non-paralytic population in terms of bc donation. the literature reviews of blood donation suitability criteria among these people are scarce and the vegetative lability that they may present depending on the type of their sci it's obvious. in daily practice these potential donors are often rejected for donation with no specific criteria related to their sci. the objectives of this study are to establish the selection criteria for bc donation in people with sci based on medical criteria. to evaluate the rate of adverse donation blood reactions of these donors against a donor control group without sci. study design/method: our organization regularly organizes a donation campaign at a rehabilitation center for patients with sci. in this campaign some donors with sci as donors without (professionals of the center, relatives, etc.) donate blood. from january to december we analyzed the number of donors who came to give blood, the number and reasons for exclusion of those who could not make the donation, whether or not they had sci and number and typology of adverse reactions to the donation detected in both groups. donors with sci higher than t due to the high risk of autonomic dysreflexia were excluded for donation. donors with sci below t and less than one year of evolution were set as temporary exclusion criteria. the presence of neurogenic bladder was not considered a reason for exclusion. results/finding: in the analyzed period, donors came to give blood, of these, ( %) were excluded for donation for various reasons. two of the donors excluded suffered sci higher than t excluding them due their high risk of dysreflexia. another one donor excluded suffered sci lower than t but his hemoglobin levels were lower than our selection criteria. of the donors selected for donation ( . %) had sci lower than t and t . adverse reactions to donation ( . %) were recorded in our haemovigilance program, none of them in donors with sci. conclusion: according to our experience donors with sci lower than t have not had any type of adverse reaction to the blood donation. there should be selection / exclusion criteria based on the donor's paralytic conditions. the vagal syndrome that could appear as a complication to the donation in these sci donors should be approached differently to the usual protocols that we use. blood donor center's experience with changing from manual to automated blood pressures kimberly j duffy*, sandra bryant, audrey e traun, kristine i borth, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: blood pressure (bp) is important for determining the health and suitability of blood donors. the manual method of reading bp can result in variability due to minor variances in the way staff perform the manual procedure. automated bp devices are able to reduce the variability in bp determination. in december of , automated bp devices were validated and replaced the manual bp method in our blood donor center. the objective of this retrospective study is to determine if the change from a manual to an automated bp process has impacted the average systolic and diastolic pressures and, additionally, if a differences in the deferral and reaction rate can be observed. study design/methods: data for the manual bp process was accumulated for an month period from january to november . the same information was assembled for the automated bp process for the month period of january to november . the automated bp process implemented in mid-december ; so the december data for both and has been excluded from the study. bp, bp deferrals, reactions, donor weights and demographics were evaluated for each time period. a donor may be included multiple times in each year and could be in both sets of data. generalized estimating equations were used to assess differences between automated and manual bp with significance defined as p < . . results/findings: significantly more people were deferred using automated bp compared to manual bp readings (p . ). both systolic and diastolic bp measured significantly higher by automated bp method than by manual method. although donors in the automated bp group experienced fewer reactions than those in the manual bp group, the reduction was not large enough to reach statistical significance. even after adjusting for gender, weight and age at donation, bp deferrals, systolic and diastolic bps all remained significantly higher (all p < . ) with the automated bp while and reactions remained non-significantly lower (p . ). conclusion: automated bp devices have improved convenience for both staff and donors. with a statistically significant increase in deferrals and marginal decrease in reactions, the use of automated bp devices may play a minor role in the safety of blood donors. for the purpose of this study, only the hemoglobin values that were below . g/dl will be compared as a surrogate for deferral. to adjust for multiple visits per donor, generalized estimating equations were used to assess significance between lancet a and lancet b, using the appropriate distribution for the data type, defining statistical significance as p-value < . . results/findings: the average hgb was slightly lower with lancet b but there was a larger change with the number of donors under . . statistically more visits with hgb less than . g/dl used lancet b than lancet a. additionally, fewer first time donors were seen during the lancet b time than during the lancet a time. after adjusting for the effects of both gender and first-time donation by using logistic regression, the risk of hgb under . was . % higher with lancet b than with lancet a. conclusion: donor's hgb was slightly lower with lancet b than lancet a, but not clinically different. slightly more lancet bs were used per visit than lancet as. in addition, more hgb deferrals were obtained using lancet b than lancet a. even after adjusting for the effects of gender and repeat donors, we saw more potential deferrals with lancet b than lancet a. the slight difference in the gauge of the lancet may have some association to free-flowing amount of blood and may affect hgb levels. prior to implementing materials at a lower cost, an evaluation of downstream consequences would be recommended. blood donors' acceptance and response towards implementation of automatic appointment booking yi lin ang*, ching lian toh and william choon hong sim. health science authority background/case studies: with surges in demand for blood due to an aging population and more hospitals being built, it is becoming increasingly important to be able to ensure that donors return on a regular basis to improve blood supply and blood stock management. disliking the obligation imposed by appointments, singaporean donors generally prefer "walk-ins" as opposed to appointment bookings. blood services group (bsg) singapore, has made a move to change donors' mindset by introducing automatic appointment scheduling. this paper aims to study donors' level of acceptance towards this initiative. study design/method: to determine the donors' acceptance rate, data was collected from january to march . after completing their donation, donors were automatically given the next earliest eligible date for their next donation. those who do not wish to accept the recommended appointment can either decline this arrangement or log into the blood bank's donor appointment booking system (donor-care) to make changes to the appointment offered. a reminder will be sent to their phone via sms and/or email to their account three days before the appointment date. data was collected from donor-care and was used to measure the number of appointments made and declined over the three months period. donors who declined appointment scheduling were verbally interviewed for their reasons. results/finding: a total of donors who has donated blood in the blood bank's main branch were used as the baseline for this study. % of donors (n ) accepted automatic appointment booking, whereas some donors (n ) were not comfortable with it. % of those who declined still preferred walk-ins (n ) based on their own time schedule, the rest decided that variable situations (n ), donation frequency (n ) and choice of preferred donation locations (n ) were reasons for declining automatic appointment booking. prior implementation of appointment booking at other blood bank branches showed that donors who booked appointment through donor-care was %. a comparison was made and found that this study shown a significant increase of acceptance rate by %. conclusion: generally, the results were positive and the automatic appointment booking system enabled bsg to predict donor attendance, ensure better manpower management to reduce donor turnaround time and thus hopefully improve donor retention. bsg is still monitoring this automatic appointment system and future study are still required to determine the effectiveness of automatic appointment booking, donor return and retention rate. currently bsg has collection centers, each managing its own appointment system. the eventual aim is to be able to have a centralized appointment booking system whereby donors can book appointments and still be able to donate at any collection site. ) , . . poisson distribution, normal distribution, logistic distribution, lognormal distribution a transfusion (p> . ) in donor and reference populations except in younger ( - yrs) male donors (p< . ; donor . %, reference . %). mean donor sbp, dbp, and pulse were . mmhg, . . mmhg, and . . bpm, respectively. screening blood pressure levels consistent with hypertension ( . % male; . % female) in the - year donor group, significantly (p< . ) higher than the reference population ( . % male; . % female). no differences were observed in the - year groups. conclusion: normal source donor demographic and physiologic characteristics often paralleled those of the reference usa populations. however there were differences including lower cholesterol levels and a higher rate of high blood pressure in younger donors and higher weights in - year old females. developing blood donor educational materials gay wehrli* , susan rossmann , louis m. katz and dan a waxman . university of virginia health system, gulf coast regional blood center -sugar land, americas blood centers, indiana blood center background/case studies: donors must have sufficient information to make a decision, time to consider options before making a decision and an opportunity to make a choice of whether to proceed with or decline donating. donor education (de) materials must address mandates set forth by regulatory agencies. these materials must be accessible and understandable by the general population. the goal of this non-experimental, qualitative design study was to evaluate knowledge acquired through standardized de materials. this study was irb approved as an exempt protocol. study design/method: we developed a de document written at an th grade comprehension level. a convenience sample of volunteers was identified for this two-part study. a focus group (fg) incorporated a pre-and post-quiz for knowledge acquisition from reading the four-page de document. the quiz was followed by a group discussion for feedback. the preand post-quiz contained the same multiple choice questions with single best answers including the option to answer, "i don't know." the de document was revised based upon the fg feedback and quiz results. the revised, . page, de document was then tested using the same pre-and post-quiz during individual interviews (ii). results/finding: demographics and quiz results are summarized in table . results from the fg and ii revealed a lack of knowledge in four areas: a donor might be asked not to donate at any time during the donation process, the need for photo identification to donate, iron helps increase a low red blood cell level, and not to donate for the sole purpose to obtain hiv testing. post-quizzes from the ii group revealed an improvement in knowledge acquisition for all four areas. feedback from both groups reiterated that the document was too long. conclusion: developing de materials requires a complicated balance of providing critical information, concisely and at an appropriate comprehension level ( th grade). testing de materials is an essential step in the development process to ensure the intended knowledge is acquired by the end user population. the next steps for this group will be to pilot the further revised, two-page de document at donation sites. effect analysis of the 'rh(-) blood supply program' establishment hyesung han*, deokja oh, buja hur and chulyong kim. korean red cross blood services background/case studies: the rh(-) blood supply program was developed in for the purpose of prompt and stable blood supply. based on the computerized system, the program operates the emergency contact/ communication. this program has major functions such as the request of the emergency blood, the recruitment and management of the rh(-) blood donors for the emergency blood donation, real-time blood supply status monitoring program and statistics program. the aim of the research is to validate the effect of rh(-) blood supply program operations and the responsiveness of the emergency blood supply under the rh(-) blood supply program. study design/method: researchers investigated the database from to after the rh(-) blood supply program was developed. investigators analyzed and compared the recruitment and blood donation of the rh(-) blood donors for the emergency blood donation and securing the blood supply upon request. results/finding: the data shows that the number of voluntary blood donors who pledge to give blood for the emergency blood donation has increased from . % to . % in and , respectively. also, the actual participation rate of rh(-) blood donations among the group who pledge to give blood for the emergency blood donation has increased from . % in to % in . moreover, the data has indicated that the blood supply has fully met the demand for the emergency blood request. conclusion: the result showed that the rh(-) blood supply program was effective for the recruitment/management of the rh(-) blood donors for the emergency blood donation. this system contributes to recruiting and managing rh(-) blood donors who pledge to donate blood and securing rh(-) blood in emergency situation . the institution that needs to meet the demand of rare blood type could possibly use the rh(-) blood supply program which leads to securing special type blood. hanwei chen*. wuhan blood center background/case studies: in china, volunteer blood donors can donate platelets by apheresis (ap) up to times per year. however, the awareness and knowledge of ap donation is much lower than whole blood donation among the chinese population. there are approximately . million doses of ap transfused within . billion people each year in china; it is one challenge to recruit new ap donors and retention them as frequency ap donors in china. study design/method: one stratified recruitment and retention strategy established and applicate at wuhan blood center since . firstly, "one-to-one" telephoning model for whole blood donors instead to donate platelet; secondly, group message for permanent ap donors and had not donated with an interval of more than days in low inventory. thirdly, specific recruiter telephone for those ap donors who had donated aps for more than times and had not donated for more than days or less than times with an interval of more than days from the last donation; the last one is preparing one letter of thanks for those ap donors who gave more than times annually which advise them to voluntarily come to the blood center for ap donation when they were available. results/finding: over the past decade, the overall donation time of ap donors increased by . times from to and the doses of ap increased by . times from to within years. the aps collected fulfilled the clinical needs. according to the donation frequency, ap donors were divided into groups: those who donated ap once, those who donated - times, - times, - times, and those who donated more than times, respectively. it was found that the number of permanent ap donors who donated ap more than times was only ( . %), but they denoted a total of doses of ap ( . %) from to . conclusion: aps increased at a rapid and steady pace in wuhan blood center from to , which not only met the clinical needs but also were supplied to other region outside wuhan. and in addition, the permanent ap donors who gained more attention donated the greatest percentage of platelets. in conclusion, stratified recruitment is one effective approaches to meet clinical needs for platelets and worth to popularize to other region. years were evaluated at sites on consecutive donations for finger stick (fs) hemoglobin (hb) per site policy. venous (ven) and capillary (cap) zpp and ven ferritin (fer) were performed per manufacturers' direction. donors were assessed for subclinical iron deficiency using ranges (fer < ng/ml and zpp levels > umol/mol heme) at hb levels. participants completed an online survey between donations to collect data on symptoms of anemia. univariate linear regression analysis was used to determine relationship between tests. results/finding: subclinical iron deficiency was present among first-time and repeat blood donors at all hb levels with both genders and all age groups. (table) there was a highly significant correlation between fs zpp and ven zpp . % (r . ) at first and . % (r . ) at second donations. at first donation when compared to fs hb, only . % (r . ) of variation could be explained by variation in fs zpp, . % (r . ) by ven zpp and . % (r . ) by ven fer. at second donation, when compared to fs hb, only % (r . ) of variation could be explained by variation in fs zpp, . % (r . ) by ven zpp and . % (r . ) by ven fer. for each donation, variation among tests (fs hb, ven fer, ven zpp and fs zpp) was significant (p< . ) suggesting strong evidence against correlation. % ( ) responded to the survey of which % ( ) reported not feeling well after donation. it should be noted that noted that % ( ) female study participants reported feeling unwell after the first donation and had ferritin levels below ng/ml but the zpp levels were less than umol/mol heme. of the % ( ) male participants that reported not feeling well none had ferritin levels below ng/ml nor ven or fs zpp levels above umol/mol heme. conclusion: subclinical iron deficiency was present at all hemoglobin levels. there was insufficient correlation with fs hb and ven fer to support use of fs or ven zpp analysis as measurement of iron stores for blood donors. symptoms reported by study participants were not consistent with laboratory results. the minimum male hb was raised from . to . gm/dl. fda imposed specific vs ranges for acceptable pulse (p) and blood pressure (bp), removing center-by-center discretion. a survey of members of america's blood centers (abc) was performed to assess the impact on donor deferrals resulting from these changes. study design/method: online survey software (surveygizmo, boulder, co) was used to solicit collections and deferral information from blood centers over two intervals, july-dec. and july-dec. (i.e., before and after the implementation deadline for the final rule respectively). information on deferral at presentations for whole blood (wb) donations and apheresis platelet (ap) donations was requested for hb thresholds and vs. the information was stratified by gender (male m, female f), and abo type. statistical analysis included t-tests for numerical and chi-square for categorical data (minitab . , chicago il). p <. was considered significant. results/findings: data were provided by of centers invited, representing , , and , , wb donations and , and , ap donations in aggregate during the two intervals respectively. gender and abo distributions appeared representative of the us donor base. among m wb donors the rate of deferral rose from . % to . % in the two intervals among aggregated donation attempts (p<. ), and for m ap from . to . % (p<. ). the mean "by center" deferral rates (table) were similar to that and significant (p<. ). mean by center hb deferral rates among f donations during the two intervals were . and . % (p . ) for wb, . and . % (p . ) for ap, respectively, absent any change in their acceptable hb thresholds. data on vs deferrals were much sparser. for p deferrals, only centers could provide specific high vs. low vs. irregular pulse deferrals; provided only a summary (i.e total pulse deferrals), and could provide none. for bp, provided detail (high vs. low), summary and none. p deferrals increased in the successive intervals among f wb donors from a center mean of . to . % (p . ) and for m wb donors from . to . % (p . ). where details were available, high and irregular pulses were responsible for most of the changes for both genders. bp deferrals were not significantly increased among wb donors, regardless of gender. the data sets and deferral rates re: vs in ap donors were quite small, possibly reflecting culling during their prior donation experience. conclusion: substantial additional donor deferrals attended the increased hb thresholds for m in the final rule, for both wb and ap. changes were more modest among female donors, consistent with the absence of changes in allowable hb levels. modest but significant changes attended more stringent requirements for vs, though data limitations restrict this aspect of the analysis. background/case studies: diabetes mellitus is reaching potentially epidemic proportions in india. given the disease is now highly visible across all sections of society within india, there is now the demand for screening of diabetes and urgent research and intervention -at regional and national levels -to try to mitigate the potentially catastrophic increase in diabetes that is predicted for the upcoming years. due to its ease of use, several studies have found that hba c testing can identify patients in the community who might otherwise go undiagnosed. we took an initiative to find out the incidence of diabetes by random blood sugar (rbs) measurement among indian blood donors and measure the hba c levels among those with rbs > mg/dl study design/methods: a prospective study was done at department of transfusion medicine and department of biochemistry from st march to st march . total of , blood donors were tested for rbs. those with rbs > mg/dl were further tested for hba c by gold standard hplc method using variant ii biorad. blood donors with > mg/dl rbs and hba c > . % were advised to consult a physician for further evaluation. results/findings: of the , donors tested, ( . %) donors showed a rbs of > mg/dl. forty two ( . %) were males and ( . %) females with a mean age of . years ( - years). of these, ( . %) were known case of type-ii diabetes mellitus (dm) on oral medications and were excluded. of the remaining , ( . %) of them had a family history of dm. of these donors, donors did not give a consent for testing for hba c. among the donors tested for hba c levels, ( . %) had hba c > . %. all the donors were counselled and referred to a physician for further management. the overall incidence of donors having dm in the population is . % ( of donors). conclusion: screening for blood glucose level by targeting the blood donors can go a long way in curbing the diabetes burden on the society. incidence of low ferritin levels in regular male blood donors with acceptable hemoglobin levels in singapore ramir alcantara* , hwee huang tan and ai leen ang . health sciences authority blood services group, health sciences authority, blood services group background/case studies: iron deficiency is a known complication of regular blood donation. in order to protect the donor's health and prevent iron deficiency, aabb increased the minimum acceptable hemoglobin level for male whole blood and apheresis donors from . to . g/dl last may . the current minimum acceptable hemoglobin for male donors in singapore is . g/dl. the aim of the study is to determine the incidence of low ferritin levels in regular whole blood and apheresis male blood donors with acceptable borderline hemoglobin levels ( . - . ) and in donors with hemoglobin g/dl and above. study design/method: during a month period, serum ferritin testing was performed on regular male whole blood and regular male apheresis donors who made at least donations in the last two years with an acceptable hemoglobin level. the donors were divided into groups according to donation type and hemoglobin range; group a (whole blood with hemoglobin . - . ) group b (whole blood with hemoglobin ! , group c (apheresis with hemoglobin . - . ) and group d (apheresis with hemoglobin ! ). the serum ferritin levels of the four donor groups were compared and analyzed. a ferritin level below ug/l is considered low and levels below < ug/l are considered having absent iron stores. results/findings: . % of donors in the study have ferritin levels below ug/l. there were more donors with low ferritin in group a compared to group b, % and % respectively (p< . ). in apheresis donors, low ferritin rates were higher in group c donors compared with group d, % and % respectively (p . ). ferritin results for the groups can be seen in table . conclusion: more than half of the donors in the study have low ferritin and of the donors with low ferritin, more than half or . % have absent iron stores. donors with low ferritin were immediately informed of their result, given iron supplements and advised to come back for donation after months or more. since donor health and safety is of paramount importance, measures to limit and prevent iron deficiency in blood donors must be implemented. due to the high incidence of low ferritin levels in whole blood and apheresis donors with hemoglobin . - . g/dl, it is recommended that the minimum hemoglobin level cut off for male blood donors in singapore be increased to . g/dl. other measures to be implemented includes better donor education on the risk of iron deficiency and the need for iron supplementation using our website and social media. background/case studies: safe blood is a crucial and irreplaceable component in the medical management of many diseases. the voluntary nonremunerated blood donation is the ideal sources of quality blood, which forms less than % of the demand of the blood in pakistan. motivation among the youth, particularly students, is essential to make voluntary blood movement more successful. to assess the knowledge, attitude and practice regarding the voluntary blood donation among the young student population of karachi so that an effective approach can be made regarding motivation enrolment of voluntary non remunerated blood donors in future in pakistan study design/method: a cross sectional prospective study was conducted among students from different universities and colleges of karachi. a well-structured and pre-tested questionnaire, in english, was used to access the knowledge, attitudes and practices about voluntary blood donation. a scoring mechanism was used to understand overall knowledge level. obtained data was analyzed. results/finding: the sample population consisted of % male and % female students in the age group of - years. only % of the students have heard about voluntary blood donation and % of the students have given blood once in their lifetime and among them % are blood donors at the moment. % of the participants believed that there is a specific reason why they don't donate blood and % believed that there is a risk involved for the donors, when donating blood. % students wanted to promote voluntary blood donation. fear and lack of awareness on blood donation are the reasons for not donating blood. students gather information about voluntary blood donation from several sources mostly schools, colleges, family and friends. ( ); miscellaneous effects were reported in courses. side effects led to interruption of supplementation in instances. ferritin levels (mgt sd) at entry into the program and at the last visit were . and . . mg/l in participants, vs . . and . . mg/l in controls. the positive impact of iron supplementation on ferritin levels was observed only in those who took ! % of the tablets. ferritin levels< mg/l were found in , % of participants and . % of controls. deferral for low hemoglobin was below % in both groups. conclusion: an iron supplementation program in a drbcd program is feasible.however, when taking into account acceptance to participate and compliance with supplementation, only % of donors obtain full benefit from such a program. using an iron preparation which is better tolerated may increase compliance. background/case studies: hereditary hemochromatosis (hh) patients are permitted to donate blood for the allogeneic blood supply as long as they are eligible for donation under cfr . and the collection is a physician-ordered therapeutic phlebotomy. blood collections establishments do not need an exception or alternative under § . to make a collection under this provision if the requirements set forth in § . (a)( ) are met. the objective is to describe current hh donors and long-term contributions of to our hospital-based donor center and hospital blood supply. study design/method: in , an irb protocol was approved for the enrollment and therapeutic phlebotomy of hh patients/subjects. this required filing an fda variance to permit hh donor blood for use in our allogeneic supply without disease labeling. the frequency of therapeutic bleeds are guided by routine clinical assessment, mcv/hemoglobin, serum ferritin, and transferrin % saturation monitoring. serum ferritin levels of - ng/ ml are targeted for maintenance phlebotomy. operationally, a custom, computerized database application is employed to ease phlebotomy management. results/finding: since inception, the cumulative number of hh subjects enrolled in the hemochromatosis protocol reached , of whom ( %) are c y homozygotes. without active recruitment, accrual rate is about per quarter, with % of subjects qualifying as allogeneic donors. the mean current age is . years, % male, % caucasian. the majority of hh donors ( of an active cohort of ) are in the maintenance phase of therapy with an average of . donations/year and a % deferral rate. over the last years, hh donors contributed approximately - % of the hospital's allogeneic blood supply, averaging whole blood units for transfusion per year. moreover, hh donor's whole blood (wb) donations provided - % of blood for in vitro research at our institution with an average of wb research donations/year. there have been no hh donor-derived transfusion-transmitted infections over years. since / / , with an increase in male hgb deferral threshold to g/dl, there has been only hh male deferral from blood donation. conclusion: a simple, safe system for donor evaluation, phlebotomy management, and transfusion of blood drawn from hh subjects was established. blood donated by hh donors remains an important resource at our hospital. hh donors benefit from careful medical follow-up of their iron status. this mutually beneficial relationship is feasible and sustainable. testing for accuracy of non-invasive blood hemoglobin methodology in a blood donor setting michele walker*, sharon garcia and mythili ram. gulf coast regional blood center background/case studies: the objective of the study was to assess the accuracy of hemoglobin (hb) levels measured on the orsense nbm- non-invasive occlusion spectroscopy device by comparing them to hb levels measured on venous samples with a laboratory hematology analyzer. in addition, the study examined operator ease of use and donor satisfaction with a finger stick-free method. study design/method: study procedures and protocol, including acceptance criteria, were defined in conjunction with the device manufacturer to determine the standard deviation (sd) of the difference between the nbm- non-invasive sample results and the sysmex hematology analyzer venous sample results. staff were provided training on the use of the nbm- non-invasive occlusion spectroscopy device. over a span of days, eligible blood donors, both male and female, were first screened by the nbm- non-invasive occlusion spectroscopy device followed by performance testing utilizing a capillary blood screening method. a venous sample was collected from each of the blood donors for the performance of hb measurement on the sysmex hematology analyzer within - hours of collecting the venous samples. results/finding: the sd of the difference between the nbm- non-invasive sample results and the sysmex hematology analyzer venous sample results was not to exceed . g/dl. the hb measurements obtained from the nbm- and the sysmex hematology analyzer were analyzed using the statistical software minitab and the sd of the difference was reported to be . g/dl. the precision of the nbm- yielded a co-efficient of variation of . g/dl and a standard deviation of . g/dl. conclusion: the operators found the nbm- easy to install, maintain, and operate with minimal training. the nbm- non-invasive occlusion spectroscopy technology showed accurate performance compared with the venous sample results. it was comparable to the capillary finger stick method and deemed suitable for screening donors. donors were satisfied with the process and appreciated the safe, painless methodology. ronel swanevelder , ravi reddy , dhuly chowdhury , don brambilla and edward l. murphy* . sanbs, rti international, ucsf/bsri background/case studies: to maintain an adequate blood supply, south african blood centers need to collect more blood from their majority black african population. success in recruiting first-time black blood donors has been tempered by lower suboptimal return rates. study design/method: we performed a prospective cohort study of firsttime, black blood donors donating during a four-month period in and followed them for one year. within days post donation, a questionnaire including questions on blood donation motivators and deterrents was administered by telephone. questions used -point likert scales to assess agreement with statements relating to domains of altruism, collectivism, selfesteem and marketing derived from local focus groups (muthivhi et al. ) . linking questionnaires to a blood donation database allowed logistic regression analysis to predict return for a second donation within one year. results/finding: we included , first-time black donors with median age and female predominance ( %). within one year, , donors ( %) attempted at least one additional donation. when likert scales were analyzed as an ordinal variable ( strongly agree to strongly disagree), donor return was associated with the following motivators "blood donation is an easy way to make a difference" (odds ratio for each likert increment (or) . , % ci . - . ), "i donated in response to adverts/campaigns on the radio, tv or newspapers" (or . , % ci . - . ). responses to altruism-associated statements were not associated with return. among deterrents, donors were less likely to donate if they agreed with the statement "i am afraid of the sight of blood" (or . , % ci . - . ) and "i wasn't treated well by the <blood center> staff" (or . , % ci . - . ). surprisingly, donors were more likely to return if they agreed with the statement "i was afraid of finding out about my hiv status" (or . , % ci . - . ). a secondary analysis treating the likert scales as -level categorical variables revealed generally similar results, with the additional finding that donors who disagreed with the statements "if i give blood then blood will be available when i need it" and "i don't know where the nearest blood collection point is" were more likely to return. conclusion: this novel design allowed us to study the link between donation motivators and deterrents and actual rather than intended return for donation. it is interesting that self-esteem and marketing predicted return better than altruism. fear and poor customer experience are recognized deterrents which could be addressed. we plan to use these data to construct black donor recruitment interventions which may be tested using randomized trial designs. willingness to donate blood during the summer christopher d bernard , ramya ghantasala , obhijit d hazarika , nicole leonard , cori a polonski , zachary b wunrow , michelle heleba , jan k carney and mark k fung* . university of vermont larner college of medicine, american red cross blood services background/case studies: each year donation rates fall in the summer months straining blood banks' capacities to meet local demands. in hopes of identifying factors to increase summer donations, our study investigated donor reported barriers which influence summer donations habits. study design/method: an anonymous question survey investigating various donation factors was administered across multiple blood donor centers in a state-wide region. questions addressed donor demographics, frequency of blood donation, preference in appointment making modalities including smartphone app use, summer travel habits, willingness to donate during vacation, and factors that deter donors from donating on vacation. results/finding: a total of surveys were received. survey respondents across multiple demographic groups cited similar barriers to summer donation, namely "too busy" ( . %) and "traveling is a time for me to relax." ( . %). of the respondents who travel in the summer, very few reported donating while traveling ( . %). summer donation rates between summertime travelers ( . %) and non-travelers ( . %) were essentially equivalent. the most preferred methods of scheduling appointments were via the regional blood donor center website ( . %) and phone ( . %). willingness to use a regional blood donation smartphone app was highest among respondents ages of to ( - %) and lowest among ages and older ( - %). of respondents with no prior knowledge of summer seasonal shortages ( %), / rds indicated newfound motivation to donate. background/case studies: viral infections (adenovirus, ebv, cmv, bk, hhv , and rsv etc.) have been implicated as major contributors to posttransplant morbidity and mortality in hematopoietic stem cell transplantation (hsct) from unrelated donors. investigators have shown that in-vitro expanded virus specific cytotoxic t lymphocytes (ctls) generated from donors with specificity for one or more viruses are safe and effectively treat viral infections in the hsct setting in recent clinical trials. present clinical trials have shown that ctls can be rapidly produced by a single stimulation of donor peripheral blood mononuclear cells (pbmcs) with a peptide-mixture spanning the target antigens in the presence of potent prosurvival cytokines interleukin- (il- ) and il . others have used banked third party epstein barr virus (ebv)-specific ctls generated from third party ebv-seropositive blood donors with encouraging results. study design/methods: eligible and consented blood donors were tested for cmv antibodies by serology. cmv-seropositive whole blood (wb) units underwent buffy coats processing from non-leucocyte reduced wb units collected in fenwal triple blood-packs tm that underwent hard spins at rpm for minutes with separation after each spin on a compomatev r g . plasma and buffy coat was separated from red cells after the first spin. the second spin lead to the separation of the buffy coat from plasma. the buffy coats were submitted to the gmp stem cell lab for processing of cytomegalovirus-specific ctls. hla typing at high resolution for hla-a/-b/-drb loci was obtained for all donors. results/findings: forty five eligible healthy blood volunteers ( m [ %]: [ %] f); median age years (range - ) donated a unit ( ml) blood from which buffy coats (average volume ml) were processed. the buffy coat process was previously validated on wb units. the mononuclear cells (lymphocytes and monocytes) recovered from the buffy coats are listed in figures and . all of the buffy coats received by the gmp stem cell lab were adequate in cell numbers to be processed. the processing of buffy coats from whole blood is a viable option for the concentration of pbmcs specifically for production of viral specific ctls as third party off the shelf products as well as use in other research projects that require pbmcs from healthy adults. background/case studies: the goal of this presentation is to describe the journey and challenges towards tjc, patient blood management (pbm) certification. transfusion-related health risks and increasing economic pressures have driven hospitals to recognize evidence-based blood management as an important cost-saving strategy. providence holy cross medical center (phcmc), as the providence california region alpha site, has embarked on this journey. our goals are pbm certification and reduction of the number of unnecessary transfusions by % within months of the program launch while improving patient outcomes. this paper will discuss our journey toward certification and the various hurdles being overcome. study design/method: tjc, aabb, and the society for the advancement of blood management have served as our primary resources for identifying current evidence-based transfusion practices and management methods. we needed to identify our organizational gaps in data gathering and analysis. then we could determine baseline performance and set improvement targets. from our internal assessment, we learned we had to start from scratch as we had no easily accessible data metrics and gaps in education to our staff. we took the following steps to develop our pbm program: formed an interdisciplinary pbm team consisting of physicians, nurses, blood bank staff, and data analysts constructed a report on rbc transfusions to help identify outliers and opportunities background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed at a hospital along with a recommendation for pre-transfusion testing and rbc allocation before each surgery. the extent to which hospitals have an msbos and its design was explored in this survey. study design/methods: the survey was designed, piloted and refined by members of the best collaborative and invited colleagues. it was then encoded in online survey software and the link distributed to best members and colleagues who were encouraged to respond and to further distribute it. the survey was open for days. results/findings: there were completed responses, of which ( %) indicated that their hospital had an msbos and ( %) did not. the majority of hospitals without an msbos were academic centers ( / , %) from oceania ( / , %) or europe ( / , %), had between - beds ( / , %); the majority of these hospitals transfused between , - , rbcs ( / , %) per year. / ( %) are going to implement an msbos in . of those with an msbos, the majority / ( %) were from north america. the majority were academic hospitals ( / , %) with - beds ( / , %) that transfused ! , rbc units per year ( / , %) offering a wide range of surgical services. on average there were procedures listed in the msbos'. the msbos recommended no pre-transfusion testing for a mean of % of the procedures listed, a pre-operative type and screen for %, crossmatching rbc units for %, and for % of procedures a different recommendation was made. most ( / , %) of the msbos' were created by a combination of obtaining consensus between the surgical services and blood bank and use of procedure-specific transfusion data; only / ( %) of msbos' were created solely by using procedure-specific data, and most ( / , %) do not use patient-specific data in making a testing recommendation. most msbos' are updated less frequently than annually ( / , %), and the hospital transfusion committee is often ( / , %) involved in updating it. the msbos' are generally available electronically in both the operating rooms and in the blood banks. it was the opinion of the majority of respondents ( %) that the msbos was used regularly by only a limited number of surgeons and anesthesiologists, % of respondents felt that it was regularly used by all surgeons and anesthesiologists; % felt that it was not used at all at their hospital, % did not respond. conclusion: an msbos was available in only about half of the respondent's hospitals and in only the minority of cases was it felt to be regularly used. however, % of the hospitals currently without one indicated that it would be implemented in suggesting that these hospitals perceive the value of having one in place. implementing and following an msbos can be an important step in peri-operative patient blood management and in streamlining the operations of the blood bank vis-a-vis pre-operative testing. blood management -one hospital system experience leana serrano rahman*, mallika gupta, susan solometo, ronald walsh and joan uehlinger. montefiore medical center background/case studies: our system, a pioneer aco, is a -bed tertiary-care referral center dedicated to serving patients from across the new york city area and beyond. the comprising four hospitals see , hospital admissions and nearly , emergency department visits annually. we have active programs in high risk ob, stem cell transplant, solid organ transplant (heart, liver, and kidney), ct surgery, ecmo, oncology and critical care. transfusion medicine plays a key role in the support of these services. blood product spending in was approximately $ . m. in nov. , an interdisciplinary committee was created in an effort to improve patient care (by reducing blood product exposure) and reduce blood product expenditures. the vice president-sponsored multidisciplinary committee was composed of representatives of: surgery, anesthesia, blood bank, pediatrics, perfusion, cardiothoracic surgery, critical care, medicine, and emergency department. study design/method: first important step: "know your numbers"-although the committee had multiple sources of data, there was no "one report" that could display all of the pertinent information. baseline numbers were imperative to the committee's ability to effect change. a home grown one time only report revealed which services and clinicians were the highest volume users. the initial plan was to target their use with education. an initial goal was set to reduce expenditure by $ . m. the journey continued with regular bimonthly meetingsto brainstorm strategies and monitor utilization. utilization was analyzed using a home grown crystal report "transfused patients by location". this report was further compared to utilization patterns ( and ), by "dollars spent" and "total units per patient" by the project manager using excel. key initiatives developed by the committee . development of evidence based transfusion triggers. . education on evidence based transfusion triggers across multiple campuses, specialties and resident programs . clinical information system (cis) "soft stops" when ordering blood products outside guidelines. rbc order set defaulting to " " unit instead of " " units. . updated guidelines posted to easy to find internal intranet spots results/finding: despite higher patient volumes and a more complicated patient mix in , we were still able to reduced blood product expenditures by $ , when compared to . conclusion: in spite of limited resources, the committee was able to effect change by capitalizing on current stakeholders fully supported by leadership and project management. cord blood pathway to reduce iatrogenic blood loss in neonatal intensive care patients tracy shachner* , anna w rains and christopher t clark . university of tennessee graduate school of medicine, univeristy of tennessee medical center, univeristy of tennessee graduate school of medicine background/case studies: anemia due to iatrogenic blood loss in preterm and low birth weight infants is a major contributory factor leading to red blood cell transfusion in this patient population. methods to reduce phlebotomy for laboratory testing can reduce iatrogenic anemia. at a universitybased teaching hospital, a pathway to collect cord blood samples on all newborn deliveries was established. the cord blood sample is used for initial blood bank laboratory testing on newborn patients transferred to the neonatal intensive care unit (nicu), preventing need for additional blood draw. the blood tubes are saved for week post-delivery, with cost of $ . per delivery tray for sterile tubes. with an initial negative antibody screen on cord blood sample, no additional phlebotomy is required for blood product selection or compatibility testing in this population until four months of age. study design/method: labor and delivery data from our facility in was analyzed, and the gestational age and birth weight of all infants transferred to the nicu was collected. from this data, we were able to calculate the total blood volume of these infants using medcalc system. by using the blood volume values, and assigning a value of . ml as the minimum amount of blood that would be drawn to perform an antibody screen, we calculated the percent of an infant's blood that would have to be drawn if the cord blood pathway was not established. transfusion results/finding: in , there was a total of , infants delivered at our facility. out of all the deliveries, ( %) infants were transferred to the nicu. of those infants, % received at least one red blood cell transfusion and % received at least one platelet transfusion. of the infants transferred to the nicu, ( %) had a percentage of blood volume that would have had to be drawn for blood bank testing greater than or equal to % (which we considered to be significant), had the cord blood pathway not been in effect. the percentage of blood volume preserved in these infants ranged from . % all the way up to . %. in those infants, the birth weight ranged from - grams, and the gestational age ranged from weeks to weeks and days. conclusion: the established cord blood pathway has proven to be a relatively cost-effective method to prevent iatrogenic blood loss secondary to blood bank testing in a population of nicu infants who are most susceptible to iatrogenic anemia. the infants that were most likely to benefit from this policy are premature infants who are low birth weight (less than grams). development of a standardized response team for massive hemorrhage events outside of an operating room setting james burner* , shannon davis , suzan new , vaishali patel and oren guttman . university of texas southwestern medical center, ut southwestern medical center background/case studies: managing a massive transfusion protocol (mtp) in an operating room (or) is a relatively frequent occurrence with team members well trained in their specific roles. however, in the event of mtp activation outside of an or, sufficient and/or appropriately trained individuals may not be present. this can lead to a scene of confusion and chaos with potential for patient harm. study design/method: a failure mode effects analysis was performed to develop a standardized process for managing mtp outside of an or setting. with participation from anesthesia, surgery, transfusion medicine, patient safety and quality and nursing, every step of the hospital's mtp was analyzed for potential errors. the results were used to create a "code hemorrhage" team trained to respond to any massively hemorrhaging non-or patient. results/finding: code hemorrhage represents a multi-system team critical event requiring coordination of different sub-teams (primary resuscitation, surgical/interventional, transfusion services, blood preparation, equipment management, medication management, and lab requisition/monitoring). our code hemorrhage protocol utilizes critical care trained nurses from the hospital's rapid response team who play two key new coordination roles: hemorrhage coordinator and electronic medical record (emr) coordinator. their combined roles serve to reduce the cognitive load of the various teams, prevent duplication of resources/efforts during mtp and enable enhanced closed loop task performance. the hemorrhage coordinator establishes reliable : communication between the primary resuscitation team and transfusion services, and aids in multi-team on-site coordination. the emr coordinator enters all orders into the emr, sends/communicates laboratory results and ensures blood products are available to the resuscitation team. the primary resuscitation team includes a team leader (medical decision making and cardiac life-support management); a proceduralist (establishing venous and/or arterial access), event documenter (real-time documentation of actions, medications, events, etc.), medication manager (registered nurse who prepares and administers medications) and equipment technologist (managing rapid blood product infusion devices). additional secondary roles will also be assigned, such as blood product checker(s) (verifies blood product prior to transfusion) and blood bank runner (courier sent to retrieves blood product shipments). conclusion: the code hemorrhage protocol is designed to ensure timely, efficient delivery of blood products to massively bleeding patients outside of an or setting. future work will assess its overall effectiveness by comparing blood product utilization/wastage and patient outcomes before and after implementation. background/case studies: preoperative anemia affects up to % of surgical patients and increases the risk of red blood cell (rbc) transfusion. both preoperative anemia and perioperative rbc transfusion are associated with increased risk of adverse outcomes following surgery. preoperative treatment of anemia includes oral and intravenous (i.v.) iron and erythroid stimulating agents (esa) such as erythropoietin (epo); however, the optimal treatment strategy for preoperative anemia remains to be established. our objectives were to evaluate the efficacy and safety of esa and iron therapy based on their effects on the prevalence of rbc transfusions and adverse thrombotic events. study design/method: we searched the cochrane central register of controlled trials, medline and embase from inception to july ; reference lists of published guidelines, reviews and associated papers, as well as conference proceedings. no language restrictions were applied. we included randomized controlled trials in which adult patients undergoing surgery received either an esa and/or iron before surgery, versus iron or no intervention. three authors independently reviewed the studies and extracted data from included trials. risk of bias was assessed for all included studies. where applicable, we pooled risk ratios of dichotomous outcomes and mean differences of continuous outcomes across trials using randomeffects models. our primary outcome was the number of patients transfused with red blood cells. secondary outcomes included risk of mortality and other thrombovascular events (stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism). results/finding: a total of randomized controlled trials ( , conclusion: amongst patients undergoing surgery, the administration of an esa in addition to oral or i.v. iron was associated with a reduction in patients requiring rbc transfusion. intravenous iron was less effective at reducing rbc transfusion. neither treatment was associated with any clear increase in risk of adverse thrombotic events. additional large prospective randomized controlled trials are required to determine the optimal management strategy for patients undergoing surgery with iron restricted anemia. evidence based blood therapeutics scott neeley* and stephanie rogers . dignity health st joseph's medical center, dignity health background/case studies: over million units of packed red blood cells (prbc) are transfused annually in the united states and there is no clinical basis for as many as half of these transfusions. no randomized prospective trial has ever demonstrated a clinical benefit for transfusion in mild to moderately anemic patients and yet there is a large body of evidence which has shown that due to a variety of reasons including an immunomodulatory effect and the storage lesion, blood transfusions can cause considerable harm, including higher risk of hospital acquired bacterial infections, transfusion related acute lung injury/acute pulmonary edema, acute myocardial infarction, higher recurrence of rebleeding and higher cancer recurrence. study design/methods: a system wide goal was launched across hospitals to decrease the number of prbc transfusions given to clinically stable patients with hemoglobin (hgb) levels > . g/dl. the numerator consisted of all prbc units transfused to patients with a hgb of . g/dl or greater prior to transfusion and the denominator consisted of all prbc units transfused. exclusions included cardiac surgery, nursery, nicu, pregnancy, post-partum hemorrhage, massive transfusion protocol and transfusions in which or more prbc units were transfused in one episode. data was extracted directly from the electronic medical record and hospitals received patient level detail every month for all prbc units transfused to patients with a hgb of . g/dl or higher prior to transfusion. an extensive educational campaign re: evidence-based transfusion practice was launched for physicians and nurses, including the development of a blood therapeutics toolkit, development of standardized dignity health blood therapeutics guidelines, a one day blood therapeutics advanced training symposium, on-site visits to hospitals including cme presentations, online physician and nursing educational videos, communication tools including infographics and " is the new " buttons, development of a patient education resource and bi-monthly webinars with various educational topics and speakers. additionally, the ehr powerplans were revised to ensure available selections for "transfusion indication" (required field) were aligned with evidence based guidelines. facilties were encouraged to develop multi-disciplinary blood therapeutics committees to review all transfusions given to patients with pre-transfusion hgb > . g/dl on a routine basis, providing feedback to providers whose transfusions were deemed not in accordance with current evidence-based guidelines. results/findings: from fy to fytd , there was a % reduction in prbc units transfused to patients with hgb > . g/dl, starting at a baseline of % down to %. this represents an fy annualized savings of $ . m, from a baseline of units per , patients days down to an average units and approximately , fewer units transfused per month. conclusion: blood transfusions, while life saving, should be regarded as an organ transplant and as such they carry considerable risk. transfusions to stable, non-bleeding patients with hgb levels > . g/dl are not in accordance with evidence-based guidelines and should be avoided due to the associated potential harm. furthermore, this potential harm is dose dependent, so if the decision to transfuse is made, one unit of prbc should be transfused rather than two. three af studies (sdm - . ) reduced rbc units and two studies decreased the percentage of patients transfused (or . ). forty-three studies showed that intravenous tranexamic acid reduced the percentage of patients (or . ) and rbc units transfused (sdm - . ). qualitative/meta-analyses were translated into recommendations by an expert panel and approved by the lmbp workgroup for reducing rbc transfusion. recommendations are: early assessment and effective am; rt, hb alerts in cpoe/cds; reduction of blood loss and af assessing the percentage of patients and rbc units transfused across cases, physicians and service areas over discrete periods of time with feedback to physicians for continuous quality improvement. conclusion: conclusion: the lmbp a- method led to evidence-based recommendations for reducing transfusion. critical laboratory support is needed to achieve continuous quality and patient safety. background/case studies: reducing the inappropriate use of blood products via the implementation of evidence based guidelines is a main tenet of patient blood management. the use of electronic decision support tools such as best practice alerts (bpas) to enforce red blood cell (rbc) transfusion thresholds have been shown to reduce use by informing ordering providers when or when not to transfuse. the tools in use to date have not provided a dose of rbcs to transfuse, so in fact providers can continue to over-transfusion based on the number of units of rbc given. a therapeutic hemoglobin/hematocrit (hgb/hct) targeted approach to rbc indications/ orders allows for the calculation of a dose of rbcs to achieve the desired target and could further reduce the use of rbc units. our group has developed a computer algorithm to calculate rbc dose based on patient specific data drawn from the electronic medical record (emr) that has been used in select patient populations but has not been prospectively applied to hospital wide clinical practice. this study describes our initial experience with the use of this algorithm in non-surgical rbc transfusion. study design/method: the blood utilization calculator (buc) is a mathematical formula that draws patient specific information including index hgb/ hct and calculates a dose in number of units of rbcs to transfuse in order to achieve a selected target hgb/hct. hgb/hct target based indications for rbc transfusion were designed and used as the basis for rbc order set with in the ethe buc was embedded within the emrs rbc order set to provide a recommended transfusion dose in number of units when any nonsurgical rbc indication was selected. the target hgb/hct for these indications was g/dl/ % or g/dl/ %. the number of rbc units ordered and transfused were tracked prospectively for each of the orderable indications. comparison of units transfused per month before and after the buc implementation was performed using student's t-test. results/finding: historically, the three non-surgical rbc indications represented approximately % of the total rbc transfused. prior to the buc the mean number of non-surgical rbc units transfused was units/ month. after the first months of buc activation the mean number of units was units/month a reduction of units/month or % of nonsurgical blood use (p . by t-test). non-surgical rbc use now represents approximately % of the total rbc use hospital wide a % reduction. this change represents a significant cost savings in rbcs over time. conclusion: the use of target based transfusion indications and an electronic decision support algorithm to calculate a recommended transfusion dose can significantly reduce the non-surgical rbc transfusion rate providing enhanced patient blood management and potential cost savings. implementation of patient blood management at a community hospital - month report card richard gammon*. oneblood, inc. background/case studies: a collaboration between blood center between (bc) as consultant and three hospital ( beds) healthcare system (hcs) to implement a patient blood management (pbm) program was undertaken. this is a review of the first months. study design/method: during year one pbm working group was established. achievements included physician engagement programs, creation of transfusion committee and providing nursing education. auditing processes were implemented with nonconformance letters sent to physicians and nurses when compliance with informed consent, transfusion tags and thresholds and discharge instructions was not achieved. in year two, it created best practice alerts (bpa) when an order did not meet transfusion threshold criteria. bpa showed first line of associated procedure, link to the full procedure, three most recent lab results (e.g., hemoglobin & hematocrit for red blood cells (rbc)) and allowed ordering physician to cancel order after review. a blood administration video was created. it was mandatory that all physicians granted privileges complete within six months. low vital sign compliance required action that included reducing requirement from five to three during transfusion and formation of working group (wg) to address knowledge and practice gaps. in year three, as historically at this hcs very few jehovah's witness patients (jwp) presented, pbm wg was involved with implementation of a bloodless medicine program. all steps of care were addressed including identifying jwp at registration, creating a transfusion special arm bands, forming a bloodless medicine physician group, implementing nursing bpa in the electronic medical record, creating advanced directives and marketing to the public. results/finding: the following were monitored for compliance ( q vs. q ): present and completed consents ( vs. %), present and completed nursing flow sheets ( vs. %), transfusion thresholds supported ( vs. %), discharge instructions provided ( vs. %); ( q vs. q ) vital sign compliance ( % vs. %). jwp increased from to ( / - / ). cost savings were realized by decreased utilization and implementation of bpa. (table - q ) conclusion: pbm implementation at a hcs is a continuous and multiyear process. even with a robust program challenges such as vital sign compliance remain. improving patient outcomes in the golden-hour beatrice lebeuf*. medical city plano background/case studies: in emergency medicine, "the golden hour" refers to the critical one-hour time period following traumatic injury in which the patient has a higher likelihood of survival. nearly half of all trauma related deaths occur in the first hour after injury -half of those deaths are the result of major hemorrhaging. rapid administration of blood products is vital to the survival of these patients. we implemented bloodtrack emerge (haemonetics, braintree, ma) in our trauma emergency department (ed) as part of a quality improvement initiative to more efficiently provide group o rbcs and thawed/liquid plasma for incoming trauma patients to support ratio-based transfusions and ensure the proper handling and traceability of this regulated resource. study design/methods: we treat approximately - trauma patients monthly. an assessment of our current blood supply chain revealed a multistep, manual process that took about minutes to prepare and physically transport a cooler from the blood bank to the ed. coolers of blood were provided for incoming trauma patients, whether they ended up needing transfusions or not. this practice worked to ensure available blood supplies during critical moments, but resulted in inefficiencies and unnecessary inventory tie-ups, with only percent of coolers fully used. it also consumed valuable staff time as technologists typically made - trips per month from the blood bank to the ed. plus, there was no effective way to maintain traceability, control access to coolers or monitor usage. results/findings: since our november implementation, bloodtrack emerge has freed up technologists to perform important tasks, tightened traceability and inventory control procedures and contributed to the medical city plano's verification as a level trauma center. rather than preparing coolers of blood in case they may be needed in emergency situations, bloodtrack emerge provides ed staff ready access to emergency units whenever they're actually needed -and frees up an estimated - hours of tech time per month during which they can perform other tasks. audio and visual alerts notify the blood bank when emergency units are removed, allowing a quick response. plus, by stocking emergency blood supplies in the ed, the blood bank isn't unnecessarily tying up group o rbc units. today, the blood bank stocks and maintains - units of group o rhd negative, units of o rhd positive, and units of group a thawed plasma/ liquid plasma in bloodtrack emerge. conclusion: implementing bloodtrack emerge has enabled us to more effectively provide blood products for incoming trauma patients to support ratio-based transfusions, improve staff efficiencies and proactively respond to emergency situations. background/case studies: platelets are a limited resource for which the benefits of transfusion must be weighed against the risks. in , the aabb published platelet transfusion guidelines to assist providers. at our academic medical center, a computer provider order entry (cpoe) system combines institutional transfusion guidelines with a patient's most recent lab results to guide transfusion decisions. discordant information activates an "override" system, in which providers are prompted to select a prefixed indication for transfusion (e.g. count < k/ml [prophylaxis]) with the option to add a free-text comment. the order is placed and data is stored for later review. study design/method: override platelet orders placed from june -october were reviewed using the following data: prefixed indication, most recent platelet count, free-text comment, and ordering service/department. one of five "codes" was assigned to each order: i-indicated or ni-not indicated (based on institutional/aabb guidelines); nmi-need more information; p-protocol (e.g. liver transplant), and nic-non-indication comments (e.g. reserve for or). free-text comments were categorized and assigned one or more keywords in order to determine the common reasons for overrides. results/finding: over a -month period, , cpoe override platelet orders occurred. the percentages of code assignments by month are provided in table below. overall, ( %) were assigned as not indicated (ni). the top keywords assigned to free-text comments were "platelet count less than. . ." ( ), "active bleeding" ( ), "platelet count of . . ." ( ), and "downtrend" ( ), many with specified platelet count goals. certain platelet count goals and reasons for transfusion (e.g. "downtrend," "anticipate drop," or "per service,") are not included in institutional or aabb guidelines. of note, ( %) of overrides were placed by hematology-oncology providers. conclusion: a majority of override platelet orders were determined to not be indicated based on institutional and aabb guidelines. of concern were keywords such as "downtrend" and "anticipate drop," as these are not indications for transfusion and expose patients to unnecessary transfusions. it is unclear whether trainee progression throughout the year had any effects on ordering practices and associated override patterns. this review suggests the potential benefits of provider education initiatives at all levels of experience (with particular emphasis on hematology-oncology) in order to improve blood product utilization practices. background/case studies: early diagnosis of iron deficiency anemia (ida) by clinical laboratories (cl), with effective prevention and treatment in primary care may have an impact on packed red blood cell (prbc) transfusion, as well as intravenous iron therapy and, most importantly, applying lower transfusion triggers. they all help to avoid not essential transfusions, but also promote health and wellbeing by improving iron status in the population. results are described after implementing a process to prevent ida, its early detection and treatment for years - . study design/methods: performance measure after educational and organizational intervention. setting: public integrated healthcare system located in north africa bordering morocco, isolated by km sea distance to nearest continental spain airport, with a general hospital blood transfusion service and a establishment for blood donation and component production. cl involved in anemia detection and diagnosis receives four primary care centers and hospital based samples, and shares common leadership with both blood establishments. process: guidelines for first step cl diagnosis of ida and call for attention, primary oral iron prevention and treatment in first level care, and early intravenous iron complex for inpatients (sucrose) and outpatients (carboxymaltose). transfusion was avoided for stable ida patients without active bleeding or coronary heart disease, with a safety hemoglobin (hb) threshold of , g/dl. severely anemic patients were closely followed to asses hb increase and referred for etiology studies when hb> g/dl. background/case studies: bedside nurses are critical in safeguarding the delivery of appropriate patient care. more recently, nurses have also begun to play an important role in patient blood management (pbm) programs at the administrative level, although to our knowledge little has been published on the influence nurses may have on transfusion practice at the bedside. the goal of this study was to evaluate the impact nurses have on patient expectations and physician ordering practice. study design/method: a short electronic survey ( questions) was prepared to assess how often bedside nurses discussed transfusion necessity and the persons (patient or physician) with whom they discussed it with, as well as what was discussed, and what they felt were appropriate lab thresholds for transfusion. the survey was distributed to all registered nurses via email from floor leaders. responses were also solicited by hospital volunteers and lab staff with electronic tablets and included coverage of the night shift. results/finding: there were a total of complete responses ( %). the nurses had a range of experience from less than one year to forty years. ninety percent stated they discussed transfusion necessity with patients, % with physicians, and of these, % reported doing so proactively before an order was placed. ninety-six percent said they would discuss transfusion to suggest their patient required a blood product; only % responded that they would suggest product was not needed. nursing perception of acceptable transfusion thresholds had a wider distribution, with the most commonly reported values being hemoglobin of - g/dl ( %), platelet count of - , ( %), and inr of greater than . ( %). conclusion: this study demonstrates that nurses are willing to discuss transfusions with both patients and providers, although they appear to be most comfortable doing so in the setting of perceived transfusion necessity. the limited number of survey responses suggests a discomfort with their level of education in transfusion practice. this, along with the distribution of perceived thresholds and the reluctance to recommend against transfusions, presents an opportunity for education to further empower nurses in providing appropriate patient care within the guidelines of pbm programs. background/case studies: the use of red blood cell per , inhabitants may vary folds between european countries, revealing that there may be substantial room for blood optimization strategies. patient blood management (pbm) is an evidence-based, multidisciplinary approach aiming to preserve and optimise patients' own blood in order to improve clinical outcomes. the objective of our study was to assess the effect of a nationwide pbm program on public health in portugal. study design/method: the first phase of this research project involved a group of key opinion leaders (kol) in a stated preference inquiry to assess the relative value of specific pbm strategies, grouped in pbm pillars, to highlight the need for strategy prioritization in the implementation of a nationwide pbm policy. adaptive conjoint analysis techniques were used to elicit kol preferences. in the second phase a decision analysis model was used to estimate the impact of pbm implementation in the following therapeutic areas: surgery (orthopaedic, cardiac and urologic), cardiology, oncology, gastrointestinal bleeding, abnormal uterine bleeding, haemodialysis, inflammatory bowel disease and pregnancy. model inputs included effectiveness data regarding transfusion utilization, health resource consumption and mortality obtained from portuguese national health databases and literature review. the public health value of pbm implementation in portugal derives from the comparison of two scenarios: "current clinical practice" and "with pbm implementation". results/finding: kol elicited iron administration followed by restrictive transfusion of red blood cell as the most preferred pbm strategies ( . % and . %), for the remaining strategies weights varied between . % and . %. we estimate that , patients would be eligible for pbm strategies in one year time horizon, resulting in premature death avoided ( . % reduction) corresponding to a gain of approximately , life years and a reduction of , ( . %) disability adjusted life years (daly) relative to the current clinical practice. a decrease of , in-hospital days is expected mainly due to a . % reduction in hospital length of stay and a . % reduction in -day readmission rate. in this population the overall transfusion rate could decrease to . % from the current . % ( . % reduction) implying , blood transfusion avoided and , red blood cells units spared. conclusion: we anticipate that the implementation of a nationwide patient blood management program will represent a paramount improvement in clinical outcomes in terms of morbidity and mortality and may have a substantial public health impact while contributing a more efficient use health resources. results/finding: adult liver transplants were performed during the evaluation period. preoperative hemoglobin, creatinine, meld score, spontaneous bacterial peritonitis (sbp), preoperative hemodialysis, gender, and portal vein thrombosis (pvt) gave the strongest model predicting rbc usage. if the model predicted < ml of rbcs, all cases with ml transfused were captured and only . % of the time > ml were used. if - ml rbcs were predicted to be transfused, > ml were used % of the time. if predicted usage was > ml, % of the time it exceeded ml. conclusion: a model using specific preoperative factors can be used to predict intraoperative rbc usage. patients at risk for > ml of rbc transfusion can be identified with reasonable accuracy using this model at our institution. use of this model might help improve preparation and utilization of the blood bank. review of blood ordering practice for elective surgeries in a maternity hospital qi raymond fu*. kk women's and children's hospital background/case studies: pre-operative over-ordering of blood is common, resulting in waste of blood bank resources. blood units are withdrawn from the pool, leading to constraints in allocating the limited blood resources to meet the needs of other patients. the cross-match to transfusion (ct) ratio is often used in benchmarking efficient blood utilization within the hospital blood transfusion service. according to the american association of blood banks (aabb), a ct ratio of less than . is favorable, and anything above indicates over-ordering and cross-matching of blood. to achieve this, it is necessary to review pre-surgical blood ordering practice in a maternity hospital. study design/methods: data on elective surgeries requiring blood for standby was collected retrospectively over a month period (jan to mar ). details of total blood cross-matched, issued, transfused and returned were analyzed along with the ct ratio. results/findings: during the month period, there were patients undergoing obstetrics and gynecology procedures requiring blood on standby. a total of units of blood were requested. units were crossmatched, of which units were sent to the operating theatre (ot). only . % of blood issued to ot were transfused (n ) while the rest were unutilized. the observed ct ratio was . . conclusion: although only % of total blood requested was crossmatched, the ct ratio remains above the recommended guideline of ! . , with almost % of cross-matched blood unutilized. there is a need to improve and standardize the blood ordering practice to achieve costeffectiveness and reduce unnecessary workload. establishing and adhering to a maximum surgical blood order schedule (msbos) could help in conserving blood and prevent over-ordering of blood. background/case studies: total knee arthroplasty (tka) is a major orthopaedic procedure with increased perioperative blood loss. this perioperative blood loss could be more significant in patients undergoing bilateral tka in a single stage. the increased blood loss in bilateral tka often requires blood transfusion which results in high post-operative morbidities. study design/methods: in this retrospective study patients who received tranexamic acid (txa) (study group) and patients who did not receive txa during surgery (control) were evaluated for blood loss and transfusion requirement. the study group received a single bolus dose of txa gm iv before tourniquet deflation on first side knee. statistical background/case studies: blood product utilization is an increasing concern for hospital systems attempting to reduce transfusion-associated risks. one strategy to optimize utilization is to employ clinical decision support in the form of alerts to clinicians ordering blood products. we investigated whether an alert targeted to a patient's transfusion indication could alter provider ordering behavior. study design/method: this retrospective, observational study over the course of seven months included the inpatient adult medicine floors and intensive care units at a large academic hospital. each time a crossmatch for packed red blood cells (prbcs) was ordered via the hospital's electronic ordering system, an indication (e.g. "hemodynamically stable with hemoglobin < . g/dl") must be selected. if the indication selected contains a threshold hemoglobin concentration, and the patient's most recent hemoglobin on record was greater than this threshold, an interruptive alert displaying the patient's hemoglobin was activated. ordering providers were then given three options: cancel the order, select a more appropriate indication from a list, or provide an explanation via free text as to why transfusion was being requested outside of approved indications. an alert encounter was defined as all activations on a patient within a six hour period without an intervening transfusion results/finding: over seven months, there were unique alert encounters. of these, ( . %) led to a crossmatch being ordered while ( . %) led to the order being canceled. providers were more likely to cancel transfusions in response to alerts for hemodynamically stable patients with lower hemoglobin thresholds ( . g/dl) than for more complicated patients (bleeding, cardiovascular disease, or preoperative) with higher hemoglobin thresholds ( . or . g/dl background/case studies: the maximum surgical blood ordering schedule (msbos) is a list of surgical procedures performed along with a recommendation for the extent of pre-transfusion testing to be completed before the surgery begins. with improved patient data management systems it is now possible to create an msbos based on actual red blood cell (rbc) utilization data on a per-patient basis. this study investigated the transfusion patterns at academic hospitals with data-derived msbos. study design/method: the hospitals were in groups, with one shared msbos for each group. three of these hospitals were large academic centers while one was a children's hospital. at each center the msbos recommended no pre-transfusion testing if % of patients had been transfused for a specific procedure in the previous year, a pre-operative type and screen (t&s) if - % of the patients had been transfused, and a crossmatch of the median number of rbcs transfused if ! % of the patients had been transfused. data were collected at each center over a month period between january to march and included a maximum of cases per hospital during that one month to ensure equal representation between centers results/finding: between these centers there were a total of cases analyzed. some of the more frequently performed surgeries included orthopedics ( % of cases), general surgery ( %) and cardiac surgery ( %). there were t&s ordered for these cases, of which were positive for antibodies on the day of surgery. of all the t&s ordered, % were ordered in accord with the msbos recommendation, % were ordered when the msbos did not recommend one, and in . % a t&s was not ordered when the msbos recommended one. background/case studies: peripartum blood transfusion is more common in south africa than in the usa and recent studies have demonstrated that antenatal anemia is a strong risk factor for such transfusion (odds ratio . for prenatal hemoglobin (hgb) - . ). we therefore analyzed the etiology and characteristics of antenatal anemia according to hiv status at a large hospital with a hiv prevalence of % among obstetric patients. study design/method: we studied a sample of anemic (hgb< . g/dl) pregnant women who were referred to an antenatal anemia clinic at a large hospital in south africa. clinical information was abstracted and blood was sent for laboratory studies. t-tests were used to compare continuous variables between groups. results/findings: a total of women were enrolled, with median age (interquartile range - ) years, median gravida / para and median gestational age weeks. mean hgb before referral was . g/dl and most were already taking oral iron therapy. a total of women were hiv positive with mean cd lymphocytes counts of cells/ul; ( %) of hiv positive subjects were on anti-retroviral therapy (art) prior to the pregnancy and ( %) were on art during the current pregnancy. iron deficiency anemia was the overwhelmingly prevalent diagnosis, present in ( %) of women. there was concurrent chronic disease (n ), infection (n ), vitamin b deficiency (n ) and antenatal hemorrhage (n ); had other/unknown/missing causes of anemia. there were few pregnancy related complications. hiv positive women had higher levels of c-reactive protein but slightly lower levels of transferrin, soluble transferrin receptor and rbc folate than hiv negative women (table) . conclusion: iron deficiency is the overwhelming cause of antenatal anemia among south african pregnant women. compared to hiv-negative women, hiv-positive women had evidence of increased inflammation, relatively little differences in iron studies after early treatment with iron and lower red cell folate. a high proportion of hiv positive women were receiving art, consistent with national guidelines. future studies will examine longer-term responses to iron therapy to assess its potential in decreasing the incidence of peripartum blood transfusion. background/case studies: a month old boy presented to our institution after a month hospitalization in japan. he was admitted there, several weeks after his unremarkable term birth to an ab rh positive woman, with lethargy, failure to thrive, bloody mucoid stools with eosinophilia, and an elevated serum white count. he was found to be anemic and thrombocytopenic and required multiple transfusions. also, he had a diffuse, scaling, erythematous rash over his inner thighs. study design/method: initial workup was suspicous for an allergic/necrotizing enterocolitis. the patient had an elevated ldh and potassium, and concern was raised for leukemia with possible tumor lysis syndrome. a sample sent to our blood bank showed an anti-e, with a positive dat (igg and complement), and was positive for e, e, and c antigens. concern for a maternally-induced antibody was raised, as was the possibility of a red cell antigen passively transfused from blood products administered at the japanese hospital; both possibilities were excluded. further workup revealed no infection or hematologic proliferation. biopsy of his rash showed spongiotic dermatitis. his clinical course deteriorated, and he developed hepatomegaly and jaundice. a concern for wiskott-aldrich syndrome was raised, and workup showed normal immunoglobulin levels, but with elevated ige ( ku/l; rr: - . ). anti-platelet antibodies were identified. three days after admission, testing was sent for genetic alterations of foxp , while a japanese-speaking physician at our institution read a prior flow cytometry study showing a deficiency of foxp cd lymphocytes. the majority of these indications are seen in adults and for which a reported plasma wastage is $ . %. fortunately in pediatrics the incidence of these indications is low despite the heterogeneity of the patient population. during the utilization review process at our primary pediatric institution, we noted a mean wastage of . % over the last years. with recent changes in clinical practice (liver transplants and increased trauma) and recent evidence that faster plasma improves massive transfusion protocol (mtp) outcomes, our facility decided to implement the use of thawed plasma and benchmark mtp plasma wastage. study design/method: blood utilization review revealed an increase in the overall percentage of plasma wastage from to , with a peak of . % (range . %- . %). a single cause could not be readily identified prompting us to query children's hospital association (cha), as our initial external pediatric benchmarking, to determine if our wastage was comparable to other children's hospitals in addition to reviewing our "time of plasma availability" for mtps. results/finding: in , mtp was activated times. in cases the patient did not receive any blood product and in cases plasma was already available at the time of rbc allocation/issue. this left cases to evaluate. the median time to plasma availability was minutes (range minutes - minutes). the mean plasma wastage for mtp activations was % (range - %). of the cha replies, were using thawed plasma and their wastage was </ %. conclusion: increasing clinical evidence supports the prompt transfusion of plasma in the setting of mtp to decrease morbidity and mortality. our brief review suggests that implementing thawed plasma will allow a potential decrease in plasma availability of minutes. our implementation began with thawing a single unit of plasma for "level neuro trauma" alerts, and we are now keeping a single unit of ab plasma thawed at all times. plasma is now available at the same time as rbcs thus providing more effective and balanced blood product support while reducing wastage (preliminary estimates are . %) to a level consistent with external benchmarking. prospective evaluation of this practice is ongoing at our institution and includes collaborative clinical assessment with the trauma team. ultimately prospective, multi-institutional studies in pediatric institutions are necessary to define best clinical practice and effectively benchmark blood bank practice. background/case studies: bacterial and fungal infections remain a significant cause of morbidity and mortality in severely neutropenic patients with hematological disease receiving high-dose chemotherapy and hematopoietic stem cell transplantation (hsct). granulocyte transfusions (gtx) have been used for over years, although effectiveness, indications, and both patient and donor safety remain debated, particularly in children. to contribute to this discussion we demonstrated cases of gtx in pediatric patients with age from months to years old, neutropenic, with severe infection resistant to antibiotics and/or antifungal therapy, undergoing hsct and chemotherapy. study design/method: donors: data concerning a total of granulocytes donations from to were collected from health donors ( male/ female). donors had matched blood type and serology status for cytomegalovirus when the patient was negative. granulocytes were mobilized with a single dose of rhu-g-csf (filgrastim) subcutaneously, mcg/kg/dose (donors up to kg, maximum of mcg) and oral dexamethasone ( mg), h prior to apheresis. apheresis was performed using cobe spectra v r or spectra optia v r with citrate as anticoagulant. all products were irradiated with gy. results/finding s: granulocytes number increased times from basal levels and the median of cells collected were . x (range . - . ). in general donors reported no significant adverse reactions. from donations, only in was reported light paresthesia during the procedure. patients: all patients were medicated prior to transfusions with diphenhydramine and acetaminophen. the product was transfused within hours after collection, with efforts to transfuse as soon as possible. details about the transfusions are described on table . adverse reactions for patients were minor and not frequent ( in gtx) including vomiting, hypotension and headache, which resolved without serious or lasting complications, assuming that gtx is well tolerated. conclusion: our study provides further evidence that gtx transfusions can be safely performed on pediatric patients. irina kumukova* , elena kurnikova and pavel trakhtman . russian institute for paediatric haematology, oncology and immunology, instituteffor pediatric hematology, oncology and immunology background/case studies: infections cause major mortality among persons with prolonged neutropenia related to hsct and chemotherapy and among patients with granulocyte disorder. the granulocyte transfusion therapy, a logical approach in treating patients with infections, who are refractory to antibiotics. we want to demonstrate our granulocyte transfusions experience in children who have neutropenia or defects of the immune system, with severe infections study design/method: we analyzed the retrospective data for the period / - / . donors underwent granulocyte mobilization with g-csf in a dose - mg/kg s.c. - hrs prior to donation; granulocytes were collected with cobe spectra, caridianbct. the analysis was performed through microsoft excel, nonparametric mann-whitney's and spearman tests results/finding: was performed granulocytes collections in donors ( f, m), ages to . the mean increment of wbc after stimulation was , x /l ( , - , x /l; f- , x /l; m- . x /l); in the age groups - years (n ) and over years (n ), the mean increment of wbc was respectively , x /l ( , - , x /l) and , x /l ( , - x /l). the mean volume of treated blood was , a , ) . the mean number of total wbc in the product was , x /l ( . - x / l; f- , x /l, m- , x /l). only three donors ( , %) had apheresisrelated adverse effects the analysis included granulocyte transfusions in cases of infectious complications in patients. the mean number of transfusions was . ( - ). each patient received transfusions from mean donors ( - ); the granulocyte transfusions started at the mean days ( - ) after infection; the mean dose of wbc on the transfusion was . x /kg ( - . x /kg). wbc increment was able to estimate after transfusions. wbc increment was recorded after transfusions ( . %); the mean increment was , x /l ( , - , x /l). efficacy was determined by the number of patients who survived an infection episode and amounted to . %. in the cases of severe sepsis, the efficacy of transfusions was . %. the frequency of post-transfusion reactions was %, (n ): febrile nonhemolytic reactions , % (n ); pulmonary complications , % (n ); the anti-hla antibodies formation , % (n ) conclusion: no significant differences in the mobilization of granulocytes and products' volume, between men and women, or between age groups and we did not find significant relationship between baseline wbc and their increment after stimulation. the mean total wbc in the collected product and the mean volume of treated blood from men and women were significantly different. perhaps the reason for lower cell products from women is to treat smaller blood volume. the lack of increment wbc after transfusion is not equivalent to lack of its efficacy. we found a significant association between transfused dose and increment of wbc. we deliberately did not estimate our data on the treatment of patients with sepsis and other severe infections between patients receiving and not receiving granulocyte transfusions because the need for transfusions of granulocytes indicates more severe patients, when they have infections in a neutropenia or dysfunction of the immune system, and refractory to antibiotics cp hemolytic disease of the newborn due to anti-rh keegan barry-holson* and jennifer andrews . stanford university, dept of pathology, stanford university, dept of pathology & pediatrics background/case studies: anti-rh is a rare antibody against a high incidence red blood cell (rbc) antigen (ag) present in people with common rh phenotypes. this ag is missing in individuals who are rh null or have rh deletion phenotypes, including d--/d--. d--individuals strongly express d and lack c, c, e, and e ags. the clinical relevance of anti-rh has primarily been reported in pregnant women, causing mild to fatal hemolytic disease of the newborn (hdn). we present a rare case of hdn due to anti-rh alloimmunization during pregnancy in a d--mother. study design/methods: an o-positive full term infant was born to an opositive g p > mother with a negative st trimester antibody screen and no prior transfusions. she had two prior pregnancies, the first resulted in a normal term singleton, and the second resulted in a spontaneous miscarriage during the st trimester. father's blood type is unknown but presumably he has rh antigens. the infant was transferred to our institution at hours of life because he was found to have anemia (hemoglobin . g/dl), severe hyperbilirubinemia (total bilirubin (t bili) . mg/dl), reticulocytosis ( %) and a positive direct antiglobulin test (igg ). he was admitted to our neonatal intensive care unit for potential need for exchange transfusion given concern for hdn. he was treated with intravenous immunoglobulin and triple phototherapy on the day of admission, temporarily blunting his hemolysis. t bili rose to a maximum of . mg/dl on day of life and phototherapy was restarted. his t bili subsequently stabilized and he was discharged home and followed in clinic. meanwhile, his mother donated blood given there were no compatible red blood cells available in the united states via rare donor query. nine days after discharge, he was readmitted for worsening anemia (hemoglobin . g/dl) and was given steroids and washed maternal red blood cells. he was discharged and followed in clinic for several months with ultimate resolution of his anemia and hyperbilirubinemia. results/findings: at delivery, the mother's antibody screen was positive and anti-rh was identified; no other alloantibodies were detected. antibody identification was performed using polyethylene glycol, low ionic strength solution and ficin enhancement. maternal serum was pan reactive against panel cells and non-reactive against d--cells. anti-rh sera did not react against maternal rbcs. phenotyping of the mother revealed that she was d c-e-c-e-. molecular testing confirmed her d--genotype; molecular beadchip test yielded no type due to low signal for e, e, v and vs ags. genotyping for rh variant and targeted genomic rhce testing failed to detect several rhce exons. father was unavailable for further testing. conclusion: we report a rare case of hdn due to anti-rh antibody in a d --mother. we hope to obtain further laboratory studies in maternal relatives given the rarity of this phenotype in the general population. these studies have important implications for genetic counseling for mother's sisters. management of severe autoimmune hemolytic anemia: a case report of an infant treated with manual whole blood exchange with rapid clinical improvement yunchuan delores mo* , cyril jacquot , valli criss , philippe p pary , jay greenberg , naomi lc luban and edward cc wong . children's national medical center, quest diagnostics background/case studies: management of severe autoimmune hemolytic anemia (aiha) presenting with life-threatening anemia is challenging, particularly in the pediatric population. mortality rates in aiha are typically low; however, in children, the rate may be as high as - %. although corticosteroids and immunomodulatory therapies are first line modalities, several case reports describe the use of manual whole blood exchange (wbex) to successfully treat aiha in older children and adults refractory to first line treatment. to our knowledge, this is the first case report in which an infant with severe aiha has been successfully treated with manual wbex in an acute care setting. study design/methods: case report format. results/findings: a month-old previously healthy female patient presented to the emergency department with hemodynamic instability and a nadir hemoglobin (hb)/hematocrit (hct) of . g/dl/ . %. wbc counts ( x /l) were mildly elevated and platelet counts ( x /l) were within normal limits. her history was notable for upper respiratory tract infection days prior to the onset of anemia. laboratory studies on admission showed hyperbilirubinemia (total . mg/dl, direct . mg/dl), normal ldh ( u/l), and undetectable haptoglobin (< mg/dl) indicative of ongoing hemolysis. clinical symptoms included diffuse jaundice, hemoglobinuria, lethargy, and emesis. she was admitted to the pediatric intensive care unit for further management, including right internal jugular central venous catheter placement due to poor peripheral vascular access. the patient's blood group was o, rh (d)-negative with a positive antibody screen and panel demonstrating a strong panagglutinin ( - reactivity) with positive autocontrol. dat was positive for anti-igg and negative for c despite a positive cold antibody screen. the patient weighed . kg with an estimated total blood volume of ml. she initially received simple transfusions totaling ml/kg of least incompatible group o rh(d)-negative rbcs with no incremental response. manual wbex was then performed with ml of reconstituted whole blood consisting of o, rh(d)-negative rbcs and ab fresh frozen plasma (ffp) to an hct of %, utilizing the central venous catheter. no adverse events took place over the course of the hour exchange. her one hour post-exchange hb was . g/ dl and a subsequent antibody screen demonstrated reduced intensity of the panagglutinin ( ). after initiation of steroid therapy (methylprednisolone, mg/kg/day), she continued to improve clinically. one week later, the patient was discharged home with a hb of g/dl. one month later, she experienced recurrent hemolysis requiring re-hospitalization, at which time she had normal igm and iga levels with markedly elevated igg levels ( mg/dl). at a subsequent follow-up visit months after her initial presentation, her anemia had resolved and she had been completely weaned off steroids. conclusion: we demonstrate a case of severe neonatal aiha successfully treated with manual wbex. the main advantages of wbex include removal of both autologous rbcs and plasma as well as infusion of allogeneic rbcs. in this case, manual exchange transfusion avoided the need for an automated apheresis procedure requiring citrate anticoagulation. in summary, manual wbex is a potentially safe procedure that may be performed in young children with severe aiha. abstract operating room, each experienced blood-colored urine, laboratory evidence of hemolysis, and acute kidney injury. clerical and serologic investigations revealed no cause for hemolysis. mechanical hemolysis from transfusion rate, catheter gauge, or a recently introduced one-way valve was considered. study design/methods: in vitro simulated transfusions were performed via syringe. measurements included hematocrit (hct), free hemoglobin, and visual hemolysis index. washed and unwashed red blood cells (rbcs) were tested with or without a one-way valve, using a or gauge (g) intravenous (iv) catheter. each one-way valve was used to test three identical samples. constant pressure was applied manually (rapidly, . /- . ml/ second) or with a mechanical syringe pump (slowly, ml/min). a subset of the manual transfusions was timed. control samples for baseline measurements were collected by gravity drip, without passing through the one-way valve or catheter. results/findings: the one-way valve increased hemolysis markedly during rapid transfusion using both catheters as well as both washed and unwashed rbcs (see table) . with the g catheter, the mean change in hct was - . /- . % with the one-way valve and . /- . % without (p< . ). comparing the one-way valves tested, differences in hemolysis were observed (change in hct; p< . ). during rapid manual transfusion with a g catheter and unwashed rbcs, hemolysis was greater for samples that took longer to transfuse . ml when using a one-way valve (change in hct versus time: r - . , p< . ) compared to a significantly different (p . ) slight increase in hemolysis for samples that took less time to transfuse . ml when not using a one-way valve (change in hct versus time: r . , p . ). correlations between time and hemolysis were similar, but insignificant using g with washed rbcs and the g iv catheter. conclusion: mechanical hemolysis should be considered when investigating possible hemolytic transfusion reactions, especially with high rates of transfusion and use of a one-way valve. during rapid manual transfusion with the one-way valve, greater resistance was associated with increased hemolysis. background/case studies: gerbich (ge) antigens expressed on glycophorin c are present in . % of the population. ge antibodies cause delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). ge antibodies also suppress erythropoiesis resulting in late-onset anemia. we report a case of hdfn due to anti-ge . study design/methods: a woman of paraguayan origin with prior terminated pregnancies presented at weeks gestation with passive anti-d and an anti-ge titer of . she was d-and ge:- ,- , by antigen typing. her obstetrician scheduled maternal blood collection near her due date for possible neonatal transfusion, but the woman went into labor at weeks. cord blood was dat positive for igg; the eluate confirmed anti-d and anti-ge . the birth hemoglobin (hgb) was . g/dl, reticulocyte (retic) was . %, bilirubin (bili) was . mg/dl; the infant was discharged. on day of life, the infant was referred to pediatric hematology for lethargy and poor feeding, with hgb . g/dl, retic . %, and bili . mg/dl. ge -blood was not available from the blood center or rare donor registry. the mother was b rh-and baby was b rh . obstetrics had to authorize maternal blood donation due to her hgb of . g/dl. maternal blood collection and rbc washing was expedited and the infant received ml of maternal rbcs within hours, at which time his hgb was . g/dl. post-transfusion hgb was . g/dl. one week later, the infant was symptomatic with hgb . g/dl, retic . %, bili . mg/dl. a nd aliquot of ml washed maternal cells was transfused. two weeks thereafter, the infant had hgb . g/dl, retic . %, anti-ge titer , and needed another transfusion. the maternal blood stored for just weeks had hemolyzed necessitating a nd maternal donation for baby's rd transfusion. at weeks, the infant's anti-ge titer was , hgb . g/dl, retic . %; no transfusion was necessary. at weeks of life, hgb was . g/dl, retic was . %, and the baby was thriving. results/findings: serologic studies at the hospital and reference blood center confirmed the antibodies and risk of anti-ge hdfn. molecular analysis revealed that the mother was homozygous ge -negative ge* .- , the father had homozygous wild type ge* , and the infant was heterozygous ge* /ge* .- . conclusion: the infant had hdfn due to antibodies to the high prevalence ge antigen. the continued need for transfusion was consistent with hemolysis and suppression of erythrocyte production caused by anti-ge . hemolysis of stored maternal blood was consistent with the absence of glycophorin c. this case demonstrates that cooperative multidisciplinary care among the blood bank, donor center, obstetrics, and hematology in a rare case of hdfn resulted in a successful neonatal outcome. background/case studies: patient blood management is a collaborative approach to optimize transfusion therapies to improve patient outcomes. in pediatrics, blood management is not 'one size fits all' given the paucity of clinical trials to guide evidence-based practice. in addition, pediatric care encompasses a very heterogeneous patient population such that applying one set of guidelines is difficult. because there are no standard, evidence-based clinical best practices regarding blood product usage in all children, unnecessary variation is occurring at our institution. we designed a robust analytics process to study baseline clinical practice and examine blood product usage, and plan to target the three pediatric sub-specialties with highest usage to establish standards in order to decrease variation/unnecessary transfusions. study design/methods: a data base encompassing all admissions and outpatient visits to a large, tertiary care academic children's and women's hospital was established, and included all relevant patient demographics, diagnostic and procedural codes, attending physician and specialty for each visit/admission, relevant hematology/coagulation laboratory results and blood product orders. we focused on rbc orders given the tripicu randomized clinical trial results ( ) supporting a hemoglobin trigger of g/dl in stable critically ill children and ffp since anecdotally we noted many children receiving this product for only minimally elevated international normalized ratio (inr) values without bleeding. results/findings: in , , rbc orders occurred and the top three patient groups were: % in congenital heart disease patients, % in hematology/oncology patients and % in neonates in the neonatal intensive care unit (nicu). average hemoglobin of every patient was . g/dl as measured in the hours prior to rbc order placement. in , ffp orders occurred and the top three patient groups were: % in neonates in the nicu, % in congenital heart disease patients and % in pediatric intensive care patients. average inr of every patient was . as measured in the hours prior to ffp order placement. conclusion: we have designed a robust data base that is continually updated for children in a large, tertiary care academic children's hospital. this serves as an important benchmark in pediatric blood utilization, and we plan to leverage usage patterns to make relevant practice changes in the care of children with a heterogeneous set of illnesses. background/case studies: bacterial contamination of plts remains an ongoing threat to transfusion recipients. recently, a psoralen-based pr technology that reduces the replication potential of pathogens in stored plts was fda approved. we describe our approach to phasing pr-plts into our inventory, including preliminary results of an ongoing qa study of neonatal and pediatric (peds) recipients of pr-plts. study design/methods: before the arrival of pr-plt, we undertook an educational campaign for hospital administrators, it staff, laboratory staff, clerical/clinical aides, nurses, and physicians. we also contacted risk management and the hospital ethics committee. phototherapy devices used at our hospital were confirmed to be compatible with the psoralen-based pr-plt product. shortly following the arrival of pr-plt, we introduced day bacterial "safety measure" testing of our conventional (c-plt) supply. a peds qa study monitored plt utilization and adverse transfusion event reporting relating to both pr-and c-plt transfusions. this study evaluated neonates ( - months of age), infants (> - months of age) and children (> months- years of age) who received at least one transfusion of pr-plts. results/findings: risk management and the ethics committee agreed that both pr-plts and bacteria tested c-plts would be the hospital standard of care. pr-plts were phased in and transfused to patients based on abo compatibility and expiration date, per routine, without regard for patient age or medical condition. after months, pr-plt represented % of our platelet inventory (average daily plt inventory: units). we encountered no complications with the pr platelet phase-in, either from a clinical, informatics or logistical perspective. due to the dual inventory, many peds patients in all age groups were transfused with both pr-and c-plts (table) . two potential transfusion reactions (trs) were reported over the study period in teenage recipients, one associated with a c-plt and the other with a pr-plt. in both cases, the symptoms were ultimately attributed to an underlying medical condition. no rashes were observed among transfused neonates ( - m) who received any pr-plts and phototherapy. background/case studies: packed red blood cell (prbc) transfusions are believed to improve oxygen delivery particularly in vulnerable patients such as neonates and children. however, evidence shows that hemoglobin (hgb) in prbcs has increased oxygen affinity and thus reduced oxygen delivery to tissues due to decreased , dpg levels. standardization of prbc transfusion practices in this population and the scientific evidence on which current practice is based is limited. additionally, due to small transfusion volumes, infants may be exposed to multiple blood donors, increasing their potential for adverse events. study design/method: medical records of pediatric patients receiving prbc transfusion over a month period were retrospectively reviewed. a total of patients were identified as receiving allogeneic prbc transfusion. patients who received autologous blood (cell salvage) were excluded. patient characteristics, length of stay, prbc transfusion volume, pre-and post-transfusion hgb, and adverse events were collected. results/finding: the average pre-transfusion hgb was . g/dl with post-transfusion hgb rising to . g/dl. the mean prbc volume transfused was . ml using a dose of ml/kg for all patients. complications noted were; volume overload, thrombosis, fever/infection, hemolysis, necrotizing enterocolitis (nec), and death (table) . conclusion: evidence based transfusion guidelines are lacking in neonates and infants. a typical dose of - ml/kg in a kg patient, for instance, would translate into full prbc units (about ml) in an average size adult. the current standard dose of - ml/kg yields very high increases in hgb and may put these patients at risk of adverse outcomes, especially thrombosis due to increased blood viscosity. additionally, many of these patients received volume reduced products which delivers a higher hgb concentration per transfusion. dosing should be based on goal hgb and patient condition rather than weight based, though the hematocrit level at which the benefits outweigh the risks remains unclear. pneumoniae has rarely been associated with warm autoimmune hemolytic anemia, with only case reports suggesting this association. however, each of these cases is confounded by other findings in addition to a mycoplasma infection. we describe a unique case in which a pediatric patient has clear evidence of severe hemolysis, a very strongly reactive warm autoantibody, and clinical and laboratory evidence of a mycoplasma infection without a detectable cold agglutinin. study design/methods: the patient is a month-old, previously healthy female infant who presented to the hospital with a -week history of fever, fatigue, decreased appetite, and pallor. she was only treated with acetaminophen. she also developed clear rhinorrhea the day before hospital admission. at the time of her admission, laboratory testing (outside hospital) revealed a hemoglobin and hematocrit of . g/dl and . %, respectively, platelets of , , and a reticulocyte count of . %. all other elements of the complete blood count were within the normal reference range for age. a complete metabolic panel revealed no abnormalities except for a total bilirubin of . mg/dl with a direct fraction of . mg/dl. a filmarray respiratory panel (biofire diagnostics; salt lake city, ut) detected mycoplasma pneumoniae, while all other pathogens ( total) were non-detectable. the patient was started on a -day course of azithromycin (zithromax). results/findings: prior to rbc transfusion, blood bank evaluation revealed that the patient was o-positive and had a stronglyreactive antibody screen. further testing demonstrated an antibody reactive with all reagent red blood cells. the dat was strongly reactive for igg but very weakly reactive for c . an eluate was reactive with all reagent red cells tested. finally, a cold agglutinin study was negative with undiluted serum. in addition to starting azithromycin, the patient was given iv methylprednisolone. during her -day hospital course, the patient received rbc transfusions on the day of admission and several rbc transfusions thereafter (see table ). despite transfusion, her hemolytic process persisted, so she was infused with a dose of iv immunoglobulin on hospital day . her hemoglobin rose to . g/dl on hospital day and increased to . g/dl on hospital day . at that time, the patient was discharged from the hospital with instructions to wean her oral steroid dose over the next weeks. she was followed closely by the hematology clinic and was found to have a stable hemoglobin (up to . g/dl on day after her hospital admission) with no recurrence of her hemolytic process. conclusion: m. pneumoniae infection is a typical cause of cad and has only rarely been associated with warm autoimmune hemolytic anemia. our case demonstrates clear evidence of severe warm autoimmune hemolysis in a previously healthy infant. with the increasing use of multiplex respiratory viral and bacterial pathogen detection systems, the once rare phenomenon of a m. pneumoniae infection associated with warm autoimmune hemolytic anemia may become a more recognized entity. ) and may serve to more reliably reflect when the neonates at risk for hyperbilirubinemia. the difficulty in eliminating the cord blood testing is the neonatologists' reliance of using abo incompatibility as part of the neonates risk assessment rather than using the point of care bilirubin testing. currently the ts requires all positive dat tests to be communicated to the nursing staff immediately. given that the dat strength positively correlates with the percentage of neonates diagnosed with hyperbilirubinemia, the ts staff may also consider notifying nursing staff only for those patients whose dat is or . platelet and leukocyte immunohematology, testing and genetics table . of pairs, pairs were complete match ( / ), pairs were partial match ( / ), pairs were complete mismatch ( / ). the matching rate of hla-dpb in our study is %. conclusion: the matching rate of hla-dpb in / hla matched unrelated hematopoietic stem cell transplantation is low and the gene frequency of hla-dpb in unrelated hematopoietic stem cell transplantation was obtained ,which will help to study on the relationship between hla-dpb and unrelated hematopoietic stem cell transplantation. this work was sponsored by national science foundation of china ( ) background/case studies: thrombotic thrombocytopenic purpura (ttp) is caused by severely reduced activity of the von willebrand factor-cleaving protease adamts . therapeutic plasma exchange (tpe) as well as immunosuppression minimize the morbidity and potential mortality of this presentation. absolute immature platelet counts (a-ipc) have been shown to help diagnose and follow ttp patients' responses to therapy. we report the case of a man with relapsing ttp, low adamts with high inhibitor, treated with mycophenolate mofetil in which a-ipc-indicated an unexpected response to therapy. study design/method: a year old male with a -year history of ttp, presented with status epilepticus complicated by acute respiratory failure admitted with suspicion for relapsing ttp. patient had been treated in prior admissions with tpe, prednisolone, rituximab, and cyclophosphamide with clinical improvement. he was on mycophenolate mofetil maintenance therapy which he last received just prior to day of admission due to consistently low platelet counts, adamts < % and inhibitor of . . on day of admission platelet count was x /l which decreased within five days to x /l leading to initiation of daily tpe along with mycophenolate mofetil discontinuation just prior to tpe start. immature platelet fraction (%-ipf) and calculated a-ipc (%-ipf x platelet count) were obtained with daily pre-tpe cbc. a-ipc ratio was calculated from baseline. abstract results/finding: a-ipc and platelet count were x /l and x /l respectively. counts improved rapidly post-tpe initiation and after one tpe his a-ipc tripled to . x /l achieving the ratio of previously shown to be diagnostic of ttp. on day his a-ipc and platelet counts had improved to . x /l and x /l respectively. absence of anti-pf antibodies ruled out heparin-induced thrombocytopenia at this time. on day he had an unexpected decrease in both a-ipc and platelet count to . x /l and x /l respectively, worsening by day to . x /l and x /l respectively despite daily tpe. patient received additional tpes that failed to improve a-ipc or platelets which on day were . x /l and x /l respectively. a-ipc had remained at this level for days suggesting that the observed decrease was irreversible. adamts activity remained < % low with a high inhibitor. patient's clinical condition continued to deteriorate and family placed patient on comfort care. conclusion: ttp patients have low a-ipc and plt counts at presentation, with the former improving first post-tpe initiation. despite appropriate therapy leading to early improvement of platelet count, patient's counts declined rapidly leading to suspicion for platelet production suppression as indicated by the sustained very low a-ipc. in the setting of ttp, or relapsing ttp use of immunosuppression should be closely followed and a-ipc may aid in establishing early if therapy is affecting platelet production. application of luminex bead technology to detect hpa- a, hpa- a, and hpa- a antibodies su-dan tao*, ying liu, yan-min he, ji he and fa-ming zhu. blood center of zhejiang province, key laboratory of blood safety research, ministry of health background/case studies: detection of antibodies against human platelet antigens (hpas) is crucial for patients' refractory to platelet transfusion therapy. in the text, luminex bead coupled with anti-gpiib/iiia and anti-gpia/iia monoclonal antibody was implied to detect hpa- a, hpa- a, and hpa- a antibodies, and the sensitivity of luminex bead technology was compared with monoclonal antibody immobilization of platelet antigens (maipa) assay. study design/method: monoclonal antibodies p and gi , specific for platelet glycoproteins gpiib/iiia and gpia/iia, were separately coupled to luminex xmap beads. four standard sera, containing anti-hpa- a, anti-hpa- a, anti-hpa- a and anti-hpa- b respectively, were bought from nibsc; three negative sera without hpa antibodies were prepared from ab type blood donors. platelets (containing hpa- aa, hpa- ab and hpa- aa) were collected and reacted with anti-hpa- a, anti-hpa- a, anti-hpa- a and anti-hpa- b standard sera respectively, then the antigen-antibody reaction complexes were lysed and the lysates were incubated with luminex beads to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins. the beads-antigen-antibody complexes were then subjected to flow cytometric analysis on a luminex . the hpa- a serum was diluted to serial dilutions (from neat to / ) to test the sensitivities of maipa and luminex beads assay. the two methods were then used to test five blinded samples which were collected from fmait patients. results/finding: luminex bead technology showed that the mfi values of hpa- a, hpa- a, hpa- a standard sera samples reacted with the coupled beads were significantly higher than the negative controls ( . vs . ), which implied that the luminex bead technology could specifically identify negative and positive sera of anti-hpa- a, anti-hpa- a, anti-hpa- a. furthermore, because the platelet was hpa- aa, the hpa- b serum did not react with the coupled beads with mfi was comparable to negative control ( . vs . ). the sera were re-tested by maipa and the results of which were comparable to luminex bead technology, illustrating that detecting hpa antibodies by luminex beads technology was successful. the sensitivity of luminex bead assay and maipa to detect anti-hpa- a was / ( . iu/ml) and / ( . iu/ml), respectively. no cross-reactivity was observed with the samples containing hla, abo or other platelet antibodies. all results of five blinded samples tested by luminex assay showed that four sera were positive for gpiib/iiia antibodies which were consistent with maipa results. conclusion: the luminex beads coupled with gpiib/iiia and gpia/iia monoclonal antibodies could be successfully used to detect hpa- a, hpa- a and hpa- a antibodies via the epitopes on platelet glycoproteins. the sensitivity of luminex technology was higher than maipa technology. (ahus) is a thrombotic microangiopathy (tma) characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia and renal failure in the absence of infectious toxin. the literature suggests the presence of pathogenic mutations in complement proteins in % of cases of ahus. there is a lack of well-defined recommendations regarding testing for genetic ahus. complement pathway mutation analysis is an expensive test so appropriate utilization is crucial to prevent undue health care costs. we reviewed the indications for genetic testing to understand physician ordering practice and determine the frequency of pathogenic mutations in the population. study design/method: we performed a retrospective review of all cases referred for complement pathway mutation analysis to a national reference laboratory from january to december . clinical history was solicited by genetic counselors. cases were classified by the authors as primary ahus (tma and renal failure without identifiable cause), secondary tma (tma and renal failure with identifiable cause previously associated with tma) or non-tma. the test panel identified variants in complement proteins (cfh, cfi, mcp, factor b, c , c bp, thbd, dgke, cfhr , cfhr , cfhr and cfhr ) that were classified as vus (variances of uncertain significance), pathogenic or benign by the american college of medical genetics. chi square analysis/fishers' exact test was used to determine differences in proportion of patients with pathogenic mutations and primary ahus versus secondary tma. independent sample t-test was used to compare differences in continuous variables between primary ahus and secondary tma. results/finding: of patients tested, pathogenic mutations were detected in % ( / ) and vus in % ( / ). % ( / ) of patients did not fulfill criteria for tma; no pathogenic mutations were found in this group and ( %) had vus. % ( / ) of patients had primary ahus; of these, % ( / ) had pathogenic mutations and % ( / ) had vus. % ( / ) of patients had secondary tma; of these, % ( / ) had pathogenic mutations and % ( / ) had vus. in patients with pathogenic mutations, % ( / ) were children, . % ( / ) had a positive family history of ahus and % ( / ) had recurrent disease. patients with primary ahus had a significantly lower age at presentation ( vs. yrs; p-value: . ) and a higher proportion of pathogenic mutations ( % vs. % p-value: . ) compared to patients with secondary tma. gender distribution, hemoglobin nadir and serum creatinine levels were similar between the two groups. conclusion: we found a lower frequency of patients with pathogenic mutations compared to reported literature. our data suggests that patients with secondary tma should be carefully evaluated prior to ordering genetic testing and those without tma should not undergo this test. counting of platelets in platelet concentrates on hematology analyzers pentraxl and sysmex xn compared with a flow cytometric method farshid ezligini , kjersti roen eriksen , annette vetlesen , thomas larsen titze and geir hetland* , . oslo university hospital, university of oslo background/case studies: hematology analyzers are made for counting of whole blood samples but are often used for quality control of blood components such as platelet (plt) concentrates (pcs). a flow cytometric method for counting of plt in pcs has been developed as validation tool (van der meer et al, transfusion ). therefore, it is pertinent to evaluate plt counting in bcs on hematology analyzers with this validation method in a flow cytometer. study design/methods: samples from ten apheresis pcs and buffy coat-derived pcs were subjected to plt counting on hematology analyzers pentraxl (horiba abx, montpelier, france) and xn sysmex toa (kobe, japan) (both impedance score), and additionally, diluted and stained with anti-cd a fitc in truecount tubes (bd biosciences)(internal bead standard) for measuring in a gallios flow cytometer (beckman coulter, indianapolis in, usa). results were analyzed by paired samples test and shown in bland-altmann plots. results/findings: mean plt values x /l sd were , (<) , (<) and for counting by sysmex toa, pentraxl , and the gallios flow cytometer, respectively. sysmex count was the very lowest a transfusion vol. supplement s abstract ( . % less than for flow cytometry), but all plt counts were significantly different (p< . ), although least so ( . %) between pentra and flow cytometry. conclusion: as validated by the flow cytometric method, pentraxl seems suitable for routine quality control of pcs both because of the small difference and lower counts compared with flow cytometric method, which is too cumbersome in a routine setting. the much lower plt count on sysmex may reflect its optimization for plt counting in whole blood rather than in pcs. fast, precise & easy hpa typing with real-time pcr jonathan downing , arishma lata , roland russnak , zachary antovich , heather dunckley and thierry viard* . new zealand blood service, linkage biosciences background/case studies: the interaction of membrane-bound plateletspecific glycoproteins with the extracellular matrix plays a significant role in hemostasis. human platelet antigens (hpa) found within these glycoproteins can stimulate production of antibodies in recipients of transfused platelets or in fetus of mothers with incompatible hpa. thus, platelet incompatibility is associated with various forms of thrombocytopenia, posttransfusion purpura and other blood disorders. the new zealand blood service performs hpa typing on a pool of platelet donors to provide compatible transfusions where the need arises. the molecular basis of most hpas has been characterized as generally caused by a single-nucleotide polymorphism (snp). hpa typing has typically been performed using pcr-ssp, a method that utilizes time-consuming post pcr analysis steps. the aim of this study was to evaluate the use of real-time pcr-based techniques in a transfusion laboratory setting. study design/method: we evaluated a commercially available solution which consists of reactions that identify both variants of relevant snps located within hpa genes (hpa- through hpa- , and hpa ). genomic dna purified from blood samples, previously genotyped for hpa- ,- ,- ,- ,- and - by our in house pcr-ssp method were used in this study as validation samples. results/finding: results of the validation samples were % concordant with typing obtained by pcr-ssp. the real-time pcr approach overcomes the major challenges of hpa molecular typing by providing an automated solution resulting in increased laboratory productivity and decreased turn-around time. the analysis is facilitated by a software which generates the results. with less than minutes of hands-on set-up and no further operator intervention with the reagents, complete molecular genotyping results are provided in approximately minutes. further, since amplified products are never handled, the risk of laboratory contamination is significantly reduced. the real-time pcr approach with automated analysis was implemented by the new zealand blood service tissue typing laboratory in late and to date has tested dna samples from blood donors ( donors were tested in duplicate). concordance between the sample replicates was %. there were occasions where the assay had to be repeated, giving a repeat rate of . %. occasionally a reaction peak was insufficient to trigger the software automatic allele call and a manual interpretation was required. this occurred most commonly with the hpa- ( . %) and hpa- ( . %) assays. conclusion: real-time pcr with automated analysis provides an effective, robust an accurate method for molecular hpa genotyping. with its minimal hands-on time workflow, it is also very easy to implement and offers a cost effective alternative to classical methods used in a transfusion laboratory setting. genetic variation of cd antigen deficiency expression in jiangsu chinese han population qing chen* , jianyu xiao and chengyin huang . jiangsu province blood center, jiangsu province blood center background/case studies: cd has been implicated in the platelet refractoriness, neonatal alloimmune thrombocytopenia, and posttransfusion purpura, especially in the non-caucasian. cd deficiency varies widely among different ethnic populations, with the frequency of - % in asians and . % of african americans, respectively. however, there is little information on the molecular basis of individuals with cd deficiency in jiangsu chinese han population. study design/method: to investigate platelet cd expression levels and to determine the molecular basis of cd deficiency on the platelet surface of the han population in jiangsu region. cd expression levels on platelets were detected by flow cytometry among blood donors in jiangsu region. donors without cd antigen expression on their platelet surface were further to be determined the expression of cd antigen on their peripheral blood monocyte cells. the coding exons of cd gene and adjacent introns were amplified and sequenced in cd deficient individuals. results/finding: among these blood donors, cd -deficient and cd -expression individuals were . % ( / ) and . % ( / ), respectively. the frequencies of type i and type ii cd deficiency among the study population were . % ( / ) and . % ( / ), respectively. among individual with platelet cd expression, according to mean fluorescence intensity (mfi) value, , and individuals showed low, moderate and high expression levels of cd , respectively, and their mfis were . . , . . and . . (p< . ), respectively. the type i cd deficiency individual were heterozygous for - a>g and - c>g, respectively. among type ii cd deficiency individuals, two harbored a t insertion at position in exon which caused frameshift at codon ; one has a t>c exchange at position in exon which resulted in a tryptophan to arginine substitution at codon ; one has a a insertion before the th bp of the start codon atg in the promoter region; one were heterozygous for t>c and t>g, respectively. conclusion: platelet cd surface expression levels were diversified in the jiangsu chinese han population. the frequency of the type ii cd deficiency was higher than that in type i. the study findings indicated that the frequency of cd deficiency in the chinese population is slightly lower than that in other asian countries. background/case studies: cd -deficient phenotype can be immunized by pregnancy or transfusion, and involved in neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and other disorders. the frequency of platelet cd -deficient individuals widely varies among ethnic groups, with % to % in japanese, % in sub-saharan africans, . % in african americans, and . % in caucasians. although some studies of cd deficiency are focused on the asian populations, relatively little information has been reported in the chinese population. here we investigated the cd expression on platelets in large samples of the eastern chinese donors. study design/methods: peripheral blood samples were collected from unrelated platelet-apheresis donors in the eastern china. the expression of cd antigen on platelets was determined by flow cytometry using fluorescein conjugated monoclonal antibodies (fitc-anti-cd and peanti-cd ). the isotype control (fitc-mouse igg) was also analyzed to calculate a reference range of cd -nagtive phenotype. for those donors with cd -negative platelets, cd antigen expression on monocytes was analyzed further to distinguish between cd type i and type ii deficiency. flow cytometric parameters were statistically analyzed by mann-whitney test. the work was supported by national natural science foundation of china ( ) and zhejiang high-level innovative health talents. results/findings: the mfi (mean fluorescence intensity) of platelet cd in all samples showed a continuous distribution profile, and no obvious fluorescence-gap could be utilized to distinguish negative from positive phenotype. on account of this limitation, we classified the cd phenotypes using the (mean sd) of the background mfi observed in isotype controls. forty-three samples were detected as cd deficiency on platelet, in which one sample was cd negative both on platelet and monocyte. the frequency of cd type i and type ii deficiency in the eastern chinese donors was . % and . %, respectively. the average mfi of cd deficiency samples was significantly lower than cd positive samples ( . . vs . . , p< . ). conclusion: the frequency of platelet cd deficiency in the eastern chinese donors was close to japanese and african americans. it means that the possibility of cd antibody occurred by pregnancy and transfusion in this population is existed. it is useful to find and register cd -deficient donors by large-samples screening for potential immune thrombocytopenia patients with cd antibody. background/case studies: cd (gpiv, chromosome q . ) is an kda glycoprotein expressed on multiple cell types including platelets (plts), monocytes (mono), & erythroblasts. although rare among whites, cd deficiency (cd -n) is observed in - % of africans (t g) & is classified as either type i (cd -n plt, cd -n mono) or type ii (cd -n plt, cd mono). an acquired type ii cd -n phenotype can also be observed in the setting of myelodysplastic syndrome (mds). type cd -n individuals can develop anti-cd alloantibodies with plt refractoriness & neonatal alloimmune thrombocytopenia. we report a case of profound plt refractoriness caused by anti-cd in a patient with newly diagnosed mds. study design/method: hla antibody testing was performed with a commercial bead-based fluorescent assay. cd phenotyping (plt, mono) of patient & family members was performed by flow cytometry (fc). cd staining of bone marrow was performed by immunohistochemistry. plt crossmatching (plt-xm) was performed by the american red cross. pltspecific alloantibody testing & cd dna sequencing were performed at a commercial reference laboratory. results/finding: the patient was an year-old, group o african-american male who presented with blurry vision & lightheadedness. complete blood count findings were significant for hemoglobin . g/dl & plt count k/ml. bone marrow biopsy & cytogenetic analysis revealed multilineage dysplasia, - % blasts & a complex karyotype with del( )(q q ) consistent with mds. plt refractory work-up was initiated after repeated plt transfusion failures with corrected count increments (ccis) < . hla antibody testing was negative (class i panel reactive antibody (pra) %). the patient was plt-xm-incompatible with most donors ( / ). a trial of group o, plt-xm-compatible plts was unsuccessful (cci ). subsequent testing for plt-specific alloantibodies identified anti-cd . fc-phenotyping showed no cd on patient's mono or plt, consistent with type i cd -n. preliminary dna results show that the patient is heterozygous for t g. because cd -n apheresis plt were unavailable from blood suppliers, the patient's children & grandson were screened as possible donors: all showed normal cd expression on plts. trial of eltrombopag & romiplostim was attempted with no improvement in plt count. repeat hla antibody testing (day ) demonstrated new class i alloantibodies (pra %) in response to transfusion ( apheresis plts, rbcs). given his plt refractoriness & poor prognosis, the patient opted for hospice. conclusion: we describe a patient with cd -n & severe plt refractoriness in the setting of new mds, and q-chromosomal abnormalities. the absence of cd on plt & mono support congenital type cd -n although a contribution by the patient's underlying mds cannot be excluded. rapid platelet donor classification: hla & hpa profiles by "leansequencing" without dna purification dipika patel , kristopher fernandez* , eric senaldi , pascal george , michael seul and ghazala hashmi . biomolecular analytics, central jersey blood center background/case studies: prophylactic platelet transfusion is the standard of care for managing thrombocytopenia. in the emerging paradigm of personalized medicine, the selection of cellular products in accordance with patient immunomolecular signatures has the potential to reduce the rate of antibody-mediated platelet clearance and thus to improve treatment efficacy. while the benefits of customizing transfusion therapy have long been recognized (gmur http://bit.ly/ q heq), the routine, real-time selection of platelets by immunogenetic profile has remained impractical by current methods of dna analysis. to address this issue, we evaluated a process of platelet donor classification using buccal swab samples from apheresis platelet donors for determining the combined hla class i and hpa signature without dna purification using a novel "leansequencing" process. study design/method: under a study protocol and informed consent, we evaluated a process for collecting and classifying buccal swab samples from $ adult donors who had made ! donations in the previous months. samples (labeled with study barcodes) were shipped weekly to biomolecular analytics ("bmx") for preparation of "crude extracts" for leansequencing: this novel process combines a proprietary sample pooling strategy with a protocol that eliminates many traditional sample "clean-up" steps. briefly, after preparation of crude extracts, samples were amplified, pooled and analyzed (in separate runs) for , , , , , , , , , and for hla class (a,b,c) , the latter using a proprietary design that limits analysis to informative alleles in the hla sequence; this "information-theoretic" design permits direct allele and haplotype reconstruction using bmx-proprietary software. a subset of crude extracts was purified and analyzed side by side with positive and negative controls. results/finding: crude extracts from buccal swabs produced viable profiles for hpa as well as hla class i with significant savings in time-to-result. as an illustration, the table reports allele frequencies for platelet-antigens ("hpa") that are consistent with a predominantly caucasian or hispanic platelet donor population (http://bit.ly/ pdplf ) in hw equilibrium. similarly, hla-class i haplotype frequencies were determined. conclusion: leansequencing lends itself to the rapid determination of hla-class i and hpa signatures of platelets; the process with its streamlined lean protocol achieves additional time (and cost) savings by accommodating crude extracts produced from buccal swab samples collected and handled in accordance with the process validated in this project. the process could be readily implemented to another site using the elements and process developed. the "pool & plex" process and the early donor recruitment enables economies of scale for matched donor procurement. the serological characteristics and heritage background of a novel hla allele, hla-a * : chuan-fu zhu*, yong-hong song, xiang-min nie and wen-ben qiao. blood center of shandong province background/case studies: there are , hla alleles documented according to the imgt / hla sequence database in janury , and more than % of them were identified in the last years. besides sequences many of the novel hla alleles have not been analyzed their serological reactivities. hla-a * : allele was fist detected in our laboratory during our hla typing for china bone marrow donor program(cmdp). for further study, the serological characteristics and heritage investigation were performed. study design/methods: the routine hla tying for the potential donors from cmdp were performed by bi-allelic sequence-based typing method,using a commercial kit (rose europe gmbh, frankfurt, germany). in the case of no full matched hla typing results, group specific hlassure-se sbt typing kit (texas biogene inc., taipei, taiwan) was employed to identify the nucleotide sequences of the novel allele. fresh blood samples were collected of the proband and his family members with the consent, in order to nanalysis the serological reactivities and the possible haplotype associations to the novel allele. the hla serological specificity was indicated by one lambda(asn d)hla kit. results/findings: no full matched result was obtained at hla-a locus in hla typing for a donor,which suggested the possible existence of a novel allele. the latter nanalysis indicated that the proband have a nove nucleotide sequences at hla-a locus, the new sequences was most close to those of hla-a * : : : , but nucleotide substitution in exon , by nt c-a (codon acc-aac), which resulted in one aminoacid substitution ,thr-asn. the novel hla-a allele was officially named as hla-a background/case studies: anti-d is a frequent cause of hemolytic disease of the fetus and newborn (hdfn). as a rule, immunization occurs in d negative pregnant women, but occasionally anti-d is also observed in carriers of d variants. currently, maternal plasma analysis for determination of the fetal rhd status became an exciting new tool for the management of d-negative pregnant women, but one of the challenges in non invasive fetal rhdgenotyping is the presence of d variants in the pregnant women. we present a case of a year-old pregnant woman typed as ab , who delivered a baby affected by severe hdfn. the newborn was typed as b and presented a positive direct antiglobulin test (dat) with an anti-d identified in the eluate. the baby was treated by exchange transfusion and the mother's sample was investigated. study design/method: serologic testing was done by hemagglutination in gel cards. genomic dna was extracted from whole blood by spin column and all rhd exons were sequenced by sanger sequence method. results/finding: the mother's rbcs reacted with the four monoclonal anti-d used (igm clones p x and rum and the blends clones th ms and d d ) and were typed as c-c e-e . an anti-d was identified in her serum. molecular analysis showed the c>t and a>c in exon , the snp t>c changes in exon and the t>g nucleotide change in exon . the set of snps found is similar to the molecular background of dol , except for a>c change. conclusion: this novel set of snps found in this mother is related to a novel rhd allele leading to a partial d antigen involved in the production of an anti-d that can cause severe hdfn. this finding shows the need to elucidate the clinical significance of different rhd genotypes in various ethnic backgrounds. the and erytra v r (the routine reference platform) was performed. a total of immuno hematological tests ( abo/d grouping (including newborn samples), extended erythrocytic phenotype, antibody screening, antibody identification, dat) and crossmatches were performed on patient's whole blood samples. the erytra eflexis v r performance was evaluated according to a protocol that was designed to simulate the routine workload using the system in its two different configurations. concordance between systems was assessed and discrepancies were analyzed. the following performance metrics were assessed: time to first result (ttfr), turn-around time (tat) for the total workload from first result to last result (throughput, results/h), and manual "handson" time required as well as walk-away time, considering the two different configurations of the system. for the ease of use evaluation, different usability features were ranked and the number of steps and timing of the following activities were tracked: sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. a threshold for in vitrodetection of anti-d gamma globulin was also determined. v r analyzer and the reference method were obtained in . % of the abo/d tests (n ), , % of the antibody screening tests (n ), , % of the antibody identification tests (n ) and % of the dat tests (n ). there were discrepancies ( abo/d for the same patient, for antibody screening and antibody identification: in both cases, the erytra eflexis v r could conclude whereas erytra could not due to a poor reaction. use of the stat mode (incubator is reserved for urgent tests) proved its usefulness when testing several samples (time saving was more than min). detection threshold of the d antibody was assessed at . ng/ml ( . ui/ml) whereas the french recommendations are ng/ml. the possibility of interchanging the trays (reagents/sample) makes also possible to optimize the analyzer operation. the impressions of the technical staff were positive regarding esthetic and functional design, intuitive and easy use, as well as flexibility. v r results demonstrated velocity, sensitivity, as well as the ability to easily perform the routine workload of a medical analysis laboratory. erytra eflexis v r meets both the requirements for french regulatory in immunohematology and for iso accreditation. background/case studies: kell system antibodies inhibit erythropoiesis causing severe anemia in hemolytic disease of the fetus and newborn (hdfn). we report a case of hdfn secondary to anti-kpb that resulted in multiple intrauterine transfusions of kp (b-) donor cells and hemolytic anemia upon birth. case: a year old g p presented during her fifth pregnancy with anti-kpb with an initial titer measured of . by history, the anti-kpb developed during her third pregnancy which ended in a spontaneous abortion before antibody titers could be initiated. the patient's antibody titers peaked at during the fourth pregnancy which resulted in a healthy male without anemia or jaundice. . in the latest pregnancy, ultrasound was initiated with elevated middle cerebral artery doppler exams ( . moms) peaking at weeks. this resulted in three intrauterine transfusions. due to potential labor and the finding of reversed diastolic flow on middle cerebral artery doppler studies, a finding that has been associated with impending intrauterine fetal demise, caesarean delivery was performed at weeks gestation. the baby boy required phototherapy for hyperbilirubinemia. the indirect bilirubin at birth was . mg/dl with . g/dl hemoglobin. the baby typed as o positive, kp (b ) with a micro positive dat. the antibody workup revealed an anti-kpb. continued hemolysis required one more transfusion at weeks of age. the positive dat and passively acquired anti-kpb were no longer detected by weeks of age. his hemoglobin recovered to . g/dl with an indirect bilirubin of . mg/dl at weeks of age. all clinical signs of hemolytic anemia were resolved. study design/method: serologic testing included peg iat by tube methods. acid elution was performed using immucor gamma elu-kit ii. molecular testing was performed using immucor bio-array hea platform. results/finding: antibody identification on the mother was performed as well as alloadsorption studies to rule out other underlying alloantibodies. a new weakly reacting anti-s was detected on the day of the delivery. the baby typed as s positive however the anti-s was not detected in an eluate prepared from the baby's red cells. all of the intrauterine transfusion units were s negative. conclusion: to our knowledge only five case reports have been described for anti-kpb which resulted in moderate to severe hdfn. pregnant mothers with anti-kpb detected should be monitored closely. background/case studies: in some clinical cases, the c d-specific dat may be too insensitive to detect low, but significant levels of c d, or it may be inconclusive due to spontaneous red cell (rbc) aggregation. further, the dat is not well suited to quantify the number of immunoprotein molecules on rbcs, since a " " reaction corresponds to about molecules/ cell. a number of flow cytometric methods for the detection of rbc-bound c d have been published. however, these are mainly designed to quantify the fraction of rbcs with c d-sensitization. the aim of this study is to present a flow cytometric method for the quantification of the level of rbcbound c d. study design/method: ten microliters (ul) of : (after documenting experimentally that this amount ensured maximum binding of anti-c d) mouse monoclonal anti-human anti-c d (abcam, clone c ) were added to ul of a . % rbc suspension. after incubation for minutes at c, samples were washed x , and ul of : diluted anti-mouse-f(ab) -pe (ro , dako) were added. after incubation at c, samples were washed and resuspended before being acquired on a flow cytometer (becton dickinson facscanto ii). to enable calibration of fluorescence signals in antibody binding capacity (abc), a calibration standard (dako qifikit) stained with ro was run in parallel with all experiments. background fluorescence (in abc) was subtracted to yield net abc values corresponding to specific staining with anti-c d. the assay, in parallel with our routine dat (dc-screening i, id-card, gel card, biorad) was applied to a series of a rbcs stained with levels ( fold dilution, : - : ) of o serum with high titer anti-a. to estimate the normal range of rbc-bound c d, edta-stabilized samples from healthy donors were tested. finally, the assay was applied to a sample from a patient with clinical aiha with an inconclusive dat due to unspecific dat polyreactivity. results/finding: the correlation of the net level of rbc-bound c d (values ranging from to , abc) with level of -serum dilution (used to sensitize a rbcs) proved to be highly linear (logarithmic vs. logarithmic plot; r . , p < . ). compared with dc-screening , the sensitivity of the flow cytometric assay was superior. it detected c d sensitization at least dilution steps further. the median normal level of rbc-bound c d was abc (range - abc, n ). the assay enabled demonstration of specific c d-sensitization in the patient; the level of rbc-bound c d in the sample was significantly elevated ( , abc). conclusion: the presented flow cytometric assay is capable of quantifying the level of rbc-bound with a high degree of linearity and analytical sensitivity. further, it is capable of quantifying the level of rbc-bound c d in dat polyreactive samples. background/case studies: abo blood group system of red blood cells (rbcs) consists of a and b oligosaccharide antigens and anti-a and anti-b antibodies against these antigens, which are present in the sera of individuals who do not express the antigen(s)(landsteiner's law). because of the expression of those antigens on some epithelial and endothelial cells in the body, the abo matching is critical not only in blood transfusion, but also in cell/tissue/organ transplantation. in spite of the fact that both antigens and antibodies are involved, these genetic traits are specified by a single genetic locus of abo. forssman (fors) system is another rbc blood group system which consists in a glycosylation polymorphism specified by the gbgt gene. in humans, the abo and gbgt genetic loci are located on chromosome q , and the functional alleles encode a and b glycosyltransferases (at and bt) and forssman glycolipid synthase (fs), which catalyze the last biosynthetic steps of a and b, and forssman (fors ) oligosaccharide antigens. the molecular genetic bases for allelism of those two systems in humans have been well-elucidated. the abo and gbgt genes are also present in some other species in addition to humans. however, the presence/absence and functionality/non-functionality are species-dependent. molecular mechanisms/forces that created this species divergence, including human polymorphism, were unknown. study design/methods: utilizing genomic information available from gen-bank and ensembl databases, the gene maps of the chromosomal region surrounding the abo and gbgt genes have been constructed of vertebrate species. results/findings: extensive similarities were observed in the kinds, numbers, and orders of genes, as well as their chromosomal locations. however, numerous differences were also identified. these include chromosomal rearrangements, as well as the insertions and amplifications of specific genes. interestingly, the abo and gbgt genes were found located at the boundaries of chromosomal fragments that seem to have undergone frequent inversions/translocations during species evolution. conclusion: genetic alterations, such as deletions and duplications, are known to be prevalent at the ends of rearranged chromosomal fragments. therefore, the species-dependent divergence and polymorphism within species of those clinically important glycosyltransferase genes may have been resulted, at least partially, from unstable chromosomal structures neighboring those genes. alloimmunization despite phenotype matching in a patient with sickle cell disease and a complex rhce genotype jessica kneib* and emily coberly. university of missouri health care background/case studies: red blood cell transfusion plays an important role in the treatment of patients with sickle cell disease. sickle cell patients have a significantly increased risk of alloimmunization compared to the general population, and the standard of care is to provide phenotypically matched units for at least c, e, and k antigens to reduce this risk. unfortunately, the genotype and true alloimmunization risk may not always be accurately represented by the red blood cell phenotype, particularly in patients with complex partial rhce variants. study design/method: a year old female with a history of sickle cell disease, stroke, and iron overload presented for routine exchange transfusion. transfusion vol. supplement s the patient's blood type was o positive and her red cells had been previously phenotyped as c-, c , e-, e and k -. an antibody screen was positive, and antibodies against c and e antigens were identified in the plasma. the patient had only received phenotypically matched units negative for c and e antigens for all previous transfusions at our institution, based on her known red blood cell phenotype. blood samples were sent to a reference laboratory for molecular testing to look for partial rhce variants that might explain the antibody development. results/finding: molecular testing was performed to reveal the presence of two different partial rhce alleles, resulting in a predicted phenotype of d , c-, e-, partial c , partial e . the probable rhce genotype, rhce*ce-jal/rhce*ce g, results in partial expression of both c and e antigens. in addition to the known risk of alloimmunization against the absent c and e antigens, this result indicates that the patient is also capable of forming alloantibodies against the absent portions of both c and e antigens. based on these results, the patient's anti-e was determined to be an alloantibody and not an autoantibody. conclusion: although phenotypically matched units are standard of care for patients with sickle cell disease, the red blood cell phenotype may not accurately represent the alloimmunization risk in patients with complex partial rhce genotypes. in this case, molecular testing confirmed that the patient is at risk of developing alloantibodies against c, c, e, and e antigens. as the patient had already made alloantibodies against c and e antigens, it was determined that she would require units that were molecularly matched to her rhce variants for all future transfusions. this case demonstrates that phenotype matching for sickle cell disease patients may not be adequate to prevent alloimmunization in individuals with partial rhce variants. altered splicing in the rhd*weak d type allele associated with the skipping of exon in a pregnant woman and her newborn carolina bonet bub* , maria giselda aravechia , thiago costa , marilia sirianni , eduardo bastos , leandro santos , lilian castilho and jos e kutner . hospital israelita albert einstein, hemocentro unicamp background/case studies: rhd*weak d type is a variant commonly found in caucasians associated with a weak d phenotype. as previously reported (vege et al, transfusion ) the c. g>c change (p.g a), which characterizes the rhd*weak d type allele is a splicing variant that induces skipping of the whole rhd exon . we report an altered splicing in the rhd*weak d type allele associated with the skipping of exon in a pregnant women and her newborn with weak d expression. study design/method: the d antigen expression was evaluated with commercially available monoclonal anti-d reagents: blended igm/igg (clones th- /ms- ), igm (clones ms and p x ) and igg (ms ) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. rhd genotyping was performed with the rhd beadchip platform from immucor. direct automated sequencing of the rhd exons and flanking intron regions was performed by the sanger dideoxy method. results/finding: both pregnant women and newborn samples were phenotyped as d w c-c e e . the samples showed weak hemagglutination reactions ( / ) with all anti-d clones used. rhd beadchip results showed the ls* signal indicating a possible deletion of exon in both dna samples. sequencing showed the c. g>c change and the intronic c. - t>a and c. - t>c substitutions, which are associated to the rhd*weak d type allele. conclusion: our results showed that c. g>c associated with c. - t>a and c. - t>c variations had probably a functional impact on splicing inducing exclusion of exon in both dna from mother and newborn. this finding is important to develop assays and interpret genotyping results, as current guidelines do not recommend anti-d igg prophylaxis for women with weak d type . background/case studies: sickle cell disease (scd) patients require red blood cell (rbc) transfusions to minimize disease-specific symptomatology. previous studies have shown that more than % of children with scd receive at least one rbc transfusion in their lifetime. both simple transfusions and erythrocytapheresis are associated with increased risk of rbc alloimmunization. published literature is lacking on the frequency of alloimmunization and geographical associations in pediatric populations, which is made difficult to compare due to lack of standardized categorization of what represents a pediatric patient population across studies. therefore, we looked at the alloimmunization rates of pediatric patients with scd in the unites states (us) and other countries. study design/method: a literature search was performed for studies published on alloimmunization rates of scd pediatric patients including hbss, sickle beta-thalassemia and hbsc. we evaluated the overall alloimmunization rates as number of alloantibodies per transfused patient and alloantibodies per transfused units across world literature and compared them using chi-square analysis. results/finding: fourteen studies reporting data to derive alloimmunization rates of pediatric scd patients were found. these included eleven us studies with , patients and studies from other regions (brazil, egypt and france) with patients. majority of patients included in the studies had hbss disease. patients received either episodic, chronic simple transfusions or erythrocytapheresis. age range for the us studies was to years and for the other countries to years. available data from us studies included a total of alloantibodies, the most frequent of which were antibodies to c, e, kell, m, s and kidd antigens ( . %, . %, . %, . %, . % and . % respectively). alloimmunization rates were calculated as antibodies per patient in some studies and antibodies per transfused units in other studies. we evaluated rates using both approaches as per available data. us had an alloimmunization rate of . % ( . to . , % ci) vs. . % for non-us studies ( . - . , % ci) (p . ) and a transfusion vol. supplement s more alloantibodies per transfused patient ( . vs. . , p . ). similarly, the number of alloantibodies per transfused units in the us, evaluated from five studies, was higher compared to a large french patient cohort ( Á vs. . , p Á ). average number of rbc units transfused per patient in the us was also higher compared to data from france ( vs. , p . ). conclusion: despite limited studies available to compare alloimmunization rates in pediatric scd patients in the us and other countries, the overall rates are higher in the us. though no definitive reasons could be concluded from the available data, limiting the number of rbc exposures, i.e. units transfused in non-critical conditions could lead to lower alloimmunization rates. results/findings: a post-transfusion sample was referred to the irl for a trxn investigation. there were no clerical errors; however, hemolysis was present in the post-transfusion plasma/serum. abo/rh and crossmatches using lo-ion tm were repeated on the pre-and post-transfusion samples with no discrepancies. the post-transfusion dat was positive with a negative eluate. the hospital requested another unit before the investigation was complete. antibody identification on the post transfusion sample with lo-ion tm was negative. suspecting a weak antibody, additional investigation using peg tm on both samples revealed an anti-c. no additional clinically significant alloantibodies detected in the pre-or post-transfusion samples using peg tm . conclusion: the patient experienced an acute hemolytic transfusion reaction due to anamnestic interaction of anti-c in the patient's plasma/serum against c antigen on the transfused cells. anti-c was not detected by our routine antibody identification techniques. the mma confirmed anti-e, -m and -c were clinically significant. laura bailey* , melissa grohotolsky , lisa deblass , bala carver and kip kuttner . health network laboratories, miller keystone blood center background/case studies: the en a antigen is a high prevalence antigen in the mns blood group system. the antigens of the mns system are carried on glycophorin a (gpa) and glycophorin b (gpb). anti-en a is a rare immune igm/igg antibody made by individuals who lack all or part of the gpa protein. anti-en a has been implicated in fatal htr and hdfn. the en(a-)phenotype can result from either a rare deletion of the gpa protein or the even rarer m k phenotype. because individuals with the m k phenotype lack both the gpa and gpb protein their red blood cells type as m-, n-, s-, s-, u-, en(a-), wr(b-) and have reduced sialic acid. study design/method: year old white mennonite female g ,p presented to her midwife for prenatal care with the intent of home delivery. she had a positive antibody screen by solid phase at the hospital transfusion service. an antibody identification panel was done in gel. testing for antibodies against selected cells (u-and u var ) in tube with peg enhancement and phenotyping was done. based on mns phenotype, anti-en a was suspected. the specimen was referred to an immunohematology reference laboratory (irl). the testing included phenotyping with unlicensed antisera, ficin treated panels by tube technique, allogeneic adsorptions for antibody exclusion and identification and antibody titration. following identification of anti-en a by the irl the midwife was advised to refer the patient to a maternal fetal medicine specialist at an academic center close to the patients' home. the midwife was also advised to consider autologous blood donation and /or testing of siblings. results/finding: testing by the hospital blood bank demonstrated positive reactivity in the antibody screen. the gel antibody panel ahg phase resulted in panagglutination and a negative autocontrol, suggesting a high prevalence antibody. the phenotype was performed and determined to be m-, n-, s-, s-, u -. outdated u variant reagent cells reacted in peg igg phase ruling out anti-u. anti-en a was suspected and the sample was referred to the irl. allogeneic adsorptions were performed to rule out antibodies to common red cell antigens. lack of reactivity on a ficin panel eliminated the presence of anti-u,-wr b . phenotyping with unlicensed anti-u was negative and unlicensed glycine soja demonstrated reactivity, suggesting that the patient is en(a-). the patient's phenotype is consistent with the m k phenotype. based on the lack of reactivity on the ficin panel, the antibody was identified as anti-en a fs. since anti-en a is extremely rare, this specificity could not be confirmed due to the lack of en(a-) cells and appropriate antisera. the baseline antibody titer was at igg phase without enhancement. conclusion: this case study describes the workup of a rare antibody in a prenatal patient at a tertiary care hospital. studies performed after the patient was transferred closer to home confirmed the anti-en a (fs) and genotyping was performed. three titers were performed for the remainder of the pregnancy and held at . although anti-en a has been implicated in hdfn, a healthy infant was delivered without complications. this patient should be monitored closely through future pregnancies. autologous donation and/or sibling testing should be considered in order to provide compatible blood for intrauterine transfusion or transfusions at or after delivery. background/case studies: a year old caucasian male diagnosed with hemolytic anemia and no previous transfusions was referred to the immunohematology reference laboratory (irl) for antibody identification and rbc genotyping. initial serologic testing by the referring facility and the irl demonstrated anti-d, anti-c and/or anti-g specificity with a positive auto control and igg dat. anti-g has an anti-d, -c specificity and is most frequently found in rr individuals exposed to r'r cells. the g antigen is present on rbcs expressing either rhd and/or c and very rarely on d-c-g (r g r) cells. both rhce*c and rhd genes encode ser which determines g expression. rare rhd variant antigens lacking ser are g-. study design/methods: serologic evaluation included tube testing using gamma lo-ion tm (immucor, inc., norcross, ga) enhancement, elution studies (gamma elu-kitv r ii (immucor, inc.)), edta glycine acid treatment (gamma ega tm kit (immucor, inc.)), allogeneic adsorptions with papain treated intact rbcs, reagent and patient-derived rbcs and antisera. molecular testing was performed with bioarray precise type ivd hea assay (immucor, inc.). results/findings: molecular testing revealed an rhce*ce genotype (with a c-e c e-predicted phenotype) and an otherwise unremarkable rbc typing report. serologically, the antibody(ies) demonstrated an anti-d, -c, -g specificity in the serum and eluate using r o r, r r , r'r, r g r and rr cells. this patient is predicted to be r r (dce/dce) therefore, anti-c is possible but an allogeneic anti-d or -g is exceptionally unlikely. allogenic adsorptions using papain treated r o r and r'r cells excluded anti-c and anti-d, leaving anti-g as the only explanation of the initial findings. reactivity with the patient's ega treated (dat negative) cells against the "neat" serum, eluate and anti-g antisera confirmed auto anti-g. conclusion: warm autoantibodies are common findings and often have an rh specificity; however, these antibodies usually demonstrate a broad but weaker specificity in the eluate or in the serum when enhancements are used. this anti-g had no reactivity with g-cells. the differentiation of anti-g from anti-d and anti-c is generally academic as transfusion recommendations are the same: provide rhd-, c-units. it is relevant and clinically important to determine the presence or absence of anti-d in rhd negative women of childbearing age who present with an anti-g specificity. if anti-d is a transfusion vol. supplement s excluded these women should receive rhig as part of their prenatal care. in this case differentiating anti-d, -c from an auto anti-g was necessary to provide transfusion recommendations. providing rhd-and c-units to give serologically compatible rbcs could result in formation of an allogeneic anti-e. automated eluates: comparison of solid-phase red cell adherence and gel automated eluate testing jayanna slayten* , christa voliva , kathy fletcher , heather vaught and tracie ingle . indiana university health, indiana university health (iu health) background/case studies: acid eluates (elu kit ii. immucor. norcross, ga) are to be tested via tube iat method in parallel with the recovered last wash per the manufacturer's package insert. finck et al (immunohematology ; : - ) demonstrated acid eluates may be tested in other platforms such as manual gel microcolumn assay (id-mts.igg card. ortho clinical diagnostics. raritan, nj) and automated solid-phase red cell adherence systems (echo. immucor. norcross, ga). our study looked to compare the use of the automated gel microcolumn analyzer (vision, ortho clinical diagnostics. raritan, nj) to the solid-phase red cell adherence analyzer (echo, immucor. norcross, ga) for the testing of acid eluates in a regional midwestern transfusion service. study design/methods: twenty patient samples, less than days from collection and drawn in edta, were used to prepare acid eluates (elu-kit ii. immucor. norcross, ga) while retaining the last wash to be tested in parallel. two samples were > dat positive, were weakly dat positive and were dat negative. the prepared eluates were observed for color (bluegreen/bg, blue-brown/bb, blue-purple/bp), and the ph was documented for the prepared eluate (whatman . - . ph. whatman international. maidstone, england). the prepared eluates and last washes were tested on the vision and echo against an antibody screen. if the antibody screen was positive, the sample was tested against an antibody panel to determine specificity/pan-reactivity. prior to the eluates being tested on the automated platforms, they were spun for minutes twice to remove any rbc debris which could cause false positive reactions. results/findings: the eluates prepared ranged in color: bb, bg and bp. the ph of all eluates ranged from . - . with the highest percentage of eluates at a ph of . ( %). sixteen of the eluates tested yielded the same results in both automation platforms (concordance of %). four eluates with different results are summarized in table . conclusion: the study demonstrated that both analyzers may be used for eluate investigations. both methods yielded apparent false positive results on samples which were initially dat negative. the echo was more sensitive, yielding false positive results ( ) when the vision was negative, while the vision was false positive with one eluate with echo negative. there was no apparent association in the non-correlating eluate results in relation to color of eluate, age of sample when eluate was prepared, or ph of the eluate. a larger study may be able to better elucidate the apparent false positive results noted in this study between echo and vision eluate study. background/case studies: blood agglutination observed by landsteiner in led to the discovery of human blood groups. in the abo system > alleles have been described. the glycosyltransferase encoded by most results in weakened expression of a or b or the null (group o) phenotype. as testing methods and reagents improve, donors may appear to change their abo type. here we describe a frequent group o blood donor ( units over years) who is actually a w . study design/methods: donations were tested with the pk instrument (beckman coulter inc.). routine forward and reverse abo testing was used to investigate the discrepancy. molecular studies were performed by dna sequencing of abo introns , and and exons and . specific primers located in the flanking intron regions of the blood group gene were used to amplify relevant exons by pcr. the template used is genomic dna extracted from whole blood collected in edta. pcr-amplified exons are subjected to bidirectional dna sequence analysis using standard sanger dideoxy chemistry. seqscape software (abi) was used to analyze sequence data by comparing the obtained sequence to a reference sequence from ncbi. results/findings: serologic results are shown in table . tests with anti-a, -a , -b anti-a,b were negative as were the a cells in reverse testing. the results of dna sequencing of abo introns/exons are shown in table . the significant changes were found in exons and . in exon there was a nucleotide (nt) deletion of g which resulted in a shortened transcript due to a stop codon, and another nt substitution lead to the amino acid change gly ala. mutations in exon included a nt substitution causing a pro -leu change and a nt deletion c resulting in shortened transcript. conclusion: serologic testing of the donor plasma with a cells was nonreactive revealing the abo discrepancy. molecular testing confirmed the donor genotype is heterozygote a/o [abo*o. . /abo*aw. ] which predicts a w phenotype. normally, donor rbcs are tested with anti-a and -b and the reverse type confirmed by testing the with a and b cells. this abo discrepancy was caused by the presence of anti-a in the plasma causing the forward and reverse type to be interpreted as group o. according to fda guidelines, the donor is technically group a, and as such all donations need to be labeled as group a. the donor was contacted and instructed to cease donating blood for transfusion. if donations continue, the unit labeled group a would likely test as group o at the transfusion facility resulting in an fda reportable error. there are numerous reports in the literature of the relative insusceptibility of a cells to destruction by anti-a, however, there is one hemolytic transfusion reaction to a x blood transfused to a patient with a potent anti-a titer > : . (schmidt, nacarrow et al. ) . a review of transfusion recipients of the donor reported here did not reveal any untoward reaction after transfusion. a transfusion vol. supplement s extraction of gdna from edta-anticoagulated whole blood from pilot tubes derived from the unit. dna extraction from whole blood is performed on up to blood tubes using the biorobot universal system (qiagen). there is no information on the maximum acceptable age of the blood for this purpose, either from the vendor or in peer-reviewed literature. we set out to assess if blood up to days post collection yielded suitable gdna for downstream rbc genotyping. study design/method: edta blood tubes collected from random blood donors were used to extract dna from microliters of whole blood on day , and days post collection. blood samples were stored at - c before and after extraction. tubes were brought to room temperature and rocked before loading on the biorobot. extraction was performed using the mdx blood minikit (qiagen). resulting dna samples were assessed for gdna yield and absorbance a /a using a nanodrop (thermo scientific). the extracted gdna was tested using precisetype hea molecular beadchip ("hea", immucor) and failure rates on both the biorobot and the hea were assessed. results/finding: all three extractions were successful with no invalids (result ) on the biorobot universal report. no evidence of visible clots or splatter during extraction was noted by the technologist. out of the samples, samples were chosen at random and concentrations were measured using nanodrop for each of the extracted plates. dna concentrations ranged from . to . ng/ul. all readings with the exception of ( . ng/ ul) had concentrations > ng/ul. interestingly, the one that was < ng/ ul on day , yielded > ng/ul on day and post collection. over the next months, sets of samples were extracted and tested by hea. eighty-three ( . %) failed extraction and ( . %) failed hea. none of the samples that failed extraction were or days post collection; none of those that failed hea were days post collection; . % were > < days post collection. conclusion: based on these results it can be concluded that edta blood tubes up to days post collection can be used as a source of gdna for rbc genotyping without negatively effecting the concentration of the resulting dna samples and the validity of the resulting genotyping. case study: investigation of persistent negative antibody screens on patients receiving daratumumab raeann thomas , carlos villa , rachel davis-rauser* , helen carpenter and vrunda patel . university of pennsylvania, hospital of the university of pennsylvania background/case studies: daratumumab is an anti-cd monoclonal antibody therapy that received fda approval for treatment of multiple myeloma in . communications suggest all patients receiving therapy would have a positive antibody screen because cd is a common antigen expressed on red blood cells. currently, patients have been treated with daratumumab at a large academic medical center. a wide variation of reactivity was observed, including patients who were found to have consistently negative antibody screens. while there are several potential causes, neutralization of anti-cd antibodies could easily be tested by applying established techniques used for neutralizing antibody reactivity. study design/method: samples received were drawn as a standard of care. indirect antiglobulin testing was performed using solid phase red cell adherence and gel. neutralization was performed by adding equal volumes of negative daratumumab treated patients' plasma with positive daratumumab treated patients' plasma. a dilution control was made by adding saline to each positive patient's plasma. samples were incubated for hour at room temperature and antibody screens were repeated. serial two-fold dilutions were also tested to determine if the neutralization could be titered. testing was repeated using various positive patient samples to determine if negative/positive combinations resulted in different reactivity. results/finding: all control samples remained positive. positive/negative samples were negative in solid phase testing across all patient combinations at : dilutions. variable reactivity was observed in gel. serial dilutions showed that neutralization for negative patients was observed up to a : dilution. conclusion: results suggest that patients' plasma may have a substance that neutralizes the antibodies. there is a possible correlation with patients who have persistent negative antibody screens and patient response to daratumumab. additional studies are necessary to uncover how this correlates to patient outcomes. further studies using a standardized daratumumabspiked sample will be conducted. background/case studies: the mns blood group is a red cell antigen system located on glycophorin a (gypa) and glycophorin b (gypb). individuals lacking gypa or both gypa and gypb on their red blood cells may develop a rare antibody against the en (a) antigen. the en (a) antigen is a highprevalence antigen, located on gypa. we present a case with a rare red cell phenotype and alloimmunization to the en (a) antigen. a y/o g p at approximately weeks gestation was discovered to have an anti-en (a) antibody in her plasma on a prenatal type and screen. this was worrisome for both mother and fetus, as the en (a) antibody is of igg isotype and has been implicated in both acute and delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn) [ , ] . further testing with red cell antisera revealed that the patient lacked m, n, s, s, and u antigens. a multiplex, allele-specific, pcr platform we commonly use to detect the presence or absence of red cell gene sequences failed to amplify genes specific for the m, n, s, s, and u antigens. these findings were consistent with a null phenotype for both gypa and gypb antigens, i.e. m (k) m (k) phenotype. the patient's husband and father of her unborn baby demonstrated a m n-s s phenotype by the same serological and molecular means. given the exceedingly rare incidence of en (a-) individuals (positive frequency > . ), clinical encounters with alloantibodies to this antigen are limited in our experience and in the literature [ , ] . however, the existing data gives credence to its association with transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). the consensus in this case was to work her up as a high-risk pregnancy with frequent intensive monitoring which involved frequent monitoring of antibody titers. if transfusions were required for the mother or fetus, our options were to either search for rare units lacking the en(a) antigen via rare blood donor registries or directed donations from family members who match the patient's phenotype. at term, the patient underwent induction of labor and successfully delivered a health baby boy by vaginal route. the delivery was without event. no transfusions were necessary antepartum or postpartum. study design/methods: n/a results/findings: n/a conclusion: anti-en(a) is a rare antibody and there is limited data about its potential clinical sequelae, which is concerning in a pregnant woman. providing this patient with rare en(a) negative red cells via national or international blood donor registries would have been an arduous task if needed. this patient had many compatible family members available and willing to donate blood. the m(k) null allele (s) within this family is likely due to a genetic recombination among the gypa and gype genes rather than a mutation in both the gypa and gypb genes [ ] . this results in the absence of glycophorins a and b and the constitutive antigens of the mns blood group system. our patient was exposed to the en(a) present on glycophorin a on her unborn baby's red cells (inherited from father) in utero with subsequent alloimmunization. in conclusion, this case report demonstrates a clinical approach in identifying a rare anti-en(a) antibody in a prenatal sample. the clinical finding of a rare antibody in which there is limited data requires leveraging every resource available in order to predict its behavior and provide safe blood products to patients who may require it. background/case studies: transfusions are essential for patients with scd and thalassemia to maintain growth and development during childhood and to sustain good quality of life during adulthood; however, the development of red blood cell (rbc) alloantibodies and autoantibodies complicates transfusion therapy in such patients. routine phenotyping of blood recipients and the use of phenotype-matched blood units for transfusion has been useful to lower the occurrence of red cell alloantibodies in chronically transfused patients with thalassemia and scd. nevertheless, extensive phenotyping is expensive, laborious and cannot be performed in certain situations. the molecular understanding of blood groups has enabled the design of assays a transfusion vol. supplement s that are being used to better guide matched red blood cell transfusions and to maintain an inventory of units dna typed. based on this, our aim was to evaluate the clinical outcomes of molecular matching performed at different levels during years for patients with scd and thalassemia. study design/method: blood group genotypes were determined in dna samples from chronically transfused patients with scd, in patients with thalassemia and in dna samples from blood donors. laboratory developed tests (ldts), hea beadchip tm , rhd beadchip tm , rhce bead-chip tm , and sequencing were used to determine the genotypes among patients and donors. molecular matching was performed in levels: ( ) rh and k matching; ( ) extended matching and ( ) extended matching including rh variants. we considered the total of red blood cell units requested for each patient and a number of donations per year for the compatible donors. results/finding: according to the patients needs we performed molecular matching for % of our thalassemic and scd patients at level , % for scd patients and % for patients with thalassemia at level and % for patients with scd and % for patients with thalassemia at level . the patients were transfused with a median of . rbc units. after three years of molecular matching, we observed that this transfusion strategy avoided new alloantibodies development and hemolytic transfusion reactions in all studied patients. conclusion: molecular matching has shown clinical benefits to the patients with scd and thalassemia, contributing significantly to reduce the rates of alloimmunization to - % with c e k matching and < % with extended matching. improvements in the clinical outcomes of the patients have also been observed as shown by an increase in their hb levels and reduction in the % of hbs in scd patients, better in vivo rbc survival and diminished frequency of transfusions. allahna lilly elahie* and sandra fazari. hamilton regional laboratory medicine program background/case studies: the ideal manual backup method for an automated antibody detection system is an important choice. currently, our backup method is saline tube ( drops plasma, minutes incubation). the change to either a low ionic strength solution (liss) or polyethylene glycol (peg) method would reduce incubation time to minutes and specimen volume to drops, both important laboratory considerations. objectives of this study were to compare the relative sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of peg and liss, and to determine the most appropriate manual backup method for the existing automated solid phase system. study design/method: a total of specimens were compared utilizing: automated solid phase red cell adherence assay (sprca) with manual tube peg and liss, some samples were not sufficient quantity to test in liss. identification panels were used to determine: clinically significant antibodies, warm autoantibodies, and nonspecific reactions. calculations were based upon comparison to sprca. results/findings: a total of clinically significant antibodies were detected using sprca technique, as well as warm autoantibodies and nonspecific reactions. peg demonstrated the highest sensitivity and lowest specificity while liss was least sensitive and most specific for clinically significant antibodies. for warm autoantibodies, liss was more sensitive than peg with both being % specific. both reduced the detection of nonspecific reactions. while peg had more nonspecific reactions ( versus ), it identified more clinically significant antibodies ( ) than liss ( ). (table) conclusion: ultimately, the decision to choose a manual backup method must be based upon the highest sensitivity for clinical significant antibodies so as to minimize failure to detect one. peg was selected as the backup manual method even though peg has a higher sensitivity to nonspecific reactions. this study clearly demonstrates the interplay and tradeoffs between methods, which are important to understand and consider when making method choice decisions. comparison of thiol reagents in denaturing cd on rbcs patricia a arndt* , anthony salazar and regina m. leger . american red cross blood services, long beach memorial medical center background/case studies: monoclonal anti-cd , e.g., daratumumab (dara), which is used to treat patients with multiple myeloma, causes positive indirect antiglobulin tests (iats) due to expression of cd on red blood cells (rbcs). this serologic reactivity cannot be removed by adsorption so other methods have been developed to detect/identify underlying alloantibodies. one popular method is to denature the cd antigen by treatment of rbcs with thiol reagents, e.g., dithiothreitol (dtt) or aminoethylisothiouronium bromide (aet). chapuy et al described ( ) and validated ( ) ), and % aet (ph . ) as per the aabb technical manual, th ed. these treated and untreated rbcs were stored in alsevers at c and tested on days , , , and by two methods: ) polyethylene glycol (peg) iat using plasma from two myeloma patients who had received dara (plasmas from total dara patients were tested with reactivity - ), and ) flow cytometry using phycoerythrin (pe)labeled anti-cd . rbcs were also tested on days and or with a serum containing anti-k by peg iat. results/findings: the . m dtt in ph . pbs had a final ph of . and the ph of the commercial . m dtt was . . results are in table ; flow cytometry results from days , and (data not shown) were similar to those from days and . rbcs treated with . m dtt (both sources) or aet were nonreactive with anti-k and plasma from all dara patients and gave very low results (% positive events) with pe anti-cd by flow cytometry for up to days after treatment. rbcs treated with . m dtt reacted similarly to untreated rbcs with anti-k and dara plasmas, and showed only some weakening ( - %) of reactivity with pe anti-cd . background/case studies: clinically significant hemolytic disease of the fetus and the newborn (hdn) is often caused by feto-maternal rhd incompatibility. with the discovery of cell-free fetal dna (ccfdna) in maternal plasma, it became possible to determine the rhd genotype of the fetus using non-invasive techniques. however, the reliability of the non-invasive prenatal rhd test (nip rhd) is dependent on sufficient amounts of cffdna in the maternal plasma sample. recent studies show that the fraction of ccfdna in maternal plasma varies significantly between pregnant women and is inversely related to maternal body mass index (bmi). thus, high maternal bmi, may impair the validity of nip rhd. the aim of this study was to examine the effect of maternal bmi on the correctness of nip rhd and the correlation of maternal bmi with fraction of ccfdna to total free dna in the sample. study design/method: measurements of body height and weight of pregnant rhd negative women in gestational week were obtained from patient records and used for the calculation of maternal bmi. data on bmi were combined with the results from nip rhd (real-time pcr targeting rhd exon and ) and sample fraction of ccfdna (measured as threshold cycle [ct] value of rhd) to total free dna (measured as ct of ccr ) in gestational week . the correctness of nip rhd was determined by correlation with postnatal serological rhd determination. results/finding: a total of pregnant women were included. nip rhd was positive in / ( %), negative in / ( %) and inconclusive in / ( . %). compared to the postnatal rhd type, / ( . %) of nip rhd results were false positive (fp) and / ( . %) were false negative. in / ( %) of inconclusive nip rhd, the postnatal rhd type was positive. mean bmi (n ) at gestational week was . ( -and -percentiles: . - . ). there was no difference in mean bmi between individuals who tested inconclusive or false negative by nip rhd compared to the remainder (p , ). the fraction of ccfdna was calculated for randomly selected nip rhd true positive cases. median ccfdna ratio was . (the distribution had a highly positive skew, -and -percentiles: . - . ). there was no statistical correlation between bmi and fraction of ccfdna to total free dna (r , ; p . ). conclusion: neither the correctness of nip rhd test result nor the fraction of cffdna to total free dna appear to be correlated to maternal bmi with regard to maternal plasma samples drawn in the th gestational week. delayed hemolytic transfusion reaction due to anti-lan antibody: a case report. adla dh angelina*, suneeti sapatnekar and suzanne bakdash. cleveland clinic background/case studies: lan is a high-prevalence antigen and the sole member of the lan blood group system. anti-lan is a very rare igg antibody, with conflicting information regarding its clinical significance and potential for hemolysis. we report a case of delayed hemolysis in a patient with anti-lan antibody. study design/method: the patient's medical record and available literature were reviewed. results/finding: an year old man, o-positive, with a history of heart disease and bladder cancer was admitted for radical cystectomy. the antibody screen and panel were panreactive by multiple test methods (gel, liss, peg) with negative autocontrols and dat and a saline antibody titer of , suggestive of an antibody to a high-frequency antigen. anti-lan antibody was identified by a reference laboratory. only in , donors are lan-, but two frozen rbc units were locally available and transfused postoperatively. the patient's siblings were tested; one o-positive, lan-sibling was identified. nine months later, the patient was admitted for surgical management of metastases. at this time, the antibody screen was weakly reactive with cell and new antibodies were ruled out. blood conservation measures were instituted, including limited blood draws and cell salvage for surgery. due to bleeding during and after surgery, lan-rbc units were transfused over days, including rare donor units and units from the sibling donor. another surgical procedure was then performed; by post-operative day , the patient had symptomatic anemia with hemoglobin (hb) . g/dl and serially increasing troponin. no lan-rbc units were available. four rbc units untested for lan were transfused without adverse event; the units were presumed lan but crossmatch compatible and phenotypically matched for the patient's other antigens. a post-transfusion hb of . g/dl was maintained for days. the antibody screen was negative on day post-transfusion, but strongly panreactive on day , with a positive dat (igg , c ) and anti-lan antibody identified in the plasma and eluate. there was also evidence of extra-vascular hemolysis, including a progressive decrease in hb from . g/dl on day to . g/dl by day with no bleeding identified, and increase in total bilirubin and ldh (peak . mg/dl and u/l on day ) with normal haptoglobin. the patient was febrile with leukocytosis, but had negative cultures and no other evidence of infection. a lan-rbc unit was transfused on day with good response (hb . g/dl). the patient remained stable and was discharged to a skilled nursing facility days later. conclusion: transfusion of lan rbcs caused a resurgence of anti-lan antibody and a delayed hemolytic transfusion reaction days after transfusion. the rarity of lan-units may require a patient with anti-lan to be transfused with lan units, but close monitoring for delayed hemolysis is necessary even if the antibody is not demonstrable at the time of transfusion. delayed serologic transfusion reaction caused by auto-anti-f. karen yunker* , andrea gerner , lynne stewart , carol sostok , mollie bell and gregory r halverson . st. elizabeth healthcare, hoxworth blood center background/case studies: anti-f was first described in by rosenfield and coworkers in the serum of a hemophiliac who had been multiply transfused. the f antigen is comprised of the c and e antigens alligned in cis on the same chromosome, and is the th antigen assigned to the rh blood group system (isbt rh ). it is capable of causing significant transfusion reactions and mild hdfn. we report in this case a year old caucasian male, admitted for evaluation of suspected t-cell lymphoma, who appears to have had a delayed serologic transfusion reaction (dstr) due to auto anti-f. abstract study design/method: antibody screen and compatibility testing was performed by automated solid phase (echo and neo, lmmucor, inc). red cell phenotyping was done by standard tube testing with commercial reagents following the manufacturers instructions. molecular genotyping was performed using the bloodchip assay (grifols, san marcos tx). elution studies were performed using the elu-kit ii (lmmucor, inc.) results/finding: the initial antibody screen (as) was negative and the patient was transfused unit o-rbcs. two weeks later the patient received an additional o-rbc. within days the hgb had decreased from . to . g/dl, the as and direct antiglobulin test (dat) were now positive, and ounits were incompatible. anti-f was identified in the patient's plasma and eluate. three additional units were requested for transfusion. due to the rarity of o-f-rbcs, the patient was transfused r r (dce/dce) rbcs with no reported complications. the patient was discharged to follow up in clinic. molecular genotyping showed the patient was rhd deleted (rho* del) and had normal rhce (rhce*ce/rhce*ce) genes which predict a d-c-e-c e f phenotype. the rh phenotype and as was repeated on a sample collected days later. the c typing was micro positive, mixed field only after minute incubation. the other rh antigens were not mixed filed, and the as was non reactive. however, the dat was weakly positive with anti-lgg. no elution study was performed. conclusion: the expected post hour hgb increment from the receipt of a standard unit of blood should be near g/dl (or % hct.) throughout this patients hospitalization, the post-transfusion increments did not fully achieve this expectation. the first transfuion resulted in a . g/dl increase, and the second unit was only . g/dl. the last transfusion of units increased by only . g/dl. less than three weeks later, the rhc antigen typing was microscopic/mixed field only after extended incubation, indicating the removal of r r units was nearly complete. in a case from , ohto and kariyone (transf. ; vol , no. ) reported a cr Ásurvival study of f rbcs in a patient with anti-f. they showed that the initital survival of f cells was fairly normal, however, after days, there was a sudden increase of red cell destruction, and by day all f cells were cleared from the circulation. it is not unusual to find auto-anti-f as many have been reported, however, it is unusual to find the auto-antibody has caused the clearance of three units of f-negative blood. this patient will be monitored to see if the autoantibody recurs and determine if it still has anti-f specificity. background/case studies: use of dithiothreitol (dtt) treated reagent red cells (rrbc) is increasing in blood banks as an effective way to negate the interfering panreactivity caused by daratumumab, an anti-cd drug for treatment of multiple myeloma. daily preparation of dtt-treated rrbc for testing of individual patients is burdensome for the laboratory and may delay patient care. we evaluated the effectiveness of batch-prepared dtt-treated rrbc, stored up to days after treatment, in antibody detection tests. study design/methods: in-date rrbc (ortho clinical diagnostics, raritan nj) were selected based on phenotype to match the antisera to be tested. rrbc were treated with . m dtt (sigma-aldrich, st. louis mo) and stored in reagent red cell diluent. rrbc were tested with commercial antisera (ortho clinical diagnostics, raritan nj and immucor, norcross ga) per the manufacturer's instructions for specificities from the rh, duffy, kidd and mns blood groups (see table ). patient source antibodies (anti-d, anti-c) were also tested. testing was performed before dtt treatment, on the day of dtt treatment and up to days following the dtt treatment of rrbc. reactions were graded using standard serological grading of (negative) to (positive) reaction strength. stored dtt-treated rrbc were also observed for hemolysis during the storage period. results/findings: see table for a summary of results. commercial monoclonal and human source antisera, and patient source antibody, were reactive with the dtt-treated rrbc throughout the storage period. reactivity decreased by less than one reaction grade for all antisera and patient source antibodies tested. mild to moderate hemolysis was noted in the dtttreated rrbc's during the storage period. conclusion: dtt-treated rrbc showed adequate reactivity with various red cell antisera after storage for up to days. this suggests that dtttreated reagent red cells can be stored for at least days and used for the detection of alloantibodies with minimal effect on detection ability. batch preparation and storage of dtt-treated rrbc can increase testing efficiency and decrease turn-around-time when performing pre-transfusion testing for patients receiving anti-cd therapy. interference: more than just kell? marilyn stewart*, angela treml and geoffrey wool. university of chicago background/case studies: daratumumab (dara) is an anti-myeloma and anti-lymphoma agent that is known to interfere with routine blood bank antibody screening tests. dara is an igg monoclonal antibody that binds cd that is present on the red cell surface. at the university of chicago blood bank, we have seen many patients treated with dara and were showing this interfering reactivity. it has been well described that cd is a disulfidelinked molecule and its immune epitopes are disrupted by reducing agents such as dtt. we performed a validation of dtt-treatment of reagent rbc to abrogate dara interference. study design/methods: the validation was done to prove that dtt treated red cells could be used to screen patients receiving dara and still detect clinically significant allo-antibodies. screening cells and panel cells selected for dtt treatment were those rbc homozygous for clinically significant antigens, therefore allowing rule-outs of clinically significant antibodies in patient plasma. several patients that had received the dara drug protocol were selected for testing as well as many patients that had allo-and auto-antibodies (but not dara treatment). reagent screening cells and panel cells were treated with . m dtt prepared using the sop from judd's methods in immunohematology and the aabb technical manual. the treated cells were preserved between testing episodes using alsever's solution, stored at abstract - c, and observed for hemolysis (none was seen) for up to days. all immunohematology testing using dtt-treated cells was performed using gel methodology. untreated and dtt treated cells were tested with anti k before any patient testing was done. the untreated cells reacted - with the anti k, and the treated cells were negative. these controls were run and tested each time dtt treatment was done. thirty eight patient samples, including six dara patient samples were tested. results/finding: of the six patients who had dara interference in their untreated antibody screens, all samples had negative reactions with the dtt treated cells except one patient, which had weak reactions in one cell. this specimen was repeated three times and all repeats had weak positive reactions in the same cell. this sample was sent to the arc reference lab for dtt treatment and all clinically significant antibodies were ruled out. patients with allo-antibodies present in their plasma did react with the dtt treated cells as would be expected based on the underlying alloantibody, with the exception of newly formed anti-e antibodies in patients. plasma from these four patients with a nascent anti -e all showed no reactivity with dtt treated cells. plasma from fourteen patients with a long history of anti-e (greater than months) did react with the dtt treated cells. conclusion: dtt treatment eliminates dara interference as previously described, but also unexpectedly lessens the ability of treated cells to react with nascent anti-e. because of the negative testing with some of the alloanti e antibodies, dara-treated patients at ucm will be given both kell and e negative blood if they have immunohematology testing performed using dtt reagent cells. mahboubeh rahmani* , monique scott , garcia curtis , ellice wong , alexa j siddon and christopher a tormey . yale-new haven hospital, va connecticut healthcare background/case studies: benign ethnic neutropenia (ben) seen in approximately % to % of persons of african descent is characterized by neutrophil count of < . x /l with no obvious cause and no increased susceptibility to infection or any other adverse effect. at present, there is no laboratory assay used to identify this condition and it is generally diagnosed on a clinical basis. in this study, we investigated whether duffy (fy) blood group phenotyping would be a potentially useful modality to help identify patients with ben; such testing could potentially be used as a surrogate test to prevent unnecessary further work up including bone marrow biopsy in the correct ethnic and clinical setting. study design/method: cases included patients clinically diagnosed with ben; and controls were chosen randomly from the pools of patients from whom a cbc and type and screen were checked for any other reason. cases and controls were tested for the rbc antigens fy a and fy b phenotype using serologic methods. the fy phenotype, absolute neutrophil count (anc), white blood cell (wbc), hemoglobin level, platelet count, and medical diagnoses were extracted from the medical record. where appropriate, data were compared statistically using the mann-whitney u test with significance set at p< . . results/finding: subjects who were clinically identified as having probable ben included patients (mean age . ; all self-identified as african-american; / were male) and controls included patients (mean age . ; self-identified as african american; ( / male). all of the cases ( %) diagnosed with ben had fy(a-b-) phenotype. mean anc ( . x /ul) and wbc counts ( . x /ul) were significantly lower in the cases with ben and fy(a-b-) phenotype (p . and . , respectively) compared with controls (mean anc . x /ul ; mean wbc count . x /ul). there was no significant difference in mean platelet counts ( x /ul vs x /ul; p . ) or mean hemoglobin levels ( . g/dl vs . g/dl; p . ) between the two groups. none of the patients with ben had an accompanying marrow-suppressive hematologic disorder based on record review; however, subjects in the control group had accompanying conditions that were potentially marrow-suppressive including hepatocellular carcinoma, acute myeloid leukemia, and myelodysplastic syndrome. conclusion: testing for fy phenotype could potentially be used as a surrogate test in patients with chronic neutropenia in a correct ethnic and clinical setting for the diagnosis of ben. further studies regarding fy phenotyping comparing controls with neutropenia for any reason to our ben population are in progress to better determine the positive predictive value. these tests were compared to the provue for concordance. additional samples tested with anti-igg,-c d were correlated against tube testing for the dat and antigen typing for: c, c, e, e, and k. results/findings: the ih- had % concordance for all blood grouping assays. for ahg assays, the ih- detected an anti-jka e, anti-fya warm antibody, antibody to a high incidence antigen and a warm antibody that were missed by the provue. the ih- identified one additional anti-e not identified on the provue. discrepancies were also noted with the non-cord dat results. five samples were positive on the ih- with anti-igg,-c d vs. tube testing; reflecting the increased sensitivity of gel methodology over tube. the table below summarizes the results. conclusion: this study demonstrated that the ih- analyzer and associated ih-system tm gel cards are equivalent to the ortho provue. with random access capability, minimal operator touchpoints, broad test menu and excellent assay performance, the ih- is an ideal immunohematology system for the hospital transfusion service environment. chris elliott*, susan barnes, fiona lisle, debra smith and whitehouse natalie. background/case studies: the erytra eflexisv r (grifols) is a new fully automated, mid-size analyzer that performs pre-transfusion compatibility testing using dg gelv r technology. erytra eflexisv r analyzer performance, usability and adaptability to different workflows was evaluated in the routine environment of a large uk acute hospital transfusion laboratory. study design/methods: a comparison study was performed between the erytra eflexisv r and erytra (our routine system providing the reference platform). a total of tests were performed on , adult patient samples and donor red cell units. erytrav r eflexis performance was evaluated according to a series of scenarios designed to simulate routine workload using the system in different configurations. concordance between systems was assessed and discrepancies analyzed. time to first result (ttfr), overall turn-around time (tat) total workload from first result to last result (throughput, results/h), manual "hands-on" time and walk-away time were all recorded. for ease of use evaluation, we ranked usability features with number of steps and timing of activities including sample sort and loading, routine testing, post-run procedures, consumables used, and space requirements. fault recognition and messaging was assessed by simulating failures e.g. reagent absence. results/findings: blood grouping, antibody screening, antibody identification (using panels), direct antiglobulin test, red cell phenotyping and serological crossmatching were successfully tested. concordant results between the erytra eflexisv r analyzer and reference method were obtained in . % of samples tested. there were discrepancies, all antibody screening ( false positives, failure to detect a very weak prophylactic anti d and positive reaction not detected on the erytra but panels on both systems suggested a genuine anti cw). ttfr and tat depended significantly on a number of factors including; number and variety of tests requested and whether the stat functions were activated. the analyser seemed to prioritise antibody screening prioritization of the group, especially for stat samples, was considered preferable the laboratory team found the software easy to use with some improvements over existing ertyra software. physical design of the analyser was considered good with easy access to almost all areas. probe changing was quick and simple. while the analyser successfully flagged all error scenarios some messages were considered misleading and could be better phrased. conclusion: results showed the erytra eflexisv r offered a robust automated solution for routine transfusion testing. the device could comfortably deal with a medium laboratory (processing - group and screens per day). it is very flexible being able to deliver grouping, antibody screening and identification, dat, phenotyping and serological crossmatching ,compensating for its' single probe and wash station by clever use of incubators, centrifuges and design features. this allows a compact design with maximum flexibility without compromising on turnaround times cp evaluation of two monoclonal anti-e as reagents for the detection of the rh e antigen and its variants gregory a. denomme* , kathleen bensing , michael schanen , cindy piefer , randall w. velliquette , christine lomas-francis and connie m. westhoff . immunohematology reference laboratory, versiti/bloodcenter of wisconsin, immunohematology and genomics laboratory, new york blood center background/case studies: monoclonal antibodies are used as reagents for automated and manual phenotyping. false negative phenotypings have implications for variant antigens; e.g. altered c antigen mistyped as a cblood unit stimulating anti-c in a c-recipient. the development of new / / / cecf / / / rhce*ce or rhce*ce compound heterozygotes ce g ce g or ce c, g or ces or ceti / / / ce g ce c, g or c, g / / / ce g ces or cemo or ceek or ceek(var) or cern / / / ce c, g ce c, g or cemo or ceti / / / ce c, g/ce g/ g; ces/ceti; cear/ceek; ceek/cejal; cemo/cebi / / / total / / / a transfusion vol. supplement s reagents should include an evaluation of antigen variants to confirm fidelity. we evaluated two monoclonal anti-e reagents with comparator reagent using a large panel of molecular confirmed rh e variants. study design/method: two monoclonal anti-e clones, rd / and rd / , and a licensed comparator anti-e (p gd ms ), all from diagast (loos, france), were evaluated. rbc samples were either recovered from frozen storage (n ) or edta blood from donors (n ) and were tested using a manual tube method or on a pk automated platform. a score ( ) or greater was deemed acceptable for manual tube and a positive call for automated testing. results were tabulated by complexity of rhce*ce alleles (table ) . results/finding: the specificity of the monoclonal anti-e were confirmed using common rhce haplotypes: r r , r r , r r, and rr. twenty-one different rhce*ce alleles were included in the extensive panel: were rhce*ce that were in trans to rhce*ce; were various rhce*ce plus rhce*ce c compound heterozygotes; were rhce*ce or rhce*ce homozygotes; were various rhce*ce and rhce*ce compound heterozygotes. the comparator reagent was negative or unacceptably weak for rhce*ce alleles in trans to rhce*ce (rhce*cear, rhce*cemo, rhce*-cejal, rhce*cehar), with rhce*cear/rhce*ce c compound heterozygote, and with rhce*cecf homozygote. rhce*cear, rhce*cemo, rhce*cejal, and of rhce*cecf homozygotes were detected using the comparator reagent. rd / and rd / failed with and e variants, respectively (table ) . failure to detect the e variants was observed using both manual tube and automated methods for the comparator and the rd / clone. none of the reagents detected e antigen variant expressed on example of rhce*cehar/rhce*ce. conclusion: rd / and rd / anti-e reacted with more e variants than the comparator reagent. the e antigen encoded by rhce*jal and rhce*ar is not always detected when in trans to rhce*ce. however, double-dose expression was detected suggesting that the monoclonal reagents bind weakly to the respective altered e antigen epitopes. the e antigen encoded by rhce*cehar continues to be a challenge to detect. meihong liu*, teresita mercado, orieji illoh, maria rios and zhugong liu. obrr, cber, fda background/case studies: extended molecular typing of a large number of blood donors can increase the likelihood of identifying donor red blood cells (rbcs) that match those of the recipient. this is especially important in the management of chronically-transfused patients and patients with rbc alloantibodies. several high-throughput multiplex blood group molecular typing platforms have been developed to determine blood group antigen phenotypes. targeted next-generation sequencing (ngs) provides comprehensive sequence information focusing on specified genomic regions, and allows the simultaneous detection of genetic variants from multiple genes in a large number of samples. we developed and evaluated targeted ngs assays using two different target enrichment platforms for extended blood group genotyping. study design/method: two custom design platforms sureselect and halo-plex were used independently for preparation of probes that target the entire genes of blood group genes associated with the expression of blood group antigens from blood group systems. we used the illumina's hiseq / system to perform next generation sequencing first on sureselect-enriched genes from dna reference samples with average target design coverage of . %, and then on haloplex-enriched genes from dna reference samples with average target design coverage above . %. twelve samples were enriched and sequenced in both methods to allow a direct comparison. all reference samples were previously characterized for blood group genetic variants in these genes using taqman snp assay and sanger sequencing assay. serological data were also available for these samples. the ngs data were analyzed by clc genomic workbench. sequencing variants were detected and annotated using dbsnp database. blood group genotype calls by the two targeted ngs methods were compared with the reference results. results/finding: for the two targeted ngs methods, we evaluated and compared the target enrichment efficiency, off-target enrichment, quality of ngs, sequencing coverage, and genotype concordance. a higher percentage of the haloplex reads ( . %) were mapped to the target regions relative to the sureselect reads ( . %). the mean sequence coverage depth of the targeted bases was around x for sureselect method and x for haloplex method. some exons, such as rhd exons and , , rhce exon , ermap exons and , cd exons and , cr gene (most exons) and gypb exon , are consistently covered with less than x coverage by both sureselect and haloplex targeted ngs methods. both methods detected rhd gene deletion in a few representative samples. the genotype call concordance on blood group genetic variants was assessed by comparing ngs results to taqman genotyping and sanger sequencing results, and more than % concordance was obtained for both targeted ngs methods. incorrect calls were restricted to four complex blood group genes: mns, rhd, rhce and abo, and involved mainly heterozygous variants and indels. conclusion: using two targeted ngs methods, we have correctly detected more than % blood group genetic variants in selected genes. evidence rhce*cehar does not encode for rh (hr b ) antigen debra j bailey* , trina horn , paul mansfield , najmi qazi , pamela nickle , jessica keller , margaret a keller and jan r hamilton . background/case studies: the rhce gene has many variant forms, yet for many, the phenotypes encoded by these variant alleles is unknown or incomplete. new information can be elucidated when two altered alleles or haplotypes are expressed in an individual with subsequent alloantibody formation. the rhce*cehar allele was first described in and has a phenotype of c e c e w f w , g , hr w , hr , hr s , rh: , rh: with a partial d antigen expression. we describe new information regarding an rh haplotype that includes an rhce*cehar allele and its apparent rh: (hr b ) expression. study design/method: rbc typing was performed by standard tube methods with polyclonal and monoclonal antisera. antibody identification studies were performed by standard tube hemagglutination methods by published techniques. molecular immunohematology testing was performed on genomic dna extracted from whole blood and included hea, rhd and rhce beadchips (immucor) and pcr-rflp analysis for rhce c. c>g and rhd c. c>t. results/finding: a sample from an african american female with a history of an anti-e and anti-k was evaluated for unexpected antibodies. her red cell serologic rh phenotype on an untransfused sample was d c e c e . her plasma contained an alloanti-s and an antibody that reacted strongly with all random e k s reagent red cells except her own. the unidentified reactivity persisted following ficin and dtt pretreatment of reagent red cells. only d and dc red cells were non-reactive in initial tests. differential adsorption studies excluded antibodies to all other common antigens and hr b except e, s and k. when subsequent examples of e s k red cells homozygous for the rhd*diiia-ce( - )-d, rhce*-ce c, g, t haplotype (i.e., r' s /r' s ) and rhd*diiia, rhce*-ce c, g, t/ rhd*diiia-ce( - )-d, rhce*ce c, g, t (i.e., bastiaan genotype) were found to be non-reactive with the patient's plasma, the antibody specificity was determined to be anti-hr b . the patient's red cell antigen genotype identified the following probable rh haplotypes: rhd* , rhce*cehar and rhd*diiia-ce( - )-d, rhce*ce c, g, t. additional antigen typing of the patient's red cells with unlicensed antisera indicated an hr ( of sources) and hr b ( of sources) phenotype. conclusion: the rhd*diiia-ce( - )-d, rhce*ce c, g, t haplotype is one of the rh haplotypes expressed by the original hr b individual bastiaan. the hr b antigen status of red cells of individuals with the rhce*-cehar allele has not been described. we report an individual with the probable rhd* , rhce*cehar and rhd*diiia-ce( - )-d, rhce*ce c, g, t rh haplotypes and production of alloanti-hr b . the specificity of the alloantibody produced and the red cell hr b serologic antigen type supports the conclusion the variant allele rhce*cehar does not encode for the hr b antigen. excluding clinically significant alloantibodies in the presence of interfering antibodies with high-titer, low-avidity characteristics. background/case studies: high-titer, low-avidity antibodies (htla) are a group of clinically insignificant antibodies (ab) directed against highprevalence red cell antigens. they interfere with the exclusion of clinically significant red cell ab and crossmatch testing, leading to long work-ups and potential transfusion delays. we often use automated solid phase red cell adherence assay antibody panels (sp) when htla interference is seen by other methods, and undertook this study to determine its efficacy. study design/methods: a search of the laboratory information system database was conducted for patients with htla between / / and / / . all patient samples with available records of the full serological investigation were reviewed for testing method and results, with specific attention to the value of a given test method in permitting exclusion of clinically significant ab (rule out). results/findings: over approximately years, patients had htla established at least once by titration studies. serological investigations on a total of samples using a combination of gel, sp, and peg and liss tube methods, and occasional dtt and ficin panels, found that htla interference noted most frequently in gel (primary method) was, indeed, less often seen with sp. however, the proportion of cases achieving rule out on sp was no greater than that with peg testing (table) . for samples where rule out could not be performed with a combination of methods, patients were assigned to phenotype-matched transfusions, or testing was referred to a reference laboratory. reference testing on samples was successful in rule out in % of cases. in an additional patient samples, with negative antibody screens, htla were identified upon work-up for incompatible crossmatches. conclusion: sp is useful in avoiding interference from htla, but this conclusion is limited because sp was performed in only % of samples, and the inability to use select cell panels with sp made it difficult to complete rule out on samples containing multiple ab. peg testing was available for % of samples, and was at least as effective. further, manual testing allowed flexibility in selecting test cells when other ab were present. both sp and peg testing may be used alone or in combination to avoid interference due to htla, and can potentially decrease the number of patients requiring phenotype-matched units due to incomplete serological evaluations. background/case studies: the dau family of rhd alleles is characterized by c. c>t (p.thr met). the dau allele harbors only this change, is not associated with depressed or altered d antigen expression, and is the ancestral allele from which other dau alleles are purported to have evolved. srivastava et al (transfusion , : ) recently summarized serologic characteristics and associated anti-d alloimmunization for dau family alleles. we investigated two samples with the c. c>t change referred with weak d antigen expression. study design/method: serologic testing was performed by standard tube methods using licensed anti-d reagents and the albaclone partial rhd typing kit. genomic dna was isolated from wbcs and used in manual and array assays and for amplification and sequencing rhd. results/finding: sample was from a yo multiracial female. her rbcs reacted s at immediate spin (is) and in iat with immucor gammaclone and series and , and mi at is and in iat with ortho bioclone anti-d. rbcs did not react with of (lhm / & / ) anti-d in the partial d typing kit. this pattern did not match any of the defined partial d epitope patterns. rhd beadchip found no changes but rflp detected c. c>t characteristic of dau. rhd sequencing confirmed c. c>t and identified two adjacent changes, c. g>t and c. g>t (c. _ delinstt), in exon encoding p.gly leu. sample rbcs reacted w at iat with both ortho bioclone and quotient albaclone delta, but were non-reactive with immucor gamma-clone, series and , and quotient albaclone blend and alpha anti-d. papain treated rbcs were s in iat with ortho bioclone. these results suggested a d el like phenotype. rhd beadchip found no changes but rflp detected c. c>t. sequencing confirmed c. c>t and found a new c. c>t change (p.ser -leu) in exon . the c. t has not been reported, but c. g encodes a stop codon (p.ser stop) in japanese (vox sang , : ). conclusion: we report two new alleles: rhd with c. _ delinstt (p.gly leu) and rhd with c. c>t (p.ser leu), both also carrying the c. c>t (p.thr met) characteristic of the african dau cluster. d antigen associated with p. leu is a partial d antigen with a novel epitope pattern. the p. leu change is associated with a del-like phenotype, the first observed to our knowledge for a dau allele, and d antigen on the rbcs is not detected in iat by / commercial anti-d. the rhd nucleotide changes reported here are not in dbsnp database. this study brings the dau family of alleles to . the number and diversity of alleles in the dau cluster supports that the c. c>t change is a major ancestral african background allele (wagner et al, blood , : ). tae eun kim*. krc btri background/case studies: there have been the cases of anti-d alloimmunization caused by the transfusion of serologically d negative blood component. by analysis of genotype of the blood component, all of them were confirmed as asian type del. for that reason, the application of genetic analysis for the blood donor has been required in addition to serological assay. we established the algorithm for the genetic analysis of rhdin blood donors. in this study, we would introduce the experience of the application of the algorithm and the results in the preliminary test. study design/method: from september to present day we got samples of repeated blood donors who are known to be d negative, c positive and/or e negative from blood centers. we obtained the consent for the test from all of the donors who provided samples. as a genetic analysis, we accomplished polymerase chain reaction with sequence-specific primers (pcr-ssp) for the region of promoter, exon , exon and exon in rhd gene. based on the results of pcr-ssp, we discriminated the results into total rhd deletion, rhd-ce-d hybrid and rhd variant. when the results were discriminated to be rhd variant, we additionally analyzed the sequence of exon to confirm the existence of c. g>a and c. t>a variations. for the sample with indeterminate results, we performed sequencing for the full region of exon. when the result was confirmed to be rhd deletion or rhd-ce-d hybrid, the blood components were regarded as rhd negative. when the result was confirmed to be rhdvariant, the blood components were regarded as rhd positive. blood components were not supplied until the final results were obtained. results/finding: for the sample, we identified cases ( . %) of total rhd deletion, cases ( . %) of rhd-ce-d hybrid, and cases ( . %) of rhd variant. of rhd variant were determined to be asian type del with c. g>a variation. cases of rhdvariant were regarded to be unknown variation. conclusion: the frequency of rhd variant in this study was % higher than that of the general d negative donors not considering rhce phenotype in a previous study. for that reason, we considered that the genetic analysis of rhd targeting the donors of d negative, c positive and/or e negative is more efficient approach to identify rhd variant and better way to improve blood safety in the transfusion medicine related with rhd negative blood donors. lei fang tsai*, ping chun wu, shu hui feng, yi wen tsai, ming hung chen and shun chung pai. taipei blood center, taiwan blood services foundation background/case studies: certain abo subgroups or physiologic conditions may lead to mixed-field agglutination on abo typing among blood donors. the b phenotype was found to be the most common subgroup in taiwanese. however, it is hard to distinguish the b phenotype from other b subtypes also with mixed-field agglutination using routine serology without the genotype. this study aimed to evaluate if flow cytometric method could alternatively differentiate different b subtypes with mixed-field agglutination rather than using molecular genotyping. study design/method: blood samples from taiwanese blood donors exhibiting known common abo phenotypes were included to establish normal flow cytometric patterns and genotyped. blood samples (n ) from b subtype donors with mixed-field agglutination by routine serology (tube method and gel card) were further analyzed by flow cytometry and genotyping. flow cytometric method was performed by facscalibur flow cytometry using the gamma-clone anti-a and -b. for genotyping, exon and exon of the abo gene were amplified and sequenced. the abo*b . allele was confirmed by pcr-rflp analysis. results/finding: among subjects with b or ab phenotypes, were genotyped as abo*b . . the abo*b . group performed similar characteristic flow cytometric pattern and the profile was reproducible over time. the pattern showed the main population of cells expressed no b antigen, while a percentage ( . . ) of the rbcs exhibited b antigen levels diminishing with increasing of fluorescence. other subjects with b or ab subjects, genotyped as abo*b . (n ), abo*bw. (n ), abo*bw. (n ), abo*bw. (n ) and abo*bw. (n ), displayed flow patterns differed from the abo*b . group. the abo*bw. , abo*bw. and abo*b . subjects also showed a main population of cells expressed no b antigen and, however, less percentage of rbcs exhibited b antigen levels (< % in abo*bw. and abo*bw. subjects and < % in abo*b . subject). both abo*bw. and abo*bw. displayed a wedge-shaped pattern. conclusion: the flow cytometric method for the detection of b antigens on rbc might be useful in discriminating between b subtypes with mixed-field agglutination, especially abo*b . genotype. this approach could assist the serological abo subgrouping in clinical reference laboratory. frequencies and specificities of "solid-phase only" detected erythrocyte antibodies: is solid phase testing worth the headache? karen finegan*, karen gray, jill adamski, theresa kinard and qun lu. background/case studies: an effort to re-evaluate automated testing platforms (automated solid-phase red blood cell adherence vs automated gel column agglutination) was recently initiated due to the perception of excessive equivocal reactions from the solid-phase resulting in "unnecessary" workup at one site of a hospital system. the data available from parallel testing on solid-phase, gel, and peg performed at another cite of the same hospital system was collected and evaluated to determine the frequencies and specificities of "solid-phase only" detected erythrocyte antibodies and to see if solid-phase only antibody workup is necessary for patient care. study design/methods: throughout , the transfusion service used automated solid-phase red blood cell (rbc) adherence as the primary method for antibody screening and identification. all solid-phase antibody screen positive samples were re-tested using both gel column agglutination and peg method manually in order to determine which method should be used for antiglobulin phase crossmatch of rbc products. all antibody screen results on three methods and final antibody identification results were transcribed into a spread sheet and analyzed. results/findings: a total of patients were positive on solid-phase antibody screen and re-tested on gel and peg antibody screen. in % (n ) patients antibody reactivity observed in solid phase only and the concurrent gel and peg testing were completely negative. of them clinically significant rbc alloantibodies, warm autoantibodies, clinically insignificant antibodies were identified in % (n ), % (n ), and % (n ) of the cases, respectively. rbc alloantibodies identified in solid-phase only included anti-e (n ), anti-jka (n ), anti-k (n ), anti-jkb (n ), both anti-e and anti-c (n ) (see table ). conclusion: solid-phase only rbc antibodies are clinically important in a significant portion of cases (roughly in cases). workup for solid-phase only antibodies is not "unnecessary" workload. transfusion of corresponding antigen negative rbcs to these patients prevented possible hemolytic transfusion reactions. full-length nucleotide sequence of ackr alleles encoding duffy (fy) antigens in africans of ethiopia qinan yin*, kshitij srivastava, addisalem taye-makuria and willy a flegel. background/case studies: the human ackr gene (previously known as darc), comprising two exons and a single intron, encodes a multi-pass trans-membrane glycoprotein expressing the duffy blood group antigens (fy). the duffy protein acts as a chemokine receptor for various proinflammatory cytokines and for the malaria parasites plasmodium vivax and p. knowlesi. the study of fy variants in the low altitude and tropical gambela region is important, as malaria is endemic and the endogenous population is living in this region for a long time. in the present study, we determined the full length nucleotide sequence of the ackr gene encoding fy antigens in donors from ethiopia's southwestern gambela region. study design/method: edta-anticoagulated whole blood was collected from study volunteers in the gambela region (nct ). the whole ackr gene was amplified in one reaction covering , base pairs (bp). this primary amplicon was re-amplified using nested primers covering nucleotides. nucleotide sequence was obtained by sequencing reactions and manually annotated using ncbi refseq ng_ . . the sequencing covered bp of both exons, bp of intron, bp of '-flanking region, bp of '-utr, bp of '-utr and bp of '-flanking region and encompassed all the variations present in dbsnp and nhlbi esp databases. results/finding: among the samples, a total of snps, including one novel snp in '-utr were observed. snps occurred in the exons, in 'and 'flanking region, in '-utr and in the intron. all individuals carried the snp indicative of the common fy: phenotype; while individuals were homozygous and was heterozygous for the gata box mutation. no splice site mutation was detected. as individuals were observed as being homozygous or heterozygous for snp, we could unambiguously assign distinct alleles. in the remaining individuals with or more heterozygous snps, allele specific pcr is required to identify the alleles. conclusion: we sequenced more than . kb of the ackr gene and identified at least different alleles. the present study found that the vast majority of alleles ( / ) in the gambela population as defined by snps, were similar to the clinically relevant fy* n. allele, which in turn is defined by only snps at positions c. - t>c and c. g>a. out of the remaining alleles, were similar to fy* with the fy(b ) phenotype and was similar to fy* w. with the fy x phenotype. the high frequency of fy* n. ( %) in this study is similar to other studies conducted in western, central and south-eastern regions from gambia to mozambique ( %- %). a more detailed analysis, including other regions of ethiopia, will be useful to support transfusion care in the us for ethiopian-americans, the majority of whom may be of mixed ethiopian ethnical background. judith aeschlimann*, sunitha vege, christine lomas-francis and connie m. westhoff. immunohematology and genomics laboratory, new york blood center background/case studies: the homology, proximity, and inverted orientation of rhd and rhce on the chromosome favor gene conversion events. regions of rhd are transferred into rhce and conversely, resulting in hybrid alleles that encode novel or the absence of high prevalence antigens. rhd*diiia-ce( - )-d is the most common hybrid and is found in african blacks. it arose by conversion of exons - of rhce*ces into rhd*diiia and no longer encodes d antigen, rather (somewhat confusingly) encodes partial c antigen from the rhd locus. this hybrid allele is in cis to rhce*-ces, together known as the r's haplotype. we investigated atypical rh genotyping results in three samples; two associated with weak d typing and one patient with sickle cell disease (scd). study design/method: serologic testing was by standard methods. genomic dna was isolated from wbcs. all samples were investigated by hea precisetype, rhd and rhce beadchip, rflp, and rh-cdna sequencing. snp-specific sequencing was used to establish linkage/phasing. results/finding: sample (male) and sample (multiracial female), both c c e e (presumed r r ), presented with weaker than expected d typing; is and / at iat. rhd beadchip identified the common african rhd*diiia-ce( - )-d hybrid encoding partial c antigen with apparent conventional rhd in trans. these results did not provide an explanation for weak d antigen. hea indicated rhce*ce /ce, concordant with the rh phenotype, but c. c>g and c. g>t (heterozygous) was also detected. as rhce*ce with g and t has not been reported, rh-cdna analysis was done. transcripts from the rhce locus included one conventional rhce*ce in trans to rhce*ces with exons and replaced with rhd*diiia, and from the rhd locus, one conventional rhd and the hybrid rhd* diiia-ce( - )-d were found in both samples. sample (scd male), d c e c e , by rh beadchip had one conventional rhd and rhd*diii type , and rhce*ce g/ces. as rhd*diiia type has never been found with either of these rhce alleles, rh-cdna analysis was performed. transcripts representing a unique conversion event at the rhd locus, specifically rhce*ce( c) exons and had replaced those exons in the common hybrid rhd*diiia-ce( - )-d and expression of partial c antigen was lost these unique hybrid alleles have been deposited as genbank#: ky and ky . we report two different and novel complex rh rearrangements: two samples thought to be r r had a unique rhce locus representing a gene conversion into rhce*ces, designated rhce*ces-diiia( - )-ce. in kind, a sample genotyped as diii type rather had a novel rhd locus representing a gene conversion into the common hybrid, designated as rhd*ce c( - )-diiia( )-ces( - )-d . these represent novel events on the r's haplotype that can confound rh genotyping interpretations. interestingly, samples and have weaker than expected d antigen typing, despite the presence of a conventional rhd with rhce*ce [r haplotype (dce)]. it is important to further investigate samples with unconventional results when interpreting rh genotypes. high-frequency antibodies anti-lu(b-) and anti-yt(a-) in a multi-transfused patient: a case study nadia baillargeon*, carole ethier, cynthia parent, jessica constanzo-yanez, maryse st-louis, marie-claire chevrier and andre lebrun. hema-quebec background/case studies: a -year-old caucasian female was referred to our immunohematology reference laboratory (irl) for serological investigation. she was diagnosed with anemia, renal failure and cardiac history. her hemoglobin level was recorded at g/l. her pregnancy history was not provided. she had received units of packed red blood cells (rbcs) in the past including unit within the last months. none of the transfused unit was phenotypically-matched. the referring hospital obtained panreactivity in gel with liss-suspended rbcs and ficin-treated rbcs and negative direct antiglobulin test (dat) and autocontrol (at). study design/methods: abo/rh, dat and antibody identification were performed by h ema-qu ebec's irl according to approved techniques. in addition to liss-suspended rbcs and papain-treated rbcs, trypsin-treated and chemical-treated reagent rbcs such as dithiothreitol (dtt) were tested. alloadsorption were done using papain-treated allogeneic rbcs (r r , r r , rr). id core xt platform (progenika biopharm / grifols, vizcaya, spain) was used to analyse polymorphisms which determine antigens including carthright and lutheran blood groups. pcr-ssp (sequence specific primer) and pcr-rflp (restriction fragment length polymorphism) were also performed to verify the absence of the high frequency antigens yt a and lu b . sibling samples were also requested to conduct a family study. results/findings: initial serologic testing showed strongly reactive panels in gel with liss suspended rbcs, papain-treated rbcs as well as trypsintreated rbcs and dtt-treated rbcs but negative at in each media leading to a probable antibody directed against high-frequency antigen. alloadsorption procedure allowed the identification of an anti-jk a . a select panel of high frequency antigens absent in caucasian population was tested. the patient's sera react weakly with one jk(a-), lu(a-b-) reagent cell. in the meantime, genotyping results confirm the probable phenotype of the patient as jk(a-) lu(b-) yt(a-). additional testing in gel using trypsin and dtt differential effects on antigens lu(b) and yt(a) were performed to confirm antibody specificities. no rbcs unit jk(a-) lu(b-) yt(a-) were available for transfusion. selected units were jk(a-) and lu(b-) as alloanti-yt a are known to cause none to moderate transfusion reactions. her daughter' sample were types as yt(a ) and lu(b ). conclusion: serological study showed the presence of an anti-jk a in addition to two antibodies directed against high prevalence antigen namely anti-lu b and anti-yt a . the association of various selected serologic procedures combined with ethnic clues and genotyping results serves to solve this uncommon antibody combination. background/case studies: the kel blood group system, consisting of antigens encoded by the kel gene, is organized into exons. there are approximately kel alleles associated with a kell null phenotype (k ) in which no kell antigens are expressed, and alleles associated with a kell mod phenotype (k mod ). individuals with the k mod phenotype express very weak amounts of antigen on the surface of the rbc, and expression levels vary based on the allele present. here we describe the molecular and serologic testing that was performed in the case of a year-old hispanic male blood donor whose rbcs phenotyped k-k-js(b-) kp(b-). study design/method: the blood donor was phenotyped for k, k, kp b and js b antigens using standard tube agglutination methods. adsorption and elution studies of the donor red cells were performed using commercial anti-k antisera (american national red cross). genomic dna (gdna) was isolated from an edta blood tube using standard techniques. dna was genotyped for human erythrocyte antigens using the precisetype tm hea molecular beadchip (immucor). exons , , , and and flanking intron sequences were amplified and sequenced. total rna was extracted using rneasy lipid tissue mini kit (qiagen) and kel cdna was amplified and the resulting pcr product was subjected to sanger sequence analysis and aligned using sequencher (genecodes). results/finding: precisetype tm hea molecular beadchip testing predicted the sample to be k-k kp(a-b ) js(a-b ). kel-cdna sequence analysis was performed and detected a single transcript species with c. c, c. c, t, and missing the sequences corresponding to exons , and . amplification of the exons from gdna did not identify any nucleotide changes when compared to the reference sequence and the splice sites were intact. cdna analysis was repeated and the same aberrant transcript was detected. adsorption and elution studies of the k antigen demonstrated weak anti-k reactive after c incubation at the peg-igg-agt phase. conclusion: here we describe a donor homozygous for a novel kel* allele. this donor was presumed to have a k phenotype based on serology, but after molecular testing, has been reclassified as a k mod phenotype with extremely weak expression of k. the discovery of the aberrant transcript led to adsorption and elution studies to confirm the presence of weakly expressed k antigen on the red cells. the variant alleles reported to date (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-bloodgroup-terminology/) are associated with missense mutations. in contrast, the allele reported here is associated aberrant mrna transcript. we propose that this allele be named kel* m. . here we report a case of a possible novel b subgroup observed in a pregnant black female. the patient specimen was referred to our reference laboratory to investigate a possible abo discrepancy. the referring facility reported the patient's red blood cells were nonreactive with reagent anti-a and anti-b and the patient's plasma was reactive with a cells, but nonreactive with b cells using automated gel methodology. study design/methods: serological testing of the patient's red blood cells was performed using routine and enhancement methods. molecular testing by pcr-rflp was performed to determine the patient's genetic abo typing and predicted abo phenotype. results/findings: serological testing of the patient's red blood cells is summarized in table ; similar results were obtained with multiple sources of antisera. enzyme treatment failed to enhance reactivity. patient sera strongly agglutinated a and a cells, but failed to agglutinate multiple sources of b cells at all phases of testing. molecular testing by pcr-rflp resulted in an uncommon banding pattern and indicated the presence of c. deleted g, characteristic of o alleles, c. t, characteristic of a and some uncommon o alleles, and c. a and c. a, characteristic of b alleles. genomic sequencing of exons and confirmed the presence of an o allele, abo*o del g, t, t), and the presence of a b allele ( g, g, t, a, a, c, and a), but did not reveal any changes associated with previously reported weak subgroups of b. conclusion: while serologic abnormalities in pregnancy have been reported due to decreased antigen expression, the unusually weak reactions observed when testing this patient are unlikely due to pregnancy alone. additional abo gene sequencing is required to determine the specific allele mutation responsible for this weakened antigen expression. carine arnoni* , tatiane vendrame , janaína muniz , diana gazito , afonso cortez , lilian castilho and flavia latini . associa, associac¸ão beneficente de coleta de sangue, hemocentro de são jos e do rio preto, hemocentro unicamp background/case studies: after the elucidation of the molecular basis of vel, molecular tools have been used to explain the reduced expression of vel antigen in different populations. negative or weak reactions are generally related to the -bp deletion in smim in homozygous or heterozygous status. however, other nucleotide changes have been described to reduce the vel expression, as for example, the major a allele of the snp rs located in the second intron of the gene, a regulatory region in erythroblasts. this study aimed to characterize the genetic changes related to atypical vel expression in a brazilian population. study design/method: a total of blood donor samples from the southeast region of brazil were typed for vel with an anti-vel serum from our inventory in gel-iat. samples typed as vel-negative were further analyzed by adsorption-elution. molecular study was performed in samples with negative results, in samples reacting and in samples with reactivity of . dna was isolated from peripheral blood and smim was sequenced. results/finding: from donor samples studied, were serologically vel negative by gel-iat but positive by adsorption-elution, presented a reaction and the remained samples showed a reactivity of . genotyping results showed that the samples with negative results and of samples that presented reaction were heterozygous for the bp deletion and presented the a allele rs in homozygous status. from the of remaining samples with reactivity of , ( %) had the a allele of rs and ( . %) had the a allele of rs . in contrast, in the samples with stronger reactions we found the a allele of rs in ( . %) samples and the a allele of rs in ( . %) samples. conclusion: the molecular changes rs and rs are located in intron distancing nucleotides. this study reinforces the association of the a allele of rs with reduction of vel expression and suggests the involvement of a new rs change in vel expression. in conclusion, the several patterns of vel expression found in different populations can be influenced by different molecular changes. background/case studies: the d antigen is the most immunogenic antigen after abo. consequences of misclassification of the d-antigen in patients or donors can be severe. some persons inherit mutations resulting in quantitative reductions of d antigen on the cell surface (weak d), some inherit rh haplotypes that result in biochemical effects that reduce the availability of the d antigens to reagent anti-sera (ceppellini effect), and others inherit d genes which are qualitatively different than wild type d. these latter individuals are often not identified until after they have formed anti-d. we hypothesize that some of these persons at risk of forming anti-d might be uncovered if they have weak and/or disparate d typing results with reagents that recognize different epitopes of the d antigen. study design/methods: all testing was performed using microtiter-well direct agglutination on the galileoneo or galileoecho (immucor, norcross,ga). any specimen that did not react as (rh negative), or ! on the neo or ! on the echo (rh positive) for both series and series anti-d antisera were included. patients with discrepant historical types also were evaluated. any specimens meeting the inclusion criteria were tested on the neo, echo, and by saline tube method using series and series anti-d antisera. genotyping was performed from whole blood samples sent to immucor genotyping laboratory in warren, nj using an algorithm of: rhd beadchip, rhdxp (prototype assay), rhd zygosity, and rhce beadchip. results/findings: patients met inclusion criteria for molecular testing for the d antigen. weak or rhd variants were identified in of ( . %) of the samples. ceppellini effect (i.e. c in trans to rhd) resulting in weak d reactivity was seen in of ( %) of samples. of ( . %) of the samples that resulted in weak or discrepant reactivity had some type of genetic cause that was resolved by using our algorithm. of ( %) of tested samples had results indicating weak/variant d proteins with the potential to cause alloimmunization to the d antigen. the remaining of ( . %) samples did not have identified genetic cause for the weak and/or discrepant d test results and were presumptively classified as wild type d. conclusion: transfusion services that use the galileoneo or galileoecho to perform rh typing should consider molecular testing of patients whose rh typing results are discrepant, or positive but < on the galileoneo or positive but < on the galileoecho, as about half of these patients can develop anti-d. this is particularly relevant for females of child-bearing potential where avoidance of d-positive transfusions and administration of rhig during pregnancy is prudent until their d typing can be confirmed by molecular testing. carine arnoni* , tatiane vendrame , janaína muniz , rosangela person , lilian castilho , afonso cortez and flavia latini . associa, associac¸ão beneficente de coleta de sangue, hemocentro de são jos e do rio preto, hemocentro unicamp background/case studies: rhd and rhce, are major protein constituents of red blood cell membrane, composing a complex together with rhag. many variant rh proteins have been described and most of them affect the integration of rh proteins in the membrane. d antigen expression can be affected by several molecular changes and also by the rhcehaplotypes. the present study investigated the score of reactivity of samples presenting a strong reduction in d expression. study design/method: a total of samples were included in the study, being previously genotyped as rhd*dar . , rhd*dar . and rhd*dau . the samples were phenotyped in neov r (immucor) to d, c/c and e/e antigens by direct agglutinationin microplate. results obtained in neov r were expressed in a score from - corresponding to the reaction intensity. zygosity assay was performed by a multiplex real-time quantitative pcr using a set of rhd-specific primers in rhd exon . rhce genotyping was performed by pcr-rflp and ssp-pcr. the presence of a d-cehybridexon was identified by amultiplex pcr. sequencing and identification of rhce variants were also performed when necessary. results/finding: zygosity results showed that of samples ( dar . , dar . and dau ) had rhd genes, were phenotyped as c e-c e and genotyped as rhce*ce/rhcece. rhd and rhce genotyping in these samples showed the presence of the d-ce-d s hybrid gene. rhce variants investigated in dar . samples showed the rhce*-cear/ce s genotype, in dar . samples the rhce*cevs. /ce s genotype and in the dau sample the rhce*ce s /ce genotype. table describes the differences found in the reactivity of d among the samples carrying the (c)ce s allele and in the samples homozygous for rhce*ce. the results showed that the presence of rhce*(c)ce s significantly reduces the expression of d antigen, probably due to the expression of the partial c partial antigen in trans to rhd. additionally, the samples with reduction on d expression carrying rhce variant alelles phenotype can be useful to provide compatible blood to some patients with rarerh variant alleles. background/case studies: drugs are known to interfere with routine blood bank testing. a novel monoclonal humanized f antibody (hu f -g ) that binds human cd has been entered into clinical trials for patients with acute myeloid leukemia, non-hodgkin lymphoma and solid tumors. we describe two cases of patients treated with hu f -g (anti-cd ) who had abo discrepancy with extra-reactivity in the reverse typing and a panaggutinin in the plasma. study design/method: this is a retrospective review of two cases with immunohematology work-up showing abo discrepancy and plasma panagglutinin. the first case is of a year old female with progressive follicular lymphoma who was enrolled in phase b/ trial of hu f g in combination with rituximab designed for patients with relapsed/refractory b cell nhl. she had no prior transfusion history and her historical blood type was not known. two rbc units were requested in anticipation for a surgical procedure. the second case is of a year old male with refractory diffuse large b cell lymphoma enrolled in hu f -g clinical trial. his historical blood type was a rh d positive with a negative antibody screen. he received three rbc units within the past month prior to testing and receiving the anti-cd therapy. results/finding: the abo typing in the first case showed a discrepancy between the forward typing ( with anti-a, non-reactive with anti-b) and the reverse typing ( with both a cells and b cells). rhd typing was positive. the extended reagent rbc panel tested with the patient's serum reacted with all cells tested at the immediate spin (is) phase ( to ), at liss- c ( to ), at liss-polyspecific ahg (m ), and at peg-anti-igg (m to ). plasma reactivity at is persisted with dtt or ficin treated red cells and was not removed by cold autoadsorption, cold alloadsorption, or rest adsorption. repeat testing, which avoided the is and c readings, was non-reactive in the antihuman globulin (ahg) phase using both liss and peg enhancements, ruling out clinically significant alloantibodies directed toward common red blood cell antigens. the direct antiglobulin test (dat) and autocontrol were negative. the rbc units issued to the patient were crossmatch compatible at o c ahg phase. the abo typing of the second case performed after anti-cd administration showed a discrepancy between the forward ( with anti-a) and the reverse ( with both a and b cells). rhd typing was positive. the antibody screen performed in solid phase technology was positive with all reagent red cells. his plasma reacted with all reagent red cells at is ( ), at c in liss ( ), and liss-polyspecific ahg (m ). the dat and autocontrol were negative. his genotype was determined to be a /o and full rbc phenotype by dna analysis was obtained. repeat testing which avoided the is phase did not show reactivity at peg-ahg excluding all alloantibodies directed toward common red blood cell antigens. conclusion: anti-cd therapy interferes with blood bank testing by causing abo discrepancies and panagglutinin reactivity in the plasma at is, c liss, but not at ahg phase using gamma-clone anti-igg, unlike the anti-cd interference. knowledge of patient's blood type and phenotype before starting this therapy is critical for providing safe blood. background/case studies: a middle-aged male with discrepant abo typing results was investigated. initial forward typing was group o but no anti-b was seen in the reverse typing. an unexpected reaction was noted with an anti-a,b reagent. genotyping surprisingly showed abo*o. . / o. . , consistent with group o. after initial testing at the referring center, samples were sent for extended analysis. study design/method: standard serological methods and flow cytometry were used. a panel of abo reagents (n ) and lectins were tested with both native and papain-treated red blood cells (rbcs). lewis phenotyping was performed, as was genetic testing for abo, gbgt and a galt. papain-treated patient rbcs were used to screen donor plasmas (n ) and two reactive plasmas were dtt-treated. results/finding: positive reactions were obtained with polyclonal anti-a,b and a monoclonal anti-b (clone g / ) when tested with the patient's papain-treated rbcs. a panel of lectins gave negative results. genetic testing confirmed the predicted group o and ruled out the presence of fors or nor antigens. the patient was le(a-b ) and thus a secretor. a positive crossmatch was seen with % of group o plasmas, while no reactivity was obtained with a or b plasmas. dtt treatment of crossmatchpositive plasmas indicated the antibody to be mainly of igg type. this was confirmed by positive flow cytometry cross match using anti-human igg secondary antibodies. reactivity remained after b-zyme treatment, thus excluding the normal (type or ) b antigen to be the underlying reason. inhibition with lewis substance significantly decreased reactivity. enzyme activity assay showed the patient's plasma to contain a fully functional b glycosyltransferase. on the suspicion that the patient had non-erythroid cells producing breactive type chains, a sample from a hematopoietic stem cell transplant (hsct) patient (group b secretor receiving group o donor cells) was included as a control and gave the same type of reactions. conclusion: the medical history of the patient was queried and he had indeed undergone an hsct $ years earlier. the reactions are likely due to uptake of recipient-derived ble b (type ) antigen (isbt no. ), which is the dominant lewis antigen in the recipient's original blood group, b le(a-b ). interestingly, b-zyme did not affect ble b . anti-ble b is not simply anti-b plus anti-le b but an inseparable and rarely reported specificity, which appears to be common among group o donors. the phenomenon reported here has unknown clinical implications but highlights the complexities of carbohydrate blood groups. background/case studies: the provuev r and visionv r (ortho scientific, raritan new jersey) automated analyzer use mts-gel tm card technology to perform immunohematology testing. benefits of automated testing include improved efficiency and enhanced reliability. after eight years of using the provuev r our transfusion medicine service switched to the ortho visionv r analyzer in january of . shortly after implementation, technologists reported increased time spent performing manual resolution of indeterminate (designated as "?") results. additionally, some test columns were noted to be visually negative but called positive ( ) by the analyzer. the objective of this study was to investigate the cause of "?" and apparent false positive results on visionv r three-cell antibody screens. study design/methods: with assistance from ortho diagnostics, analyzer archives were queried to identify the number of gel card columns used for screens, the number of columns with "?" results, and the gel card lot numbers used for testing from / / to / / . reactivity was determined to be false positive based on supervisory review of digital images and antibody panel results. investigation also included review of daily qc records, instrument maintenance, instrument diagnostics, and camera calibration. results/findings: of , columns run as part of antibody screens, , ( . %) columns generated "?" results. assuming seconds of technologist time per "?", we estimate that . hours were needed to resolve and update these results. among all potential causes investigated, only the gel card lot number was associated with the number of "?" generated (table ) . in cases, all three columns were visually negative but the analyzer reported reactivity with of cells. all cases had mts-gel tm antibody identification panels performed, of also had a mts-gel tm ficin panel. the yield for the panels performed was two routine panels with weak reactivity against hla cells, and four ficin panels with weak reactivity with no apparent specificity. fourteen patients coincidentally had a subsequent type and screen; were negative. one patient newly demonstrated anti-jka. fifty percent ( / ) of visually negative but analyzer positive samples were tested with gel card lot number , % ( / ) with lot , and % ( / ) with lot . conclusion: the incidence of "?" and visually negative analyzer positive results is dependent on the specific lot of mts-gel tm cards used. the difference between the lots is being investigated by ortho diagnostics, and remains to be explained. to avoid unnecessary waste of technologist time and other resources, with assistance from ortho diagnostics, we have results/finding: of the methods evaluated, the dtt method proved the most useful for mitigating dara interference. cord cells were effective but in limited supply and alloadsorption was ineffective. of the three different dtt methods evaluated, the tube method initially failed which led to re-evaluation with the addition of liss (passed). the gc method was the most sensitive method. following release of dara, samples from patients ( cross-match samples, units issued) were tested using both liss tube and gc iat methods. despite dtt treatment, the gc method remained positive by iat in / patients. further testing was performed in / . eight were tested for the presence of antibodies at c and confirmed in / . rouleaux formation was observed in / patients, / had reactivity detectable at c. no transfusion reactions have been reported to date nor has alloantibody formation been observed to date. conclusion: as previously reported, the dtt method was the most useful for mitigating dara interference. the observed interference seems to be due to rouleaux and/or cold reactive antibodies -seen least in the liss tube iat. this may be due to the washing phase in this technique which dissipates rouleaux formation. reactivity due to cold reactive antibodies can be eliminated by performance at strict c. our practice is now to use both dtt iat methods on initial patient referral, if residual reactivity in gc is observed use liss tube in preference thereafter in these patients. a further observation is that investigation of pan-agglutination could include the use of cord cells to confirm/exclude dara use if suspected. wendy beres* , sandra nance , david moolten and p. dayand borge . ( ): - ). our laboratory tested random allogeneic blood donors, and autologous donors (which were intended to represent a hospitalized patient population), to determine a mean and range of "normal" levels. this . year retrospective study was performed to assess levels of rbc-bound igg, iga and igm in normal donors. study design/method: residual edta-anticoagulated aliquots from random allogeneic and autologous blood donors were sequestered and tested per institutional review board approved protocol. the samples were tested by fc with fluorescein isothiocyanate (fitc)-labeled anti-human igg and iga (jackson immunoresearch lab, west grove, pa) or fitc-labeled anti-human igm (life technologies, carlsbad, ca) at optimized dilutions in dulbecco's pbs containing . % bsa. the becton dickinson facscalibur tm or facscan tm (san jose, ca) fc analyzed k rbcs from each sample. edta-anticoagulated samples and ig coated control rbcs were tested to determine fc settings and control for validity and cross-reactivity. controls reacted as expected. there were less autologous donors tested and with a mean age of these donors could have been older than the allogeneic donors, but the mean age of the allogeneic donors was not captured. despite the relatively small number of samples tested there was a higher than expected instance of allogeneic donors having elevated rbc-bound iga, igg, and igm levels. this emphasizes the need to include testing of normal donor populations in establishing expected reactivity, thus normal and abnormal ranges for flow cytometric testing. long range pcr reveals the genetic basis of an antibody in pregnancy to a high prevalence mns antigen judith aeschlimann* , anna burgos , virginia lew , sunitha vege , susan veneman , christopher j gresens , jonathan hughes and connie m. westhoff . immunohematology and genomics laboratory, new york blood center, blood centers of the pacific, bloodsource background/case studies: recombination events have generated many gypa and gypb hybrids giving rise to glycophorin (gp) variants that express low-prevalence antigens (e.g. mia, miny, mur). in rare individuals who are homozygous these alleles are associated with lack of highprevalence antigens (e.g. enkt, eneh, enav). complex hybrid recombination events can make it challenging to elucidate specific alleles present in samples, particularly heterozygotes. we investigated samples from a pregnant asian (hmong) woman with an antibody to an unidentified highprevalence mns antigen, and samples from her sister. study design/method: standard methods were used for rbc typing with licensed and unlicensed reagents and for antibody identification. dna was isolated from wbcs, and hea precisetype, exon-specific amplification and sequencing gypb exons - , and long range sequencing of exon - ( . kb amplicon) were performed. snp-specific primers were used to associate changes (phasing) to specific alleles. results/finding: rbcs of the pregnant proband typed s-s-(gammaclone anti-s), and the plasma reactivity was consistent with an antibody to a high background/case studies: the rhd antigen is clinically significant and immunogenic and therefore individuals who develop anti-d are at risk of haemolytic transfusion reaction. rhd polymorphism shows substantial ethnic variability and at least rhd variants associated with weak d alleles have been reported. in this study, we report two new rhd alleles in brazilian blood donors associated with weak d antigen expression. study design/method: the d status was evaluated with commercially available monoclonal anti-d reagents: blended igm/igg (clones th- / ms- ), igm (clones ms and p x ) and igg (ms ) in tube and on gel cards. c, c, e and e phenotyping were performed in gel. most common weak d and partial d alleles were investigated by allele specific (as) pcr and with the rhd beadchip platform from immucor. direct automated sequencing of the rhd exons and flanking intron regions was performed by the sanger dideoxy method. in order to determine rhd allelic combinations, we also performed rh-cdna cloning and sequencing. background/case studies: donors negative for multiple common antigens or lacking a high prevalence antigen are efficiently identified using a red blood cell (rbc) genotyping panel. when serology is used to confirm antigen negative status, discrepancies are identified, albeit rarely. investigation of the discrepancy often leads to identification of variant antigens. it is known that the set of gyp variants associated with expression of the st a antigen can also be associated with n typing discrepancies in m n-individuals (meyer et al. br j haematol. ; : - ) . the st a allele, also described as gyp* , is a hybrid gypb-gypatranscript with the crossover in intron . we sought to investigate five n typing discrepancies for which alternative genotyping methodology was performed and found to be concordant with the initial panel. background/case studies: a sample from a years old pregnant, african american female g p was sent to the blood bank for abo/rh and antibody screen. the sample was analyzed using the provue analyzer (ortho diagnostics). the patient was typed as o pos with no reverse type discrepancy. a retype of the same sample was performed using tube method with biorad reagents per hospital policy due to no previous abo/rh history on file. the retype showed that the patient was a subgroup with anti-a antibody present in the plasma. the sample was referred for genotyping, with the suspicion of a like phenotype. genetic testing did not support the serological findings of a subgroup and a new abo allele, abo* c that has never been reported in correlation with an a like subgroup was detected study design/method: the patient rbcs were typed with anti-a (immucor) and anti-a,b (biorad and grifols dg gel). an anti-a antibody work up was performed using three different lots of a cells and three lots of a cells, as well as a type o screening cell and auto control . the tubes were read at is and also incubated at rt and c for min. the patient 's initial antibody screen using ortho gel was negative. conclusion: the patient delivered a healthy baby boy at weeks of gestation. the baby cord was sent to the laboratory. the baby serological type showed an a b phenotype and it was referred for genetic testing. the baby rbcs showed the same abo* c found in the mother. the previously reported abo* a allele encoded an aspartic acid to asparagine change at position p. consistent with an a weak phenotype. also, at least five other alleles encoding an a phenoytpe consisted of polymorphisms at positions c. through c. , giving special characteristics to this new and unreported abo allele. from the data collected, it can be concluded that this a / aweak phenotype is encoded by the variant allele abo* c. this highlights the clinical relevance of confirming the serology of abo subgroups by molecular methods. philip berardi*, jacqueline cote, gwen clarke, vito scalia, robert liwski and mindy goldman. canadian blood services background/case studies: elucidation of the molecular basis of blood group expression has led to the development of high throughput molecular methods for predicting blood group antigens. the commonly used single nucleotide polymorphism (snp) arrays require nucleic acid isolation which is typically achieved by extracting genomic dna from whole blood. this method requires venipuncture and may not be an ideal approach for severely anemic patients or potential donors that are unable to provide a sample of whole blood due to their remote location. dna extracted from buccal swab samples offers a noninvasive alternative to venipuncture and may provide a safe and efficient means of transporting samples from remote locations to reference laboratories for extended blood type prediction. canadian blood services (cbs) has performed large scale dna extraction and hla genotyping for the onematch stem cell and marrow registry using buccal swabs since ; buccal swabs are also used by other unrelated donor stem cell registries, such as the us nmpd. we sought to assess the accuracy and reliability of using dna extracted from buccal swabs in predicting blood group antigen expression. study design/method: we performed parallel red cell genotyping on an automated typing platform, the progenika/grifols idcorext assay (progenika biopharma-grifols, bizkaia, spain) using dna extracted from blood and buccal tissue from volunteers. for antigen systems with available serologic reagents, we also compared results with serologic typing. we evaluated three different methods of dna extraction and performed testing regardless of dna yield or purity. two buccal swabs (puritan medical products, guilford, maine) were used for each test. swabs were stored at room temperature, and dna extraction was performed within six days of collection. in the initial phase of the study, buccal swab samples (n ) were processed with the automated biorobot m robot using the magattract dna mini m extraction method (qiagen, venlo, the netherlands). extracted dna had a mean concentration and purity of . ng/ml and . respectively. in the second phase of the study (n ), dna extractions from buccal swabs were performed using methods available in our national red cell immunohematology reference laboratory: the qiaamp dna mini kit, using either manual or an automated qiacube robotic workstation (qiagen, venlo, the netherlands). results/finding: the manufacturer's recommended analytical range for dna concentration was - ng/ll and the recommended purity was an absorbance ratio of . - . (a / ) for use of the id corext platform. dna extraction from buccal swab samples did not meet these specifications in several cases. however, in most cases, a lower concentration of dna was adequate for prediction of phenotype. the dombrock system was the most susceptible to failure of interpretation in the samples with a low dna concentration, with "no call" results reported. there was % concordance in genotyping results when source dna was extracted from whole blood or buccal tissue; there was also % concordance between predicted phenotype and serologic testing results. conclusion: this study supports the use of genomic dna extracted from buccal tissue on the id corext for predicting rbc phenotype with high accuracy. extraction methods may require optimization to achieve dna yields within the recommended analytical range of the assay. performance evaluation of id rhd xt, a genotyping assay for the detection of high-prevalence rhd negative and weak d types araitz molano , izaskun apraiz , maría azcarate , miguel angel vesga , montserrat rubia , mercedes piedrabuena , fernando puente , barbera veldhuisen , ellen van der schoot and m onica l opez* . progenika biopharma, a grifols company, centro vasco de transfusi on y tejidos humanos, banco de sangre y tejidos de arag on, sanquin blood supply research background/case studies: it is well established that weak d , and phenotypes are not at risk for forming allo-anti-d, whereas a few weak d and all partial d and negative phenotypes are. routine serologic d typing does not distinguish among them, consequently rhd genotyping is recommended, especially in patients. id rhd xt (progenika, grifols) is a qualitative, pcr/luminex v r xmap hybridization-based genotyping test for the identification of the following rhd gene allelic variants: rhd*weak d type , rhd*weak d type , rhd*weak d type , rhd deletion, rhd*pseudogene and rhd*diiia-ce( - )-d and itgb gene: hpa a and hpa b, in genomic dna extracted from whole blood specimens. in this study the performance of id rhd xt genotyping assay was evaluated in terms of whole system failure rate, call rate and accuracy for rh and hpa- blood group typing. study design/method: a cohort of previously serotyped samples for d antigen obtained from three european blood centers were analyzed with id rhd xt at progenika. samples were distributed as recommended by the annex of the common technical specifications / /ce for a ivd product of list a (! % clinical samples, > % neonatal specimens and ! % weak d donors). for the intended use of the product, weak d serotyped donors were enriched (n , %). commercial serology tests for d antigen predicted phenotype and bi-directional-sequencing (bds) for weak d type confirmation and hpa- predicted phenotype were used for comparison. transfusion results/finding: no system failure, % call rate and no inconclusive results were obtained. discrepancies were found for d antigen between serology and id rhd xt predicted phenotype results, although a % concordance was obtained when analyzed by bds, considering id rhd xt result correct. concordance between id rhd xt and bds results for the weak d type was %. the following id rhd xt predicted phenotype results were obtained: d negative (n ), no amplification variant (n ), weak d type (n ), weak d type heterozygous (n ), weak d type (n ), weak d type heterozygous (n ), weak d type (n ), weak d type heterozygous (n ), weak d types , or not detected (n ). regarding hpa- blood group, the predicted phenotype results obtained by id rhd xt were % concordant with bds results: hpa- a positive (n ) and hpa- a negative (n ), hpa- b positive (n ) and hpa- b negative (n ). conclusion: id rhd xt genotyping assay performed as a reliable and accurate method for predicting the genotype and phenotype of high prevalence rhd negative and weak d types ( % specificity and % sensitivity for d antigen, hpa- a and hpa- b antigens). that makes it a useful tool for the implementation of the rhdgenotyping recommendation on patient blood transfusion and anti-d prophylaxis. background/case studies: scd patients form red blood cell (rbc) antibodies at higher rates than other transfused populations. multiple predictors of alloimmunization have been reported but not well replicated in large scd cohorts. we investigated the clinical, laboratory and genetic predictors of alloimmunization. study design/method: a large scd cohort was established in brazil to investigate disease outcomes. at participating sites, patients are currently transfused with abo/d/cc/ee/kell matched rbcs prophylactically and extended phenotypically matched rbc after first antibody forms. policies for matching are center-specific and evolved to increased levels of matching over the exposure period included in this study. transfused subjects with rbc alloantibody of defined specificity within the cohort were compared to transfused antibody negative subjects using chi squared to compare categorical variables and t-test or wilcoxon rank-sum tests as appropriate to compare continuous variables. backward elimination multivariable logistic modeling was used to generate odds ratios (or) and identify independent predictors of alloimmunization using results of univariate analyses. all subjects had peripheral blood whole genome snp typing performed using an affymetrix array, which included enhanced content for blood related snps. genome wide association (gwa) analyses were conducted using a logistic model to identify additive genetic effects associated with alloimmunization. a p value < . (clinical analysis) or < x - (gwa) was considered statistically significant. results/finding: of the cohort patients, ( . %) transfused subjects were included with alloimmunized children < years ( . % of ) and alloimmunized adults ( . % of ). in multivariable logistic regression models, age (or . , p . , for age compared to - ), gender (or . , p . , for female compared to male), transfusion history (or . , p< . , for transfusions compared to - ), site, hemolysis (or . , p . , for log transformed lactate dehydrogenase) and presence of autoimmune disorders (or . , p< . ) were independent predictors of alloimmunization. gwa identified a single snp of unclear biologic significance associated with alloimmunization (eefsec gene responsible for incorporation of selenocysteine into proteins). conclusion: rbc alloimmunization is primarily driven by transfusion burden in this scd cohort. hemolysis remained significantly associated with alloimmunization after controlling for transfusions. presence of an autoimmune disease was also associated with rbc alloimmunization, indicating more systemic immune dysregulation may be present in scd patients who develop rbc alloantibodies. however, the gwa did not identify snps in immunoregulatory genes significantly associated with rbc antibody formation in the study population. background/case studies: physiologic anemia is more severe in preterm infants and worsened by the blood loss required for laboratory tests. to reduce iatrogenic anemia, placental blood, which otherwise would be discarded, can be used for laboratory testing. mother and infant blood are mixed in the placenta during delivery and pre-transfusion test results potentially can be altered due to fetal-maternal hemorrhage. there has been no published study to show if pre-transfusion test results of placental blood give the same result as the heel stick samples, which is the standard of practice. study design/methods: transfusion service tested sample pairs from newborns less than , gr birth weight. one of the samples was collected from the newborn as a heel stick sample, the other from the placenta. the following tests were performed on the sample pairs: abo, rh, antibody screen and direct antiglobulin test with igg (dat). results/findings: abo, rh and dat tests were performed on sample pairs. dat test was negative on sample pairs and two were positive. there was % concordance with the abo, rh and dat tests performed on these sample pairs. antibody screen was performed on placental blood samples and heel stick samples. twenty eight sample pairs were negative with the antibody screening test. there was one positive heel stick sample, which was also positive using the placental sample. one heel stick sample was negative for the presence of an antibody but found to be positive with the placental blood sample. this antibody which was detected only in the placental sample was a passive anti-d mother received during pregnancy. this discrepant result indicates that the placental blood sample was more sensitive to detect a weak antibody. conclusion: this study shows that placental blood sample is not inferior to heel stick sample regarding abo, rh and dat testing. based on this comparison study placental blood can be used for pre-transfusion testing for < , g birth weight newborns. o-( . %), ab ( . %), b-( . %), and a-( . %). among the tested donors, . % were d positive with r r being the most common rh phenotype. in the kell blood group system, . % of the donors were k positive, while the k antigen was found to be . %. the most common phenotype in the duffy blood group system was fy(a-b-), while the fy(a b ) was found at a higher frequency compared to what has been reported in the black population. (table) the commonest phenotypes for the kidd and mns blood group systems were jk(a b ) and m n-s s at % and . % respectively. the le a and le b alleles were seen in . % and . % of donors respectively, while lu b -phenotype was found in . % of the donors. the frequencies of the rare phenotypes jk(a-b-), le(a b ) and lu(a-b-) were . % , . % and . % respectively, while the m n-s-s-and m-n s-s-phenotypes were not found. the frequency of the p antigen was found to be at . % similar to what has been reported in caucasians. conclusion: this is the first study to examine the frequencies of rbc blood group phenotypes among the omani blood donors. the results show higher frequencies of the rare null phenotypes fy(a-b-), jk(a-b-) and lu(a-b-) compared to what has been reported in caucasians. the frequencies of the duffy blood group system resemble what has been reported in the black population. this data is helpful in understanding the influence of the arab ethnic background on the rbc blood group systems and warrants large genotype-phenotype studies in the region. quantitation of anti-d in serum using flow cytometry amanda whitelonis*, izekial butler, karen leighton, scott jones and anand srinivasan. qualtex laboratories background/case studies: rh(d) antibodies (anti-d) are developed in rhnegative individuals when exposed to d antigens. this scenario is commonly observed in alloimmunized antenatal and volunteer immunized patients. quantitation of anti-d in serum is important in the clinical setting to predict the risk of hemolytic disease of the newborn. quantitation of anti-d is also performed in quality control operations of organ procurement organizations and plasma fractionators. it is a common practice to report the strength of anti-d in serum as antibody titer values but quality control operations require a quantitative value. we have developed a screening assay using flow cytometry to quantitate anti-d in serum. study design/method: we have developed a method to quantitate anti-d in serum using flow cytometry, by modifying the protocols of christensson et al., and hilden et al.. red blood cells from rh-positive blood samples were washed three times in phosphate-buffered saline (pbs) at ph . and the supernatant was discharged. a dilution buffer containing % human serum albumin (v/v) in phosphate buffered saline was prepared. serum samples or who anti-d standards, suspended in dilution buffer were mixed with ll of washed red cells. the cell suspensions were incubated for min at c. following incubation, fitc-labeled anti-igg diluted in buffer was added and the mixture was incubated for an additional min at c. the samples were then analyzed by flow cytometry using gates for a typical red cell based on the forward and side-scatter signals. green fluorescence was collected using a band-pass filter set for - nm. events were recorded at a frequency of cells. results/finding: multiple dilutions of who anti-d reference standard were tested against rh-positive red blood cells from five different donors. the reproducibility of the assay was determined by measuring the change in coefficient of variance due to dilution procedure, machine variation and sample storage condition. after optimizing these factors, a linear regression was calculated to establish the standard curve. the fluorescent intensity emitted by probes demonstrated a linear correlation with the concentration of rh(d) antigens in reference standard. serum from thirty rh(d)-immunized volunteers were analyzed for concentration of anti-d and the results were benchmarked with antibody titer values. conclusion: based on our study, we conclude that the quantitation of rh antigens by flow cytometry can be used as a reliable assay to measure the concentration of anti-d antibodies in serum. the method is reproducible and advantageous over reporting antibody titer values. the operations of this platform can be translated to a well-plate based high-throughput flow cytometry. sarah k harm* , mark yazer , nancy m. dunbar and biomedical excellence for safer transfusion (best) collaborative . university of vermont medical center, university of pittsburgh, dartmouth-hitchcock medical center background/case studies: the use of emergency issued group a plasma and uncrossmatched group o whole blood (wb) in patients without a valid abo group is becoming increasingly common in the usa. it is unclear if low titer products should be provided in this situation and indeed a universally agreed upon threshold that would qualify as "low titer" has not been established. this study was designed to determine the rate of high titer donors using a titer threshold of . study design/methods: three academic hospitals that routinely issue group a plasma units for emergency issue participated in this study. before issuing this plasma to patients, a : dilution of the donor's plasma in saline was produced and added to group b reagent red blood cells (rbc). if any degree of macroscopic agglutination after immediate spin was observed, the unit was considered high titer and it would only have been issued to group a or o recipients. at these three centers no temperature, plasma volume or time enhancements were performed in the titer procedure, and anti-human globulin was not added. at one center samples were taken from the plasma of group o wb units and the same procedure was followed using a and b reagent rbcs; if at least one antibody demonstrated macroscopic agglutination after immediate spin, the wb unit was considered high titer and it was then centrifuged into an rbc unit for transfusion while the plasma and platelet components were discarded. two centers provided plasma testing data for a -year and -year period, respectively. one center provided plasma and wb testing data for a -year period. results/findings: in total there were group a plasma units tested and ( . %) had a high titer anti-b. the range of high titer group a plasma units between these three centers was . %- . %. of the wb units tested, ( . %) units had a high titer; / ( . %) of the units had a high titer anti-a, / ( . %) had a high titer anti-b, and / ( %) had high titers of both anti-a and anti-b. background/case studies: dithiothreiol (dtt) is a sulfhydryl reagent that denatures selective blood group antigens. reagent red blood cells (rbcs) treated with . m dtt is used as a tool in identifying antibodies to high frequency antigens. recently, dtt has become widely used in destroying cd on reagent rbcs and render them free from plasma anti-cd drug interference. procedures for the preparation of . m dtt has been published advocating the use of buffered saline at different ph levels. in this study, an effect of ph on . m dtt treatment time is investigated. study design/methods: non-buffered saline (nbs, thermo fischer scientific inc, middletown, va), used in the preparation of . m dtt, was adjusted to ph . , ph . , ph . using sodium phosphate dibasis (sigma aldrich, saint louis, mo). reagent rbcs (immucor, norcross, ga)(n ) were treated with the . m dtt solutions in parallel by mixing : ratio of packed rbcs to . m dtt solution followed by incubation at c. for up to minutes during treatment, the expression of k antigens was measured every minutes by tube method using different sources of anti-k. to assure uniformity, all reactions were graded by the same investigator. each reaction grade (in each rbc and each antiserum) is converted into a semiquantitive score and an average score was calculated every minutes for each ph level. the reduction in average scores between different phs were also calculated at every minutes to measure the impact of . m dtt reagent ph on the rate of k antigen destruction. results/findings: the expression of k antigen, measured by agglutination grades with two different k antisera, is significantly weakened (by ! ) after minutes of dtt treatment at ph . ; minutes at ph . and minutes at ph . . complete loss of k expression was seen after minutes of dtt treatment at ph . ; minutes at ph . and minutes at ph . . the reactivity patterns of k antigen tested with sources of anti-k correlate with each other. the reductions in average scores were seen between to minutes range of dtt treatment time when ph . was raised to ph . ; to minutes range when ph . was raised to ph . ; and to minutes range when ph . was raised to ph . . conclusion: the use of higher ph buffered saline may shorten the treatment time it takes to weaken or destroy k antigen. based on the comparison of reaction scores between different ph levels, the ph levels did not have an impact on dtt treatment up to minutes and/or beyond minutes of incubation. the ph of the . m dtt reagent relative to the treatment time is a factor to consider during the validation of dtt-treatment process and qualification of . m dtt reagent in a laboratory. background/case studies: data on the characteristics and frequencies of clinically significant red cell antibodies within the prenatal population have not been well established in the united states. the aim of this study was to determine if frequencies of red cell antibodies differed between geographically distinct regions within the continental united states. study design/ method: the aim of this retrospective study was to evaluate a cohort of prenatal patients (n , ) drawn between july , and june , . these patients were divided into united states census bureau regional and divisional categories according to their place of residence. prenatal blood work was collected which included an abo, rh(d) and a screen for unexpected alloantibodies. samples found to be positive for red cell antibodies were sent to one of nine regional laboratories for identification. results/ finding: in total, , patients were found to possess clinically significant red cell antibodies for an overall incidence of . percent. the three most commonly encountered antibodies were anti-d (n ) . %, anti-m (n ) . %, and anti-e (n ) with a frequency of . %. a total of ( . %) prenatal women were found to possess two or more antibodies. in general, the combination of anti-d and anti-c proved to be the most common, with instances ( . %) followed by anti-e and anti-c with ( . %), and anti-c, anti-e with ( . %). of the multiple antibodies identified, ( . %) included at least one antibody from the rh blood group. the south region had the largest number of antibodies identified with or . % of the total. the west had ( . %), the midwest ( . %) and the northeast with ( . %). a contingency table, using the two-sided fisher's exact test, was performed comparing the northeast, south, midwest and west regions. the p value of anti-d was calculated to determine nonrandom associations and values of . and below was deemed significant from a region-to-region perspective. with regard to anti-d, the pacific division comprised of california, oregon, washington, and alaska, had p values below the . thresholds when compared against seven of the eight other divisions. the west south central division (texas, oklahoma, arkansas, and louisiana) did not show statistically significant results when compared against the pacific division (p . ). conclusion: depending upon the antibody, statistically significant variations between geographical regions and divisions within the united states were observed. this relationship between antibody and locality requires further investigation but may be attributed to the presence or absence of red cell antigens among different racial and ethical populations. reduction in repeat testing using gel technology amy mata* , lindsy rich , sherry stern , sharon wangen and camille van buskirk . mayo clinic, mayo clinic rochester background/case studies: our institution currently uses the immucor neo (immucor, inc., norcross, georgia) to perform abo/rh and antibody screen (absc) testing utilizing solid phase technology. when results are unable to be obtained from the immucor neo, testing is repeated on the manual testing bench using tube agglutination. this repeat testing can lead to significant expenses including reagents, supplies, and technologist time. it was decided by leadership in our laboratory that it would be beneficial to observe how other methodologies perform in this regard. a side-by-side evaluation was performed between the immucor neo and the ortho vision (ortho clinical diagnostics, rochester ny) to determine if there was a significant difference in the amount of repeat manual tube testing that needed to be performed. the evaluation looked at abo/rh and absc testing as those are the only tests that are currently automated in our laboratory. study design/method: thirty specimens that were processed on the immucor neo and resulted in no type determined (ntd) for abo/rh testing were selected to be tested on the ortho vision. twenty-three specimens that were processed on the immucor neo and produced positive results for absc testing were selected to be tested on the ortho vision. all specimens were edta tubes and were collected within the previous days. the timeframe between when the specimen was tested on the immucor neo and the ortho vision was to days. results/finding: of the ntd specimens from the immucor neo, resulted in valid abo/rh typings on the ortho vision. three results were flagged indicating possible extra reactivity. upon performing a visual review of all results, it was determined that there was no reactivity and a valid result was present. the other samples required manual tube testing to interpret the abo/rh and were due to mixed field, weak isoagglutinins, unexplained extra reactivity, and hemolysis. of the absc specimens that were resulted out as positive on the immucor neo, specimens produced a negative result on the ortho vision and were confirmed to be negative with manual tube testing using peg as the enhancement media. one specimen was flagged for fibrin, but upon performing a visual review, was determined to be negative. nine specimens that were positive on the immucor neo were also positive on the ortho vision. one specimen proved to be an anti-m that was seen in gel but not in tube and one specimen displayed unexplained reactivity in gel as it was negative in tube and all clinically significant antibodies were ruled out. all showed discrepant results with monoclonal anti-c reagents, with a similar pattern of reactivity: - with ms (n ), - s with ms (n ), no reaction with ms , dgc , p x (n ). samples tested with a polyclonal anti-c showed a - reactivity. d c e c e cases tested with a polyclonal and monoclonal anti-e (ms , ms , ms , ms ) showed no weakened reactivity. rhce sequencing (genomic dna or cdna) showed a c. g>a mutation in exon , predicted to encode the p.gly ser substitution. for apparent r r donors, a f-negative type allowed the prediction of a rhce*ce a/rhce*ce genotype. altogether, our results are consistent with the presence of a very likely rhce*ce a allele (c and e in cis) in all samples. d c e c e individuals were reactive s with the original source of anti-rh , slightly weaker when compared to rhce*ce a/rhce*ce rbc samples available from our cryobank ( ). conclusion: our results confirm that the c. g>a mutation alters the conformational properties of the rhce protein, either on a ce or ce background, and encodes the low-prevalence locr antigen (rh ). the locr reactivity appears to be rather similar when coded by rhce*ce a or rhce*ce a alleles. this was quite an unexpected finding, since the p.gly ser substitution is close to the critical amino-acid for c/c expression (p.pro ser). none of our cases made anti-c and/or anti-e but few were subject to a potential alloimmunization background. however, as rhce*-ce a was reported to code for a partial c (rh:- ), we consider that rhce*ce a likely encodes partial c and e, this being also supported by the predicted localization of the p.gly ser change on the second extracellular loop of the rhce protein. background/case studies: weak d genotyping is recommended for transfusion recipients, pregnant women, and newborns who had a rhd typing discrepancy, or a serological weak d phenotype, to determine if they carried the weak d genotypes , or . the purpose of this study was to analyze the underlying rhd genotypes of the patient samples received for weak d genotyping since published recommendations, in particular those found to not carry the weak d , , or genotypes. study design/methods: between / and / samples were received for weak d genotyping. testing was performed using pcr-rflp targeting the sequence variants in the rhd gene that have been previously defined. samples that did not have weak d types , , or genotypes, but a transfusion vol. supplement s had evidence of rhd genetic sequences in exon and/or intron in preliminary testing were evaluated by sanger sequencing for rhd and rhce exons - to determine the underlying rh genotype. when provided, the patient's ethnicity and presence of anti-d was recorded. results/findings: the majority of the samples were from obstetrical patients ( %) followed by transfusion patients ( %); % had no clinical indication provided. samples ( %) were found to be weak d type , , or ( , , and samples, respectively). samples ( %) appear to be genetically rhd negative. genetic sequencing was performed on samples; had rhd genetic variants that were not weak d types , , or (table) . all of these variant rhd samples also showed some variation in the rhce gene. two samples ( %) had wild type rhd alleles; further evaluation is ongoing. conclusion: most samples tested by weak d genotyping were found to be weak d types - . of the samples that had evidence of an rhd gene and did not carry the known weak d types - polymorphisms, ( %) of were found to have other rhd variants, and ( %) did not have underlying genetic variation detected in the rhd gene. the majority of the non weak d types - variants were dar alleles, which are often associated with anti-d production. background/case studies: rhd genotyping has been recommended to guide transfusion of d-negative rbcs and administration of rh immunoglobulin to patients with discordant or weaker than expected d typing, particularly for females and ob patients . the recommendation is based on observational evidence, primarily from europe (flegel , curr opin hematol : ) , that individuals with weak d types , , and are not at risk for clinically significant anti-d. the implications and utility of this approach for the diverse u.s. population are not yet clear. here we report months experience with rhd genotyping on samples referred with discrepant or weak d typing investigated from january to april . study design/method: serologic testing was performed by standard tube agglutination with licensed reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assays and rhd sequencing for some. ethnicity was known for samples ( . % caucasian, . % african american/african, . % multiracial, . % hispanic, % asian, and . % other). results/finding: rhd genotyping identified weak d types , , and in / ( %) and alleles known to encode partial d phenotypes in / ( . %) (table) . uncommon or rare weak d alleles including types , , , , , , (n ), , , , , and were found in ( . %). the partial d alleles found were diverse, but the largest number included partial rhd*d . (n ) and *dar ( conclusion: in a multiracial cohort of individuals with weaker than expected d typing % were due to weak d types , , or and would not be considered at risk of clinical significant anti-d, but for % there is potential or unknown risk. these studies are important to gain insight into the prevalence of specific alleles in the u.s. multiethnic population and to continue to evaluate and refine rhd genotyping for clinical practice. cp rhd* . allele causes discrepant genotyping results for rhce small c sabine scholz* , sandra schneider , sabrina k€ onig , susanne helmig and vicky van sandt . inno-train diagnostik gmbh, rode kruis vlaanderen background/case studies: in the human rh blood group system the c, c, e, e and d antigens are expressed by the two highly homologous genes rhce and rhd. after d, c is the most immunogenic rh antigen. the difference between c ( c) and c ( t) is caused by the snp on position on the rhce gene. the rhd* . allele (also known as rhd cat vii type ) carries the snp t>c on the rhd gene and additionally the snp t>c. this rhd* . allele has been described to partially express rhc on the d polypeptide (faas, transfusion, ) . aims: genotyping was performed to clarify the cause of the weak c expression. serology of a patient sample (male, ) indicated a partial c phenotype with a cde. study design/method: rhd and rhce phenotyping was done by accredited routine protocols (monoclonal ab id card: diaclon rh subgroups, seraclone anti-c). genotyping was performed with a taqman probe assay (rbc-fluogene veryfy, inno-train diagnostik gmbh), sso (rbc-lifecodes, gen-probe inc.), in-house ssp-pcr (hila, rode kruis-vlaanderen) and commercial ssp-pcr (rbc-ready gene cde, inno-train diagnostik gmbh). sanger sequencing of the rhd gene was performed using an inhouse method (inno-train diagnostik gmbh). results/finding: discrepant genotyping results were generated by different test systems: the taqman probe based assay showed in repetition a ccee genotype, while the sso system rbc-lifecodes predicted in repetition a ccee phenotype. in ssp-pcr the sample showed a weak c band with the in-house method, while there was no band visible with the commercial test kit. the parallel analysis of the rhd gene with rbc-ready gene cde test system revealed a variant d cat vii rhd allele. sequencing of the dna sample identified two snps on one of the rhd alleles ( t>c, t>c) confirming a rhd* . and one rhd* allele. hispanic female in preparation for surgery resulted in variable reactivity and weakly positive d reactions when using microtiter-well agglutination versus tube testing. determination of whether the d antigen expression represented a weak d or a variant d could not be resolved by serologic testing alone. here we report the characterization of a novel rhd gene mutation identified by rhd gene sequencing. study design/method: serologic typing was initially performed by microtiter-well agglutination by automated analyzer platforms galileo neo and galileo echo (immucor, norcross,ga) and by standard tube testing using the immucor series and anti-d reagents. further immunohematologic evaluation was performed by standard tube testing (immediate spin -is, and indirect antiglobulin -iat) using orthobioclone, immucor gammaclone, immucor series and series , and albaclone anti-d reagents. dna isolated from wbcs was used in manual rflp and rhd beadchip assay (immucor, bioarray) and rhd sequencing. results/finding: rbc reactivity is summarized in the table. dna testing detected a hybrid rhesus box associated with the rhd gene deletion, indicating the patient was hemizygous for rhd. rflp assay and rhd beadchip did not identify any changes. rhd gene sequencing identified a new c. a>g change in exon encoding an amino acid change p.met val. the predicted location of this change is within the fourth transmembrane segment of the rhd protein. conclusion: we identified a novel rhd allele with c. a>g (p.met val) change in exon . several snps, deletions, and insertions have been reported with changes in exon . phenotypes of these genetic variations result in rh negative, weak d types, and variant d. since this change has not been previously identified, we are unable to determine if this confers a risk of anti-d alloimmunization, but the rhd c. a>g snp results in serologically weak phenotypic expression of d antigen when tested by microtiterwell agglutination on the neo/echo platforms. in this case the combination of microtiter-well agglutination and dna sequencing helped identify a new allele which would be missed by standard tube serologic testing and the current commercially available array assays. serologic and molecular detection of an antibody to a high incidence antigen in patient with history of chronic transfusions georgia spanos* , juan merayo-rodriguez , christopher lough and nancy eckert . lifesouth community blood centers, life south community blood centers, lifesouth community blood center-headquarters background/case studies: the jo a antigen is one of three high incidence antigens in the dombrock system. the prevalence of this antigen is % in most populations and greater than % in the black population. the jo a antigen can be resistant or enhanced with enzyme treatment (ficin/papain) and typically variable with dithiothreitol (dtt), w.a.r.m. tm (immucor) and zzap treatment. anti-jo a is an igg antibody that demonstrates at ahg phase. hemolytic transfusion reactions to the jo a antigen vary from none to moderate/severe. hemolytic disease of the fetus and newborn (hdfn) has not been observed with any antibody associated in the dombrock system. there are two common phenotypes present in the black population:hy negative/ jo a negative and hy weakly expressed/jo a negative. study design/methods: an antibody identification and red blood cell (rbc) units were requested for an o positive, year old, african-american female with a history of sickle cell disease and no history of pregnancy. the patient was not recently transfused, however, had a history of chronic transfusions. last reported transfusion was three years prior to the current specimen. there were no known rbc antibodies at the time of the request. facility reports that the patient's hemoglobin(g/dl)/hematocrit(%) (hgb/hct) is . / . and does not appear to be in sickle cell crisis. a request for phenotypically matched units, as per hospital policy, for c, e, k and s was received by our immunohematology reference laboratory (irl). results/findings: anti-jo a was detected in patient plasma reacting with liss and peg (tube method) and manual gel-iat. the antibody was resistant when tested with dtt treated red cells. in-house frozen reagent rbcs negative for the jo a antigen (positive for hy) were used to serologically prove the presence of the antibody to this high incidence antigen. an allogenic peg adsorption was performed to rule out other common clinically significant antibodies. anti-kp a was identified using this adsorbed plasma. further testing with molecular genotyping (grifols idcore xt ) confirmed the patient's genotyping as antigen negative for the jo a , kp a and positive for hy. conclusion: molecular testing is frequently performed on patients and retained donor samples from our local community donor pool throughout florida, georgia and alabama. staff is able to search our database for any combination of antigen negative phenotypes using the internal (k) blood establishment computer software (becs) integrated blood bank information system (ibbis). this enabled us to locate one refrigerated and three cryogenically preserved jo a negative rbc units. we found eligible blood donors that could be recruited via an automatically generated call list. the request for rbcs was cancelled. patient's clinical symptoms improved without transfusion and repeat hgb/hct increased to . / . the patient's sibling is historically negative for the jo a antigen and should future transfusions be required, it was recommended that a directed donation be made on the patient's behalf. in order to continue having blood components available to meet all our patient's needs, irl staff is consistently screening and searching our inventory for blood components that are negative for rare antigens to retain for patients needing antigen negative units in a timely fashion. rbcs of two females whose samples were referred for rhd genotyping with previously reported alleles for which serologic reactivity had never been investigated. study design/method: serologic testing was performed by automated analyzer, galileo echo and neo (immucor, norcross, ga), and by standard tube testing with licensed anti-d reagents and the albaclone advanced partial rhd typing kit. genomic dna isolated from wbcs was used for immucor rhd beadchip assay, pcr-rflp, and rhd sequencing. results/finding: patient was a yo female, c e c e , whose rbcs reacted by echo and by neo with anti-d , and '?' with anti-d . testing with d and d by tube gave and w on initial spin (is) respectively and by indirect antiglobulin test (iat). rbcs were non-reactive at is with ortho bioclone and biorad seraclone, and w with immucor gammaclone anti-d, and all were at iat. rbcs did not react with two (lhm / & / ) of anti-d in the alba partial d kit. this pattern did not match any partial d identified by these clones. rhd beadchip detected an inactive rhd pseudogene in trans to rhd. gene sequencing confirmed the presence of the pseudogene, but rhd had a c. c>a change encoding p.his gln. patient was a yo pregnant female, c e c e , whose rbc were w at is and at iat with immucor series and and gammaclone, and moderately reactive, is and iat, with alba alpha, alba blend and delta anti-d. rbcs did not react with two (lhm / & / ) anti-d in the partial d kit with no known partial d pattern. dna testing predicted she was rhd hemizygous and rhd beadchip detected markers for rhd*dar but exon gave low signal (ls). sequencing found a hybrid dar with ce-specific nucleotides in exon from c. to c. encoding amino acid changes p.ile leu and ser asn. conclusion: we found two previously reported rare alleles: rhd with a c. c>a (p.his gln), previously found in france (lefloch et al. genbank ku ), and rhd*dar with part of exon replaced by rhce, reported in sub-sahara africa (granier et al. transfusion : ) designated rhd*dar(ce :v v-s n) with an allele frequency of . to . . blood samples were not available to test for alterations in d expression for either allele. we provide serologic evidence that these alleles, found in two females evaluated by rhd genotyping, inform transfusion and rh immune globulin prophylaxis, as they encode partial d phenotypes with novel epitope expression patterns, meaning these patients are at risk of forming allo anti-d. background/case studies: hu f -g is a human monoclonal igg antibody recognizing cd that is in clinical trials to treat hematologic or solid malignancies. cd is a transmembrane glycoprotein that binds to signalregulatory protein a (sirpa) on macrophages and functions to regulate phagocytosis. blocking cd is thought to enhance phagocytosis and promote anti-tumor responses. cd is also highly expressed on rbcs, and the purpose of this study was to evaluate anti-cd drug interference in blood bank testing. study design/method: serologic testing was performed by standard methods. serial samples (n ) from patients were tested over the course of month treatment. plasma was tested at immediate spin (is) and by iat with r r , rr, d--, rh mod and rh null rbcs, as cd expression levels vary depending on rh phenotype. dtt and enzyme treated rbcs were also tested. both immucor gamma-clone anti-igg (does not detect igg ) and ortho bioclone anti-igg (total igg) were used. for titration plasma was diluted in pbs. allo-adsorptions were performed with papain treated rr rbcs and eluates were made using gamma elu-kit ii. results/finding: anti-cd was observed in plasma as soon as hour post infusion. plasma reacted to at is and with all panel cells in peg iat using ortho anti-igg. d--, rh mod and rh null rbcs were nonreactive at is and weaker ( and ) in peg iat with ortho reagent. reactivity with all panel cells by ortho igg gel card was . in contrast, iat reactivity using gamma-clone anti-igg was only w to , and this reactivity was confirmed to be carry-over agglutination. d--, rh mod and rh null were non-reactive in peg iat using gamma-clone anti-igg . the anti-cd titer was at is and peg iat with gamma-clone anti-igg, but was ! with ortho anti-igg. plasma reacted with dtt, trypsin, papain, a-chymotrypsin or w.a.r.m. treated rbcs. somewhat unexpected, autocontrols were negative and dats were non-reactive or microscopic only. acid eluates (n ) were reactive with ortho, and non-reactive with gamma-clone anti-igg. plasma reactivity was removed after x allo-adsorption with papain treated rr cells, but in some samples low level (micro- ) reactivity remained. peg adsorption was invalid due to precipitation/complexing of antibody. robust plasma reactivity interferring in abo reverse typing was observed, and weak spontaneous agglutination of the rbcs in the abo forward and rh typing. conclusion: hu f -g anti-cd therapy interferes with routine pretransfusion testing, not only antibody screening and crossmatch, but abo and rh typing. high levels of cd expression on rbcs results in plasma agglutination at is, mimicking reactivity observed with igm antibodies although hu f -g is igg . reactivity was observed in all phases and with all test methods. cd is not cleaved from rbcs by dtt, trypsin, papain/ ficin, dtt with ficin (w.a.r.m.) or a-chymotrypsin, and treatment of rbcs with these does not mitigate interference. numerous adsorptions with papain treated rr rbcs were required to remove anti-cd reactivity from plasma. use of immucor gamma-clone anti-igg, which does not detect igg , can mitigate interference in iat although carryover reactivity may be observed. due to blocking by anti-cd on the patient rbcs, dat and autocontrols were weak or non-reactive; however eluates prepared from the dat rbcs were strong and pan-reactive using ortho anti-igg. background/case studies: a caucasian woman with history of a caesarean section and a rbc tx in . in august , she was admitted to hospital for trauma surgery, ab screening was negative and two units were transfused without transfusion reactions. five days later she was referred to a tertiary care trauma center due to a severe postop infection and need for a reoperation. ab screening was now positive, with an antibody reacting with all panel cells detected. because of the urgent need for rbc tx, two weakly cross-match positive rh k matched units were transfused with a warning of possible alloantibodies. the patient got acute hemolysis. study design/method: a gel technique was used in the hospital transfusion laboratory. in addition, various antibody identification panels and special serological and genotyping methods were used in the reference laboratory. kel sequencing was done by the international immunohematology center. results/finding: the hospital transfusion laboratory results were o rhd neg, dat neg, and the ab identification was with untreated and with enzyme-treated cells, with weakly positive autocontrols. a sample was submitted to the reference laboratory for additional investigation. dat was weakly positive, while ab identification results were similar to the hospital results. different pheno-and genotyping methods were used in addition to several identification panels to exclude rare blood groups. after pk, vel neg, jk:- etc. had been excluded, k-phenotyping revealed a k -phenotype. a total of silencing mutations are known for the kel gene and the genotyping kits used did not recognize these. the anti-ku antibody reacts with all cells apart from the k -phenotype. the presence of dtt-sensitive anti-ku was confirmed with dtt-treated panel cells. anti-ku may cause immediate and delayed hemolytic transfusion reactions. samples were taken from the patient's two siblings and daughter. kel sequencing revealed kel* n. with c. t encoding p. ter (reported in an individual from austria in ). there are two known k -patients in our country, both homozygous for c. t. the daughter was a c. t heterozygote, while the siblings did not have this variant. a new operation is necessary but no k -donors are available in our country. with the help of the isbt rare donor working party, a k o rhd neg donor was found in japan and one unit was delivered to us for use in the next operation. conclusion: an alloantibody should always be suspected when autocontrol is weaker than panel cell reactions, even if the direct coombs is positive. a combined serological and genotyping approach offers the best solution for problematic antibody cases. compatible blood is not always available in rare blood group cases, but international co-operation may be of help in finding a suitable donor. transfusion strategy for the serologic weak d phenotype in tunisia based on rhd alleles and rh haplotypes mouna ouchari* , kshitij srivastava , houda romdhane , saloua jemni yacoub and willy albert flegel . nih, dtm/cc/nih, regional blood transfusion center sousse, regional blood transfusion center sousse, tunisia background/case studies: d antigen variants have been studied molecularly in many arab populations, including gaza, tunisia, egypt and libya, a transfusion vol. supplement s since . the tunisian population has the largest known prevalence of weak d type . alleles, occurring in of rh haplotypes, compared to in , or less in europe. a systematic study was missing for samples with the serologic weak d phenotype routinely found in blood donor and patient testing in tunisia. the study was designed to obtain data on weak d type . in a population known to harbor the greatest prevalence of such allele worldwide. study design/methods: a total of , random blood donors were serologically screened for the d antigen using routine techniques. samples with weak reactivity were tested with a panel of monoclonal anti-d (partial rhd-typing set) to identify partial d phenotypes. the rhd gene was sequenced in all samples with serologic weak d phenotype. the rhce gene was also tested molecularly by either direct sequencing or using the rhce beadchip kit to ascertain the rhce allele linked to the rhd allele. results/findings: a total of discrepant samples ( . %) were observed and expressed the serologic weak d phenotype. among them, carried an allele of the weak d type cluster ( . %), of which samples ( . %) showed the weak d type . allele. only sample each was found for the weak d types , and and the dvii, while samples showed the consensus rhd sequence. no mutation in any of the rhd exons was detected in another samples. the molecular analysis of the rhce gene showed that out of samples with serologic weak d phenotype ( . %) had a variant rhce allele and the most common associations were: weak d type . linked to rhce*cevs. . ; weak d type . . with cear; and weak d type . to rhce*cevs. , while the other rhd alleles were linked to one of the common rhce alleles. conclusion: almost % of the weak d phenotypes in tunisia were caused by alleles of the weak d type cluster, of which % represented the weak d type . allele. based on established rh haplotypes for variant rhd and rhce alleles and the lack of adverse clinical reports in tunisia, we recommend d positive transfusions for patients and no rhig administration for pregnant women with weak d type . in tunisia. we propose this strategy as a pragmatic clinical decision, even if eventually a rare allo-anti-d immunization would occur in tunisia associated with weak d type . phenotype. there is a possibility that the rhce*cevs. . allele, typically associated in tunisian individuals, may protect from allo-anti-d immunizaton and other rhce alleles, such as rhce*ce more often associated in individuals of other ethnic groups, may not. however, we conclude that this conjecture has not much evidence in support at this time and would need corroboration by experimental and clinical data, before used to guide clinical recommendations. martha rae combs* , heather simmons , christine lomas-francis , gayane shakarian , sunitha vege , lauren hutelmyer , sandra nance , jessica poisson , nicholas bandarenko and connie m. westhoff . duke university hospital, immunohematology and genomics laboratory, new york blood center, arc pennjersey, american red cross, immunohematology reference laboratory, biomedical services background/case studies: plasma from a transfused, a , year old white female, post liver transplant with rbc aplasia, reacted at rt and in peg iat with all rbc samples tested except her own. study design/method: standard hemagglutination methods were used for antibody id and antigen typing. acid eluates were prepared using gamma elu-kit ii (immucor). genomic dna was isolated from wbcs and used for hea precisetype array and kel and sc gene sequencing. samples from the proband and her mother were tested, as applicable. results/finding: the patient's dat was negative. her plasma reacted with . m dtt-treated and papain-treated rbcs, all available rbc samples lacking high-prevalence antigens, and with phenotypically similar rbc samples [c , k , fy(a ),s ]. reactivity was detected to a titer of ; it was not removed by prewarm technique or by x peg alloadsorption. the adsorbed plasma reacted with . m dtt-treated rbcs. extensive rbc phenotype results were unremarkable except for the following: k , k , js(b ), kp(a b ) and sc: , . her plasma reacted with k o , mcleod, sc: , rbc samples and dtt-treated sc: rbcs at rt and peg iat but her diluted plasma and pretransfusion eluate showed relative kp b specificity. the patient was transfused aliquots of crossmatch incompatible kp(b ), s rbcs. her post-transfusion dat was with anti-igg, with anti-c d. the eluate reacted with all rbc samples except kp(b ) sample. she tolerated additional aliquots from phenotypically similar rbcs untested for high-prevalence kell or scianna antigens. the hea precise-type predicted k , k , kp(a b ), js(a b ) and sc: , , discordant with her rbc phenotype. kel gene sequencing identified a homozygous change, c. a>t (p.glu val) (kel* . ) encoding the low prevalence antigen, ul a , but no changes associated with lack of kell system antigens; however, her rbcs typed ul(a ). sc sequencing found heterozygosity for a '- g>a change (rs , to % prevalence) and conventional sc* , predicting sc: , , . kel and sc results on the mother were kel* /kel* . , heterozygous for the sc change '- g>a, and her rbcs typed k k kp(a b ), sc , ula , consistent with dna predictions. plasma collected months later was nonreactive at rt and in peg iat. her rbcs were dat and now typed k , kp(a b ), ul(a ) sc and sc ,concordant with predicted kell and sc phenotypes. conclusion: we report an example of kell and scianna antigen suppression or blocking in the presence of autoantibody or an alloantibody in the kel system. to our knowledge, this is the first report of a ul a kel* . homozygote. the rbcs may lack a high-prevalence antigen antithetical to ul a . without dna testing and gene sequencing, the patient would be presumed to have kell null and sc null phenotypes, a search for k o , and/or sc: , rbc units would be performed and we would not have been prompted to re-type her rbcs when the dat was negative. background/case studies: anti-jka is a common antibody identified in the blood bank and providing phenotypically characterized red cells lacking this antigen is important in avoiding an acute or delayed hemolytic transfusion reaction. in nearly all cases, this antibody is identified in the context of a phenotypically homozygous jkb patient, jk(a-b ). other scenarios are quite rare. we present two cases of anti-jka in which this phenotype was not observed. study design/method: patient a is a -year-old multiparous female with no known transfusion history. her blood typed as o positive with a positive antibody screen, negative dat, and a clearly identified anti-jka in plasma. the patient phenotyped as jk(a-b-). genotyping revealed the presence of the jk*b allele, but not the jk*a allele. complete sequencing of the jk gene showed an intron polymorphism in homozygosity. specifically, the patient showed a jk*b(ivs - a)genotype, associated with a jkb null phenotype. anti jk was not identified. the conclusion was an allo-anti-jka in a jk null patient. the patient did not receive any transfusions. patient b is a multiply transfused old female. her blood typed as a positive with a positive dat and antibody screen. both the plasma and eluate revealed an anti-jka. despite the recent transfusion, the patient phenotyped as jk(a ) and jk(b ) . genotyping showed the presence of both jk*a and jk*balleles. whole gene sequencing was not performed. there was no hematologic or biochemical evidence of hemolysis. the patient was considered to have an auto-anti-jka and jka negative cells used for transfusion. results/findings: patients a and b both developed anti-jka while having uncommon phenotypes/genotypes. conclusion: it is common for jk null patients to develop anti-jk . however, we speculate that expression of the kidd glycoprotein with the jkb epitope was below the threshold of serological detection, but enough to prevent the formation of anti-jk or anti-jkb. auto-anti-jka is usually reported in the context of an active hemolytic process, but patient b illustrates an auto-anti-jka without hemolysis which is more commonly observed with autoantibodies exhibiting specificity for rh epitopes. these rare cases of anti-jka require phenotypic and genetic analysis for the jkb epitope and jk*b allele respectively, and in more complex cases whole gene sequencing. background/case studies: donor genotyping for red blood cell antigens has become common practice in many blood bank laboratories. package inserts for commercial assays indicate false negative results may be generated when unexpected rare mutations affect primer or probe binding and cause allele dropout or failed amplification. these outcomes may go unrecognized unless serological results are available for comparison. study design/method: a routine blood donor, self-identified as african american, was selected for red blood cell genotyping. dna was extracted and genotyping was performed using two commercial platforms a transfusion vol. supplement s (precisetype, bioarray, warren nj; idcore xt , grifols, emeryville, ca). genotype results were compared to historical serological results. discrepancies were resolved by sanger sequencing (grifols ih, san marcos, tx). results/finding: genotyping results showed variants in both the duffy (fy) and kell (kel) blood group systems. the donor's genotype was concordant on both platforms, fy*a/fy*b_gata, kp*a/kp*a, for a predicted phenotype: fy(a b-); kp(a b-). when genotype results were compared to historical serology, it was noted that the donor previously typed fy(a-) on separate donations. no previous kpa or kpb serotyping was available. sequencing of fy exon revealed a g>a mutation, fy *n. , known to silence fya. sequencing of kel exons - exposed a silent polymorphism in exon , g>c. this polymorphism causes a dropout artifact yielding a false negative kpb interpretation. conclusion: the discrepant fy*a result, as well as the unlikely kp(b-) type prompted the request for sequencing. the rare fy *n. mutation has been reported in people of caucasian descent. this is the first example of this fy mutation identified in this regional population. the kpb antigen is present in nearly % of all populations. however, kp(b-) is most frequently seen in people of caucasian descent. to date, self-identified african american donors have been genotyped as kp*a/kp*b at this blood center. given the diversity of regional heterogeneity, it is feasible to identify a kp(b-) donor, self-reporting as african american. red blood cell genotyping offers an abundance of information, but cannot replace serology as the sole means of red cell antigen characterization. donor ethnicity continues to play a key role in selection for genotyping and the search for rare and unusual red cell types. in this case, a donor selected for genotyping based on ethnicity was initially thought to have genetic variants not previously reported in those of african descent. only was proven to be present. this case acts as a reminder that genotype limitations must be considered even when using licensed methodologies. this case report presents two group o pediatric patients who had been on enteral feeds and had absent/weak anti-b that became strong over time in patient . study design/methods: patient was a year-old male born prematurely with short gut syndrome who underwent a small bowel and liver transplant at years of age. anti-b changed from undetectable/weak to strong at the age of years. patient was a month-old female with a metabolic urea cycle disorder who underwent a liver transplant. anti-b was / . both patients were on total parenteral nutrition (tpn) since birth and had strong anti-a and normal immunoglobulin testing. abo typing with enhancing techniques is presented in table . results/findings: both patients typed as group o on forward typing. anti-a was strong in both patients. anti-b varied in strength in patient with - reactions up to years of age. thereafter, abo typing showed mainly strong anti-b. patient had / anti-b. conclusion: intestinal bacteria stimulates production of anti-a and -b. unexpected changes in anti-b that caused abo discrepancies are reported here for children on long-term tpn. patient had absent/weak anti-b since birth up to years of age, then developed strong anti-b with no change in feeding regiment and medications. patient had consistently strong anti-a and absent/weak anti-b. these findings support the notion that normal colonization of the gut is important in the development of anti-a and -b and suggests that microflora of the gut in patients on prolonged tpn is different leading to the delayed formation of these antibodies compared to individuals on normal enteral diet. difference in strength of anti-a and-b could be due to stronger a than b antigen expression on gut bacteria. results/finding: a daratumumab protocol was established that incorporated use of the cord panel. multiple myeloma patients selected as candidates for daratumumab treatment were baseline tested for blood type and antibody screen, dat and genotype. after daratumumab infusion, a two unit crossmatch was order as a precaution in the event the patient developed a reaction to the medication. repeat of the antibody screen demonstrated panagglutination which served as a positive control for the medication. the cord panel ruled out underlying alloantibodies. selected red cell units were crossmatched at immediate spin phase to avoid expected indirect antiglobulin reactivity. conclusion: the cord panel was used times over a five month period to rule out underlying alloantibodies. tests for the daratumumab protocol consisted of a routing antibody screen followed by a cord panel for resolution. the daratumumab protocol significantly reduced testing time and allowed for the provision of compatible blood products in an efficient and cost effective manner. teresa gorey* and elizabeth hart . brigham and women's faulkner hospital, university of massachusetts-dartmouth background/case studies: the purpose of performing a pre-transfusion antibody screen is to detect clinically significant unexpected antibodies and to decrease the probability of detecting clinically insignificant antibodies. several antibody detection methods (polyethylene glycol (peg), liss, and albumin) are routinely used in small transfusion services. the utility of peg is to enhance the sensitivity of detecting clinically significant antibodies by the indirect antiglobulin procedure. the code of federal regulations, title , cfr part . (a), states the manufacturer's instructions are followed when testing for unexpected antibodies. the package insert for gamma peg tm (immucor inc., norcross, ga), states that negative reactions may be examined with an optical aid. based on these directions, our institutional policy is to confirm all negative reactions using the microscope. study design/method: a one-year retrospective document review was performed on all patient samples in which a positive antibody screen (absc) triggered the antibody identification (abid) to be performed in . a total of samples were evaluated. each abid was subcategorized; ( ) as being a new antibody for our facility or in the patient's shared electronic health record within the partnersv r healthcare system and ( ) whether a microscopic absc result triggered the abid. also, patients with known antibodies were grouped according to a microscopic absc result. a comparison of the new patients and the previously known antibody patients with microscopic results were reviewed to determine if the antibodies were clinically significant. results/finding: a total of abids were performed on new patient samples. of the new abid samples, ( %) had microscopic absc results. for the previously known antibody patients, there were which accounted for % of the total abids performed. when reviewing the total abid workups, a total of ( %) of the abscs had microscopic results which resulted in an abid being performed. the antibodies identified in the new antibody samples were: conclusion: a total of % of the new antibodies identified based on a microscopic absc were clinically insignificant. the manufacturer's directions were followed but they do not state that an optical aid is required to confirm all negative results. due to the results of this study, a decision will be made to: ( ) discontinue the use of the microscope, ( ) switch to a peg manufacturer whose directions indicate to observe macroscopically for agglutination, or ( ) define the use of the agglutination viewer as the optical aid. decreasing the number of abids will save time and money while providing potential rbcs for transfusion in a timely and efficient manner. anton") has a prevalence greater than % in all populations. hereditary absence of anwj has only been described once (in a single family). however, red cell expression of anwj may be markedly decreased to near undetectable levels in blood donors of the in(lu) (or "dominant lutheran inhibitor") phenotype. similarly, anti-anwj antibody formation is rare, with only cases reported in the literature. the antibody developed in the context of hereditary absence of anwj (i.e., a true alloantibody) in only one of the cases. in the other nine cases, the antibody occurred in the context of autoimmune or lymphoproliferative disease, where, in this context, it is believed to have developed secondary to transient anwj antigen suppression. most of the reported cases lacked clinical or laboratory evidence of hemolysis. however, in the most recently reported case, involving a -yearold woman with aplastic anemia, the antibody was associated with acute hemolytic reactions after rbc transfusions, necessitating transfusion support with anwj-negative and in(lu) rbcs. the case was also unique in that the anti-anwj resulted in a direct antiglobulin test (dat) that was positive for complement only, rather than igg like all previous cases in which the dat was performed and was positive. study design/method: a -year-old woman with severe aplastic anemia experienced acute hemolytic transfusion reactions (ahtr) with development of a panagglutinin on indirect antiglobulin test (iat) screens. prior to identifying the specificity of the panreactive antibody, the patient received rbc transfusions and showed signs of hemolysis with six of them. the first three transfusions were prior to her positive iat and were electronically crossmatched. the next seven transfusions were incompatible by antihuman globulin (ahg) phase crossmatch, but were extended phenomatched for clinically significant antigens. the patient's ahtr signs and symptoms included fever, rigors, nausea, vomiting, dark urine, flank pain and "impending doom" anxiety; while her laboratory findings included hemoglobin decreasing below pre-transfusion levels, and increased total bilirubin and ldh. the dat, while initially negative during the immediate posttransfusion workup of the transfusion reactions, eventually became positive for igg only ( - ), and negative with anti-c b, c d reagent. the antibody showed a peak gel-igg iat titer of . results/finding: the antibody was identified as having anwj specificity. the patient's pre-transfusion sample showed weak anwj expression (w ), altogether suggesting an auto-anti-anwj. monocyte monolayer assay testing using the patient's plasma and rbcs from the ahtr-implicated units yielded monocyte indices ranging from to %, consistent with the clinical hemolysis observed. given the patient's group o, rh d negative blood type and continuing transfusion dependence, in order to avoid further ahtrs, international collaboration was necessary in order to procure and provision group o, rh d negative rbcs that were also serologically negative for anwj. the patient was successfully transfused three such units without further incident. conclusion: this is the second documented case of anti-anwj in a patient with aplastic anemia and, overall, the third anti-anwj case associated with ahtr. this case also underscores the importance of international collaboration. cold auto-antibody anti-p anti-m anti-sd a anti-le b anti-jk a anti-k anti-e anti-c results/finding: three hundred and ninety weak d genotypes have been determined to this day with frequencies of % (type ), % (type ), % (type ), % (type ) and % other than , , or . further investigation was conducted to determine the molecular identity of the «others». out of samples, ( %) were confirmed to be legitimate serological weak or partial d, mainly deletions of exon or both exons and . a surprising amount of samples were discovered to be normal rhd. conclusion: along with sandler et al. ( ) data, our findings highlight the difficulties hospitals face in interpreting serological weak d. trend analysis was conducted regarding the reagents and technologies used by each hospital, the origin of the request and the ethnicity of the concerned patient, but no significant correlation could be identified at this point. altogether, our findings allow to share the frequency of weak d types , , and obtained in serological weak d, years old quebec's women, and also highlight the need for further investigation of standard practices amongst hospitals regarding the management and interpretation of atypical d typing. were classified as fnhtr. taco incidence was , %. no trali happened in the period. prophylaxis were used in % of patients. conclusion: fnhtr is described as the most common adverse event related to transfusion, but our data showed a higher incidence of allergic reactions. fnhtr occurred times less than allergic reactions. this might be explained by universal leukoreduction and universal prophylaxis adopted at our institution. further studies are necessary to evaluated the benefit of this approach. that cannot be associated with a specific rbc unit or were deemed unrelated to transfusion, rbc transfusion aes were analyzed. chi-square test and logistic regression were used to compare the ae incidences among transfusion groups. results/finding: univariate and multivariate logistic analyses showed that irradiated rbcs were associated with a significantly increased incidence of transfusion-related aes (p < . ). there was a significant difference in febrile non-hemolytic transfusion reaction (fnhtr) ( . % vs . %, p < . ) or aes with a non-allergic type inflammation etiology ( . % vs . %, p < . ) including transfusion-related acute lung injury, transfusion-associated dyspnea, but not transfusion-associated circulatory overload, infections or hemolytic transfusion reactions, between irradiated rbcs and non-irradiated rbcs. in contrast, the incidences of allergic aes ( . % vs . %, p . ) were similar between these two groups. the incidences of inflammation aes after transfusion of irradiated rbcs that were stored for , , , and weeks were . %, . %, . % and . %, respectively (p . , logistic regression) but there was a significant difference in the incidence of inflammation aes caused by irradiated rbcs stored for a week ( . %) and longer than a week ( . %) (p < . ). conclusion: irradiated rbcs associated with a higher incidence of transfusion inflammation aes compared to non-irradiated rbcs and this risk increased when rbcs were stored longer than week after irradiation. while it is likely the patient population is a factor in ae caused by irradiated rbcs, it is also possible that rbc radiation damage, as shown in previous studies, contributed to this increased ae incidence. a list of patients with one of these icd codes was generated. the emr was searched to find the clinical scenario in which trali was mentioned. these patients' records were then searched within our laboratory information system (copath), to determine if they had a transfusion reaction reported to our transfusion medicine service. results/finding: the search of our electronic medical record found patients from - , who had trali mentioned in their chart as a diagnosis or possible/likely diagnosis. one patient was excluded from our study because trali was mentioned as a past medical history from an outside hospital. only the patients who had trali listed as a diagnosis or possible diagnosis were included in this study. these patients had clinical scenarios in which a transfusion of a blood product occurred which was followed by various forms of respiratory distress. the clinical teams caring for these patients were either giving a diagnosis of trali or considering trali as a possible diagnosis. of these cases, only of them were reported to our transfusion medicine service as transfusion reactions. of the reported cases, one was determined to be trali and the other one was consistent with taco. eight out of those cases were never reported. background/case studies: despite diligent efforts to transfuse the safest product available to patients, undetected alloantibodies may cause delayed hemolytic transfusion reactions (dhtr). this transfusion reaction is seen in as many as out of transfused products. therapeutic plasma exchange (tpe) may be employed to mitigate ongoing immune mediated hemolysis, but few reports in the literature describe tpe for clinical management after profound hemolysis. study design/method: case review of a patient was performed after diagnosis and treatment of severe dhtr. results/finding: a man with a history of gastrointestinal bleeding presented to the emergency room with shortness of breath and "hematuria". he had a known history of anti-d and anti-c, and was transfused two units of crossmatch compatible rbcs seven days prior during a previous admission. readmission hemoglobin (hb) was . g/dl but declined to . g/dl the next day. an antibody screen was consistent with anti-d, anti-c, and direct antiglobulin test (dat) was negative. he received three units of crossmatch compatible rbcs over days and with poor responses. on day , routine labs could not be reported due to marked hemolysis, he had "worsening hematuria", creatinine rose from . mg/dl to . mg/dl (reference . - . mg/dl), and lactate dehydrogenase was above reportable linearity, > u/l (reference - u/l). testing revealed additional anti-e, anti-jkb, dat c , plasma free hb . mg/dl (reference - . mg/dl), and hemoglobinuria. four of five transfused rbc units were jk(b ), one of which was also e . one volume tpe was performed to remove free hb on days , , and using fresh frozen plasma as replacement fluid for haptoglobin supplementation. creatinine peaked at . mg/dl on day , decreased to . mg/dl before discharge on day results/findings: twenty three cases were identified, of which had medical records available for analysis. ten ( %) patients were male, the mean age was . years (range - years), ( %) had an underlying hematologic malignancy or bone marrow disorder, and ( %) had a history of coronary artery disease (cad). the implicated units included ( %) red blood cells and ( %) platelets; ( %) patients received a single unit, and ( %) received two or more within the previous hours; the mean volume transfused was . ml (range - ml). the mean time to onset of chest pain was . minutes (sd minutes), with % of patients presenting within . hours and % within hours of starting the transfusion. chest pain was present as the only symptom in % of the cases, and for the other cases the accompanying symptoms included dyspnea ( %), fever ( %), back pain ( %), and hypo-and hypertension ( %). a post-transfusion chest x-ray was performed in % of cases, and all showed no evidence of pulmonary edema to suggest possible volume overload/transfusion associated circulatory overload (taco). electrocardiogram was performed in % of cases and showed no findings to suggest acute ischemia. three ( %) patients had a minimal increase in their troponin levels, although had a history of chronically elevated troponin due to stress cardiomyopathy. fourteen ( %) patients received some form of treatment, including increased oxygen supplementation, metoprolol, acetaminophen, morphine, and oral calcium carbonate; the pain resolved after more than minutes in the majority of patients ( %). no cases resulted in new admission to the icu or procedure cancelation. conclusion: chest pain associated with transfusion was infrequent, but several such cases were identified during the review period. this symptom is not a diagnostic criterion for any of the other hemovigilance categories and merits further characterization to determine whether blood product transfusion could be the cause of the chest pain. larger observational studies to power clinical characterization could help to further inform hypotheses regarding a transfusion-related mechanism, which could be interrogated by translational research studies. background/case studies: thrombotic microangiopathy (tma) in children is most commonly seen in the form of hemolytic uremic syndrome (hus). however, tma may be seen in the presence of streptococcus pneumoniae (spn). the action of bacterial neuraminidase of spn results in exposure of the normally "hidden" thomsen-freidenreich antigen (t-antigen) found on erythrocytes and other tissues. ultimately, this may lead to spn induced hemolytic uremic syndrome (phus) with subsequent hemolysis and end organ damage by naturally occurring anti-t antibodies against the exposed t antigen. specific lectins or anti-sera can confirm exposure of the t antigens in phus. alternatively, phus can be identified by minor crossmatch incompatibility resulting from agglutination of exposed t antigens on recipient's erythrocytes to anti-t antibodies in the plasma portion of blood products. we present a case of suspected phus that resulted in a compatible minor crossmatch leading to concern and eventually diagnosis of atypical hus (ahus). study design/method: a months old boy presented with respiratory failure. he was found to have blood cultures positive for spn as well as hemolytic anemia, thrombocytopenia, and acute renal failure. he was shiga toxin negative and had normal levels of adamts . based on the findings, the clinical team was concerned for phus. therefore, he received washed erythrocytes. for his thrombocytopenia, our institution does not routinely provide washed platelets due to decrease quality of the platelet product. as a result, a minor crossmatching was suggested and performed to determine if t activation was present. results/finding: minor crossmatch was performed with patient's erythrocytes and plasma of abo-identical platelets to be transfused. no agglutination was seen at immediate spin, degree, or anti-human globulin phase. check cells were found to be . these findings were conveyed to the clinical team and platelets were issued without washing. due to the lack of identification of t activation by minor crossmatching and poor clinical response despite appropriate antibiotic treatment, additional studies were performed by the primary team for complement mutations and found to be consistent with ahus. the patient was then treated with eculizumab with clinical and laboratory improvement. we present a case clinically consistent with phus. confirmation of this diagnosis is done with lectins or anti-sera that are not readily available. an alternative means of identifying phus is by minor crossmatch incompatibility. by demonstrating minor crossmatch compatibility, we further elucidated a definitive diagnosis of ahus with appropriate management. background/case studies: orthotopic liver transplantation (olt) is a complex and technically challenging procedure that can be complicated by severe intraoperative bleeding. we report a case of massive transfusion in an olt patient necessitating an abo blood group switch (from o to a ) to sustain transfusion support and minimum group o rbc inventories. study design/methods: type & screen (ts, gel) and anti-a titers (tube) were performed using routine methods. a chart review was performed for pertinent medical and laboratory findings. results/findings: the patient was a -year-old o man with cirrhosis secondary to nonalcoholic steatohepatitis and alpha- antitrypsin deficiency who presented for olt (donor o ). during olt, the patient endured substantial bleeding from retroperitoneal collateral vessels complicated by post-transplant coagulopathy. he required rapid high volume rbc and plasma support, which strained hospital inventories. after receiving units of o rbcs and units of o plasma with ongoing severe hemorrhage, he was switched to group a products. ten units of a plasma were transfused to wash out anti-a antibody prior to transfusion of a rbcs. due to difficulty controlling the bleeding, biliary reconstruction and fascial closure were delayed for hours post-transplant. the patient's total estimated blood loss was > l. he received a total of units of rbcs (including a ), units of plasma (including a ), units of cryoprecipitate, and units of platelets. towards the end of the second procedure, the patient's hemorrhage was stabilized and the final two rbc units he received were o . on postoperative day (pod) , a ts showed predominantly a rbcs with trace o rbcs, as well as very low anti-a igm and igg titers (table ) . he received two additional o rbc units ( each on pod and pod ) with increasing o rbcs on ts and rising anti-a titers. his blood type was unequivocally o by pod . the patient showed recovery of liver synthetic function on pod (factor activity %) complicated by cholestasis. conclusion: this study shows successful switching of a group o patient to group a in the setting of rapid hemorrhage and massive transfusion. by pod , the patient had reverted to o with recovery of anti-a titers. at months post-olt, the patient is alive with signs of improving biliary graft function. a new rfid transfusion safety system anna millan* , alfred mingo , maria isabel gonzalez , antoni mena and juan pedro benitez . bst, at-biotech background/case studies: a new transfusion safety system (tss), based on processes and technologies, especially, identification by radio frequency (rfid), is currently implemented in two hospitals, a general one (h ) and an oncology center (h ). the tss is fully effective in protecting against incidents, and specifically offers mechanisms to detect near misses (nm) by using procedural and physical barriers, assuring that the pretransfusional sample extraction (pse) and the blood components administration (bca) only take place at bed side, using a location control and interacting with the clinical and transfusion information systems (tis). the tss allows to analyze the transfusional activity information in real time to project organizational changes in both transfusion services and hospital units, and to create a new classification of nm. study design/method: retrospective analysis of transfusion activity in both h and h shows pse and bca, out of and respectively, since the tss deployment in . retrospective analysis and classification of security events has been done. results/finding: activity results for both hospitals are shown in the table below. the safety events have been classified in pretransfusion sample extraction (pse), blood component assignment (bcas) in the transfusion service and blood component administration (bca) near misses (nm). for h , nm related to pse accounted for . % of all, being the mistake in concordance between patient identification and prescription order the most frequent ( . %). the nm detected in bcas were . % of all and mostly ( . %) occur when the patient information in the tis does not match the one registered in the tss. the nm detected in bca are . % of all and mostly ( %) the systems detects a not assigned bracelet. for h , nm related to pse accounted for the . % of all, being the error in concordance between the transfusion security number in the bracelet and in pretransfusion sample the most frequent ( . %). the nm detected in bcas accounts for . % of all and in . % occurs when the patient information in the tis does not match with the one registered in the tss. the nm detected in bca are . % of all and in . % of them the blood components were assigned to another patient. ( , , , , , , , , , , , , , ) were analyzed via a commercially available elisa. comparison of adequate response to ppv , defined as ! mcg/ml for > serotypes, was perform based on alloimmunization status. statistical significance was determined by comparing means of subgroups using paired and non-paired t-tests. results/findings: pre-vaccine sp titers were available in patients (alloimmunized, ); pre-and post-vaccine titers were available for patients (alloimmunized, ). of the patients, were on chronic transfusions, were on hydroxyurea, were surgical splenectomized, patients had no history of surgical splenectomy or status was unknown. forty-four patients had a previous history of ppv in the previous years; / also reported previous history -valent sp conjugate vaccine within the last years. baseline pre-vaccination titers (n ) showed no difference between alloimmunized and non-alloimmunized patients (all p-values > . ). in the group with pre-and post-vaccination (n ) titers available, out of ( %) non alloimmunized patients had an adequate response versus out of in the alloimmunized group ( %, p ns background/case studies: blood transfusion is the most common procedure performed in the hospital setting and the transfusion process is monitored to ensure regulatory compliance. to safeguard safety, efficacy and regulatory compliance, transfusion services actively benchmark transfusionrelated errors (tres) as they occur from "vein-to-vein", i.e. from collection of pre-transfusion sample to final infusion of product -with the goal of ensuring that the right product/dose goes to the right patient at the right time. multiple over-lapping error documentation processes are needed to capture and report tres from within and outside of blood bank (bb). we present a comprehensive error management program along with data on five years of benchmarking tres at a large academic medical center. study design/method: tres were detected by capturing and reporting of sample suitability, testing variances and biologic product deviations. in addition, tres as observed and reported by providers and clinical staff (i.e. blood delays/undertransfusions, transfusions without consent, infusions with wrong fluids) were reported to the bb and hospital quality through the veritas system, a hospital based reporting system that enables reporting any occurrence with potential for causing patient harm. all serious errors were reviewed daily and summation of tres was discussed on a monthly basis. mapping tres within the "vein-to-vein" was performed by reviewing the fiveyear of transfusion medicine quality records (from to ). patient harm events recorded within the veritas system from january to july were investigated in depth. transfusion reactions were excluded in this analysis. results/finding: an average of tres per month and per year were found over five years. % of tres are associated with pre-bb activities, % occur within bb, and % are post-bb events. sample collection and handling represent % of total tres. most tres ( %) were reported by bb staff, % were reported by non-bb staff. patient harm analysis revealed an average of four level (near miss), three level (no known harm), and . level (patient harm) per month. no deaths related to tre were detected over the seven month january to july period. patient harm was associated with tres occurring in the bb ( %) and post-bb ( %). these events were reported externally ( %) and by bb staff ( %). conclusion: although most tres were detected in the pre-bb phase, no patient harm was associated with these events indicating an efficient capture prior to causing patient harm. the tres causing patient harm, including near miss events, were mostly reported externally and they occurred entirely in the post-bb and bb phases. these results suggest that significant opportunities for quality improvement may be achieved in two areas: the pre-bb phase aimed at reduction of waste associated with sample collection and handling, and the post-bb and bb phases aimed at improving tre detection and decreasing patient harm. background/case studies: uncrossmatched red blood cells (rbc) and emergency issued platelets (plt), plasma and cryoprecipitate (cryo) are lifesaving in a bleeding patient without a valid type and screen. collectively termed "emergently issued products" they are issued as a bridge until pretransfusion testing is completed. this study evaluated the utilization and wastage rates of blood products during pregnancy-related hemorrhage where the first products issued were emergently issued. study design/methods: a list of patients on whom blood products had been emergently issued between january , and march , was obtained from the blood bank at a regional maternity care hospital. patients who were not experiencing a pregnancy-related bleed (e.g., postpartum hemorrhage or bleeding relating to a complication of pregnancy such as a ruptured ectopic pregnancy or bleeding post spontaneous or therapeutic abortion) were excluded. the total number of products (emergently issued plus crossmatched or non-emergently issued products) that were transfused, returned back into the blood bank's inventory, and wasted within hours of the first emergently issued products were enumerated. apheresis plt units were multiplied by and added to the number of individual whole blood plts; apheresis plasma units were multiplied by and added to the number of whole blood plasma units. results/findings: seventy women who received emergently issued blood products during a pregnancy-related hemorrhage were identified. average age was . the majority of these patients with pregnancy-related hemorrhage who received at least one unit of emergently issued blood products received at least one unit of the product that was issued to them and few units were wasted. that plt wastage was higher than the other products was likely due to the -hour post-pooling room temperature shelf life. keeping wastage rates low while meeting the clinical needs of these patients is the ideal situation for the blood bank. . patient blood platelets were higher before prophylactic than therapeutic transfusions ( [ /l vs. [ /l, p . ). there were no significant differences in the frequency of effective therapeutic ( % vs. %, p . ) and prophylactic ( % vs. %, p . ) transfusions between the prcs and gypcs. we did not find significant differences between prcs and gypcs in cci after prophylactic ( . . vs. . . ) and therapeutic ( . . vs. . . ) transfusions, in cc after prophylactic ( . . vs. . . ) and therapeutic ( . . vs. . .) transfusions. there were no significant differences between prcs and gypcs also in ma after prophylactic ( . . vs. . , p . ) and therapeutic ( . . vs. . . , p . ) pc transfusions. reduction of the severity of bleeds was obtained in ( %) of the cases after prpc transfusions and in ( %) of cases after gypc transfusions. there were no significant differences in the frequency of adverse post-transfusion reactions between the groups (respectively, and cases). background/case studies: an 'end-to-end' electronic transfusion management process including a bedside administration system was developed and implemented in this large multi-site academic center in . it enables the safe administration of blood components at the patient bedside and provides an audit trail for all blood components. an error was identified in the electronic bedside transfusion process which was reported to our national hemovigilance scheme in under the category 'errors relating to information technology'. this error was the incorrect use of the emergency transfusion process for non-emergency transfusions. the standard (non-emergency) process requires a scan of the barcode on the patient's wristband containing their identification details which is verified against the same details from the barcode on the compatibility label attached to the blood bag. the emergency transfusion option is only intended for use with 'emergency group o rhd negative blood units' which, unlike non-emergency units allocated to specific patients, do not have a compatibility label. the emergency transfusion option skips the compatibility label barcode scan as the emergency units can be transfused to any patient needing urgent transfusion. it was found that the emergency blood option was being misused for non-emergency transfusions, leading to blood units not being checked to ensure they were for the correct patient. study design/method: this center worked with the software supplier to develop a solution which corrects the weakness in the process. the revised process involves providing a universal compatibility label for emergency units so that all units (emergency and non-emergency) require a scan of the compatibility label on the blood bag and the patient's wristband at the bedside before transfusion. the use of the emergency process was audited pre and post implementation of the new process to determine whether it was being used correctly or not. results/finding: there were units administered using the emergency transfusion process in the months before the change was implemented. it was found that / ( %) units were non-emergency units administered incorrectly without a bedside compatibility check. following the implementation of the change there were no instances of incorrect administration of non-emergency units in the next month ( components administered), / ( %) were emergency units which were administered correctly. users of the system reported the revised process was quicker, safer and unified with other functions on the device. conclusion: the improved process for the administration of blood in an emergency now prevents users from following the incorrect procedure for non-emergency transfusions and missing the essential final bedside electronic check. this report indicates the need for continued vigilance of the functionality of electronic transfusion processes, and the correction of any weaknesses compromising patient safety. background/case studies: recent recommendations indicate one red blood cell (rbc) unit should be transfused at a time with reassessment after each transfusion to determine the need for more. however, the practices of canadian transfusion medicine (tm) experts and what constitutes a reassessment are unknown. therefore, we conducted a survey of tm experts across canada to gather information on their practices and criteria for reassessment. study design/method: tm experts were identified and contact information obtained from the canadian national advisory committee (nac) and from contacting least one tm expert per province. each respondent was assigned a unique study id after consenting to the survey, allowing for anonymity on analysis. the survey contained demographics, general practice questions, and questions regarding transfusion in: ) a stable anemic inpatient, ) a stable anemic inpatient to be discharged, and ) an asymptomatic post-operative inpatient. results/finding: we identified canadian tm experts: ( . %) provided a response and most had a primary place of practice in a laboratory setting ( / ; . %). for a stable, non-bleeding, anemic inpatient, . % of respondents recommended transfusing one rbc unit, then reassessing. recommendations were more variable in outpatient settings, with . % generally recommending transfusing two rbc units then reassessing. recommendations for reassessment were mainly functional status/symptoms and vitals within a short time period ( - hours), a repeat hemoglobin > hours later dependent on the clinical scenario, and a search for an underlying cause of anemia in outpatient settings. lab practitioners emphasized volume status, cardiac examination, and transfusion at lower hemoglobin thresholds. with an asymptomatic patient to be discharged, fewer respondents chose to transfuse ( . %) compared to an inpatient potentially symptomatic due to anemia ( . %). none of the respondents suggested transfusion in an asymptomatic post-operative patient who had a hemoglobin trending down. conclusion: tm experts generally recommend transfusing one unit at a time in stable inpatients. assessment for transfusion should focus on patient symptoms, pertinent physical exam, hemoglobin levels, and an underlying cause. "top-up" transfusions were not recommended. these recommendations may help guide clinicians, but further research is needed to generate higher quality evidence around the clinical benefits and cost effectiveness of these practices. background/case studies: current evaluation of red blood cell (rbc) post transfusion recovery is based on ex vivo labeling of stored rbcs with radioactive chromium- ( cr). this method has several limitations including the risks associated with radioactivity, and the inability to evaluate multiple rbc populations in the recipient. rbc labeling with s-nhs-biotin (bio-rbcs) overcomes many of these limitations and offers safe and longitudinal tracking of multiple transfused rbcs in vivo. the purpose of this study was to scale up and optimize the biotinylation procedures to the current good manufacturing practice (gmp) environment. study design/method: packed rbc units (n ) were divided into two ml aliquots, which were labeled with selected concentrations of s-nhsbiotin ( and lg/ml) in a cgmp closed system (average bio-rbcs hematocrit of . . %). optimization of labeling efficacy was determined by flow cytometric analysis of bio-rbcs using fluorochrome-conjugated streptavidin (sa). approximately million rbcs were measured in triplicate. quantum simply cellular beads were used to quantify fluorochrome (molecules of equivalent soluble fluorescence, mesf) and infer number of biotin molecules per rbc. the lower limit of detection was determined for rbc labeled with varying amounts of biotin. product quality and safety were evaluated by endotoxin and sterility testing, and by determining the levels of spontaneous hemolysis before and after rbc biotin labeling. results/finding: investigation of different fluorochromes, laser excitation wavelengths and laser power to maximize the signal to noise ratio of labeled and unlabeled rbcs revealed that nm excitation of phycoerythrin (pe)-sa and high laser power ( mw) provided the best separation between the two bio-rbc populations, and between labeled and unlabeled rbcs. labeling with lg/ml of biotin resulted in $ , mesf/rbc, and were detectable among unlabeled rbc at a lower limit of detection (lld, % ci) of in , ( . %). the lld for rbc labeled with biotin at lg/ ml was $ in million ( . %). biotinylation was not associated with increased levels of hemolysis ( . . % before labeling versus . . % after labeling; p . ) or bacterial contamination. conclusion: the resulting manufacturing process produces large volumes ( ml/transfusion) of bio-rbcs with low risk of contamination or hemolysis. the flow cytometry assay can detect bio-rbc in unlabeled blood at very low frequency. we plan to use this technology to study the impact of donor characteristic on rbc storage stability and post-transfusion survival. background/case studies: blood products offer resuscitation benefits in trauma over crystalloid/colloid volume expanders (which provide no hemostatic benefit or oxygen delivery), but usage is often hampered by supply or storage needs. hemoglobin-based oxygen carriers (hbocs) are not red cell replacements but may supplement oxygen delivery and expand volume during transport until blood is available. since hemostasis is critical in resuscitation, this study evaluated bovine hemoglobin glutamer- (hboc- ) effects on coagulation parameters alongside freeze-dried plasma (fdp) in an in vitro model of hemorrhage/resuscitation. study design/method: whole blood (wb) was collected from healthy donors under an approved institutional standard operating procedure. in the first study (limited resuscitation), samples were: ( ) wb, ( ) wb % hboc volume (model of two units in an adult), ( ) wb % fdp, and ( ) wb % hboc % fdp. samples ( )-( ) simulated autoresuscitation by adding % plasmalyte to - . susceptibility to lysis was tested with ng/ml tissue plasminogen activator (tpa). follow-up studies were performed with severe resuscitation simulations of %, %, %, and % volume replacement with hboc and/or fdp, with or without prior % plasmalyte dilution. coagulation parameters were obtained with a coagulation analyzer and thromboelastography (teg). rbcs/hemoglobin were measured on a hematology analyzer. thrombin generation was quantified by thrombogram. platelet aggregation was measured in multiplate and adhesion to collagen under shear in bioflux. viscosity was evaluated by rheology. results/finding: a limited resuscitation model with hboc and/or fdp had no effects on fibrinogen, pt, aptt, ph, hct, or hemoglobin. in teg, wb, wb hboc, and wb hboc fdp had reduced clot strength with dilution and tpa. there was increased susceptibility to tpa-induced lysis between wb and wb hboc in autodilution simulation (mean lysis . % vs. . %; p<. ). hboc and fdp had no statistically significant impact on thrombin generation. no effects on platelet aggregation were observed; no significant differences within diluted v. undiluted groups were seen in platelet adhesion under flow. hboc ( %) did not significantly change viscosity. severe resuscitation simulations had increased pt/ptt and reduced clot strength, particularly in hboc-only resuscitation; however, even % hboc volume replacement produced clots with acceptable teg parameters. conclusion: in a limited resuscitation model with hboc- , there were no significant in vitro effects on hemostatic parameters (except increased susceptibility to lysis); more severe resuscitations impacted coagulation parameters but did not prevent clotting. considering the large impact healthy platelets have on coagulation function, further in vitro studies with impaired platelets are warranted alongside in vivo studies of hboc plasma as initial resuscitation of hemorrhagic shock. therapy in patients with acute major bleeding. while published literature has largely focused on the efficacy and safety of pcc, actual usage practices are less characterized. our aim was to describe the pcc usage practices within a tertiary care center. study design/method: we conducted a retrospective review of the electronic medical records of patients who received pcc between its addition to our institution's formulary in / and / . we compiled information about the usage of pcc in these patients. descriptive statistics were generated with microsoft excel. results/finding: of patients, were on warfarin. pcc was most frequently prescribed for hemorrhage due to surgery ( %). pcc was given for warfarin reversal in % of cases. a subset of patients received plasma within hours prior to pcc ( %) or hours after ( %). pcc was most frequently ordered in the or/perioperative service ( %). conclusion: the majority of pcc usage was "off-label" in terms of being prescribed for indications other than warfarin reversal. the most frequent indication was hemorrhage due to surgery, and pcc was most often ordered in the or/perioperative service. although guidelines recommend the use of pcc as a plasma alternative, plasma was administered within hours of pcc in a notable subset of patients. background/case studies: in emergent situations, when a patient's life may be jeopardized by delaying transfusion, a physician may decide to transfuse blood emergently. however, in some cases, poor communication and lack of clear expectations between the blood bank and patient care areas can lead to frustration and delays in the timely provision of blood products. an incident prompted an appraisal of our emergency release protocol (erp), which revealed gaps in communications and expectations by both the blood bank and nursing personnel. thus, it is imperative that there is a standardized er protocol with clear communications for both the blood bank and nursing personnel. reported here is the outcome of a process improvement that resulted in improved communication, expectations, and turnaround (tat) for our er protocol. study design/method: in , several meetings were conducted with stakeholders (critical care units (icu), emergency department (ed), internal medicine, interventional radiology (ir) etc.) in an effort to identify process gaps, improve communications, and expectations for er episodes. the goals was to design a process for emergent blood product request and release in life threatening situations that will; ) simplify and expedite the process; ) improve communication and expectations to decrease tat; ) improve patient safety and meet compliance. in order to achieve these goals, a series of activities were conducted. these included meetings with all stakeholders to ensure process improvement meet the needs intended. a series of training sessions with nurse educators in icu, ed, ir and surgery managers were conducted. during the meetings, communication goals, and expectations were defined and agreed upon. training sessions included powerpoint presentations to educate staff members and performance of dry runs, to identify weaknesses and strengths with the process flow. the impact on the current process was analyzed and, as a result, led to the revision of the current sop, addition of pre-labeled emergency pack blood ( units of o neg rbc's) and implementation of an electronic emergency blood order set. results/finding: in the ten months post implementation of our improved, standardized er pack protocol, a total of er episodes were received. the average tat from order to delivery at the bedside was reduced by % ( . minutes compared to minutes previously), while the compliance rate for er orders and physician documentation was % ( / ), with no current wastage of blood products. conclusion: the implementation of the improved standardized er protocol significantly improved communications and expectations, decreased tat and delays in transfusions while ensuring patient safety and compliance to regulatory requirements. background/case studies: massive transfusion (mt) in the trauma setting has been extensively studied. yet, the literature in non-trauma areas, especially oncology is rather sparse. the following study was conducted to understand the background and outcomes of mt in cancer patients. study design/methods: this was a single center retrospective study performed at a large cancer center between february -february . mt was defined as the transfusion of ! rbc units in a -hour period. the following data were collected included: age, gender, primary diagnosis, surgery or acute care type, amount and type of blood components transfused, whether or not a massive transfusion protocol (mtp) was activated, and survival at days. results/findings: thirty mts occurred during a one year period. a total of , blood products were transfused during that time period. gender distribution was / ( %) males, and the average age of all patients was with a range of to years of age. surgical patients accounted for / ( . %) mts, and / ( . %) were critical care patients. tumor categories included carcinomas ( / ), sarcomas ( / ), leukemias ( / ) and lymphoma ( / ). resection of tumor followed by complex reconstruction was the cause of the majority of mts. metastatic renal cancer ( / ) was the most common disease seen followed by sacral chordoma ( / ). mtps were activated in only / ( . %) cases. thirty-day survival was seen in / ( . %) patients. only of mortalities was a surgical case (peritoneal mesothelioma), and the remainder were caused by gi hemorrhage ( / ) or perisplenic hematoma ( / ). the overall ratio of rbc:ffp in the entire patients ( background/case studies: plasma is a straw-colored supernatant of blood that is used for type and screen (t&s) and crossmatch. in the analytic phase of testing, plasma is examined prior to processing. plasma occasionally becomes discolored, interfering with crossmatch procedures. timely identification of the etiology allows for corrective actions and minimizes delay in transfusion. study design/method: during the analytic phase of blood bank testing, samples were evaluated for t&s and crossmatch; this identified three samples with discolored plasma. we present a series of cases that illustrate the testing process. results/finding: a -year-old woman diagnosed with breast cancer presented for mastectomy with sentinel lymph node biopsy. a preoperative t&s specimen contained bright green plasma. review of her preoperative case revealed exposure to intravenous methylene blue. this dye is known to alter the color of urine, tears, and blood with no known pharmacologic effects. alternative causes of green plasma include other dyes used to locate sentinel nodes and oral contraceptive use. although not ideal, this sample could be used for crossmatch by tube method, but not automated gel technique. a specimen drawn one week later contained clear plasma. a -year-old woman diagnosed with a warm autoimmune hemolytic anemia was refractory to blood transfusions secondary to alloantibodies. administration of a synthetic blood product resulted in dark maroon colored plasma. the most common cause of a dark red color is hemolysis of the sample, which is usually discarded. in this instance, the hemoglobin color was due to the infused product, an experimental bovine pegylated carboxyhemoglobin that affects colorimetric evaluation of blood samples. with this in mind, the sample was not discarded and testing was completed by tube method. a -year-old woman admitted with acute stroke was treated with a thrombolytic. her t&s revealed cloudy white plasma that could not be used for the crossmatch procedure. common causes of white plasma include purulence, hypertriglyceridemia, and sampling of blood drawn proximal to administration of radiopaque agents such as propofol. although an etiology could not be identified a repeat specimen drawn several hours later was clear. conclusion: these cases highlight the importance of an appropriate evaluation of discolored plasma. once a discolored sample is identified, a repeat sample is required to confirm the change in color. in the first two cases, the discoloration persisted, prompting further clinical investigation. once the etiology was identified, need for further testing and eligibility for further transfusion was determined. testing by tube method could be performed in two cases. in the third case, repeated sampling revealed a clear sample and the transfusion process continued without delay. decisions regarding the analytic phase of testing must include reevaluation of the sample, identification of the etiology, and comprehension regarding how to proceed when discoloration persists. caleb wei-shin cheng* , , rebecca ross , christopher a tormey , , and amit gokhale , . yale university school of medicine, yale-new haven hospital, va connecticut healthcare background/case studies: daratumumab (dara) is a igg monoclonal antibody therapy that specifically targets cd , a glycoprotein highly expressed on plasma cells, where it has been successfully used in patients with refractory or relapsed multiple myeloma. dara interferes with blood bank testing as it binds to cd expressed on red blood cells, causing pan reactivity. the dara interference can be overcome with the use of dithiothreitol (dtt) treated reagent red blood cells. to minimize alloimmunization and to provide crossmatch compatible blood to treated patients, we instituted a dara protocol in our blood bank. the purpose of this retrospective study was to identify the outcomes of our protocol, with a particular focus on the development of de novo alloantibodies during dara treatment at our institution. study design/method: all dara patients' antibody workups were completed using dtt pre-treated reagent red blood cells. if the antibody screen was negative, k antigen negative rbc products are provided. if an antibody is identified, k negative along with that particular antigen negative blood is provided. our electronic medical record (emr) was searched for patients who received dara over the past eight months. study subjects were examined to see if they had pre-existing alloantibodies before dara treatment and whether they formed new alloantibodies during dara treatment. the age, gender, type and screen pre-dara treatment, type and screen post-dara, intervening blood transfusions, and the date of first dara treatment was recorded. results/finding: overall, subjects were identified for analysis. their mean age was . years, with male and female subjects; all were diagnosed with multiple myeloma. we found an alloimmunization rate of % ( / ) prior to administration of dara. of these patients, were transfused with red blood cells (rbcs) after initiation of dara therapy. following our testing/matching protocol, none of these ( %; / ) patients formed a confirmed, new alloantibody during dara treatment; each of these patients underwent at least one follow-up screen after their first rbc unit. we also found no complications in providing crossmatch compatible units to any of the patients. conclusion: to our knowledge, this is the largest case series reporting on results of overcoming dara interference with blood bank type and screen testing. the protocol implemented in our laboratory appears to be successful in providing compatible units and preventing alloimmunization in patients receiving dara therapy. it is possible that the drug, targeting antibody forming cells, may have an immunosuppressive effect on the humoral response; further studies of this effect may be warranted. background/case studies:a multi-facility transfusion service began stocking liquid plasma in september of for use in massive transfusion and trauma situations. due to the infrequent occurrence of these incidents, the liquid plasma would outdate before use. a policy to use liquid plasma in nonemergent situations when the units were nearing their expiration dates was implemented. this study evaluated the effects of that policy on inr values of plasma recipients. study design/methods:a retrospective analysis was developed to compare the effectiveness of fresh frozen plasma (ffp) and liquid plasma (lqp) in changing inr values of recipients. all plasma units transfused within the facility from september , through april , were identified. the following data was obtained from the hospital and laboratory information systems for each unit: the recipient, primary reason for transfusion of plasma, number of plasma units transfused, type of plasma transfused, preand post-transfusion inr values, and whether or not vitamin k was administered. patients were divided into groups based on the type of plasma units transfused and were evaluated based on primary reason for transfusion, number of units transfused, and administration of vitamin k. the change in inr for each recipient was calculated, along with the average change in inr for each group. background/case studies: in gynaecological settings, most but not all relatively young anaemic women are iron deficient due to blood loss associated with menstruation. transfusion could generally be avoided in those without haemodynamic instability. the oral antifibrinolytic drug tranexamic acid is an effective and well tolerated treatment for menorrhagia. besides, iron replacement is often necessary for a prolonged period of time after normalization of haemoglobin (hb). the present study attempted to look into transfusion appropriateness and the use of iron and tranexamic acid in transfused women in hong kong. study design/methods: anonymous data of gynaeological patients age was retrieved from a central database of public hospitals which included age, number of units of red cell transfused, pre-and posttransfusion hb, the use of iron and/or tranexamic acid during hospitalization and upon discharge. all transfusion episodes associated with surgical operations during same admission are excluded. results/findings: in , , unique women receiving a total of , units of red cells (rc) in , transfusion episodes were identified. their median age was (range - ). the distribution of pre-and post-transfusion hb and units of rc transfused were summarized below: in this cohort, pre-and post-transfusion hb were absent in ( . %) and ( . %). ( . %) transfusion episodes were associated with the use of units or more rc. as a result, ( . %) episodes resulted in a post transfusion hb ! g/dl. parenteral iron or tranexamic acid was uncommon during hospitalization and was given (< . %) and ( . %) women respectively. upon discharge, ( . %), ( . %) and , ( . %) women were prescribed with oral iron alone, oral tranexamic acid alone or both respectively. however, neither were given to ( . %) women. conclusion: in the present study, it is observed that . % transfusion episodes were given at hb ! g/dl. a substantial number of episodes ( . %) were transfused with multiple units and resulted in almost half having a post transfusion hb level (! g/dl). for iron replenishment and bleeding control, up to . % transfused women were not given iron or tranexamic acid at discharge. the results indicate that awareness of both transfusion appropriateness and iron deficiency anaemia management have to be improved. it is recommended that in-depth education and training should be provided for a better gynaecological patient blood management. background/case studies: granulocyte transfusions may be utilized to boost the immune response in patients with life-threatening neutropenia or neutrophil dysfunction and evidence of treatment-refractory bacterial or fungal infection. however, granulocytes are rarely administered due to uncertainty regarding efficacy, difficulty in collection, and increased propensity for adverse reactions. we report a case of granulocyte transfusion therapy following chimeric antigen receptor t-cell (car-t) therapy in a patient with severe neutropenia and multiple infections in the context of relapsed b-cell acute lymphoblastic leukemia (b-all). study design/method: granulocytes ( . - . x per unit) were collected from abo-identical unstimulated donors at a regional blood center. each unit was irradiated with gy and transfused over - hours within hours after the time of collection. the patient's response and laboratory data were reviewed in the medical record. conclusion: this data suggests that a diagnosis of aml is associated with anti-hla antibodies. an increased frequency of blood group a in patients with aml has been reported, but here no statistically significant difference between abo blood group frequencies was found in any category except the patient's with hla antibodies. blood group b has a significant association with hla alloimmunization in the studied patients. it has been reported in a large study of female blood donors that no difference in hla antibody frequency was observed based on abo blood group at centers using the flow-based assay. although the reasons for the higher rate of group b blood type among patients with anti-hla antibodies and hematologic malignancies is unknown, this could be due to variation in immunizing events (pregnancy vs transfusion) or immune dysregulation related to the hematologic malignancy, especially aml. females with aml who are blood group b appear to be most likely to have hla alloimmunization among patients with hematologic malignancies. implementation of electronic solution to reduce risk of mistransfusion in a regional transfusion service debra lane* , lee grabner , brenda herdman , robert fallis , amin kabani and charles musuka . canadian blood services, kenora rainy-river regional laboratory program, diagnostic services manitoba background/case studies: patient misidentification and improper sample labeling has been an ongoing risk for the safety of blood transfusion. the rate of mistransfusion has remained unchanged in over years. attempts have been made to reduce mistransfusion including barrier devices, barcoding and rfid. within a regional background/case studies: the role of donor age and sex on hemoglobin content and susceptibility to hemolysis during storage of red blood cell (rbc) units is receiving increased attention. however, the impact of donor characteristics on efficacy of rbc transfusion has not been studied in largescale donor-recipient outcomes databases. study design/methods: we conducted an analysis using blood donor data routinely collected by a blood center and transfusion recipient data from a large community hospital network between and before patient blood management initiatives. linkage was performed between blood donor characteristics and hospitalized rbc transfusion recipients who received a single rbc unit. studied exposures for this analysis were blood donor sex and age in addition to rbc storage age. the wilcoxon test was used to examine changes in hemoglobin level following rbc transfusion, and , and % were male. recipients of rbc's from male and female donors had similar pre-transfusion hemoglobin levels ( . g/dl; p . ); however, transfusion recipients of male donor rbc units had higher post-transfusion hemoglobin levels and larger increments in hemoglobin compared to those of female rbc units ( . vs . g/dl; . vs. . g/dl; both p . ). female recipients had a larger rise in hemoglobin per rbc unit compared to male recipients ( . g/dl vs. . g/dl; p< . ). female sex of the recipient remained a significant predictor of change in hemoglobin after accounting for recipient age and estimated circulating blood volume in multivariable analysis (p . ). rbc storage age and the age of the donor were not significant factors in changes in hemoglobin levels in multivariable analysis, p . and p . , respectively. conclusion: rbc units from male donors resulted in a larger rise in hemoglobin levels compared to those from female donors, and these changes were more apparent in female recipients even after accounting for effective circulating blood volumes. this suggests that the dose of hemoglobin is lower in female than male rbc units. this analysis demonstrates the feasibility of using this approach to study the association between donor characteristics and rbc efficacy, hemolysis and other donor-component-recipient interactions. background/case studies: people who identify as jehovah's witnesses (jw) comprise less than % of the population of the united states. however, as a group they can present a special challenge in medicine due to a religious aversion to blood products, based on biblical readings. the degree of this religious refusal can vary from individual to individual, but as institutional policy, a conservative approach is warranted. however, in large institutions where multiple teams manage a single patient, blood refusal information can be lost or poorly communicated from provider to provider. as such, a system to alert providers of patient blood refusal was recently implemented through the electronic medical record in a large west-coast institution. study design/method: the electronic medical record (emr) utilized in this study in an institutionally modified version of epic ea best practices alert (bpa) was designed to trigger each time an end user attempted to place orders related to blood transfusion, transfusion-related lab testing, or human-derived pharmacy items on patients with blood refusal codes in their history, problem list, or religion (jehovah's witness) discrete data fields. the alert constitutes a "soft-stop" in which the ordering provided is prompted to either cancel the triggering orders or acknowledge the blood refusal/religious history and override the warning with an option to select a reason for the override. data on the triggers are automatically collected through the emr systems and generated into a report by informatics personnel. results/finding: the available data covers triggers in the two month postimplementation of the bpa. the bpa triggered times in total, affecting patients and users. stratified by location, the majority of triggers occurred in the perioperative areas ( times) and the liver icu ( times) with a minority occurring on the regular hospital floors and emergency department. nurses, attendings, residents, pharmacists, and nurse anesthesiologists were the users affected. orders that triggered the bpa included type & screens, human albumin % iv solution, human albumin % iv solution, immune globulin (human) solution. conclusion: despite the limited and very preliminary data, the user action findings seem to indicate that the bpa is effective in halting up to half of the contraindicated orders for blood-derived products and type & screens orders. given the limited types of orders that the bpa is triggering on, the pattern suggests that the bpa is potentially alerting some previously unaware providers of the patient's religious status and/or the fact that certain pharmacy items are blood-derived, and therefore unacceptable to many jw patients. despite these positive initial findings, this is an ongoing study to track the efficacy of the bpa and more data needs to be collected for better metrics of the institutional sensitivity to patient blood refusal. intervention to address inappropriate cryoprecipitate-ahf orders at a tertiary medical center sirisha kundrapu* , , mahmut akgul , , hollie m reeves , , robert w maitta , , marcie pokorny , anne capetillo and katharine a downes , . background/case studies: although introduced for the management of hemophilia a, now cryoprecipitate is primarily indicated for low fibrinogen levels. at our institution the transfusion medicine service (tms) reviews and makes recommendations to clinicians for all inappropriate cryoprecipitate orders. we aimed at analyzing the effectiveness of this intervention in reaching target fibrinogen levels in under-estimated and over-estimated orders. study design/method: we conducted a -month retrospective study (january-july ) of adult cryoprecipitate order quality assurance forms. the reference range for fibrinogen was - mg/dl with critical value of mg/dl. cryoprecipitate orders for massive transfusion protocol, from operating rooms and for extracorporeal membrane oxygenation were not reviewed by the tms. during the study period, tms evaluated orders for appropriateness of dosing and agreement with estimated required doses. post-transfusion fibrinogen levels due to intervention were compared with hypothesized no intervention levels. statistical analysis was performed using chi-square and t-tests. results/finding: there were adult (> years) orders reviewed by tms out of which were approved. of the approved orders, ( . %) were in agreement with tms's estimated dose. of ( . %) orders that were not in agreement with the tms's estimate, ( %) were underestimated and ( %) were overestimated. seventeen of orders had no post-transfusion fibrinogen levels. without intervention, there would have been a median deficit of . mg/dl (range . to mg/dl) and a median excess of . mg/dl (range . to mg/dl) of fibrinogen from the target. median difference between target and actual post-transfusion fibrinogen level was mg/dl above target, which is significantly higher with intervention than without (which could have been mg/dl below the target; p< . ). median differences between target and post-transfusion fibrinogen levels for the group with agreement between approved and requested units was not significantly different from possible differences without intervention ( vs. . mg/dl, p . ). median differences between target and post-transfusion fibrinogen levels for the group with non-agreement between approved and requested units was significantly different from possible difference without intervention ( vs. - . mg/dl, p< . ). seven of ( ) ( ) orders were for critically low fibrinogen (< mg/dl) and of these were under-estimated requests and reached target fibrinogen with tms's estimate and approval of required units to be transfused. overall most frequent orders were and units ( . % and %) i.e. and pools and the most frequent orders in the disagreement group were , , and units ( %, %, % and %). there is a significant difference between agreement and disagreement groups based on clinical service ordering the units (table) . conclusion: intervention by tms to review and approve cryoprecipitate orders was associated with increased accuracy of orders and achievement of desired target fibrinogen levels. further studies are needed to develop multidisciplinary strategies for accurate cryoprecipitate dosage. patient characteristics, medical records, vitamin k administration, and adverse events, were collected (table) . results/finding: the average pre-transfusion inr was . and posttransfusion was . . only % of patients had their inr corrected to . , while % had no change, or had increased inr. (table) . the majority ( %) of patients received units of plasma. the mean plasma dose was ml/kg. there were transfusion reactions reported, non-hemolytic and transfusion associated circulatory overload reactions in which required admission to the icu. two patients experienced bleeding during ir procedures (tips) and developed a hematoma (tunneled central line). the median of inr correction in this study was . with no relationship to the number of units of plasma transfused and/or if vitamin k was administered. this study suggests it may not be beneficial and may be harmful to transfuse plasma for correction when inr is . . randomized trials are needed to assess whether the inr is a rational tool to measure bleeding risk, and whether prophylactic treatment with plasma yields any benefit. of the patients experienced bleeding complications indicating that inr of . may be considered safe in some lower risk procedures. current practices may provide little or no benefit, with substantial risk of life threatening complications. background/case studies: group ab plasma, which lacks anti-a and anti-b antibodies, is considered to be the universal plasma donor and is used in the emergency setting before the patient's blood group is available. approximately % of the population is group ab, which limits the available inventory of group ab plasma. of group ab population, only plasma from male donors are considered suitable for transfusion since females, especially multiparous female donors, have a greater propensity to develop antibodies that can cause transfusion related acute lung injury (trali). this makes type ab plasma a limited resource. our hospital is a level one trauma center, where a significant amount of plasma transfusion is required for severely bleeding patients before their blood type is known. group o individuals make up % of the population and have no a or b antigens on their cells. group a is the second most prevalent blood group in the us population ( %) and has no b antigen on their cells. so, group a plasma is compatible with both group o and a patients, approximately % of the patient population. before patient's blood type is known, type o red cell units are transfused with a plasma, which decreases the chance of hemolysis. to conserve ab plasma, we instituted a policy effective july , as follows: units of group a plasma and units of group ab plasma is provided for the massive transfusion protocol (mtp) along with units of o negative rbc until patient blood type is known. study design/method: this prospective study is designed to monitor the use of group a plasma in mtps at our institution and to evaluate the risk and severity of hemolysis in patients transfused with incompatible plasma. direct antiglobulin test (dat) is performed if patient received incompatible plasma. if dat is positive, lactate dehydrogenase (ldh), haptoglobin and bilirubin levels are obtained to detect possible hemolytic transfusion reaction. results/finding: we reviewed mtps at our institution between july and march . twenty patients ( . %) were transfused with incompatible group a plasma ( group ab and group b patients). five patients died due to severe injury, and follow-up testing of these patients could not be performed. the remaining patients had negative dat, indicating the lack of significant amount of antibody coating their red cells, which could lead to hemolysis. none of these patients developed acute hemolytic reaction, or any other adverse effects of incompatible plasma transfusion. conclusion: our study adds more evidence of the safety of group a plasma transfusion in trauma patients requiring emergent massive transfusion before the patient blood type is known. based on this and other recently published studies, starting in april , our institute will provide only group a plasma for emergency release and mtp cases before the patient blood type is known. average ( background/case studies: in , bonfils immunohematology reference lab (irl) sent out approximately special platelets for patients with hla antibodies. by , hla platelet orders increased dramatically and the irl sent out over special platelet products. the purpose of this abstract is to illuminate the methods used to fulfill increased client need that occurred in a short period of time. study design/methods: bonfils blood center has over , donors in the database with historical hla typing. however, only approximately of those donors actively donate. in the denver area, one of the most common hla types is a a b b . only of the , donors have this type ( . %). therefore, to fill an hla platelet order request for a common hla type, only donors in the system would be a perfect hla match. with that low number of donors, it is not likely that there would be a platelet on the shelf ready to fill the order. after a donor is recruited and donates, it takes at least two days to fill an order. for a less uncommon hla type like a a b b , there is only out of , donors ( . %) that match perfectly. in those cases, there are no donors to recruit to fill such an order. in some complicated cases, the irl was provided with an hla antibody list or panel reactive antibody test (pra). in order to find product for these patients, lists of platelets in inventory with corresponding hla types were printed. if a patient had an antibody to a for example, all of the a positive platelets were crossed off the list. this cross-out process would continue manually until the only platelets on the list were the ones positive for hla antigens to which the patient did not have antibodies. these platelets are pra matched to the patient. in order to automate this process a report linked to the donor database was created to find both pra platelets in inventory and donors for recruitment. the blood center medical director began suggesting that hospital clients order a pra for each patient with platelet refractoriness. the pra test is fast and it is a definitive method to discern hla antibody mediated refractoriness from platelet refractoriness due to other causes. results/findings: in all but the most complicated cases with rare hla patient phenotypes, it was much easier to find a pra patient matched platelet on the shelf than an hla match donor. in , approximately % of these special order platelets were pra matched and the remaining % were hla matched by donor recruitment. by , approximately % of special platelets sent are pra matched. this change resulted in a . fold increase of finding product in inventory to fill orders quickly. conclusion: developing a system to provide pra matched platelets is a faster alternative to finding hla matched platelets thus contributing to better patient care. background/case studies: in urgent cases where large amounts of blood products are needed quickly, maintaining a standard massive transfusion protocol (mtp) is critical to the timely delivery of these products. each mtp pack at ucm contains packed red blood cells (prbcs), fresh frozen plasma (ffp) units, and plateletpheresis pack; a unit of prepooled cryoprecipitate is also given if the patient is in labor and delivery (l&d) or if one is requested. at ucm, blood products are generally transported through the pneumatic tube system (pts). we undertook a review of our mtp issuing practices and efficiency patterns over the last three and half years. study design/method: the electronic archives of the blood bank laboratory information system and electronic medical record at our institution were queried for patients who had mtp activations. the archives were correlated to paper copies of these activations to collect data pertaining to the relevant information such as where the order originated from, how quickly the first product was sent out, how many products were transfused, and so on. results/finding: between august to march mtps were activated at ucm, of which orders could be traced to the origin: on inpatient floor (including icus), in the operating rooms, in the emergency department, in labor and delivery, and in other procedure rooms. of the prbcs that were issued, were transfused ( % utilized); of the units of ffp that were issued, were transfused ( % utilized); of the platelet packs that were issued, were transfused ( % utilized); of the units of cryoprecipitate that were issued, were transfused ( % utilized). since march , the time of first product issue after the initiation of an mtp has also been tracked. of the events that fall within this time period, ( %), had the first product issued in minutes or less. another ( %) were issued between - minutes, resulting in over % of patients being issued their first blood product within the first minutes. only of ( %) events had an initial time greater than minutes and none were greater than minutes. conclusion: the majority of our activations currently come from inpatient floors (primarily icus). as our institution anticipates the introduction of an adult level trauma center, we anticipate this balance will shift. in addition, the data shows that (with the exception of cryoprecipitate) the utilization rate is nearly identical among the blood products sent during mtp activations ($ - %). again, we anticipate utilization rate of issued mtp products to increase with the introduction of a new adult trauma center. we have recently begun tracking time to last product issued during an mtp, but cannot report on that variable at this time. overall, our data show that our transfusion service is generally performing adequately to issue the first product within minutes of mtp protocol activation. this data only reflects time to issue in the pts; patient care areas can experience additional minutes delay in pts delivery and arrival of product at bedside. we must continue to collaborate with our clinical colleagues to collect accurate data to provide the best and most efficient mtp care. mehreen yasin* , shailesh macwan , arline stein , jane fischman , nancy nikolis , matthew bank , lennart logdberg , alexander indrikovs , sherry shariatmadar and vishesh chhibber . north shore university hospital, northwell health background/case studies: massive bleeding is generally defined as any patient who requires blood volume replacement within hours and/or receives transfusion of greater than or equal to units in one hour with a transfusion vol. supplement s ongoing bleeding. our mtp was officially implemented in in preparation for an initial verification as a level trauma center by acs. our mtp has the following packages: st pack has a ratio of : : (rbcs, plasma & platelets) and subsequent packs a ratio of : : . our mtp also includes prothrombin time (pt), activated partial thromboplastin time (aptt) and fibrinogen testing after each pack is transfused. this data is used to assess the patient and allows the transfusion service and clinical team to identify coagulopathies. however, attempts to supplement mtp packs with cryoprecipitate (cryo) and prothrombin complex concentrate (pcc) were challenging to accomplish in a timely manner. study design/methods: due to challenges in timely supplementation of mtp packages with cryo and pcc, the protocol was modified in march to add cryo and pcc at a defined point in the mtp (cryo is included in the rd pack and pcc in the th pack). in order to validate this modification of adding these products at defined intervals regardless of laboratory data, we decided to review all patients that received > rbc at our institution as these massively hemorrhaging patients would receive pcc based on our current protocol. we reviewed the blood products received by these patients and their available laboratory data. results/findings: we had patients who received > rbc in and . mtp had been activated for all patients and all patients received between . to unit of plasma for each rbc unit transfused. despite receiving these ratios of blood products, all patients had elevations of their pt > seconds and many had elevations of the aptt and fibrinogen levels less than our institution's target of mg/dl (table ) . as anticipated, improvement in the coagulation parameters was noted with cryo and pcc supplementation. conclusion: our data on massively hemorrhaging patients supports a role for supplementation of our mtp with cryo and pcc in patients who require transfusion of > rbc. our current protocol with the addition of cryo and pcc at defined intervals has streamlined the process and improved timely provision of these products in bleeding coagulopathic patients. background/case studies: red blood cell hemolysis is a key finding for a diagnosis of transplant-associated passenger lymphocyte syndrome (ta-pls). however, whether a hematopoietic stem cell or organ transplant recipient experiences hemolysis when a transplant contains unintended antibody-forming passenger lymphocytes depends, by chance, on the recipient's blood group phenotype. a living donor liver segment transplant resulted in a case of ta-pls with donor-derived anti-d that had the potential for causing a clinically significant hemolytic event. the donor's plasma contained anti-d. anti-d was absent in the recipient's pre-transplant plasma, but present in the recipient's -day and -day post-transplant plasma. although these findings established a diagnosis of ta-pls, hemolysis did not occur because the recipient's blood group phenotype was d-. the conventional focus on hemolysis, rather than on the transfer of antibody-forming lymphocytes, is a diversion from the primary pathophysiology of pls and limits capturing the true scope of the syndrome. study design/method: to determine the standard of practice for detecting and diagnosing ta-pls, a retrospective -year pubmed search for peerreviewed english-language journal articles was conducted using key words "passenger lymphocyte syndrome." cases were categorized according to the presence or absence of hemolysis and whether there was a routine antibody screen to detect donor-derived, passenger lymphocyte-formed blood group antibodies. results/finding: of published cases ( reports) of ta-pls, ( reports) were stem cell and ( reports) were organ transplants. all ( %) stem cell transplants and ( %) organ transplants were associated with hemolysis, reflecting an overwhelming bias for identifying ta-pls associated with hemolysis. of the reports of stem cell ta-pls, actively screened for antibodies in the immediate post-transplant period, and of the reports of organ ta-pls, actively screened for antibodies. these screens detected cases of stem cell ta-pls before hemolysis became apparent and cases of organ ta-pls with antibodies without hemolysis. it can be inferred that ta-pls is currently under-diagnosed, because hemolysis is not consistently present and/or antibody screens are not performed routinely. conclusion: a new category of "non-hemolytic ta-pls" is recommended to capture otherwise undiagnosed cases where ta-passenger lymphocytes form blood group antibodies in the recipient, but hemolysis does not occur, as in our aforementioned case. to ensure including the full scope of ta-pls, an antibody screen should be performed routinely one week after transplant and repeated as clinically indicated. occult hemolytic anemia due to anti-mur in a patient receiving blood from a region with a prominent asian donor population jean oak* , rosario mallari , marc de asis , elaine shu , jonathan hughes and tho pham , . stanford university, stanford health care, stanford blood center, bloodsource background/case studies: mur antigen is present in - % of individuals in southeast asia, taiwan, and parts of southeastern china, but is rare elsewhere. antibodies against mur antigens are clinically significant, hence many countries in asia routinely screen for it while other countries, including the us, does not include mur in the standard screen. we describe a case of an occult anti-mur antibody causing anemia and donor ethnicity distribution in a regional blood center with a large asian donor population. year old hispanic male with chronic myelomonocytic leukemia and plasma cell dyscrasia developed anemia. initial antibody screen and dat were negative, and the patient received - rbc units every - weeks to maintain a hemoglobin (hb) level of g/dl. the patient remained stable for months when his hb level acutely dropped to . g/dl. the antibody screen remained negative for an additional months when it became positive for anti-jka and anti-mur. donor ethnicity data was available for of the rbc units he received. units were from an asian donor, and a unit transfused days prior to the hb drop was from a caucasian/chinese donor. study design/method: we reviewed the ethnicity data of , donors at a hospital-associated blood center located in a region where asians comprise approximately % of the population. results/finding: . % of donors identified as chinese, vietnamese, filipino, or other southeastern asian. these donors account for of ( . %) rbc collections. conclusion: identification of anti-mur in this patient was triggered by the presence of a concurrent anti-jka alloantibody. since over % of the rbc supply in the local blood center was collected from chinese or southeast asian donors, chronically transfused patients are at risk of developing anti-mur-mediated hemolysis that could be missed on a standard screen. this finding raises a possible need for blood banks located in regions with a prominent asian population to implement screening for anti-mur. brian adkins* , princess maynie , carol chandler , shelia garret and pampee young . vanderbilt, vanderbilt university medical center, department of pathology, microbiology and immunology background/case studies: antibody titration is a testing modality vital to both obstetric and transplant services. manual direct tube testing is associated with variability in results (poor reproducibility/precision) and is also time and resource intensive. in fact, studies have shown a three-to eightfold inter-institutional difference between the antibody titers from the same samples using manual tube method. the orthovision automated analyzers offers automated titering of patient plasma using gel technology. although there is intense interest in adopting automated testing technology for titering, it is well-appreciated that titers obtained in manual gel testing are much higher than those obtained by manual/direct tube testing. the higher titer results lack clinical fetal anemia and outcome correlations, which is a barrier to their implementation. moreover, despite the increased sensitivity of gel testing, prior studies have found variable results with regard to reproducibility and precision. [ ] [ ] [ ] there is minimal information on the comparisons of tube titers to orthovision automated titers or assessment of the reproducibility of this automated method. study design/method: rh and non rh minor rbc antibody titrations were performed by manual direct tube method on clinical samples and the same samples were analyzed on three different ortho vision analyzers to assess precision and inter-instrument reproducibility. results/finding: a total of samples have been analyzed (table) , rh and non-rh antigens. titers via automated testing on orthovision resulted in a mean titration being . (range - ) times higher. the average fold change for rhd/c/e antibody titers were . , whereas the average fold change for non rh titers was . (range - ). the range for anti d titers was particularly variable, - , whereas for c/e, it was - . the overall reproducibility/precision of the automated analyzer was $ %. to correlate the a transfusion vol. supplement s increased titers observed with some classes of antibodies, particularly anti d, we will be performing parallel testing of obstetric samples and correlating with pregnancy outcome/fetal testing the obtained values. conclusion: automated titration of antibodies using the orthovision analyzers resulted in highly reproducible results between different instruments using the same sample. however, the automated analyzers consistently yielded higher values, particularly with rh d, with results $ times higher than in manual tube testing. interestingly, the difference in titers of non rh antibodies between manual tube and automated testing was not statistically significant, although our n thus far is small. in order to leverage the efficiency and reproducibility benefits of automated titering we will need to establish "critical titer ranges" which require active monitoring of the fetus. platelet additive solution reduces the isoagglutinin titer in apheresis platelet units maxim tynuv*, elizabeth j furlong and willy a flegel. dtm/cc/nih background/case studies: isoagglutinins in the plasma of apheresis platelets are a concern during transfusion, as high titer anti-a and/or anti-b may cause a hemolytic transfusion reaction (htr) in a recipient with cognate antigen. apheresis platelet collections are usually reconstituted with donor plasma, however most facilities do not test for high titer of isoagglutinins, exposing recipients to the risk of htr due to plasma incompatibility if given based on short outdate and not abo type. at our facility testing is performed on all apheresis platelets with a cutoff titer of . units above the cutoff are marked as "high titer" and only given to abo plasma-compatible recipients or washed with saline to reduce plasma. however, washing platelets is a time consuming process that results in a loss of up to % of the platelets. platelet additive solution (pas) is used as an alternative collection and storage solution, replacing approximately % of donor plasma in the final product. the goal of this study was to determine what affect pas has on isoagglutinin titers and whether using pas could lead to a revision of one facility's procedure for management out of group platelet transfusions. study design/method: isoagglutinin titers of whole blood edta samples were compared to the final apheresis platelet unit collected in pas (intersol, fresenius kabi, lake zurich, il). using two-fold dilution steps, plasma was tested with pooled red cells (equal mix . % suspension of a and b cells, ortho, raritan, nj) in a gel matrix test (mts buffered card, ortho, raritan, nj) with min incubation (room temperature) prior to centrifugation (mts ortho workstation). fifty two donors were group o, group a, and group b. results/finding: of the whole blood edta samples tested, ( group o and group b) exceeded a high titer threshold of . when the pas samples of these donors were tested, only one (group o) exceeded the same threshold. pas specimens showed a consistent two-fold decrease in titer compared with whole blood specimens. nearly half of the group o donors exceeded a titer of when whole blood specimens were tested. conclusion: only one sample from apheresis platelets collected in pas exceeded our clinically applied titer threshold of , a % decrease from the number of whole blood specimens exceeding the threshold. testing the platelet bag collected in pas instead of plasma from whole blood specimens would lower the number of units exceeding the high titer threshold, and reduce products needing to be washed. furthermore, facilities not collecting platelets on site or without access to whole blood specimens from donors could implement the process described here and screen platelet apheresis collections for potentially clinically adverse isoagglutinin titers, whether collected using pas or not. other components. the majority of blood components in israel are collected and distributed by magen david adom (mda), from main locations. several hospitals in israel also collect platelets in-house. as part of an effort to understand plt utilization, a nationwide survey of plt transfusion and expiration was conducted. study design/methods: data on the disposition of all plt units, acquired from mda and collected in-house, during the calendar year was requested from all hospitals in israel. the number of plt distributed to hospitals by mda was also collected. plt wastage was defined as the sum of plt that were returned and not reissued from the hospital blood banks and plt that expired on blood bank shelves. results/findings: sixteen of the ( %) hospitals in israel, along with mda, participated in the survey, listed as a to p. the results are presented in the table along with each hospital's distance from the mda facilities. for some hospitals, the sum of transfused and wasted plt was slightly less than the number of plt supplied by mda; this is likely due to the small number of plt that had not either been transfused or expired by the time the data collection period ended. three of the largest hospitals (c, b and a) collected plt in-house in addition to acquiring units from mda. these hospitals had a lower overall rate of wastage including their own donations than the other hospitals that did not collect in-house plt. the other hospitals had wastage rates ranging between - %. no correlation was apparent between the hospital's distance from the mda facility or its number of beds and the plt wastage rate. conclusion: there is considerable platelet wastage in israel. large hospitals in israel with in-house donations had the lowest overall wastage rates in comparison to the other hospitals. factors known to affect plt utilization and wastage such as patient diagnosis mix, policies about how plt are issued and accepted back into hospital inventory, plt inventory size and the time of pooling of whole blood platelets relative to the time they are issued and returned to the blood bank need to be investigated and optimized in order to reduce wastage rates. possible immune-mediated hemolysis due to platelet transfusion masked by underlying hemolysis in a patient with blast crisis sirisha kundrapu* , , christopher j gresens , anne capetillo , hollie m reeves , and katharine a downes , . case western reserve university school of medicine, university hospitals cleveland medical center, bloodsource background/case studies: transfusion-related hemolysis with abomismatched platelets is rare with a reported incidence of < . %. most commonly in such cases group o platelets having high titer anti-a result in clinically significant hemolysis when transfused to a group a or ab recipient. we present a patient with a possible hemolytic reaction following transfusion of abo mismatched platelets presenting in the setting of underlying disease associated hemolysis. study design/method: a -year-old male with chronic myelogenous leukemia in blast crisis was evaluated for possible transfusion reaction to a single donor platelet (sdp). two hours post transfusion he developed chills, rigors, and increased blood pressure ( / mm hg to / mm hg) followed by hematuria ( ml). chills and rigors resolved; blood pressure stabilized after min with diphenhydramine, solumedrol, and acetaminophen. negative. patient abo group, rh (d) type and antibody screen on pre-and post-transfusion specimens showed no discrepancies. laboratory indicators of hemolysis are summarized in table. notably, while total/ indirect bilirubin increased and hemoglobin decreased after transfusion other tests were indeterminate for hemolytic transfusion reaction with abnormal pretransfusion levels. despite underlying disease associated hemolysis, the blood supplier of the unit was contacted to investigate into the possibility of high titer donor anti-a. this revealed donor anti-a titer results of (igm) and , (igg); donor was deferred from future platelet donations. conclusion: while the post-transfusion sample had no visible hemolysis and a negative dat, increased total/ indirect bilirubin after transfusion and high titer donor anti-a are supportive of immune mediated hemolytic transfusion reaction. the key unique aspect in this case is baseline underlying hemolysis, which may mask needs for further investigation of donor for high titer anti-a. ana paula hitomi yokoyama* , leila patricia de sousa fontenele , isabel nagle reis , carolina bonet bub , araci sakashita , raffael zamper , cristiane nakazawa , tatiane almeida omura paula , patricia silva batista , marcio dias almeida , fernanda loureiro de andrade orsi and jose mauro kutner . hospital israelita albert einstein, hemocentro unicamp-universidade estadual de campinas background/case studies: orthotopic liver transplantation (olt) is a high complex procedure, fundamental to therapeutic approach for end-stage liver disease. despite improvements in hemostatic , surgical, and anaesthetic techniques, liver transplantation is still associated with massive blood loss and high rates of transfusion requirements. peri and intraoperative transfusion of red blood cells (rbc) have been previously reported as major predictors of post -operative mortality . identifying predictive factors for transfusion requirements may help optimise patient blood management strategies in olt. we conducted a single center retrospective analysis of cases of olt performed between and in brazil in order to identify predictive factors for red blood cell transfusion study design/method: a retrospective analysis in a single institution was performed, and charts of consecutive patients submitted to liver transplantation between and were reviewed. the following variables were collected for each patient: gender, race, primary diagnosis, presence of hepatocellular carcinoma, age, body mass index, corrected model for end-stage liver disease (meld), duration of warm and cold ischemia. categorical variables were analysed using pearson chi-square test. continuous variables were analysed using t-student test. a forward logistic regression model was used to analyse data in a multivariate fashion, to identify independent contribution of variables previously found to be significant. results/finding: in univariate analysis, female patients, absence of hepatocellular carcinoma (hcc), primary diagnosis, corrected meld and warm ischemia time were significantly associated with consumption of rbc use in the intraoperative period. multivariate logistic regression of these factors showed that female patients (or , - % ci: , - , , p: , ), absence of hcc (or , - % ci: , - , , p: , ), cirrhosis of any cause (or , - % ci , - , -p: , ), miscellaneous diagnosis (auto-immune, metabolic diseases, familial amyloid polyneuropathy, vascular complications) (or , %ic , - , ) and retransplantation due to primary non function of the graft (or , %ci , - , , , p: , ) were independently associated with rbc transfusion requirements. conclusion: in this study, female patients, absence of hcc, specific primary diagnosis and retransplantation due to primary non function of the graft were significantly associated with rbc consumption in intraoperative period. determination of rbc transfusion predictors before surgery might provide important information regarding management of blood components and help optimise utilisation of resources for blood conservation strategies. prevalence of high-titer anti-a /b in group o platelet products. charles k. childers* , mark destree , ashley rose and theresa nester , . madigan army medical center, bloodworks northwest, bloodworks nw, dept of laboratory medicine, university of washington background/case studies: with platelet substitution policies, minor aboincompatible platelets (where donor's plasma may contain antibodies to recipient's red blood cells) are often issued in an effort to best utilize the community supply. however, rare reports of acute intravascular hemolysis have been reported from such transfusions, and can be attributed to high anti-a or anti-b titers, typically in a group o donor. one method to reduce the risk of hemolysis is to identify high titer platelet units prior to transfusion with a subsequent intervention. the percentage of high titer anti-a /b in group o platelet products is presented from a large regional blood center collected over - months. data from both pre-storage pooled platelet units (pspp) and apheresis derived platelet units (aplt) is shown. study design/method: platelet component samples were collected in ml edta sample tubes. a single : dilution of plasma was prepared using a hamilton microlab series dilutor using . ml saline diluent and . ml platelet component sample. using a standard transfer pipette, two drops of diluted sample were transferred to each reaction tube along with one drop of a or b red blood cell reagent. reaction tubes were centrifuged immediately in a serological centrifuge at rpm for seconds. reactions were read using a lighted agglutination reader. the presence of macroscopic agglutination (weak or greater) with either the a cells or b cells was recorded as a positive reaction, indicative of a high titer anti-a or anti-b. retesting of samples was performed to confirm high titers. results/finding: the above results indicate that, when a titer cut-off of is used, approximately % of group o apheresis platelets will have a high titer, most commonly with anti-a . less than half of a percent of pspp units will have a high titer. testing units for the titer can help to change abo out-of-group platelet substitution policies. in our example, the bloodworks transfusion service was able to change from a policy of volume reducing any group o apheresis platelets being issued to a group a or ab patient, to giving high titer products to only group o patients. the subsequent decrease in episodes of volume reduction helped to improve overall availability of apheresis platelets, by maintaining their day outdate. after months of testing pspp units and verifying that the products rarely had a high titer ( . %), the blood center stopped performing this testing for pspp units. rh ) ] started complaining of worsening back pain two and half hours after receiving one unit of rbc for a drop in hematocrit to % (from % on the previous day). his hematocrit did not increase ( %), and over the ensuing hours, he became anuric and jaundiced. clerical checks confirmed that his forward type was a positive, which was also the type of the rbc unit transfused, but revealed anti-a at a titer of in his plasma. furthermore, the direct antiglobulin tests (dat) were positive for c in the pre-and post-transfusion blood samples. anti-a was not detected in his plasma collected three days earlier, however. although his plasma color was amber, he had signs of intravascular hemolysis: undetectable haptoglobin, increased lactate dehydrogenase (ldh) and total and indirect bilirubin results/finding: the positive dat in the pre-transfusion sample pointed to ongoing hemolysis prior to the transfusion of the a rbc unit. in the setting of recent abo-mismatched transplant, his picture was consistent with hemolysis from newly formed anti-a by proliferation of donor lymphocytes, or pls. we performed an emergent rbc exchange using o rbcs with a goal hematocrit of % while reducing the number of a rbcs in his circulation by approximately %. his pain improved rapidly thereafter, and he had complete recovery of renal function. conclusion: pls should be in the differential diagnosis when suspecting/ investigating clinically significant hemolysis in abo-mismatched hpc transplant recipients, especially when the hpc source is from peripheral blood. as in our patient, it usually takes - days for antibodies to develop and they are short-lived ( - weeks). due to the severity of his manifestations, we performed an emergent rbc exchange successfully. furthermore, this patient's event exposed a vulnerability in our system of issuing the proper blood type for abo-mismatched transplant recipients, which has since been remediated electronically background/case studies: group o rhd negative (oneg) red blood cells (rbcs) are a precious resource. to conserve the oneg inventory while minimizing the risk of rhd alloimmunization in oneg females of childbearing age, transfusion services may automatically provide group o rhd positive (opos) rbcs to rhd negative males and/or rhd negative postmenopausal females during bleeding emergencies. despite these conservation strategies, shortages of oneg rbcs occur. the goal of this study was to determine how the utilization of oneg rbcs can be optimized using agebased opos switching for routine transfusions in oneg patients. study design/methods: recipient age and abo/rhd group were obtained for all allogeneic rbc transfusions during the calendar year from hospitals. an additional hospital* provided data for august-december . rbc transfusions in patients < year of age, and in patients whose age and/ or abo group were unknown, were excluded from analysis. the abo/rhd group of each rbc unit was compared to that of the recipient to determine the number of oneg rbcs transfused to all patients, the number of rbcs transfused to oneg patients and the number of oneg rbcs transfused to oneg patients. the number of oneg rbcs transfused specifically to oneg patients >/ years was also determined. results/findings: see table . the fraction of all transfused rbcs that were oneg ranged from - % (row f). the percentage of oneg rbcs transfused to oneg patients ranged from - % (row g); thus, non-oneg patients received - % of the oneg units transfused (row h). hospitals differed widely in the practice of issuing oneg rbcs to oneg patients ( %- %; row i). overall use of oneg rbcs could have been reduced by %- % if opos units had been given to all oneg patients >/ years old (row j). conclusion: during times of oneg shortage, age based opos switching rules may be applied for routine transfusions. this would help to ensure the availability of oneg rbc units for oneg females of childbearing age. rasha eldeeb mohammed* , nehad mohammed , marwa aly and nashwa fahmy . national blood transfusion services, nbts background/case studies: sensitization to the transfused red cell may complicate further transfusion& make it increasingly difficult to find compatible blood components for those patients. splenectomy has been shown to increase human leucocyte antigen immunization. the aim of the study is to evaluated the effect of splenectomy on the occurrence of red cell alloimmunization in humans. study design/method: this study was conducted on multitransfused patients who received blood transfusion chronically at our central blood center. they were thalassemia patients ( bthalassemia patients, one patients with a thalassemia), sickle cell anemia patients and immune hemolytic anemia patients ( auto immune hemolytic anemia patients, one paroxysmal nocturnal hemoglobinemia patient, one immune thrombocytopenic purpra patients). oncology patients, chronic diseases patients. history and demographic data were documented. all the patients who received blood are examined for the presence of the spleen.our patients were subjected to direct & reverse blood grouping (abo& rh) tests, alloantibody screening and detection. results/finding: statistical study is done to determine what is the effect of splenectomy in increasing the rate of red cell sensitization in chronically transfused hemolytic patients. the study revealed that: out of ( %) alloimmunized patients and out of ( %) non alloimunized patients(p< . ) . statistical analysis show that there is high statistical significant difference between patients who performed splectomy& who did not perform splenectomy as regard form conclusion: patients who had splenectomy had a higher alloimmunization rate removal of the spleen is not recommended in those patients who are periodically in need of blood and blood components. restrictive transfusion triggers rather than specific evidence. therefore, two systematic reviews of a) rbc transfusion guidelines and review articles to determine if single or multiple unit transfusion strategies are recommended and b) to identify studies comparing strategies were performed. study design/method: methods medline, embase, cinahl, web of science, national guideline clearinghouse, and the trip database were searched from inception to june . screening and data abstraction were done independently by two assessors. for review a, the proportion of articles with recommendations and articles recommending single unit strategies were assessed; stratified by guidelines, systematic reviews, and other review articles. for review b, the primary outcome was rbc utilization. secondary outcomes included proportion of units transfused using a single unit strategy, length of stay, and mortality. meta-analysis was done using the mantel haenszel random effects model. results/finding: review a identified articles for data abstraction, where articles were transfusion guidelines. there were guidelines ( %) that made a recommendation, for a single unit and for multiple unit transfusion strategy (table ) . review b identified retrospective cohort studies that were eligible and data abstraction was performed. all utilized a policy encouraging single unit transfusion strategies and compared a pre-implementation period to a post-implementation period. meta-analysis could only be performed on the secondary outcome of the proportion of units transfused using a single unit strategy, which was higher after the policy intervention (or . , % ci . - . ), although heterogeneity was high (i %). conclusion: our systematic reviews demonstrated a lack of recommendations amongst guidelines pertaining to transfusing single units of rbcs and only a few retrospective cohort studies to support benefits of the use of single unit transfusion strategies. additional high quality studies are needed to identify the benefits of a single unit transfusion strategy and when it should be used. guidelines groups should review research in this area to determine if a recommendation can be made. background/case studies: platelets made with platelet additive solution c (pas c) and treated for pathogen reduction (pr) have been shown to have decreased post transfusion platelet counts from platelets stored in all plasma. with the advent of multiple types of platelets, we are evaluating whether a mixed platelet inventory has had an effect on component use. the literature from europe has shown that platelet and red cell use does not increase when pr and pas products are used. evaluation of rbc use at our institution has shown no change in the number of products transfused per patient per month. we are evaluating whether the mixed inventory has led to more platelet transfusions. study design/method: we looked at occasions when patients received all of their platelet transfusions on a single day. by doing this we were able to exclude refractory patients from the analysis. the information obtained from routine quality management audits of transfusions between december and february was used for this analysis. the information included the ordering service, product release time, product code, pre and post counts. statistical analysis was performed using minitab. results/finding: during the months, units of platelets were transfused to recipients. over the months, a median of units was given to each patient with a range of to . the overall distribution of products used was % plasma, % pr, % pas f and % pas c. thirty percent of patients (n ) received all of their products on a single day. single units were given to patients while , and received , , and units respectively. the distribution by product type was % plasma, % pr, % pas c and % pas f. this same percentage was present for single and multiple products and was not statistically significantly different from the overall distribution of the products given during the month period (p . ). the distribution by service was different for the groups receiving multiple units. for single units the distribution was % hematologic malignancy, % infusion clinic (nos), % solid tumor medicine, % surgery, and % pediatrics. for those receiving multiple units the distribution was % surgery and % each for solid tumor, hematology and infusion (nos). the chi-square test for associations showed the increase in multiple units to surgical patients to be significant with a p value of . . conclusion: the distribution of the type of platelets given during a single event of transfusion was not significantly different from the overall distribution of platelets given during the month period. the patient's clinical service was a better predictor of the use of multiple products than the type of product given. this suggests that surgical losses or the need to have a higher platelet count during a procedure was the leading factor in the use of multiple products in this transfusion scenario. the effect of red blood cell transfusion on iron metabolism in critically ill patients margit boshuizen* , , yvemarie b.o. somsen , maike e. van hezel , marleen straat , robin van bruggen and nicole p juffermans . sanquin research and landsteiner laboratory, academic medical center background/case studies: anemia of inflammation (ai) has a high prevalence in critically ill patients. in ai, iron metabolism is altered, as high levels of inflammation-induced hepcidin reduces the amount of iron that is available for erythropoiesis. ai is treated by red blood cell (rbc) transfusions. it is known that rbc transfusions increase iron level in neonates and thalassemia patients, but the effect of rbc transfusion on iron metabolism during inflammatory processes is unknown. since one unit of rbcs contains mg of iron and % of the rbcs are cleared by macrophages within hour following transfusion, rbc transfusion could increase iron levels and iron availability for erythropoiesis. we investigated the effect of rbc transfusion on iron metabolism in icu patients, and additionally compared the effect in septic patients to non-septic patients. study design/method: in a prospective cohort study in icu patients who received one rbc transfusion, different iron parameters were measured before and hours after transfusion, to determine the effect of a rbc transfusion over a period of time. next, the impact of a rbc transfusion on plasma iron parameters in septic patients compared to that in non-septic patients was analyzed. plasma iron concentration, transferrin (saturation), ferritin, haptoglobin, hepcidin and il- levels were determined. results/finding: in this cohort, serum iron levels were low and did not change following transfusion ( . vs. . mmol/l, p . ). also, the transfusion had no effect on transferrin saturation ( vs. %, p . ), ferritin ( . vs. . mg/l, p . ) and il- levels ( . vs. . pg/ml, p . ). hepcidin levels increased in these icu patients after rbc transfusion ( vs ng/ml, p . ). in septic patients, rbc transfusion induced a decrease in haptoglobin levels compared to baseline, which did not occur in non-septic patients (- . vs. . % change, p . ). other iron parameters did not differ between septic and non-septic patients. conclusion: transfusion of one unit of rbcs does not increase iron levels in icu patients. the increase of hepcidin suggests rbc transfusion induced upregulation of hepcidin, despite the absence of a significant increase in il- or plasma iron levels. this increase in hepcidin levels after transfusion can potentially further hamper iron availability for erythropoiesis. in sepsis, rbc transfusion decreases haptoglobin levels, suggestive of hemolysis. in conclusion, rbc transfusion might have a negative effect on erythropoiesis, due to the increase in hepcidin levels that are observed after transfusion. the effects of pas and pr on platelet use barbara mendez, judith delmonte, elizabeth mccabe and joanne becker*. roswell park cancer institute background/case studies: with the anticipated release of the fda guidance: bacterial risk control strategies for blood collection establishments and transfusion services to enhance the safety and availability of platelets for transfusion, the use of pathogen reduced platelets (pr) which are often produced from products made with platelet additive solution (pas) may become more common. our institution has been transfusing platelets made with additive solutions since and pathogen reduced platelets have been available since . in our data validating pas and pr, the post counts from transfusion of pas-c and pr products have been statistically lower than platelets in all plasma (pp) or pas f products. our study looks at whether this difference has led to a corresponding increase in the number of units of platelets transfused. study design/method: the data was obtained from the routine quality reports produced for the blood utilization committee at our facility between and . during this time pas c, pas f and pr went from % to % of all platelet products given. all recipients had an oncology diagnosis. the data collected included the service, unit number and product code. the number of unique recipients was determined monthly. the data was converted to plt/month/recipient for analysis. statistical analysis was performed using the two sample t-test results/finding: the data was normalized to plt/recipient/month. in patients received an average of . units/recipient/month and in the average was . units/recipient/month. the intervening data points for , , and were . , . , and . respectively. the year average was . . the slope of the graph for all points was y - . . . the two sample t-test showed that the plt/recipient/month from to was not statistically different with a p value of . . conclusion: the implementation of pas and pr platelets in the oncology environment has not increased in the number of platelet transfusions given. in additional analysis, the red cell use has decreased (data not shown). this can be interpreted as indicating that patients have not had increased episodes of bleeding. although the post platelet count from pas/pr platelets may be lower, we do not have evidence from our platelet transfusion data that this is leading to clinical outcomes necessitating additional products to be given. background/case studies: it is reported that the incidence of alloimmunization in aml patients is unrelated to the number of transfusions the patient receives and most patients who have hla antibodies do not exhibit platelet refractoriness. many cases are also found not to have any anti-platelet antibodies detectable by standard laboratory tests. recent data in leukemia and hematopoietic stem cell (hsct) recipients transfused exclusively with leukoreduced products show that % to % develop alloimmune platelet refractoriness. objective: to determine an improvement in platelet count with the match grade and/or the abo blood group of the hla matched platelets in highly alloimmunized patients with concomitant non-immune causes for platelet destruction. study design/method: clinically documented platelet refractory patients, who received hla matched irradiated sda platelets with their hla typings for hla-a/-b and hla antibody identification were reviewed. there were two strategies utilized, the hla strategy (matching recipient and donor hla-a/ -b types) and the antibody specificity prediction (patient provided with platelets from donors lacking only those hlas to which the patient had antibodies) strategy. statistical analysis: a one sample t-test using minitab statistical software was performed comparing the mean against a platelet increment of a hypothetical difference of at least k/ul. the analysis revealed that the mean of . k/ul (n ) had a percent lower bound confidence interval platelet increment of k/ul (p< . ) results/findings: (median range [ - ]) hla matched leucoreduced irradiated sda platelets were transfused to ( m/ f) patients, median age years (range - ). / ( %) patients showing broad alloimmunization to hla class i/class ii antigens. / ( %) patients had anti-hpa antibodies (gp iib/iiia and gp iib/iiia and gp ia/iia). the majority / ( %) had a diagnosis of hematologic malignancy (aml/mds/mpn/ cmml/mm); / ( %) female patients had prior exposure via pregnancy and / ( %) had a history of hsct. ( %) platelets were abo identical-platelet increment median k/ul (range - to ), ( %) were abo compatible -platelet increment median of k/ul (range - to ) and ( %) were abo incompatible with platelet increments median k/ul ( range - to ). platelet counts were performed within hours in ( %) transfusions. the hla match grade of the transfused platelets were as follows: the use of massive transfusion protocol (mtp) in a community hospital rohini patel* , renee leblanc , dongfu xie , alice cabe and yanyun wu . overlake hospital, bloodworks northwest the use of massive transfusion protocol (mtp) in a community hospital background/case studies: the establishment and use of massive transfusion protocol (mtp) have become common practice, especially in trauma centers and tertiary hospitals due to significant number of patients with massive bleeding. however, it is not well established if the use of mtp also has value in small hospitals and community hospitals, and how mtp is used in fig. these settings, such as indication for mtp, blood products used, and the outcomes of these patients. study design/method: retrospective review of transfusion data from a community hospital with a bed size of about for years (from to ) was performed. patients with mtp requested are included in this study. results/finding: please see the table below for the summary of data. notably, patients with gi bleed and ob bleed are the two most common indications for mtp, and % of patients survived with the support of mtp. in one case, no blood product was used. the establishment and readiness of mtp can be very important in supporting patients who experience massive bleed in small hospitals and community hospitals. in these settings, mtp is most commonly used for patients with massive gi bleed and ob bleed. if the patient develops antibodies to a high incidence antigen, finding compatible units may become impossible. included in the mns system, and residing on glycophorin b (gpb), the u antigen is absent in less than . % of the black population. those with altered forms of gpb, known as u variants, can produce a diverse group of antibodies capable of causing mild to severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (hdfn). this case illustrates the balance between the need to transfuse and avoiding complications thereof. a -year-old ghanaian woman with scd and history of chronic transfusion presented with diffuse pain and a hemoglobin value of . g/dl (baseline - g/dl). she is known to be e, c, k, fya, jkb, s, s negative, u variant, and has anti-e, c and u antibodies. there were no eligible family donors and a nationwide search for compatible blood yielded four crossmatch compatible u variant units. the decision to transfuse was made. the patient had no change in symptoms or vital signs during transfusion but post-transfusion hemoglobin was . g/dl. a transfusion reaction work-up was ordered. post-transfusion serum sample was negative for hemolysis and no new antibodies were identified. the post-transfusion dat was weakly positive only with complement and laboratory data revealed a decrease in total bilirubin ( . to . mg/dl). two additional u variant, crossmatch compatible units were transfused over the next two days restoring her hemoglobin to . g/dl. the patient was discharged to home in stable condition and follow-up hemoglobin levels continued to rise back to baseline. study design/methods: molecular genotyping was used in donor unit selection prior to compatibility testing by transfusion services. conventional methods were used to monitor the patient's condition pre and posttransfusion. results/findings: each donor unit came from a different donor but all were the same gpb genotype as the patient. the patient did not experience an acute or delayed hemolytic transfusion reaction and genotype matching successfully facilitated donor unit selection in this case. conclusion: transfusion of u variant red cells to a u variant patient should be undertaken with great caution due to epitope and antibody heterogeneity. this case highlights the importance of genotype compatibility in selecting donor units for a chronically transfused, scd patient with anti-u. sound transfusion management of such patients requires planning and good communication on the part of clinicians and the laboratory staff. background/case studies: patients with decompensated waiha may require transfusion with red blood cell (rbc) products that are cross-match incompatible due free autoantibodies. the feasibility of blood transfusions in waiha patients is controversial because of difficulty in cross-matching and increased risk of transfusion reactions, since transfused rbcs may be destroyed more rapidly in patients with active hemolysis. to study the actual vs. theoretical risk of increased hemolysis in waiha patients, we investigated the post-transfusion (post-tfn) hematocrit (hct) change in waiha patients who were transfused compatible rbcs compared to those who received li blood. we further hypothesized that a post-tfn hct would be inversely related to the degree of ahg-phase incompatibility. study design/method: we reviewed all transfusions to patients in our quaternary-care hospital with a history of waiha from october to march . patient hcts were ordered by prescribing physicians for clinical purposes. a transfusion episode was defined as all units released in the interval before a post-tfn cbc. ahg-phase crossmatch was tube tested in saline per clinical procedure. transfusion medicine physicians determined the release of least-incompatible units. statistical tests were performed with statcalc (epiinfo, cdc) and www.socscistatistics.com. results/finding: there were rbc products transfused to waiha patients. twenty-three ( . %) patients received at least incompatible unit. the mean age was . years (range - yrs) with % women. ethnic composition was % african-american, % caucasian, and % patients of mixed/other ethnicity. one hundred fourteen ( %) of these products were released as li products and ( %) were compatible. ninetythree ( . %) of the li product transfusions had a post-tfn hct change of < % whereas only ( %) of the compatible product transfusions resulted in a post-tfn hct change of < % (p . , v ( ), exact methods). the mean hct increase in the compatible group was . % per unit vs. a slightly lesser per-unit increase of . % in the li group (p . , t-test, -tailed) within the li group, there was no difference in the per-unit hct change according to strength of incompatibility (table) . strength of ahg incompatibility was not available for units. units that were incompatible had a lower mean post-tfn hct rise compared to all other li units ( . % vs. . %); however, this difference was not statistically significant (p . ). conclusion: the post-tfn hct change for transfusions of li units to patients with waiha was less than the expected % per unit more frequently than it was for waiha patients who received compatible products ( . % vs. %). however, likely due to our small sample size, the mean differences were not statistically significant. interestingly, there was no difference in the per-unit post-tfn hct according to differing strengths of incompatibility in our sample, although the mean increase for the li products was less than all other li products combined. the increase was unexpectedly low for weaklyincompatible units, which we are further studying. future work includes consideration of inpatient vs. outpatient clinical status, effect of co-incident alloantibodies, comorbidities, and medications. transfusion management was summarised by individual hospital, type and total cases. in-hospital mortality (adjusted for age, sex, comorbidity, bleeding context and number of rbcs in the first -hours from mt onset) was calculated with % and . % control limits to indicate potential outliers. data were analyzed using statistical software (stata). results/finding: there were mt cases from hospitals ( tertiarylevel, smaller/medium sized acute-care and specialist women's). number of mt cases per hospital ranged from to . patient median age was years (iqr , ), % were male and % required admission to intensive care. the most common clinical groups were cardiac surgery ( % cases), trauma ( %) and gastrointestinal hemorrhage ( %); however there was marked variation between hospitals. ratios of transfused products, analyzed according to bleeding context, varied between hospital types. the pooled average adjusted in-hospital mortality for the tertiary-level hospitals was % (range % to %) and / ( %) were within the % control limit. cb that required ! rbcs within -hours of mt onset occurred in % of cases. comparison of transfusion management for this subset of mt cases showed that patients treated in smaller/medium sized acute-care were less likely to receive cryoprecipitate than patients treated in tertiary-level hospitals ( % versus %; p . ). conclusion: patient characteristics and transfusion practice varied between hospitals and hospital types, however in-hospital mortality outcomes were comparable. results are made available to participating hospitals in the anz-mtr to initiate discussion, practice review, and examination of compliance with national standards, patient blood management guidelines and to highlight areas for further investigation. data are also available for review by governance and policy bodies at state and national level to support practice improvement activities and highlight priority areas for future research. background/case studies: in hospitals and medical centers, in case of big traumas often an intraosseous entrance via a bone needle is combined with a fast flow fluid warmer. with this, infusion fluids, including blood products, are administered under pressure. this is done because veins of trauma patients are often not suitable for infusion of fluids. suppliers of pump and needles describe the possible transfusion of blood products, but this is mainly limited to plasma and erythrocytes. there is no information available concerning transfusion of platelets under pressure via a bone needle. the aim of the study was to investigate the effects of warming and administration of a platelet concentrate (pc) under pressure via a bone needle on the in vitro quality of platelets. study design/method: pools of bcs and ml of platelet additive solution iii (pasiii) were used to produce pcs (n ). pcs were stored on a flatbed agitator ( cycles/min) in a temperature-controlled cabinet at c for - days. to mimic hospital conditions, pcs were warmed using a blood warmer and transfused via a bone needle to a transfer bag. on the pcs a pressure of mm hg was applied. using clamps, a flow velocity of - ml/minute was realized. platelet quality before and after pressurized simulated transfusion was determined by means of various in vitro parameters. results/finding: due to priming of the transfusion disposable with saline, the pcs were diluted - %, resulting in a significantly increased pc volume and decreased platelet concentration after simulated transfusion. because of loss of platelets in the disposable set, also the total number of platelets was decreased after simulated transfusion. after simulated transfusion, the pcs still fulfilled the requirements for platelet concentration ( . - . x /l) and number (> x /unit). simulated transfusion had no effect on the percentages of cd p and annexin v positive cells, indicating no activation or induction of apoptosis. ph was not influenced by simulated transfusion. due to the dilution effect, glucose and lactate concentrations were slightly lower after simulated transfusion. conclusion: warming and simulated transfusion of pcs under high pressure via a bone needle has no negative effect on the in vitro quality parameters of platelets. transfusion of warmed pcs via an intraosseous entrance via a bone needle is not expected to have a negative effect on the in vivo functionality of platelets. it is recommended to study the in vivo effects in a limited clinical study. alesia kaplan* , , joan sevcik and joseph e. kiss , . university of pittsburgh, blood systems inc. background/case studies: low titer a plasma has been safely used as a substitute for ab plasma in trauma patients. low inventories of ab plasma can cause a delay in life saving therapeutic plasma exchange (tpe) procedures in ab patients needing plasma replacement. here, ab non-bleeding patients are presented who safely received ab and low titer a plasma for tpe. one ab patient who received ab plasma only was used as control to compare hemolysis laboratory data over tpe course. study design/method: a retrospective review of tpe procedures for patients was conducted from medical records. number of procedures, volume replaced, total number of plasma units, number of a plasma units, quantity of a plasma and hemolysis laboratory data were recorded. average quantity (ml) for a plasma and % of a plasma out of total volume of plasma used were calculated. all a plasma units were low anti-b titer units. in the laboratory, plasma dilution : is prepared and tested with reagent b cells. if agglutination is not observed, the unit is labeled as "low titer anti-b". hemolysis laboratory data was traced with linear graphs and trends were compared between patient and and (control). results/finding: all patients were ab blood type. patient , a year old female with recurrent adamts deficient ttp, received courses of tpe (total tpe procedures) for relapse and exacerbation. ten out of procedures were performed with ab and a plasma (average ml of a plasma or % of total plasma volume for tpe procedures). patient , a year old female with thrombocytopenia, schistocytes and presumed ttp, received a total of tpe procedures. four out of procedures were performed with ab and a plasma (average . ml of a plasma or % of total plasma volume for tpe procedures). patient , a year old female with adamts deficient ttp who served as a control, received a total of procedures with ab plasma only. haptoglobin, ldh, hemoglobin and total bilirubin were graphed and compared between patients. the trends of hemolysis laboratory data for patient and were comparable with patient . all patients had negative dat. only patient received rbc transfusions. all patients had a favorable clinical outcome with tpe treatments and adequate platelet recovery. conclusion: in this study, tpe was effectively performed without evidence of increased hemolysis using up to % of low titer a plasma. this approach can reduce strains on limited supplies of ab plasma while providing a vital treatment alternative for ab patients undergoing tpe who require plasma replacement. when cd negative platelet unit is not available for a patient with anti-cd antibodies sameer khatri* , charles harmon , brian r curtis and chisa yamada . background/case studies: refractoriness to platelet (plt) transfusion can be caused by antibodies (abs) against human leukocyte antigen (hla) class i antigens (ags) or less frequently against plt specific ags (psas). glycoprotein iv (cd ) is one of the identified plt surface ags and deficiency is rare, but found in asians ( - %), sub-saharan africans ( - %) and also in some people from mediterranean descent. two types of cd deficiency have been described. type deficiency is the complete lack of cd on both plts and monocyte-macrophages whereas type deficiency lacks cd on plts with variable expression ( - %) on monocytemacrophages. transfusing plts in a patient with cd deficiency is challenging given the rarity of cd negative phenotype and risk of further immunization when giving ag non-matched platelets. study design/method: a patient with cd negative phenotype who received multiple plt units was reviewed in the electronic medical record. results/finding: a year old man developed aplastic anemia following liver injury possibly due to a supplement for body building and required multiple plt and rbc transfusions. he received more than units of apheresis plt units over a week period without any significant increase in plt count. cross-match compatible plt unit found in of units and hla matched units were tried without success. at that point, a cd ab was identified in the serum and the patient's type cd deficiency was confirmed by flow cytometry. his hla class i panel reactive ab (pra) was % due to multiple plt transfusions, although all abs were low levels. the patient initially received high-dose prednisone and thymocyte immune globulin infusions without significant improvement in plt increase. following three doses of ivig, he received a cd- negative (but blood type different and hla a transfusion vol. supplement s unmatched) plt unit from his relative with only a slight increase in plt count. however, he started to respond to cd non-tested apheresis plts after receiving a fourth ivig and two rituximab infusions. since then, he has received ivig every weeks. other medications include filgrastim, eltrombopag, and cyclosporine for treatment of aplastic anemia. the mean corrected count increments (cci) when post-transfusion plt count was available are shown in table. with desensitization therapy, his cd antibody positive reactivity in serial dilutions has reduced from : to : dilutions and his hla class i pra has decreased to %. he is currently receiving apheresis plt units twice a week and rbc units periodically. his bone marrow (bm) has been slowly recovering evidenced by increased wbc count from zero to up to . k/ml and slow increase of reticulocyte counts. current plan is rbc/plt transfusion support until bm recovers or a haplo-identical transplant if bm recovery fails. conclusion: we report a case with anti-cd abs that received multiple plt transfusions. this case demonstrates that decreasing ab level with immunomodulation can be an alternative option for successful plt transfusion when compatible plts are not available for patients with rare or multiple abs to plts. table: mean available cci for plt transfusions a blood center's experience screening donations for babesia microti using enzyme-linked immunoassay methodology nancy van buren*, jed gorlin, vanessa reynolds and deborah anderson. background/case studies: our blood center, located in an area considered to be moderately endemic for babesia microti, implemented universal screening of red cell collections from minnesota and wisconsin under an investigation new drug (ind) study in oct utilizing the immunetics investigational enzyme-linked immunoassay (elisa) performed by creative testing solutions (cts). this test was selected as the most cost-effective approach for universal screening of blood donors, as opposed to the investigational ifa/pcr test combination. study design/methods: we performed a retrospective analysis of our screening test results and deferral rates for to evaluate for seasonality, donor abo bias, deferral rates, and outcomes of lookback investigations. since an opt-out of this research test was originally offered, we report donor opt-out rates. results/findings: from jan through dec , , blood donations were screened for b microti by immunetics elisa. of those, ( . %) were positive. the percent of positive donations was evaluated monthly revealing a variable reaction rate between . % and . %. no patient babesia transmission has been reported since implementing this test, but we only had documented babesia ttd cases from - . donors who previously tested negative demonstrated an increased seroconversion rate during the summer months, consistent with historical seasonal variation corresponding with tick season in minnesota and wisconsin. test performance characteristics were analyzed by abo group with no demonstrable differences in positive rates. the opt-out rate of donors who chose not to be tested significantly decreased over time, reflecting an increased acceptance of this test. of positive test results, lookback investigations were initiated representing % of positive donations. lookbacks were only performed when there was a donation within months of the new positive screening test, according to ind protocol. no confirmatory testing was performed per ind protocol or for donor counseling, so the true positive rate is unknown. in the prior ind trial, up to % were unlikely to transmit infection in our region, i.e. were pcr and blood smear negative. although a small number of antibody positive, pcr negative donors may be actively infected, no transfusion-transmitted babesia infections were identified by lookback investigations. notification of blood donors with positive screening results was also performed and information provided for healthcare provider followup. overall, donor deferral represented . % loss of eligible donors during this follow-up period. deferred donors were invited to participate in other research collections not requiring volunteer donor eligibility. conclusion: testing for b microti may help improve blood safety, particularly in endemic regions. although only . % of donors have a positive reaction, this represents a significant loss of eligible donors over time, most of whom are unlikely to transmit infection. a direct test capable of detecting babesia in individuals with very low levels of organisms without the need for concurrent antibody testing would be ideal. a reinstatement protocol for donors who test positive should also be considered. nonetheless, the current method of screening is inexpensive compared to pcr-based methods. background/case studies: human anelloviruses are the smallest in particle size, smallest in genome size, and least complex in genetic organization of all human pathogens. they establish a chronic persistent infection in infancy or early childhood and produce a constantly detectable load in plasma thereafter. some studies suggest they are ubiquitous, present in > % of the human population, and that immune surveillance is required to control the level of the virus load. study design/methods: we have developed a quantitative dna pcr assay for the most conserved region of the anellovirus genome that detects all known genotypes of the virus. we used this assay to examine viral loads in the plasma of us blood donors and transplant recipients pre-transplant and three months post-transplant. results/findings: for blood donors, were positive with an average load of . x copies/ml of plasma, a median value of . copies/ml of plasma, ranging from to . x copies/ml. pre-transplant viral loads were similar. for transplant candidates, were positive with an average of . x copies/ml of plasma, a median value of copies/ml of plasma, ranging from to . x copies/ml. post-transplant viral loads were remarkably different. for transplant recipients, all were positive with an average of . x copies/ml of plasma, a median value of . x copies/ml of plasma, ranging from to . x copies/ml. conclusion: these results validate the pcr assay that was developed and confirm that detectable viral loads of around - copies were present in > % of the blood donors surveyed. in addition, the effect of post-transplant immunosuppressive therapy has caused an increase in the viral load of at least orders of magnitude above that of non-immunosuppressed individuals. background/case studies: the screening of blood donors and travelers returning from endemic/epidemic areas has highlighted the importance of multiplex diagnostic approaches for the simultaneous analysis of various pathogens. furthermore, in the context of similar clinical signs, the differential diagnosis of arboviruses during acute infection is essential to discriminate the causative agent for patient management and epidemiological surveillance. the development of a flexible diagnostic approach is a key challenge to face the continuing emergence of arboviruses, belonging to flavivirus and alphavirus, such as dengue virus (denv), west nile virus (wnv), zika virus (zikv), yellow fever virus (yfv), usutu virus (usuv) and chikungunya virus (chikv). study design/method: an innovative diagnostic approach combining generic rt-pcr amplification and identification on low cost microarrays has been developed. we have patented original polythiolated probes grafted on maleimide-activated microplates for the robust, sensitive and specific mean cci pre-ivig: all plt ( ) . post-ivig: all plt ( ) . post-ivig: cd -negative plt from relative ( ) . post-ivig: single donor apheresis ( ) . post-ivig: cross-match compatible ( ) . post-ivig: flow cross-match compatible plt ( ) . a transfusion vol. supplement s detection of the viral genomes. analytical performances of the test were evaluated on viral standards and on clinical samples: denv ( / / / ), wnv, zikv and chikv. forty human plasmas from blood donors with no history of contact with arboviruses were used as negative controls. we have designed two sets of degenerated primers for the generic rt-pcr amplification of all flaviviruses and for chikv. biotinylated amplicons were captured on complementary grafted polythiolated probes on microplate. after addition of streptavidin-europium label, the molecular hybridization events were detected by time-resolved fluorescence using a microplate reader. results/finding: one original generic probe for denv and specific probes designed for each of the four denv serotype, wnv, the two zikv lineages and for chikv, were validated. the use of our methodology combining the amplification of the viral genomes and their identification using polythiolated probes shows % of specificity, with no false positive results on the control samples, and no cross reactions. using viral reference standards, we have observed sensitivities of tcid /ml for denv- , denv- and chikv and of tcid /ml for denv- , denv- and zikv. finally, the first results obtained on denv( ), zikv( ) and chikv( ) clinical samples show %, % and % correlation respectively between our approach and commercial or in house real time rt-pcr methods. conclusion: this innovative strategy allows the development of flexible, highly sensitive and easy to handle platforms dedicated to the multiplex screening and identification of emerging viruses. this methodology is adapted for the easy inclusion of additional molecular targets to improve the surveillance and the prevention of arboviral infections. babesia microti serological testing with pooled samples: a feasibility study laura tonnetti*, aaron thorp, letitia dixon and susan l stramer. background/case studies: blood donation screening for babesia microti, a tick-borne intraerythrocytic parasite endemic in the northeast and upper midwest us, is performed under an investigational study using nucleic acid and immunofluorescence assays (ifa). however, ifa is a time consuming and labor intensive procedure. with the possibility of an fda licensed screening assay(s) in the near future, we investigated if b. microti testing by ifa in pools of plasma or serum could be a feasible screening approach. study design/method: to test if the increased amount of plasma or serum interferes with background fluorescence, pools of , , and were prepared from plasma or serum samples determined to b. microti-negative by individual ifa screening. the pools were tested by ifa with or without a blocking step using bovine serum albumin (bsa) and goat serum to minimize background fluorescence. potential interference from multiple pooled plasma or serum samples on the endpoint titer of positive samples was investigated by including positive samples with endpoint titers from : to : ( -fold dilutions) in the pools. results/finding: non-specific fluorescence was visible in pools of or higher and was not eliminated by the addition of a blocking step. pools of or samples did not show significant increased background. there was no difference between testing of pooled serum or plasma samples. when one single positive sample was included in the pools of or samples, the pool tested positive and the final titer was the same as the positive sample tested individually. when two or more positive samples were included in the pools, the final titer of the pools was equal to the sample with the highest titer. conclusion: this study represents a proof of concept that serological testing for b. microti by ifa in pools of up to plasma or serum samples does not increase false positivity while maintaining the sensitivity of the test. background/case studies: the rapid detection of bacterial contamination in platelets is key to reducing the risk of infection in transfusion of blood components. the bact/alert virtuo* is an advanced, next generation system with improved automation, connectivity and with data management systems. the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert (bta) bpa (aerobic) and bpn (anaerobic) bottles. as plasma is known to be bactericidal, a study was completed to evaluate plasma susceptibility/resistance for organisms considered for virtuo studies. study design/method: human plasma (thawed and pooled) and saline controls were seeded with $ cfu/ml of organisms associated with platelet contamination and incubated at room temperature for - hours. colony counts were performed initially and after incubation. plasma resistance was determined if the colony count of the seeded plasma was equivalent or higher ( log) than the colony count of the seeded saline after incubation. results/finding: the serially diluted strains and all bioball tm strains except p. aeruginosa, nctc , were determined to be plasma resistant. the bioball tm p. aeruginosa was susceptible to the antimicrobial effects of human plasma, but when spiked into ml of leukocyte reduced apheresis platelets (lrap) and inoculated into bta bpa bottles and loaded into the bta d and virtuo the organism was recovered % . conclusion: results confirm that previously tested organisms and additional strains are plasma resistant with the exception of p. aeruginosa, nctc . however, the bpa bottles still recover p. aeruginosa in the presence of lrap. bpa/bpn bottles inoculated with select organisms from this panel in the presence of ml lrap demonstrated % recovery when loaded onto the virtuo and d ( table ) . further studies may be required to determine if higher test volumes of lrap could affect the recovery of plasma sensitive strains. * virtuo is not fda cleared for platelet testing a. notoscriptus, identified as a major urban vector of rrv, is also capable of transmitting dengue virus - , and has recently been found in los angeles, illustrating an expansion in range. with the growing geographical distribution of aedes species mosquitoes, the potential for rrv to enter local transmission cycles outside of australia is significant. in , a probable transfusiontransmission (tt) was confirmed as the cause for an rrv infection in australia, validating the reality that rrv tt can occur. rrv morbidity leads to clinical manifestations that are similar to chikv infection, with varying degrees of arthralgia, which can become debilitating. various asymptomatic to symptomatic infection ratios have been reported, but this further increases the risk of additional tt in endemic areas and could mask the spread of the disease globally. study design/method: platelet concentrates (pc) prepared in pas were inoculated with rrv, amotosalen was added to final concentration of mm and the units were treated with uva light. pre-and post-treatment illumination samples were collected for titration. as- rbc units were contaminated with rrv, mixed with processing solution/glutathione (gsh) and treated with amustaline at a final mm concentration. pre-and post-treatment samples were removed prior to amustaline treatment and hrs after amustaline addition, respectively, for titration by plaque assay on vero cells. log reduction was calculated as the difference between the mean infectious titer in pre-vs. post-treatment samples. results/finding: inactivation of rrv was achieved to the limit of detection in pc and rbc. in pc, > . log or log /ml of rrv was achieved, with > . log or > . log /ml of rrv inactivated in rbc. conclusion: these studies illustrate that amotosalen/uva and amustaline/ gsh treatments are effective at inactivating rrv in pc and rbc, respectively. these data corroborate previous results achieved with other alphaviruses, including chikv and mayaro virus which are inactivated at high titers in pc and rbc, demonstrating the ability for these systems to mitigate tt potential and maintain safe blood component availability in endemic areas. (data have not been submitted for fda review and intercept for red blood cell is not approved for commercial use). background/case studies: the interceptv r blood system for platelets is designed to inactivate pathogens and contaminating leukocytes. this photochemical treatment process utilizes amotosalen and low energy ultraviolet a (uva) light. the current available sets include small volume (sv; - ml), large volume (lv; - ml) and dual storage containers (ds; - ml) designed to treat platelet doses between . and . x . the new triple storage (ts) set was designed to expand the dose range to . x and the maximum volume to ml, generating either or doses of pathogen reduced platelet components (pc). the objective of this study was to evaluate the effectiveness of the system by performing log reduction assays using representative gram positive and negative bacteria and enveloped and non-enveloped viruses in platelets suspended in pas, or % plasma using ts set. study design/methods: for each experiment, a platelet pool was prepared either in % plasma/ % pas or % plasma with a final volume of $ ml and a dose of - platelets. these conditions represent inactivation using the lowest amotosalen concentration ( mm) and highest concentration of platelets. platelet units were inoculated with high titers of viruses, or bacteria and treated. control (pre-uva) and test (post-uva) samples were serially diluted and cultured. plates with suitable media were used for bacteria, whereas viral titers were determined using plaque assays. log reduction was calculated as the difference between the log titers in control (pre-uva) and test (post-uva) samples. conclusion dromedary camels were identified to be the reservoir of mers cov, transmission to humans occurs through direct and indirect contact. mers cov has been detected with high genomic titers of - logs in respiratory secretions of mers patients, and with lower genomic titers of - logs in blood. the presence viral particles in the blood of acute patients gives rise to concerns, especially in endemic areas. the high mortality rate, especially for critically ill patients, which often require blood transfusion, raises the need for a method to safely exclude mers cov contamination of blood products. pathogen reduction with amotosalen/uva technology is a widely established technology with a broad range of data supporting clinical efficacy and safety of amotosalen/uva treated blood products. the aim of the study is the assessment of the mers cov inactivation efficacy in human plasma with amotosalen/uva pathogen inactivation technology to safely exclude the presence of infectious virus in human plasma units. pre-uva titer post-uva titer log reduction/ml (log /ml) %plasma/ % pas e .coli . <- . > . e. cloacae . <- . > . k. pneumoniae . < . > . s. aureus . <- . > . blue tongue virus . <- . > . bovine viral diarrhea virus . <- . > . adenovirus- . <- . > . %plasma k. pneumoniae . - . > . s. aureus . <- . > . adenovirus- . <- . > . n a transfusion vol. supplement s abstract study design/method: four therapeutic human plasma units were spiked with a fully characterized mers cov clinical isolate followed by pathogen inactivation with amotosalen/uva (intercept blood system, cerus corporation) at four different days. pathogen reduced samples were taken preand post-pathogen reduction after various processing steps to assess the infectious titer by plaque assay titration and the genomic titer by real-time-pcr. samples post pathogen reduction have been passaged times up to days, assessing the infectious titer and genomic titer every rd day to exclude the presence of low-titer infectious particles. results/finding: all viral particles in the plasma units were completely inactivated with an average efficacy of ! . log infectious titer. no viral replication was observed after days of passaging post inactivation. the genomic titer was only slightly affected by pathogen inactivation, which is designed to target the infectious titer, but not the physical titer. conclusion: amotosalen alone had a slight effect on the infectious titer while amotosalen/uva effectively inactivated all infectious mers cov viral particles in the plasma units with an inactivation efficacy above logs infectious titer, giving evidence for improved blood safety of amotosalen/uva treated plasma in mers cov endemic regions. estimating the prevalence and incidence in a national blood service in taiwan for hcv eradication program yun-yuan chen* , jen-wei chen , chi-ling chen , sheng-nan lu and pei-jer chen . department of research, head office, taiwan blood services foundation, graduate institute of clinical medicine, college of medicine, national taiwan university, division of hepatogastroenterology, department of internal medicine, kaohsiung chang gung memorial hospital background/case studies: world health organization (who) has set a goal to eliminate hcv by , and the epidemiological indicators generated from a national blood service is useful to monitor the effectiveness. this study aimed to evaluate the prevalence and incidence of hcv infection in taiwan. study design/method: in taiwan, anti-hcv (since ) and -sample mini-pools triplex nucleic acid test of hcv, hbv and hiv (since ) have been used in the routine blood screening. prevalence of anti-hcv and hcv rna were estimated in the first-time donors during - and - , respectively. age-standardized prevalence and its % confidence interval ( % ci) were calculated with adjustment of who world standard population - . for the incidence study, donors who have donated blood two or more times during - and who were without a history of anti-hcv positive before the follow-up period were included. the incidence and its % confidence interval was estimated from the number of new hcv rna positive cases divided by the person-years of follow-up. results/finding: the crude prevalence of anti-hcv in the first-time donors was dramatically decreased from . per , donors ( % ci: . - . ) to . per , ( % ci: . - . ) during - , and the agestandardized prevalence was also decreased from . per , donors ( % ci: . - . ) to . per , ( % ci: . - . ). the agestandardized prevalence of anti-hcv was generally higher in female donors before , but it was significantly higher in male donors at (p-value . ). a total of , hcv rna positive cases, . % of them were anti-hcv negative, identified from , first-time donors during - , and the crude and age-standardized prevalence of hcv rna was . per , ( % ci: . - . ) and . per , ( % ci: . - . ), respectively. crude prevalence of hcv rna was significantly higher in female donors (p value < . ), but no significant difference was found after age standardization (p value . ). both the prevalence of anti-hcv and hcv rna were increased with age (p for trend< . ). in the incidence study, a total of new hcv rna positive cases, . % of them were anti-hcv negative, found from , , donors followed for , , person-years. the incidence of hcv rna was . per , person-years ( % ci: . - . ), and no significant difference was observed between both genders (p-value . ) and between age groups (p for trend . ). conclusion: the prevalence of hcv infection has been dramatically decreased by . % during - . it becomes significantly higher in male donors and that needs to monitor in the future. incidence of hcv rna is low in repeat blood donors and it needs to identify more incident cases to observe the epidemiological characteristics. dalia ashour* , sahar muhmmad and dalia el dewi . national blood transfusion services, azhar university background/case studies: blood safety is a challenge in egypt because of the high prevalence of hcv and hbv. nucleic acid amplification test (nat) technologies have the potential to detect viremia earlier than current screening methods, which are based on seroconversion. the primary benefit of nat is the ability to reduce residual risk of infectious wp donations. the estimated reduction of the wp utilizing nat for hcv is - days, hiv from to days, and hbv from - days. study design/method: this cross sectional study was conducted in national blood transfusion center (giza, egypt) from to , the total number of donor samples to be screened is , the age of the donors ranged from to years, and they were of both sexes (m: f : ).screening by nat ulterio assay (grifols diagnostics; formerly novartis diagnostics) was done in parallel with eia testing for hbsag, hcv-ab and hiv ag/ ab. using individual donation nat (id-nat). multiplex nat yield samples are further tested using the discriminatory assay in order to ascertain which viral nucleic acid is present in the donor sample. statistical analysis chi-square (v ) test was used to measure the association between two qualitative variables. results/finding: nat screening detected a total of nat yield donations among ( . %) seronegative donors. among these nat yields cases, ( . %) were reactive for hbv, ( . %) were reactive for hcv and ( . %) were reactive for hiv- . we stratified the age of the donors into groups; group a ( - years), group b ( - years) and group c ( - years). the prevalence of nat yield to the three viruses was significantly higher in either group b or c, compared to group a (p . ; with % confidence interval (ci) . - . & p . ; with % ci . - . respectively). prevalence of nat-hbv; was significantly higher in age group b, as compared with group a (p . ; with % ci . - . ). on the other hand, there was no statistically significant difference between groups c and a and between groups b and c. comparing groups b and c combined with group a found a significantly higher prevalence of hbv in the former (p . ; with % ci . - . ). nat-hcv; did not differ significantly between the three groups (p . ; with % ci - . to . between groups a and b & p . ; with % ci - . to . between groups a and c & p . ; with % ci - . to . between groups b and c). nat-hiv; did not also differ significantly between the three groups (p . ; with % ci - . to . between groups a and b & p . ; with % ci - . to . between groups b and c). in either group a and c, no nat-hiv detected. nat yield to the three viruses was significantly higher in males than in females (p . ; with % ci . to . ). nat hbv was significantly higher in males (p . ; with % ci . - . ), but the prevalence of either hcv or hiv did not differ significantly between males and females (p . ; with % ci - . - . & p . ; with % ci - . - . ; respectively). conclusion: in this study the nat yield of in assumes more significance when one considers the fact that single donation is used for generating components that can be used by recipients. hence, in effect the nat yield becomes times that is, in . saving recipients from tti out of ( . %) is indeed very significant. results/finding: of the , donors who were tested by our donor center, , ( . %) were repeat reactive. a seasonal pattern in the prevalence was observed with the highest number of donors being positive in summer, and then progressively declining during the fall and winter months and increasing again in spring. there was a single case of transfusion transmitted babesiosis reported from our center during this period. a patient who was transfused with two units of packed red blood cells (rbcs) from two donors in the beginning of july presented in august for further transfusion and was found to have parasitemia in the peripheral blood smear and was subsequently diagnosed with babesiosis. the donors were called back, however one of them could not be tracked. samples were sent to the state for further testing: an immunofluoresence assay was performed (combination of igg, igm and iga). the test was positive at : titer. the screening elia s/co of this donor was . . both donors were indefinitely deferred as blood donors. conclusion: our data confirm a decreased risk in transfusion transmission with the use of a screening assay. prior to implementation of the screening there were - transfusion transmitted babesia cases per year from - (table ). in the months after implementation of pre-transfusion babesia screening, one break through case of transfusion transmitted babesia was observed ( in , donors tested). thus the babesia eia screening test effectively prevents ttb. however, there was a substantial loss of donors due to being screen positive. four years of experience with id-nat at a tertiary care centre in north india: implications for transfusion transmission and donor screening. jasmeet singh*, amarjit kaur, gurpreet kaur, rajesh kumar and sonia gupta. dayanand medical college and hospital background/case studies: transfusion transmitted diseases are a challenge for transfusion medicine specialists and patient care providers around the globe. blood safety is a formidable task especially in a high population country like india. newer technologies like id-nat equip us to screen and prevent transfusion transmitted viral infections and prevent their transmission by improving over the sensitivity and specificity of conventional methods. this study aims at examining the effect of id-nat as an additional test on the safety of blood supply. study design/method: a retrospective observational study was conducted to analyze the data of years of additional nat testing at blood bank, dmch, ludhiana from september to december . results/finding: results . % ( of ) units were initially nat reactive. these units were further tested, of which . % were discriminated ( hiv, hcv, hbv and co-infections). the remaining . % ( ) were repeat non-reactive and . % ( ) could not be discriminated. overall, nat yield rate was one in , whereas virus-specific nat yield rates were one in , for hiv, one in for hcv, one in for hbv and one in , for hbv/hcv coinfections, respectively. conclusion: id-nat screening of all blood donations at our institution over past years has increased the screening sensitivities to check viral load and prevented transmission of probable transfusion transmitted viral infections. assuming % component preparation it saved transfusion recipients from harm. implementation of nat along with routine serological tests for screening of the blood donations definitely improves the transfusion safety and should be mandated across all transfusion centers. min xu , , wei mao , tao he , yashan yang , , zhan gao , , chunhong zhang , hongmei liao , jingxing wang , and miao he* , . institute of blood transfusion, chinese academy of medical sciences & peking union medical college, sichuan blood safety and blood substitute international science and technology cooperation base, chongqing blood center background/case studies: many emerging infectious pathogens are known to be existed in heathy blood donations, and could be transmitted via transfusion with potential hazardous consequences against recipients. with more convenient application of high through put sequencing, it becomes much easier to investigate uncultured microbiome in qualified blood donations. therefore, metagenomics analyses were used to reveal emerging and re-emerging infectious diseases in healthy donations which might potentially threat the blood safety. study design/method: pooled plasma sample were collected from , voluntary blood donors from chongqing, china. total dna and rna were extracted and amplified with random primers pcr respectively in order to construct a pe library to peform deep sequencing by illumina miseq. all reads were trimmed to remove low quality bases and adapter sequences. the fully overlapping paired-end reads passing the quality filter were concatenated using pear. we classified the final reads using kraken and a kraken database made from complete refseq bacterial, archaeal and viral genomes, along with the grch human genome. the unclassified reads by kraken were aligned to ncbi nt database using blastn with cut-off evalue as e - . the best alignment hits were used to classify the reads. krona was used to generate all taxonomic distribution plots. finally, the potential emerging and re-emerging infectious pathogens were identified out of the classified microbiome by experience. abstract results/finding: . gb raw data with , , reads were generated in the dna library. meanwhile, . gb raw data with , , reads were generated in the rna library. after cleaning the human background, reads from bacteria, reads from viruses, and reads from parasites were identified (table ) . no hazardous viruses were identified as potential threats to blood safety. except for viruses and bacterias which would do limited hazards to blood safety, plenty of parasites were identified in which some were already considered as threats to blood safety in some developed countries were also discovered such as plasmodium sp. and leishmania infantum (table ) . conclusion: the investigation has revealed the metagenomics of the qualified blood donations in chongqing, china. the results showed a thoughprovoking discovery of genomic fragments of some microbes which might threat the blood safety. the displayed serious results let us have to think about regulating some reasonable screening methods as well as donor recruitment strategy in certain epidemic areas or seasons to ensure the blood safety. however, on the contrary, the results should be considered more cautiously because the existing of genomic fragments could not represent the existing of infectious pathogens. the validity of the metagenomics hints were suggested to go through epidemiological investigations and specifically tested under laboratory ways such as bacteria or virus culturing to ensure the vitality of those pathogens. background/case studies: the caribbean has become an endemic region for several emerging viruses in the last decade. after a chikungunya outbreak in most recently zika was shown to be endemic on the caribbean island of curacao. to effectively provide safe blood products in an endemic region the conventional international recommendations of donor exclusion and testing do not seem a viable option and could severely affect the local blood supply. pathogen reduction (pr) is considered an important new approach with potential benefits. the introduction and experience of use of pr platelets in the dutch caribbean over a period of one year is presented. study design/method: pathogen reduction of thrombocyte concentrates by use of riboflavin and ultraviolet treatment (mirasol prt, terumo, belgium) was introduced. all thrombocyte concentrates provided to the general hospitals on the dutch caribbean islands of curaçao, bonaire and sint maarten were pr and data collected over the period of february to february . thrombocyte concentrates are prepared out of single donation units by the buffycoat method. results/finding: over the period platelet concentrates were provided to adult and pediatric patients. these included patients on the intensive care and neonatal intensive care departments. no adverse events were reported and the cci for each transfusion was within the expected outcome. introduction of pr had minimal impact on the logistics of thrombocyte concentrate preparation and availability. furthermore no transfusion related bacterial contaminations were reported. conclusion: pr of platelet concentrates seems viable and safe for use in a small scale caribbean setting with endemicity for emerging viruses like chikungunya and zika. it offers a realistic alternative for conventional recommendations of donor exclusion and testing, thereby helping to maintain sufficient labile blood product availability. michael phillips* , germ an leparc , phillip c williamson , lani palmer , ben reynolds , maria noedel and lindsey houghton . creative testing solutions, oneblood background/case studies: due to the risk of travel and sexually transmitted zika infections, the food and drug administration issued a guidance document on february , recommending that blood centers in puerto rico cease distribution of locally collected blood products unless donors are tested or products are pathogen reduced by march , . with the high incidence of zika virus (zikv) in puerto rico and uncertainty of the impact to the continental u.s. blood supply, there was intense pressure to implement a donor screening test for zikv. the project was initiated on february , and included clinical trial requirements, client onboarding and laboratory operations. stakeholders consisted of clients, the manufacturer, institutional review boards (irb), informational technology (client and lab based), the food and drug administration (fda), the centers for disease control cdc, and the florida department of health. clinical trial requirements included development of instrument and assay validations, sop creation, result reporting, assay and clinical trial training, deviation management, donor notification, and follow up sample handling. client onboarding began with confidentiality agreements between the client and the sponsor. a zika based webinar was created to provide an overview of the sponsor protocol, lab test system and client responsibilities. the complexity of the project increased when mosquito borne zika transmission was identified in two counties in florida. this required zikv testing to be performed on collections in both florida and puerto rico. the zikv-nat is performed in singlet, unlike the mpx and wnv assays which are run in minipools. this had a significant impact on instrument capacity. despite these obstacles and the changing regulatory requirements, the zikv screening test was implemented within six weeks. study design/method: one metric used to measure client service levels is our ability to meet established upload time goals for individual clients. the percentage of samples released on time is evaluated daily with a running monthly total. our upload time goals were negatively impacted from july through september due to the unexpected increase in zikv testing, the requirement to perform testing in singlets and the resulting instrument capacity issues. additional instruments were sourced in october and operations stabilized. conclusion: on february , , the project to implement a zikv ind test was initiated. six weeks later, testing was performed on the first batch of samples. despite the changing regulatory requirements over time, the implementation was extremely successful. initiating a new ind testing within weeks is unprecedented and required exceptional collaboration between all participants and stakeholders. background/case studies: plasmodium falciparum (pf), an intraerythrocytic protozoan parasite, is accountable for nearly all malaria mortality in africa. in , who reported $ million new cases worldwide, resulting in > , deaths. malaria prevalence is highest in sub-saharan africa, home to % of all infections accounting for % of mortalities. both the incidence and prevalence of malaria in africa significantly increase the potential for transfusion-transmission (tt), with little to no screening of products in developing countries. the objective of this study was to evaluate the inactivation of pf in whole blood (wb) using a system specifically developed for the realities of the developing world and in support of the swiss red cross humanitarian foundation for whole blood pathogen inactivation for africa. the inability to consistently supply blood components leads to routine wb transfusion, and as transfusion-transmitted diseases are prevalent in the developing world, the establishment of a robust wb pathogen inactivation system is desirable. the approach uses the small molecule amustaline to form covalent adducts and crosslinks within nucleic acids of leukocytes and contaminating pathogens to prevent replication. the process includes addition of . mm amustaline and mm glutathione (gsh) and a h at room temperature (rt) incubation after which the treated wb unit is suitable for storage up to days at rt. study design/method: for each experiment, a wb unit was spiked with ring-stage pf-infected red blood cells (irbc). a pre-treatment sample was removed prior to addition of amustaline and a post-treatment sample was removed h after amustaline addition to determine the pre-and posttreatment titers to calculate the level of inactivation. these samples were serially diluted in flasks containing medium with % fresh rbcs. the diluted samples were used to inoculate flasks in quadruplicate and monitored for parasitemia by counting irbc in blood smears and by flow cytometry. pretreatment cultures were terminated after reaching > % parasitemia, while no residual pf was detected in post-treatment cultures. log reduction was calculated as the difference between the mean titer in pre-and posttreatment samples. results/finding: robust inactivation of pf in wb was achieved to the limit of detection, at > . log or > . log /ml. conclusion: pf was inactivated to the limit of detection in wb after treatment with amustaline/gsh, illustrating that the system has potential to mitigate the risk for pf transfusion transmission in endemic regions that lack testing capacity and operate under the constraint of a very limited blood component supply and rely on wb transfusion. (this system for wb is not approved for commercial use). increased patient safety and improved inventory management with day apheresis platelets nancy m. dunbar* and zbigniew m. szczepiorkowski. background/case studies: a pathway currently exists for apheresis platelet (ap) outdate extension from to days using an fda cleared rapid test (rt). in february , our hospital based transfusion service implemented the use of rt on day , and to routinely extend ap shelf life to days. prior to this, we tested aps by rt on day and transfused day or day units with physician approval when deemed medically necessary. this report describes changes observed in transfusion practice and platelet inventory management one year following routine use of day platelets. study design/methods: data were obtained for two -month study periods: october -september (pre-implementation) and february -january (post-implementation). the interval transition period was intentionally excluded. for each study period, we determined the total number of aps transfused, rt status on the day of transfusion, total number of rts performed, expired ap units, and aps obtained from suppliers using ad-hoc ordering. we also obtained hospital data including inpatient admissions, surgical volumes, average length of stay and case mix index. results/findings: data are shown in table . the number of ap transfusions increased by % post-implementation, comparable to a % increase in inpatient admissions and an % increase in surgical volumes. the hospital length of stay and case mix index were similar for both periods. the average number of platelet transfusions per patient was not statistically different ( . pre; . post, p . ). the number of rts performed increased by %. the percentage of transfused units tested at least once by rt prior to transfusion increased by % (p< . ). the outdate rate decreased from % to % (p< . ). ad-hoc ordering decreased from % to % (p< . ). conclusion: use of an approved rt for routine ap outdate extension to day was associated with increased patient safety as more transfused units underwent secondary testing prior to transfusion. increased cost of rt was offset by reduced ap waste and less frequent need for ad-hoc ordering. sheila o'brien* , vito scalia , carla osiowy , michael carpenter , anton andonov and margaret fearon . canadian blood services, public health agency of canada background/case studies: the rates of hepatitis b (hbv) and hepatitis c virus (hcv) positive donations are low ( . and . per , donations, respectively) and most are among first time donors. we aimed to determine the frequency of various genotypes of hbv and hcv in canadian blood donors confirmed positive for hbv and hcv. study design/methods: in the roche multiplex assay (hcv/hiv/ hbv) was implemented in minipools of units. hcv nat was in place since (using minipools of ) but this is the first time donors have been screened by hbv nat. hbsag, anti-hbc and anti-hcv were tested using the abbott prism assay. confirmatory testing for hbsag was by the prism neutralization assay. anti-hcv repeat reactivity was confirmed by the inno-lia hcv score line immunoassay. since march all samples testing hbv nat positive, or confirmed positive for hbsag and all hcv nat positive or anti-hcv confirmed positive samples were considered positive and samples were sent to phac for sequencing. a sample from each positive donation was aliquoted and frozen at - o c. genotyping was carried out by sequence and phylogenetic analysis of the hbv surface antigen coding region. hcv viral rna was extracted and subjected to reverse transcription and pcr amplification in the ' ntr-e and ns b regions. sanger sequencing of these regions represents approximately % of the genome. results/findings: all confirmed positive donations were whole blood donations. there were hbv positive donations. of these, had tested hbv nat positive. genotypes were type a, b, c, d and e. there were samples hbv nat negative but hbsag positive ( were anti-hbc reactive). of these, could not be sequenced and one was genotype a (also anti-hbc reactive). there were samples considered hcv positive. of these, samples were hcv nat positive. genotypes were type a, b, c, b and a. there were also samples hcv nat negative but anti-hcv positive. none of these could be sequenced. conclusion: the first months of molecular surveillance show a range of genotypes for hbv and hcv for samples identified as nat positive. to date no samples that were nat negative anti-hcv reactive could be sequenced, however one nat negative sample that was positive for hbsag and anti-hbc reactive was hbv genotype a. surveillance over a longer period is background/case studies: the bact/alert d microbial detection system (bta d) is currently fda cleared for the quality control testing of leukocyte reduced apheresis platelets (lraps). the bact/alert virtuo microbial detection system (virtuo) (biom erieux, st. louis, mo) is a new generation of bact/alert instrumentation. the underlying colorimetric technology used in previous generations of bact/alert is used in the vir-tuo and incorporates new instrument architecture to improve temperature stability, workflow improvement via automation of processes that are currently performed manually, an improved user interface and an enhanced algorithm to shorten time to detection. the objective of this study was to compare the performance of the virtuo and bact/alert d (bta d) instruments, using bact/alert bpa (aerobic) and bact/alert bpn (anaerobic) bottles, for the detection of a range of typical bacterial contaminants seeded into leukocyte reduced apheresis platelets (lraps).* study design/method: the study was performed at two institutions, one in the us and the other in the uk. aliquots of lraps were seeded with low levels ( - cfu/ml) of bacterial species commonly associated with platelet contamination, and replicates ( per instrument) of ml aliquots per bottle were inoculated into bpa and bpn bottles. one set of bottles was loaded into bta d and the other into virtuo and incubated until signaled positive by the instruments or for up to days. overall detection rates and time to detection of bacterial contaminants between instruments were compared. additionally bottles were tested in each instrument (lraps only, no organism) to evaluate differences in the overall negative agreement rates (detection of false positives) between instruments and to serve as sterility controls for the platelet preparations. background/case studies: the implementation of nucleic acid testing (nat) blood screening is still a challenge in resource-limited countries. at the same time, in these countries, higher to similar proportions of replacement to voluntary blood donors are recruited. a higher prevalence of infections is observed in relation to developed countries. as a consequence, more incident cases of infections can be expected. in our country, some hospital blood banks could not afford nat due to high costs, but belong to a net that centralizes nat in a reference blood center. the process to consolidate small blood banks in regional blood centers, which will be able to implement nat, is not yet complete. although efforts to reduce replacement /familiar blood donations are in progress, these goals have not been completely achieved. the aims were to compare the prevalence of hiv, hcv and hbv by nat screening in a blood center recruiting only voluntary blood donors with the prevalence in centers recruiting replacement and voluntary blood donors, and describe the nat yield rates for hiv, hcv and hbv in a period of three and a half year experience. study design/method: a regional blood donor center (rbdc) has centralized nat screening from centers in different regions of the country due to since august . this process required to achieve adequate laboratory conditions and staff qualification and a development of software to assure sample traceability and interface for transmission of results. when a window period was suspected, the nat screening was repeated from the plasma unit and a second sample of the blood donor was required to confirm nat results. this rbdc have also been developed a % voluntary donor program since and is the only center in the country that has achieved this goal. results/findings: a total of , blood donations were studied from august to december . in the rbdc, where only voluntary blood donations are recruited, the prevalence was per , donations for hiv (ic % - : , ); per , for hbv (ic % - : , ) and per , for hcv (ic % - : , ). in all other centers together, where voluntary and replacement blood donations are recruited, the prevalence was per , donations for hiv (ic % - : , ); per , for hbv (ic % - : , ) and per , for hcv (ic % - : , ). window period infections were detected only in centers recruiting voluntary and replacement blood donations, giving nat yield rates of : , for hbv; : , for hiv and : , for hcv. conclusion: the hiv, hbv and hcv prevalence was lower in a center where the tasks to sustain a voluntary blood donor program were developed. nat yield rates could be reduced in the region if this program could completely be applied in all centers. mechanisms leading to obi include various factors such as imperfect host's immune response and viral variation factors. this study was to determine the viral loads of obi under currently recruitment and screening among blood donors in five blood services of zhejiang province, china. study design/method: before donation, the donors were screened and precluded with hbsag preliminary test positive and alt level abnormal. following, the samples were detected for hbsag twice using different elisa reagents and hbv dna using tma or qt-pcr techniques. then, the samples with hbv dna positive and elisa negative were tested for the viral loads using taqman technique in cobas s system. hbv s region was also sequenced. results/finding: obi were found in the , donations. in the viral loads assay, samples were negative and samples' viral loads were lower iu/ml. the mean viral loads was . . (log ) iu/ml in other samples,while the mean viral loads with hbsag /hbv dna samples was . . (log ) iu/ml. samples of obis have analyzed the hbv genotype, which b was the most prevalent subtype ( . %) and the other was hbv c genotype( . %). compared the samples with hbsag /hbv dna ,we found two obi samples carrying with t>c mutation, which could cause an amino acid s f. conclusion: in this study, the viral loads of obi infection in donors was much low than hbsag /hbv dna , and some unique variation was identified in the obi individuals. a transfusion in general population. screening of blood donors for hbv in india is primarily based upon detection of hepatitis b surface antigen (hbsag) in donor's sera. the current study was undertaken to determine the prevalence of occult hbv infection (obi) in voluntary blood donors and to analyze the burden of hbv window period donations. study design/method: this is a prospective, observational, mono-centric study performed in a national accreditation board for hospitals (nabh) accredited apex blood bank, located in maharashtra state, india. monolisa hbsag ultra (bio-rad, france)sandwich type elisa using monoclonal and polyclonal antibodies was used for hbsag detection in donor's sera. all the elisa non-reactive samples were also tested by an additional real time multiplex polymerase chain reaction (mpx-pcr) by cobasv r taqscreen mpx test. the donors which were found to be positive for hbv dna were followed upat th days, month, months& months by monolisa hbsag ultra (bio-rad, france) to analyze interval of window period and to delineate the window period donations (wpd) & true obi. background/case studies: occult hepatitis b infection (obi) is characterized by hepatitis b virus (hbv) dna-positive, but hbv surface antigen (hbsag) -negative. since may , we have been testing apheresis donors for hbv nucleic acids and improvements in laboratory testing have reduced the risk of transfusion-transmitted infection. the number of apheresis collections increased significantly year by year, however, data on hepatitis b virus marker rates among these donors continue to be lacking. the aim of this study is to evaluate the epidemic characteristics, incidence and estimate the risk factors of obi among apheresis donors in a region of central china. study design/method: apheresis donors' data from may to dec was retrospectively analyzed. all samples were tested for hbsag, hbv dna, and other markers. nucleic acids testing (nat) was performed on the roche cobas s platform using pools of serologically negative samples and any pools positive would undergo nat again individually. hbsag negative, but hbv dna positive were further tested for hbv dna quantitative pcr, antibody to hepatitis b surface antigen (hbsab), antibody to hepatitis b core antigen (hbcab), hepatitis b e antigen (hbeag) and antibody to hepatitis b e (hbeab). results/finding: in the evaluation, seronegative donations were screened by nat and a total of hbv dna-reactive/hbsag-negative donors were detected. no hiv rna -reactive or hcv rna -reactive sample was detected. complete serologic screening of the index donations indicated that the majority of these donors had an occult hbv infection and the majority of which were married men and the fixed donors with many whole blood or apheresis donations. age distribution of the age group - years old showed a large proportion, who accounted for % of reported infections. most of the hbv dna cases (about . %) reached senior high school education. the average hbsag dna positive rate was . % ( / ). incidence among apheresis donors in this period for hbsag dna were . / . these estimates were comparable to those among repeat whole blood donors. we developed pathogen reduced (pr) cryo derived from ffp and pf with day stability at c. study design/method: six replicates of type-matched pools of whole blood derived (wbd) and apheresis (aph) plasma were split to produce conventional control ( ml) and test components ( ml ml). test components were pr with amotosalen and uva light. aph and wbd ffp were produced by freezing plasma within hr and wbd pf within hr. cryo was manufactured according to site sops and frozen at - c (test ml, control ml ). test and control cryos were thawed at c, and characterized immediately post thaw (t ), and after d storage at c and tested for fb and fviii function, thromboelastography (rotem) and thrombin generation (cat). results/finding: pr cryo retained sufficient fb and fviii activity post thaw and over d at c (table) for hemostatic capacity. rotem (extem) showed retention of fibrin formation (a angle) and clot quality (mcf) (table) . thrombin generation was robust as demonstrated by multiple parameters (lag time, peak thrombin, endogenous total thrombin potential (etp), and time to peak (tt) despite lower fviii levels. these parameters were maintained through d storage at c. conclusion: pr cryo can be processed from plasma sources, including pf , and stored at rt for days. pr plasma provides adequate levels of fb with hemostatic capacity equivalent to control as demonstrated by rotem and cat. use of pf with stability over days can increase the availability of cryo with a reduced risk of transfusion-transmitted infection. cryo produced with psoralen-treated (pr) plasma is not approved for use in the us. performance of a new automated alinity s assay for antibodies to t. cruzi darwin smith* , ed bakker , anton van weert , jane bryant , mark paradowski , lynne fleischmann , mirjana sarac , george chen , george schlauder and gregg williams . abbott diagnostics, sanquin diagnostics, abbott gmbh & co. kg background/case studies: the parasite, trypanosoma cruzi (t. cruzi), is the cause of chagas disease which is endemic to the americas and infects - million people. in order to prevent transfusion mediated transmission of this parasite in endemic countries, blood collection centers require high throughput anti-t. cruzi assays with good specificity and sensitivity. in nonendemic countries, selective testing of at risk donors is a strategy to avoid temporary donor deferrals. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of the new automated chemiluminescence immunoassay for the detection of antibodies to t. cruzi was evaluated on the alinity s automated platform and compared to another onmarket chemiluminescent immunoassay. precision was assessed over days using a panel of positive and negative samples. sensitivity was evaluated on presumed antibody positive specimens and specificity was evaluated on random blood donor samples. results/finding: precision was % cv or less for positive samples over days. the overall specificity in a blood donor population was . % ( / ). sensitivity was . % for presumed antibody positive samples. conclusion: these results indicated that the new automated alinity s chagas assay provided very good performance in sensitivity and specificity, comparable to the current on-market anti-t. cruzi assay, and is equally suitable for use of universal screening in endemic and selective donor screening in non-endemic countries. performance of a new automated alinity s assay for hepatitis b surface antigen and hepatitis b surface antigen confirmatory randal makela* , anton vanweert , ed bakker , jane bryant , mark paradowski , lynn martin , daniela kaleve , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, abbott gmbh & co. kg, abbott diagnostics background/case studies: despite the development of sensitive nat methods, blood transfusion in many parts of the world relies on serologic screening for hepatitis b surface antigen (hbsag) to prevent transfusion transmitted hbv infection. sensitive hbsag assays must be capable of coping with a wide range of mutants while exhibiting an uncompromised specificity. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection and confirmation of hbsag was evaluated on a next generation automated platform, abbott alinity s. precision was assessed over days. sensitivity was evaluated using known positive samples, commercially available seroconversion panels, the who standard, hbsag mutants, and hbsag genotyped specimens (a through h). specificity was evaluated on random blood and plasmapheresis donors. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for presumed positive samples. sensitivity was % for all genotypes. % of the mutants were detected vs % for the comparator assay. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. analytical sensitivity ranged from . to . iu/ml. the alinity s hbsag confirmatory assay confirmed all known positive hbsag specimens, including hbsag mutant samples that were not confirmed by the comparator hbsag confirmatory assay. conclusion: the new automated alinity s hbsag assay provided precision, specificity, and seroconversion sensitivity comparable to the current onmarket comparator assay. however, the alinity s hbsag assay demonstrated a gain in sensitivity over the comparator assay through the detection and confirmation of a wider range of mutants. performance of a new automated alinity s immunoassay assay for hiv darwin smith* , ed bakker , anton van weert , jane bryant , mark paradowski , kevin callear , susan sullivan , george chen , george schlauder and gregg williams . abbott diagnostics, sanquin diagnostics background/case studies: blood donations are commonly screened to detect the presence of antibodies (or antibody and antigen) to human immunodeficiency virus types and (anti-hiv- / ). blood centers require very high throughput anti-hiv- / assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have evaluated an improved automated assay for the detection of anti-hiv- / antibodies and hiv- p antigen. study design/method: the performance of the new chemiluminescence combination immunoassay for the detection of anti-hiv- / antibodies and hiv- p antigen was evaluated on the abbott alinity s system. precision was assessed over days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors and plasmapheresis donors. sensitivity was evaluated using presumed positive samples for hiv- , hiv- and hiv group o antibodies and hiv- p antigen. seroconversion sensitivity was evaluated with commercial seroconversion panels. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for presumed antibody positive samples comprised of hiv- , hiv- and hiv- groups o, n, p, crf and urf samples. also, sensitivity was % for antigen positive viral isolate samples comprised of hiv- , hiv- and hiv- groups o, n, p, crf and urf samples. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s hiv ag/ab combo assay provided acceptable performance in specificity, sensitivity and precision, while providing similar seroconversion sensitivity as the comparator assay. performance of a new automated alinity s immunoassay for the detection of anti-hbc antibodies randal makela* , anton vanweert , ed bakker , jane bryant , mark paradowski , joyce siregar , angela vockel , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, abbott gmbh & co. kg, abbott diagnostics background/case studies: in countries with a low prevalence of hepatitis b, blood donations are commonly screened to detect the presence of antibodies to hepatitis b core antigen (anti-hbc) alongside hbsag and hbv nat to detect donors with occult hepatitis b infections (obi). blood centers require anti-hbc assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in the response for the need for such screening assays, we have developed an improved automated assay for the detection of anti-hbc on the alinity s system. study design/method: the performance of a new chemiluminescence anti-hbc assay for the detection of anti-hbc antibodies was evaluated on the next generation automated abbott alinity s system. precision was assessed over days evaluating positive samples. specificity was evaluated on samples obtained from random blood donors. sensitivity was evaluated using specimens characterized as anti-hbc positive by means of serologic methods. analytical sensitivity was assessed using the who st international standard. seroconversion sensitivity was evaluated using commercial seroconversion panels. results/finding: precision was less than % cv for positive samples over days. the blood donor specificity was . % ( / ). sensitivity was % for samples presumed to be anti-hbc positive. analytical sensitivity results on the alinity s anti-hbc assay ranged from . to . iu/ml. seroconversion detection was equivalent to the comparator assay with reactive samples detected with the alinity s assay and reactive samples detected by the comparator assay. conclusion: these results indicate that the new automated alinity s anti-hbc assay provided good performance in specificity, sensitivity and precision versus the comparator assay. performance of a new automated alinity s immunoassay for the detection of htlv i and htlv ii antibodies melanie anderson* , anton vanweert , ed bakker , mark paradowski , jane bryant , tuan bui , joyce siregar , george chen , george schlauder and gregg williams . abbott laboratories, sanguin diagnostics, abbott diagnostics background/case studies: in endemic countries, universal blood screening is necessary to prevent transfusion transmitted htlv infections (anti-htlv i/htlv ii). in non-endemic countries, selective testing may avoid unnecessary temporal deferrals for donors at high risk, such as returning travelers from or donors born in countries with a high htlv prevalence. blood centers require high throughput anti-htlv i/htlv ii assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining a safe blood supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response for the need of an assay with high specificity on a high throughput instrument we have developed a new assay for the detection of antibodies against htlv-i/ii antibodies for the alinity s system. study design/method: precision was assessed over days using htlv i and htlv ii positive samples. specificity was evaluated using , blood donor specimens from europe and diagnostic samples obtained from the united states. sensitivity was evaluated using preselected htlv i and htlv ii positive samples. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using the mp diagnostic htlv blot . . results/finding: imprecision was less than . % for positive samples over days. clinical sensitivity was . % ( / ) on preselected htlv i and htlv ii positive samples. the specificity was . % ( , / , ) on a blood donor population and . % ( / ) on diagnostic samples. conclusion: these results indicate that the new alinity s automated htlv i/ ii assay provided very good performance in specificity, sensitivity, and precision. sensitivity and specificity were comparable to the comparator assay. claudia ramirez , michel garcia* , fernando palomino and guillermo orjuela-falla . national blood bank colombian red cross, universidad del rosario, fuats background/case studies: current hepatitis c virus (hcv) supplemental testing algorithm for blood donations in colombia, requires that an immunoblot assay be performed on every hcv enzyme immunoassay (eia) repeatreactive sample. a higher proportion of indeterminate (ind) results by immunoblot assays has been documented for non-us donor samples, affecting donor counseling and eventually increasing costs and opportunity for the notification of infected donors. this work aimed to establish the distribution of immunoblot results in colombian repeat-reactive samples, as well as the frequency of band detection in both positive and indeterminate blots. study design/method: in total, anti-hcv-reactive donor samples (signal-to-cutoff (s/co) ratio greater than . ; abbott architect i sr) underwent supplemental testing by immunoblot (either chiron riba hcv . sia or hcv blot . test, mp diagnostics). negative (neg), indeterminate (ind) and positive (pos) blot results were grouped by s/co ranges as follows: - . , - . , > . band detection and intensity were independently analyzed for indeterminate and positive results. results/finding: immunoblot results were negative in . % ( / ) of samples, indeterminate in . % ( / ) and were positive in . % ( / ). a direct relationship was observed between positive immunoblot and increased s/co. the proportion of ind results were higher in the s/co group - . ( . %) compared with the - . ( . %). in samples with indeterminate results, ns _ was the most frequent band detected ( , %). in contrast, the most frequent band in the group of positive results was core ( , %). only one sample from the indeterminate group ( . %) had a strong band intensity ( ), compared with samples from the positive group ( . %). conclusion: the proportion of indeterminate immunoblot results in this sample of colombian donors is one of the highest ever reported, being twice as much as the proportion found in larger samples of us donors. the high proportion of ind results found in the s/co group ( - . ) suggests that the optimal s/co ratio for predicting a confirmed anti-hcv result in this population should be higher than the one recommended by the cdc for us population (> ). overall, these results suggest that the supplemental testing algorithm for blood donations in colombia could be improved not only by using high s/co ratios as an alternative to immunoblot, but also by introducing hcv genomic assays instead of immunoblots, at least for samples with intermediate s/co ratios. ns _ and ns _ cross-reactivity in colombian population warrants further investigation. performance of the alinity s immunoassay for the detection of syphilis antibodies melanie anderson* , ivanka mihaljevic , manuela miletic , miljana stojic vidovic , irena jukic , jane bryant , mark paradowski , angela vockel , george chen , gregg williams and george schlauder . abbott laboratories, croatian institute of transfusion medicine, abbott gmbh & co. kg, abbott diagnostics background/case studies: blood donations are commonly screened for syphilis in order to detect the presence of antibodies to the bacterium treponema pallidum. in addition, continued pressures on laboratory operations demand that the full panel of ttid assays perform on a single platform capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. in response to those needs, we have evaluated a new automated immunoassay for the detection of antibodies to t. pallidum. study design/method: performance of the new automated chemiluminescence immunoassay for the detection of antibodies to treponema pallidum was evaluated on the alinity s system. precision was assessed over days using positive samples. specificity was evaluated on samples obtained from , blood and plasmapheresis donors from the united states and europe and diagnostic samples obtained from the united states. sensitivity was evaluated using preselected positive samples. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm with confirmatory assays, inno-lia tm syphilis score, and mikrogen recomline treponema igg and igm blots. results/finding: imprecision was less than . % cv for positive samples over days. clinical sensitivity was . % ( / ) on preselected syphilis positive samples. the specificity was . % ( , / , ) for blood donor specimens and . % ( / ) on diagnostic samples. conclusion: these results indicate that the new automated alinity s syphilis assay provided good performance in precision, specificity and sensitivity in line with data found for the comparator assay. performance of the new automated alinity s assay for anti-hcv melanie anderson* , ed bakker , anton vanweert , jane bryant , mark paradowski , tuan bui , lynn martin , george chen , gregg williams and george schlauder . abbott laboratories, sanguin diagnostics, background/case studies: serological screening for antibodies to hepatitis c virus (hcv) often in conjunction with nucleic acid testing (nat) is used worldwide to prevent transfusion transmitted hcv infections. while nat provides improved sensitivity and detection of hcv in the pre-seroconversion window, serological testing provides continued detection of hcv in infected individuals and individuals with resolved infections with no detectable hcv rna. blood and plasma centers require very high throughput anti-hcv assays with high specificity and sensitivity to prevent unnecessary donor deferrals while maintaining the safety of the blood and plasma supply. in addition, continued pressures on laboratory operations demand that assays perform on platforms capable of increased walk away time and enhanced automation in areas of reagent management, retest options, and commodity/waste management. study design/method: the performance of a new automated chemiluminescence immunoassay for the detection of antibodies to hcv was evaluated on the alinity s system. precision was assessed over days evaluating positive samples. sensitivity was evaluated using preselected positive samples and seroconversion panels. specificity was evaluated on samples obtained from , blood and plasmapheresis donors from the united states and europe and diagnostic samples obtained from the united states. sensitivity and specificity samples were split across reagent lots during testing. confirmation of repeatedly reactive samples was done using a testing algorithm consisting of the inno-lia tm hcv score and nat/hcv discriminatory nat assays. results/finding: imprecision was less than . % cv for positive samples over days. overall clinical sensitivity was % on preselected anti-hcv positive samples. seroconversion sensitivity was better than the comparator as evidenced by the new anti-hcv assay identifying more bleeds than the comparator assay. the specificity was . % ( , / , ) for blood donor specimens and . % ( / ) background/case studies: zika virus (zikv), which has been outbroken in south america and the united states since middle of , was declared as the public health emergency of international concern by who in feb . in addition to mosquito, zikv can be transmitted via maternalneonatal relationship, sexual intercourse or blood transfusion. the potential for transfusion-transmitted zika virus was shown in french polynesia where . % of asymptomatic blood donors tested were positive for zika virus rna using nucleic acid test (nat). several case reports have confirmed that zikv can be transmitted by transfusion. it has been shown that among blood donors, . % of the zikv infections were asymptomatic and the ratio of symptomatic to asymptomatic patients observed in micronesia was approximately : to : . thus zikv has raised a great challenge to transfusion safety. measures should be taken to prevent transfusion-transmitted zikv, including temporary deferral of blood donors in epidemic locations, donor self-reporting of zikv symptoms after donation with or without quarantine of blood components, supply by blood collected from non-endemic areas to epidemic regions, nat of blood donations, and pathogen inactivation of blood products. in this study, we evaluated zikv inactivation in plasma by using methylene blue photochemical treatment (mbpt). study design/methods: plasma units from randomly selected healthy donors were collected and spiked with zikv. samples were added by mb at a final concentration of lm and assayed after illumination with visible light from both sides for , , and min. viral infectivity and zikv rna loads (reverse transcription pcr) were measured in spiked plasma before and after mbpt and confirmed using repetitive passages in cell culture. control was zikv spiked plasma without photochemical treatment. results/findings: zikv titer of control sample was . log % tissue culture infectious dose (tcid )/ml. no viral infectivity was detected after mb photochemical inactivation treatment for min, min or min and the losses of the infectivity were further demonstrated by repetitive passages of cell culture. meanwhile, zikv rna loads decreased significantly during the initial min of treatment whereby ct-value jumped from . (control) to . (mbpt for min) (table ) . conclusion: it showed that mb photochemical treatment could effectively inactivate zikv in plasma. rna lesions were induced during mbpt process so that nucleic acid reverse transcription and amplification were inhibited. mbpt is proved to be an efficient method to prevent plasma transfusiontransmitted zikv infections. gilles delage* , margaret fearon , susan l stramer , megan l nguyen , france bernier , sheila o'brien , vito scalia , sakina smith , yves gr egoire and boris hogema . h ema-qu ebec, canadian blood services, american red cross, sanquin background/case studies: hepatitis e virus (hev) is known to be transfusion-transmissible. as part of the risk assessment for this infection, a study was carried out in , canadian blood donors in . in a subset of , donor samples the seroprevalence was . %. however, no donor samples were positive for hev by an in-house nucleic acid test (hev-nat). since that study suggested exposure to hev in canada but used an hev-nat with a limit of detection of iu/ml, a larger study was performed using a more sensitive hev-nat assay. study design/method: donors were informed about the study in the predonation reading materials. linked samples from approximately , canadian whole blood donors including , from canadian blood services (cbs) and , from h ema-qu ebec (hq) were collected. clinics were selected to ensure representative sampling of the donor population. all a transfusion vol. supplement s donations with available plasma samples were tested by individual donation nat at the american red cross laboratory in gaithersburg, md, using the cobas v r hev test ( % lod . iu/ml, % ci . - . ) for use on the cobas v r / system. this test is not currently approved in canada or the usa, but is available as a ce marked test. all nat-reactive donors are questioned concerning risk factors for recent hev infection (travel, animal contact, food and water exposure), undergo confirmatory testing (alternate nat, viral load, genotyping and igm/igg serology), are notified by letter, and deferred from donating for months; in-date products collected from the donor, and any frozen red blood cells or plasma from the previous months are destroyed. recipients will be traced in the event of any products transfused in the previous months. results/finding: as of april , , of , ( , cbs, , hq) tested samples with valid results have been found hev-nat reactive: donors have been confirmed by further testing to date. confirmation is pending in donor. of the donors, were from quebec, and one each from nova scotia and alberta ( male, female). ages ranged from to years. only two donors reported non-specific symptoms (fatigue). in terms of risk factors: ate pork (including who ate pork liver), ate shellfish, ate venison, and drank well water. one donor had no identifiable risk factor. viral loads ranged from to iu/ml, of which were < , were - , and were > iu/ml; were anti-hev igm positive and anti-hev igg positive at index (wantai assay). conclusion: the prevalence rate of acute hev infection in this donor population appears to be around / . the data from this study will contribute to the ongoing risk assessment of transfusion-transmitted hev infection in canada. prevalence of malaria parasite in donated blood at nakasero blood bank, uganda gerald nsubuga* and musiisi ezra. uganda blood transfusion service background/case studies: introduction infectivity of donated blood with malaria is a significant health problem facing humanity. in uganda, screening for malaria parasite is neither routinely done in blood banks, nor stipulated in the current uganda national blood transfusion service (ubts) guidelines by the ministry of health. as a result, the proportion of donated blood that is infected with malaria is largely unknown. malaria infection places more than half of the world's population at risk and in majority of the tropical and sub-tropical regions of the world and about to million cases and to million death occur per year. however the study aimed at determining the prevalence of malaria parasites in donated blood at nakasero blood bank, kampala, uganda study design/method: a cross sectional study was carried out in nakasero blood bank, kampala, uganda in four hundred and seventy randomly selected donor samples at the blood bank between june and august . both thin and thick glass stained blood smears of blood samples with giemsa was examined using microscope. results/finding: of the donated blood samples, ( . %) tested positive for malaria parasite (p. falciparum), although there was no significant difference in occurrence of plasmodium in relation to sex, age and blood group (p> . ), majority of the blood donors that tested positive belonged to blood group o ( . %). the prevalence of malaria parasite in the study was . %. regardless of the prevalence, the presence of malaria parasite (plasmodium falciparum) in donated blood from donors that were presumed to be healthy raises a serious concern on the safety of donated blood in uganda. the ministry of health should review the existing guidelines for screening malaria and mandatory universal blood donor screening policy for malaria, for exclusion of blood donors with plasmodia parasitaemia. using methods like pathogen inactivation compared to tedious microscopic procedure to screen donated blood to be introduced to further enhance blood safety in our communities. components. the bact/alert virtuo* (virtuo) is an advanced, next generation system with improved automation, connectivity, and with data management systems. most importantly, the virtuo's new algorithm significantly reduces the time to detection (ttd) of microorganisms during quality control testing of platelet preparations using bact/alert bpa (aerobic) and bpn (anaerobic) bottles. bpa and bpn bottles were tested on virtuo and bact/ alert d (bta d) to evaluate repeatability to detect growth in seeded leukocyte reduced apheresis platelets (lrap) without platelet additive solution (pas), throughout platelet shelf life ( , and days after collection). study design/method: pooled lrap were seeded with low levels of organisms commonly associated with platelet contamination at , and days post collection. the seeded lrap were inoculated into bpa and bpn bottles on different days (not consecutive) alternating between teams of people each. seeded bottles were loaded into a virtuo and a bta d and incubated until declared positive or negative (up to days). additionally, bpa and bpn bottles inoculated with ml of unseeded lrap were tested on the virtuo and the bta d ( and bottles respectively), to serve as negative controls, sterility controls, and to evaluate the risk of false positives caused by lrap results/finding: the repeatability of the virtuo to detect organisms in lrap was demonstrated by a recovery rate of seeded bottles of . % for the virtuo and . % for the bta d. the virtuo demonstrated an average improved ttd of . hours, when compared to the bta d in the presence of ml lrap platelets. the lrap did not cause false positives. additionally, the age of the lrap units (within day expiry),did not impact the ttd when seeded with organism background/case studies: zika virus (zikv) is an emerging flavivirus that is transmitted by the aedes aegypti mosquito and sometimes a. albopictus mosquito. most infections are asymptomatic. zikv nucleic acid testing (nat) became a required test for blood donors per the fda guidance entitled, "revised recommendations for reducing the risk of zika virus transmission by blood and blood components". based on our geographical location, implementation of this testing began weeks after this guidance was issued. we performed zikv nat for donors of whole blood and blood components under an investigational new drug (ind) study (sponsored by hologic, inc.). we performed a retrospective analysis on all nat results as there is a potential to defer donation due to false positive screening results. study design/method: donors that consented to donate blood and be tested for the zikv were obtained from three blood banks in colorado and nebraska. nat was performed using the procleix virus assay which is a qualitative in vitro nucleic acid assay system that detects zikv rna in plasma specimens. the assay was performed on the automated procleix panther system. all testing was performed according to the manufacturer package insert. results/findings: in the event of a reactive result, donors would be retested by nat in addition to other testing (igm antibody testing, neutralization test). donors are deferred for days barring continued zikv testing and nonreactive results. a total of , donors were screened for zikv. all donors screened for zikv were nonreactive by nat. no invalid test results were obtained. in addition the number of failed test runs due to instrument or assay issues were experienced were quite low ( . %). this data indicates that both the assay and instrument are robust. there was a low frequency for additional testing which allows the laboratory to publish timely infectious disease results for our blood bank customers. conclusion: the reactive rate data presented here demonstrate that there is a low/zero incident rate in our region for whole blood and blood component discard due to reactive results. this screening is important to continue to ensure blood safety in the united states. robust inactivation of the yellow fever virus d strain can be achieved using amotosalen and uva light for pathogen reduction treatment (prt) of platelet components andrew laughhunn , felicia santa maria , yvette girard , peter bringmann , marion lanteri* and adonis stassinopoulos . microbiology department, cerus corporation, scientific affairs department, cerus corporation background/case studies: yellow fever virus (yfv) is known to cause explosive outbreaks, such as the one in angola in . the rapidly increasing number of infections in brazil, with hundreds of fatalities since december , is of concern. yfv is a flavivirus transmitted by aedes mosquitoes and could spread, like zika virus, to other parts of the americas where the vector is endemic. with no effective antivirals and only supportive therapy available, the best mitigation strategy is through vaccination with live attenuated vaccine strains, like the d-yfv strain. yfv vaccine is considered an effective and safe vaccine; however major adverse events have been reported including neurologic and visceral adverse effects. in addition, transfusion transmission (tt) of live attenuated yfv has been reported with severe clinical outcomes, especially in immunosuppressed patients. in order to prevent tt by yfv vaccine strain, the aabb recommends a weekperiod deferral after yfv vaccination. yfv outbreaks and vaccination campaigns may therefore reduce blood availability. this pilot study evaluated the ability to inactivate d-yfv using amotosalen (s- ) and uva light prt of platelet components (pc). study design/method: pc in %pas (n ) or % plasma (n ) were spiked with high titers of d-yfv and treated with s- /uva prt. samples were taken pre-and post-uva illumination and infectious titers were determined, by plaque assay using vero cells. the extent of inactivation was quantified by comparing titers before and after inactivation. results/finding: pre-prt infectious titers were . . log pfu/ml for pc in % plasma and . log pfu/ml for pc in % plasma while titers in post-prt samples were <- . . log pfu/ml for pc in % plasma and <- . log pfu/ml for pc in % plasma. inactivation to the limit of detection of > . . log or inactivation of > . . log pfu/ml was achieved for pc in % plasma. inactivation to the limit of background/case studies: the use of biotin as a supplement has increased in recent years and many health care professionals may not be aware of the high dosage intake by their patients. this high dosage has resulted in an increased prevalence of individuals being exposed to biotin levels much greater than the recommended daily dose and as a consequence, has led to inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. although abbott's alinity s assays do not utilize this free capture biotin-streptavidin methodology, eight assays developed for blood screening on the alinity s system were evaluated for biotin interference to ensure there are no unknown consequences of high biotin levels. study design/methods: the purpose of this study was to determine if the eight developed abbott alinity s assays would be susceptible to biotin interference by evaluating their performance in the presence of a high concentration of biotin. for each of the alinity s assays evaluated (hiv ag/ab combo, htlv i/ii, anti-hcv, chagas, hbsag, anti-hbc, syphilis, and cmv igg), samples spiked with a concentration of biotin at approximately ng/ml were tested against a control (unspiked) sample preparation to determine if there was a difference between the control and biotin containing samples. two samples, one negative and one positive, were tested with all assays, except the hiv and htlv assays, which each tested two positive samples ( hiv- antibody and hiv- p antigen, and htlv-i antibody and htlv-ii antibody, respectively). results/findings: for the negative samples, the sample to cutoff (s/co) differences between the biotin spiked and control were . for hcv, hbc, syphilis, cmv igg, and chagas, . for hiv ag/ab and htlv i/ii, and . for hbsag. for the positive samples, the mean s/co % differences between the biotin spiked and control were . % (antibody sample) and . % (antigen sample) for hiv ag/ab combo; . % (htlv i antibody sample) and . % (htlv ii antibody sample); - . % for anti-hcv, - . % for chagas, - . % for hbsag, - . % for anti-hbc, - . % for syphilis, and - . % for cmv igg. conclusion: eight abbott alinity s assays were evaluated to determine if they were susceptible to biotin interference. these results indicate that the eight alinity s assays do not show susceptibility to biotin interference at an approximate concentration of ng/ml. robustness of the abbott prism methods to biotin interference c fischer , r schneider , w leonard , m cobb , g schlauder , g williams , m zuske m janulis* . transfusion medicine, abbott diagnostics, wiesbaden, germany, add diagnostics, transfusion medicine, abbott laboratories, chicago, united states background/case studies: the use of biotin as a dietary supplement has increased significantly in recent years and many health care professionals do not realize their patients are taking high doses. the increase has resulted in an increased prevalence of people being exposed to biotin levels much higher than the recommended daily dose and as a consequence, potentially inaccurate lab results for assays that utilize the free capture biotin-streptavidin methodology. the purpose of this study was to identify any abbott prism assays that may be susceptible to biotin interference based on assay design and then evaluate the performance of those assays with high concentrations of biotin. after a comprehensive review of abbott's current on market prism assays, no assays were identified that utilize biotin-streptavidin capture; however, assays were identified for subsequent testing as they contain biotin in their assay design. background/case studies: bacterial contamination of platelets is the highest residual infectious risk in transfusion despite the current preventive strategies. while bacterial contamination may affect any blood component, the ambient storage temperature conditions for platelets make them most likely to facilitate bacterial growth. based on all the precautionary measures, the final platelet concentrates include in the worst cases a very limited viable bacteria number estimated from to colony forming units (cfus)/bag (i.e. . to . cfu/ml). one major difference between viruses and bacteria is that bacteria have the ability to grow up to a concentration of - cfu/ml over the days product shelf-life. moreover, a large diversity of strains is found in contaminated platelets representing a key challenge for the development of a generic bacterial test. the aim of this study was to develop an economic and easy diagnostic approach for the early, rapid, sensitive and generic detection of bacteria in platelet concentrates. the adaptability of the process with the blood transfusion services requirements was of major concern. hence, attention was focused on an easy to automate technique able to deliver results on day after collection. study design/method: a large panel of bacteria involved in transfusion reactions including clinical isolates and reference strains was established and used for mouse immunizations, antibody screening and platelet spiking steps. an original approach was used to produce and select monoclonal antibodies directed against bacteria to develop our generic immunoassay. as recommended, hours (day ) after collection a sampling volume of spiked platelets ( . - cfu/ml) was tested after a short generic culture, lysis and capture of bacteria on magnetic microparticles in a microplate format. an immunoassay was performed for the detection of the captured bacteria. results/finding: this approach was tested on a panel of bacterial strains involved in transfusion reactions. the pre-analytical steps and the capture of bacteria on microparticles were improved to avoid false negative results and to enhance the sensitivity of detection. the full test developed in this study combining a pre-analytical culture step followed by an there are many stakeholders are involved in hcv eradication program, including government authority such as centers for disease control and prevention, national health insurance and health promotion administration, and private property like hospitals, medical societies, pharmaceutical and vaccine industries, npos and academia. results/finding: tbsf is a private nationwide single blood services program in taiwan, and performs anti-hcv screening test and nat confirmatory test on every collected blood, which is a large-scale population screening of hcv in taiwan because of its high blood donation rate ( . %). tbsf confirmed positive test result of repeated blood donors, and can identify hcv rna seroconversion cases as recently-infected hepatitis patients. those infected patients would be referred to physician for further medical care and deferred permanently by tbsf to secure blood safety. by interviewing the newly-infected cases, the risk factors of hcv patients can be studied and then help identifying and eliminating sources of hcv infection. tbsf also contribute to health education by teaching our donors being aware of potential risks of hcv infection and keep monitoring every parameters of hcv epidemiology to evaluate the efficacy of hcv eradication program. conclusion: in hcv eradication program, tbsf can not only secure blood safety but also participate in health education, disease screening, etiology finding and prevention, surveillance and evaluation. thus, among all stakeholders, tbsf is particularly important and can play a pivotal role in eradicating hcv by in taiwan. the theraflex uv-platelets technology efficiently inactivates transfusion-relevant bacteria species in contaminated platelet concentrates ute gravemann , frank tolksdorf , wiebke handke , thomas h. m€ uller and axel seltsam* . german red cross blood service nstob, maco pharma international, gmbh background/case studies: the theraflex uv-platelets system (macopharma) is a uvc-based pathogen inactivation system for platelet concentrates (pcs). inactivation efficiency has been shown for a broad range of viruses, bacteria, and protozoans. previous studies with the first set of bacteria species of the who international repository of platelet transfusion relevant bacterial reference strains revealed a high inactivation capacity for clinically relevant bacteria. aim of the current study was to investigate the bacteria inactivation efficacy of the theraflex uv-platelets system for enterobacter cloacae, pseudomonas fluorescens, staphylococcus aureus and streptococcus bovis which have recently been added to the who international repository. study design/method: pcs were produced from buffy coats using the additive solution ssp (macopharma) with a residual plasma content of %. for inactivation kinetics, pcs (n ) were spiked with bacteria to a final concentration of approx. colony forming units (cfu)/ml and irradiated with increasing doses until the full uvc dose was achieved. samples were taken for the bacterial titer determination after each irradiation step. for sterilization studies, two pcs were pooled and inoculated with bacteria to a final concentration of approximately . cfu/ml. bacteria were allowed to grow for h in the pcs at c under agitation. after splitting, one pc remained untreated (growth control) while the other one was uvc-treated. after storage for seven days, samples were taken from both bags for sterility testing by bactalert (biomerieux) and for determination of the bacterial titer in the untreated control units. results/finding: bacteria in pcs were inactivated in a dose-dependent manner by treatment using the theraflex uv-platelets system. mean log reduction factors ranged from to for enterobacter cloacae ( . . , pei-b-p- ), pseudomonas fluorescens ( . . , pei-b-p- ), staphylococcus aureus ( . . , pei-b-p- ), and streptococcus bovis ( . . , pei-b-p- ). pcs (n for each species) spiked with these different bacteria species were efficiently sterilized ( out of ). treated pcs remained sterile during storage for days, while bacteria in non-treated pcs grew to high titers of - cfu/ml. the theraflex uv-platelets system efficiently inactivates a broad range of different bacteria species, including the who reference strains. sterility is maintained over a storage period of days. these results suggest that the uvc-based pathogen inactivation technology will significantly improve the bacterial safety of platelet transfusions. transfusion transmissible infections among blood donors and strategy on direct laboratory testing cost of blood screening at national blood bank center, addis ababa, ethiopia abraham zewoldie*. national blood bank service background/case studies: blood and its components are life saving; however, they are also associated with life threatening hazards such as transfusion transmitted infections (ttis). hepatitis b virus (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv) and syphilis are the most serious infections transmitted during blood transfusion. serious of blood shortages especially in developing countries and reliance on unsafe family replacement or paid donors also contribute to an increased risk of ttis. knowing the current prevalence of ttis among blood donors will be crucial in donor program strategy development and cost effective alternative strategies of blood screening are highly required especially in resource limited setup. study design/method: a retrospective analysis of blood donors' record covering the period from july , to july , was conducted. the data was collected from the nation al blood bank (nbb) center donor data base. in addition, direct laboratory costs of parallel versus sequential strategy of blood screening were compared using the current price of the laboratory costs. data was first exported to spss version software for analysis. data analysis was performed using scores and odds ratio using same software to look for an association between dependent and independent variables. p values less than . were considered significant. results/finding: a total of , consecutive blood donors were screened between and . the overall seroprevalence rate of hbv, hiv, hcv and syphilis of blood donors was . %, . %, . % and . % respectively. the hiv-hbv co-infection was higher among blood donors ( . %) followed by hbv-hcv co-infection whish accounts about ( . %). significantly increased sero-prevalence of ttis was observed in among family replacement donors, factory workers, daily labors and the age group of - . in this study the difference in cost between the current in use strategy (parallel) versus the newly proposed designed sequential testing algorism was , . ethiopian birr. conclusion: a significant percentage of the blood donors harbor ttis. the nbb center should work on voluntary blood donor mobilization and develop culture of voluntarism. the direct laboratory cost analysis using current in use strategy (parallel) was higher than the newly designed sequential testing algorithm. thus, the new strategy can be implemented to make screening of ttis cost effective in nbb center. transfusion transmitted malaria in a month old infant patricia davenport* , geeta paranjape and laurie sutor , . carter bloodcare, ut southwestern medical center background/case studies: in at a large pediatric hospital, a month old infant was supported for days by extracorporeal membrane oxygenation (ecmo). over this time blood products were transfused. about days after end of ecmo support, a routine blood smear examination revealed inclusions in some of the patient's red cells. the patient had also been having intermittent fever. malaria was confirmed by pcr as plasmodium ovale (p. ovale). because the patient had no other risks, the infection was suspected to be transfusion related and was reported to our blood center which had supplied all transfused products. study design/method: the investigation began by focusing on donors of red cell products, since the chance of an apheresis platelet product transmitting malaria is relatively small, and that of a frozen product is remote. we identified donors of red cell products. each donor was contacted and was asked four questions. additional questions were asked for clarification if needed. based on donor response, risk for active malaria infection was assessed. we also considered areas where p. ovale is, or is not found. donors identified as having possible risk were tested for antibodies and parasitic dna. results/finding: the five donors who had been ill all had common cold or bronchitis like symptoms. donors who traveled went only to non-risk areas. three donors were former residents of another country and may have risk because they lived in malaria endemic countries since birth and came to the u.s. as adults. it was discovered that one of these three did not meet all donor criteria. the donor had failed to disclose that he had not completed years stay in the u.s. after emigrating from cameroon, an area endemic for p. ovale. he had not travelled anywhere after coming to the united states in october and answered "no" to travel. antibody tests on this donor were positive for p. ovale and p. falciparum, but pcr tests were negative. another possible at-risk donor, a former resident of iran was tested and was pcr and antibody negative. the third donor has not yet been tested but the country of residence does not have p. ovale malaria. conclusion: while it could not be definitively proven that the donor with antibodies to p. ovale had active malaria at the time of donation, the donor was indefinitely deferred and referred to an outside physician for treatment. transfusion-transmitted babesiosis outside an endemic area: a case report german felix leparc*. oneblood background/case studies: an y.o. male patient was admitted to the emergency room for severe acute gastrointestinal bleeding, caused by an arterio-venous malformation later located in the proximal jejunum that was clipped endoscopically. during this admission, he received a total of units of red blood cells. approximately weeks later, he was re-admitted due to another episode of gi bleed manifested by melena. as part of his routine evaluation, a cbc was performed in which a blood smear revealed the presence of intraerythrocytic parasites consistent with babesia sp. study design/method: upon notification of a suspected case of transfusion-transmitted babesiosis, lookback of all donors involved in prior transfusion event was initiated. results/finding: to confirm the presumptive diagnosis of babesiosis, pcr was performed and babesia microti dna was detected. an evaluation of the patient's risk factors revealed that prior to the gi bleed episode for which he received transfusions, eight months earlier he was also transfused during open heart surgery. no travel history to the us midwest, and while he travelled to new england two years ago he did not spend time outdoors. he was splenectomized in his mid 's. donor lookback identified a donor who lived in new london county, connecticut but spent the winter season in central florida, where the blood donation (double rbc collected by apheresis) took place. he had never been diagnosed with babesiosis, but participated regularly in outdoor activities in connecticut that put him at risk for tick bites (although he never noticed being bitten or showing signs of it). upon testing, he was found to be negative for b. microti on pcr as well as igm antibodies, but had igg antibody titers of : . the recipient of the other rbc unit collected in the same donation was deceased within hours of transfusion, so no follow up could be performed. during phone interviews, none of the remaining donors had risk factors for babesiosis, and all but four were tested and found serologically negative. conclusion: while transmission of babesiosis through the zoonotic route is confined to regions were the appropriate hosts and vector coexist, people from areas where it is endemic may establish temporary residency and donate blood in non-endemic locations facilitating transmission through transfusion as illustrated in this case. once licensed assays for babesia microti become available, testing schemes will have to be formulated through policies that take this issue into consideration. transfusion-transmitted stenotrophomonas maltophilia from a red cell unit: a case report ashley c gamayo* , andrea j linscott and donny dumani . background/case studies: transfusion-transmitted bacterial infections (ttbi) are rare, but serious complications of blood product transfusions. from - , % of transfusion-associated fatalities reported to the fda were attributed to bacterial contamination. red cell units are rarely implicated in severe and fatal ttbi. when present, contaminants are often gram-negative rod (gnr) bacteria with psychrophilic properties. we present a case of a sickle cell patient who developed definitive sepsis after receiving a red cell unit contaminated with stenotrophomonas maltophilia (s. maltophilia). study design/methods: a -year-old female with sickle cell disease was admitted to the hospital for possible pain crises. pre-transfusion blood and urine cultures collected on day of hospitalization showed no growth after five days. on day , the patient required a blood transfusion for which she was issued a cmv-safe, irradiated, hbs-negative, crossmatched, o-negative red cell unit. the ml unit had been aliquoted via sterile connecting device days prior for a pediatric patient. all ml of the pediatric aliquot were transfused without adverse effects. the patient's pretransfusion temperature was . c. within minutes of starting the transfusion, the patient's temperature increased to . c and subsequently reached a maximum of . c. the transfusion was stopped and the blood bank notified immediately. gram stain of the remainder of the transfused component revealed gnr bacteria. blood was collected from the patient for culture and antibiotic treatment initiated. results/findings: initial transfusion reaction work-up revealed no evidence of clerical errors with negative post-transfusion antibody screen and direct antiglobulin test. blood cultures from both the patient post-transfusion and the implicated red cell unit grew gnr bacteria identified as s. maltophilia. further microbial testing revealed the cultured pathogen was able to proliferate at - c; a finding not characteristically observed in s. maltophilia. conclusion: this is the first definitive case of ttbi with s. maltophilia. this bacterium is a globally emerging gnr that is widely spread in the environment, causing both community-acquired and nosocomial infections in immunocompromised and debilitated patients. contamination was unlikely due to an asymptomatic donor. there was laboratory evidence of the pathogen in both the transfusion recipient and the transfused component. the patient was not infected with the pathogen prior to transfusion, and no other potential exposures could be identified. the patient recovered following appropriate antibiotic treatment, but endured prolonged hospitalization. the transfusion reaction was classified as definitive, severe tti of definite imputability. validation of commercial immunoassays for detecting hbsag and hiv antibodies in production pools karen leighton, izekial butler and scott jones*. qualtex laboratories background/case studies: plasma fractionators test plasma production pools for hbsag and hiv antibodies as a qualitative limit test for the control of impurities, to safeguard against errors in donation testing or pooling. the european medicines agency (ema) has published guidelines for the validation of immunoassays for the detection of hbsag and hiv antibodies in production pools. the aim was to validate commercial immunoassays for the testing of production pools for hbsag and hiv antibodies utilizing the ema guidelines. study design/method: a lower calculated cutoff value for the abbott prism hbsag and hiv o plus assays was determined by calculating the mean signal-to-cutoff ratio (s/co) plus standard deviations of four different types of plasma production pool samples. the calculated cutoff values were utilized for the rest of the validation. the detection limit was determined by testing in triplicate, serial dilutions of who hbsag and hiv antibody standards diluted in plasma. a normalized detection limit was calculated for the hbsag assay using production pools containing low, typical and high anti-hbsag titers. intra-assay variability was determined by testing a minimum of determinations of a low positive control in run. inter-assay variability was determined by testing at least representative negative production pool samples, at least low positive sample (about s/co) and a titration series of who standard spiked into plasma production samples. runs were performed on six separate days using two different instruments and two different lots of assay reagents. results/finding: the lower calculated cutoff values for the hbsag and anti-hiv assays were both below the manufacturer cutoffs of . and were . and . respectively. the hbsag assay detection limit was . iu/ ml for source plasma and . iu/ml for recovered plasma samples. the normalized detection limit study demonstrated that one and a half hours was the maximum amount of time the pool samples could sit at - c where all samples were still reactive for hbsag. the anti-hiv lowest positive dilution for all replicates varied between : , to : , , depending on subtype and group. the % cv of the s/co values of the replicates of the intra-assay variability validation were less than % for both assays. the %cv of the s/co values of the panel of samples of the inter-assay variability validation were less than %. conclusion: a lower calculated cutoff value could be determined for commercially available immunoassays for hbsag and anti-hiv. these immunoassays could meet all of the recommendations in ema validation guidelines. the abbott prism hbsag and hiv o plus assays can be utilized to test production pool samples. was performed on donors ( - days after the index donation) - donors in the follow up study and tested by the doh. no donors tested by the doh participated in the follow up study. follow up testing was negative for all donors. denv antibodies were negative in donations and equivocal in . our initial reactive rate is higher than that reported to date for the procleix zikv tma of per , [p. williamson, et al transfusion, in press] . conclusion: universal testing under ind was successfully implemented and incorporated into blood center operations. we have noted an initial reactive other demographics that should be analyzed for their potential to be used to predict cmv seroconversion rate include gender, age, race, ethnicity or a combination of these. background/case studies : growing the geographic footprint has been a priority for the organization since . over a four year period, the organization doubled the number of blood centers, with continued growth expected. with the current challenges in the blood industry, the audit program needed to be flexible, maximizing efficiency and capacity utilization, and without increasing compliance risk. the internal audit function was centralized in late , for which the program consisted of types of audits, an operational compliance audit and a support systems compliance audit. each type was performed twice per year at each main center. this model was no longer serving the changing organization. study design/methods: lean six sigma concepts were applied to this project. survey results and brainstorming aided in capturing the strengths of the current program, opportunities for improvement, and ideas for a redesigned program. this information was the primary input to the swot analysis (strengths, weaknesses, opportunities, and threats) for the purpose of understanding performance of the current program, as well as elements that could impact the future design. potential solutions were placed into a pugh matrix, which was used to facilitate a disciplined, team-based process for concept generation and selection. each potential solution was compared to criteria for evaluation and selection of the best solution. results/findings: the program was re-designed to perform internal audits annually as a single, team-based comprehensive audit. remote auditing was incorporated to require less on-site time, less disruption, improved auditor work/life balance, and cost savings. a formula was created to determine on-site audit time that included adjustable risk factors. the audit reporting process was also automated for simplification, efficiency, and to meet stakeholder needs. the team-based approach leverages auditor strengths, fosters a learning environment, and increases detectability of organization-wide concerns. conclusion: the comprehensive team-based approach, and other program improvements, has been effective in responding to organizational growth without sacrificing quality or increasing compliance risk. external inspection performance has achieved record performance levels the past year. diversity of auditor skills led to a stronger skill presence, which was consistently applied across system. auditing is more efficient and effective. stronger collaboration among audit team members provided stronger objectivity, fairness, and consistency across the system. auditors and auditees have increased in knowledge, and the internal quality audit program has improved. background/case studies: in many places, blood banking is using semiautomated systems to perform fractioning in different blood components (red blood cells, platelets and plasma). banc de sang i teixits (bst), adopted the fully automated reveos system (terumo bct inc, lakewood, co) few years ago to manufacture blood components. in june , bst started a validation of new blood bags manufactured by terumo bct with different variables on platelet volume after processing and a kit to perform platelets pools with a new filter. study design/method: to perform this validation, blood donations were used under different conditions (see table below ). the current filter evaluated for the platelet pool (lrf-xl, haemonetics corporation) was compared to a new filter (terumo bct inc.). the new blood bags were manufactured using a new vinyl supplier. a portion of these processed blood components (red blood cells, platelets and plasma) was used for different quality control (qc) tests (routine qc performed at bst following european directorate for quality of medicines & healthcare; cytokine analysis, such as p-selectin and platelets recovery through the filter). results/finding: the results are very similar between both bags, current and new one, as well as filters. all the analysis done to evaluate the quality of the blood components were similar in all conditions. also, it was shown a better performance on platelets pools, when they came from bags centrifuged with ml of plasma, vs. ml of plasma and additive solution. conclusion: these new bags and filter have shown a similar behavior when using them for manufacturing blood donations with reveos system in our blood bank. regarding the new platelets pooling kits, a better manipulation by the operator was observed; although the tubing is shorter and it meant being more difficult when manipulating the pools. no issues should be found if they are implemented in routine use. it's planned to start this implementation during this year, ; so then there will be larger results in order to have a proper procedure qualification. conclusion: patients requiring rare blood products are rare, and those lacking high prevalence antigens are the most challenging for whom to obtain antigen negative blood. it is clear that some requests for exquisitely rare types are not able to be filled with current donors. molecular testing of large numbers of donors has likely helped to identify more rare donors in recent years. it is recognized that commercial platforms do not include many of these making these rare types even more challenging to find. consideration should be given to testing more donors of all ethnicities to identify more rare donors. recommendation # : updating donor educational material to provide more comprehensive information on risks of iron deficiency and recommendations on iron supplementation. updating our educational materials will likely have a minor impact. recommendation # : implementing strategies such as iron supplementation, ferritin testing or increasing interdonation intervals for all donors or those groups most at risk for iron deficiency. initial implementation would likely be either iron supplementation or ferritin testing for at risk groups only and implementation of either one of these strategies would potentially affect over , donors. the recommendation to limit the number of donations would have a substantial impact. for this analysis, the focus was on - year olds and premenopausal women (ages - ) donors. on average, - year olds donate . times a year and premenopausal women donate . times a year. if both of these groups were limited to donating once a year, a total of , donations from - year olds and , donations from premenopausal donors would not be collected. conclusion: after analyzing the impact of the aabb association bulletin # - , the bulletin will have a significant impact on both donors and our local blood supply. more than half of donors would receive either ferritin testing or iron supplementation. if the only measure employed is limiting the number of times a donor could donate for - year olds and premenopausal women, this recommendation would have a substantial impact on our ability to provide blood products to local hospitals. background/case studies: transfusion medicine knowledge deficits are apparent among medical students, residents and practicing physicians. these deficiencies may be due to the frequency and type of education. the majority of medical students in the united states receive four or fewer hours of transfusion medicine education. the transfusion medicine academic award group published educational content guidelines for medical school, residency and fellowships. however, the frequency and educational methods remain poorly evaluated and with little guidance. we investigated the effects of different educational techniques on transfusion medicine knowledge acquisition in novice learners. study design/method: three educational pathways were developed to teach principles of transfusion medicine while allowing learners to recognize problems and develop solutions for transfusion medicine complications. the simulation group received all educational activities within a . hour inperson, high-fidelity live session. the hybrid group received some educational component online and also attended an in-person high-fidelity simulation session. the online only group received all educational materials online, including a pre-recorded-video simulation session. the learners were second year medical students enrolled at one institution. the same faculty members taught all live sessions and developed all online materials ensuring the content was the same. a pre-and post-test was created to address blood groups, blood donation, blood testing, blood component indications and transfusion complications. the educational session was evaluated by the likert scale survey which ranges from zero (poor/unsatisfactory) to five (outstanding). results/finding: % ( / ) of the simulation group students improved their post-test scores and had an average likert scale rating of . (very good). % ( / ) of hybrid group students improved their post-test scores and had an average likert scale rating of . (very good). % ( / ) of online only students improved their post-test scores and had an average likert scale rating of . (good). the average changes in scores were statistically significant within all training groups (p value < . ). additionally, the simulation group had a larger increase in average post-test scores when compared to the online only group (p< . ) and the hybrid group (p< . ). conclusion: our study demonstrated that a faculty taught high-fidelity transfusion medicine simulation curriculum consisting of an in-person didactic session and simulation session for second year medical students produces greater knowledge acquisition compared to an online only or hybrid curriculum. the high-fidelity simulation curriculum is also preferred over the online only education as indicated by the likert survey results. aaron j wyble*, yeon mi kim and barbara j bryant. university of texas medical branch background/case studies: diagnostic management teams (dmts) are an innovative way to bridge the communication gap between the laboratory and clinical services thereby facilitating the delivery of improved patient care. dmts employ a multidisciplinary approach which integrates clinical and laboratory data into succinct interpretations and recommendations. the interpretations must be of moderate to high complexity in order to be clinically valuable. recommendations are made regarding future testing, timing of testing prior to blood component needs, and other pertinent concerns to allow for improved coordination of patient care. the timeliness of the dmt reporting is vital to patient management. the inherent design of a dmt also provides an educational opportunity for trainees at academic centers. study design/method: the transfusion medicine service at a large university-based academic medical center implemented a dmt in . all cases involving complex antibody identification workups, transfusion reactions, deviations from standard operating procedures, consultations for blood component utilization, and massive transfusion protocols from july through january were evaluated by transfusion medicine residents. the electronic medical record (emr) of each patient was also reviewed to determine relevant clinical history. all significant findings were presented at the transfusion medicine dmt conferences. the dmt was comprised of physicians from transfusion medicine, hematology/oncology, anesthesiology, transfusion service technical staff as well as visiting clinical staff from surgery, obstetrics and gynecology, transplant services, and pediatrics. the dmt integrated the clinical and laboratory data to formulate relevant interpretations and recommendations. the final dmt reports were placed into the emr for access by health care providers. financial benefits of a transfusion medicine dmt were also evaluated. results/finding: in a -month period, cases of complex antibody identification workups ( %), transfusion reactions ( %), consultations for blood component utilization ( %), and deviations from standard operating procedures and massive transfusion protocols ( %) were presented at the transfusion medicine dmt conferences. the placement of dmt narratives in the emr as progress notes and laboratory reports provided informative and timely communications. residents participating in dmts demonstrated improved clinical and laboratory correlation skills. as a result, resident competency in transfusion medicine was enhanced. over $ , of revenue was generated utilizing the standard professional component cpt codes. conclusion: dmts encompassing multiple aspects of transfusion medicine improved patient care through enhanced communication between laboratory and clinical services. additional benefits of a dmt program include resident, clinician, and technical staff education and the generation of revenue for the institution. streamlining a blood center and hospital transfusion service supply-chain with an informatics vendor-managed inventory solution hamilton c. tsang* , david lancaster , dianne geary , robert scott , anh thu nguyen , adam garcia , raina shankar , leslie buchanan and tho pham . stanford health care, stanford blood center background/case studies: inventory management is both a major challenge and an integral part of hospital transfusion service (hts) and blood centers (bc) operations. the current process at our institution involves twice-per-day shipments from the bc to the hts, with each shipment predicated upon current stock levels at hts. manually obtaining inventory levels for each product is time-consuming. the manual determination is also errorprone. we aim to enhance inventory management operations by developing an informatics solution to ( ) streamline the ordering process to accurately reflect inventory status and transfusion practices and ( ) re-allocate valuable hts tech time. study design/method: at our hts, the general inventory accounts for over product categories broken down by component, blood type, irradiated status, and cmv-serology status. we therefore sought to establish an electronic method to reliably infer the general inventory level. since the raw electronic inventory report comprised both the general inventory and physically sequestered units (e.g. special antigen units, cross-matched units), over a -month calibration period we performed linear regression between electronic and the gold-standard manual count to impute from the electronic census the number of units of each product category in the general inventory. once we had a reliable electronic method to determine inventory levels, we implemented a -month pilot period. we analyzed various metrics pre and post pilot implementation to ensure non-inferiority of our electronic system: ( ) the ratio of units transfused per week to the number stocked (t:s), ( ) the number of products ordered as stat, and ( ) the number of expired products. we created in-house programs on visual basic for applications (microsoft, redmond, wa) for both the calibration and pilot periods. lines of code were written for both programs, including class modules and distinct subroutines. results/finding: during the pilot period, we investigated our system's noninferiority. the average weekly t:s ratio for cryoprecipitate, plasma, and rbc, respectively, were . , . , and . before the pilot period compared with . , . , and . during the pilot period. these differences did not reach statistical significance (p . ). we also monitored the number of stat ordered products before and during the pilot period, which were and stat units per week, respectively (p . ). lastly, we also monitored the number of monthly wasted products due to expiration as an indicator of inventory mismanagement before and during the pilot period, which were and units, respectively (p . ). an estimated hours per week of technologist time was reallocated to other tasks once the electronic census was adopted. this translates to . fte and $ , per year saved from labor costs per year if permanently adopted. conclusion: we created an in-house electronic ordering system to enhance information fidelity, re-allocate technologist time, and further standardize ordering. our system showed non-inferiority to the labor-intensive manual system, by not changing the number of stat orders, having the same t:s ratio, and not increasing the number of expired products. this is achieved while freeing up over hours of staff time per year. future directions include full automation with involvement from hts informatics department. transfusion practice improvement: gaining traction through the use of a provincial transfusion quality improvement plan denise evanovitch* , yulia lin , troy thompson , allison collins and sheena scheuermann . ontario regional blood coordinating network, sunnybrook health sciences centre background/case studies: a provincial regional blood coordinating network (prbcn) held a "quality focus day" (qfd) in to explore transfusion quality indicators to be included in a province wide quality improvement plan (qip). the plan's main goal is to reduce patient harm by improving transfusion practice in hospitals through the reduction of inappropriate use. the following recommendations were made: select a blood component that most hospitals could monitor display progress in a public forum so that hospitals could compare themselves to peers strike a province-wide transfusion qip committee to guide the development of the plan, supporting resources and ongoing improvement initiatives study design/method: a provincial transfusion quality improvement plan (ptqip) committee was formed and included broad representation: the provincial patient blood management coordinators, physicians, technologists, nurses, administrators, clinicians, quality/risk managers from all regions of the province and the provincial blood advisory committee, the blood supplier and a patient. there was further collaboration with other organizations such as the provincial health quality division, choosing wisely after the launch, an informal survey indicated that of the province's hospitals were interested or had already adopted portions of the ptqip. to further assist hospitals in advancing their qips, a technologist prospective screening educational module was developed in addition to an electronic tracking tool with which hospitals can enter their baseline data and subsequent audit data and track their success. both hospital and provincial reports can be generated from the tracking tool. a more formal survey conducted in indicated that % plan to implement or already have implemented the ptqip and % of the respondents already have put prospective order screening by technologists in place. conclusion: helping hospitals through the development of standardized templates, instructions, education and other tools for transfusion quality improvement increases the ability of hospitals to uptake quality improvement initiatives. taking a standardized approach across the province allows for both aggregate and hospital data comparison analyses. background/case studies: military and civilian trauma-based studies have demonstrated the advantages of transfusing blood products prior to a patient's hospital arrival, a process known as pre-hospital transfusion (pht). helicopter emergency medical services (hems) worldwide have implemented this protocol with great success, despite a current lack of guidance or advisory publications. there is a need for literature that addresses the regulatory requirements and logistical challenges associated with developing a pht program. herein, we report our experience as a large hospital system embarking on the development of a multi-state pht service. study design/method: in october a work group was formed to establish pht services for the hems providing care to over thirty regional hospitals. composed of flight care staff, emergency physicians and transfusion medicine specialists, the group identified the major tasks to be addressed: federal/state regulations; inventory structure/management; product storage/testing; tracking/traceability; emergency release protocol; and staff training. while there are no specific regulations governing pht, the regulations pertaining to blood product storage, validation, and monitoring apply. the fda, aabb, and state agencies were each consulted to ensure compliance with all directives. results/finding: the largest hospital within this system, already acting as a reference site, was designated to perform all confirmatory testing on products supplied to the multi-state hems. similarly, this hospital was tasked with remote monitoring of all blood refrigerators at the helipad sites. the system's fda licensed blood supplier was deemed responsible for product consignment and transport between the four hems sites. the blood inventory at each site was designed to contain: group o positive rbcs, group a low anti-b titer liquid plasma, and four-factor prothrombin complex concentrate. a military-tested in-flight medical record system will be used to transfer transfusion information to non-affiliated hospitals as needed. validated inflight coolers, protocol for product emergency release, inventory tracking system, and re-stocking schedule were also requisite to this plan. staff competencies regarding emergency release guidelines, transfusion reactions, and the handling/storage of products are maintained by the hems medical director with additional oversight provided by transfusion medicine physicians. conclusion: our work group successfully identified the challenges associated with a multi-state pht helicopter based service, which spans blood product management, adaptation of existing transfusion procedures and operating policies, licensing requirements, and personnel training. our pht service will go live in . publishing this experience may benefit future sites as they launch similar pht initiatives. blood transfusion during humanitarian emergencies yetmgeta e. abdella* , rana hajjeh and cees th. smit sibinga . world health organization regional office for the eastern mediterranean, international quality management (iqm) consulting background/case studies: more than million people are affected by humanitarian emergencies in the eastern mediterranean region of the world health organization (who), where some of the most affected countries in the world are located. in these countries, the health systems have been weakened or destroyed and health workers provide health services under difficult circumstances. humanitarian emergencies increase the demand for blood transfusion and make its delivery challenging and complex. despite these obvious needs, across the region, there is a lack of information on the emergency preparedness and response capacity of blood transfusion and on the challenges countries and health responder's face in meeting the needs of the patients during emergencies. study design/method: we searched pubmed and index medicus for the who eastern mediterranean region for data on availability and safety of blood transfusion in humanitarian emergencies. we conducted a structured survey of blood transfusion services (bts) in all countries in the region to identify the following: type of humanitarian emergencies between and ; current strategies to ensure availability and safety of blood transfusion during emergencies; coordination and collaboration between countries; and gaps and challenges. additional information was collected during a regional consultation (eastern mediterranean region) held in may in tunisia. results/finding: we found publications on disaster from five countries in the region and publications on disaster preparedness and blood transfusion in casualties and severe trauma outside the region. however, none dealt with the questions of availability and safety of blood transfusion during emergencies. twelve countries ( . %) responded to the survey. armed conflicts and terrorism are the commonest types of emergencies with estimated - % of the injured requiring blood transfusion. nine countries have emergency preparedness and response plans for bts. potential blood donors are mobilized through public calls, besides a direct appeal on regular and replacement donors. seven of the responding countries keep an emergency blood stock. collaboration between the different stakeholders exists in seven countries. lack of adequate and competent human resource, transport and cold chain deficits, shortages in supply of consumables and maintenance of equipment, lack of reliable power supply, and shortage in finances are the gaps identified. conclusion: there is a need to integrate bts in the overall national emergency preparedness and response, collect and disseminate updated information on factors affecting provision of blood transfusion in humanitarian emergencies, provide technical and financial assistance to affected countries, strengthen mechanisms for coordination and collaboration among different parties, and develop a regional emergency blood services system and management expertise. ( , , and for - ) . the number of collections per registered trt donor varied significantly, ranging from to therapeutic draws/donor per year. excluding those that didn't present for a therapeutic blood collection, the average number of trt collections/donor per year decreased from . to . between and . conclusion: our blood center has experienced an increasing number of therapeutic phlebotomies, as well as individuals on trt referred for therapeutic phlebotomy due to elevated hemoglobin values from through . it is not clear from information provided by the ordering physician whether this is intended as a temporary measure to decrease the hemoglobin while the patient is on trt, or whether the dose was being adjusted or discontinued due to the known risk factor of cardiovascular disease in patients with polycythemia; however, the average number of donations per trt donor decreased during this timeframe. the percentage of men on testosterone who present as regular blood donors at our blood center is not known, since this hormone is not reason for deferral. our findings raise the concern, however, that regular phlebotomy is necessary to reduce the risk of testosterone-associated polycythemia in this population. as it is our duty to provide a safe and adequate blood supply, our blood center also has concerns about perpetuating the misperception that repeat phlebotomy, particularly if required more frequently than days, is sufficient to mitigate the risks of testosterone therapy. hence, we have made the decision to discontinue offering phlebotomy services to this population of donors other than for those on testosterone that meet all donor eligibility requirements. approaches involving the use of a vein illumination device in a blood donor center sara matheson*, kimberly j duffy, audrey e traun, mary m benike, james r stubbs and justin d kreuter. mayo clinic background/case studies: venipuncture is a critical step in blood collection and locating a suitable vein for this procedure can be a challenge. unacceptable vein selection or incorrect needle placement can lead to incomplete collection or infiltration. in a blood donor center, the primary selection of a vein is done by palpation within the antecubital area. prior to needle insertion, the skin at the site must be prepared and contact avoided until after needle is placed. vein illuminator (vi) devices are available to aid in visual display of potentially suitable veins. such a device was made available to staff in march of . after an initial testing and instructional period, the vi has since not been used by staff. the objective of this study is to discover reasons why staff does not use the vi to identify potentially suitable veins. study design/method: a staff survey was developed and distributed to staff in march to inquire about usage of the vi and obtain feedback about the device. at the time that the survey was sent, the device had been available for several years. the survey included questions involving frequency of use, adequacy of training, comfort with using the device, knowledge of the device's storage location, willingness try the device, and general feedback. results/finding: the survey had a % response rate (n ). of these, . % have never or very rarely utilized the vi. self-reported reasons for low utilization focused on two dominant themes. first, that the device is not needed and second that it doesn't accurately show veins. . % of respondents are aware of where the vi is stored and a more accessible location to share the device was not identified. although . % of respondents have been provided training on using the vi, the group was mixed regarding their comfort level in using the device independently. only % of the group was willing to try vi. conclusion: infiltration and incomplete collection account for approximate % ( units/year) of qualified blood donors, yet vi does not appear to be a viable solution for our blood donor program. there seems to be both an opportunity and challenge with vi implementation. the opportunity is to create critical awareness of problems with vein cannulation. the challenge is to identify a device that is more effective at visualizing deeper veins necessary for blood donation. benefits of converting from mcs to alyx penny schroeder* and elizabeth parker. indiana blood center background/case studies: in , apheresis red cells (arc) represented . % of total red cell collections at our center. hae mcs ln was utilized to collect arc. due to the age of the instruments, challenges with collections on mobiles as well as the need to increase collection of right type products, the decision was made to change technologies. study design/method: fresenius kabi demonstrated the fenwal alyx technology as well as the business case to the primary stakeholders. all implicated departments were involved in the initial impact assessment. a multidepartment kick off meeting was held and project team formed. due to product demands, the decision was made to validate arc and plasma apheresis. the primary departments affected were blood collection and production. fresenius kabi provided sample validation plans, sops, training and training materials for use. four mobile-carts were purchased for easy transportation of alyx and quick-connect feet for installation on mobile buses. the lead trainer and the bc technical administrator traveled to an affiliate blood center to observe their alyx program and identify best practices. a team of blood collection trainers and preceptors were the initial group trained and validation performed. this team also served as the subject matter experts and field preceptors. fresenius kabi returned for advanced alyx operator training. the training plan targeted previous mcs operators first and then operators new to apheresis with a training goal of % of mobile staff. validation of the alyx began / / and took approximately days to complete. during this time, fresenius kabi conducted alyx education and apheresis recruitment training to all collection and recruitment staff. the mcs machines were removed from service / / . alyx go-live occurred / / . additional operator training continued through september . results/finding: due to ease of mobility and use of alyx, reduced procedure time compared to mcs and donor conversion training we increase components collected. alyx disposable kit includes pre-attached solution containers reducing ancillary items required to pack and carry to mobiles. this decreased kit cost by $ . each providing an estimated annual savings of $ , . conclusion: with the multiple alyx donation types we were able to increase our collection of right type procedures by approximately . % and decrease our kit costs by %. with alyx the collection plasma on mobile blood drives is now possible. due to ease of use, operators have embraced this technology and we have consistently met our monthly collection goals from october -march . background/case studies: high frequency of donation is a risk factor for iron deficiency. because females' iron stores are generally lower than males' before they start their donation career, females who donate frequently are particularly high risk. minimum hemoglobin (hb) has long been the same for males and females at g/l, but for males this falls below the normal limit. as a first step to mitigate iron deficiency, criteria for whole blood donors were modified for males (minimum hb increased to ! g/l) and for females (minimum interdonation interval increased from to days). the longer interdonation interval in females was gradually implemented, starting with donor messaging in october , changes in rebooking of donation appointments in december and culminating with eprogesa criteria changes on march , . both these changes are expected to initially result in donation loss, but may be partly counteracted by a decrease in hb deferral rates in female donors. we aimed to assess the impact of these changes on hb deferral rates. study design/method: percentages of hb deferrals were calculated as the number of donation attempts that resulted in hb deferral divided by the number of successful donations plus hb deferrals multiplied by . percentages were calculated for male and female donors before and after changes were made. results/finding: the percentage of hb deferrals increased in male donors from . % in the weeks pre-implementation to . % in the weeks post-implementation of the change in the hb criterion. hb deferral rates for female donors were . % in september, . % in october/november, and . % from december to march, . conclusion: hb deferral was more frequent in male donors after the minimum hb was increased to g/l. the gradual implementation of increased interdonation interval for females resulted in a reduction in deferrals, thus the initial donation loss associated with this change may be partly offset over time by decreased hb deferrals. a longer observation period is necessary to confirm these findings and assess impact on phenotyped blood and donor retention. in the past years, , blood products, derived from , procedures, were distributed to different investigators in over laboratories. whole blood was the most common product ( . %), followed by unmanipulated mononuclear cell collections ( . %), and elutriated monocytes or lymphocytes ( . %). less common requests included platelets ( . %), plasma ( . %) and granulocytes ( . %). adverse donor reactions were infrequent ( . % of procedures). conclusion: we report the feasibility of a program for collecting and distributing blood for investigators to obtain blood components for in vitro research use, utilizing the staff and resources of a hospital-based blood bank. research blood donation is essential to support laboratory research and to maintain positive relationships with donors who have been deferred from allogeneic transfusion. hospital-based blood donor center's experience with implementing platelet pathogen reduction system kimberly j duffy*, mary m benike, james r stubbs and justin d kreuter. background/case studies: the safety of platelet products has been continually improving due to testing despite the continued emergence of microbial threats. the recent fda approval of platelet pathogen reduction technology will protect transfusion recipients regardless of the new microbial dangers. in order for platelet products to use the pathogen reduction technology, the volume, platelet yield (dose), and concentration must be collected within tight specifications. the objective of this study was to determine the optimal collection settings to enable % collection of pathogen reduced platelets while limiting the loss of products. study design/methods: the collection instrument evaluated for this study has fda approval for platelets suspended in % plasma. the corresponding pathogen reduction system used for the study has kits with different collection specifications. all apheresis collections occurred at a fixed site and pre-platelet counts were performed on a hematology analyzer. the yield scale factor has been established for correlation between the hematology analyzer and apheresis collection device. in order to determine the optimal collection targets, the apheresis collection instrument had a variety of multiple yields and volumes established for collections. staff was instructed to collect the highest available yield per donor. after collection, volume, platelet yield, and concentration data was obtained. this data was used to determine if the product met the specifications for one of the available kits, and if the actual platelet yield was higher than . x , thus meeting the criteria for a double product. results/findings: a higher platelet concentration product is ideal to produce a double product, but targeting products with a platelet concentration greater than x /ml was more likely to be outside the specification of the pathogen reduction kit. the platelet concentration target of x / ml results in discarding products and was quickly removed from instrument settings. collections with a platelet yield as low as of . x and platelet concentration of x /ml were more likely to produce a product that was not within the specification of the pathogen reduction kit. abstract conclusion: the loss of both triple platelet products and lowered postprocessing platelet recovery requires the collection of platelets to be far more precise. the goal of platelet collection has shifted from simply maximizing each platelet collection to an approach that considers optimal collection within the limits of kit specifications. final collection instrument configurations are platelet yield of . x and . x at the volume of mls and platelet yield of . x , . x , and . x at the volume of mls. moving from subjective to objective donor eligibility screening platforms: a blood center's journey angela dirr* and steve cihura . bonfils blood center, bbc / bsi background/case studies: in , the device used by bonfils blood center to determine donor hemoglobin and donor eligibility was reaching its end of life, and bbc needed to define a path forward for a reliable replacement device. study design/method: bbc evaluated devices with the following criteria in mind: ) device disposable costs, ) reagents/controls/quality control, ) objective hgb/hct measurement, ) portability and durability for a mobile environment, ) ease of use, ) donor experience, ) battery life, ) validation requirements plans, ) blood center suitability, and ) ability to link to becs. multiple departments including donor care, equipment management and validation, quality, and regulatory affairs were involved in the evaluation and product selection. bbc tested donors per each device at both a fixed and a mobile site. bbc also considered donor feedback for the choice of replacement technology. the project started february with a targeted implementation date of july . after creating necessary sops and adopting existing sops, bbc successfully completed the validation of the devices, and chose the compolab technology from fresenius kabi as the new device for bbc blood bank. results/finding: the compolab was selected as it met project scope and selection criteria. it was important for bbc to reduce paperwork and daily tasks. the compolab eliminates daily qc reducing paperwork, time and improves error management. after converting to the new technology, bbc donor deferral rates increased by approximately %. as a consequence to this increase, bbc conducted reminder training with bbc staff to ensure proper sampling technique and higher sample quality. over time, bbc deferral rates stabilized to . % in and . % in . during this time period, bbc also successfully recruited new blood donors to bbc program, which may have contributed to an increase in deferral rates. in , the deferral rate increased again, probably due in part to the fda final rule "requirements for blood and blood components intended for transfusion or for further manufacturing use", which went into effect in may . conclusion: during the evaluation for new equipment, bbc learned that it is critical to understand the equipment's life cycle and the effect the equipment has on all aspects of the business. after comprehensive evaluation of multiple donor eligibility screening platforms, the compolab device was selected at bbc facility. it met the majority of all aspects of the project scope and qualifying criteria. bbc also learned that continuous refresher training of the staff ensured optimal device performance, and how external factors such as changes to the regulatory environment may impact deferral rates. flowmetry on platelet apheresis. tetsu yamamoto* , ayumi araki , hiromi sanyoshi , hiromi kanai , hiroya kikuchi , katsushi tsukada and kazuhide mure . hokkaido red cross blood center, japanese red cross hokkaido blood center background/case studies: vasovagal reaction (vvr) is known to be the most common adverse reaction to blood collection, but effective measures for preventing vvr have not yet been developed. effective timing of interventions during apheresis donations in particular should hold the key to predicting vvr, but no research has been done on the topic. study design/methods: this study investigated the potential to predict vvr from fluctuations in peripheral blood flow measured by laser doppler flowmetry in platelet apheresis donors, a population highly likely to experience vvr. data were collected from individuals who donated platelets during the -month period between february and august , and data from the donors who experienced vvr were analyzed. to calculate the level for issuing vvr alert, the percent decrease in blood flow (dbf) and the percent decrease in heart rate (dhr) were calculated, the time from alert to vvr was estimated for three dbf levels, and the detection performance of each alert level was calculated. results/findings: eight of the men ( . %) and of the women ( . %) experienced vvr. one donor did not experience vvr during blood collection, but had a delayed reaction while resting afterward. mean maximum dbf in the donors in the vvr group was . . %, which was significantly higher than the . . % in the non-vvr group. at a maximum dbf threshold of %, sensitivity for discriminating between vvr and non-vvr donors was . % and accuracy was . %. when % dbf was used as the alert level, alerts were issued for donors, including in the vvr group. therefore sensitivity for predicting vvr was . % and specificity was . %. mean time from alert to diagnosis in the vvr group was . . minutes, and accuracy of the alert was . %. some of the vvr could not be predicted even the value of maximum dbf exceeded %. the reason was supposed to be the difference of donor susceptibility on dbf. conclusion: we investigated whether vvr in platelet apheresis donors can be prevented by prediction and found that it is possible to predict vvr early enough before onset to intervene by monitoring dbf in real time during blood collection using laser doppler flowmetry. future research must also investigate whether the incidence of vvr can actually be reduced by interventions such as adjusting extracorporeal circulation. the risks of alloimmunization in sickle cell patients using c, e, k negative blood: experience of a hospital apheresis and transfusion service grace banez sese* , , salam abdus and shabrina shah . inova blood donor services, inova fairfax medical campus, inova fairfax medical campus transfusion services background/case studies: red blood cell (rbc) transfusion is often a lifesaving measure for patients with sickle cell disease (scd). it is critical in the management of scd complications such as splenic sequestration, stroke, priapism, iron overload and acute chest syndrome. a wellrecognized complication of chronic transfusion in scd patients is alloimmunization to rbc antigens. to prevent alloimmunization, transfusion with rbcs negative for c, e, and k antigens has been advocated. this has led to reports of reduction in the rate of alloimmunization and a decrease in hemolytic transfusion reactions. we report a summary of our three year experience with the prophylactic transfusion of rbc units negative for c, e, k antigens for scd patients during red blood cell exchange transfusions (rbcx). study design/method: retrospective review of scd patients with a history of stroke, refractory sickle pain crisis and priapism was done. rbcx was performed every to weeks from december to march . blood bank work-up used the mts gel method for antibody screen and identification. our hospital-based donor center proactively works with the hospital blood bank in preparing these units in a timely manner. results/finding: a total of patients, females and males, who underwent a total of rbcx from october to march , using an average number of rbc units per rbcx. rbc units negative for c, e, and k antigens were used during rbcx for patients. two patients positive for c antigen underwent rbcx, using e and k antigen negative rbc units. review of the antibody screen test results performed prior to each of the rce showed that no new clinically significant alloantibodies were formed after exposure to multiple rbc units. conclusion: although there is no consistent standard of care in transfusion practice related to the extent of antigen matching for scd patients, studies suggest that the standard of care for transfusion of all patients with scd is to provide rbc negative for c, e, and k antigens. this ability to find these rare units is also affected by the characteristics of one's institution and blood supplier. it is an advantage to have a hospital based donor center to work with, as we proactively collaborate with them to provide these rare units. the approach by our institution to transfuse rbc units negative for c, e, k or study design/method: venous blood specimens of healthy volunteers were collected before blood donation and after blood donation immediately, day, week, weeks, and weeks among men and weeks among women. immunoglobulin g (igg), immunoglobulin m ( igm) , immunoglobulin a ( iga)and complement component ( c ) , red blood cell (rbc), white blood cell count ( wbc) , hemoglobin (hb), hematocrit (hct), and serum iron (fe) , were measured to monitor he dynamic changes of these biomarkers and blood quality. results/finding: the level of igg slightly decreased after blood donated immediately, iga and c decreased significantly but still within their normal ranges, igm did not change after blood donation. the level of iga significantly decreased at weeks among men and weeks among women, while c significantly increased at the same time period. igg, rbc, hb, hct and fe started to recover week after blood donated and reached their levels before blood donated within weeks among men and weeks among women. conclusion: the biomarkers mutually changed over the course of weeks among men and weeks among women. donating ml blood will not significantly affect overall blood quality. utilizing amicus dxt relay data managment solution to increase platelet split rate and improve amicus productivity janelle wilhelm* and jennifer kaluza. memorial blood centers background/case studies: with the increase in platelet demand and the opportunity to export products we set an initiative to increase the platelet products collected form our existing donor base. we also faced the challenge of managing multiple collection sites in multiple states. the decision was made to implement amicus dxt relay data management solution to provide us insight into procedure details to make data driven decisions. day to day variability previously dipped as low % split forcing reactive planning. study design/method: incorporate dxt to strategically plan our day to day operations. dxt reports were monitored by management and with the fresenius kabi team for productivity by site, phlebotomist and device. reports measured target vs actual yield, donor parameters, and procedure events to perform a donation opportunity analysis. this allowed us to adjust configuration settings when appropriate to improve the accuracy of the yield prediction. reports by phlebotomist were utilized for training on how to optimize the donor's gift to donate an additional platelet or plasma product(s) and increase procedure success rate. results/finding: the monthly dxt report analysis resulted in device configuration improvements, phlebotomist and center manager accountability, effective training, and donation optimization we increased our overall platelet split rate percent and increased concurrent plasma collections by percent. with utilization of the dxt reports we are able to take a proactive approach allowing us to predict product availability, with day to day variability dropping no lower than percent split. phlebotomist qns rates were easily monitored regularly (daily, weekly monthly) resulting in a decrease in our overall qns rate to consistently below percent. conclusion: dxt was easy to implement, is very user friendly and will continue to help improve our platelet collection and process improvements between donor centers. dxt provides invaluable tools for the operational supervisors to monitor their staff and improve productivity at their multiple sites. next step is to develop the plan for implementation of paperless documentation with dxt and healthcare-id. the ability to immediately review data directly from amicus was key in the productivity improvements realized. evaluating the impact of a background/case studies: as blood and blood products are limited and expensive resources, they are prescribed, handled, stored and transfused according to hospital guidelines established to ensure that the best practice standards are maintained for patient safety. it is a prerequisite for all registered nurses (rns) involved in blood and blood product administration to possess fundamental knowledge of transfusion practice. aim: the aim of this study is to evaluate the impact of a hospital-based transfusion practice training program among registered nurses, through administration of a knowledge-based questionnaire before and after implementation of the program. the results gathered would identify gaps in assimilation of knowledge and suggest improvements to the design and implementation of specific content in the nurse-led transfusion training programme. study design/method: all rns from various units and departments were invited to participate in the blood transfusion knowledge questionnaire in october . after which, a formal transfusion practice training programme was introduced, consisting of an online learning platform and in-service training sessions. the same questionnaire was administered to the rns one year later in september for post-training programme evaluation. individual item scores and proportion of nurses with perfect scores was compared pre-and post-implementation. results/finding: in and , a total number , rns and rns completed the questionnaires, giving a response rate of . % and . % respectively. the overall mean score in was . points (range to ). the mean score in was . points (range to ). the percentage of rns having perfect scores of increased from . % in to . % in . table i below shows the results for each question item. the implementation of a hospital-based, nurse-led transfusion practice training programme has led to encouraging improvement in blood transfusion knowledge amongst rns. further training may be needed in the preparation of blood sets and management of fever. background/case studies: clinical use of blood has shown to be the least developed part in the vein-to-vein transfusion chain. this global survey was therefore carried out in order to investigate the level of awareness, accessibility and utilization of e-continuous learning and quality of blood use among blood prescribing clinicians and nurses. study design/methods: a descriptive 'ex-post facto' survey design was used; purposively selected blood prescribing clinicians and nurses from hospitals in countries of the human development index (hdi) groups (low, medium, high, and very high) participated. three research questions were answered, while seven null hypotheses were tested at . level of significance. descriptive statistical tools (frequency counts and percentage) were used to analyze the demographic backgrounds, while inferential statistics -pearson product-moment correlation coefficient (ppmc), analysis of variance (anova), were used to analyse the hypotheses. results/findings: quality of clinical use of blood was positively and significantly correlated with levels of awareness (r . ; p . ; df ) and accessibility (r . ; p . ; df ) to e-continuous learning among blood prescribing clinicians/nurses. there was significant difference in levels of awareness [f( , ) conclusion: today e-continuous learning has become a conditio sine qua non to effective and quality clinical use of blood. the higher the hdi level the better the awareness, accessibility and utilization of continuous education, both through e-learning and conventional programs. there is a better awareness among clinicians routinely prescribing blood as compared to others involved only incidentally in blood transfusion. accessibility of e-learning depends highly on the presence of a sustained societal infrastructure which is less guaranteed in the low and medium hdi countries; reliable power supply, maintenance of hardware tools and updated software programs, together with the necessary knowledge and skills of e-technology are prominent factors. the results are used for policy and strategy recommendations to improve knowledge and clinical practice through continuous e-learning programs eg, starting at undergraduate medical and nursing schools and continuing at postgraduate vocational medical specialization institutes, principles of clinical transfusion practice should be comprehensively included through appropriate and timely curricula; creation of a technical climate to guarantee access to e-learning courses and materials; stimulation of national and international exchange of e-learning programs focused on continuing education; creation of an e-learning mentoring network through professional societies, associations and education institutes. background/case studies: transfusion medicine (tm) didactic teaching materials for pathology residents are not widely available to share among residency training programs. the advancing blood knowledge (abo) leaders project is a novel approach wherein education materials are created collaboratively through a community of practice (cop). educational theorist etienne wenger defined cops as groups of people who share a concern or passion for something they do and learn how to do it better as they interact regularly. study design/method: as a pilot project, junior faculty co-investigators from west coast institutions each had months to create a minute powerpoint presentation on a fundamental tm topic, after which other members had months to review and edit. therefore, each member created and reviewed presentations (three total steps). during each step, members wrote multiple-choice questions for those particular topics. in the end, each topic would have quiz questions to assess learning. at completion, evidence-based, peer reviewed presentations would be available for all members to use for teaching pathology residents. three methods were planned to measure effectiveness of these materials: ) pre and postlecture abo leaders exam using the questions made for each topic to assess learning; ) pre and post-lecture question validated examination (best collaborative) to assess learning; ) resident in-service examination trends specific to tm. results/finding: six presentations were developed as of the abo leaders members continue to participate in this cop for tm education. abo leaders and best pre-test results are shown in tables and . abo leaders pre-test data could not be obtained for institution b, and trainees declined to participate in the examinations at institution a. challenges experienced by the cop have included heterogeneity between institutionsÕ resident schedules, balancing time dedicated to the group given busy schedules, and difficulty in giving all presentations during the defined institution-specific teaching period. post-test results will be included when assessments are complete. conclusion: despite logistical and organizational challenges, it is feasible to create a multicenter cop for tm education. the impact of such a group on resident learning will be assessed and plans for growth will be evaluated. background/case studies: the traditional educational curriculum for the pathology residency program is primarily based on didactic lectures, casebased presentations, and discussion of on-call cases. the use of dramatic vignettes has proven to be an effective educational tool to illustrate complex and multidisciplinary topics in medicine. our goal is to use and evaluate the relevance of this approach in resident education. study design/method: a clinical vignette based on a placenta accreta case was written by a pathology resident during the transfusion medicine rotation. during a two-week laboratory management course, residents prepared for the dramatic vignette performance with a focus on transfusion medicine and laboratory management topics. each resident completed a question preand post-test on topics related to the vignette. several meetings for review and adaptation of the script, topic discussion, and rehearsals were held. there were several commonly encountered problems and deviations from the standard operating procedures that the residents in the audience were asked to identify prior to the performance. during the skit, each resident presented at least one major transfusion management teaching point. results/finding: the educational activity, including the minute vignette performance and the minute discussion, was completed with a focus on: communication between the operating room and the blood bank during surgery, maximum surgical blood order schedule, pre-transfusion testing, transfusion safety, informed consent, massive transfusion protocol, emergency release blood products, thromboelastometry interpretation, patient safety, adverse events, and root cause analysis. all performers significantly improved their scores in the post-test (mean %) when compared to the pre-test scores (mean %) ttest p< . . during the vignette discussion, residents together identified all the intended non-conformances and answered related questions. residents in the audience actively participated in the post skit discussion and % reported a satisfactory learning experience. conclusion: dramatic clinical vignettes can illustrate multidisciplinary complex interactions that are of pivotal importance in the daily activities and professional development of pathology residents. with specific structured goals, clinical dramatic vignettes can be used as a complementary educational tool to illustrate challenging topics in an integrative way that is enjoyable and easy to understand and remember. the skit performers benefit from the activity further by preparing and extensively studying the topics to deliver a multifaceted and coherent presentation with emphasis on the integral role of the laboratory and transfusion medicine in patient care. hannele sareneva*, susanna sainio, inna sareneva, tiia kivipuro and taru jaske. finnish red cross blood service background/case studies: the finnish red cross blood service (frcbs) is the nationwide blood service provider in finland, responsible for collection, testing, processing and distribution of blood products to all hospitals and health care providers. the frcbs serves as the national blood group reference laboratory and provides a wide range of other laboratory services e.g. tests for hemostasis and tissue typing for possible donors as well as patients waiting for organ or stem cell transplantation. frcbs also performs antenatal blood group and rbc antibody tests covering whole country. as a sole national operator we are providing educational services to ensure the safe use of blood products as well as accurate use of our laboratory services. study design/methods: we have performed customer surveys to healthcare professionals to assemble the needs for education. based on these results and continuous feedback frcbs provides hospital customers in blood banks and clinics the following additional services: * regular education * e-learning application of transfusion medicine * handbook for blood products on the web site * reports to hospitals for their use of blood products * annual national blood safety reports regular elements of our educational program are the practical, problem solving course for blood bank personal and safe transfusion training day for clinicians. for every education we collect numerical feedback as following: "how did the education responded your expectations" and "can you utilize the knowledge in practice". we also inquire "how likely you would recommend the training for your colleges" indicating net promoter score (nps). results/findings: more than healthcare professionals participate training days at frcbs annually. in addition our experts give tens of lectures at hospitals across finland. feedback from educations has been very good, varying between . to . (in the range of - ). nps varies between and . according to customer surveys frcbs provides appropriate education to healthcare professionals. this score has increased - from . to . . conclusion: feedback, nps scores and surveys ensure that education and training program of frcbs responses to customer needs. hospitals can utilize annual courses of frcbs in their own initiation programs. together with clinical contact persons in hospitals our aim is to ensure patient blood management (pbm) and to optimize use of blood products. we also have plans to increase e-learning applications and the courses of transfusion medicine for nurses and medical students. educational outreach and effect on reporting septic transfusion reactions kathleen m grima* , anne eder , beth a. dy and mary o'neill . american red cross, georgetown university background/case studies: hemovigilance programs to monitor adverse events after transfusion depend on clinicians' ability to recognize and report reactions to the blood center. about in , apheresis platelet donations are implicated in septic transfusion reactions (strs), but this could underestimate the risk because of the difficulty in recognizing delayed or mild reactions. a large blood center designed an educational outreach program to increase awareness of strs and assessed its effect on the rate of str reporting to its national hemovigilance program. study design/method: in dec. , a large blood center developed a web based course on strs for cme/ceu credit. letters were sent to , hospital customers about recognizing and reporting strs, and alerting them to the availability of the course. blood center physicians and staff in sales and marketing also engaged hospital customers directly in discussions about recognizing and reporting strs, using the online educational content. the physicians tracked their interactions. the blood center's national hemovigilance program compared the number of strs reported in the months before and after launching the educational outreach. results/finding: the web based course was completed by more than participants; were physicians. based on a review of the evaluations, the course was highly valued with % of participants rating it excellent or very good. the blood center physicians gave over presentations to hospital customers. reporting of suspected strs in increased by % compared to the prior year. the increased reporting came from specific regions. the total number of strs that met the hemovigilance definitions for definite (culture-confirmed) and probable strs in the nationwide system increased but did not change significantly compared to the previous years. the educational initiative was designed to deliver a consistent message on the risks, recognition and reporting of strs. while the number of reports of suspected strs in two regions increased, there was no meaningful change in the overall reporting of suspected or confirmed strs across the national blood system. this finding could reflect that hospitals already recognize and report medically significant reactions or that the target audience was laboratory personnel and physicians in transfusion medicine, but not the clinicians closest to patient care at the bedside. more targeted educational efforts provided by personnel who interface with hospitals could be used to address identified professional practice gaps in transfusion medicine. implementation of subscription-based cgmp e-learning laurie mcgraw*, courtney saphier, helene belton, sallie bittner and ward scott. gulf coast regional blood center background/case studies: previous cgmp e-learning courses we developed required - minutes for learners to complete. while feedback was positive, manufacturing areas struggled to schedule time for staff to complete courses within their assigned schedules. at the same time, a shift in design trends suggest that subscription-based learning is more effective (thalheimer, .) subscription-based e-learning utilizes - minute modules, delivered at regular intervals. this changes the learning process from a singular event to a regular interaction that reinforces learning and keeps the content at the top of the learner's mind. study design/method: we began developing cgmp subscription-based elearning in by selecting our first five series topics: equipment, personnel, labeling, sops, and records. the first topic, equipment, was divided into modules on selection, validation, calibration, quality control, and maintenance. these modules, and pre-and post-quizzes for the equipment series, were developed and assigned to employees in manufacturing-related jobs using our learning management system. the pre-quiz was assigned to employees in june , with a new equipment module assigned each month for the following five months. the series concluded in december with the post-quiz. results/finding: using surveys, assessments and incident reports, we evaluated the training effectiveness using three of the four kirkpatrick levels. while our previous cgmp courses received good ratings from learners, the equipment series received the highest rating of . on a -point scale. of employees who completed all versions of our cgmp courses, the majority preferred the equipment series over all previous courses combined. comments clearly demonstrated that learners preferred the short, subscription format over the previous courses with positive and negative comment. level : learning the average score of users increased % from the pre-test to the posttest, with the greatest improvements noted in the scores from laboratory employees. a two-sample t-test determined the result to be statistically significant with a t-critical value of . and a t-stat value of . . level : results while equipment-related errors decreased by % after training, there is not enough data to demonstrate a statistical significance. conclusion: our level and evaluation data validated that the subscription approach was effective. knowledge increased from the pre-to postquiz, learners reported that they appreciated the shorter training, and they completed the modules without special scheduling requirements. as a result, we are continuing development of the remaining series. background/case studies: the interdisciplinary nature of transfusion medicine requires the collaboration of multiple work units for efficient patient care, but departmental "silos" impede collaboration between transfusionrelated care teams. we hypothesized that regular educational meetings would improve knowledge and awareness of each department's role, so in october , a multidisciplinary educational meeting called friday blood conference (fbc) began as a collaborative, interprofessional forum involving frontline staff of our transfusion practice. during these monthly meetings, which are also broadcast online for those unable to attend in person, presenters from different work units share background information and patient cases before opening the floor to constructive discussion. study design/method: a survey was sent to fbc participants (n ) to retrospectively capture the effect of fbc on interdepartmental collaboration. the survey was structured to obtain formative feedback using the published interprofessional collaborative practice competencies (icpc) as a guide. these core competencies target maintaining a climate of mutual respect, communicating within and between departments, fostering teamwork, and understanding everyone's role in patient care. results/finding: our survey response rate was %. of those, % endorse that fbc creates a climate of respect within our transfusion practice, % believe it has improved communication between work units, and % feel that fbc leads to increased understanding of interdepartmental processes. notably, laboratory scientists and transfusion nurses have the highest attendance rate. furthermore, those attending via the online broadcast report the lowest satisfaction, with only % responding positively. the main reasons individuals attend fbc are to increase knowledge about transfusion medicine, interact with and learn from other departments, hear about patient case studies, and understand the "big picture" of one's role in patient care. suggestions for improvement include preparing questions to help initiate discussion, increasing representation of other areas for broader perspectives during interdepartmental dialogue, and posting recordings of fbc for later viewing. conclusion: the application of icpc in transfusion medicine was an effective lens to assess the value of interprofessional collaboration. although there is room for improvement, the results support that fbc has contributed to better communication between transfusion-related care teams and has increased understanding of interdepartmental processes within our transfusion practice. novel approach to curriculum development: demystifying transfusion medicine ritcha saxena* and ananya saxena. all saints university school of medicine background/case studies: transfusion medicine is an essential element of education required for the future physicians in various disciplines like surgery, internal medicine and anesthesiologists to work effectively with the blood bank personnel. transfusion carries considerable advantages as well as risks. consequently, educational initiatives are required to identify the particular knowledge deficits in transfusion medicine and subsequently, bridge the gaps. and the challenge is to update the undergraduate medical curriculum to reflect the latest enhancements in transfusion medicine. study design/methods: students of undergraduate semester and students of semester participated in the study. self-directed learning resources combined with modules of interactive instruction were implemented in a tbl course design. five education modules focusing on quality management, blood collection, transfusion reactions, precise utilization of blood products and innovations in component safety were designed for the students. the students' reaction to tbl in transfusion medicine was evaluated using qualitative and quantitative assessment tools to analyze knowledge attainment and critical thinking development along with team continuity. the participants were first assessed with readiness assurance testing (rat) to guarantee that they understood the concepts and their application followed by case study based test questions. results/findings: students' reaction to tbl was primarily positive, with % of students giving a positive feedback. evaluation through readiness assurance testing (rat) illustrated improved team knowledge acquisition in implementation of effective quality management systems over knowledge acquired through individual study. students grasped a conceptual knowledge of principles of transfusion medicine and achieved confidence in dealing with transfusion-related complications. anecdotally, students significantly attained perception in blood component preparation, storage and their optimal utilization along with developments in safety techniques in blood donation. conclusion: our study suggests that reforming the medical curricula for undergraduate medical students, with specific educational modules designed to focus on blood banking and blood transfusion principles and latest advances in transfusion medicine, is much required in the interest of patient care and safety, by the future physicians. tbl is an interesting and efficient way to deliver the key aspects of transfusion medicine to the students. results/finding: open house attendees were given tours of the bb, led by a bb attending, bb residents, bb supervisor, or bb quality coordinator. the patient blood management nurse was also in attendance to answer attendee questions and educate about patient blood management. light refreshments were offered to the attendees in the bb break room. the first bb open house was held on wednesday, / / from - am. there were attendees, including a second-year medical student, four regular blood donors at the hospital blood donor center (who were also employees in facilities management and the university office of admissions, respectively), a hospital senior vice-president, six apheresis nurses, two clinical laboratory staff, two medical laboratory science students, and one additional staff member from the university office of admissions. the second bb open house was held on thursday, / / from - am. there were attendees, including regular blood donors (who were also employees in the office of international affairs and supply chain, respectively), a hematology/oncology fellow, and surgical residents. background/case studies: simple, partial, and exchange transfusions are routinely performed in patients with sickle-cell disease (scd) with the goal to increase the oxygen carrying capacity of the blood and reduce the relative percentage of sickled cells. it is essential for clinicians to be able to rapidly estimate the effects of the available therapeutic modalities using clinical information to minimize the risk of red blood cell exposure. given that the formulas for these calculations are complicated, we developed and validated an online calculator to assist physicians with such tasks. study design/method: a web application was generated (www.phamcalcs.com). the performance of the simple transfusion and partial manual exchange calculators were validated by comparing the predictions to clinical data. the performance of the automated and depletion rbcx calculators was validated using the terumo bct (lakewood, co) calculator up to a fraction cells remaining (fcr) % as patients with fcr % may benefit from delaying the procedure for performance in the future. validation process included ( ) a deming regression to globally assess the predicted vs. actual results and ( ) an individual comparison wherein validation was contingent on the (predicted-expected results)/(expected results) demonstrating |d| %. validation was performed for hematocrit (hct) and hemoglobin s (hgbs) level post-transfusion for simple and partial manual exchange and volume of replacement fluid for automated and depletion rbc exchange. results/finding: see table background/case studies: with the focus on new technologies the modern medicine requires more expenses. despite the increase in the target impact on patients there is still a risk of adverse reactions to medical treatment. the issues that are currently under discussion: the use of standardized or personalized approach, for doctors -being multidisciplinary or having a narrow specialization, integration of new technologies, the need for more trainings resulted from knowledge deficiency. in russia, the development of insurance medicine creates the demand for more intensive and cost-effective treatment programs. as a multidisciplinary approach, pbm optimizes the transfusion practice reducing the risk of adverse effects and improving the financial performance of a health care institution. however, the prosperous implementation of pbm also requires supplemental medical competencies that provide harmonization of dialogue logistics: administrator -clinician -transfusiologist. study design/method: at the medical simulation centre of hospital there has been a unique opportunity to launch an educational program for the medical specialists practicing blood components transfusion. the main innovative features of the training course are an interdisciplinary approach, intensive learning performance, comprehensiveness of learning methods. during days ( academic hours) the trainees can attend lectures, discuss the methodical materials, participate in seminars, interactive clinical discussions, a master class and a game that presents the modelling of working processes. since initiating the project in june, with the group capacity a transfusion vol. supplement s of up to people the number of medical specialists who have attended the training is nearly . results/finding: the medical competencies gained: knowledge of modern recommendations on the use of blood components the ability to interpret all parameters of the haemogram, coagulogram and tromboelastogram the ability to unveil the indications and contraindications for urgent and scheduled blood component transfusion personalization of the blood transfusion risks using a personalized approach on selecting the type and the dosing of transfusion habitat predicting the efficacy of transfusion the correction of anemia and hemostasis system malfunctions using the medicinal treatment performing the macroscopic assessment of blood component before the transfusion procedure performing the differentiated diagnostics and ability to prescribe the adverse effect treatment ability to carry out the auditorial check of health cards conclusion: the launch of the program "guidance for safe and effective blood use in adult patients of multi-field hospitals" is aimed to meet the educational and professional needs of medical specialists, develop the algorithmic thinking and a range of useful motivations in case of patient blood management and reach the compliance in practice. the effect of emergent situation drills on technologist teamwork and comfort levels abigail neils*, raeanne stensgard, rebecca wren, elisabeth greer, amy mata and camille van buskirk. mayo clinic rochester background/case studies: teamwork and composure are essential for technologists when dealing with emergent situations in a large hospitalbased blood bank where multiple situations can occur simultaneously. in an effort to reduce errors and improve emergency response, a group was formed to evaluate the effectiveness of emergency situation drills (esd). the esd were based on common emergent situations encountered in the lab and were run once per month per shift. the main goal of esd was to improve teamwork and comfort level during real emergent situations; therefore reducing the amount of unplanned standard operating procedure (sop) deviations. study design/method: prior to esd implementation, a survey was sent to all technologists to determine baseline comfort levels associated with various emergent situations. one year post esd implementation the same survey was sent to all technologists to reassess the comfort levels for the same situations. the surveys asked employees to rate satisfaction and comfort level on a grading scale of - ; being least satisfied/comfortable and being most satisfied/comfortable. the pre and post survey results were evaluated by calculating lab average comfort levels per situation and survey. in addition, unplanned sop deviations related to emergent situations were counted for one year before and one year after esd implementation. results/findings: out of total technologists, technologists took the pre esd survey and technologists took the one year post esd implementation survey. table shows the lab averages from the pre and post surveys as well as the percent difference. out of the employees who responded to the post survey, ( . %) answered "true" to the statement "esd have improved my comfort level with emergent situations." in the year prior to esd implementation there were unplanned sop deviations; in the year after esd implementation there were only deviations. conclusion: all but one area increased in comfort level post esd implementation. also most technologists agreed that the esd helped improve their overall comfort level with emergent situations. the goal of implementing esd has been met based on the unplanned sop deviation decrease and technologist satisfaction increase; therefore esd were deemed effective. monthly esd will continue to be run with the hope of continual improvement in teamwork, comfort levels and deviation levels. therapeutic background/case studies: category i indications for red blood cell exchange (rbc exchange) in children with sickle cell disease include following acute stroke and for stroke prophylaxis, as well as for iron overload prevention. as described in the first installment of this series about therapeutic plasma exchange (tpe), the challenges of access, volume management, and instrumentation persist, as along with the need to address the psychological and emotional well being of this population. rbc exchange is a complicated procedure to explain to adults and becomes an even more intimidating task when translating into the language of childhood. nevertheless, pre-treatment education is shown to decrease the anxiety associated with medical care. providing age appropriate specific treatment information to pediatric patients decreases negative behaviors, reduces stress and promotes faster recovery. a previous project explaining tpe to the pediatric population revealed the lack of age specific literature for apheresis procedures in general, including tpe and rbc exchange. study design/method: in collaboration with a child life specialist, an ageappropriate story-driven explanation of the rbc exchange procedure was adapted from a previously implemented project related to tpe. artwork was produced with the aid of a medical illustrator to complement the story-line. results/finding: the story board addresses why rbc exchange is performed, the steps involved in preparing for and performing the procedure, and strategies for coping before, during and after the procedure. the idea of long-term therapy is also briefly addressed, to prepare these children for the concept of ongoing therapy. the booklet is in production in concert with our hospital's medical illustrator and will be available on our hospital website for patient use. conclusion: using the previously illustrated story as a guide, an explanation of red cell exchange was created to provide education and reduce anxiety. this second installment continues the pediatric series helping to explain apheresis procedures to pediatric populations in the hopes of reducing patient stress and promoting age appropriate coping strategies. transfusion safety officer resource manual leonor de biasio*. it is also intended to be utilized by hospitals that do not have a formal tso position but which have delegated the responsibilities to other staff. the resource provides helpful information to assist with education in transfusion safety, adverse event investigation and reporting, product administration guidelines or monographs, and links to information about the equipment used for infusion of blood. the resource manual will serve as a useful reference tool to assist with a healthcare professional's transition into the tso role. turning on pathogen reduction: a case of flipping the switch kassandra poffenberger*, darla wendt, jennifer vrieze and james r stubbs. mayo clinic background/case studies: a critical aspect of implementing a new method in manufacturing blood products is to develop a training plan that adequately prepares staff but doesn't interfere with production or cause delays in patient care. with the implementation of pathogen reduction technology (prt) using interceptv r blood system for platelets it was understood that we would need more collections to make up for the loss of products, specifically our triple collections. our institution collects the majority of its blood products and supplements inventory from a major blood collection center. it was crucial for the component laboratory to maintain daily processing levels while learning the new method in order to sustain optimal platelet inventory levels without relying on purchasing additional platelets from external vendors. our approach in introducing prt for apheresis platelets was to "flip the switch" and process all products with the new method rather than a step wise roll out with a dual inventory. study design/method: it was essential to prioritize who would be trained first. collections occur monday through friday from to . the first group to be trained was those who would be performing training (a two person team) and product validation; they were trained by cerus deployment team. the second group was those who would process platelets on weekends and evening hours without direct management support. the last group was the technologists who would be working during normal hours with direct management support. prt processing for platelets in % plasma is broken up in to two days. on day platelets are treated with amotosalen and placed in a compound adsorption device to remove residual amotosalen for - hours. on day products are removed from the cad and modified into final product codes and labeled. each technologist was trained one on one, over a one week period. the trainers alternated training processing days for day and day . in the weeks following training it was important that each technologist rotated back thru prt processing to maintain proficiency. results/finding: of employees were trained in a two month time period. prior to "flipping the switch" the daily average of products collected was . for the two month training period the daily average rose to . conclusion: our "flip the switch" training plan for implementing prt platelets in % plasma has been highly successful for our laboratory; training while implementing the new technology did not create a bottle-neck in the process. it was imperative to prioritize who would be trained first to insure complete coverage during off hour shifts. technologists were able to become proficient with the new process while maintaining daily processing expectations and sustaining an optimal platelet inventory. accepted depending on each individual's conscience. due to these unique medical challenges, it is important for caretakers to have an understanding of their beliefs in order to provide optimal care. we describe the process of identifying jw in our hospital and communicating treatment needs to staff. study design/methods: proper treatment of jw requires the ability to identify the patient and his/her needs. when a jw is admitted to our hospital, our electronic medical record (emr) triggers several processes based on the patient's listed religion. one process creates an order that reminds caretakers to complete the declining blood consent (dbc) with the patient. the dbc contains language declining mabf and reviews the mibf with the patient to identify any that would be accepted. the emr order regarding the dbc provides educational links that include a bloodless policy, step-by-step instructions on obtaining the dbc, and information on alternatives to transfusion. a second emr process triggers a stop-gate to prevent the completion of any mabf order or mibf order for a product that the patient has declined. a third enrolls patients in the minimal blood volume labs protocol which uses microtainers, partial-fill vacutainers, and blood reservoir sets to reduce blood loss during draws. additionally, at registration, a bloodless packet is added to the patient's paper chart. this packet contains the dbc, a glossary of dbc terms, a bloodless sign to be placed over the patient's bed, a bloodless wristband to be worn by the patient, and two bloodless chart stickers that are added to the outside of the chart. these steps remind the caretakers of the patient's special requests. finally, the patient blood management (pbm) department receives emr developed reports which identify jw presenting to the hospital. these patients are followed by the pbm nurses and medical director during the duration of care. treatment plans to optimize hemoglobin, oxygen carrying capacity, and hemostasis are discussed with the bedside caretakers and implemented as needed. results/findings: nearly % of jw that enter our hospital have a dbc completed. this has resulted in increased education of the medical staff. in addition, patients have reported better communication with caretakers leading to a more inviting environment for the patients. conclusion: our hospital has found success by using an education-based team-oriented approach involving emr, pbm, and caretakers when caring for the jw patient. this approach has set up a foundation for treating other bloodless medicine patients. background/case studies: transfusion services should provide safe blood components from vein to vein with donors acting as suppliers and patients as final customers. this process involves labor-intensive activities, critical materials, human resources, facilities and highly coordinated processes. cost management has a great impact on technical processes guiding decisions upon supplies and technical staff. activity-based costing (abc) is a method to determine cost drivers within activities and determine process or product final cost allowing managers to take precise decisions. we demonstrate how an effective abc approach can result in financial savings without compromising process quality in a mid-size transfusion service. study design/method: materials costs can represent as far as % of an activity. in we had a central storage supplying satellite storages at each department and replacement was done independent of residual stock. purchases were performed on demand. at the end of we performed a supply inventory on all departments to plan future purchases and control residual stocks. in , we implemented annual purchases and satellite storages were supplied only to replenish programmed stock. cost drivers were defined upon activities on standard operational procedures (sops) resulting in a cost estimate. technical staff was involved in cost driver calculations to indicate possible changes to sops, supplier and deliveries. to minimize seasonal fluctuations we compared last quarter (q / ) with last quarter (q / ). in this work we present activity data from blood collections to illustrate abc method. results/finding: in q / blood bags were used compared to in q / , demonstrating an activity " . %. price negotiation resulted in . % readjustment. both indicated an estimated cost " . % with a possible impact of over us$ , . we have identified a real cost # . % in q / , representing an overall # . % and us$ , . (r$ , . ) savings. conclusion: economy had deteriorated in our country in with higher inflation and exchange rate variations, directly impacting imported materials, most of them critical. even with adverse economy, abc showed to be an effective tool that allowed cost decrease without significant changes in critical materials and processes. cost drivers calculations demanded review of sops and suppliers by technical staff resulting in optimization of activities. also, staff involvement reduced discharged materials since costs were wellknown to area supervisors and satellite stocks were reviewed briefly. automated verification of immunohematology results and the impact to donor testing barbara j bachman* , candace williams , carmen meyer , paul lamonby , anne cleverley and silke milbradt-pohan . bio-rad laboratories, diamed gmbh title: automated verification of immunohematology results and the impact to donor testing background/case studies: staffing challenges in today's blood banks require instrumentation with minimal operator intervention. technology advances have developed where every immunohematology result does not necessarily require operator visual review. this study evaluates the impact of automated result verification on the bio-rad ih- tm immunohematology system through the ih-com tm data management system (dms) for donor processing laboratories. study design/method: a multi-center study was performed on donor samples as shown in table a evaluating two of the most commonly used ih-system gel cards available in the us. workflow data was analyzed using process modellar app (ipad). this study focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (# operator touchpoints, visual result review occurrence), result accuracy, and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v , statistical the transfusion team collaborated with multiple user groups to educate them regarding the new processes. a gap analysis was performed to determine the optimal delivery process for blood products, with key stakeholders invited to review the options. the use of the pneumatic tube system to deliver blood throughout the entire campus was investigated to determine whether it would be a viable option given the expanded size of the new campus. results/finding: user groups requested additional training sessions as questions arose regarding use of the ehr for blood ordering. because the pneumatic tube system would be heavily used, and due to concerns that blood products could become "lost", it was decided this would not be the best route for delivery of blood. department educators requested support to create job aids specific to workflow changes impacting their departments, such as how to order rh immune globulin, a cord blood workup, etc. conclusion: leadership was challenged to provide a stable and positive environment during a complex set of changes. the simultaneous hospital move/merger and implementation of a new ehr constituted an arduous task that would not have been possible had substantial preparations not been initiated a year in advance. training is essential to the success for a scope of change this big and should not be minimized. while training was thorough prior to the move, gaps were nonetheless discovered following the move. abstract conclusion: strategic development partners funding and support based on newly developed government strategy on blood service with commitment of the government has brought a positive impact in establishing sustainable and safe national blood service program in ethiopia. even though the identified positive impacts mentioned are achieved, the bts remains with multiple challenges and needs continuity of funding and more partner support and government commitment. pilot implementation of a comprehensive hybrid performance management system at national blood service zimbabwe blessing mukwada*, judith j parirewa and tonderai mapako. national blood service zimbabwe background/case studies: the national blood service zimbabwe (nbsz) introduced its first performance management system (pms) in . in the - nbsz strategic plan it was noted that the current pms lacked objectivitety and there was no relationship between perfomance and remuneration. in order to revise the pms, the nbsz set up a three membered committee at the executive management level to spearhead the revamp of the nbsz pms. the aim of the new pms was to achieve a shared vision of the purpose and objectives of the organization, helping each staff member to understand and recognize the contribution to the strategic plan. in this paper, we share how nbsz revamped and implemented its new hybrid pms that derived its inputs from established pmss and nbsz monitoring and evaluation (m&e) process that have been linked together. one-selected departmental results for one quarter are shared to demonstrate how the system works. study design/method: pms committee developed and shared with executive management a pms conceptual and implementation framework. consultations including field visits were done on three established pms to assess suitability for nbsz adoption. a hybrid pms was adopted for nbsz and a pilot application for one quarter on selected department was done. review of policy, procedures to including hybrid pms templates and forms were done. pms committee trained all staff on how to implement an integrated scorecard, how to conduct appraisal, how to develop scorecards, how to measure performance using the new pms, how weighted performance reward systems based on all layers of performance for bonus payments works using standardised tools. throughout the process risk assessment were done. results/finding: the nbsz hybrid pms is based on five levels of planning namely strategic, departmental, branch, sectional and individual. the fourcoloured traffic light reporting system is central in uniformly assessing performance at all levels. the levels of accountability were properly defined for each level of planning. a weighted overall integrated individual scorecard (iis) is determined based on % individual and % for the other four levels ( % for each). the bonus (%) is calculated based on the iis as follows; category a: % (iis > %), category b: % (iis: -< %), category c: % (iis: -< %) and category d: % (iis < %). on the pilot implementation, the individual scores for staff ranged from % to %. the iis were % to %. the number of staff in each bonus categories were , % (category a) and , % (category b). conclusion: the new hybrid pms was generally accepted by all staff and it was easily implemented at various staff levels. this provides a basis for the full implementation of the new pms and this simplified pms can be easily be adapted in similar settings to ensure all staff contribute sufficiently and objectively to the realisation of the organisation strategic vision. rare donor engagement with american rare donor program (ardp) margaret c manigly* , deborah r fludd and sandra j nance . background/case studies: rare donors are defined as a blood type occurring in less than in people in a given population. these donors are discovered by testing new donors in a random or targeted way and require testing many donors to find one rare donor. once found, if the facility is a member of the american rare donor program (by being an aabb accredited or american red cross accredited irl), the donor is registered in the ardp database as a rare donor. in , there were , active rare donors in the ardp. with the mobility of the population in the usa, it is important that as donors relocate, that they are recognized as a rare donor when they donate and their unit can be identified and used for a patient with a rare blood need. in addition, when recruitment is needed for a patient need, correct contact information on the donor is required. study design/method: the ardp procedure for ardp members requires that donors be contacted every six months to ensure that ardp (or the facility) has their latest contact information. the timing is determined by the postal service time limit of six months to forward mail to a new address. this contact ensures that if recruitment is required to obtain blood for a patient with a rare blood need, the donor can be contacted by the collecting center to donate. this contact is achieved by ardp sending a contact card by postal mail twice yearly to all donors for whom the ardp has address demographics. results/finding: the ardp reports on the information obtained from the contact cards returned in the ardp annual activity report to the ardp members at the aabb annual meeting. of the ( . % of total active donors) returned contact cards alerting ardp of changes in calendar year , ( . %) were donors moving from one ardp facility to another, ( . %) were donors no longer eligible to donate, and an additional ( . %) were address changes. other changes were ( . %) reactivated donors and ( . %) donors who we were notified were deceased, or did not want to be listed in the ardp. in , new rare donors were submitted to ardp for registration. the number of donors that could potentially be lost to follow-up in was ( ), which would be . % of the new donors submitted. conclusion: with nearly a % response rate for donors receiving the mailed contact cards, it is clear that rare donors (and their families) are responsive to the ardp contact card, and inform ardp of address changes and changes in their health status that affects their ability to donate. this is evidence of the importance of the card in ensuring correct donor contact information. in , donors changed their addresses which often are not known to the collecting facility until the donors donates again, after their move. the ardp contact card is effective in retaining the relationship with the ardp registered donors and keeps the address information of rare donors current. workflow comparison of two gel analyzers in a large transfusion service j peter pelletier* , barbara j bachman , mike leamy , susan olson and candace williams . university of florida college of medicine, bio-rad laboratories background/case studies: vendor-assisted workflow studies are becoming more popular as analyzer choices and capabilities vary in the market. the purpose of this study was to evaluate the provue (ortho clinical diagnostics) against the ih- (bio-rad laboratories, inc.) in a large volume transfusion service using lean process flow. study design/method: twenty-two ( ) runs of one to six ( ) samples per run were observed for two ortho provues alternating testing at a large transfusion service performing , types, screens, type & screens (t&s) annually. the workflow patterns observed were then repeated on the ih- and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (testing process steps, biohazardous exposure episodes, and maintenance tasks), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v , and statistical significance was assessed using the paired t-test, with p values of < . considered significant. regardless of quality or speed metrics evaluated, the ih- demonstrated a significant reduction (improvement) in process steps and associated times when compared against the ortho provue (p < . ). ih- process steps and time studies addressed in the table below did not account for the ih- reagent storage capacity. in reality, the improvements would be greater than what was displayed here in a real-life operation. evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance, there was a significant reduction on the ih- ( % reduction, a difference of hours/year). conclusion: this study verified the ih- provided significant efficiencies and cost avoidance over the ortho provue for a large volume transfusion service. workflow comparison of two high volume, high throughput analyzers aaron samson* , kimberly monnin , barbara j bachman , kyla warren , susan olson and candace williams . clinical pathology labs, bio-rad laboratories background/case studies: few workflow studies have been performed on high volume, high throughput blood bank analyzers in large volume testing facilities. the purpose of this study was to evaluate the galileo v r neo (immucor) against the ih- tm (bio-rad laboratories, inc.) using lean process flow. study design/method: a total of separate test runs of or samples per run were observed over a three day period on the galileo neo at a reference laboratory annually performing approximately , type & screens (t&s). the workflow patterns observed were then repeated on the ih- and compared. each process was mapped in detail by direct observation using process modellar app (ipad). the evaluation started at sample centrifugation completion and drop-off in testing area and ended with results sent from analyzer to lis (lab information system). each was evaluated for quality (process steps, biohazardous exposure), speed (operator/analyzer time) and cost (testing/maintenance personnel hours recaptured). time studies were analyzed using minitab v , and statistical significance was assessed using the paired t-test, with p values of < . considered significant. results/finding: detailed process steps, biohazardous exposures, and published analyzer maintenance tasks were evaluated/compared (table, part a) . time studies focused on operator time, analyzer time, and maintenance time (table, part b). regardless of quality or speed metrics evaluated, the ih- demonstrated significant reduction (improvement) in process steps and associated times when compared against the galileo neo (p < . ). evaluating the total number of maintenance tasks required annually, as well as the times associated with maintenance performance and downtime, was a significantly reduced on the ih- (difference of hours/year). conclusion: this study verified the ih- provided significant efficiencies and cost avoidance over the galileo neo for high volume/high throughput testing facilities. workflow impact of automated result verification for patient and donor blood typing barbara j bachman* , candace williams , carmen meyer , paul lamonby , anne cleverley and silke milbradt-pohan . bio-rad laboratories, diamed gmbh background/case studies: immunohematology facilities face many challenges including standardization, process control, productivity, staffing and patient safety. to alleviate these challenges, the ih- tm instrument and complementary ih-com tm data management system (dms) were designed to provide lean automation to enhance blood testing facility workflow. the purpose of this study was to focus on the lean functionality of automated result verification on the ih- and ih-com dms and determine its impact on workflow. study design/methods: internal and external studies using the ih- with the ih-com dms were performed with patient and donor samples. assays included abo/rh blood grouping and antibody screening (abs) as shown in table a . workflow data was analyzed using process modellar app (ipad). the evaluation focused on post-analytical steps of result verification, evaluating with and without automated result verification to determine the impact on quality (operator touchpoints, visual result review occurrence, result accuracy), and speed (time from result interpretation to lis data transfer). operator touchpoints during the post-analytical phase are only required when doing visual result verification and are software defined. speed metrics were analyzed using minitab v . statistical significance was assessed using the paired t-test, with p values of < . considered significant. results/findings: using automated result verification, only . % out of , samples evaluated for abo/rh testing would require visual verification, resulting in a % reduction in operator touchpoints (p < . ) and a labor saving of minutes ( : hh:mm) for abo/rh testing. for , antibody screens, automatic validation of results would result in . % reduction in operator touchpoints (p < . ) and a labor savings of minutes ( : hh:mm). no false positive or false negative typing results or false negative screenings occurred with results auto-verification. (rbc) has remained, and in fact is proportionally increasing while blood usage has notably declined in the era of patient blood management. over the past years a steady increase in demand for o neg rbc compared to other blood types has been observed at our blood center. utilization metrics for hospital customers are monitored monthly for overall trending and forecasting and the data shared with them during regular visits. despite heightening awareness, percent o neg rbc sales continued to rise by % annually and peaked at % in mid . to better understand this increased demand a survey was conducted to gather insight for improved utilization. we speculated that during the survey an observer effect, or change in the staff behavior, would result in reduction of o neg rbc sales. study design/methods: a tie tag was designed as a survey tool and attached to each o neg rbc distributed to hospital customers for an week period in late . hospital transfusion service staff were asked to record the final disposition of the o neg rbc (transfused, wasted, returned) on the tie tag. information on the survey objective and instructions for tie tag completion were communicated via customer meetings, emails and reminders sent by blood center drivers. completed tags were returned to the blood center. customers are allowed to return rbc units with greater than day shelf life remaining. units with tie tags attached were in hospital inventories for up to months due to the shelf life of rbc. return rates and percent of net sales (gross sales minus returns) by abo/rh type were tracked monthly before, during and after the survey. results/findings: participation was % of the hospitals surveyed. mean percent o neg rbc gross sales for a month period before, during, and after the survey was . %, . % and . %, respectively. mean percent o neg net sales during the -month survey fell to . % compared to an average of . % in the months prior. during the -month survey period o neg rbc monthly return rate increased to an average of . % compared to an average of . % in the months prior. for the months after the survey the average o neg rbc return rate further increased to . % while mean percent o neg rbc net sales trended slightly upward to . %. when customer hospitals were queried whether any process changes occurred, no major changes to policy or inventory levels were reported. conclusion: during and after the survey percent o neg rbc gross sales was fairly constant indicating that target inventory levels and transfusion service staff ordering practices remained unchanged. however, during the same period the increase in o neg rbc return rate and corresponding decline in percent net sales suggests improved o neg rbc utilization. increased awareness from participating in the survey and staff knowing they were being observed likely played a role in the lowering of percent o neg rbc net sales. tracking of monthly metrics will provide ongoing review to determine if the effect is transient or sustained and identify other opportunities for improving o neg rbc utilization. acoustophoretic separation of platelets from whole blood: a relevant and practical alternative to centrifugation pierre bohec* , jeremie gachelin , veronique ollivier , thibaut mutin , xavier telot , benoit ho tin noe and sandra sanfilippo . aenitis technologies, hôpital bichat, inserm u background/case studies: shear-induced platelet activation is an unwanted side effect of the centrifugation-based procedure currently used in blood banks to prepare platelet concentrates. transfusion of partly activated platelets could indeed increase the risk of adverse transfusion reactions. aims: here we evaluated the effectiveness of an innovative acoustic-based fractionation device by carrying out a qualitative and functional in vivo analysis of isolated human platelets. study design/method: whole blood was obtained from donors and fractionated using an acoustic-based device. platelet recovery and purity were determined by quantifying blood cell subpopulations in the microchannel outlet samples. quality of isolated platelets was evaluated using the surface expression of two activation markers (p-selectin, pac ) using flow cytometric methods while their procoagulant ability was investigated using in vivo experimentation. platelets isolated using a soft-spin protocol, were used as inactivated control. results/finding: fractionation using the acoustic-based device led to a red blood cell clearance ratio from whole blood greater than % (p< . ) and a purity of platelets close to . %. we did not find any difference in terms of quality and functionality of platelets from the same donors isolated using the acoustic device versus the soft-spin protocol. conclusion: this acoustic-based blood processing method led to excellent preservation of platelet quality and functionality providing a novel promising technique for whole blood fractionation in clinical settings. automation in blood bank processing: where we go? robert fernandez, lluis puig, pilar ortiz, joan ovejo, nuria martinez, elena valdivia and susana g gomez*. banc de sang i teixits background/case studies: nowadays, blood banking is requiring new strategies to manufacture blood components, due to the increase on their production. at banc de sang i teixits (bst), we have implemented during the last years automation manufacturing, including lean management methods, to be able to process our needs of over . blood donations for an area with more than million people. study design/method: the automation of blood donations process, bst has done different changes on the equipment. in , orbisac (terumo bct) was the equipment to obtain buffy coats and from this product, we got platelets concentrates. it was in , when we moved from this equipment to atreus c (terumo bct), to get red blood cells, buffy coat and fresh frozen plasma. then we did some updated on atreus; in we changed to atreus c (terumo bct) and finally in , we moved to reveos system (terumo bct). since the changes in , our blood components were red cell concentrate, plasma, platelets and a leukocyte residue. while all these changes in processing equipment, we added also some automation in our registration (donation id, weight and temperature) and labeling steps, implementing two homemade robots. and finally, to get better results and more efficacy in our production, in , bst incorporated an engineer to introduce lean manufacturing methods. these methods are based on the identification and analysis of problems, and then chose all these activates that add some value to the procedure. results/finding: once all these changes have been updated, we have evaluated the quality of blood components, such as red cells and platelets, also the number of donations that we were missing and working hours that were necessary to process our blood donations. this evaluation was done for processes during and . conclusion: with these results, it's obvious that automation in blood banking makes more efficient the manufacturing of blood components, getting better quality of them and also in a cheaper way. we encourage maintaining lean philosophy in order to keep improving our methods and identifying those activities that add value to our processes and get rid of those ones that are not necessary. in a globalized and industrialized world, where everything changes very fast, these improvements are necessary to be on top of the field and be a state of the art blood bank. background/case studies: the laboratory envisioned an automated blood product delivery system that extended blood access to the bedside through the use of remote blood allocation devices, or "smart blood refrigerators" to improve patient safety, provide timely access to blood products, and potentially reduce laboratory workload. as part of this initiative, bloodtrack haemobanks (hb) (haemonetics, braintree, ma) were installed and interfaced to the existing safetrace tx (haemonetics, braintree, ma) laboratory information system. one hb was installed in the methodist hospital (rmh) campus which includes a busy outpatient infusion therapy center (itc). study design/method: an assessment of the current blood supply chain revealed improvement opportunities for both nursing and blood bank staff. frequent daily trips to and from the blood bank take nurses away from the patient beside and can create congestion at the blood bank window during peak times. for the itc, with a daily outpatient volume of - patients and an average, round-trip travel time of approximately -minutes, even small delays waiting in line at the blood bank window would produce profound ripple effects. itc nurses faced the additional challenge of maintaining nurse-to-patient ratios and providing timely patient care. about % of patients in the itc have same-day transfusion orders, adding to the blood bank workload and creating unpredictability in workflow. often for patients in the itc, nurses had to repeat pre-transfusion vital signs because too much time had elapsed between gathering vitals and obtaining the blood. these inefficiencies resulted in longer patient wait times and, ultimately, a longer stay in the itc. results/finding: hb devices allow nursing staff to access red blood cells (rbc) for the majority of their patients at the point of care. since implementing in november , the hb has significantly improved the turnaround time of rbc issue -from -minutes to less than -seconds-and helped maintain nurse to patient ratios and reduced traffic at the blood bank issue window. prior to hb implementation, blood bank staff at rmh were issuing approximately rbc per month out of the window for non-surgical patients. this has been reduced to approximately rbc per month, a % average monthly reduction. conclusion: having the hb located in the itc has helped to expedite the care of patients and more easily manage blood products for patients with same-day orders. the use of hb devices has not resulted in a reduction in blood bank fte, but rather a shift in workload; from issuing products to monitoring inventory and restocking. consists of registered paramedics that are pararescue specialists and helicopter personnel. when in combat, the squadron conducts personnel recovery operations and rescues downed airmen. when stationed in the us, they mitigate in state emergencies and perform aeromedical evacuations. in , they supported a civilian medical emergency and the patient needed a transfusion in the field. they procured blood products from a distant air force base with adjacent medical facility. at the debriefing, members of the st rescue squadron ( rqs) decided to find a local civilian blood supplier. the master sergeant contacted our blood center and set up a contract for blood supply. study design/method: blood center representatives met with the rqs master sergeant in january . we asked what rqs's order and delivery expectations were. he said sporadic use and the blood order would be rbcs. we wrote a procedure for consignment and packaging, using standard blood transport boxes. we developed a communication template for staff to anticipate the rqs needs. staff was trained based on data from january meeting. we contacted the rqs in september to perform a trial run. at that time, we learned the master sergeant was shipped out to military theater. we invited his replacement to the blood center. this pararescue senior airman had just returned from syria and was assigned to civilian duty. he had no prior knowledge of the rqs association with a civilian blood center. based on his field experiences, he changed the blood order from to rbcs. he introduced blood transport containers, used in military operations, saying they were easier to carry during water and land pararescue missions. we rewrote the procedures, incorporated his transport containers, and made a pictorial job aid to assist staff on packaging blood using these containers. the blood center and rqs performed a mock run on october , and we felt prepared for any future events. results/finding: on november , , the rqs was deployed to a civilian aeromedical evacuation. we anticipated a rbc order. the actual order was rbcs and ffp. staff was preparing frozen ffp to ship, as was their norm for filling hospital orders. realizing that they could not thaw plasma in flight, we contacted the rqs and offered liquid plasma instead, which they accepted. product was consigned and picked up at : am by the rqs. the patient was transfused in the field and then taken to a nearby hospital. at our joint debriefing on november th , we established a maximum blood order of rbc and liquid plasma, noting future orders may request fewer products, yet meet the preferred rbc; plasma transfusion ratio. conclusion: military personnel are adapted to instantly adjust to an ever changing environment. regulated blood centers are not as adaptable. with clear and comprehensive communication and anticipation on the blood center's part, we now supply civilian blood products to the air national guard. (table ). the highest mean fib concentration was mg/donor unit; lowest mean fib concentration was mg/donor unit. all sites had a mean fib concentration at least mg/donor unit above the fda minimum requirement of mg/donor unit. fifteen of blood centers completed the manufacturing process survey. one used a leukocyte reduction filter with ahf destined plasma. all blood centers manufactured single donor cryoprecipitate; manufactured pooled donor cryoprecipitate. most froze plasma in a - c or colder blood bank freezer. one froze plasma using dry ice, and one used a blast freezer. two blood centers method of thawing frozen plasma took longer than hours. conclusion: blood centers consistently met the overall fib minimum requirement with a mean of mg/donor unit, over double the fda requirement. however there is variability in fib levels amongst blood centers. in general, manufacturing processes were similar with a few exceptions. blood centers should inform their hospital customers of their average fib level in cryoprecipitate in order to most appropriately care for patients receiving this product. compliance & productivity improvement via engineered-staffing/ scheduling calculator application (app) mary deck, mark angelelli and kevin lee*. american red cross background/case studies: the healthcare industry, particularly the blood banking industry continues to experience tremendous pressures not only with ensuring patient safety and quality daily, but managing and maintaining an efficient operation with a cost competitive structure. applications of basic industrial engineering tools, coupled with lean-six sigma techniques such as time study analysis, bottleneck elimination & process standardization to transfusion reduce variation has been transformed into an application (for short "app"), which can be utilized to determine process and staffing optimization and provide flexibility to the dynamic nature of changing needs in blood banking. study design/methods: a time study analysis offers valuable data about the process requirements. once this baseline has been established, translating the data into a user-friendly app would enable ease and practical use to facilitate business decision-making as well as effectively manage daily operations. important concepts such as lean-pull production system, bottleneck elimination, work-load balancing together with basic development of the app using ms excel software will be demonstrated. results/findings: successful rollouts and implementations of the staffing/ scheduling calculator app across pilot facilities, then onto facilities nationwide, has yielded improved productivity together with a sustainable compliance scorecard. the app interactive-based approach, programmed via a commonly used software, ms excel, was used to analyze how to optimize staffing requirements together with staff-scheduling (i.e. match incoming volume/work content to staffing availability). the staffing/scheduling calculator app has been utilized by executives to evaluate "what-if" scenarios (sensitivity analysis) as well as a planning toolkit to proactively manage the changing demands of blood banking. conclusion: besides providing a key mechanism for increased productivity and sustained compliance -a top priority for blood banks -the staffing/ scheduling calculator app will highlight continuous improvement opportunities and spring-board to system-wide acceptance and standardization. all coolers were prepared in a walk-in refrigerator. two scoops of wet ice or two ice packs were placed at the bottom of large/medium or small coolers, respectively, with rbc units on top of the ice. a quality-controlled thermometer was placed on top of the rbc units. a control thermometer was place at the interface between the ice and the rbc units in one large and one medium cooler. the start temperature was recorded and then the temperature was recorded every minutes for a hour period or until the temperature exceeded c. results/finding: the temperature recorded from the thermometer on top of the units in all five coolers reached > c in minutes as shown in table . the control thermometer recorded temperatures maintained at - c for the entire hour observation period in both the large and medium cooler. conclusion: when units are placed on top of the ice in a cooler, the temperature is not reliably maintained at - c for more than minutes. these data support a policy of wasting units that are returned to the blood bank with rbc units on top of the ice. background/case studies: an fda draft guidance has highlighted the need to reduce the risk of bacterial contamination of platelet components (pc) via pathogen reduction (pr) or secondary rapid testing (rt). hospitals must understand the cost implications that may result. our objective was to create an interactive model to analyze the budget impact for different pc types across the range of existing us hospitals. study design/methods: an excel model was built and populated with base case costs and probabilities identified through literature search as well as through a survey administered to us hospital transfusion service directors. the model was reviewed and refined by a panel of seven transfusion medicine physicians. the model allows base-case assumptions to be overwritten with values specific to the institution. three scenarios were generated to compare annual costs of plt acquisition, testing, wastage, dispensing /transfusion, adverse events (ae), shelflife, and reimbursement for a hospital that purchases all of its pcs: % conventional (c-pc), % pr-pc, and mix of % c-pc/ % pr-pc. the model predicts a modest ($ %) cost increase for pr-pc compared to c-pc depending on the degree of pr conversion; this takes into account cost offsets such as elimination of bd and irradiation, decreased waste due to increased shelf-life, and outpatient reimbursement. the effective pc shelf-life is potentially increased with pr due to elimination of bd, and is dependent on nat turnaround time. benefits not captured by the model include transfusion-transmitted infectious risk mitigation from emerging pathogens, which may impact cost/benefit analyses. future iterations of this model will also enable hospitals to consider scenarios in which rt is used. this model can serve as an important tool for hospitals considering pr adoption. in january . a report was created to identify donors previously classified as rare according to the american rare donor program (ardp) criteria. donors are classified as rare by meeting one of the following: highprevalence antigen negative, multiple common antigen negative, or iga deficient. the new process utilized the report and involved sending a letter to the donors notifying them of their rare donor status and encouraging them to continue to donate. a database was created to track the letters sent to rare donors. in august , inventory reduction efforts were implemented to gradually decrease the number of allogeneic red blood cells (rbc) collected to minimize unit age at transfusion. the inventory reduction occurred in phases and was completed by january . a study was performed to determine the impact of the inventory reduction on the number of rare donor donations. study design/methods: the total number of allogeneic rbc donations, rare donor donations, and number of rare donor letters sent was analyzed from to (see table) . the percentage of rare donor donations per year was calculated. background/case studies: blood centers (clients) often carry low inventory of blood and blood components. laboratories performing donor screening therefore, have limited time to determine the presence or absence of infectious disease within these products. in order to measure and ensure expedited donor screening we implemented a daily performance metric consisting of upload time goals for release of results to clients. in , zkv-nat testing was implement for travel deferral donors (july), followed by universal individual donor screening in september and november in response to the fda recommendations for "reducing the risk of zkv transmission by blood and blood components". per the fda guidance we implemented mandatory zkv testing for clients with proximity to areas with locally acquired mosquito-borne cases of zkv within weeks (sept. phase ) and nationwide within weeks (nov. phase ). zkv testing is performed on individual samples, unlike all other nat tests that are performed in minipools ( -donations). therefore zkv testing has a disproportionate impact on the turnaround times for testing, which we analyzed in this study. study design/method: within two regional testing labs, participating in the same clinical trial, lab had % and lab had % of clients requiring universal zkv testing. we evaluated a -month test result upload performance period to determine the impact of zkv test implementation. results/finding: during , lab upload time performance ranged from % to . % from january to july; upload time performance fell between august through november, returning to . % performance in december. lab upload time performance ranged from . % to . % january to august. performance fell september through december . % - . %. lab experienced a low of % upload time performance during phase when there was a rapid implementation; % clients required zkv nat. improved performance was observed during phase , with a % increase in zkv clients. for lab : phase experienced a modest decline of upload performance ranging from . % to . % with . % of clients implementing zkv nat. performance was . % in phase , when an additional . % of clients implemented zkv testing. conclusion: with an unprecedented rapid implementation of zkv testing our laboratories experienced a short period of reduced ability to maintain our upload time performance metric. enhanced platelet bacterial screening in an eight-hospital system robin larson* and colleen a. aronson . advocate lutheran general hospital, acl laboratories/ advocate hospitals background/case studies: in response to two platelet-related septic transfusion reactions and the draft fda guidance released in march regarding bacterial risk control strategies for transfusion services, an eight-hospital system implemented the verax pgd enhanced platelet bacterial screening test in of the hospital transfusion services. the sites that did not implement the test arranged for fresh platelets to be rotated in from the blood supplier. the sites which implemented the verax pgd test perform testing on all day and day platelets to be issued for transfusion. this abstract summarizes the data collected for the first weeks of testing. study design/methods: platelet bacterial testing logs were reviewed over the entire time period studied for platelets tested on day , day , and those that were tested twice. inventory reports were reviewed for platelets issued on day or day that did not require testing, and for the total number of platelets issued over the time period studied. results/findings: in the month of february ( week of performing the test), . % of all platelets issued by the participating transfusion services were day or day platelets. in march that number dropped to . %. it is expected that this number will level off at some percentage at or below . % with further data collection. in february . % of platelets were tested twice prior to final issue from the transfusion services. in march conclusion: the percent of platelets issued fresh (day or day ) will likely level off at some number at or below . % due to inventory management from both the blood supplier and the individual transfusion services. testing platelets twice is undesirable. ideally, no platelets would be tested twice as this represents a high cost for both the test reagents as well as the staff time to complete the testing. in addition, of the sites performing testing are level trauma centers and need to have tested platelets available at all times. this will require some amount of double testing, but the goal is to have this number be as low as possible, so that the percent of tested vs issued platelets does not exceed %. as the transfusion service staff becomes more comfortable with judging inventory levels and performing testing, it is expected that the amount of double testing will decrease. background/case studies: in order to make up for the deficiency of the apheresis platelets in clinical application, and also to improve the comprehensive utilization of blood, we investigate the feasibility of preparation of pooled platelet concentrates(pcs) for providing a reliable source for clinical application. to speed up the storage research of pooled pcs in china, we evaluate the changes in platelet function after filtering leukocytes with leukocytes filter for pcs and the quality changes during storage in pvc-bthc blood bags. study design/method: pcs were prepared from ml virus free whole blood by platelet-rich plasma (prp) method. five or six bags of abomatched pcs were pooled and filtered with leukocytes filter for pcs(n ). the swirling phenomenon, ph, automatic blood count, platelet aggregation, hypotonic shock response (hsr), the extent of shape change(esc), cd p expression, atp level in platelet, glucose and lactate concentration were detected before and after filtering, and on days , , and of storage, respectively. results/finding: the platelet recovery ratio of a therapeutic dose of pooled platelet concentrates after filtering leukocytes was ( . . )%, relative change rate of hsr was ( . . )%, the residual leukocytes were ( . . ) . the ph, hsr, and the cd p expression of pooled platelet concentrates before and after filtering were ( . . ) vs ( . . ), ( . . )% vs ( . . )% and ( . . ) % vs ( . . )%. there is significant change for wbc after filtering (p< . ). during storage in pvc-bthc blood bags, the biochemical parameters of pooled platelet concentrates changed with increasing storage time, as shown in table . conclusion: storage in pvc-bthc blood bags for five days, the quality of pooled pcs met the requirements of chinese standards (gb - ) . it can be a complementary source for apheresis platelets supplement in china. evaluation of samplokv r segment sampler to obtain and measure samples from blood component tubing segments abbejane blair*. ajblair laboratory consulting background/case studies: current methods used to obtain samples from blood component tubing segments are cumbersome and present a significant risk for exposure to biohazards, sharps injury and cross contamination. itl biomedical has developed samplokv r segment sampler (ss), a device for obtaining measured samples from sealed tubing segments that is less cumbersome and offers improved safety, eliminating the need to manually cut and squeeze tubing segments. ss was evaluated with the goals of reducing the number of steps required to obtain a measured sample, and, reduce biohazards and sharps exposure. study design/method: ss obtains fluid samples from sealed tubing segments into a needleless syringe. it consists of two chambers with recessed internal needles located at the top of the device and a female port located at the bottom of the device. a needleless syringe is attached to the female port, the sealed tubing ends are then aligned with the ss chambers and, gently pushed onto the needles to pierce each end of the segment. the sample from the segment is then withdrawn into the syringe. the study was performed at rhode island blood center (providence, ri) using tubing segments from three bag manufacturers to demonstrate ease of use on the following processes: segment alignment over needles and piercing, ability to draw sample into syringe, ability to expel air bubbles from syringe, fluid leaks, ease of transfer of sample from syringe to tube and to collect user feedback. two lengths of tubing segments were filled to contain sample volumes of ml and ml. two users then evaluated the ss tubing segment types with ml or ml samples for a total of data points. samples were collected into the attached ml or ml syringe then a measured sample was transferred from the syringe into a test tube or microcentrifuge tube. results were tabulated as pass or fail. results/finding: a total of ss were evaluated by two users. all samples were successfully collected and transferred into tubes. insertion of the segment edge requires observation to ensure placement onto the needles. any air bubbles collected into the syringe could easily be moved to the top by background/case studies: the management of platelet inventory is crucial due to a number of factors including the day product outdate, the allocation of staff due to the lengthy donation process, the increasingly small donor pool, and the high cost of production (e.g. platelet collection kits, testing, product processing). the use of a platelet inventory management tool has the potential to enhance the understanding of units transfused, optimize inventory, increase efficiency, and reduce waste. the objectives of this assessment were to decipher if the platelet inventory management tool has reduced the amount of outdated platelet products, total cost of platelet production, and full time equivalent (fte) allocation. study design/method: in january , a platelet inventory management process was implemented which uses a spreadsheet based tool to predict the amount of platelet collection procedures needed to be scheduled each day. the tool uses daily historical transfusion data from the last five weeks. additional calculations are included to account for deferrals, no shows, incomplete collections, and product split rate. the number generated from the calculations correlates to how many platelet collection procedures to schedule for the specific day of the week considering testing release and historical daily transfusion trends. the effectiveness of the tool was verified by comparing platelet collections, platelet products outdated, and fte information for a one year period prior to the implementation of platelet inventory management to one year period following implementation. results/finding: by implementing a platelet inventory management tool, collections have been lowered or shifted to accommodate the transfusion needs. the staffing adjustments and targeted collections have lowered fte and outdate cost by %. the platelet outdate rates dropped after implementing the platelet inventory tool from % ( units) to % ( units); a % decrease. fte was able to be monitored closely with the donor schedule and lowered from a yearly average of fte to . fte, lowering fte by %. conclusion: considering historical transfusion data for potential platelet demand has had a positive impact on scheduling platelet collections. staffing requirements and outdating products have decreased since implementation of the platelet management spreadsheet tool, leading to less waste both in terms of staffing and platelets. given these positive results, we are beginning to develop a similar tool for our whole blood collections. identifying opportunities to right-size hospital inventory using compotrace radio frequency id inventory management system nanci fredrich* , jaclyn mckay , jennifer curnes and rowena punzalan , . bloodcenter of wisconsin, children's hospital of wisconsin background/case studies: the ability to track inventory of blood components in real time is challenging for both hospital transfusion services (ts) and blood centers (bc) using current blood bank information systems (bbis). in addition, determining if established par levels of individual components meet or exceed daily transfusion needs is difficult to ascertain. a pilot was designed to track and monitor all blood components from distribution at the bc to issue in a hospital ts using fresenius kabi compotrace radio frequency id (rfid) enabled inventory management system. the objectives were to determine feasibility of the compotrace system and analyze compotrace data for real-time usage and optimal inventory levels. study design/method: a month pilot was conducted at a pediatric hospital and its bc using both bbis and compotrace systems to track all adult-size blood components. staff were trained on use of compotrace system. upon receipt of order from pilot hospital, bc staff applied rfid tags to all component bags and scanned components into the compotrace system. components were transported and delivered to ts following established procedures. upon receipt at the hospital, components were scanned into inventory using both the ts bbis and compotrace systems. dual scanning of components occurred upon issue to or return from floor, component modification or return to bc. products for emergency use or at time of high demand were not rfid-scanned. a priori, the pilot would stop if the compo-trace system hampered current workflow, component issue was delayed or if ts errors increased. no inventory changes were made during the pilot. results/findings: real-time data from compotrace system provided actual usage for all blood components including component disposal and shipment to and from bc. average daily rbc inventory levels and usage for selected blood types is shown in table. lessons learned related to equipment and workflow: ( ) use of smaller irradiation canister may damage rfid tag, which was resolved by relocating tag, ( ) ts workflow and stat orders challenged consistent use of dual processes to track component status. however no increase in ts errors or delay in issue of components occurred. conclusion: use of rfid to track blood components from bc to final disposition is feasible. real-time data from compotrace system identified optimal inventory levels for rbc at the pilot ts. use of real-time rfid to track inventory and adjust target levels based on actual daily usage over time may reveal seasonal influences that affect target inventory. background/case studies: physicians expect blood to be available at all times. following a national appeal in july for donors based on a predicted summer shortage with high likelihood of extending into the fall, our transfusion service (ts) recognized a potentially dire situation given the institution's patient acuity. our hospital-based ts supports a full range of services: a level i trauma service; stem cell and solid organ transplant services; a brisk cardiothoracic surgical program; a high risk obstetrical service; and high acuity medical/surgical services. a regional donor center supplies our blood products. to insure appropriate response to patient needs, the ts created a management plan, with input from multiple stakeholders, to assist with product management in times of extreme shortages. the approach is described herein. study design/method: at the direction of the transfusion committee (tc), ts directors presented the concern for impending shortages to the hospital quality directors (qd) committee. the qd committee consists of clinicians and non-clinicians trained in health care quality/regulatory affairs who are responsible for institutional health care quality (hcq) activities. the qd recommended creation of a multidisciplinary team: "the blood shortage task force (bstf)", analogous to an existing task force started for management of drug shortages. results/finding: with hcq and tc support, the ts created the bstf and blood shortage management algorithm (bsma). standing members of the bstf include ts medical director (chair), senior vice president (svp) of hcq, svps of clinical services director of regulatory affairs, legal counsel, and representatives from ethics, social work, pharmacy, patient referrals, and communications. ad hoc members include those whose patients would be most impacted by the specific shortage. the bsma designed by the bstf provides a framework for ts's to conduct operational and therapeutic assessments of potential impact and defines criteria for convening the bstf. trigger criteria include: marked ts concern; essential product; high likelihood of inventory depletion; broad patient impact. once convened, the bstf is responsible for situational assessment and formulation of a management plan, with a goal of maintaining quality patient care. conclusion: faced with the potential for limited blood supply, the ts reached beyond the laboratory and engaged the tc and members of hcq to assemble a robust, multidisciplinary task force. this resulted in an inclusive plan which can be activated at any time to address shortages, and assist in management of impacted patients. abstract background/case studies: cryoprecipitate (for short "cryo") plays a critical role in clotting and controlling hemorrhaging, and is often used in the treatment of massive trauma and major diseases, including metastasized cancers, cardiac diseases, hepatic failures, and organ transplants. the collection process of cryo is particularly challenging; due to fact to be processed into cryo units, the collected whole blood has to be shipped to the production facility and be processed within -hours after collection. this tight hour time constraint between collection and production can only be satisfied with precision collection planning and extra courier services; which makes the collection for cryo units more costly than other products. study design/methods: the american red cross (arc), in partnership with researchers from the georgia institute of technology (gt), has developed a blood collection model to increase the amount of whole blood that can be processed into cryoprecipitate. after reviewing blood collecting and processing schedules, collection locations, and other factors, arc-cryo subject matter experts together with gt researchers were able to analyze the problem structurally with several analytic/dynamic programming properties, and developed a near optimal solution algorithm or mathematical model. results/findings: to facilitate implementation, a decision support tool (dst) was developed to systematize the selection of the collection sites; determining when and from which mobile collection sites to collect blood for cryo production and how to schedule the courier services such that the collection targets are met and the total collection costs are minimized. the implementation of the dst led to an increase in the number of whole blood units satisfying the tight -hour completion time constraint for cryo production (capacity expansion). in particular, during the th -quarter of , a blood processing region was able to process about more cryo units/month (an increase of %) at a slightly lower collection cost (cost avoidance), resulting in an approximately % reduction in the per unit collection cost for cryo. conclusion: by utilizing operations research toolkits, a mathematical model or near-optimal algorithm could be developed to optimize the cryoprecipitate collection process, ensuring the time constraints and product consistency levels are achieved. this interdisciplinary improving cryoprecipitate collections collaborative project has been selected as a finalist on the -the franz edelman award, recognizing outstanding achievements and practices in operations research. inventory management and transfusion practice before and after -day apheresis platelets sarah k harm*. university of vermont medical center background/case studies: the shelf life of apheresis platelet (ap) units stored in plasma may be extended from to days in the usa using an fda cleared rapid test (rt). in august , our hospital based transfusion service began using a rt on day and to routinely extend ap shelf life to days. this report describes changes in platelet inventory management and transfusion practice six months following routine use of -day ap. study design/methods: data were obtained for two study periods: september -february (pre-implementation) and september -february (post-implementation). the study periods were intentionally made to span the same months of the year due to seasonal variability in platelet transfusion rates in our region. the transition period from -day to -day ap inventory was excluded. the following data was collected for each study period: the total number of ap transfusion recipients, ap units transfused, expired ap units, ap units ordered ad-hoc from suppliers, inpatient admissions, surgical volumes, and average length of stay. results/findings: data are shown in the table. the number of ap transfusions decreased by % post-implementation while inpatient admissions and surgical volume increased by % and %, respectively. the hospital length of stay was similar for both periods. ap inventory decreased by % post-implementation and the outdate rate decreased from % to % (p< . ). ad-hoc ordering was not statistically different between study periods (p . ). the average number of ap transfusions per patient between pre-and post-implementation periods was not statistically different ( . and . , respectively, p . ). furthermore, a new "rejection threshold" for lipaemic products will be implemented. this threshold represents the tg concentration above which viral marker testing for donor screening will be affected. in kcbb abbott's prism assays are used for: hbsag, anti-hcv ab, anti-hiv ab, anti-htlvi/ii . results/finding: using data management system and file records in kcbb as regard discarding blood components due to lipaemia during the last five years ( ) ( ) ( ) ( ) ( ) , it was demonstrated that number of discarded rbcs due to lipaemia during the whole period was units. number of discarded different plasma, platelets, and cryoprecipitate components during the last two years due to lipaemia was , , and units respectively. the mean number of discarded rbc units of the five years of the study exceeds % of the tested ones. literature about guidelines on the management of lipaemic donations were reviewed in order to minimize donation loss, and establish an accurate rejection threshold for lipaemic donations. by reviewing sample requirements for viral marker testing in kcbb, the accepted level for tg in blood samples is below mg/dl, and so the rejection threshold for lipaemia is level equal to or more than mg/dl. conclusion: many blood product units are discarded needlessly in kcbb due to lipaemia in the last five years (including rbcs, plasma products and apheresis platelet units). in an effort to reduce the waste of potentially lifesaving products, the rejection threshold for lipaemic products is recommended to be changed from mg/dl to mg/dl which does not affect blood safety. a follow up study is recommended after applying the new threshold to evaluate the new policy. logistical management of the incorporation of pathogen reduced single donor platelets (pr-sdp) into inventory at a u.s. tertiary care medical center eric gehrie* , , rebecca ross , debra mraz , anne baker , zenna neal , melanie champion and edward l. snyder , . johns hopkins university school of medicine, yale university, yale-new haven hospital background/case studies: the approval of pr-sdp by the fda provided an opportunity to improve the safety of our platelet inventory across all patient demographics. we outline our approach and address issues we faced during the first months of pr-sdp availability. study design/methods: our nursing education team provided presentations to the nursing and clinical unit support staff. a company-sponsored trainer staffed sessions for the evening/night shifts on the clinical wards. presentations to physicians were made by the blood bank medical staff. information technology personnel created a new product type in the blood bank computer system, tested the abo/rh truth tables, and ensured that billing codes were in place. the necessity for transiently supporting a dual inventory of pr-sdp and conventional platelets led to consultation with the ethics committee and risk management, to confirm that pr-sdp and conventional platelets (c-plts) tested for bacteria ("safety measure" testing) could both be considered the hospital standard of care. we chose to not gamma irradiate any unit of pr-sdp, consistent with the package insert. results/findings: the ethics committee and risk management confirmed that informed consent was not needed for transfusion of pr-sdp. pr-sdp available from our blood supplier incremented monthly. over the first four months of pr-sdp availability, pr-sdp were transfused at our hospital (out of a total of platelets transfused). after months of scale-up, pr-sdp were approximately % of inventory. questions received during the nursing and medical conferences related to: the risk of bacterial contamination with c-plts vs. pr-sdp; toxicology of the pr process; scanning pr-sdp labels into the electronic medical record; and the need to irradiate pr-sdp. our use of a "safety measure" addressed concern over bacterial contamination of c-plts. published pr-sdp toxicology data comparing the content of psoralens in food products such as grapefruit ($ mg per g) to the content in pr-sdp (< ng per ml) addressed toxicology concerns. nursing/it allayed concern over scanning issues with a simple demonstration. finally, we ensured that all parties were aware that fda did not require irradiation of pr-sdp. presentations at the medical conferences were also used as an opportunity to provide transfusion-transmitted disease training and information on platelet utilization. company personnel did not present at medical or nursing conferences per institutional policy. no background/case studies: ensuring platelet supply capability represents a challenge in terms of donor recruitment and inventory management operations. in september , the apheresis collection process (acp) was completely revised to increase the number of products per donation by maximizing the rate of double-platelet donations (dpd). the process review has led to several changes, including the substitution of the pre-donation platelet (plt) count measurement before donation type allocation, in favor of the use of the donor's past donation records. multiple processing steps were eliminated, and the evaluation of plt concentration as a function of time, deduced from complete blood count (cbc) measurements, allowed the centralization of the analysis at the qc department. finally, introducing the concept of non-optimal donations has led to an increase in the proportion of dpd. study design/method: at the donation centers, whole blood (wb) from donors was collected in k edta tubes. plt concentrations were determined at the qc department using the coulter act diff hematology analyzer (beckman coulter). sample tubes were stored at - c and measured at , and hours post-collection. single platelet donations (spd) or dpd were collected using the trima accel. units were pooled and split in elp (extended life platelet, terumo bct) storage bags to mimic spd ( ml; n ) or dpd units ( ml; n ). plt pools were stored at - c under mild agitation for seven days except for dpd, which were split in two -ml bags after h. samples were taken on days and . ph, po and pco , hypotonic shock response (hsr), extent of shape change (esc), cd p expression, atp content, lactate and glucose concentrations were used as in vitroquality markers. results/finding: plt concentration as a function of time, determined from wb cbc measurements, showed no significant difference at h ( pltx /l), h ( pltx /l) and h ( pltx /l) postdonation. dpd can be stored in the same collection bag for h after donation without any significant impact on plt quality markers. plt concentrations were within the manufacturer's acceptable limits ( - pltx / l) before splitting. on day , lactate and pco concentrations increased, and po decreased in dpd. however, these values normalize to those of control units at the expiration day. conclusion: this project was approved by health canada and implemented in our organization in march . there are numerous operational and cost benefits from this process optimization initiative, without significant impact on safety and quality. post-implantation efficiency data will be compared to the targeted % increase in the targeted number of plt units per donation ratio. phased implementation of pathogen-reduced platelets in a health system elizabeth s. allen* , colleen vincent and patricia kopko . university of california -san diego, american red cross background/case studies: pathogen reduced platelets (prp) provide improved safety compared to conventional apheresis platelets, but collection and manufacturing are complex. early evidence shows only - % of double platelet collections meet requirements for pathogen reduction treatment. blood centers need hospitals to implement prp to start manufacturing, but hospitals may not wish to use prp until they can provide the product to all patients. scaling up manufacturing at the blood center and phasing in prp across patient populations meets both parties' needs. we evaluated this strategy at our university health system (transfusion volume: , apheresis platelets annually), which includes two hospitals ( inpatient beds) and an outpatient cancer center. study design/method: before initiation, approval and funding were obtained from the hospital quality council and administration, and stakeholder groups such as hematology/oncology were educated and consensus gained. live training was provided for nurses in the outpatient cancer center (week ) and the bone marrow transplant (bmt) ward (week ). an e-mail communication explained the change to all physicians and nurses. in phase , we implemented prp in the outpatient cancer center. these patients are immunocompromised and do not have access to the immediate advanced critical care of the inpatient environment should a septic reaction occur. in phase , we expanded usage to include the inpatient bmt ward. in phase , we lifted all restrictions so prp could be used throughout the health system, with the goal to reach % prp within months. results/finding: in phase (weeks - ), we requested prp products weekly, based on typical usage in the outpatient cancer center. our blood supplier provided an average of prp weekly (range - ), and prp constituted % of platelet transfusions in the cancer center. in week , excess prp inventory required use of prp in the inpatient bmt ward ahead of schedule, a practice which continued throughout phase . in phase (weeks - ), we formally expanded issuing of prp to include the inpatient bmt ward and requested prp products weekly. our blood supplier provided an average of prp weekly (range - ), and prp constituted % of platelet transfusions in the phased-in areas. in phase (weeks - ), we began issuing prp throughout the health system. our supplier provided an average of prp weekly (range - ), and prp constituted % of all platelet transfusions. scaling-up is ongoing. conclusion: phased implementation of prp by patient group prioritizes patients who stand to benefit most from the product, and allows time for the blood center to scale up manufacturing. background/case studies: maintaining adequate inventory of platelets without significant outdating and waste of product is a constant challenge for many institutions, especially for smaller community hospitals. our health system comprises hospitals including smaller community hospitals (sch) and larger tertiary care medical centers (tcmc). for several years, we have been using a limited internal process of platelet sharing between some of our institutions to successfully reduce platelet wastage. this encouraged us to analyze platelet usage throughout our health system and devise an expanded novel concept of platelet distribution, in partnership with our blood supplier that would allow us to maintain an inventory of apheresis platelet (ap) units at our smaller community hospitals without significantly increasing platelet waste and the associated cost. study design/methods: a "round robin" (rr) transportation system for platelet delivery and pick up was strategically developed with the regional blood center to align with routine delivery of red blood cell (rbc) standing orders. an efficient delivery system was implemented so that the regional blood center would realize reduced supplemental and emergency deliveries of blood components to our hospitals. platelets are transferred at the time of rbc standing order delivery based on a predetermined route schedule. each day, ap are delivered to the sch and the previous day's platelets retrieved (if not transfused), packed in blood center transport boxes, and then picked up by the blood center driver. these platelets typically have a hour shelf life remaining. the same process occurs at the next sch on the route. all retrieved platelets from the sch are delivered to the tcmc which is the last stop on the route. thus, the sch has adequate number of units available for regular transfusion and massive transfusion protocol. results/findings: review of our rr process revealed a significant benefit to our smaller community hospitals as we were able to routinely maintain an ap inventory for patients requiring urgent platelet transfusion. an additional benefit was further decrease in ap waste (table ) resulting in a cost savings of $ k. an additional cost savings of approximately $ k was noted due to decreased cost of emergent platelet transportation. conclusion: our novel rr process of platelet distribution has resulted in improved platelet availability at our smaller community hospitals while maintaining the reduced level of ap waste at our health system from our previous platelet sharing process. we anticipate additional decreases in ap waste as a transfusion vol. supplement s we further streamline our process. with the trending merge of health delivery systems, we predict that other health systems will adopt similar processes to improve platelet availability and reduce waste. post implementation adjustments of our pathogen reduction process jacqueline carlson* , james r stubbs , scott a hammel and manish gandhi . mayo clinic, mayo clinic-rochester background/case studies: the implementation of pathogen reduction for apheresis platelets using cerusv r intercept system for apheresis platelets was a substantial endeavor encompassing many different areas. as with any process change, adjustments and modifications can occur along the way. after implementing % pathogen reduction technology (prt) for apheresis platelets, we made two additional adjustments to our sampling processes to ensure accurate labeling/categorization/branding of our final products. study design/method: our prt validation consisted of apheresis platelet products. each product was tested pre-processing for white blood cell (wbc) content and platelet yield, along with post processing platelet yield. this data was used to calculate our yield and volume retention during processing. we anticipated products with preprocessing yields of . , . , and . x may end up below a . in the final storage bag and would need a post-processing sample to ensure the product met criteria at ! . x platelets. results/finding: during the validation, we discovered one collection was not leukoreduced and two collections started at a . yield but ended with a yield below . . these two discoveries led to adjustments in our prt platelet process. with the wbc failure, we reviewed the wbc count on the sysmex xe- d preprocessing report to see if it would alert us to a potential wbc failure. the review discovered that of results were . or . x / mcl with the exception being the wbc failure with a count of . . further monitoring of the wbc counts discovered a result of . which was tested on the adam r-wbc for wbc count and determined to not be leukoreduced. we decided all sysmex wbc results from the pre-processing sysmex report would be reviewed prior to processing and a wbc result of . will be tested on the adam to confirm a leukoreduced product. we also discovered of ( %) of the . preprocessing yields products ended with a post processing yield < . . we decided to increase the yields requiring post processing samples to include the . . conclusion: we are continuing to sample all collections for a post processing yield so we can be confident that we are releasing products into inventory with a yield of ! . x platelets and to have enough data to accurately determine our volume and yield loss during processing background/case studies: the university of kentucky medical center (ukmc), a large academic hospital with level i trauma center, is supplied with blood products by the kentucky blood center (kbc) on a consignment agreement-based contract. ukmc is kbc's largest consumer of blood products. as platelet usage can vary widely day to day platelet usage projections are provided to kbc by the ukmc blood bank, thereby allowing kbc to act accordingly with a given day's stock (i.e. import vs export). daily platelet projections are based on phone calls asking clinicians working in high-demand locations to estimate their needs. this process can be easily confounded by multiple factors and has undergone multiple adjustments to improve its accuracy. study design/method: daily platelet projection forms from / - / were retrospectively reviewed and compared to actual usage data over that same time. the prediction system used in the ukmc bb up to that time (estimated clinical need ) was evaluated for effectiveness based on: total number of days under-predicted, number of days with large underprediction, average number of units under-predicted, and average difference between prediction and usage. the prediction system was subsequently changed based on this data in ; the revised prediction method (estimated clinical need ) was then evaluated retrospectively using the same data sources covering / - / and then compared to the prior method. results/finding: the average number of platelets transfused from / - / was . u/d with a standard deviation of . u/d; the predicted amount was . u/d. the difference between the predicted amount and the number of units used was - . u/d. % days ( d/month) were under-predicted (average: u/d). % of days ( ) were under-predicted by ! u (average: u; max: u ( x)). the average number of platelets used from / - / was . u/d with a standard deviation of . u/d; the predicted amount was . u/d. the difference between the predicted and units used was a . u/d. % days ( d/ month) were under-predicted (average: . u/d). one day ( %) over this period was under-predicted ! u ( u). conclusion: review of clinical platelet usage over this time identified a relatively stable average daily clinical demand. adjustment of our prediction system to ensure that no, or as few as possible, days were projected for less than that average has markedly reduced catastrophic shortages ( % a %), reduced the number of days under-predicted ( % a %), and decreased the discrepancy on those under-predicted days ( . u a . u). these improvements in estimating usage allow for an increased ability to handle unpredictable events without suddenly straining kbc's supply flexibility or severely limiting ukmc clinical settings. rapid implementation of zika virus (zkv) nat blood donor screening joan dunn williams* , maria noedel , nancy haubert , kenneth hudson , larry morgan , robert shaw , tracy fickett , jamie jue , valerie winkelman , sally caglioti , german leparc , and phillip c williamson . background/case studies: on / / , fda issued a guidance document for "reducing the risk of zkv transmission by blood and blood components". in response, a plan was implemented for mandatory zkv testing for all clients with locally acquired mosquito-borne cases of zkv within weeks; nationwide in weeks. this organization performs testing for clients (blood centers, hospitals) across the country. we report on of manufacturers' (sponsor) provided investigational new drug (ind) protocols. a single project management (pmo) system was used to control all required processes. study design/method: project focus included: clinical trial requirements, client onboarding, lab operations (labs). our objectives were to implement zkv testing for clients within weeks, and an additional clients within weeks. to minimize the impact to labs a staggered implementation was used with tracked/streamlined communications from stakeholders: vendors, institutional review board (irb), it (client and lab based), client services and labs. results/finding: clinical trial requirements increased the complexity of implementing an unlicensed test. documents included donor notification, informed consent, protocol training, staff certification, deviation management, and result reporting. multiple irb documents were required. to ensure accuracy in ind commitments a principle investigator was assigned to labs with client sub-investigators. deliverables were multiple including client requirements, vendor responsibilities and labs. client onboarding included confidentiality agreements between client and sponsor. an immediate zkv based webinar provided materials and understanding of sponsor protocol, lab test system, and client/donor based responsibilities. to facilitate and ensure effective communication, twice weekly conference calls were held. clients sent questions which were facilitated by labs and directed to sponsor. specific to clients were irb documents, it updates/validation for zkv test ordering and result receipt. labs were multifaceted: vendor instruments, assay materials, package inserts, staff training. assessments included: zkv sample volume, throughput, instrument capability/capacity. work requirements included vendor installation, equipment, assay and reagent qualifications, staff training, competency assessments, result reporting. all clients were provided with zkv testing within required timeframes. conclusion: the success in meeting a rapid implementation of zkv testing was largely due to a centralized pmo system which provided a controlled process for sponsor, client, vendor and labs. within lessons learned strength was found in a multi-client onboarding process. a weakness was in understanding instrument test volume capacity throughput which was exceeded during the -week period but overcome during the -week cycle. red blood cells baby units traceability and discard in kuwait central blood bank and five hospitals marwa moemen al deeb* , hala samuel boules , fatemah saleh al matroud , rabab hussien ali dashti , hanan alawadhi and reem al radwan . kuwait central blood bank, kuwait central blood bank, kuwait central blood bank background/case studies: ill children are more likely to receive red blood cells (rbcs) transfusion than any other patient age group. rbcs are the component most often transfused during neonatal period. small volume aliquots are used to limit donor exposure, prevent circulation overload and decrease donor related risk. traceability is the ability to trace each individual unit from donor to recipient or disposal. blood component should be fully traceable from collection to final disposition. the kuwait central blood bank (kcbb), is preparing baby units and distributing it to all hospitals all over the country. kcbb, being accredited by the american association of blood banks (aabb), is following the aabb's regulations in tracing every component. study design/method: this is a retrospective study to assess final deposition and the percentage of discard of prepared packed rbcs baby units in the kcbb and five hospital blood banks (hbb). also, to assess the levels of traceability as a reflection of the improvement in the efficient use of these blood products. methods: a total of rbcs baby units were randomly chosen to be traced to their final deposition from the year till . half of them ( units) were traced in kcbb. tables showing the numbers of the chosen units were distributed to the five governmental hbb ( units for each year of the study period). results/finding: preliminary results show that the tracing of rbcs baby units in the kcbb is % efficient. results from other hospitals are under process. statistical analysis of the traceability will be done as soon as the data is collected. the study will analyze the usage of the baby units in different departments and the percentage of discarded units. the traceability of rbcs baby units in the kcbb is excellent, this is due to good management and training of the working staff and the use of an electronic system in registration and issuing. most of the kuwait governmental hospitals are using electronic systems, so the traceability should be up to the recommended levels. the percentage of discard of the baby units in the hospitals is very high. this may be due to the practice of using fresh blood (< days of donation) and the reservation of the baby units of the same donor to the same baby to reduce the hazards of multiple donor exposure. the creation of a national policy for using rbcs baby units is highly recommended to reduce the discard of such units. we also calculated the number of false positive results. the study traced all products through mid-march . results/findings: a total of products were tested. fifteen units ( %) had a false positive result and could not have their life span extended. of the fifteen reactive units, two repeat donors were identified and their charts were marked to not test subsequent donations. cross-reactive antibodies were identified in all by the vendor and none were true positives by re-culture. of the units that were successfully tested, were tested again on day for use on day ( %). there were platelets transfused ( %) and expired after day ( %). the cost to test the products including controls was $ , and our calculated cost to produce products would be $ , . if we had needed to import products to meet needs, the cost would be roughly $ , without shipping costs which are estimated at $ , . . we averaged expired platelet products per month (range - ) before verax testing and (range - ) after implementation. conclusion: using verax point-of-care testing saved platelet products from discard. the cost savings were $ , . from importing and $ , from producing a replacement for those products. the average discard rate per month went from to after verax implementation. extending platelet shelf life to days more than paid for the cost of testing and ensured products were available for patients who needed them. secure text messaging in transfusion medicine: can texting decrease wastage? melanie estrella* and elsie lee. george washington university hospital background/case studies: secure text messaging in hospital settings allows for quick, easy, and hipaa compliant communication between members of patient care teams. it works on a mobile phone or computer, and provides read-receipt confirmation and a temporary record of team communications. secure texting has potential to be a useful management tool in transfusion medicine in reducing blood product wastage. for example, it provides a relatively low-burden means for busy clinicians to provide feedback to the transfusion service about scenarios of potential wastage. this information can be used to identify areas in which management strategies could be developed. it also allows for personalized educational opportunities between clinicians and the blood bank about usage guidelines and how to reduce future wastage. the goal of this study is to use secure texting to investigate wastage, evaluate the responses from clinicians, and evaluate the potential effects on reducing wastage. it is hoped that the results will identify secure texting as a useful management tool in transfusion medicine. study design/method: wastage records that were investigated without the assistance of secure texting from july to december were reviewed to identify the most common scenarios of preventable blood product wastage. wastage records from january to april were reviewed, and wasted products that were considered preventable were investigated using secure texting to communicate with the ordering physician. results/finding: for data, units were investigated without the use of secure texting. of these, units were identified as preventable wastage, and wasted units were considered beyond the control of the clinician. the categories for preventable wastage were defined as follows: ) product not released after procedure/ or when patient stabilized ( ) ) product returned outside of appropriate temperature range ( ) ) clinician unaware product was assigned ( ). thus far in , wastage records have identified units of preventable wastage. secure texting was used by a transfusion service physician to investigate. twelve responses provided useful feedback for future management strategies, responses thanked the transfusion service for the information, and in instances, the message was read with no reply. conclusion: secure text messaging has the potential to improve communication in transfusion medicine. it is easy to use, hipaa compliant, and helps identify strategies for reducing wastage by improving communication and allowing personalized educational opportunities between ordering physicians and the transfusion service. sequence of reagent adding for cryopreservation freezing solution guoling chen*, xu zhao, andrew tiss, sasha turner, devin emerson, manijeh shemirani, sharon novak, david garvin, john eng and wanxing cui. medstar georgetown university hospital background/case studies: dimethyl sulfoxide (dmso), plasmalyte-a (plas-a), human serum albumin (hsa) are widely used to prepare cryopreservation freezing solution. some use autologous plasma instead of plas-a and hsa. this study is to identify the choice of reagents and the optimal sequence of adding these reagents when making freezing solution. study design/methods: materials: . % dmso, plas-a, % hsa, autologous plasma extracted. containers: transfer pack (bag) and polystyrene tubes. the freezing solution recipe used in this study is (volume ratio) . %dmso: plas-a : %hsa : : . plas-a and hsa are kept at room temperature ( - c, rt) and refrigerated at c, plasma at rt (to simulate the end-of-centrifuge temperature), dmso at rt (due to high freezing point . c). different combinations of the reagents choice, storage temperature, adding sequence, are tested with photo taken. total tests. at least minutes cooling after dmso, before adding the next reagent. see table: ( ) after directly adding . % dmso alone to bag, the bag turned from transparent to white, so dmso should not add first. ( ) in tube, autologous plasma first, dmso next, powder-like precipitates. ( ) in tube, dmso first, hsa next, precipitated instantly, a layered appearance. ( ) & ( ) in tube, plas-a first, then hsa, dmso at last, precipitates formed; rt plas-a and hsa combination formed a thicker precipitate than those kept at c. ( )&( ) in tube, hsa first, dmso next: precipitation formed heavily, sculpture shape. precipitation in the c group is slightly milder/slower than rt group. so hsa should not be added first. ( )&( ) trace of hsa(< ml) was mixed into the plas-a bag ( ml). in tube, such "hsa-contaminated" plas-a was added first, then dmso, small fragments of precipitates formed, so dmso should not add last. background/case studies: maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care for all major hospitals. however, daily blood product use is difficult to anticipate. platelet products are the most variable in daily usage, have short shelf lives, and are also one of the more expensive products to produce, test, and store. due to the combination of absolute need, uncertain daily demand, and short shelflife, platelet products are also frequently wasted due to expiration. sophisticated data analysis has the potential to accurately predict hospital wide platelet needs and therefor reduce wastage. study design/method: we have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive -month period. using a convex statistical formulation, we have found that platelet usage is highly dependent on several factors. these include day of week, number of abnormal cbc, location-specific hospital census data, and other less important factors. we exploited this relationship to develop a mathematical model to guide collection and ordering strategy. results/finding: this model minimizes waste due to expiration while never allowing for a shortage; the number of remaining platelet units at the end of any day never drops below in our model. compared with historical expiration rates during the same period, our model reduces the expiration rate from . % to . %. with an annual platelet usage of approximately , units, this reduction equates to approximately units saved from expiration annually. depending on platelet pricing in different regions, this accounts for annual savings between $ , to $ , , per institution. conclusion: to our knowledge our research is the first such use of hospital wide data to inform real-time donor recruitment strategies based on anticipated patient demand. thawed plasma implementation: signficant cost savings and decreased plasma wastage morvarid moayeri* , russell thorsen , rosaline ma , antonio g insigne , amy decourten , florence panganiban , patricia mckean , cyril jacquot , sara bakhtary and ashok nambiar . ucsf health, children's national medical center background/case studies: plasma (ffp, pf , pf rt ) stored at - c outdates hours after thawing. if collected in a functionally closed system, it may be relabeled as thawed plasma (tp), extending expiration to days from the thaw date. although coagulation factor levels decrease over this period, they remain above hemostatic levels. as tp can be safely used for the vast majority of patients requiring coagulation support, we implemented use of tp in our multi-site tertiary care system, with the aim of decreasing costs and minimizing wastage. study design/methods: the massive transfusion protocol at our instiution already allowed the use of group ab tp. following a review of literature and practice at other large centers, the transfusion committee extended the approval of tp to all patients. neonates (< months), patients undergoing plasmapheresis and those with factor deficiency or other disorders for which we also noted a significant decrease (not quantified) in technologist time and effort, as less time was expended on the following: thawing units, printing inventory reports and reporting/record-keeping for discarded units. conclusion: in many large facilities, providers frequently order more plasma units than are ultimately transfused, leading to high plasma wastage rates due to limited ( hr) shelf-life. tp has an extended shelf-life, and can be used interchangeably with ffp and pf for most patients. implementing tp in a multi-site tertiary health care system resulted in sustained decrease in plasma wastage, saving thousands of dollars and helping conserve a precious resource. the merging of immunohematology reference lab's (irl) inventories-using technology to create advanced search functions alexander delk and richard gammon* . oneblood, oneblood, inc. background/case studies: immunohematology reference labs (irls) must maintain diverse inventory of antisera to aid in antibody identification, antigen type rbc units, and meet regulatory requirements. when our current organization was established, two irl sites had independent inventory management systems. although the purpose of maintaining the antisera inventory was the same, organization, storage, & access to instructions for use (ifus) were not. our irl developed a synergistic method to organize and store antisera coupled with in-house designed custom excel spreadsheet to organize and search antisera and view ifus. study design/method: a list of similarities and differences was constructed. best practices of both methods were identified. we determined that our antisera could be broadly classified/organized into two main categories: rare and bulk for screening. sequential lab assigned numbers were given to antiserum for each category: s (rare sera) and b (bulk sera). a dynamic/static freezer box storage system that was inter-box static and intra-box dynamic was determined to be best option to combine two inventories while conserving elements of each allowing for library growth. antisera assigned to a box remained in that box, but may be moved within the box. the box itself may be moved among freezers. to track boxes, location and movement within the box, a custom excel spreadsheet was created. its location tracking feature allowed for two different storage methods to function in one spreadsheet. the spreadsheet had a tab for s and b antisera categories. abo group, desired and unwanted antibodies filters allowed quick search for appropriate antisera. the spreadsheet also had hyperlinks to scanned ifus. results/finding: sequential lab s and b numbers were assigned to new additions using a dynamic/static storage system. an excel spreadsheet with scanned ifus (hyperlinks) was used. pre-merger systems, it took on average - minutes to choose an antiserum and obtain the appropriate ifu. post-merger system was reduced to on average - minutes. (table) conclusion: the merging of two irl's antisera inventories resulted in a need for innovation to create an inventory management system with an advanced search function and hyperlinked ifus. this process saved valuable technologist time and organized the antisera more efficiently. abstract continue to flash until they are removed from shelf and their status updated in our database. 'units allocated' tab includes truncated patient name (to protect privacy), unit number, component type, allocation and expiration date/time, and time since allocation, with a flashing alert for units expiring in < hours. the xm/hla platelets tab provides patient names and status of units allocated. a 'trxn/xmplat' tab lists pending transfusion reactions and platelet cross match reports. dashboard eliminated the printing (several times/shift) of lengthy computer-generated reports, simplified thawed plasma inventory management and helped decrease plasma wastage (from % to %). conclusion: using in-house talent and minimal capital expenditure, we designed and implemented a dynamic web-based dashboard for managing blood product inventory across a multi-site transfusion service. the dashboard is stable, customizable and requires little maintenance. initially built to optimize inventory display for thawed plasma implementation, the dashboard was expanded to include all allogeneic blood products. over the past year, this tool has replaced manual processes for monitoring and rotating inventory and directly helped decrease plasma wastage. use of deglycerolized red blood cells for hospital transfusion service inventory management ronnie l. hill*, jason corley and lizabeth ostiguin. us army background/case studies: regional blood shortages have been documented across the united states during the winter holiday timeframe. deglycerolized red blood cells (drbcs) have been shown to be an effective alternative though more expensive to manufacture. this study looks into the fiscal and inventory efficacy of using drbcs to meet the needs of transfusion services during times of blood shortage. study design/method: on three separate occasions, a medium sized dod donor center used its frozen blood inventory to produce type o drbcs to meet the needs of two regional transfusion services. all frozen red cells were manufactured by an offsite facility with the haemonetics acp- using the low glycerol ( %) freezing method and frozen at - c within six days of collection. thawing occurred in a c water bath in the following order: o positive and o negative on january ; o positive and o negative on february ; and o negative on february . deglycerolization occurred on site using the acp- with all units passing internal qc requirements. drbcs were shipped the same day to a hospital transfusion service, allowing for days of shelf life prior to expiration. results/finding: during the three events, all supported transfusion services and the blood center were below minimum inventory requirements for standard type o red blood cells (rbcs). o positive rbcs were only available through nbe at $ - and had the limitation of arrival on the next business day. collection and processing time of liquid rbcs takes approximately two days including: donor screening, phlebotomy, component processing, testing, and labeling. drbcs cost the dod on average $ per unit to produce and distribute. drbcs have a shorter shelf life, days versus the days for other rbcs, but are washed during deglycerolization and thus produce fewer transfusion reactions. one tech can operate up to four acp- 's and deglycerolize four units at a time. in january and february , it took one tech four hours per iteration of eight units to include thawing, labeling, and packing for shipment. conclusion: while not as readily available as traditional rbcs, drbcs can be an effective product to bridge the inventory gap when small numbers of units are needed due to reduced inventory. collection and processing of whole blood into components takes approximately two days, but can produce greater numbers of units in that timeframe. based on this, drbcs can be ready faster than freshly collected units of blood. there is an increased cost associated with manufacturing frbcs which is compensated for by the longer available shelf life of years. having a small contingency supply of frozen red cells and deglycerolization equipment has been effective on three occasions in ensuring availability of type o red blood cells for hospital transfusion services. validation of a human anti-tetanus toxoid immunoglobulin assay performed on the abbott c izekial butler* , karen leighton , scott jones and rachel beddard . qualtex laboratories, biobridge global background/case studies: plasma fractionators require anti-tetanus quantitative testing to be performed on plasma samples collected from individual donors or plasma production pools. this testing serves as a quality control test and helps estimate the antibody potency of the product. the binding site, human anti-tetanus toxoid immunoglobulin liquid reagent kit is for use on a turbidimetric analyzer. the aim was to optimize and validate the human anti-tetanus toxoid immunoglobulin liquid reagent kit for use on a photometric analyzer. study design/method: experiments were performed in order to determine the optimal amount required of reagent buffer and latex reagent from the anti-tetanus toxoid immunoglobulin kit utilizing the abbott c instrument. precision of the new assay parameters was determined by testing replicates of a panel of samples at three concentrations of tetanus antibody in a single testing run. the panel samples were created by spiking appropriate amount of a who tetanus antibody standard into sodium citrate plasma. accuracy was determined by testing a series of samples ranging from iu/ml to iu/ml of tetanus antibody. the samples for the accuracy study were created by diluting an appropriate amount of a who tetanus antibody standard with sample diluent from the reagent kit. linearity regression was determined by using the accuracy study values within the range of . to . iu/ml. stability of samples was determined by testing samples stored at - c and - c in triplicate at various time intervals. results/finding: the %cv for the optimized anti-tetanus assay for all antibody levels determined in the precision study varied from . to . . so, precision was acceptable since the %cv for all samples tested was %. the mean values for the samples tested in the accuracy study were all % of the expected value which was much lower than the acceptance criteria which was % of the expected value. the linearity of the assay was acceptable with a r ! . %. the linearity study established that the known tetanus concentration was a statistically significant predictor of the observed concentration. the sample stability studies demonstrated the ability to quantitate tetanus antibody concentrations in samples stored up to days at - c and up to month at - c. conclusion: the data presented shows the successful optimization of the human anti-tetanus immunoglobulin reagent kit for use on a photometric analyzer. validation studies of this optimized assay demonstrate excellent accuracy, precision and linearity using samples stored for days at - c and stored up to one month at - c. a deep dive audit of intravenous immunoglobulin use for immune thrombocytopenia: is its use inappropriate? jiajia liu*. university of toronto background/case studies: intravenous immunoglobulin (ivig) is a generally safe and effective therapy for immune thrombocytopenia (itp) but is only suggested for scenarios when a rapid increase in platelet count is desired or as first line therapy if steroids are contraindicated. due to concerns regarding adverse effects, cost and resource availability, an ivig request form was implemented in our jurisdiction in to track utilization and appropriateness. a recent audit of these request forms from four academic institutions found a lack of compliance with form requirements and inadequate documentation of efficacy which led the authors to conclude that the use of ivig was broadly inappropriate (shih et al, ) . as such, we aimed to conduct an extensive chart review of patients who received ivig for itp at our institution to assess appropriateness of use. study design/method: we conducted a retrospective chart review of all patients with itp who received ivig in our institution from april , to march , . local research ethics board approval was obtained. results/finding: patients received ivig for itp at smh over the study period for a total of unique ivig infusions. the most common indications for ivig within currently accepted guidelines were: active bleeding ( , %), pre-operative or antepartum care ( , %), a platelet count of less than and contraindication to corticosteroids ( , %). additional indications that still fell within accepted guideline recommendations included: patients with arterial/venous thromboembolism or risk thereof requiring initiation of antithrombotic therapy; and patients requiring myelosuppressive chemotherapy. indications that fell outside of guidelines included: use of ivig as a diagnostic challenge where the etiology of thrombocytopenia was unclear and use prior to international travel for patients with difficult-to-treat chronic itp despite a platelet count between - x /l. patients received ivig for a likely diagnosis itp while a transfusion being investigated for alternative explanations for thrombocytopenia. three patients were refractory to all other therapy for itp and were dependent on regular ivig infusions. / ( %) of infusions consisted of g/kg over days; the remainder of infusions consisted of g/kg. of those who received g/kg, of patients ( %) had evidence of partial remission after a first g/kg dose. ivig was generally well tolerated and infusion reactions were mitigated with use of corticosteroids, antipyretics and/or antihistamines. conclusion: we found, at our institution, that use of ivig for itp was generally appropriate and carefully considered even in cases that did not meet current guideline recommendations. we believe that ivig remains an important treatment for itp particularly in the aging population where prevalence of conditions complicating bleeding risk is increasing. detailed utilization/ knowledge data inquiries are required to develop tools and policies to enhance appropriate ivig use in multiple settings. we believe that there is an opportunity to promote administration of a single g/kg dose to minimize unnecessary utilization of ivig amongst hematologists who manage itp. a process for improving crossmatch bench ergonomics janet dornfeld*, sheng-chung cheng, ann eggebrecht, beth greer, savannahsue rondeau, brian rognholt and beth taylor. mayo clinic background/case studies: a mission of our institution is to reduce the risk of work-related injuries. accordingly, each year an ergonomic survey is undertaken as a component of a general department of laboratory medicine and pathology safety audit. our survey identified potential musculoskeletal risks that suggested a redesign of our crossmatch benches. study design/methods: a seven item ergonomics survey of the working environment was sent to staff members in early february of . twenty-two technologists responded for a % response rate. the below table below reports the survey items and responses. results/findings: the most problematic area was the available workspace. of the respondents, % indicated that workspace size was insufficient and % that the chairs at the fixed height benches were problematic. problems noted were difficulty with climbing up into a chair and backing down and with the chairs holding the chosen height. our laboratory lean team operational support group was tasked to aid with the bench redesign and to choose products for improving the workspace. our goals were to design a layout to streamline testing workflow and better utilize lab space, including our plasma thawing and sink space, eliminating dead space. the configuration of the new workspace was guided by the survey findings. adjustable height workstations were recommended to replace our fixed height bench. we worked with our facilities design contactor to purchase adjustable benches and plan add-on cabinet shop work. the benches were assembled off site, which allowed a bench top layout to be determined and installation of cabinet shop add-ons of a drawer for supplies and a pull out breadboard as a writing surface. the opportunity to assemble off site streamlined the process of installation, resulting in minimal disruption of testing. conclusion: the survey was effective in identifying working areas for improvement. employee comments have been positive for the new workstations. an effectiveness assessment will follow, using the original survey, to assess the success of the project. a retrospective study of emergency department initiated type and screen testing: were patients transfused after testing? sandra lamm* , neil bangs and kimberly sanford . vcu health system, virginia commonwealth university background/case studies: type and screen (t&s) testing is often ordered on patients presenting in the emergency department (ed). if the patient does not have a historical type, a second sample is drawn with an additional phlebotomy for type confirmation. if the patient does not need a transfusion of red blood cells (rbcs), the testing and second phlebotomy is an inefficient use of resources and time. study design/method: as part of a performance improvement initiative in transfusion medicine, we performed a retrospective study of all t&s orders that were initiated in the ed from / / to / / to determine if testing was subsequently followed by transfusion of blood products. patients were stratified by ed department, time from t&s draw (tsd) to transfusion (< hours, > hours < hours), and if a second sample was required. results/finding: a total of t&s orders were initiated from the ed in this time period. ( . %) patients were not subsequently transfused any type of blood product within hours of tsd and ( . %) patients were not subsequently transfused any type of blood product within hours of tsd. a total of ( . %) patients required a second sample. of these patients requiring a second sample, ( . %) were not subsequently transfused any type of blood product within hours of tsd and ( %) were not subsequently transfused any type of blood product within hours of tsd. conclusion: routine ordering of t&s testing is not an efficient use of resources and time as many patients are not subsequently transfused. ultimately unnecessary t&s and second sample collection and testing for those patients not subsequently transfused within hours of tsd amounted to an estimated $ , in unnecessary patient charges and approximately . nursing hours for phlebotomies in a six month period. anti-d from alloimmunization versus rh immune globulin: detective work in the blood bank and transfusion medicine services (bbtms) margaret diguardo* , debra berry , yunchuan delores mo and gay wehrli . university of virginia health system, children's national medical center background/case studies: the institute for healthcare improvement triple aim incorporates enhancing patient satisfaction by providing high quality, safe care. towards these goals the bbtms is charged with communicating to obstetric physicians (obs) a patient's antibody specificity with associated hemolytic disease of the fetus/newborn risk. thus, when anti-d is detected in a female of childbearing age, it is critical to determine whether this represents rh immune globulin (rhig) or alloimmunization (alloanti-d). review of a patient's electronic health record (ehr) helps quickly identify rhig administration, but if this documentation is missing, then it is easy to assume presence of alloanti-d. rhd alloimmunization impacts mom, fetus, newborn and future pregnancies. therefore, without a national, comprehensive health information exchange (hie) system, it is imperative to investigate beyond the on-site ehr whether a patient received rhig at an outside hospital (oh). we report an irb approved (exempt) case series where detective work revealed rhig administration at ohs. study design/method: over a two month time period, anti-d was identified in four pregnant women. review of their ehrs did not reveal a history of abstract rhig administration; nor did subsequent direct communication with their obstetricians (ob) reveal a history of rhig. based on each patient's home address, the bbtms of any nearby ohs were contacted as was a primary care physician if listed in the ehr. results/finding: investigations beyond the ehr and obs revealed each of the four patients received discontinuous prenatal care with presentations at multiple sites. through phone calls to the bbtms of ohs, a history of one or more rhig administrations within the preceding three months was found for each patient. our bbtms records and ehr were amended to reflect the presence of a passive anti-d due to rhig, rather than alloanti-d. the changes were also directly communicated with the ob caring for each patient. conclusion: when a new anti-d is identified in a pregnant female, investigation is required to determine whether it is passive rhig versus alloanti-d. when neither the ehr patient history or ob reveal a rhig history, it remains in the patient's best interest to investigate further. through phone calls to oh we revealed a history of rhig administration in four patients. finding and communicating this critical information helps enhance the quality and safety of patient by ensuring subsequent rhig administrations when indicated, at our institution. future strategies for avoiding similar situations include expanding our national hie for critical information such as bbtm history and allergy history and expanding use of wallet-size patient identification cards with rhig and alloantibody histories. auditing massive transfusion protocol colleen a. aronson* , elizabeth halperin , sharon breining and mona papari . acl laboratories/ advocate hospitals, advocate health care, itxm background/case studies: a large midwest hospital system with level i trauma sites evaluated how to audit the massive transfusion protocol (mtp). the possibility of real time audits is impractical due to the unpredictability of these events. a search of the internet found an example from new zealand for post process evaluation. this was shared with a team as a starting point and then adjusted for system specific priorities. to start the audit, the initiation of the mtp needed to be determined as events are often started as a verbal request but then followed up with either downtime or computer orders. study design/method: the transfusion service (ts) was determined to be the source of truth for all of the mtp events. a tracking sheet was created to capture the patient demographics, start and stop time, number and type of products issued and wastage. this was then passed onto nursing quality staff that used the tracking form and the patient chart to enter an event into the error management data base as a focused event. the focused event was built to include patient demographics and other information from the tracking form as well as where the event was called (surgery (or), emergency (ed), labor and delivery (l&d), etc.), type of event, use of tranexamic acid (txa), calcium chloride (cacl), temperature monitoring and pre/ post lab results. a trial was started and months of data were evaluated that contained events. results/finding: there was an equal number of events that were initiated in the ed and the or ( ). male patients were involved % of the time and % of time the patients expired. trauma of some type was the majority of the cause but . % of the cases involved gi bleed and only . % were obstetric cases (see chart). the lowest hemoglobin (hgb) was found to average . with the post hgb average of . . ratios of : for red blood cells (rbc) to plasma as well as rbc to platelets (plt) and cryoprecipitate (cryo) were also determined with a target of : . it was found that the rbc: plasma was . : , rbc: plt was . : and rbc to cryo was . : . use of txa was only . % and cacl was utilized in . % of cases. conclusion: although this data is for a short period of time it has pointed out several opportunities for improvement. the use of mtp in gi cases was not previously understood but opens up a new group of people for which education and understanding of the mtp process is needed. the low use of txa needs to be evaluated and already has started conversations about how this drug should be stored and accessed for the mtp process. the product ratio numbers were suspected of being off but now that data is available, it is much easier to speak to this issue and look for improvement. the process will now be expanded to the level ii trauma sites in the system and routine evaluation will be shared with all sites. automated report significantly reduces turnaround time for rbc antibody alert jessica l dillon* , jody a barna , donald e ulinski and nancy m. dunbar . dartmouth hitchcock medical center, dartmouth-hitchcock medical center background/case studies: clinically significant antibodies should be promptly and clearly communicated to the patients' healthcare team to avoid potential transfusion delays in blood availability or complications of incompatible transfusion. at our institution, all newly identified clinically significant antibodies are immediately resulted in the electronic medical record (emr). an interpretative comment is also entered by the transfusion medicine service (tms) physician after the antibody work-up has been reviewed (this may be up to weeks after the antibody is identified). this comment describes the antibody(ies) identified, indicates the need for crossmatch compatible blood and alerts clinicians of possible delays in providing crossmatched units. since clinicians may not always review these results, the tms physician also simultaneously adds an "allergy to red blood cells" alert in the patient emr at the time the interpretive comment is entered. study design/methods: in july , we implemented an automated report to reduce the turnaround time (tat) for entry of the allergy alert. the report contains all detected red cell antibodies in the prior hours and is provided to the tms physician during daily morning rounds (monday through friday) for manual entry of allergy alerts. this study describes a three month comparison both before and after the automated report intervention, to evaluate the tat for allergy alert entry into the emr. age ( abstract results/findings: between august and november (pre-implementation) , newly identified clinically significant antibodies were resulted for patients compared to patients between the months of august and november (post-implementation). the tat for allergy alert entry for both periods is shown in table . we observed that % of allergy comments were performed within hours in the post-implementation period versus only % pre-intervention (p . ). using the new process, nearly all of the alerts were entered into the emr within hours of antibody resulting and none of the entries were missed. conclusion: there was a significant improvement in the tat for allergy comment entry following implementation of an automated report. this project illustrates how information technology can be leveraged to facilitate timely communication of antibody identification. blood bank verbal tool implementation for cardiovascular surgery rita louie* , shailesh macwan , nancy nikolis , arline stein , janelle richardson , manju bagu , lennart logdberg , alexander indrikovs , vishesh chhibber and sherry shariatmadar . north shore university hospital, northwell health background/case studies: our institution is a tertiary care facility performing over cardiovascular surgeries (cvs) in , an increase of % after the healthcare system cvs integration in . transfusion support of these patients includes preoperative preparation of prbcs according to a maximum surgical blood order schedule. additional blood components are issued as orders are placed. until december , the additional written orders were submitted to the blood bank via the pneumatic tube system without further communication. after reported events in q that resulted in delays in blood transfusion, we examined our process very closely and identified opportunities for improvements. in collaboration with cvs, the blood bank implemented a new workflow process to enhance communication with the cvs team, reduce turnaround time and improve patient safety. study design/method: . open discussions and collaboration between blood bank and cvs nursing teams . mapping the process using flowcharts for additional blood orders from cvs. . identify bottlenecks and brainstorm solutions. . a verbal cvs order process and form was implemented to improve communication between cvs and blood bank, which solidified communication by including the time of the order, patient identifiers, caller identification, ordering prescriber, staff receiving order, the quantity and kinds of products ordered, the mode of order delivery, and anticipated future orders. a read back was also documented for verification of the order. . the blood bank staff immediately processes this order while waiting for the written order to arrive. upon receipt of the written order the blood is issued to the or. . follow plan-do-check-act. the transfusion safety officer reviews each order for the following parameters: number/type of products, turn around times (tat), wastage/returned products and overall efficacy since implementation of this process. results/finding: a significant improvement was noted in communication and tat after implementation of the process described above. for the period / / - / / the blood bank has received verbal orders with varying product combinations. the table below represents average turn-around times to issue blood products: conclusion: the introduction of the verbal order tool for cvs has streamlined the blood ordering process leading to increased efficiency and lower tat. effective communication between the or team and transfusion service is the key to timely provision of blood products for these critical patients. challenge of blood type testing for multiply transfused sickle cell disease patients jayanna slayten* , tracie ingle and heather vaught . indiana university health, indiana university health (iu health) background/case studies: we report our midwestern, university transfusion service challenge of obtaining the correct blood types in rbc exchanged sickle cell disease (scd) patients tested by our primary testing method, solid-phase red cell adherence analyzer echo (immucor. norcross, ga). the echo operation manual in chapter - and appendix d it states: "warning: the galileo echo cannot reliably detect hemagglutination reactions that are graded as or less in tube methodology. the galileo echo does not generate as interpretation of mixed-field. such a mixed-field reaction will be interpreted as positive, negative, or equivocal." we report of a challenge with this analyzer limitation which impacts the assignment of the correct blood type for multiply transfused scd patients. study design/method: two scd when initially tested by the echo as o, d negative; however, each patient was historically o, d positive. both patients had received a rbc exchange transfusion with - o, d negative red blood cells over days previously. repeat testing of the samples was completed by the vision (ortho clinical diagnostics. raritan, nj), neo (immucor. norcross, ga), and by standard abo/rh manual testing (anti-a, anti-b, anti-d series , anti-d series , a cell and b cells. immucor. norcross, ga). the repeat testing was compared to verify the patient's abo/rh typing and the results were entered into the computer system to allow for assigning the patient's abo/rh typing and electronic crossmatch. results/finding: table summarizes the initial and repeat testing with the two patient samples. although the echo failed to interpret or flag the blood type as mixed-field, the other methods identified the transfusion of o, d negative blood with the detection of mixed-field in the d typing or by failing to interpret the abo/rh as not type determined (ntd). the vision and manual abo/rh typing yielded the easiest mixed-field to interpret macroscopically. conclusion: our results agree with the findings of summers et al (trans-fusion ; : - who reported the challenge detection of mixed-field with the use of the echo compared to improved detected with automated gel column agglutination. when the samples were tested by multiple automated and manual abo/rh methods, the expected mixed-field was detected. the failure of the echo to detect the mixed-field is acknowledged by manufacturer, but there is a risk that a facility may mistype the abo/rh when there is not a historical abo/rh to compare. to avoid this risk, it may be appropriate to re-type first time scd patients by other methods rather than the echo to avoid this challenge. consistent with summers do not account for regional distribution. many large hospitals acting as regional hubs for redistribution may appear to have optimized inventory based on odr and bsr, but we hypothesized that these are crude key performance indicators (kpis) requiring redevelopment. study design/method: kpi redevelopment occurred in a large tertiary care hospital blood bank in canada, responsible for % and % of transfusions in the region and province respectively. rbc supply, inventory, and disposition data were retrospectively assessed from february -june as the baseline period. a "demand-driven inventory planning policy" (ddip) was instituted to assess and implement the optimal rbc reorder quantity based on the difference between the historical maximum and minimum rbc inventories during weekdays; that would not lead to blood shortages. shelflife inventory (sli) was chosen as the main surrogate marker for the assessment of efficiency of the supply chain process, calculated by the differences between age of blood transfused (abt) and received (abr). iterative simulation modeling (r statistical software) was then performed to optimize sli in a post-implementation period from june -october . results/finding: modeling predicted observed rbc disposition. through simulation, optimization of sli was found to occur by optimizing a set of kpis for each abo blood group (table ) . this led to a reduction in observed overall sli ( . . days vs . . days, p< . ) and odr ( . % vs . %). the bsr was not significantly increased during the postimplementation period. conclusion: optimization using simulation modeling of multiple factors other than bsr and odr led to further efficiency gains in a large tertiary care hospital blood bank. hospital blood banks should use an integrative approach with a set of kpis to optimize the supply chain. this approach requires validation in other blood banks and jurisdictions. ( )) requires that the hospital make reasonable attempts to notify the patient (or the patient's physician), counsel the patient, and offer testing. the hospital must maintain records of this lookback notification as part of the patient's medical record. paper records of lookback notifications are less accessible than electronic records and are at greater risk of being damaged or lost. to facilitate the lookback process and reduce paper documentation we sought to use the electronic medical record (emr) to perform and document notifications. study design/method: representatives from transfusion medicine (tm) and information technology (it) worked together to define minimum and optimal emr solutions. minimally, a completed paper packet could be scanned into the emr. this solution had no advantages in terms of ease of use, process control, or transparency. desired optimal functionality includes the ability to send letters in the emr, document control so that original communications may not be altered, opportunity for patient's physician to electronically sign and return responses, letter and form templates that can be individualized, and the ability to track when and by whom notifications were sent and received. the emr system at our institution, epic (epic systems corp., verona, wi), has a function called "letters" with the capacity to do all these tasks. a series of five templates were developed: hiv and hcv letters to physicians, response forms for physicians to return to the transfusion service, and a blank letter template to be used for specially tailored letters. templates are opened within the patient's emr and demographic information is automatically populated by epic (eliminating many possibilities for clerical errors), the blood product transfused (e.g. rbcs or plasma) is selected from a drop-down menu, and the date of transfusion is manually entered by the sender. the completed letter is then routed to the patient's physician; it shows up automatically (and instantly) in their electronic in basket as well as in the patient's emr. physicians may electronically complete and return the response form within epic, or print it and return the form by fax. results/finding: between january and december thirty-five ( ) notifications were sent to physicians using epic letters and of those, fourteen ( ) responded to the epic notification and five ( ) used the provided electronic response form. for these cases the time to mail or handdeliver paper notifications was avoided. the remaining cases required follow-up paper notification, but the electronic letter remains as permanent, easily accessible documentation of when the transfusion service first notified the physician. conclusion: lookback notifications within the emr makes compliance with government requirements more transparent and records more accessible to caregivers, patients, and assessors. secondarily, efficiency may be improved by reducing the need to print and mail/deliver letters. evaluation of ordering practice in the operating rooms and its impact on product wastage alexandra budhai* , denden benabdessadek , annu george and alexandra jimenez . westchester medical center, new york blood center background/case studies: blood product wastage is an issue that many hospitals aim to address. the or was identified to have the highest rate of wastage within our hospital. in this study, we assessed the appropriateness of the product order and utilization by the or to understand its impact on wastage. study design/methods: data on product orders, issue, and return for two months were analyzed. the hospital cpoe and product requisition forms were used to collect this data. the surgical procedures and number of ordered units were compared to the hospital's maximum surgical blood order schedule (msbos). trends for inappropriate orders for products by physicians were evaluated. results/findings: a total of orders were reviewed. approximately, % of these products were issued to the or. we found that the physician orders were within the guidelines of the msbos for most cases ( %), but of the issued products, all were returned to the blood bank in % of cases. we observed that the percentage of products ordered and used compared with the products ordered and returned in cardiac surgeries are nearly equal. in addition, all of the products ordered for c-sections were not used; albeit ordering frequency being significantly lower than for cardiac cases. conclusion: the data analyzed demonstrates that the majority of surgeons are adhering to our institutional msbos guidelines. it was noted that surgeons are requesting products be issued for invasive procedures where rapid exsanguination is possible. our analysis revealed that the hospital's msbos does allow for an excess in blood ordering for some surgical procedures. the msbos should be updated to reduce the suggested maximum product order. in general, the data does not imply that the blood product wastage in the or is due to the ordering practices of the surgeons. a larger period of surgical blood ordering practices should be analyzed to detect blood product ordering, utilization and wastage trends in other subspecialties. background/case studies: the visionv r and visionv r max (ortho diagnostics, raritan new jersey) are id-mts tm gel card-based automated immunohematology analyzers marketed for small to medium, and high-volume [> type and screens (t&s) per day] blood banks , respectively. our laboratory which serves a large -bed multispecialty academic hospital and receives - t&s specimens per day needed to replace three provue analyzers prior to the availability of the visionv r max. we implemented three visionv r analyzers to work with our existing neov r and echov r (immucor inc, norcross georgia). a recent multicenter field application trial of the visionv r reported a mean turnaround time (tat) for t&s and abo, rh typing (abo/rh) of . . and . . minutes , respectively. the objective of this study was to determine visionv r tats under routine daily high-volume practice. study design/methods: one visionv r was in operation during a five-week period (phase i), and then two additional analyzers were brought into service (phase ii). tats are defined as the time when the order is received by the instrument to when the test is completed and available for review. three-cell screen and abo/rh tats, and number of visionv r antibody panels were collected for a nine-week period. the tat for the screen was used as the tat for the t&s because the screen is the rate determining step. all testing was performed using in-service analyzers on routine patient samples by trained technologists. samples were not deliberately batched but were placed on the analyzer based on the volume and flow of work at the time. results/findings: under the high volume conditions of our laboratory with three visionv r analyzers, the mean t&s tat was % longer and had a larger standard deviation (s.d.) than the published trial result of . . . transfusion vol. supplement s abstract during phase i visionv r performed panels. during phase ii visionv r performed of the visionv r panels. conclusion: our visionv r analyzers are used under high volume conditions more suitable for the visionv r max. when balanced with the testing menu, including ability to perform select cell panels, our tats using three analyzers were satisfactory. the large standard deviation indicates that opportunities remain for improving tats through workflow improvement. from west nile virus to the emergence of zika virus: a nationwide survey of how regulators are keeping the blood supply safe and available falisha atwell* , john roback , ronald arkin , michael bartlett , robert geiger and jaxk reeves . university of georgia, emory hospital background/case studies: with the emergence of zikv in the united states, it is important to assess the fda's response time in providing guidance to ensure the safety and availability of blood products in the face of newly emerging infectious diseases. this research compares the responsiveness of the fda during west nile virus (wnv) and zika virus (zikv) outbreaks to evaluate our current preparedness. study design/methods: the literature review was conducted to analyze fda's response time during the wnv crisis and determine if it was effective and efficient. the research survey was performed to determine if the donor history questionnaire (dhq) adequately screens donors for zikv as the sole preventive method (as per the february guidance for industry: recommendations for donor screening, deferral and product management to reduce the risk of transfusion-transmission of zika virus) and to determine if the current regulatory practices (including the august guidance for industry: revised recommendations for reducing the risk of zika virus transmission by blood and blood components) are perceived to be effective and efficient in the face of the current zikv outbreak. survey monkey was used and participation was anonymous. over , emails and web-links were sent to members of aabb, scabb, seabb, and personal network with a % target response rate. participants self-selected or deselected based on the inclusion and exclusion criteria listed in the consent letter. results/findings: the literature review revealed that the fda's response was slow during the wnv outbreak, while the zikv response is efficient thus far. a total of participants responded to the survey ( . % response rate). statistically, participant agreement with fda's decisions was performed by "t" test (with n- - df) of the null hypothesis that the mean vs. the alternative that the true mean is> . overall participants had favorable opinions of the fda's decisions. statistically, whether participants in different levels of the demographic variables (region, profession, and years of experience) answer significantly differently, one way anova models were used with likert-scale question responses as if they were continuous. the f-statistic and p-value are for the null hypothesis that all levels of the explanatory variable have the same mean for the response variable. there were no significant differences in the years of experience and profession variables for participants. region was determined to be unreliable due to undefined states for each region listed. conclusion: the research revealed that industry experts conclude that the current system of dhq and fda guidance documents, if issued timely, are adequate. background/case studies: when evaluating a new instrument solution for pre-transfusion testing, it is important to consider the operational impact of the system on the lab. there are a variety of operational, performance and system metrics that can be evaluated to determine this impact including: test workflow, hands on time, and automation time. study design/methods: the study involved a current state to a future state comparison of testing processes with an instrument ortho provuev r (pv) and manual testing vs. an instrument ortho visionv r (ov). data collection methods included direct observation, time studies, and interviews. the pv bench performs type & screens (ts) on the pv and manual abid/selected cell panels in the gel test. all other testing; cord blood(cb), dat, unit confirm(uc), patient type confirm(pc) and crossmatch(xm), etc. are done manually in tube. the future state incorporated the ov. ts, abid and uc were evaluated in both states. cycle time(ct) was averaged based on run cycles. ct was comprised of metrics; instrument time(it), standby time(st) and labor time(lt). st may be comprised of components, time that could be utilized as "walkaway" time or vigilant time (vt) which requires operator presence but not operator action. for automated instruments, vt for each cycle was measured as instrument access unavailable. instrument daily maintenance (dm) ct was evaluated as well. similarly, timing of manual tube test processes used these metrics. for repetitive activities within a process, such as uc or xm, a time per individual process was captured and then multiplied per unit. results/findings: table provides details about the metrics of current state and future state processes. tube based test timing is as follows: pc ( : ), xm ( : ), cb ( : ) and dat ( : ). by implementing the future state, an average $ . min. lt and vt is saved on each sample loaded for ts equating to a % labor reduction over the current state. a % improvement in tat on the ts was achieved in the future state. moving from manual abid to automated processing resulted in a % lt reduction. on average, a min. continuous walk-away time is achieved for each automated abid. uc had less impact on labor time with minimal difference however allowed for focus on consistency and quality metrics. conclusion: based on the metrics evaluated and compared between current state and future state, the ov has demonstrated improvement in lab operations to both the labor required and result tat delivery. opportunity exists to automate workflows on other tests that are still manually performed. background/case studies: high throughput and efficient automation of serologic tests is crucial in the workflow of a blood bank that tests $ type and screen samples per day. the erytrav r (grifols) is a fully-automated walkaway analyzer utilizing -column gel cards for pretransfusion testing. the blood bank validated and implemented the use of erytrav r for abo/d typing, antibody screening and identification of patient samples as a replacement for a solid phase testing platform. the blood bank also validated automation of donor unit retypes. the instrument has bidirectional interface to the blood bank lab information system (lis), hcll tm (hemocare life line, mediware). instrument validation and implementation were done in conjunction with the software version upgrade of hcll tm and an interface system change to maestro tm . study design/method: correlation testing of the erytrav r results with the manual tube testing (peg iat; reference method) was performed on patient samples for abo/d typing and antibody screening; of which at least had a positive antibody screen. out of the , had known antibody specificities. forty-two rbc units were also tested for abo/d confirmation; of which were d(-) and were d( ). calculations of concordance, sensitivity, and specificity were performed. precision studies were also done. interface testing of erytrav r , hcll and the hospital's information system using the maestro tm interface system was performed and validated. results/finding: concordant results between both methods were obtained in all of the patient and donor samples tested ( % concordance). all samples with positive antibody screens were obtained by both methods. all clinically significant antibodies were detected by both systems. erytrav r gave % sensitivity and specificity. the precision studies showed that both methods gave the same type and screen results for samples at different testing events. after validation of the lis upgrade and interface system change, a bidirectional interface with hcll tm was established. the instrument has been operational in our lab for over months. conclusion: erytrav r was found to be reliable and accurate and can handle the high workload of our lab. users found the instrument easy to use; hence training, proficiency, and competency of the users are achievable and manageable. the validation of the the instrument is straightforward. the major challenge and delay in the implementation experienced by this blood bank were attributed to the concurrently occurring lis upgrade and migration of the data integration system. a post-implementation workflow assessment would be ideal to perform to ensure that the instrument is being used at its full potential. implementation of a system-wide platelet inventory report optimizes platelet utilization and reduces unit wastage elly landolfi* , craig fletcher and peter millward . beaumont hospital, beaumont health system background/case studies: a sufficient number of blood components should be available to meet routine and emergent hospital needs. this must be assured while minimizing outdating of scarce and expensive blood components -an inherent challenge with platelet units which have a short -day shelf-life. we report the results of a quality improvement project implementing a custom computerized platelet inventory report designed to mitigate the most common cause for platelet wastage at our institution: high platelet outdate rates. the report includes blood type, product code, unit number, respective product attributes, supplier and availability status of all platelet units for each hospital location. all system blood banks receive a morning fax of the report which facilitates transfer of units prior to expiration and adjustments are more readily made for product orders to the supplier. study design/method: the study was conducted in the hospital-based blood bank and based on available platelet inventory and wastage quality data. the report went live october and quality data was reviewed from august to december . the collected data was then analyzed using descriptive statistical methods. results/finding: data from indicates platelet wastage comprised % of total received platelets and % of these wasted platelet units were due to expiration. other reasons included failed visual inspection, blood dispensed but not used and wasted on the floors, potential tube station problems or short-dated units transferred into our blood bank from another facility. the mean of monthly wasted platelet units months preimplementation of the report was units, compared to units months post-implementation and units months post-implementation. wastage rates improved from % (wasted yearly platelets/total received yearly platelet units) in , the year of report implementation, to post-implementation rates of % in and % in (see table) . importantly, this occurred despite a greater than % increase in platelet inventory between and and resulted in cost savings of over $ , in this period. conclusion: study limitations included restricting data collection to one campus. the option to transfer expiring platelet units to another blood bank was available to all participating sister hospitals. it would have been interesting to see the effect of the report on those hospitals which have lower transfusion rates and different ordering practices. aside from lowering platelet wastage within years of implementation, additional benefits to the report included facilitating ordering from the blood supplier. cornerstones of a successful inventory management plan include daily inventory monitoring and, ideally, coordinated system-wide efforts to share platelet units. we have shown achievement of this end is facilitated by a customized daily platelet inventory report -an efficacious and easily adaptable tool with demonstrable gains. valerie halling* , lisa marie button , lori scanlan-hanson , karen koch , janet finley , deepi goyal and camille van buskirk . mayo clinic-rochester, mayo clinic, mayo clinic rochester background/case studies: transfusions in the emergency department of a level i trauma center were ordered using a handwritten order form. the transfusion lab's (tl) management team and medical director met with emergency department (ed) leadership and it resources in to define the needs of a successful electronic blood transfusion system. the handwritten order forms had several potential error sources which could lead to a delay in filling the order pending correction (in the best of circumstances) or could lead to transfusing the wrong patient or the wrong product if the error was not detected. the potential error sources included clerical errors involving the patient's name or medical record number (mrn), writing two different names on the order form (because there were two locations to record patient name), two product types ordered on one form when the requirement is for one product type per order, no priority indicated (stat or routine), or not including the prescriber call-back information. the number of ed reported transfusion related events in and were / (events/ed transfusions - ). study design/method: electronic ordering for the ed was implemented march st . any transfusion orders generated from the ed are now electronic, unless in the case of electronic downtime. the system electronically fills in the patient's name and mrn, controls for the type of blood product being ordered, requires an order priority and provides service contact information. it was designed to accommodate transfusion ordering needs for adults, pediatric patients < kg and pediatric patients > kg. a transfusion orders had three critical fields identified that are required for the order to be processed including patient weight, product volume, and infusion rate. the electronic system was designed so that an order cannot be submitted unless all critical fields are completed. results/finding: the electronic ordering system has been in place for years (april -march ), and during that time there was instance of blood being ordered for an unintended patient . % ( / ). this was because a previous patient's medical record was accessed rather than the intended patient's medical record. there have been no instances of clerical errors (name misspelled or mrn transposition etc.), missing service contact information, missing order priority information, more than one product type ordered on a single order, or two patient names on one order. electronic ordering also provided a place for the transfusionist to chart against, leading to increased transfusion documentation compliance. prior to electronic order implementation, in , / ( . %) units were transfused in the ed but not charted in the patient's medical record. in , / ( . %) transfusions were not charted. however, in , the first full year of electronic transfusion order capability, only / ( . %) transfusions were not charted in the patient's medical record. conclusion: electronic ordering in the ed has essentially eliminated ordering errors in this area resulting in less rework for both technologists and physicians. it allowed the order to be processed more quickly by tl, resulting in a faster turnaround time. improvement in the overall quality of transfusion ordering through electronic ordering reduced the influence of human factors in order placement and provided an added benefit of having a specific order to chart against. implementation of blood bank automated attendant lok tse*, gerald motta and maria aguad. brigham and women's hospital background/case studies: the blood bank receives numerous nonemergent phone calls on a daily basis. these calls not only occupied valuable time but also made the lines unavailable when a real emergency occurred. the hospital is categorized as a level trauma center, with over inpatient beds and over operating rooms. a proposal to implement a blood bank automated attendant was recommended to decrease phone calls, minimize errors due to distraction from phone calls, free team members to perform other duties and have a direct line designated for requesting trauma coolers, massive transfusion protocol (mtp) and emergency release of blood products. study design/method: the first step was to categorize the types of phone calls received by the blood bank by creating a phone log. data were collected and analyzed for four weeks. the blood bank collaborated with nursing, hospital administrative staff and telecommunication team to evaluate the possibility of implementing an automated attendant to minimize phone calls. it was very important to maintain patient safety and quality of service at the same time. the automated attendant consist of: option (urgent) for trauma, emergency release, mtp and obstetric hemorrhage emergency release; option (verbal) for verbal orders and coolers set up; and option (staff) to speak with staff member. instructions were also given for specimen inquiry and product availability in the hospital information system. results/finding: the data in table showed that most of the incoming calls fall into three categories (specimen inquiries, product order inquiries, and other inquiries). most of the calls were from nursing staff inquiring about the length of wait time for blood products and specimen availability. there was an overall decrease in phone calls by % with the implementation of an automated attendant. conclusion: with the implementation of an automated attendant, the blood bank team was able to identify and respond accordingly and efficiently to urgent requests and verbal phone orders. the decrease in phone calls freed up team members to perform other critical tasks in the department. improved detection of wrong blood in tube errors: implementation of a two-sample blood type verification process ariana king* , steven zibrat , geoffrey wool and angela treml . university of chicago medicine, university of chicago background/case studies: our organization used a blood bank identification (bbid) band system for pre-transfusion testing and detection of wrong blood in tube errors (wbit). additionally, type & screen results were compared to patient's historical records; the specimen was retyped by a second technologist if historical results were not available. the bbid bands were prone to clerical errors and excessive specimen rejections, and believed to miss some wbit errors. in , blood bank accounted for % of all rejected clinical laboratory samples, yet comprised only % of total laboratory volume; % of rejected blood bank samples were due to bbid band issues. the wbit error rate detected by bbid-based system was . %. study design/method: a multidisciplinary workgroup was formed to review data and best practices. the decision was made to discontinue bbid bands and implement a two-sample verification process, in keeping with standards. a new laboratory test order was created in the emr system and embedded into the existing t&s order. providers are prompted to order the abo verification test only when no previous abo/rh typing results are found. education was provided to all clinical staff in the form of in-services, emails, and annual competencies completed electronically. the new process went live in september . results/finding: in the five months following implementation, four wbit errors were detected with the second sample. these may have been missed using the bbid band system. improved detection revealed a wbit error rate of . %, three times the national average of . %. under the new system, rejected blood bank samples decreased from an average of % to % of all rejected laboratory samples, a % decrease. implementation of the new process produced a net savings of $ . k. conclusion: replacing the bbid band system with two-sample verification successfully improved our ability to detect wbit errors among patients who lacked historical blood bank results. additionally, discontinuation of the bbid system decreased the incidence of clerical errors and unnecessary specimen rejections, and also saved money for the organization. next steps are for blood bank and laboratory quality leaders to partner with nursing leadership to drive down wbit error incidence. a addendum with the final culture results. we used a student's t test to determine whether there was a statistically significant difference in the mean tat for result addendum entry in the post-implementation period compared to the pre-implementation period. results/findings: in the pre-implementation period, we cultured residual products for suspected str. the tat for final culture result entry into the patient's emr was - days (mean days, sd ). in the postimplementation period, we cultured residual products for suspected str. the tat for final culture result entry into the patient's emr was - days (mean days, sd ; p . ). there were no positive cultures during either study period. conclusion: our study demonstrates that tat for documentation can improve with the use of information technology to notify the transfusion medicine physician when results are available for documentation in a patient's emr. improved turnaround time of type and screen samples michaelene hultman* , marcus holme , johnathan bakst , gunta musa and angela treml . university of chicago medicine, university of chicago background/case studies: the primary test performed in the blood bank with regard to pre-transfusion testing is the type and screen (tys). the current target for this institution's blood bank is an minute turnaround time (tat). in april of , the blood bank was forced to move to a temporary location due to building construction, which necessitated a switch from automated solid phase methodology to manual gel method. the average number of outliers increased %. tat analysis of a representative one week sampling per month showed an increase in outliers from per month to per month. average monthly tys samples performed is . these numbers did not improve even upon returning to the original facilities. study design/method: two ortho clinical diagnostics visionv r analyzers (raritan, n.j.) were purchased for the blood bank. the instruments were set up with a bi-directional interface allowing for samples to be continuously loaded without manually ordering the tests. batch testing was eliminated allowing samples to be run as received. the results were auto interpreted, and transmitted to the laboratory information system (lis) based on predetermined rules. only results in need of manual review or interpretation were held back. final verification of results was performed by the technologist within the lis. reagents and other needed consumables could be preloaded on the instruments eliminating the need to repeatedly load consumables with each sample run. key quality indicators including tat continued to be monitored throughout implementation. data was monitored for significant changes and improvements in patient care. the go-live date was / / . results/finding: the average number of outliers decreased % from per month to . further benefits include a reduction in the number of technologists needed to perform tys testing. additionally, reduced waste due to better utilization of supplies by the instruments along with less repeat testing has resulted in projected cost savings of $ , for fiscal year . conclusion: the use of gel technology, in combination with a two way interface and a continuous load instrument can result in a significant decrease in tat over manual gel method. improvements in the timely reporting of final product culture results in the patient's emr. barbara a. hewitt*. dartmouth hitchcock medical center background/case studies: in certain transfusion reactions it is required that a culture of the returned blood product be performed. these cultures are reported in our cerner operating system but those results do not cross over to the patient's emr . the finalized product culture results are entered into the patient's emr as an addendum to the transfusion reaction clinical note. a review of the transfusion reaction database revealed that there were occasions when the final product culture results were not entered into the patient's emr in a timely manner. it is important to the patient's care for the transfusion medicine service and the patient's primary provider to know if a transfusion reaction is related to a contaminated product or the patient's general overall health. this information is also crucial to the supplier of the product to determine if others have received components of the affected unit and to possibly determine if there are any quality control issues at the donor facility. study design/methods: a review of a specific month period revealed that the timeframe in which the finalized product culture results were entered into the patient's emr ranged from - days with a mean of . days. it was determined that this was not in the interest of improving patient care. in collaboration with laboratory information services a report was created in which once product culture results were finalized an email would be generated notifying the medical director and the transfusion safety officer that results were available. results/findings: data was collected for months following the implementation of this report and it was noted that timeliness of finalized product culture results being entered into the patient's emr improved to a range of - days with a mean of days. conclusion: improvements in patient care require diligence and timely reporting of finalized culture reports to determine potential causes of transfusion reactions. this process can be made easier when the correct tools are used. omer ilyas* and randy levine . northwell health, lenox hill hospital background/case studies: transfusion of non-irradiated blood in patients with hematologic malignancies and those receiving cytotoxic chemotherapy can result in life-threatening graft versus host disease (gvhd). after noting several instances where non-irradiated blood was transfused in patients requiring irradiated blood, we designed a quality improvement project with educational sessions involving the oncology unit and blood bank. study design/method: the project was separated into three parts. in the first part, data on transfusion practices was retrospectively collected over a four month period on the oncology unit. the variables collected included date and time of transfusion, pre-and post-transfusion hemoglobin, patient diagnoses, and whether or not blood was ordered to be irradiated and if so, whether or not irradiated blood was issued by the blood bank. all patients with hematological malignancies and all patients receiving cytotoxic chemotherapy were candidates for irradiated blood. the second part of this project was an educational intervention. residents, oncology floor nurses, and blood bank staff were given lectures on the importance of transfusing irradiated blood on the oncology floor. residents were also instructed to order irradiated blood for all patients on the oncology unit. in the third part of this project, repeat data was collected over a two month period to assess whether the intervention was successful. results/finding: pre-intervention, units were transfused on the oncology floor with units ( %) requiring irradiation and only of those units ( %) ordered as irradiated. since the blood bank occasionally issues irradiated blood without a specific order, additional irradiated units were issued ( / ; %). post-intervention, units were transfused on the oncology floor with units ( %) requiring irradiation and all of those units ( %) ordered irradiated specifically to prevent gvhd. eight additional irradiated units were ordered with no requirement for irradiation; thus of the ( %) total units were ordered as irradiated. again, additional irradiated units were issued ( / ; %) without a specific order by the blood bank. the results are summarized in the accompanying table. conclusion: this quality improvement project demonstrates that educational intervention can succeed in changing clinical practices. continued monitoring of ordering practices will ensure that compliance continues. we plan to expand the quality improvement project to other settings, including the emergency department and surgical floors. we expect that adherence to transfusion guidelines in this patient population will reduce the incidence of adverse events. samantha ngamsuntikul* , charlotte van dyke , dina garza van hoose and rachel beddard . biobridge global, south texas blood and tissue center background/case studies: at our blood center, apheresis platelets and red cells are collected on trima accels and double red cells on haemonetics s. in addition to routine quality control (qc), qc is performed for instrument flags on collection instruments. quality control for apheresis platelets includes: volume variance and rwbc; quality control for apheresis red cells includes: product volume, volume variance, hemoglobin and red blood cell mass. study design/methods: during the period of january , to april , , , total collections were flagged for additional qc by our trima accels and haemonetics instruments. quality control at our center is tracked by our quality control software management system, hematerra's hemacomply which allows the ability to track and retrieve this information. the majority of products flagged for instrument qc pass and are released for distribution. a small percentage, however do fail qc leading to loss of product. quality control data can be retrieved and monitored for trends using a quality control software management system. background/case studies: in , the centers for medicare & medicaid services (cms) rolled out a plan for implementing iqcp (individualized quality control plan) as a new quality control option based on a risk management plan for clia laboratories performing non-waived testing. this plan was meant for clia approved tests, but serves as a good tool for labs performing non-traditional and traditional tests on non-traditional samples. study design/method: clia clinical laboratories can either follow traditional clinical clia qc requirements according to the regulations or implement iqcp. while we perfrom traditional qc assessments on all the tests we perfrom on our cellular products we did decide to implement the iqcp program within in our quality control laboratory. we followed the iqcp process for assessing some of our qc tests used to assess the safety, purity and potency of our cell based products. one test in particular where we applied this tool was in the review of our qc sterility testing method and found it to be a very useful in improving the overall process. the tool walks you through three process requirements: ) risk assessment, ) quality control plan and ) quality assessment for the preanalytical, analytical and post analytical phases of testing. abstract conclusion: the integration of the iqcp into the quality control laboratory was determined to be a success. the iqcp tool was successful in identifying gaps within the sterility testing process. this tool will be used on additional quality control tests and manufacturing processes. the implementation of the iqcp program ensure regulatory qc requirements appropriate for testing performed. we were able to revise our procedures, reeducate those involved in the process and hopefully minimize potential sources of error. objective performance of massive transfusion protocols at a single institution gustaaf de ridder* , rachel jug , kimberly ingersoll , nicholas bandarenko , nicole guinn and jessica poisson . duke health pathology, duke university hospital, duke health anesthesiology background/case studies: hemorrhage is both a leading cause of mortality in trauma patients and morbidity in non-trauma patients. using a balanced : : transfusion ratio (tr) for massive resuscitation is recommended based on trauma data. objective performance during massive transfusion protocol (mtp) activations is poorly studied and there may be differences based on site or medical service of mtp initiation. with the impending release of a unified, redesigned exsanguination protocol (exp) at our institution, we established baseline performance characteristics for our existing mtp and obstetric massive transfusion protocol (obp). study design/method: following institutional review board approval, we performed a retrospective study on blood product utilization and outcomes of mtp and obp activations from july -december . data were manually collected from transfusion service paper records, electronic (safe-trace) records, and an automated data report from the electronic medical record (epic). conclusion: we observed considerable variability in transfusion practices during acute hemorrhage depending on the service and location of activation. trauma activations demonstrated the sharpest deficit in platelet transfusion, whereas all groups lagged somewhat in transfused plasma relative to packed red blood cells. los and mortality varied among groups, likely reflecting underlying medical conditions and indications for massive transfusion. we have identified an opportunity for improvement in mtp transfusion ratios observed in trauma cases, the specific environment from which the : : ratio was derived, and in which the impact of protocol-driven blood resuscitation is most efficacious. patient identification improvement strategy to help reduce unacceptable specimens arline stein* , nancy nikolis , linda benison , ruthmire thelusca , renee liberty , sherry shariatmadar , alexander indrikovs and vishesh chhibber . north shore university hospital, northwell health background/case studies: our blood bank (bb) processes approximately , specimens per year. bb specimens are unacceptable when they are unlabeled, unsigned or missing necessary documentation. in such cases, a new specimen is requested to be drawn as per protocol. our investigation of unacceptable specimens previously included generation of a report by the blood bank staff that was subsequently submitted to the bb supervisor for completion. following completion, the report was sent to the nurse manager of the patient care unit (pcu) for follow-up and investigation with the staff members involved. this process was cumbersome, taking a few days before the staff member of the pcu was alerted to the deviation in protocol. at times, residents or float staff involved were difficult to identify and it was often challenging to track down the staff and do the necessary investigations and in-services. study design/method: in june , a patient identification improvement strategy was implemented jointly by the department of nursing and the bb to address mislabeled, unlabeled and unsigned specimens as part of a patient safety initiative. currently, following this strategy, when an unacceptable specimen is received, the nurse manager (nm) of the pcu is immediately notified by bb staff. the nm promptly initiates a debrief process with the staff involved in drawing the specimen. a debrief form (tool) was created to guide the discussion. this process is followed / . the nm will also engage other available staff in a huddle to review the incident and reinforce the policy. the debrief form is then submitted to hospital qa and the bb with preventative actions included. we believe in using the just culture model to help us understand the reasons why the staff did not label the specimen according to policy. just culture helps promote shared accountability to ensure we have the proper systems and processes in place to deliver high quality care. results/finding: the table below represents the percentage of unacceptable specimens identified by the bb since the second quarter of . the implementation of this new process has led to a decrease in the number of unacceptable specimens up to % quarterly following its implementation. the opportunity for direct intervention by the nm with the staff involved has risen from % to %, due to the immediate debrief process. abstract conclusion: the patient identification improvement strategy allows for real time engagement of the bb and pcu staff to promptly investigate and institute corrective/preventive actions when there is a deviation from policies related to specimen collection. the heightened awareness of correcting patient specimen labeling errors can only improve patient safety and the patient experience. platelet transfusion practices among pediatric oncology patients: a single institutional experience nicole m crews* , , morgan rockwell , joseph hagan , jun teruya , and shiu-ki hui . texas children's hospital, baylor college of medicine background/case studies: despite advances in adult platelet transfusion (ptx) literature, questions persist regarding pediatric transfusion thresholds, dosage and responses. therefore, ptx are commonly guided by local institutional recommendations (ir). the aim of this study was to determine the degree of adherence of ptx practice to ir at a pediatric tertiary institution. study design/method: retrospective review of ptx practices including transfusion thresholds, responses and dosages were collected. platelet counts within hours pre and post transfusion were evaluated. patients ( - years) receiving prophylactic ptx from july to december admitted to the oncology acute care unit with diagnosis of leukemia or lymphoma were included. for prevention of volume overload, the ir for ptx were < ml/kg for patients < kg and one apheresis unit (au) for patients > kg; therefore, patients were separated into groups: < kg and > kg. a significant proportion of orders for both < kg and > kg did not meet patient platelet threshold criteria (p< . ). conclusion: ptx threshold above ir for both groups were ( kg) and % (> kg). most common reason for above ir threshold was an invasive procedure or low molecular weight heparin therapy. greater than % of ptx dosage in both groups were above ir, however the platelet response did not increase significantly (p> . ) with a higher dose vs. ir dose. this study demonstrated that there were still considerable deviations from ir in ptx practice among pediatric oncological patients. in addition, the false assumption that a higher dose will yield a better response can put patients at increased risk for transfusion related adverse events. each institution should conduct a quality assurance review to determine ptx practice. pre-surgical sample process improvement to enhance patient safety and compliance lisa marie button*, stephanie saathoff, jered luedke, benjamin colvin, umalkair amare and james r stubbs. mayo clinic background/case studies: our institution provides the option for presurgical samples (pss) to be drawn up to days prior to surgery as long as the patient reports not being transfused with a blood or blood component containing allogeneic red cells and they have not been pregnant in the preceding months from the date of pss collection. when pss patients returned for surgery, the patient's service was required to ask the patient again about their transfusion and pregnancy history to determine if there had been any new opportunities for allogeneic red cell exposure, however, there was no process to capture the information the patient reported for the time between the pss draw to the day of surgery/possible transfusion. study design/method: an electronic fix was designed that was applied to the surgical intake process. a new set of questions was added to the a.m. admit questionnaire that must be completed prior to the patient's surgical procedure. the questions ask the patient if they have been pregnant or transfused in the preceding three months and if the answer is affirmative, the computer system runs a blaze rule causing an alert in the blood bank. the blood bank techs review the alert and inactivate the patient's pss based on the new transfusion/pregnany information. one year post-implementation of the electronic fix, transfusion lab performed a retrospective review of all pss alerts generated during a three-month period. results/finding: the results of the review were analyzed and are displayed in the table. it was determined that only . % of patients with a pss alert had an active sample requiring inactivation. conclusion: implementing an electronic solution that requires documentation about pss eligibility upon return for surgery has resulted in an estimated ( x ) pss alerts in the blood bank each year. of these alerts, it is estimated that approximately patients ( x ) per year are identified as no longer eligible for pss status. once this retrospective review was performed, it was shared with the project stakeholders to determine if the electronic questionnaire could be further tailored to patient's based on age, gender, and pss status. while the benefit of having fewer false positive pss alerts ( . %) was recognized as an ideal future state, it was not compatible with the institution's current it project of implementing a new electronic medical record (emr) system. the safety enhancement provided by the current electronic fix will remain as is and the improvement suggestions were shared with the team creating the parameters for the new emr with the intention of targeting only patients with an active pss in the blood bank, rather than all surgical patients. weill cornell medicine, columbia university school of medicine background/case studies: blood ordering is a complex, high-risk process with multiple steps that have the potential for errors and delays. risks associated with this process, from ordering through pick-up, require evaluation and strategies for mitigation. given the complexity and high-risk nature of blood ordering a proactive risk assessment (pra) for blood product ordering using the fmea methodology was conducted. the goal of the project was to proactively assess the effect of a redesigned electronic order set on the quality and safety of blood ordering study design/method: to evaluate the electronic blood ordering redesign process, a pra was completed using the fmea methodology. the team identified each step and sub-step of the electronic blood ordering process, all failure modes and causes, and then scored each by severity occurrence and detectability to determine the risk priority number (rpn). all rpns with a score above the threshold were reviewed and rescored based on mitigation strategies designed to address the failure mode. results/finding: the group scored the identified failure modes by categories used in root cause analyses. the electronic blood order process has internal logic and alerts that improve communication and reduce the risk score. several mitigation strategies that will reduce the risk of the identified failure modes include type and screen status within the rbc order, streamlined alerts when the order does not meet the laboratory threshold, a nursing task list for transfusion, and a change to the pickup process that is linked to the product ready status in the laboratory information system. a transfusion history will be available to providers when ordering blood products to further reduce communication risks. categories for failure modes included clinical,communication,equipment people,process and system. the average overall failure mode rpn was reduced by % with the communication category average rpn having the greatest reduction of %. conclusion: an fmea of an electronic blood ordering process can proactively improve quality and patient safety by preventing transfusion delays and errors in blood product administration. accurate and timely information in the blood ordering process has the potential to reduce risks associated with ordering,preparing and dispensing blood. reducing turn around time for type and screens in the blood bank kimberly ouellette* , karen king and joseph sweeney . rhode island hospital, lifespan academic medical center background/case studies: expeditious turn-around times (tat) in the blood bank are critical to provide fully tested and crossmatch compatible blood in a timely manner. the blood bank at rhode island hospital, a level trauma center and teaching hospital associated with brown university, was originally designed to accommodate tube testing by all technologists. the original setup of the lab was split into three sections allowing for preparation and issuing of units in the first section, bench testing in the second, and the receipt of components in the third. as technology changed, the blood bank adopted first the manual gel station and then the automated gel system (ortho provuev r ) but did not adapt the space. the second section of the blood bank contained the manual and subsequently automated gel stations with no other changes. the process of sample receipt through completion of testing and issuing of units remained segmented and inefficient. the average tat for type and screens was minutes. study design/method: the blood bank design was remodeled to make for a more open concept to allow for collaboration amongst technologists as well as the best use of space and technology. the first section of tables were removed and replaced with a center console to allow for movement about the entire front of the laboratory. a wall was constructed to separate the main work flow, automated gel testing and issuing units, from the area for complicated workups and inventory receipt. the third section remained, but was repurposed for teaching medical technology students and residents. in addition to the remodel, the blood bank retired the ortho provuev r for the ortho visionv r , which is considered a true continuous feed machine. although the inter-device tat is not significantly different ( minutes for the provuev r and for the visionv r ) the visionv r is built with a scheduler that effectively handles the system and processes samples efficiently. the visionv r is also equipped with two centrifuges to process samples, which further reduces tat when multiple samples are onboard. a bi-directional interface was designed to allow for test orders (type and screens) to go to the visionv r and test results to go directly from the visionv r to the lis without the need to manually order the tests or transmit the results. data on tat were collected and analyzed using independent t tests and chi square. results/finding: the mean tat pre-and post-reconfiguration and implementation of the ortho visionv r and a fraction of samples with tat over minutes are shown in the table. the results show a reduction in tat by minutes with a % reduction of tat greater than minutes. conclusion: a combination of new technology and space remodeling can lead to a significant reduction of tat of testing in the blood bank. caleb wei-shin cheng* , , lorna orengo , monique scott and christopher a tormey , , . yale university school of medicine, yale-new haven hospital, va connecticut healthcare background/case studies: the type and screen (t&s) is a fundamental laboratory test that allows the blood bank to provide compatible blood for patients. despite this, erroneous blood product administration may occur as much as in , blood transfusions. to prevent errors, adequate specimens such as those lacking hemolysis and those with proper specimen labeling are necessary; otherwise the specimen is rejected, leading to a second blood draw and a delay in medical/surgical management. hemolysis rates for t&s specimens are reported to be as high as % prior to interventions, but may potentially be reduced to as little as . %. however, there is little published data on non-hemolysis-related type and screen rejections. an initiative was undertaken to reduce the rejection rate in the blood bank to a sustained rate of < %, with a particular emphasis on non-hemolysisassociated forms of rejection. study design/method: a root cause analysis (rca) was performed over the preceding months to obtain a baseline understanding of the errors involved. t&s submission at our facility involves standard completion of the specimen label plus completion of a unique witness form to confirm the identity of the patient from whom the specimen was collected; specimen and witness form must be submitted simultaneously. when a specimen was rejected, we recorded the patient name, medical record number, and the reason for rejection. following rca, an intervention was created to resolve the most common issues documented that resulted in rejection. approval for the intervention was granted by the department chair, transfusion committee, forms committee, and the medical executive committee. after implementation, prospective data will be collected for several months in the same manner as before to determine the effectiveness of the intervention. results/finding: over the study period, the t&s rejection rate averaged . %. reasons for specimen rejection were divided into groups: ) hemolysis, ) blood bank witness collection form errors, ) quantity not sufficient, abstract ) duplicate sample, and ) specimen tube labelling errors. the highest percentage of rejections was due to improperly-filled witness forms (table ) . after multiple form redesigns and approval by appropriate committees the new form was implemented. preliminary data collected thus far demonstrates a . % rejection rate with only rejection relating to witness form errors. conclusion: rejected t&s specimens are an impediment to safe clinical care as it may delay medical/surgical management. rejection rates could be reduced through simplification of blood bank specimen collection forms. care providers have multiple tasks that need to be performed in a short amount of time, therefore, simplification is often times necessary to reduce human error. future quality initiatives will aim to simplify complex healthcare processes without compromising patient care. reduction of failed whole blood donor testing runs on the roche cobas s system christopher shahan* , christina dejesus , mosi mccall , fallon hampton , tangi herring , judy davis , anjali patel , sonya gomillion and bonnie maltby . qualtex laboratories, qualtex laboratories background/case studies: as part of our quality control program, we track the number of technician related failed runs observed on the roche cobas s system. this system is used to test whole blood donor samples for human immunodeficiency virus (hiv) rna, hepatitis c virus (hcv) rna, hepatitis b virus (hbv) dna and west nile virus (wnv) rna. technician related failed testing runs can cause the laboratory to report results outside of the contractual - hour turnaround time. failed runs also cause retesting which increases reagent utilization for the system. currently % of whole blood donor testing turnaround time delays are due to issues and failed runs on the s system and we have technician related failures per week. a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failures on the s system. study design/method: the number of technician related failed runs on the s system were tracked from / / thru / / . a pareto chart was used to determine that technician related errors was the largest controllable factor causing run failures. the whys were performed to determine root causes of technician related failed runs. a gemba walk was performed on all of the lab testing processes to help identify areas for improvement. the process improvement team talked, met, observed, and worked directly with staff that operate the s system. roche was also contacted to provide guidance on how to help decrease technician related failures. results/finding: the main root cause determined was that there was no current process flow map for whole blood donor testing using the s system. counter measures implemented included creating a two phase process map. one phase was related to the processes related to start-up of the system and the second phase was related to the processes involved in processing of samples. roche provided a job aid for the technicians which provides clear steps technicians should take when handling and cleaning up crashes and failed runs on the s system. after counter measures were implemented, the number of technician related failed runs decreased from to . failures per week, which was a % decrease. conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of technician related failed runs on the cobas s system. this lean six sigma approach and counter measures significantly decreased the number of technician related failed runs by %. patients who were transfused for pre-transfusion hgb > g/dl with resulting post-transfusion hgb > g/dl were reviewed. demographics, medical history, provider identity, indication and transfusion complications were abstracted & compiled individually by volunteer internal medicine residents. group discussion for each case ensued before determination of transfusion appropriateness occurred. principal investigator/attending physician then made final determination of appropriateness of rbc transfusion. results/finding: patient charts were reviewed. were excluded for bleeding and cardiovascular instability. / ( . %) were determined to be transfused inappropriately. there was no difference in appropriateness of transfusion with respect to age or sex. patients with solid tumors ( . % vs . %, p . ) and anemia of chronic disease ( . % vs . %, p . ) were more likely to be inappropriately transfused. patients who had higher pre-transfusion hemoglobin were more likely to be inappropriately transfused (median hgb . g/dl vs . g/dl, p< . ). inappropriately transfused patients also had higher median post-transfusion hemoglobin ( . g/dl vs . g/dl, p< . ). moreover, lab evalutions revealed association with lower folate levels (median . nmol/l vs . nmol/l, p . ). / ( . %) patients were inappropriately transfused at least in part because they received more than one unit without an interval hemoglobin check in between. / providers were responsible for . % of all inappropriate transfusions. / appropriately-transfused patients experienced an fnhtr. deaths unrelated to transfusion occurred ( in appropriate, in inappropriate group). conclusion: physicians in training are interested in promulgating optimal rbc transfusion practice. this study identified patient factors (such as solid tumors and anemia of chronic disease) that correlate with a higher likelihood of receiving an inappropriate transfusion. beyond cpoe, educational intervention at individual level should be designed for specific providers responsible for more inappropriate transfusions. successful implementation of a blood bank information system in a small-scale caribbean blood bank: a structured step-wise approach. luigi sille* , willem martin smid and ashley john duits . red cross blood bank foundation, sanquin consulting services background/case studies: an important tool for complying with gmp quality standards is the effective use of a blood bank information system (bis). validation and implementation of a bis is described for centralized large blood bank and literature and guidelines are lacking for the nonautomated small scale blood bank environment. . small-scale blood banks face specific challenges for computerization in relation to economies of scale and existing processes requiring special attention. for the introduction of a bis at the blood bank of the dutch caribbean island of curaçao a specific procedure was designed based on existing guidelines and adapted to the local setting. study design/method: the red cross blood bank foundation curaçao is the sole provider of labile blood components for the dutch caribbean islands of curaçao, bonaire and sint maarten. after selection of the bis provider for implementation isbt and bcsh guidelines for validation of information systems in blood establishments were carefully analyzed to prepare the design of local procedures. these procedures were meant to evaluate and validate the features of a bis (e-delphyn, hemasoft america, miami, usa) before introduction. the outcome of the approach was entered in worksheets that were evaluated by the implementation team and management. from this the implementation plan was designed and implemented. an external auditor (sanquin consulting services, amsterdam, netherlands) was invited to evaluate the implementation and validation plan and its practical implementation. the evaluation was performed according to risk assessment of critical process steps. results/finding: based on the isbt and bcsh guidelines a process flow chart describing the relevant phases and critical steps for introduction and validation of a bis was designed. comparison of the current processes and procedures were compared to the bis characteristics making use of worksheets. with these worksheets the existing gaps with the bis procedures were carefully described. these gaps and the appropriate procedural changes for bis or blood bank were effectuated. the worksheets also provided the basis for staff training in a separate training environment before bis introduction. during the early validation phase all procedures and processes were audited by an external auditor. with the feedback of the expert several improvements were added for the validation and subsequent implementation processes. conclusion: with the use of existing international guidelines a validation and implementation plan was designed to prepare for successful introduction of a bis in a small scale caribbean blood bank. the program as designed seems well suited for small scale blood banks contemplating introduction of a bis. time and cost savings through implementation of a remote blood fridge jessica peters* , dee dee cassidy , jed b gorlin , and nancy l van buren , . hennepin county medical center, innovative blood resources background/case studies: rapid delivery of emergency release group o red blood cells (rbc) are vital to patient care. commercial remote blood fridge packages are available but have large upfront and maintenance costs. we implemented a remote blood fridge directly in our emergency department (ed) using an under counter fridge requiring id access, and a selfdeveloped ios application that scans, tracks and real-time alerts transfusion service (ts) to products used and to whom they were dispensed. prior to ed fridge implementation, rbc units were verbally requested and an ed blood runner would pick up and return the cooler. given that our ed is located in a separate building from the ts, this meant or more minutes may be required for transit of units often released in less than minutes. the net effect was that providers would routinely order products to ensure they were at the bedside for patient arrival as a precaution, only to return them when not required. implementing a blood fridge at bedside resulted in the predicted outcome of delivering emergency release rbcs more quickly, with the observed benefit of decreasing wasted staff time. study design/method: the remote blood fridge was implemented in july . data for rbc requests in coolers, rbc returns and rbc transfusions from the ed was collected and compared. baseline data included january -june , and post change included august -december . july data was excluded as it included both the pre and post processes. results/finding: baseline data shows that the ed requested an average of rbc/month in coolers. post change this dropped to rbc/month, thus less blood was requested from the transfusion service in coolers as units were being used from the fridge. baseline data also shows that an average of rbc/month were returned ( %). post change, the average rbc/ month returned was ( %), this represents an absolute % reduction in number of returned products. each rbc dispensed and returned takes approximately minutes to complete paperwork and transport, therefore this change saved an average of minutes per month. it was also noted that the average rbc/month transfused was for baseline and post change. this confirms that the decreased requests and returns were not due to decreased patient volume or severity. the fridge was also successful at decreasing delivery time of blood to patient bedside, as baseline delivery time of - minutes (estimated) was reduced to - minutes. conclusion: implementing a remote blood fridge and moving blood access closer to patient bedside ensures a faster delivery of blood to the patient. this change has an additional benefit of decreasing wasted time, and hence cost, by decreasing unnecessary product requests and returns. implementing a blood fridge can also be done at a reduced cost through homegrown processes. transfusions are everyday procedures and over patent-applications have been filed related to "transfusion medicine" and over related to "transfusion alarm", during the last years, employing numerous technical settings, aiming to support automated supervision of the mentioned actions. the aim of this contribution is to present a developed low-cost real-time individual intravenous blood-transfusion monitoring system, based on the internet of things. study design/method: the designed system is based on a commercially available pan-tilt-zoom (ptz) camera, employing an / inch color cmos sensor, providing effectively . mp, a . mm lens, ir-cut, day/night minimum illumination . lux/f and viewing angle. the camera is focused on the droplets and acts as vis/ir detector with a hz sampling-rate. custom-developed software supports droplets' ratemonitoring, causing acoustic alarm-signals if necessary (e.g. clotting, blood a transfusion vol. supplement s abstract or other suspensions depletion etc.) and enables, if necessary, wide-angle image-capturing. the video image-audio settings provide for compression h. , video frame rate (fps) - /s, refresh rate hz and audio input, through bidirectional built-in microphone. the acquires an ip-address, the connection mode is wireless, the network interface is wi-fi/ . /b/g, the supported protocols include dhcp, tcp/ip, upnp, http, smtp and p p is provided. typical v power-supply, sized x x mm and weighing g. client software is required. the ir range is - m; ir-cut filters, remote access, dual stream, motion detection, day/night and ir night vision distance of m are offered. two-way radio-link is provided, as well as, trans-flash (tf) recording and storage on a gb sd-card. pan/tilt-horizontal o and pan/tilt-vertical o movements can be performed. the system facilitates, if needed, also patient's position monitoring and readings of other monitoring displays, such as nibp, ecg, and spo , if present. results/finding: the system and is being presently tested in a laboratory (non-clinical) environment, by simulating the virtual patient, with a custommade "phantom", combining flow-rate, negative pressure and viscosity resistance regulation. conclusion: the system can measure infusion-speed with a deviation lower than %. the developed iot-system takes advantage of the existing hospital wi-fi networked environment and offers a low-cost solution, under $ for each monitoring-set. it allows for even multi-platform (ios, android, windows) smart-phone, short-range connectivity, for up to participants, for example nurse, physician etc. two potential approaches for the quality control of bact/alertv r culture media using various bioball tm organism preparations patricia rule*, michelle keener and christine crawford. biomerieux inc. background/case studies: the bact/alertv r bpa and bpn culture bottles are used with the bact/alert microbial detection system for rapid screening and detection of microbial contamination in leukocyte reduced apheresis platelets (lrap). recent changes in the clia quality control guidelines and aabb accreditation program will require additional quality control of manufactures media that is both lot specific and shipment specific to ensure recovery of bacterial growth. a study was conducted using commercially prepared organisms evaluating both a comprehensive organism panel as well as a streamlined method utilizing only two organisms from the panel. study design/method: the general protocol consisted of three replicates each of each organism inoculated into two lots each of bpa and bpn by two different analyst. the study was two part in that aspergillus brasiliensis, candida albicans, bacillus subtilis subsp. spizizenii, pseudomonas aeruginosa, escherichia coli, clostridium sporogenes, staphylococcus aureus and streptococcus pyogenes were prepared from bioball singleshot ( cfu), multishot cfu or highdose k organism preparations at a low level (< cfu) and evaluated on the same day of preparation as method validation. the second part of the study utilized escherichia coli and staphylococcus aureus prepared and frozen at a higher level and then evaluted over a day study as a stream line approach to routine quality control testing of the bact/alert culture bottles. inoculation preparations were enumerated in duplicate to confirm the level at each inoculation time point. inoculated bpa and bpn bottles were loaded into the bact/alert microbial detection system at c for automatic monitoring of growth. negative bpa and bpn bottles were included in duplicate at each day of testing. results/finding: escherichia coli, staphylococcus aureus, streptoococcus pyogenes and bacillus subtilis subsp. spizizenii were positive in both the bpa and bpn culture bottles. the aerobic aspergillus brasiliensis, candida albicans, and pseudomonas aeruginosa grew and were reported positive in only the bpa aerobic culture bottle as expected. while the obligate anaerobe, clostridium sporogenes was positive only in the anaerobic bpn culture bottles. bacterial cultures were positive in the bact/alert bpa and bpn bottles < days and the fungal organisms in < days. the overall agreement was . % in bottles tested here. no significant differences were observed in the time to detection between the different lots or between the different analyst. conclusion: the bioball prepared organisms demonstrated a reproducible method as both a comprehensive and streamline approach for the quality control of bact/alert bpa and bpn culture media. the method was simple and did not require additional microbial preparations or storage of live organisms by the laboratory. use of an electronic patient identification system for blood banking specimen labeling found to be superior over historical armband approaches annie newton* , diane schafer , debra brown , jesse cox , scott koepsell and sara shunkwiler . nebraska medicine, the nebraska medical center, university of nebraska medical center background/case studies: anticipating the implementation of the new ( th addition) aabb standard concerning the confirmation of patient abo blood typing of type and screen (crossmatch) specimens performed prior to the issue of crossmatched blood products, laboratory and organizational leadership evaluated the practical application of an electronic patient identification system to label blood bank specimen collections versus the traditional use of blood bank armbands. continued use of the armbands would require a second sample for abo confirmation of patients that did not have a historical blood type on file. concern was raised regarding the amount of increased workload of staff and delayed results availability based on the number of increased specimens that would be generated, as well the potential for increased iatrogenic blood loss and patient dissatisfaction. moreover, nd sample collection alone would not improve the rate of mislabeled specimens observed, which is of supplementary concern. study design/method: current organization employment of an electronic patient identification system for the labeling of other laboratory specimen collections made it feasible for applying this technology to the blood bank as well. an in-depth evaluation, including a failure modes and effects analysis (fmea) spanning several days, was completed to ensure that the use of the electronic system would produce comparable or superior safety results to its armband counterpart. an alternate process for specimen labeling and abo confirmation (which would satisfy the new standard) was established to support care areas that did not have the capability of using the electronic system. extensive education was provided to all staff (physicians, advanced practice providers, phlebotomist and nurses) to ensure comprehension as well rational for the new process. alerts were congruently built into the electronic health record (ehr) to supplement any information regarding crossmatch testing expiration that may not be readily available by the elimination of the armband use. results/finding: within days of implementing the new process (september , ), there was a noticeable reduction in the amount of mislabeled blood bank specimens received, totaling in months post implementation compared to in the months prior. in addition, the vast majority of specimens received into the blood bank are henceforth collected and labeled using the electronic system and thus have reduced the amount of potential nd specimen collections needed for abo confirmation. conclusion: use of an electronic patient identification system for labeling blood bank specimen collections in lieu of traditional blood bank armbands has proven to improve patient safety and department efficiency by substantially reducing the occurrence of mislabeled specimens and negate the need for nd specimen collections, reducing potential iatrogenic blood loss and improving patient satisfaction. background/case studies: based on a few small randomised controlled trials (rcts) performed in the late ' s and in early , intravenous immunoglobulin (ivig) use has been suggested as a potential treatment to avoid exchange transfusion (et) for rh hemolytic disease of the newborn (hdn). this treatment modality is now routinely used for rh-hdn and has been extended to hdn caused by abo incompatibility or by other red blood cell antibodies. however, larger rcts performed since have shown that prophylactic ivig did not reduce the need for et, the duration of hyperbilirubinemia, the maximum bilirubin levels nor the need for top-up red blood cell transfusions. the primary objective of this study was to describe the usage of ivig for hdn at a tertiary academic referral hospital. study design/methods: a retrospective chart review was performed of all neonates who received ivig for hdn in the neonatal intensive care unit (nicu) from january , to june , . data collected included patient demographics features and diagnosis, indications for ivig, neonatal laboratory results, treatment details, adverse events and patient outcomes. results/findings: ninety-seven neonates received ivig during the study period: % were female and % were less than weeks of gestational age. none had co-existing g pd deficiency, pyruvate kinase deficiency or spherocytosis. all neonates received phototherapy prior to ivig treatment. indications for ivig were abo-hdn ( %) and rhesus-hdn ( %). antibodies most often implicated in rh-hdn were anti-d ( / ), anti-d and anti-c ( / ) and anti-c ( / ). sixteen infants with rh-hdn had received intrauterine transfusions. the mean cumulative dose of ivig was g/kg (range from , g/kg to , g/kg). neonates received one to four ivig administrations. table shows the number of patients receiving ivig during two time periods. three adverse reactions were noted during ivig administration: cutaneous rash, hypotension and fever. of all neonates, required an et for rh-hdn and for abo-hdn. forty-five ( %) patients needed top-up transfusions during hospitalisation and until three months of age: with abo-hdn and with rh-hdn. the mean number of transfusions was three (range: to ). conclusion: although initially described for rh-hdn, abo-hdn is now one of the most frequent indications for ivig in neonates. the optimal use of ivig in abo-hdn needs to be better characterized. our study shows a wide variation of ivig dosing and a significant proportion of neonates requiring top-up transfusions. further research is required to evaluate whether anemia in abo-hdn might be exacerbated by hemolysis from ivig isohemagglutinins and if it is dose-dependent. background/case studies: background: one of the most serious adverse reactions to transfusion is the development of graft versus host disease. symptoms include the development of a characteristic cutaneous rash, enteritis often resulting in watery diarrhea, elevated liver function tests and ultimately pancytopenia. the clinical course is rapid with an over % case fatality rate. the patient population at risk is reasonably well-defined including patients who are immunocompromised due to disease process or therapy, the fetus and low birth-weight neonates, recipients of hla-matched cellular blood products and the recipients of cellular blood products donated by blood relatives. the basic etiology of ta-gvhd is the inability of the transfusion recipient to mount an effective immune response against donor t-lymphocytes. treatment options for ta-gvhd are ineffective, making it imperative that cellular blood components be irradiated prior to transfusion which virtually eliminates the risk of the complication. study design/methods: most transfusion service information systems have mechanisms to alert transfusion service staff to patients who have been previously identified as needing irradiated blood components. however, if these patients are not identified to the transfusion service at the time of the initial hospital visit or the time at which the qualifying diagnosis, these patients can erroneously receive non-irradiated blood components. following a "near-miss" situation, our hospital information department developed a -part program to minimize the risk that the transfusion service is not notified of patients newly requiring irradiated blood components. results/findings: our blood products ordering system has been redesigned to include specific queries to identify those patients who required irradiated cellular blood products. first, physicians have been notified to include the need for blood product irradiation in the patient problems list. once this is included in the list, the transfusion service will be notified of the need for irradiation on all subsequent transfusion orders until the problem list is modified by the clinical staff. second, if irradiated blood components have ever been requested on a patient, an alert will be generated for the ordering physician even if the requirement for irradiated products has not been included in the problem list. finally our system will automatically default to request irradiation on all cellular products ordered for children less than months of age to comply with local irradiation policies. conclusion: we believe that our approach can be further enhanced by including a list of specific diagnoses typically requiring blood product irradiation within our computer algorithm. we believe that this list will provide an additional level of safety in insuring that patients receive irradiated blood components when appropriate. using lab information system and a dynamic dashboard for labeling and tracking coolers russell thorsen, rosaline ma, peter suslow, gina giannarelli, sara bakhtary, ashok nambiar and morvarid moayeri*. ucsf health background/case studies: our tertiary-care transfusion service routinely issues blood products in validated coolers to high acuity areas such as ors, icus, cath-lab, etc. coolers are also used for emergency release and massive transfusion protocols, and for shipping products between our different hospital sites. a robot that can hold one cooler delivers products to locations not served by the pneumatic tube. on average, coolers are issued every day. cooler set-up is a multi-step, labor-intensive process. transfusion service staff track cooler location and elapsed time-in-use and notify clinical teams to return/recharge coolers to avoid product wastage. we developed a lab information system (lis)-based solution to manage cooler labelling and tracking more efficiently. study design/method: nine cooler test batteries were built; the batteries for rbc, plasma, platelet and cryoprecipitate ( each) are identical, whereas the final battery designated for the cooler delivered by robot (containing plasma and rbcs with variable expiration times) is slightly different. the second battery in each pair was built to avoid duplicate test cancellation by lis when a second cooler (for same component type) is being set up for the same patient. each battery consists of tests capturing the following information: cooler location, cooler id, number of units issued, and expiration. custom barcodes representing each test battery and different locations can be scanned from a 'quick-pick list', avoiding need for manual entry. when coolers are returned, a final entry is made in the test battery, updating lis. a dynamic cooler tracking dashboard with live-feed from lis displays data captured in the test battery. elapsed time, starting from cooler set-up (which is identical to time cooler battery is ordered in lis) is captured automatically. color codes alert users to coolers that have less than hour before expiration. a flashing alert pops up for coolers that have expired. results/finding: we replaced our manual process (hand-write patient information and expiration time on separate tags; affix one tag to cooler and retain second one to track cooler location and expiration) with a novel lis-driven labeling and tracking system. each time a cooler is set up, a test battery is ordered and resulted in lis by scanning the related custom barcodes. a single lis-generated label is printed and attached to each cooler. cooler expiration is defaulted to hours (per our current cooler validation) from the time the test battery is ordered during cooler set up. techs pay attention to expiration of each product they place in a cooler. if an individual product outdates before the cooler expiration time, this information is entered in the test battery and gets displayed on the dashboard as a cooler expiration time, distinct from the system-driven countdown. color-coded visual display and alerts greatly simplify cooler monitoring, and the elimination of some manual steps has improved staff satisfaction. conclusion: using lis for cooler set-up and deploying a linked dynamic dashboard to display cooler locations and expiration time makes cooler management more efficient. these tools reduce manual steps and decrease likelihood of wastage by aiding cooler tracking. improving cryoprecipitate collection operations using operations research and analytics-based methods american red cross, georgia institute of technology ap reduction in unnecessary use of type o-negative rbcs in a level i bellevue hospital-nyulmc our hospital is a level i trauma center serving a diverse predominately non-caucasian population. historically we stocked our trauma blood bank monitored refrigerator with o-negative rbcs. trauma requested that we stock additional rbcs to be able to initiate a mtp for multiple patients at the same time. believing that most of our trauma patients are male, elderly, or rh-positive, we agreed add type o-positive rbcs to the stock. rules for determining which units to use were established. o-positive rbcs are to be given to a) all adult males (am), b) women of non-childbearing age (wncba), and c) if both o-negative rbcs were used but not yet restocked, and o-negative rbcs are to be given to a) women of childbearing age (wcba) and b) children until the patient's aborh type are determined. we sought to assess the impact of this change on our usage and purchases of o-negative rbcs. study design/method: all patients issued emergency release trauma rbcs following the addition of o-positive rbcs were assessed %) would have needed to be o-negative. the addition of o-positive rbcs to our trauma refrigerator will enable us to markedly reduce our purchases of o-negative rbcs. ap saving apheresis platelets through use of verax point of care testing jennifer rhamy* and rebecca wride . st. mary's regional blood donor center, st. mary's regional medical center background/case studies: our rural hospital-based blood center serves hospitals and a diverse patient population including acute trauma. because of the varying need for platelet products (varies between and per day in ), we investigated the use of the verax point-of-care test to better manage our valuable inventory barrett lawson and jun teruya , . texas children's hospital, baylor college of medicine ap vision titers --easier or problematic? (table ) . results/findings: post intercept, t had volumes of - ml, with % hemoglobin (hb) recovery. t had -fold less extracellular protein than c. after days of storage t had higher atp and na than c while lactate and hemolysis were lower. hct, ph, k and glucose were equivalent between t and c on d . d hemolysis for t was . - . %, while for c it was . - . %. t and c atp was > mmol/g hb, the level of atp associated with effective rbc viability, throughout storage (table ) . hematocrit (hct, %) . . * . . . . . . hemoglobin (g/unit) not measured hemolysis (%) . . * . . . . * . . ph ( c) . . * . . . . . . total atp (mmol/g hb) . . * . . . . * . . k (mm) . . * . . . total tested total plts issued feb mar totals table: . resident reports to the intranet "drop box" increased from . % to . % to %, each over month time spans. conclusion: safe transfusion ordering requires a team approach to ensure the right information is available to the ordering provider at the right time. safe ordering prevented recurrent allergic reactions in our patient population. the tso plays a pivotal role in ensuring the full circle of communication occurs. processes that integrated the pathology resident improved with pdsa cycles and impacted the quality and timeliness of hand off. finally, the data provided from the residents enabled efficient participation in hemovigilance. decreasing results/finding: the main root cause determined was that there was no standard work process. sops were being followed but there was no standard work process that included the details so testing was not following the most efficient work flow. counter measures implemented included implementing a standard work process, visual cues were added to the work process, and a samples awaiting testing report was created for the batch release department. specific locations were identified within the work cells in the lab to place samples based on their phase/stage of testing. after counter measures were implemented, the number of exceptions decreased from . per day or , dpmo to . per day or , dpmo. this is a statistically significant difference since the p-value calculated was . . conclusion: a lean six sigma approach for process improvement was utilized to identify root causes and develop countermeasures in order to decrease the number of exceptions related to the testing of whole blood samples in the laboratory. this approach and counter measures statistically significantly decreased the number of exceptions seen in the whole blood testing process. background/case studies: our blood bank processes approximately , specimens per month. since , the requirement of having a second blood type on record was met by: . utilizing the historical blood type and the current specimen, or . having second type performed on same specimen by different technologist, and . each type and screen specimen signed by staff, one being a licensed practitioner attesting the identification of the patient was done accurately at bedside.to comply with the aabb standards th edition, # . . . a decision was made to change our practices. we considered challenges encountered at other hospitals and collaborated with nursing and it to create a streamlined and safe process. in april , the second specimen procedure was implemented addressing the following: ii. extensive education was provided to all involved in the process prior to implementation including a learning module prepared by blood bank and nursing collaboratively. results/finding: . there was a minor adjustment period with more phone calls made to blood bank to explain the process. . there was minimal impact on turn around times for release of components. . aborh retype workload decreased from to ( % to % of t&s volume) per month. . unnecessary blood draws minimized, improving patient experience. . no emergency release requests due to absence of a second specimen. the second specimen process with the conditional order has been beneficial to our blood bank as well as patient care services. overall feedback from staff on the process has been positive. our workload has decreased which results in cost savings and increased efficiency allowing us to devote more resources to the growing services at our institution. background/case studies: the hazards of transfusion are well recognized and in certain cases restrictive transfusion strategies compared to liberal transfusion strategies may be associated with better clinical outcome. with this in mind, aabb and others published guidelines for transfusion, but even with guidelines in place, rate of inappropriate blood transfusions is reported to be as high as to %. computerized provider order entry (cpoe), is a process of electronic medical order entry for medical practitioners with instructions and guidelines for treatment. the objective of this study was determination of transfusion practice quality by thorough chart and electronic medical record review, with measures in place to avoid inappropriate transfusion. additionally, factors associated with inappropriate transfusion were examined. study design/method: in our bed hospital, a retrospective chart review was performed ( / / - / / ) on hospitalized internal medicine patients. cpoe with hospital guidelines for rbc transfusions were in place. transfusion thresholds in different clinical settings were determined by a thorough literature review of studies analyzing restrictive transfusion strategy, and transfusion guidelines by various medical societies. charts for background/case studies: our midwestern university-based transfusion service (ts) evaluated the appropriateness the automated platform vision (ortho clinical diagnostics. raritan, nj) for prenatal titration studies. it has been established from previous publications that the micro-column assay, of which the vision is based, may lead to higher titer results compared to standard tube titrations. this study sought to evaluate the transition from manual to automated titer studies from a sensitivity as well as cost perspective. study design/method: twenty-three prenatal retention plasma samples were tested as part of the evaluation of titration studies of the vision. the samples were manually tested with a standard two-fold serial dilution. the titer was reported as the last tube to demonstrate a reaction by macroscopic observation. the titer studies were then repeated using the vision. the results of the manual and automated processes were compared and categorized as "< grade" or "> grade" difference between endpoints. this analysis is similar to the acceptable ranges used for evaluating college of american pathologists (cap) proficiency survey challenges. a cost analysis was completed based on the direct and indirect cost for each method, excluding the cost of an analyzer. results/finding: table demonstrates a summary of the samples tested by manual titer study and vision titration method. the vision titer results (mean, median, and mode) were higher than the manual tube titer results. less than half of the samples ( %) were > titer results higher, while the majority was titer results different ( %). the cost analysis is summarized in table . the indirect cost (labor) was significantly lower with the use of the vision. the reduction in pre-analytical technical time for manual preparation of the titration is eliminated with the vision completing the titration as part of the profile of the titer study of the analyzer. conclusion: with an estimated % decrease in the cost of a vision titer compared to manual tube method, the change in practice would clearly be a cost and efficiency measure in the blood bank. however, the vision demonstrated the expected increase in titer results compared to manual tube titer results. this would impact the critical values currently utilized. an impact assessment for clinical staff would be necessary to adequately implement the change in method. consideration must be given to changes in the computer logic for critical values on titer studies and training of physician and nurse obstetric practitioners for changes in the critical values. in addition, as part of changing to the vision an implementation period will be necessary to ensure that manual titers are compared to previous manual titers and not to vision titer results which would be higher and may be interpreted as a significant change for clinical care of the patient. what is the best practice for testing residual white blood cells in blood components for monthly routine quality control? janja pajk*. general hospital celje background/case studies: we wanted to discover what is the best routine quality control practice for testing residual white blood cells in blood components. our aim was to validate the adam device for counting thne number of residual white blood cells (wbc) in leucocyte depleted and in non-leucocyte depleted blood components (bc) and to compare with standard counting method by microscopy in fuchs rosenthal chamber (frc) used in ghc and with flow cytometry (fc). study design/method: after samples of red blood cells (rbc), platelets (plt) and fresh frozen plasma (ffp) (leucocyte depleted in top and top (t/ t) bags and non-leucocyte depleted in top and bottom (t/b) bags) were stained with propidium iodide (pi) on r-slides; adam -rwbc device was messured fluorescent images of stained wbc nucleus. data were analised by image analysis software and later compared with results of testing samples in frc by microscopy and with fc. samples of bc were microscopic tested in frc at department of laboratory medicine in ghc; another samples were measured with fc in ucc maribor. results/finding: samples ( rbc, plt, ffp-all leucocyte depleted and non-leucocyte depleted ffp) were tested in triplicates on adam and with frc once.coefficient of variation of (kv%) of samples measured on adam for leucocyte depleted bc varied for: rbc from , - , ; plt from , - , ; ffp from , - , ; and for non-leucocyte depleted ffp from , - , (table ) . samples ( rbc, plt, ffp -all leucocyte depleted and nonleucocyte depleted ffp) were tested in triplicates on adam and with fc once.kv% of samples measured on adam for leucocyte depleted bc varied for: rbc from , ; plt from , , ffp from , ; and for non-leucocyte depleted ffp from , - , (table ) .high percentage of kv was noticed in samples with low numbers of wbc (in leucocyte depleted bc; low percentage of kv in non-leucocyte depleted ffp, with higher amount of wbc was observed. conclusion: all samples tested with adam met expected criteria for wbc in bc in european union (less than x /unit for leucocyte depleted or x / unit for non-leucocyte depleted) and were comparable with those tested with fc; the correlation with microscopy in frc was worse.with use of disposable r-slides, the risk of exposure to the potential hazardous blood samples is grately reduced, the method is more precise and not time consuming.from january we changed our protocol for testing residual wbc in bc with adam device and we advise it as the best practice for monthly routine quality control.